Patent application title: Transgenic mammal carrying GANP gene transferred thereinto and utilization thereof

Inventors: Nobuo Sakaguchi
Agents: BIRCH STEWART KOLASCH & BIRCH
Assignees: Immunokick Incorporation
Origin: FALLS CHURCH, VA US
IPC8 Class: AA61K39395FI
USPC Class: 4241301

Abstract:

It is an object of the present invention to provide a high affinity antibody effective as a diagnostic or therapeutic for various diseases; a transgenic mammal for producing the high affinity antibody; and a medicine comprising the high affinity antibody or a cell producing the high affinity antibody. According to the present invention, a transgenic mammal carrying a GANP gene transferred thereinto, its progeny, or a part thereof, and a method of producing a high affinity antibody using the same are provided.

Claims:

1. A high affinity antibody or a fragment thereof produced by a process of:administering an antigen to a transgenic non-human mammal or its progeny and recovering an antibody from the resultant mammal or progeny,wherein said transgenic non-human mammal comprises a GANP gene.

2. The antibody according to claim 1, wherein said antibody has an affinity of 1.times.10.sup.-7 M or less as expressed as a dissociation constant, or a fragment thereof.

3. The antibody according to claim 1, wherein said antibody is a polyclonal antibody or a monoclonal antibody, or a fragment thereof.

4. The antibody according to claim 1, wherein said antibody is a humanized antibody or a human antibody, or a fragment thereof, and wherein said antibody comprises a V region of said antibody or a fragment thereof.

5. A pharmaceutical composition comprising the antibody according to claim 1, or a fragment thereof.

6. The pharmaceutical composition according to claim 5, wherein said antibody or a fragment thereof is a humanized antibody or a fragment thereof.

7. The pharmaceutical composition according to claim 5, wherein said antibody or a fragment thereof is a human antibody or a fragment thereof.

Description:

[0001]This application is a Divisional of co-pending application Ser. No. 10/534,043 filed on Mar. 30, 2006 and for which priority is claimed under 35 U.S.C. § 120. application Ser. No. 10/534,043 is the national phase of PCT International Application No. PCT/JP2003/014221 filed on Nov. 7, 2003 under 35 U.S.C. § 371. This application also claims priority to PCT/JP02/11598. The entire contents of each of the above-identified applications are hereby incorporated by reference.

TECHNICAL FIELD

[0002]The present invention relates to a transgenic mammal carrying a GANP gene transferred thereinto and utilization thereof. More specifically, the present invention relates to a transgenic mammal that expresses a high level of GANP and is capable of producing high affinity antibodies; a method of producing a high affinity antibody using the transgenic mammal; and utilization of the resultant high affinity antibody.

BACKGROUND ART

[0003]The functions of the immune system are classified into the function based on cellular immune responses caused mainly by the effect of T cells and the function based on humoral immunity caused mainly by the effect of antibodies. Actually, these two functions co-operate with each other to perform immune responses. Antibodies are present as cell surface receptors on the surfaces of B cells produced in the bone marrow. It is said that the number of diverse antigens recognized by the first antibody produced in the living body reaches the order of 10⁹ to 10¹¹. Such antibodies (antigen receptors) recognize all antigenic determinants that may exist in environments. However, these diverse antigen receptors are generally low in their ability to bind to antigens, and in many occasions, low affinity antibodies are produced. Such antibodies can not cause sufficient immune responses.

[0004]Lymphocytes, especially B cells/immunoglobulins (antibodies) are used in various applications based on their immune responses, e.g. they are used in kits for detecting the antigens of pathogens, or as diagnostics or therapeutics. If an antibody that has high reactivity with antigen is used in such antigen-detecting drugs or various therapeutics, sensitivity to antigen will be excellent and efficacy as a therapeutic at a same dose will be great. However, no means to enhance the affinity of antibodies have been known.

[0005]When pathogens or foreign substances have entered the living body, the body recognizes them as antigens and induces highly frequent somatic mutations in the genes of the V regions of antibodies which bind directly to those antigens. Such changes require stimulation from T cells, and it is considered that stimulation is provided from activated T cells in the germinal center region. Recently, the present inventors have found a molecule designated GANP whose expression increases selectively in activated B cells of this region (WO 00/50611). This molecule directly binds to a molecule called MCM (minichromosome maintenance) having DNA helicase activity, and has RNA primase activity. Therefore, it is suggested that this molecule GANP is involved in DNA replication. However, functions of GANP in the immune system have not yet been elucidated.

DISCLOSURE OF THE INVENTION

[0006]It is an object of the present invention to provide a high affinity antibody effective as a diagnostic or therapeutic for various diseases; a transgenic mammal for producing the high affinity antibody; and a medicine comprising the high affinity antibody or a cell producing the high affinity antibody.

[0007]As a result of extensive and intensive researches toward the solution of the above-described problems, the present inventor has found that a GANP gene-transferred transgenic animal is capable of producing a high affinity antibody when immunized with an antigen. Thus, the present invention has been achieved.

[0008]The present invention relates to the following.

[0009](1) A transgenic mammal carrying a GANP gene transferred thereinto or its progeny.

[0010]The transferred GANP gene is capable of being expressed in B cells. The transgenic mammal of the invention or its progeny may be generated from GANP gene-infected ES cells. As the mammal, mouse may be given, for example.

[0011](2) A part of the above-described transgenic mammal or its progeny.

[0012](3) A method of producing a high affinity antibody, comprising administering an antigen to the above-described transgenic mammal or its progeny and recovering the antibody from the resultant mammal or progeny.

[0013](4) A high affinity antibody obtainable by the method of (3) above, or a fragment thereof.

[0014]The antibody of the present invention is 1×10^-7 M or less as expressed as a dissociation constant. The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody.

[0015](5) A humanized antibody or human antibody, or a fragment thereof, comprising the V region of the above-described antibody or a fragment thereof.

[0016](6) A pharmaceutical composition comprising at least one selected from the group consisting of the above-described antibody or a fragment thereof, and the above-described humanized antibody or human antibody, or a fragment thereof.

[0017](7) A high affinity antibody-producing cell which is taken from the transgenic mammal according to any one of claims 1 to 4 or its progeny, wherein the transgenic mammal or its progeny has been administered an antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019]FIG. 1 shows the results of immunohistochemical analyses using anti-GANP monoclonal antibody and ALP-conjugated anti-rat Ig antibody. Scale bar is 100 μm.

[0020]FIG. 2 shows the rates of appearance of GANP^hi cells in popliteal lymph nodes of female NZB mice. Scale bar is 100 μm.

[0021]FIG. 3 shows the rates of appearance of GANP^hi cells in the spleens of female NZB mice. Scale bar is 100 μm.

[0022]FIG. 4 shows the results of the staining of plural lineage mice-derived spleen sections with anti-GANP monoclonal antibody. RP: red pulp; F: follicles. Scale bar is 100 μm.

[0023]FIG. 5 shows the identification of GANP^hi cells in the spleen red pulp.

[0024]FIG. 6 shows the identification of plasma cell markers in GANP^hi cells. Scale bar is 100 μm.

[0025]FIG. 7 shows the appearance of GANP^hi cells in the red pulp region of the spleens of C57BL/6 mice as a result of immunization with TD-Ag. Scale bar is 100 μm.

[0026]FIG. 8A-C shows somatic mutations in Daudi cell transfectants which are engineered to express mouse GANP stably.

[0027]FIG. 9A-C shows an outline of the preparation of a transgenic mouse which is engineered to overexpress GANP in its B cells.

[0028]FIG. 10 (SEQ ID NOS: 37-72) shows the results of analyses of somatic mutations in transgenic (Tg) mice overexpressing GANP and wild-type mice.

[0029]FIG. 11A-E shows an outline of the preparation of a B cell-specific GANP deficient mouse (B-GANP.sup.-/-).

[0030]FIG. 12 shows the results of analyses (flowcytometry) of cell surface staining using the B cell-specific GANP deficient mouse (B-GANP.sup.-/-).

[0031]FIG. 13 shows the results of B cell proliferation assays. Almost no difference was observed, but only the proliferation caused by anti-CD40 antibody stimulation was decreased to about 1/2.

[0032]FIG. 14 shows antibody titers in the sera from non-immunized Cre-flox/+ mice and B-GANP.sup.-/- mice. No difference was observed among the antibody titers of individual isotypes.

[0033]FIG. 15 shows the results of measurement of antibody production in B-GANP.sup.-/- mice.

[0034]FIG. 16 shows the results of the staining of GC with peanut agglutinin.

[0035]FIG. 17 shows the results of measurement of antigen-specific antibody production in B-GANP.sup.-/- mice.

[0036]FIG. 18 shows the results of measurement by differential ELISA of the degrees of maturation of affinity in mice 14 and 35 days after immunization with 100 μg of NP-GC.

[0037]FIG. 19 shows the results of flowcytometry on GC-B cells.

[0038]FIG. 20A-F shows the results of sequence analyses of V_H186.2 in Cre-flox/+ mice after PCR amplification (sequences (SEQ ID NOS: 73-88) continue from A to F in this order).

[0039]FIG. 20G-L shows the results of sequence analyses of V_H186.2 in Cre-flox/+ mice after PCR amplification (sequences (SEQ ID NOS: 89-105) continue from G to L in this order).

[0040]FIG. 21 shows the frequencies of IgG1 mutation in Cre-flox/+ mice and B-GANP.sup.-/- mice.

[0041]FIG. 22 shows the frequencies of ³³W to L mutation in V_H186.2 in Cre-flox/+ mice and B-GANP.sup.-/- mice.

[0042]FIG. 23 shows the results of measurement of activation-induced cell death (AICD) and the results of apoptosis inhibition.

[0043]FIG. 24 shows the results of measurement of the apoptosis sensitivities of cells to anti-CD40 and anti-CD95 stimulations.

[0044]FIG. 25 shows the results of detection of apoptosis cells by TUNEL assay.

[0045]FIG. 26 shows the results of detection of apoptosis cells by TUNEL assay.

[0046]FIG. 27 shows the RNA expression levels of Bcl-2 family involved in apoptosis inhibition.

[0047]FIG. 28 shows the results of production of a high affinity antibody using a GANP transgenic mouse.

[0048]FIG. 29 shows the results of production of a high affinity antibody using the GANP transgenic mouse-derived hybridoma clones.

[0049]FIG. 30 shows association-dissociation curves obtained with Biacore on culture supernatants of the GANP transgenic mouse-derived hybridoma clones.

[0050]FIG. 31 shows association-dissociation curves obtained with Biacore on culture supernatants of the GANP transgenic mouse-derived hybridoma clones.

[0051]FIG. 32 shows an outline of the structure of GANP-GST fusion protein.

[0052]FIG. 33 shows the results of a pull-down assay for determining the region of GANP which directly binds to MCM. Shown on the left side of each panel are the positions of size standards.

[0053]FIG. 34 shows the results of a pull-down assay using in vitro translated MCM.

[0054]FIG. 35 shows the binding of individual GANP constructs to MCM by immunoprecipitation.

[0055]FIG. 36A-B shows the binding of individual GANP constructs to MCM by immunoprecipitation.

[0056]FIG. 37 shows an outline of the structures of GANP constructs and their intracellular distributions.

[0057]FIG. 38 shows intracellular distributions of GANP constructs.

[0058]FIG. 39 shows the nuclear localization of MCM3.

[0059]FIG. 40 shows the cytoplasmic localization of MCM3 induced by GANP expression.

[0060]FIG. 41 shows a control protein localized in the nucleus.

[0061]FIG. 42 shows the effect of GANP construct in the localization of MCM3 mutants.

[0062]FIG. 43 shows the nucleus-cytoplasm shuttling of MCM3 detected by a heterokaryon assay.

[0063]FIG. 44 shows the localization of GANP during the cell cycle.

BEST MODE FOR CARRYING OUT THE INVENTION

[0064]Hereinbelow, the present invention will be described in detail.

[0065]The present invention has been achieved based on a finding that it is possible to obtain a high affinity antibody by preparing a transgenic animal by transferring a GANP gene into a non-human mammal and immunizing the resultant transgenic animal with an antigen.

1. GANP

[0066]GANP which is called "germinal center-associated nuclear protein" is a 210 kDa nuclear protein having homology to yeast Sac3 protein (WO 00/50611). SAC3 is characterized as an inhibitory substance against actin formation. It is known that GANP is selectively up-regulated in germinal center (GC) B cells surrounded by follicular dendritic cells: FDC), has phosphorylation-dependent RNA primase activity, and is involved in the regulation of the cell cycle of B cells (Kuwahara, K. et al., (2000) Blood 95: 2321-2328).

[0067]In the present invention, the amino acid sequence for mouse GANP protein is shown in SEQ ID NO:2 and the amino acid sequence for human GANP protein is shown in SEQ ID NO: 4. With respect to the gene encoding the GANP protein (hereinafter, referred to as "GANP gene"), the nucleotide sequence for mouse GANP gene is shown in SEQ ID NO: 1 and the nucleotide sequence for human GANP gene is shown in SEQ ID NO: 3. The above-mentioned amino acid sequences and nucleotide sequences are also described in WO 00/50611.

[0068]GANP proteins may be mutant proteins; they may be those proteins which consist of the amino acid sequence as shown in SEQ ID NO: 2 or 4 wherein one or a plurality of amino acids have been deleted, substituted or added and have RNA primase activity. For example, a GANP mutant protein may also be used which consists of the amino acid sequence as shown in SEQ ID NO: 2 or 4 wherein one or a plurality of amino acids (preferably, one or several (e.g. one to ten, more preferably one to five) amino acids) have been deleted, one or a plurality of amino acids (preferably, one or several (e.g. one to ten, more preferably one to five) amino acids) have been substituted with other amino acids, and/or one or a plurality of other amino acids (preferably, one or several (e.g. one to ten, more preferably one to five) amino acids) have been added thereto, and yet has the same RNA primase activity as that of the above-described GANP protein.

[0069]RNA primase activity" means the enzyme activity synthesizing a short primer RNA which will be a starting point for strand elongation when a strand extending opposite to the 5'→3' direction (lagging strand) is synthesized. Usually, a molecule called α primase which binds to DNA polymerase α is used. In germinal center B cells, GANP primase which is the second primase is also induced.

[0070]GANP protein includes a protein having the amino acid sequence as shown in SEQ ID NO: 2 or 4, or a mutant amino acid sequence thereof, and a protein having a part of the N-terminal sequence of those sequences (e.g. positions 1-600, preferably 139-566 of the amino acid sequence as shown in SEQ ID NO: 2) or a mutant amino acid sequence thereof.

[0071]In the present invention, a GANP gene to be transferred into an animal may be a gene encoding the above-described GANP protein, a part of the N-terminal sequence of the GANP protein, or a mutant GANP protein. Specific examples of such a gene include a gene having the nucleotide sequence as shown in SEQ ID NO: 1 or 3. A gene having only the coding region of the nucleotide sequence as shown in SEQ ID NO: 1 or 3 may also be used. Alternatively, it is also possible to use a gene that has a sequence hybridizable to a complementary sequence to the nucleotide sequence as shown in SEQ ID NO: 1 or 3 under stringent conditions, and encodes a protein having RNA primase activity.

[0072]Stringent conditions" refers to washing conditions after hybridization; specifically, the salt (sodium) concentration is 150-900 mM and the temperature is 55-75° C., preferably salt (sodium) concentration is 250-450 mM and the temperature is 68° C.

[0073]Introduction of mutations into a gene may be performed according to known techniques such as the Kunkel method or the gapped duplex method, using mutation introducing kits utilizing site-directed mutagenesis, such as GeneTailor® Site-Directed Mutagenesis System (Invitrogen) or TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.; Takara Bio).

[0074]Details of mutant genes and methods for obtaining the same are also described in WO 00/50611.

[0075]In vitro stimulation of B cells with anti-μ antibody and anti-CD40 monoclonal antibody induces not only the up-regulation of GANP expression but also the phosphorylation of a specific serine residue in the amino acid sequence of GANP protein (e.g. serine at position 502: S502). This reaction is a key reaction for the RNA primase activity of GANP (Kuwahara, K. et al. (2001) Proc. Natl. Acad. Sci. USA, 98, 10279-10283). The N-terminal primase domain of GANP protein contains a serine residue whose phosphorylation is catalyzed by Cdk2 in vitro. GANP binds to MCM3 replication licensing factor due to its C-terminal domain (Kuwahara, K. et al., (2000) Blood 95: 2321-2328; Abe, E. et al., (2000) Gene 255: 219-227).

2. Transgenic Mammal Carrying GANP Gene Transferred Thereinto

[0076]The present invention relates to a transgenic mammal carrying a GANP gene transferred thereinto. Preferably, the transgenic mammal is capable of expressing the transferred GANP gene in its B cells.

(1) GANP Gene and its Related Molecules

[0077]Complexes formed by GANP gene and its related molecules are needed directly or indirectly in the process of induction of mutations in genes. When repairing genetic mutations, GANP protein has the ability to promote induction of mutations in the V region so that high affinity antibodies are obtained. Therefore, the transgenic mammal of the invention carrying the GANP gene or a mutant thereof transferred thereinto is capable of promoting the production of high affinity antibodies of acquired immunity. Further, a transgenic non-human mammal overexpressing this GANP gene is capable of promptly producing an antibody with high binding strength to an antigen. Therefore, by immunizing the above-described transgenic non-human mammal with a specific antigen, it is possible to obtain easily an antibody with a high affinity that has been unachievable by conventional methods. As a result, it becomes possible to obtain polyclonal or monoclonal antibodies capable of eliminating obstinate pathogenic microorganisms or foreign substances. Further, by preparing humanized antibodies using the transgenic mammal of the invention, or by preparing single chain antibodies comprising the V region of the antibody produced by the transgenic mammal of the invention, it becomes possible to sharply increase the effect of antibody therapy.

[0078]Because of the GANP gene or its mutant transferred thereinto, the transgenic mammal of the invention is capable of promoting the production of high affinity antibodies in B cells, and the high affinity antibody-producing cells have resistance to apoptosis induction signals.

[0079]In order to confirm that GANP is a molecule functioning in the antibody production in acquired immune responses, the present inventors have created a GANP gene deficient mouse so that GANP is deficient B cell selectively. The results revealed that the deficiency of GANP gene did not influence the development, differentiation and proliferation of cells in the immune system and that no big change is observed in the total yield of antibodies.

[0080]It should be noted here that only when B cells have reacted with limited types of antigens, they proliferate and differentiate into antibody-producing cells without T cells. For producing antibodies to ordinary antigens, co-existence of T cells is necessary. Antigens to which antibodies are produced even in the absence of T cells are called T cell-independent antigens. On the other hand, general antigens other than T cell-independent antigens are called T cell-dependent antigens. When B cells have reacted with T cell-dependent antigens, the differentiation of B cells into antibody-producing cells is assisted by helper T cells.

[0081]Many of the antigenic determinants (also called antigenic epitopes) of pathogenic viruses are weak in immunogenicity by themselves and activated by the peptide antigens of carrier proteins recognized by T cells.

[0082]In the present invention, in order to examine that GANP gene-transferred animals are capable of producing high affinity antibodies highly frequently in those antibody-producing responses to soluble antigens where ordinary animals cannot produce strong antibodies, an antigen designated NP-CG was prepared by coupling a nitrophenyl group (NP group) (which has been extensively analyzed as a hapten) to chicken gamma globulin, followed by examination of responses to T cell-dependent antigen.

[0083]It is known that C57BL/6 mice's generate high affinity antibody to NP only when utilized a single V region. This response is dominated by only the V region of IgG heavy chain (called V_H186.2) and lambda 1 light chain of an antibody. With this system, it is possible for antibodies of IgG₁ isotype to examine genetic mutations in high affinity antibodies by analyzing the amino acid sequence of V_H186.2. Furthermore, it is reported that the highest affinity is induced when the amino acid residue tryptophan (W) at position 33 of the amino acid sequence of the heavy chain V region (V_H186.2) has been mutated into leucine (L) (W³³ to L mutation).

[0084]Then, the present inventor examined whether high affinity antibodies could be induced in GANP gene deficient mice and its defect might be associated with W33 to L mutation event or not. As a result, high affinity antibody production was hardly observed in GANP gene deficient mice, compared to the control Cre-flox/+ mice. Therefore, it has been demonstrated that GANP gene has a key function in the production of high affinity antibodies. To investigate this function further, the inventor has created GANP gene-overexpressing mice. Overexpression of GANP gene was achieved by linking a mouse immunoglobulin promoter moiety and a human immunoglobulin gene intron enhancer moiety upstream (5') of GANP gene so that the gene is expressed selectively in B cells.

[0085]The GANP-overexpressing mice were born normally, and no particular change was observed in the development, differentiation and proliferation of their lymph tissues. However, a remarkable increase was observed in the high affinity type V region gene (W³³ to L) in responses to NP-CG. Although the functional role of RNA primase activity here has not yet been established, it is believed that the RNA primase activity of GANP gene or the phosphorylation of the 502 serine residue involved in the primase activity is related to the production of high affinity antibodies in view of the following: (i) the phosphorylation of serine residue at position 502 (which is an indicator for the primase activity of GANP molecule) is high in cells present at the region of the germinal center where high affinity B cells are produced (centrocytes), and (ii) the frequency of the mutation at the V region induced by experiments to transfer a ganp gene into Daudi cells is high. These results show that high expression of GANP molecule and activation of RNA primase activity are necessary for high affinity antibody production by immune response.

(2) Mammals for Use in GANP Gene Transfer

[0086]The term "mammal" used in the present invention means any of non-human mammals such as bovine, horse, pig, goat, rabbit, dog, cat, mouse, rat, hamster and guinea pig. Preferably, mouse, rabbit, rat or hamster is used. Most preferably, mouse is used.

[0087]The transgenic mammal of the invention may be prepared by introducing a GANP gene into fertilized eggs, unfertilized eggs, embryonic cells comprising spermatozoa and protocells thereof, preferably into cells of embryogenesis stage (more preferably, the single cell or fertilized egg cell stage and yet generally before eight-cell stage) in the development of non-human mammals, by a method such as the calcium phosphate method, electric pulsing, lipofection, aggregation, microinjection, the particle gun method, or the DEAE-dextran method. Further, it is also possible to transfer a GANP gene of interest into somatic cells, organs of the living body, tissue cells, etc. by the above-mentioned gene transfer methods to use the resultant cells, etc. for cell culture or tissue culture. Further, it is possible to create transgenic mammals by fusing these cells with the above-described embryonic cells according to known cell fusion methods.

[0088]When a GANP gene is transferred into an animal of interest, it is preferred that the gene be transferred in the form of a gene construct in which the gene is ligated downstream of a promoter capable of directing expression of this gene in cells of the animal of interest. Specifically, a vector in which a GANP gene is ligated downstream of various promoters capable of directing expression of the GANP gene derived from various mammals may be microinjected into fertilized eggs of the mammal of interest (e.g. mouse fertilized eggs) to thereby create a transgenic mammal capable of high expression of the GANP gene of interest.

(3) Expression Vector

[0089]Examples of expression vectors for GANP gene include plasmids derived from Escherichia coli; plasmids derived from Bacillus subtilis; plasmids derived from yeast; bacteriophages such as λ-phage; retroviruses such as Moloney leukemia virus; and animal or insect viruses such as vaccinia virus or baculovirus.

[0090]As promoters for regulating gene expression, promoters of viruses-derived genes; promoters of various mammals (such as human, rabbit, dog, cat, guinea pig, hamster, rat and mouse)-derived genes; and promoters of birds (such as chicken)-derived genes may be used.

[0091]Examples of promoters of viruses-derived genes include promoters of cytomegalovirus-, Moloney leukemia virus-, JC virus- or breast cancer virus-derived genes.

[0092]Examples of promoters of various mammals- and birds-derived genes include promoters of such as albumin, insulin II, erythropoietin, endothelin, osteocalcin, muscle creatine kinase, platelet-derived growth factor β, keratin K1, K10 and K14, collagen type I and type II, atrial natriuretic factor, dopamine β-hydroxylase, endothelial receptor tyrosine kinase, sodium/potassium-dependent adenosinetriphosphatase, neurofilament light chain, metallothionein I and IIA, metalloproteinase I tissue inhibitor, MHC Class I antigen, smooth muscle α-actin, polypeptide chain elongation factor 1α (EF-1α), β-actin, α- and β-myosin heavy chains, myosin light chains 1 and 2, myelin basic polypeptide, serum amyloid P component, myoglobin and renin genes.

[0093]The above-described vector may have a terminator which terminates the transcription of a messenger RNA of interest in a transgenic mammal. For the purpose of achieving still higher expression of GANP gene, the splicing signal of each gene, enhancer region, or a part of an intron of an eukaryotic gene may be ligated upstream (5') of the promoter region, between the promoter region and the translation region, or downstream (3') of the translation region, if desired.

[0094]In a preferred embodiment of the invention, it is possible to allow selective expression of the transferred GANP gene in B cells by ligating the GANP gene downstream of an immunoglobulin promoter or by ligating a human immunoglobulin gene intron enhancer moiety upstream (5') of the GANP gene.

(4) Transfer of GANP Gene

[0095]The transfer of GANP gene at the fertilized egg cell stage is preferably carried out in such a manner that excessive presence of GANP gene is secured in all the embryonic cells and somatic cells of the mammal of interest. Excessive presence of GANP gene in the embryo cells of the created animal after gene transfer means that all the progeny of that animal has excessive GANP gene in all the embryonic cells and somatic cells. The progeny of this kind of animal which inherited the GANP gene has excessive GANP protein in all the embryonic cells and somatic cells.

[0096]In the present invention, first, heterozygotes which have the transferred GANP gene in one of the homologous chromosomes are prepared; then, homozygotes which have the transferred GANP gene in both of the homologous chromosomes are obtained by mating the heterozygotes with each other. Subsequently, by mating female homozygotes with male homozygotes, all the resultant progeny retains the transferred GANP gene stably. After confirmation of the excessive presence of GANP gene, the progeny may be sub-bred in usual breeding environments.

[0097]Fertilized eggs of a non-human mammal of interest (preferably, mouse) or its ancestor (back-crossing) to be used for transferring a foreign GANP gene different from the endogenous gene of the mammal of interest are obtained by mating allogenic male and female mammals.

[0098]Although fertilized eggs may be obtained by natural mating, it is preferred that female mammals after artificial adjustment of their sexual cycle be mated with male mammals. As a method for artificially adjusting the sexual cycle of female mammals, such a method may be used preferably in which follicle-stimulating hormone (pregnant mare serum gonadotropin (PMSG)) and then luteinizing hormone (human chorionic gonadotropin (hCG)) are administered by, e.g., intraperitoneal injection.

[0099]After the transfer of a foreign GANP gene into the resultant fertilized eggs by the methods described above, the eggs are artificially transferred/implanted in female mammals. As a result, non-human mammals having a foreign gene-integrated DNA are obtained. In a preferable method, fertilized eggs are transferred/implanted artificially in pseudo-pregnant female mammals in which fertility has been induced by mating with male mammals after administration of luteinizing hormone-releasing hormone (LHRH). As totipotent cells into which a GANP gene is to be transferred, fertilized eggs or early embryos may be used if the mammal of interest is mouse. As a method of gene transfer into cultured cells, DNA microinjection is preferable in view of the production efficiency of transgenic mammal individuals and the transmittance efficiency of the transgene to the subsequent generation.

[0100]Subsequently, the gene-injected fertilized eggs are transplanted into the oviduct of a recipient female mammal. Those animals which have developed from the eggs up to individuals and have been successively born are bred under foster parents. Then, DNA is extracted from a part of their bodies (e.g. the tail end in the case of mouse) and subjected to Southern analysis, PCR, etc. Thus, it is possible to confirm the presence of the transgene. Those animals in which the presence of the transgene has been confirmed are designated founder animals. The transgene is transmitted to 50% of their offspring (F1). Further, by mating F1 individuals with wild-type animals or other F1 individuals, F2 individuals which have the transgene in one (heterozygote) or both (homozygote) of the diploid chromosomes can be produced.

[0101]Alternatively, transgenic mammals expressing high levels of GANP protein may also be created by introducing the above-described GANP gene into ES (embryonic stem) cells. For example, the GANP gene is introduced into HPRT negative (i.e. lacking hypoxanthine-guanine phosphoribosyltransferase gene) ES cells derived from normal mouse blastocysts. Then, those ES cells in which the GANP gene has been integrated through homologous recombination induced in a mouse endogenous gene are selected by HAT selection. The thus selected ES cells are microinjected into fertilized eggs (blastocysts) obtained from other normal mouse. The resultant blastocysts are transferred into the uterus of other normal mouse as a recipient. Subsequently, chimeric transgenic mice are born from the recipient mouse. By mating these chimeric transgenic mice with normal mice, heterotransgenic mice can be obtained. Further, by mating the heterotransgenic mice with each other, homotransgenic mice can be obtained.

[0102]The present invention encompasses not only the above-described transgenic mammal but also its progeny and a part of the transgenic mammal or its progeny in the scope of the invention. As a part of the transgenic mammal, a tissue, organ, cell or the like of the transgenic mammal or its progeny may be enumerated. Specific examples of organs or tissues include the spleen, thymus, lymph nodes, bone marrow or tonsil; and specific examples of cells include B cells.

[0103]The transgenic mammal of the invention may be mated with a mammal that further activates B cells. As a result of such mating, antibodies of still higher affinity can be produced.

[0104]Recently, it has been reported that when B cells are activated in peripheral lymph nodes in MRL/lpr mouse, induction of mutations in the V region is further increased in the T cell region after B cells passed through the germinal center. The inventors have also found that non-immunized MRL/lpr mouse shows high expression of GANP equivalent to the GANP expression observed in ganp transgenic mouse which was created by ligating a GANP gene downstream of Ig promoter and enhancer. This suggests a possibility that, while high affinity antibodies are not produced against autoantigens normally, high affinity antibodies to autoantigens may be produced in this autoimmune disease mouse because of the abnormal activation of GANP molecule.

[0105]Still higher induction of mutations can be expected if such mouse as MRL/lpr, NZB or (NZB×NZW)F1 (all of them are considered as autoimmune disease mice) is used as the above-mentioned animal that still activates B cells.

[0106]By creating a GANP transgenic mouse from MRL/lpr mouse utilizing what has been described above, it may be possible to create a super high affinity antibody-producing mouse. In other words, by mating the GANP gene overexpressing transgenic mammal of the invention with various autoimmune disease model animals, it is possible to create mammals capable of producing high affinity antibodies.

3. Preparation of High Affinity Antibodies

[0107]The term "antibody" used in the invention means a protein having activity to specifically bind to an antigen, preferably a protein produced by B cells. In the present invention, an antibody having high reactivity with an antigen is called high affinity antibody. The term "high affinity" used herein means that the ability of an antibody to bind to an antigen is high. In the present invention, a high affinity antibody refers to an antibody which has higher ability to bind to an antigen than those antibodies prepared using conventional animals such as mouse, and which is slow in dissociating from that antigen. This means that such an antibody is high and specific in the ability to bind to an antigenic determinant (epitope) sterically and closely. Besides, the binding of such an antibody to the antigenic determinant induces changes not only in the determinant but also the structure of the antigen itself, to thereby show strong activities eventually (e.g. biological activities such as neutralization of toxicity, prevention of viral infection, deactivation of pathogens, promotion of elimination of pathogens from the body, or induction of denaturation in antigen molecules).

[0108]The binding ability of an antibody (i.e. affinity) may be measured as a dissociation constant (KD), dissociation rate constant (Kdiss) or association rate constant (Kass) by Scatchard analysis or with a surface plasmon resonance sensor called Biacore. Biacore systems in which three technologies of sensor chip, microflow system and SPR detection system are integrated are to measure the strength, rate and selectivity of molecular binding. This apparatus enables real time detection of biological molecules and monitoring of interactions among a plurality of molecules without using labels. Specific examples of useful Biacore systems include Biacore 3000, Biacore 2000, Biacore X, Biacore J and Biacore Q (all of them are manufactured by Biacore).

[0109]With the above-described Biacore system, parameters showing the affinity of antibodies, i.e. dissociation constant (KD), dissociation rate constant (Kdiss) (1/Sec) and association rate constant (Kass) (1/MSec) are measured.

[0110]Antibodies with smaller dissociation constant (KD) values are preferable because the smaller the dissociation constant value, the higher the affinity. The binding ability of an antibody (affinity) is determined by the two parameters of Kdiss and Kass, and is represented by the following formula:

KD(M)=Kdiss/Kass

[0111]Although the affinity of the resultant antibody varies depending on a plurality of factor such as the type of the antigen, generally, its KD value is preferably 1×10^-7 (M) or less. For example, preferable KD values are 1×10^-8 (M) or less, 1×10^-10 (M) or less, or 1×10^-11 (M) or less.

[0112]In the present invention, when the resultant antibody reveals any of the above-described effects or natures, the antibody is judged as a "high affinity" antibody.

[0113]Enhancement in the affinity of antibody molecules is produced by inducing somatic hypermutations (SHM) in genes of the variable regions (V region) of antibodies. Although specificities of antibodies to antigens are recognized from the beginning of immunization of the living body with antigens, most of early antibodies are IgM class antibodies; their binding affinity to antigens is not high and their ability to remove or deactivate pathogens or foreign substances is low. However, if an antigen is administered to the living body to give several boosters, the binding affinity of antibody to the antigen is enhanced. At this time, B cells need stimulation from T cells, and this activation is considered to take place in the germinal center region in peripheral lymph tissues. Recently, the RNA editing molecule AID expressed in the germinal center has been reported as a molecule necessary to induce mutations in V region genes. Further, it is reported that uracil DNA glycosidase and, as DNA polymerases necessary for DNA replication, DNA polymerases zeta (ζ) and iota () which easily produce errors are also involved in the above activation. However, the molecule(s) which control(s) these functions has/have not been elucidated. The function of GANP molecule as a novel SHM-inducing molecule has been elucidated. Increase in the expression of this molecule plays a key role in SHM induction. Among all, it has been demonstrated that GANP molecule is important in producing high affinity antibodies.

[0114]Antibodies induced by immunizing C57BL/6 mice with nitrophenyl-chicken γ globulin as a hapten carrier antigen have V_H186.2 locus as the H chain and λ1 as the L chain. In this system, it is known that antibodies obtained after boosters were given are IgG₁ antibodies, and that the mutation induced in the V region sequence of those antibodies with particularly high binding affinity among them is mutation from tryptophan to leucine at position 33. In the Examples of the present specification, this high affinity-type V region mutation is induced highly. This can be said definite evidence at the molecule level showing that high affinity antibodies have been induced.

[0115]Therefore, it is possible to obtain high affinity antibodies by administering an antigen to the above-described transgenic mammal or its progeny and letting the resultant mammal or progeny produce antibodies. Briefly, an antigen of interest is administered by conventional methods to an animal that is engineered to express high levels of GANP protein. Then, high affinity antibodies may be prepared form lymphocytes of a tissue such as blood or spleen (not limited to these tissues) of the immunized animal. These high affinity antibodies may be either polyclonal or monoclonal antibodies.

[0116]As a method for producing polyclonal antibodies, for example, polyclonal antibodies may be obtained by administering an antigen to the transgenic mammal of the invention, taking blood from the immunized mammal, and then separating and purifying antibodies from the resultant blood.

[0117]Methods of immunization are known to those skilled in the art. For example, immunization may be performed by administering an antigen once or more.

[0118]The types of the antigen are not particularly limited. All substances which may have a steric structure as an antigenic determinant fall under antigen. In addition to all biological components such as proteins, enzymes, peptides, sugars, lipids, DNAs, RNAs and prions, any substance such as cancer antigens, virus antigens, organic or inorganic synthetic antigens may be used.

[0119]The antigen may be administered, for example, two or three times at intervals of 7 to 30 days. The dose may be, for example, about 0.05 to 2 mg of the antigen per administration. The route of administration is not particularly limited. For example, subcutaneous administration, dermal administration, intraperitoneal administration, intravenous administration or intramuscular administration may be selected appropriately. Preferably, the antigen is administered by intravenous, intraperitoneal or subcutaneous injection. The antigen may be used in solution in an appropriate buffer, e.g. a buffer containing conventional adjuvants such as complete Freund's adjuvant or aluminium hydroxide, but the antigen may be used without adjuvant depending on the administration route or other conditions.

[0120]After immunized mammals have been bred for a specific period of time, serum samples are obtained from them and antibody titers thereof are measured. When the antibody titer begins to rise, boosters may be given using, for example, 100 μg to 1000 μg of the antigen. One to two months after the final administration, blood is taken from the immunized mammals and subjected to various conventional methods used for protein isolation, e.g. centrifugation, precipitation using ammonium sulfate or polyethylene glycol, and chromatography such as gel filtration chromatography, ion exchange chromatography or affinity chromatography. Thus, polyclonal antibodies may be obtained as polyclonal anti-sera.

[0121]As a method for producing monoclonal antibodies, the hybridoma method may be used. First, a peptide constituting an antigen of interest is suspended in an adjuvant. The resultant suspension is administered subcutaneously or intradermally into animals to be immunized (i.e. the transgenic mammal of the invention). The types of the antigen used here are the same as described above. Examples of the adjuvant used here include complete Freund's adjuvant, BCC, trehalose dimycolate (TDM), lipopolysaccharide (LPS), alum adjuvant and silica adjuvant. Preferably, a combination of complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) is used in view of the ability to induce antibodies.

[0122]In the production of monoclonal antibodies, preferably, animals which have undergone the first immunization with an antigen are boosted several times; after passage of appropriate number of days, blood samples are taken and antibody titers thereof are measured. Since antibodies produced by the method of the invention are high affinity antibodies, the first immunization may be sufficient without booster. Antibody titers may be measured by known methods such as enzyme-linked immunosorbent assay (hereinafter, referred to as ELISA).

[0123]Subsequently, the spleens are removed from the immunization-completed animals to obtain B cells. Obtaining B cells capable of binding to antigens is preferable because it could reduce subsequent screening. The B cells obtained at this point are high affinity antibody-producing cells, which may be used as an immunopotentiator without any processing. It is also possible to obtain V region genes directly from these B cells and to measure somatic hypermutations in the V region.

[0124]Subsequently, the resultant B cells are fused with myeloma cells by conventional methods to thereby prepare an antibody-producing hybridoma. For example, if the animal is mouse, the spleen is removed and placed in a solution such as Hanks' balanced salt solution (HBSS). Cells are pushed out with tweezers to obtain spleen lymphocytes (B cells). The resultant spleen lymphocytes are stained with trypanblue or the like to count the number of viable cells, and then fused with myeloma cells to prepare a hybridoma.

[0125]The myeloma cell used for the cell fusion is not particularly limited. Known myeloma cells such as P3-X63.Ag8 (X63), P3-X63.Ag8.U1 (P3U1), P3/NS I/1-Ag4-1 (NSI) or Sp2/0-Ag14 (Sp2/0) may be used. In the selection of the myeloma cell, compatibility with antibody-producing cells should be considered appropriately.

[0126]Cell fusion is carried out as described below. Briefly, 1×10⁶-1×10⁷ cells/ml of antibody-producing cells are mixed with 2×10⁵-2×10⁶ cells/ml of myeloma cells (preferable cell ratio of antibody-producing cells to myeloma cells is 5:1) in an animal cell culture medium such as serum-free DMEM or RPMI-1640 and fused in the presence of a cell fusion promoter.

[0127]As the method of cell fusion, any of the methods known in the art (the Sendai virus method, the polyethylene glycol method, or the protoplast method) may be selected. Preferably, the polyethylene glycol method is used in view of relatively low cytotoxicity and simple fusion operations. Polyethylene glycol with a mean molecular weight of 1000-6000 daltons may be used as a cell fusion promoter. When production of a large quantity of antibodies is desired, a hybridoma prepared by fusing antibody-producing cells stimulated with a vinyl pyridine derivative with myeloma cells is used preferably.

[0128]The resultant hybridoma is cultured in HAT medium (containing hypoxanthine, aminopterin and thymidine) for an appropriate period of time according to conventional methods, followed by selection of hybridoma clones. Subsequently, those hybridoma clones producing an antibody of interest are screened, followed by cloning of the hybridoma clones.

[0129]As the screening method, known methods for antibody detection, such as ELISA, radio immunoassay (hereinafter, referred to as RIA), the plaque method, or the aggregation reaction method, may be used. As the cloning method, known methods in the art, such as the limiting dilution-culture method, the soft agar method or FACS, may be used. The resultant hybridoma is cultured in an appropriate culture broth, or administered into the abdominal cavity of an animal (e.g. mouse) compatible with the hybridoma. From the thus obtained culture broth or abdominal dropsy, the monoclonal antibody of interest may be isolated and purified by methods such as salting out, ion exchange chromatography, gel filtration or affinity chromatography.

[0130]It should be noted that fragments and single chain antibodies of the V region of the above-described antibody are also within the scope of the present invention. A fragment of the antibody means a portion of the above-described polyclonal or monoclonal antibody. Specific examples of such a fragment include F(ab')₂, Fab', Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv) or dAb (single domain antibody). F(ab')₂ and Fab' mean those antibody fragments which are prepared by treating an immunoglobulin (monoclonal antibody) with proteolytic enzymes pepsin and papain, respectively, and are generated through digestion around the disulfide bond present between the two H chains in the hinge region. For example, when IgG is treated with papain, this molecule is cut upstream of the disulfide bond present between the two H chains in the hinge region to yield two homologous antibody fragments in which an L chain consisting of V_L (L chain variable region) and C_L (L chain constant region) and an H chain fragment consisting of V_H(H chain variable region) and C_Hγ1 (γ1 region in H chain constant region) are coupled by a disulfide bond in the C-terminal region. Each of these two homologous antibody fragments is called Fab'. When IgG is treated with pepsin, this molecule is cut downstream of the disulfide bond present between the two H chains in the hinge region to yield an antibody fragment which is slightly larger than the above-described two Fab' fragments ligated at the hinge region. This antibody fragment is called F(ab')₂. A single chain antibody has a structure in which V_L and V_H are linked by a linker.

[0131]The high affinity antibody of the invention may be a humanized antibody or human antibody. These human antibodies may be prepared by using mammals whose immune system has been replaced with the human immune system. After immunizing such mammals, human antibodies may be prepared directly in the same manner as used in the preparation of conventional monoclonal antibodies.

[0132]For the preparation of humanized antibodies, reconstructed variable regions consisting of human-derived framework regions and mouse-derived CDRs (complementarity determining regions) is prepared by transferring the CDRs of the variable regions in a mouse antibody into the human variable regions.

[0133]Subsequently, these humanized, reconstructed human variable regions are ligated to human constant regions. Portions derived from non-human amino acid sequences in the finally reconstructed humanized antibody are only CDRs and extremely small parts of FRs. CDRs are composed of hyper-variable amino acid sequences. Since these sequences do not show species specific sequences, it is possible to use humanized antibodies having mouse CDRs. Methods for preparing humanized antibodies are well-known in the art.

[0134]Human antibodies may be produced using any animal (e.g. mouse, rat, etc.) in terms of structure, though generally the antigen binding site in the variable region (i.e. hyper variable region) may raise some problem with respect to specificity and binding affinity. On the other hand, it is desirable that the structures of the remaining portion of the variable region and the constant region should be the same as the structures in human antibodies. With respect to genetic sequences common in human, genetic engineering techniques to prepare them have been established.

[0135]The isotype of the antibody of the invention is not particularly limited. The antibody of the invention may have any isotype, e.g. IgG (IgG₁, IgG₂, IgG₃, IgG₄), IgM, IgA (IgA₁, IgA₂), IgD or IgE.

4. Use of High Affinity Antibodies

[0136]The high affinity antibody of the invention is useful as a drug for diagnosing, treating or preventing diseases.

(1) Diagnosis of Diseases

[0137]Diagnosis of various diseases using the antibody of the invention is carried out as described below. Briefly, samples (e.g. sera) taken from subjects suspected of having various diseases are bound to the antibody of the invention by antigen-antibody reaction. Then, the amount of an antigen of interest in the sample is detected from the amount of bound antibody. The detection of the amount of bound antibody may be performed by conventional immunological measuring methods. For example, immunoprecipitation, immunoaggregation, labeled immunoassay, immunonephelometry, immunoturbidimetry, or the like may be used. Labeled immunoassay is especially preferable from the viewpoint of simplicity and high sensitivity. In labeled immunoassay, antibody titers in samples may be expressed directly as the amounts of label detected using a labeled antibody. Alternatively, antibody titers may be expressed relatively using as a standard solution an antibody of known concentration or known titer. Briefly, the standard solution and a sample may be measured simultaneously in the same measuring system, followed by expression of the antibody titer in the sample relatively based on the value of the standard solution.

[0138]In labeled immunoassay, any of known measurement methods, such as ELISA, RIA, fluoroimmunoassay, or chemiluminescence immunoassay, may be used. The labeling substance may be appropriately selected depending on the above-mentioned assay method; for example, an enzyme, radioisotope, fluorescent compound, or chemiluminescent compound may be selected. Specific examples of the enzyme useful in the invention include peroxidase, alkaline phosphatase, acid phosphatase and glucose oxidase. Detection sensitivity of the above-mentioned labeling substances may be increased by using avidin-biotin complex. As a specific example of the radioisotope useful in the invention, ¹²⁵I may be given at first. Specific examples of the fluorescent compound useful in the invention include fluoresceine isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Specific examples of the chemiluminescent compound useful in the invention include lophine, luminol and lucigenin. The labeling of antibodies with the above-mentioned substances may be performed according to conventional methods. Hereinbelow, labeled immunoassay using labeled antibodies will be described.

[0139]As a method of detection of various diseases according to labeled immunoassay, a method using a known non-competitive reaction system or competitive reaction system may be possible. Non-competitive reaction systems require solid phase (solid phase method). Competitive reaction systems do not necessarily require solid phase (liquid phase method), but use of solid phase is preferable since that will make measuring operations simple. Specific examples of materials for the solid phase include polystyrene, nylon, glass, silicon rubber and cellulose. As the shape of the solid phase, spheres, wells, tubes, sheets, or the like may be enumerated. However, the material and the shape useful in the invention are not limited to those enumerated above. Known materials and shapes used in labeled immunoassay may be used at discretion.

[0140]In non-competitive reaction systems, measurement operations are carried out as follows. Briefly, a sample or the antibody of the invention is immobilized on a solid support and then reacted with the antibody of the invention or a sample. Subsequently, a pre-labeled anti-immunoglobulin antibody (secondary antibody) is added to react with the above antibody reacting with the immobilized sample. With the labeling substance of this secondary antibody, it is possible to detect the amount of the antibody bound to the sample. Since the amount of the labeled secondary antibody detected is directly correlated with the amount of the antigen of interest in the sample, the amount of this antigen can be obtained from the amount of the labeled secondary antibody.

[0141]In competitive reaction systems, a sample and a specific amount of an antigen of interest are reacted with a specific amount of an antibody. For example, after immobilization of a sample on a solid support, the sample is reacted with the antibody of the invention which has been pre-reacted with an antigen of interest. Subsequently, the antibody which has reacted with the immobilized sample is reacted with a pre-labeled anti-immunoglobulin antibody (secondary antibody), followed by detection of the amount of the antibody by the labeling substance. The amount of the labeling substance is inversely correlated with the amount of the antigen of interest added. Other types of competitive reaction systems may also be used where the antibody of the invention is immobilized, reacted with a sample, and then reacted with a pre-labeled antigen of interest. The amount of the labeling substance detected is inversely correlated with the amount of GANP protein in the sample bound to the antibody.

[0142]As the method of immobilization of an antigen or antibody on a solid support, known methods such as physical adsorption, covalent binding, ionic bonding or crosslinking may be used. Physical adsorption is especially preferable because of its simplicity. As examples of the anti-immunoglobulin antibody (secondary antibody) useful in the invention, anti-IgG antibody or anti-IgM antibody may be given. These antibodies may be used as an entire molecule. Alternatively, antibody fragments Fab, Fab' and F(ab')₂ comprising the antigen binding site obtained by treating antibodies with enzymes may be used. Further, instead of the labeled anti-immunoglobulin antibody, a substance having specific affinity for antibody molecules (e.g. protein A which has specific affinity for IgG) may be labeled and used.

[0143]As a preferable example of the above-described labeled immunoassay, ELISA may be given which is an immunoassay using an enzyme as a label. Briefly, a sample or a dilution thereof is placed in 96-well plates or the like and incubated at 4° C. to room temperature overnight or at 37° C. for about 1-3 hrs so that GANP protein to be detected is adsorbed and immobilized on the plates. Then, the antibody of the invention is reacted. Subsequently, an enzyme-preconjugated anti-immunoglobulin antibody (secondary antibody) is reacted. Finally, an appropriate color-developing substrate reactive with the enzyme (e.g. if the enzyme is phosphatase, p-nitrophenylphosphate or the like) is added to thereby detect the antibody with its color development.

[0144]By using the high affinity antibody of the invention, it is possible to evaluate the efficacies of therapeutics for various diseases. The evaluation method using the high affinity antibody of the invention is performed as follows. Briefly, a drug is administered to various disease patients or disease model animals. Then, using the antibody of the invention, the amounts of the antigen (such as virus) in these living bodies are detected. By comparing the amounts, the efficacy of the drug as a therapeutic for various diseases can be evaluated based on the amounts of the antigen in living bodies.

[0145]The high affinity antibody of the invention may be provided in the form of a diagnosis kit for various diseases. This kit may be used in the diagnosis method of the invention or the efficacy evaluation method of the invention. The kit of the invention comprises as least one selected from the following (a) and (b).

[0146](a) the antibody of the invention or that antibody labeled

[0147](b) immobilized reagent in which the antibody or labeled antibody of (a) above is immobilized on a solid support

[0148]The "labeled antibody" means an antibody labeled with an enzyme, radioisotope, fluorescent compound or chemiluminescent compound. As the material of a solid support on which the antibody or labeled antibody is immobilized in the kit of the invention, polystyrene, nylon, glass, silicon rubber, cellulose or the like may be used. As the shape of such a solid support, spheres, wells, tubes or sheets may be enumerated. However, the material and the shape useful in the invention are not limited to these ones. Instead of the immobilized reagent, a solid phase and an immobilizing agent may be attached to the kit. As the immobilizing agent, if immobilization by physical adsorption is intended, a coating liquid such as 50 mM carbonate buffer (pH 9.6), 10 mM Tris-HCl buffer (pH 8.5, containing 100 mM sodium chloride) or PBS and, if necessary, a blocking liquid (which is a coating liquid containing 0.5% gelatin) may be enumerated, for example.

[0149]The antibody contained in the kit of the invention may be in a state of solution in PBS or the like, or in a state where the antibody is linked to a gel (hereinafter, abbreviated to "absorption gel"). This absorption gel may be pre-packed in 0.5-2 ml microcentrifuge-precipitation tubes for absorption by the batch method. Alternatively, the absorption gel may be pre-packed in 0.1-5 ml mini-columns for absorption by the column method.

[0150]In addition to the above-described components, the kit of the invention may contain other reagents for carrying out the detection of the invention, e.g. the substrate of an enzyme (color developing substrate, etc.), the substrate in solution, enzymatic reaction-terminating liquid or the like when the labeling substance is an enzyme, and diluents for samples. Specific examples of diluents for samples include 20 mM Tris-HCl buffer (pH 7.4) containing PBS (phosphate-buffered physiological saline, pH 7.4), 137 mM sodium chloride and 3 mM potassium chloride (hereinafter abbreviated to "TBS"); and PBS or TBS containing 0.05% Tween 20 and 0.1-1% BSA. These diluents for samples may be used for diluting other substances such as antibodies.

(2) Pharmaceutical Compositions for Treating or Preventing Diseases

[0151]When the high affinity antibody of the invention has an effect of neutralizing the activity of an antigen which will become the pathogen of a disease, the antibody of the invention is useful in a pharmaceutical composition for treating or preventing the disease. The pharmaceutical composition of the invention comprises the high affinity antibody of the invention or a fragment thereof as an active ingredient and, is provided, preferably, in the form of a pharmaceutical composition comprising a pharmacologically acceptable carrier.

[0152]The "pharmacologically acceptable carrier" used herein includes excipients, diluents, fillers, disintegrants, stabilizers, antiseptics, buffers, emulsifiers, aromatics, coloring agents, sweetening agents, thickening agents, flavoring agents, dissolution aids and other additives. By using one or more of these carriers, various forms of pharmaceutical compositions may be prepared, e.g. tablets, pills, powders, granules, injections, solutions, capsules, troches, elixirs, suspensions, emulsions and syrups. These pharmaceutical compositions may be administered orally or parenterally. Other forms for parenteral administration include solutions for external use which comprise one or more active substances and are prescribed by conventional methods, suppositories for enteric administration, and pessaries.

[0153]The dose of the pharmaceutical composition of the invention varies depending on the age, sex, body weight and conditions of the patient, treatment effect, the method of administration, time period for treatment, or the type of the high affinity antibody (the active ingredient) contained in the composition. Usually, the pharmaceutical composition of the invention may be administered to adult patients in the range from 10 μg to 1000 mg per administration, preferably in the range from 10 μg to 100 mg per administration. However, the dose is not limited to this range.

[0154]For example, in the case of injections, the pharmaceutical composition of the invention may be dissolved or suspended in a pharmacologically acceptable carrier (such as physiological saline or commercial distilled water for injection) so that the concentration of the antibody in the carrier is from 0.1 μg/ml to 10 mg/ml. The thus prepared injection may be administered to human patients in need of treatment at a rate of 1 μg-100 mg/kg body weight, preferably at a rate of 50 μg-50 mg/kg body weight, per administration once to several times per day. The route of administration may be intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection or intraperitoneal injection, for example. Among all, intravenous injection is preferable. Optionally, injections may be prepared in the form of a non-aqueous diluent (e.g. propylene glycol, polyethylene glycol, vegetable oil such as olive oil, alcohol such as ethanol), suspension or emulsion. Sterilization of such injections may be performed by filter-sterilization through a bacteria removal filter, addition of antiseptics, or irradiation. Injections may take a form that is prepared into an injection at the time of use. Briefly, a solid composition is prepared by lyophilization or the like, and this solid composition may be dissolved in aseptic distilled water for injection or other solvent at the time of use.

5. Application of the Present Invention

[0155]The present inventors have induced overexpression of GANP in B cell tumor strains and analyzed them. As a result, the B cell tumor strains showed that GANP gene transfer has a remarkable effect in inducing somatic hypermutations in V region genes. Since this effect is not observed when a mutant gene in which phosphorylation of serine at position 502 (required for the primase activity of GANP) does not occur is used, it is suggested that RNA primase activity is necessary for the remarkable induction of somatic hypermutations in V region genes. These results demonstrate that GANP has an effect of enhancing the production of specific antibodies as a clinical, supplemental immunopotentiator.

[0156]It is also effective for clinical, supplemental immunopotentiation to use a retrovirus vector as a vector and a combination of GANP and a stimulation mediated by TNF family molecules such as DC40 or BAFF. Further, by transferring a GANP gene at the bone marrow cell level, induction of high affinity binding in T cells is also expected. It is expected that this gene transfer will manifest an excellent effect in such diseases as AIDS, hepatitis C, adult T cell leukemia or Bovine Spongeform Encepharopathy where high affinity antibodies are not obtained or, even if obtained, the production of high affinity antibodies cannot be maintained because mutations promptly occur in antigens.

[0157]The GANP gene overexpressing mammal of the invention is useful in developing monoclonal antibodies useful in the preparation of biological research reagents and clinical test reagents. For example, the preparation of a monoclonal antibody to a specific signal transduction molecule in a functional domain- or functional motif-specific manner and as a high affinity antibody with high binding ability easily is very widely applicable. Since many antibodies are not screened many times, sometimes it is impossible to use them in Western analysis and immunoprecipitation. When the transgenic mammal of the invention is used for antibody production, high affinity antibody-producing cells may be selected from a relatively small number of clones. Thus, the effect of the present invention in the reduction of cost, time and labor is great. In particular, the preparation of phosphorylated antibodies and specific antibodies to mutated sites of genes is applicable to diagnostics, or the selective injection method for medicines using antibodies. The production of high affinity antibodies which selectively bind to a specific gene sequence or nucleotide portion will also become possible.

[0158]A part of the steric structure of any substance (such as inorganic substance, carbohydrate, or chemically synthesized substance) is recognized as an antigen motif. Although no high affinity antibodies have been obtained to date, mice created by mating with autoimmune mice are effective for obtaining high affinity antibodies to all antigens. There is a possibility that high affinity antibodies whose binding ability is on the order of 10^-11 M might be obtained by this method. By introducing the developed technology of ELISA, it is possible to develop a technology to detect trace substances easily.

[0159]According to the present invention, it is also possible to provide a gene therapeutic for allergic diseases or autoimmune diseases, comprising an RNA primase inactivated-type GANP gene. The "RNA primase inactivated-type GANP gene" means a GANP gene in which the RNA primase domain is deficient or mutated. Due to mutations of the serine residue at position 502 and neighboring residues in the gene, the structure and function of GANP molecule encoded by this gene has been altered.

[0160]The gene therapeutic of the invention may be prepared by combining a recombinant vector comprising an RNA primase inactivated-type GANP gene with a base to be used in the gene therapeutic. As a vector for use in the construction of the recombinant vector, a viral vector such as retrovirus vector, adenovirus vector, adeno-associated vector, vaculovirus vector or vaccinia virus vector may be enumerated. Alternatively, an animal expression plasmid may be used. Preferably, the vector is a viral vector. When an RNA primase inactivated-type GANP gene has been integrated into a viral vector, viral particles containing the recombinant protein may be produced and combined with a base for the gene therapeutic to thereby prepare the gene therapeutic.

[0161]Specific examples of the base to be used in the gene therapeutic include those bases conventionally used in injections, e.g. distilled water; solution of sodium chloride or solution of a mixture of sodium chloride and inorganic salt; solution of mannitol, lactose, dextran or glucose; solution of amino acid such as glycine or arginine; mixed solution consisting of organic acid solution or salt solution and glucose solution. Alternatively, injections may be prepared as solutions, suspensions or dispersions by combining those bases with auxiliary agents such as osmoregulator, pH regulator, vegetable oil, surfactant, etc. according to conventional methods well known to those skilled in the art. It is also possible to powder or lyophilize these injections and dissolve them at the time of use.

[0162]The gene therapeutic of the invention may be administered systemically by conventional intravenous or intra-arterial administration, or administered locally by local injection or oral administration. The dose of the gene therapeutic of the invention varies depending on the age, sex, conditions of the patient, the route of administration, the number of times of administration, and the dosage form. Generally, the gene therapeutic of the invention may be administered to adult patients in the range from 1 μg/kg to 1000 mg/kg per day, preferably in the range from 10 μg/kg to 100 mg/kg per day, in the amount of the recombinant gene. The number of times of administration per day is not particularly limited.

EXAMPLES

[0163]Hereinbelow, the present invention will be described more specifically with reference to the following Examples which should not be construed as limiting the present invention.

Example 1

Expression and Function of GANP in Autoimmune Disease Model Animals

(Materials and Methods)

1. Animals

[0164]NZB, NZW, B/WF1, MRL/lpr and BXSB mice were purchased from Japan SLC Co.

[0165]C57BL/6 and BALB/c mice were purchased from Charles River Japan. NOD mice were kindly supplied from Dr. Miyazaki, the graduate school of Osaka University.

2. Antibodies and Reagents

[0166]Rat monoclonal antibodies to mouse B220 (RA3-6B2), mouse IgM (AM/3) and mouse IgD (CS/15) were purified from hybridoma culture supernatant and labeled with D-biotin-N-hydroxysuccinimide ester (Roche diagnostics, Branchburg, N.J.). Biotin-labeled rat anti-mouse Syndecan-1 and anti-mouse CD5 monoclonal antibodies were purchased (BD PharMingen, San Diego, Calif.). Biotin-labeled peanut agglutinin (PNA) was purchased from Vector Laboratories (Burlingame, Calif.).

3. Immunization

[0167]Trinitrophenyl keyhole limpet hemocyanin (TNP-KLH) and TNP-Ficoll were purchased from Biosearch Technologies (Novato, Calif.). Briefly, 100 μg of TNP-KLH emulsified in complete Freund's adjuvant or 25 μg of TNP-Ficoll was injected into the abdominal cavity of the mouse. Fourteen days thereafter, lymph organs were removed and frozen with OCT compound to be used in immunohistological analysis.

4. Immunohistological Analysis

[0168]Six-micrometer cryosections of organs were fixed in acetone for 5 min, blocked with 3% BSA in PBS for 15 min, and incubated for 1 hr with rat anti-mouse GANP monoclonal antibody (42-23) [Kuwahara, K. et al., 2000, Blood 95: 2321-2328] or rat anti-pSer⁵⁰² GANP monoclonal antibody (PG/103) [Kuwahara, K. et al., 2001, Proc. Natl. Acad. Sci. USA 98: 10279-10283]. Sections were mounted on slide glasses, which were washed with PBS several times and then incubated with alkali phosphatase (ALP)-conjugated goat anti-rat IgG antibody (ICN Pharmaceuticals, Costa Mesa, Calif.). Color development was carried out with Vector Blue kit (Vector). Double staining was carried out using biotin-labeled antibodies in combination with horse radish peroxidase (HRP)-conjugated streptavidin (Kirkegaard & Perry Laboratories, Gaithersburg, Md.). After color development with 3,3'-diaminobenzidine tetrahydrochloride (DAB; Dojin Kagaku), sections were fixed in 1% glutaraldehyde in PBS for 1 min. For mounting, Aquatex (Merck, Darmstadt, Germany) was used. In order to detect cells with proliferation activity in vivo, bromodeoxyuridine (BrdU) (Sigma Chemicals Co., St. Louis, Mo.; 1 mg/mouse) was injected intravenously 2 hrs before slaughter. Cells which synthesize DNA were stained with a combination of anti-BrdU monoclonal antibody (BD PharMingen) and ALP-conjugated goat anti-mouse Ig antibody (sigma), followed by color development with Vector Red (Vector) for detection. PAS staining was carried out as described previously [Jiang, Y et al., 1997, J. Immunol. 158: 992-997].

5. Results

[0169](1) Appearance of GANP^hi Cells in MRL/lpr Mouse Lymph Nodes

[0170]GANP is expressed highly in autoimmune-prone, highly active B cells. High level GANP-expressing lymphocytes (GANP^hi cells) appear spontaneously in peripheral lymph nodes of MRL/lpr mice in a non-immunized state.

[0171]Immunohistochemical analysis was performed on popliteal lymph nodes from autoimmune disease model (MRL/lpr and NZB) female mice and normal C57BL/6 female mice using anti-GANP monoclonal antibody and ALP-conjugated anti-rat Ig antibody.

[0172]The results are shown in FIG. 1. While GANP^hi cells stained with Vector Blue (ALP substrate) were observed in lymph nodes of MRL/lpr mice at week 7, such cells were not observed in NZB mice of the same age and appeared at week 40 (FIG. 1). In normal C57BL/6 mice, an extremely small number of GANP^hi cells were observed throughout the period of experiment.

[0173]Compared to C57BL/6 mice, autoimmune disease model mice revealed a remarkable increase in lymphocytes but showed no GANP^hi cells under non-immunized conditions (FIG. 1). The appearance of such GANP^hi cells was examined in lymph nodes of NZB mice which develop autoimmune conditions little by little as they get older. While young NZB mice (7 week old) did not have GANP^hi cells in their popliteal lymph nodes, aged NZB mice (40 week old) had a great number of GANP^hi cells.

[0174]It is considered that GANP RNA primase activity may play an important role in the activation and differentiation of B cells. Then, inventor compared the states of phosphorylation of Ser⁵⁰² (which is a key phosphorylation site for RNA primase activity) in NZB mice using anti-pSer⁵⁰² monoclonal antibody.

[0175]The expressions of GANP and pSer⁵⁰² GANP were compared in lymph nodes of NZB mice. Briefly, pSer⁵⁰² GANP was detected with anti-pSer⁵⁰² GANP (PG/103) monoclonal antibody (blue) and all sections were stained with biotin-labeled anti-B220 monoclonal antibody, followed by detection with a combination of HRP-conjugated streptavidin and DAB (brown). Representative data obtained from two independent experiments are shown in FIG. 2.

[0176]In FIG. 2, the bottom panel (graph) shows the time course of the numbers of GANP^hi cells (black column) and pSer⁵⁰² GANP^hi cells (column with slant lines) in extrafollicular regions.

[0177]GANP expression is remarkable at week 8; GANP^hi cells were detected throughout the experiment period up to week 32 (FIG. 2, upper panel). In contrast, pSer⁵⁰² positive cells reached the peak at week 8 and then sharply decreased (FIG. 2, middle panel). The numbers of reactive cells based on peak age obtained by microscopic observation are shown (FIG. 2, bottom panel). From these results, it is understood that GANP expression is accompanied by RNA primase activity at the beginning but this activity is not regulated for a long period of time. [0178](2) Spontaneous Appearance of GANP^hi Cells in the Red Pulp of the Spleen in Autoimmune-Prone Mice

[0179]Whether or not the GANP^hi cells detected in popliteal lymph nodes of autoimmune-prone NZB mice appear in the spleen under non-immunized conditions was examined.

[0180]Immuno-staining was carried out in the same manner as described in (1) above (FIG. 2). Representative data from three independent experiments are shown in FIG. 3.

[0181]GANP^hi cells appeared in the spleen at week 4. The cell count reached its maximum at week 12 but GANP^hi cells disappeared at week 24 (FIG. 3, upper panel). The expression of pSer⁵⁰² GANP was also detected at weeks 8 and 12 (FIG. 3, middle panel). From the results of comparison with relative cell counts in the red pulp, it is understood that the GANP^hi cells which had appeared in the spleen moved to peripheral lymph nodes 12 weeks thereafter. The increase of GANP^hi cells is proportional to the yield of autoantibody prior to the occurrence of autoimmune disease (FIGS. 2 and 3; Theofilopoulos, A. N. et al., 1985, Adv. Immunol. 37: 269-390).

[0182]The appearance of GANP^hi cells may be associated with abnormalities in B cells in autoimmune-prone mice. Therefore, the appearance of GANP^hi cells was examined in various autoimmune-prone mice (8 week old) under non-immunized conditions.

[0183]The results are shown in FIG. 4. GANP^hi cells appeared remarkably in the red pulps of MRL/lpr, NZB and B/WF1 mice.

[0184]Although the number of GANP^hi cells did not show a remarkable increase in the spleens of BXSB and NOD mice (both are SLE model mice), the number showed an increase when compared to the control mice, i.e. BALB/c mouse (FIG. 4) and C57BL/6 mouse (FIG. 1). Spleen sections showed, as a GC-like structure, or immature association of PNA⁺ B cells. GANP expression in the GC-like region was not high compared to GANP expression in the GC which was created by immunizing normal C57BL/6 mouse and BALB/c mouse with T cell-dependent antigens (TD-Ags). However, GANP^hi cells appeared remarkably in the red pulp region in autoimmune-prone mice (FIG. 4).

[0185]Further, GANP^hi cell population was analyzed with markers of lymphoid cells.

[0186]Spleen sections from NZB mice were double-stained with biotin-labeled B220 monoclonal antibody, biotin-labeled Syndecan-1 monoclonal antibody, biotin-labeled IgM monoclonal antibody and anti-IgG antibody to thereby identify GANP^hi cells.

[0187]The results are shown in FIG. 5. The photographs in the left side panel of FIG. 5 show sections when biotin-labeled IgM monoclonal antibody, anti-IgG antibody, biotin-labeled B220 monoclonal antibody and biotin-labeled Syndecan-1 monoclonal antibody were used, respectively. The photographs in the central panel show GANP expression in the same sections as described above. The right side panel is a superposition of the left side panel and the central panel. Those cells which are double-stained in the right side panel indicate that GANP^hi cells are B220.sup.- Syndecan1⁺ IgM⁺. GANP expression is shown in red when IgM, IgG and B220 antibodies were used, and shown in green when Syndecan-1 antibody was used. Markers are indicated in green when IgM, IgG and B220 antibodies were used, and indicated in red when Syndecan-1 antibody was used.

[0188]GANP^hi cells show the phenotype of B220.sup.- Syndecan-1⁺ and express a large quantity of IgM within cells (FIG. 5). GANP^hi cells are negative with respect to CR1, Thy-1, GL-7, CD23 and PNA. From these results, it is shown that GANP^hi cells are B-lineage cells of late maturing stage, perhaps plasma cells. In order to examine whether or not these GANP^hi cells are proliferative plasmablast cells, BrdU (1 mg/mouse) was intravenously injected into NZB mice, which were then incubated for 2 hrs so that BrdU was taken in vivo. Subsequently, spleen sections were prepared from the resultant mice.

[0189]Sections were double-stained with anti-GANP monoclonal antibody (blue) and anti-BrdU monoclonal antibody (red). PAS staining was carried out according to conventional methods.

[0190]The results are shown in FIG. 6. GC represents germinal center (left panel). GANP singly positive cells are shown with arrows; and PAS singly positive cells are shown with arrow heads (central panel).

[0191]Also, sections were stained with biotin-labeled anti-CD-5 monoclonal antibody. The PALS region represents the periarterial sheath in lymph nodes (right panel). FIG. 6 shows representative data obtained from three independent experiments.

[0192]Since GANP^hi cells are not positive with respect to BrdU intake (FIG. 6), it is suggested that these cells are not proliferative and are more mature than plasmablast stage.

[0193]As abnormal differentiation of B-1 cells, Mott cell formation is observed in autoimmune-prone mice. Mott cell is an abnormal morphology of plasma cell; a large number of IgM molecules are accumulated in rough-surfaced endoplasmic reticulum-associated follicles which are detected as intracytoplasmic Russell bodies by PAS staining [Jiang, Y et al., 1997, J. Immunol. 158: 992-997]. GANP^hi cells are not stained by PAS staining (FIG. 6), and thus can be distinguished from Mott cells which are B-1 cell-derived plasma cells. Since the GANP^hi population in the spleen was negative in CD5 expression (FIG. 6) and peritoneal cells obtained from NZB mice (12 week) were negative with respect to GANP^hi cells, it is suggested that B-1 cells are not expressing a large quantity of GANP. From these results, GANP^hi cells are classified into highly active B cells of autoimmune sate, and it is suggested that this population is of a lineage whose origin is different from the origin of B-1 cells. [0194](3) Induction of GANP^hi Cells in Normal Mice by Immunization with TD-Ag

[0195]Whether or not the appearance of GANP^hi plasma cells in secondary lymph organs is limited to autoimmune-prone mice was examined.

[0196]Female C57BL/6 mice (7 week old) were immunized intraperitoneally with TNP-Ficoll (TI-2-Ag) or TNP-KLH (TD-Ag). Their spleens were removed on day 14. Those mice immunized with TNP-Ficoll did not show GANP^hi cells in the red pulp region when counter-stained with biotin-labeled anti-IgD monoclonal antibody (FIG. 7, left panel). Those mice immunized with TNP-KLH showed the induction of GANP^hi cells in the red pulp region (FIG. 7, right panel). In FIG. 7, GANP^hi cells are marked with arrows. WP represents the white pulp region.

[0197]GANP^hi plasma cell population is also induced in the spleens of normal C57BL/6 and BALB/c mice by immunization with TD-Ag, though the number of cells is very small (FIG. 7). Immunization with T cell-independent Ag (TI-Ag) has only a small effect in inducing such cells. The GANP^hi cell population showed a phenotype similar to B220^loIgM^hiIgD^loGL-7^loPNA^loCD5^loCD40^lo- , but was Syndecan-1⁺.

[0198]These results indicate that the generation of GANP^hi plasma cells in autoimmune-prone mice is induced by stimulation similar to the stimulation supplied for immune responses to TD-Ag. Ag-driven B cells which have undergone proliferation and differentiation in the GC may be localized in the red pulp region as the plasma cell stage for a longer period, while expressing GANP.

Example 2

Excessive Expression of GANP

(Methods)

[0199]1. Stable Transfection into Daudi Cells

[0200]Ten micrograms of linearized pCXN-2 mouse GANP or GANPS/A502 cDNA was electroporated into Daudi cells with Gene Pulser II (Bio-Rad). After 48 hrs, selection started with G418 (Promega; 1 mg/ml) to thereby obtain Daudi cells which express mouse GANP stably.

2. Analysis of the IgV_H Transcript of Daudi Transfectants

[0201]Total RNA was extracted from total cells with Trizol (Invitrogen). cDNA was obtained as described previously (Kuwahara, K. et al., Blood 95, 2321-2328 (2000)). LV_H3-C_H1Cμ transcript was amplified using the following primers and the reaction solution. For amplification, Pfu Turbo (Stratagene) was used.

TABLE-US-00001 5'-LV_H3 primer: (SEQ ID NO: 5) 5'-CTATAACCATGGACCATGGACATACTTTGTTCC-3' 3'-XbaI-C_H1-Cμ primer: (SEQ ID NO: 6) 5'-TGCATGCATTCTAGAGTTGCCGTTGGGGTGCTGGAC-3'

Composition of the Reaction Solution:

TABLE-US-00002 [0202]cDNA 0.5 μl 10x buffer 2.5 μl 10 mM dNTP mix 0.5 μl 5'-LVH3 primer (10 μM) 1 μl 3'-Xba I-CH1-Cμ primer (10 μM) 1 μl Pfu Turbo 0.5 μl dH₂O 19.5 μl

Reaction Conditions:

[0203]94° C. for 1 min

[0204][94° C. for 1 min; 62° C. for 1 min; 72° C. for 1 min]×35 cycles

[0205]72° C. for 10 min

[0206]4° C.

[0207]The resultant PCR product was digested with NcoI and XbaI, purified in a gel, and ligated to a plasmid digested with NcoI-XbaI. After transformation into competent bacterial cells, a small quantity of plasmid DNA was prepared with QIAprep kit (Qiagen). The nucleotide sequence of this plasmid DNA was determined with an automated sequencer (Applied Biosystems).

3. Preparation of GANP-Transgenic (Tg) Mouse

[0208]A transgene was prepared by inserting a 5.6 kb mouse GANP gene into the XhoI site of pLG vector. This vector having a human immunoglobulin intron enhancer domain (2 kb EcoRI fragment) is a specific vector that directs strong expression in B cells. This gene was linearized and transferred into mice. Briefly, a linearized pLG vector (Koike, M. et al., Int. Immunol. 7, 21-30 (1995)) comprising the full-length mouse GANP cDNA was micro-injected into fertilized eggs of C57BL/6 mice. The presence of the transferred gene was screened using genomic DNA obtained from mouse tail vein, the following primers and the reaction solution.

TABLE-US-00003 (SEQ ID NO: 7) 1-5' primer: 5'-TCCCGCCTTCCAGCTGTGAC-3' (SEQ ID NO: 8) 1-3' primer: 5'-GTGCTGCTGTGTTATGTCCT-3'

Composition of the Reaction Solution:

TABLE-US-00004 [0209]DNA (50 ng/μl) 1 μl 10x buffer 2.0 μl 2.5 mM dNTP mix 2.0 μl 1-5' primer (10 μM) 0.8 μl 1-3' primer (10 μM) 0.8 μl Z-Taq DNA polymerase 0.1 μl dH₂O 13.3 μl

Reaction Conditions:

[0210][98° C. for 5 sec; 59° C. for 5 sec; 72° C. for 10 sec]×35 cycles

[0211]4° C.

[0212]4. RT-PCR

[0213]Total RNA was extracted from the spleen or spleen B cells using Trizol (Invitrogen). RT-PCR was performed with two primers (1-5' primer and 1-3' primer) to synthesize cDNA (Kuwahara, K. et al., Blood 95, 2321-2328 (2000)). GANP transcript was detected by agarose gel electrophoresis. β-actin transcript was used as a control.

[0214]5. Results [0215](1) Somatic Hypermutations (SHMs) in V Region Genes of Daudi Transfectants Expressing GANP Stably

[0216]A GANP gene was transferred into various human B lymphocytes used in SHM analysis in vitro (Rogozin, I. B., et al., Nat. Immunol. 2: 530-536 (2001); Kuwahara, K. et al. Blood 95: 2321-2328 (2000); and Denepoux, S. et al., Immunity 6: 35-46 (1997)). Although a great number of B cell strains were incapable of transfection, it was possible to transfer a GANP gene into Daudi B cells which express AID that usually does not generate SHMs while maintained.

[0217]The resultant clones showed highly frequent SHMs (5×10^-4/bp) in the V regions, compared to wild-type cells and pseudo-transfectants.

[0218]V_H3-C_H1Cμ fragment was amplified by PCR and subcloned into a plasmid, followed by sequencing.

[0219]Schematic diagrams of somatic hypermutations are shown in FIG. 8: A-C. Vertical linerepresents a silent mutation (where the amino acid is not changed), and the other mark (short vertical line with black circle) represents a mutation where the amino acid is replaced. While Daudi/mock shows few mutations, four clones of Daudi/DANP-14, -15, -17 and -21 more of less show a great number of mutations. The efficiency of inducing mutations is decreased in the transfectant into which a mutant (GANP S/A) has been introduced; in this mutant, Ser⁵⁰² involved in the control of DNA primase activity is replaced with alanine.

[0220]SHMs were not induced in constant region genes (FIG. 8: A-C). The RNA primase activity of GANP is regulated by the phosphorylation of S502, and this phosphorylation can be detected with a specific monoclonal antibody (Kuwahara, K. et al., Proc. Natl. Acad. Sci. USA 98: 10279-10283 (2001)). Since both in vivo and in vitro stimulation of B cells induce the phosphorylation of Ser⁵⁰² (Kuwahara, K. et al., Proc. Natl. Acad. Sci. USA 98: 10279-10283 (2001)), whether or not this phosphorylation is involved in the generation of SHMs in Daudi B cells was examined.

[0221]When a non-phosphorylated GANP mutant (GANP-S502A) was introduced, SHMs were not induced (FIG. 8A). Therefore, it is suggested that the phosphorylation of S502 is important for the generation of SHMs in GC-B cells. [0222](2) Transgenic Mouse Overexpressing GANP in B Cells

[0223]In order to examine the involvement of GANP in immune responses, GANP-transgenic (Tg) mouse which overexpresses GANP under the control of human Ig enhancer and promoter was created (FIG. 9: A-B). Enhancement in GANP mRNA expression was confirmed by RT-PCT.

[0224]This mouse showed an increase in GANP expression in B cells (FIG. 9C), and showed normal differentiation of B lineage cells in surface marker analysis of bone marrow, spleen and lymph node cells.

[0225]In order to investigate into the in vivo role of GANP in SHMs, the V_H186.2 region was examined after immunization with NP-CG (which is TD-Ag). Briefly, 50 μg of NP-CG was administered to GANP-overexpressing transgenic (Tg) mice three times at intervals of two weeks. Then, the V_H186.2 region was amplified by PCR, followed by analysis of somatic hypermutations.

[0226]The results are shown in FIG. 10. The number of mutations was slightly increased in Tg mice. However, the somatic hypermutation of W³³ to L which indicates high affinity was increased almost 3-fold in Tg mice. "CDR" represents complementarity determining region.

[0227]The V_H186.2 locus shows SHMs of a peculiar pattern for high affinity IgG(γ1λ1)NP response. The sequence analysis on the total spleen B cells after immunization with NP-CG revealed that SHM frequency is slightly increased in GANP-Tg mice compared to wild-type mice (FIG. 10).

[0228]It has been shown previously that these somatic hypermutations are important for affinity maturation of hapten specific B cells (Allen, D. et al., EMBO J. 1995-2001 (1988)).

Example 3

Preparation of B Cell Specific GANP-Deficient Mice (B-GANP.sup.-/- Mice)

(Methods)

1. Establishment of CD19-Cre/+GANP Flox Mice

[0229]Using genomic DNA encoding GANP, neomycin resistance gene (neo) was inserted downstream of exon II to thereby construct a targeting vector. LoxP sites were introduced into the 3' flanking region to neo and the intron between exons I and II, respectively.

[0230]Briefly, flox mice in which GANP exon II is sandwiched by two loxP sequences were prepared. These mice were crossed with CD19-Cre mice to thereby establish B cell specific GANP-deficient mice (FIG. 11: A and B).

[0231]The targeting vector was linearized and electroporated into TT2 ES cells (Yagi, T. et al., Anal. Biochem. 214: 70-76 (1993)) for transfection. After selection with G418, ES colonies were picked up and incubated with proteinase K. Homologous recombinants were screened for with the following neo2 primer and CGK3'-2 primer.

TABLE-US-00005 neo2 primer: (SEQ ID NO: 9) 5'-GCCTGCTTGCCGAATATCATGGTGGAAAAT-3' CGK3'-2 primer: (SEQ ID NO: 10) 5'-GGCACCAAGCATGCACGGAGTACACAGA-3'

[0232]Homologous recombination was confirmed by analyzing the BamHI-digested DNAs of ES clones by Southern blotting using probe A. Using three positive clones showing a 4 kb band, microinjection into ICR blastocysts was carried out to prepare chimeric first generation mice. The absence of GANP expression in B cells was confirmed by Southern blotting, RT-PCR and cell staining (FIG. 11: C, D and E).

[0233]GANP flox/+ mice were backcrossed with C57BL/6 mice at least 10 times. In order to delete GANP gene in B cells, GANP-floxed mice were crossed with CD19-Cre knock-in mice (Rickert, R. C., et al., Nucleic Acids Res. 25, 1317-1318 (1997)).

2. FACS Analysis

[0234]Lymph organ-derived single cell suspensions were stained with each biotin-labeled monoclonal antibody for 1 hr on ice. After washing with staining buffer, cells were incubated with FITC-conjugated streptavidin (Amersham Bioscience) and PE-conjugated monoclonal antibody for 1 hr. Lymphocytes were analyzed with FACScan (Becton Dickinson) using Cell Quest software.

3. Purification of B Cells

[0235]Spleen cells were isolated from Cre-flox/+ mice and B-GANP.sup.-/- mice (7 to 8 week old) and treated with 0.15 M ammonium chloride buffer to remove erythrocytes. After incubation at 37° C. for 30 min on plastic dishes, unadhered cells were recovered as lymphocytes. Then, T cells were removed therefrom using Dynabeads-anti-mouse Thy1.2 monoclonal antibody (Dynal) according to the protocol attached thereto. The purity of B cells (90% or more) was confirmed by cell surface staining with FITC-conjugated B220 monoclonal antibody (BD Pharmingen).

4. In Vitro Proliferation Assay

[0236]Purified B cells were incubated in RPMI-1640 medium (with or without cell division promoter) containing 10% thermo-inactivated FCS (JRH Biosciences), 2 mM L-glutamine and 5×10^-5 M 2-mercaptoethanol in 96-well microplates at 2×10⁵ cells/well for 48 hrs. Cells were recovered after pulsing with [³H]-thymidine at 0.2 μCi/well for 16 hrs. Then, the radioactivity taken up was measured with a scintillation counter.

[0237]As the cell division promoter, affinity-purified goat anti-mouse μ-chain-specific antibody (10 μg/ml) [F(ab')₂] (ICN), rat anti-mouse CD40 monoclonal antibody (LB429; 10 μg/ml) and LPS (Sigma; 10 μg/ml) were used.

5. Antigens and Immunization

[0238]TNP-KLH, TNP-Ficoll and nitrophenyl-chicken γ globulin (NP-CG) (23:1) were purchased from Biosearch Technologies. Fifty micrograms of TNP-KLH and NP-CG (precipitated with aluminium) or 25 μg of TNP-Ficoll (dissolved in PBS) was injected into the abdominal cavities of Cre-flox/+ mice and B-GANP.sup.-/- mice.

6. Measurement of Antigen Specific Antibody Production

[0239]At day 10 or 14 after the immunization, sera were recovered from immunized mice. ELISA plates were coated with 5 μg/well of TNP-BSA (Biosearch Technology). Each well was blocked with 3% BSA in PBS, and incubated with serially diluted serum. After washing with PBS-0.1% Tween 20, each well was incubated with biotin-conjugated isotype-specific monoclonal antibody and alkaline phosphatase (ALP)-conjugated streptavidin (Southern Biotechnology). Color development was performed in the presence of substrates.

[0240]In order to determine the affinity of NP-binding antibodies in the sera, the ratio of NP2-binding antibody to NP25-binding antibody was calculated by differential ELISA using NP2-BSA (two NPs are bound to BSA per molecule) and NP25-BSA (25 NPs are bound to BSA per molecule) (Biosearch Technology) as coated antigens.

7. Immunohistochemistry

[0241]Spleen sections (8 μm) from immunized mice were fixed lightly in acetone. These samples were blocked with 3% BSA in PBS-Tween 20 and incubated with anti-IgD monoclonal antibody and ALP-conjugated anti-rat IgG (ICN) antibody. The first color development was performed with Vector Blue kit (Vector). The second color development was performed by incubating the sample with biotin-conjugated peanut agglutinin (PNA) (Vector) and horseradish peroxidase-conjugated streptavidin (Kirkegaard & Perry) and then incubating with 3,3'-diaminobenzidine tetrahydrochloride (Dojindo). Samples were fixed with 1% glutaraldehyde in PBS and then mounted with Aquatex (Merck).

8. Sequence Analysis of V_H186.2 Gene

[0242]NP-binding IgG1.sup.dullCD38^low B cells from NP-CG-immunized Cre-flox/⁺ and B-GANP.sup.-/- mice were fractioned with FACS Vantage (Becton Dickinson Biosciences) using (4-hydroxy-5-iode-3-nitrophenyl)acetyl (NIP) and incubated with proteinase K at 37° C. overnight. Using the resultant lysate, PCR was performed two times as described previously (Takahashi, Y et al., Immunity 14: 181-192 (2001)). The genetic DNA of V_H186.2 was ligated to pBluescript, followed by determination of the sequence with an automated sequencer.

9. Detection of Apoptotic Cells

[0243]B cells purified from Cre-flox/⁺ and B-GANP.sup.-/- mice were stimulated with various reagents for 40 hrs (Watanabe, N. et al., (1998) Scand. J. Immunol. 47: 541-547). For detection of AICD, anti-μ antibody (50 μg/ml) was immobilized on 24-well plates. For detection of other types of apoptosis, purified B cells was stimulated with various stimulants and the incubated with anti-Fas monoclonal antibody (Jo2; BD Pharmingen) for 4 hrs (Wang, J. et al., (1996) J. Exp. Med. 184, 831-838). Cells were incubated in propidium iodide (PI) solution (50 μg/ml PI, 0.1% Triton X-100, 0.1% sodium citrate) at room temperature for 1 hr, and apoptotic cells were calculated (percent) as sub-G1 area by FACScan. Further, apoptotic cells were also confirmed microscopically after trypan blue staining.

10. TUNEL Assay

[0244]Cre-flox/⁺ and B-GANP.sup.-/- mice were immunized with SRBC (sheep red blood cells). Spleen cryosections were prepared therefrom and fixed in 4% paraformaldehyde in PBS. Section samples were treated with MEBSTAIN Apoptosis Kit II (MBL) and counter-stained with PI. For use in an experiment conducted together with TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay, section samples were also stained with ant-IgG₁ monoclonal antibody (BD Pharmingen) and Alexa 546-conjugated goat anti-rat IgG antibody (Molecular Probes). Positive signals were detected and the results were confirmed with a fluorescence microscope (BX51; Olympus).

11. Results

[0245](1) The Role of RNA Primase GANP

[0246]In order to investigate into the role of RNA primase GANP, B-GANP.sup.-/- mice which are deficient in GANP gene in their CD19⁺ B cells were prepared using Cre-loxP system (FIG. 11: A and B). The GANP gene of the B-GANP.sup.-/- mice lacked most of exon II (FIG. 11C). B-GANP.sup.-/- cells did not express GANP mRNA (FIG. 11D) and, according to immunostaining, expressed little GANP protein (FIG. 11E). B-GANP.sup.-/- mice grew normally, showing normal numbers of lymphocytes in the bone marrow, spleen, thymus and lymph nodes. According to flow cytometry, B-GANP.sup.-/- mice showed surface marker profiles on cells of the bone marrow, spleen and lymph nodes similar to those observed in the control Cre-flox/+ mice (FIG. 12); there was no difference between B-GANP.sup.-/- mice and Cre-flox/+ mice.

[0247]The number of mature B cells expressing sIgM^lowsIgD^high (IgM⁺IgD⁺) was decreased in the lymph nodes of B-GANP.sup.-/- mice. B cells from B-GANP.sup.-/- mice showed normal proliferation responses after in vitro stimulation with anti-μ antibody, anti-μ antibody+anti-CD40 monoclonal antibody, or lipopolysaccharide (FIG. 13: white column represents B-GANP.sup.-/- and black column represents Cre-flox/+). On the other hand, B cells from B-GANP.sup.-/- mice showed a decrease in proliferation activity after stimulation with anti-CD40 monoclonal antibody (5 and 10 μg/ml) (FIG. 13). This indicates that responses to CD40/CD145 interaction are slightly impaired in B cell proliferation in B-GANP.sup.-/- mice. The amounts of serum Ig in B-GANP.sup.-/- mice were similar to those in Cre-flox/+ mice (FIG. 14).

[0248](2) Antigen Specific Antibody Production in B-GANP.sup.-/- Mice

[0249]Immunoresponses of B-GANP.sup.-/- mice after immunization of TI-Ag or TD-Ag were examined. At day 14 after immunization with a TI antigen trinitrophenyl (TNP)-Ficoll, anti-TNP antibody titers were measured by ELISA. As a result, TNP-Ficoll induced similar responses in B-GANP.sup.-/- mice and Cre-flox/+ mice; no particular difference was observed (FIG. 15).

[0250]When germinal center (GC) formation was examined, mutant mice showed delayed GC formation in response to TD-Ags such as TNP-keyhole limpet hemocyanin (KLH) or NP-CG, compared to Cre-flox/+ mice.

[0251]With respect to the peak response on GC formation, Cre-flox/+ mice showed large matured GCs stained with peanut agglutinin (a marker for GC-B cells) at day 10 (arrow marks in FIG. 16). At day 10 after immunization, GC formation in B-GANP.sup.-/- mice was slightly less. However, B-GANP.sup.-/- mice showed more GC formation than Cre-flox/+ mice at day 14, and they still showed vigorous GC formation even at day 20 (arrow marks in FIG. 16).

[0252]Since B-GANP.sup.-/- mice showed definite GC formation at day 14, their antigen specific antibody responses were measured (FIG. 17). When immunized with TNP-KLH (a TD antigen), B-GANP.sup.-/- mice did not show definite GCs until day 10 after the immunization; no difference was observed in antibody titers between B-GANP.sup.-/- mice and Cre-flox/+ mice. At day 14, however, B-GANP.sup.-/- mice showed gradual increase and expansion of GCs (FIG. 17). Mutant mice showed antibody responses to TNP-KLH similar to those shown by Cre-flox/+mice.

[0253](3) Obstacles to Affinity Maturation in B-GANP.sup.-/- Mice

[0254]In order to further investigate into the characteristic of the GC in B-GANP.sup.-/- mice (i.e. antibody response is of low affinity), antigen specific IgG1 ⁺ GC-B cells were examined after immunization with NP-CG.

[0255]By differential ELISA using conjugates of NP hapten with different molecular weights and a protein, responses to NP2-BSA conjugate were compared to responses to multi-hapten NP25-BSA conjugate.

[0256]In B-GANP.sup.-/- mice, antibody responses to NP2-BSA conjugate were of low affinity (13%) at day 35 after immunization with NP-CG. This value was remarkably lower than the value of Cre-flox/+ mice (42%) (FIG. 18).

[0257]Further, as shown in FIG. 19, NP-specific IgG1.sup.dullCD38^low B cells were remarkably decreased in B-GANP.sup.-/- mice. Specifically, while the ratio of these B cells was 1,164 cells/10⁶ cells in Cre-flox/+ mice at day 10 after immunization, it was 88 cells/10⁶ cells in B-GANP.sup.-/- mice. At day 14, while Cre-flox/+ mice had 879 cells/10⁶ cells, B-GANP.sup.-/- mice had 83 cells/10⁶ cells. This tendency was unchanged at day 20.

[0258]In contrast, IgG1^highCD38^high memory B cells were not decreased. These results indicate that the mutation of no GANP expression caused defect in B cell differentiation at the stage of IgG1^highCD38^low GC-B cells.

[0259]In order to confirm the reduced affinity maturation in antibodies of B-GANP.sup.-/- mice, the sequence of the V_H186.2 region in spleen B cells was examined after immunization with NP-CG

[0260]Since somatic hypermutations occur at this stage of B cell differentiation, a variety of purified B cells were examined on SHMs in V_H186.2 locus. It should be noted that V_H186.2 locus is used for high affinity IgG (γ1λ1) NP-responses (Cumano, A. & Rajewsky, K. (1985) Eur. J. Immunol. 15, 512-520). With respect to IgM locus, no difference was observed between B-GANP.sup.-/- mice and Cre-flox/+ mice.

[0261]Subsequently, B-GANP.sup.-/- mice or Cre-flox/+ mice were immunized with NP-CG, followed by sorting for NP-binding IgG₁ weakly positive CD38 weakly positive GC-B cells (i.e. Ag-binding IgG₁ B cells) (FIG. 19). After the sorting, genomic DNA was extracted from the resultant cells. V_H186.2 was amplified by PCR and subjected to sequence analysis. Then, V_H186.2 sequences were compared (FIG. 20: A-L). Panels A-F in FIG. 20 show comparison of V_H186.2 sequences of Cre-flox/+ mice. Panels G-L in FIG. 20 show comparison of V_H186.2 sequences of B-GANP.sup.-/- mice.

[0262]In B-GANP.sup.-/- mice, the frequency of mutations in the entire IgV region sequence was 14×10^-3, showing a decrease compared to Cre-flox/+ mice (21×10^-3) (FIG. 21). Further, the high affinity type mutation of W³³ to L (i.e. mutation of the 33rd amino acid residue tryptophan to lysine, which is observed remarkably in C57BL/6 mice) was 13% (2/15 V regions), showing a remarkable decrease compared to Ce-flox/+ mice (71%, 10/14 V regions) and the lowering of affinity to 1/3 (FIG. 22).

[0263]From these results, it was demonstrated that GANP is essential for the affinity maturation of antibodies.

[0264](4) Protective Function from Apoptosis in B-GANP.sup.-/- Mouse B Cells

[0265]It is considered that the decrease in high affinity antibody production in B-GANP.sup.-/- mice is caused because B cells after antigen stimulation are unstable. Then, in order to examine the suceptibility of B cells, the apoptosis of B cells in vitro was studied.

[0266]In normal B cells, activation-induced cell death (AICD) was induced by strongly cross-linked B cell antigen receptor, and this AICD was prevented by stimulation by CD40. In B-GANP.sup.-/- B cells, though the susceptibility to AICD stimulation was equivalent to that of normal B cells (control), inhibition of anti-CD40-mediated apoptosis was inferior to Cre-flox/+control B cells (FIG. 23). This means that B-GANP.sup.-/- mice lack the protective function for antigen-reactive B cells during GC formation.

[0267]In GCs, B cells stimulated with Ag and CD40/CD154 interaction induce the surface expression of Fas/CD95 and become susceptible to Fas-induced apoptosis. Then, the inventor measured the susceptibility of B-GANP.sup.-/- B cells to anti-CD95 stimulation.

[0268]First, spleen B cells were stimulated with anti-CD40 monoclonal antibody (LB429), anti-μ antibody+anti-CD40 monoclonal antibody, IL-4+anti-CD40 monoclonal antibody, and anti-μ antibody+IL-4+anti-CD40 monoclonal antibody for 48 hrs, and then anti-CD95 monoclonal antibody was added to the culture medium.

[0269]As a result, apoptotic responses of B-GANP.sup.-/- mouse B cells were similar to the responses of Cre-flox/+ mouse B cells; no difference was observed between B-GANP.sup.-/- mice and Cre-flox/+ mice (FIG. 24).

[0270]As described above, the stimulation with anti-CD95 after anti-CD40 (LB429) treatment did not show any difference between B-GANP.sup.-/- mice and Cre-flox/+ mice in the induction of expression. This suggests that B-G B cells may be susceptible to the apoptotic stimulation normally received by GC-B cells in vivo. Therefore, TUNEL assay was carried out using tissue sections from mice immunized with SRBC as TD-Ag.

[0271]As a result, TUNEL-positive cells increased in the GC region of B-GANP.sup.-/- mice, and most of them also showed IgG₁ expression (FIGS. 25 and 26). These results revealed that most of the apoptotic cells of B-GANP.sup.-/- mice are GC-B cells (FIGS. 25 and 26).

[0272]Subsequently, the inventor examined the RNA expression of Bcl-2 family members which are recognized to be the molecules necessary for CD40-mediated inhibition of apoptosis of various malignant lymphoma cells and normal B cells.

[0273]Stimulation with anti-μ antibody+IL-4 induced an apparent increase in bcl-2 transcription in Cre-flox/+ B cells, and anti-CD40 mAb further up-regulated this expression (FIG. 27). The B-GANP.sup.-/- B cells showed similar up-regulation of bcl-2 transcripts by stimulation with anti-μ antibody, but the response to anti-CD40 mAb (anti-CD40 mAb alone or anti-μ Ab+L-4+anti-CD40 mAb) was not as high as the response in Cre-flox/+ B cells (FIG. 27). In other words, the RNA expression levels of Bcl-2 family involved in apoptosis inhibition were decreased in B-GANP.sup.-/- B cells compared to the control (FIG. 27).

[0274]With respect to bcl-X₁, bax and bad in mutant B cells, the expression levels were equivalent to those in Cre-flox/+ B cells.

[0275]These results suggest that GANP regulates the signal transduction of CD40-mediated induction of Bcl-2 expression in GC-B cells, which greatly contributes to the survival of high-affinity BCR⁺ B cells in vivo.

[0276](5) Conclusion

[0277]The results obtained from B-GANP.sup.-/- mice and GANP-Tg mice demonstrate that GANP is involved in the generation of high affinity B cells after immunization with TD-Ag. As a role of GANP, GANP may mediate efficient recruit and regulation of DNA polymerase in GC-B cells. Once GC-B cells with V-region SHMs have acquired high-affinity BCRs, they should be positively selected and further SHMs in the V regions might be suppressed to thereby guarantee the production of high affinity antibodies in vivo. Since AID expression in GC-B cells may generate DNA mutations continuously, regulation of AID activity might be necessary for maintaining high affinity BCRs in B cells. The results obtained from B-GANP.sup.-/- mice suggest that GANP is necessary for SHM process.

Example 4

Production of High Affinity Antibodies Using Ganp Transgenic Mice

1. Comparison of Antibody Titers by Differential ELISA

[0278]Each two wild-type (WT) mice and GANP transgenic (Tg) mice were immunized with 100 μg of NP-CG. At day 28 after the immunization, serum samples were taken from them and subjected to ELISA. Briefly, ELISA plates were coated with 20 μg/ml of NP2-BSA or NP17-BSA overnight at 4° C. Then, the plates were blocked with 3% BSA/PBS-0.1% Tween 20 for 1 hr, followed by reaction with the serum for 1 hr. After washing with PBS-0.1% Tween 20 three times, biotin-labeled anti-mouse IgG₁ antibody (Southern Biotechnology) was reacted for 1 hr. Then, after washing with PBS-0.1% Tween 20 three times, alkaline phosphatase-labeled streptavidin (Southern Biotechnology) was reacted for 30 min. After washing with PBS-0.1% Tween 20 three times and with TBS once, color was developed using p-nitrophenyl phosphate as a substrate. Absorbance was measured at 405 nm.

[0279]The results are shown in FIG. 28. From these results, it is understood that a high affinity antibody is produced by using GANP transgenic mice.

2. Analysis of Antigen-Antibody Binding Affinity Using ELISA and Biacore

[0280]Wild-type (WT) mice and GANP transgenic (Tg) mice were immunized with NP-CG Cells from them were subjected to cell fusion to obtain hybridomas. Using the culture supernatants of positive hybridoma clones, binding curves of antibodies responding to the antigen were obtained by ELISA and with Biacore. The utility of Tg mice was shown from the resultant binding curves.

[0281](1) Materials

(a) Animals

[0282]Wild-type (WT) mice and GANP transgenic (Tg) mice.

(b) Antibodies and Reagents

[0283]NP16-CG (16 NPs are coupled to CG (chicken immunoglobulin) per molecule), NP2-BSA (2 NPs are coupled to BSA (bovine serum albumin) per molecule), NP17-BSA (17 NPs are coupled to BSA per molecule), HRP-labeled anti-mouse IgG, IgA and IgM were used.

[0284](2) Methods and Results

[0285]Each five wild-type (WT) mice and GANP transgenic (Tg) mice were immunized with NP16-CG three times at intervals of two weeks. After the 3rd immunization, the mice were exsanguinated, and antibody titers were compared using antisera. The results also confirmed the utility of GANP-Tg mice as the results described in (1) above.

[0286]Spleen cells from those mice which showed a high valence among them were fused with P3U1 myeloma cells, and plated at a density of 1×10⁵ cells/well based on the numbers of spleen cells from GANP-Tg mice (6.0×10⁷) and from WT mice (4.8×10⁷). GANP-Tg mice-derived 600 hybridoma clones and WT mice-derived 480 hybridoma clones were cultured in HAT medium.

[0287]At day 9 of HAT culture, the culture supernatant was recovered and subjected to ELISA using NP2-BSA (1 μg/ml) as an immobilized antigen. Upper 2.5% clones showing production of high affinity antibodies as determined by measurement of absorbance in ELISA were selected from both culture supernatants derived from GANP-Tg mice and WT mice. Then, cloning was carried out using HT medium.

[0288]At day 9 of HT culture, culture supernatants were recovered and subjected to ELISA using NP2-BSA (1 μg/ml) as an immobilized antigen. As a result, 6 hybridoma clones (G2-6, G2-9, G2-12, G2-14, G2-15 and G2-16) were established from GANP-Tg mice and one hybridoma clone (W2-7) from WT mice.

[0289]Individual clones from GANP-Tg mice and WT mice were cultured in RPMI medium, and 1 ml each of culture supernatant appropriate for use in the following experiment was secured. Using this culture supernatant, the following evaluation and examination were carried out.

(a) ELISA

[0290]For the evaluation of antibody titers, antigens different in nature (i.e. substances different in NP content per CG molecule) were used, and antibody titers were evaluated based on the ratios of ELISA reactivities.

[0291]This method is useful for measuring the affinity of NP. It is simple and capable of testing a large number of samples. Therefore, this method is appropriate and reliable as primary screening.

[0292]First, NP2-BSA (1 μg/ml) and NP17-BSA (1 μg/ml) were separately immobilized as antigens at 4° C. overnight. The antigen-immobilized plates were washed with PBS-Tween 20 and blocked with skim milk-PBS-Tween 20. After washing further with PBS-Tween 20, RPMI culture supernatants from GANP-Tg mice-derived 6 clones (G2-6, G2-9, G2-12, G2-14, G2-15 and G2-16) and WT mice-derived 1 clone (W2-7) (stock solution to 256-fold dilution) were reacted with the immobilized antigen at room temperature for 1 hr. Subsequently, the plates were washed with PBS-Tween 20. Then, HRP-conjugated anti-mouse IgG, IgA and IgM were reacted at room temperature for 1 hr. After washing with PBS-Tween 20, color was developed with ortho-phenylene diamine (OPD) for 5 min, followed by termination of the reaction with 2N sulfuric acid.

[0293]Absorbance was meatured with an ELISA reader at 490 mm.

[0294]The results of ELISA are shown in FIG. 29. From these results, it is understood that high affinity antibodies are produced by using GANP-Tg mice.

(b) High Affinity Analysis Using Biacore

[0295]Using the clone which is predicted to be most high in affinity from the results of ELISA described above, physicochemical binding ability was examined with Biacore.

[0296]Analysis with Biacore was performed as described below. Briefly, NP2-BSA (1 μg/ml) was bound to Biacore chip as a ligand. As analyte solutions, RPMI culture supernatants from clone Tg (G2-9) which was predicted to be highest in affinity, clone Tg (G2-15) which was predicted to be lowest in affinity, and clone WT (W2-7) were used. Association rate constant (k ass), dissociation rate constant (k diss) and dissociation constant KD (KD=k diss/k ass) that is an indicator of affinity were calculated for each of the above culture supernatants. The smaller the KD value is, the higher the affinity is evaluated.

[0297]As a result, the Biacore pattern of G2-9 (ELISA: 82.9% NP2/NP17 ratio) is shown in FIG. 30. Curves (a) to (e) appearing in FIG. 30 correspond to antibody concentrations of 26.6, 13.3, 6.65, 3.33 and 1.66 nM, respectively. From the above results, the following values were obtained: association rate constant (k ass)=1.48×10⁵, dissociation rate constant (k diss)=9.63×10⁴, and dissociation constant (indicator of affinity) KD (KD=k diss/k ass)=6.50×10^-9.

[0298]On the other hand, the Biacore pattern of G2-15 (ELISA: 33.9% NP2/NP17 ratio) which is predicted to be relatively low in affinity from the results of ELISA is shown in FIG. 31. Curves (a) to (e) appearing in FIG. 31 correspond to antibody concentrations of 23.0, 11.5, 5.75, 2.88 and 1.44 nM, respectively.

[0299]The following values were obtained: association rate constant (k ass)=5.33×10⁴, dissociation rate constant (k diss)=1.56×10^-2, and dissociation constant (indicator of affinity) KD (KD=k diss/k ass)=2.92×10^-7. This KD value was close to the KD value of 1.67×10^-7 shown by W2-7 which also showed an equivalent affinity in ELISA (ELISA: 31.6% NP2/NP17 ratio).

[0300]From what have been described above, it is clear that high affinity antibodies are produced by using GANP transgenic (Tg) mice.

Example 5

Association of GANP with MCM3, and Shuttling Between Nucleus and Cytoplasm During Cell Cycle

1. Outline

[0301]In this Example, the present inventor determined the MCM3 binding domain of GANP by using truncated-type mutant GANPs, and characterized the localization of GANP in NIH-3T3 cells using a monoclonal antibody specific to the phosphorylation of serine at position 502 (pSer⁵⁰²) in the GANP specific domain.

[0302]The binding of a primase to MCM is a linked function, and the molecular complex resulting from their binding has an action of unwinding the DNA double strand. Therefore, it is believed that if a GANP partial fragment has bound to MCM, that GAMP fragment also reveals primase activity and has an action of producing high affinity antibodies.

[0303]Then, the localization of GANP and partial fragments thereof, Map80 and MCM3 in the nucleus/cytoplasm compartment was analyzed by cDNA transfection and cell fusion experiment.

[0304]The resultant data show that GANP binds to MCM3 and that the localization of GANP is influenced by MCM3 expression. GANP associates with MCM3 by a binding mode different from that by which Map80 associates with MCM3. These results suggest that GANP bound to MCM3 mediates a unique function different from the function of Map80/MCM3AP.

2. Materials and Methods

2.1. Cells and Cell Cultures

[0305]NIH-3T3, COS7, HeLa and Swiss-3T3 cells were maintained in D-MEM medium (Invitrogen) supplemented with 10% thermo-inactivated FCS (Dainippon Pharmaceutical), 2 mM L-glutamine (Biowhittaker), 100 μg/ml streptomycin, 100 U/ml penicillin and 50 μM 2-mercaptoethanol at 37° C. under 5% CO₂ (Takei, Y et al., (1998) J. Biol. Chem. 273, 22177-22180; Sakaguchi, N. et al., (1988) EMBO J. 7, 3457-3464, Kimura, H. et al., (1995) Nucl. Acids Res. 23, 2097-2104). BAL17 cells were cultured in RPMI-1640 medium (Invitrogen).

2.2. Intracellular Localization of Phosphorylated GANP and MCM3

[0306]NIH-3T3 cells were fixed in 3.7% paraformaldehyde in PBS (pH 7.4) for 5 min and made transparent using 0.2% Triton X-100 (Kimura, H. et al., (1994) EMBO J. 13, 4311-4320). As primary antibodies, rat anti-pSer⁵⁰² GANP monoclonal antibody (Kuwahara, K. et al., (2001) Proc. Natl. Acad. Sci. USA 98, 10279-10283) and rabbit anti-MCM3 antibody (Kimura, H. et al., (1994) EMBO J. 13, 4311-4320) were used. As secondary antibodies, Alexa 488-conjugated goat anti-rat IgG antibody (Molecular Probes) was used against GANP and Alexa 546-conjugated goat anti-rabbit IgG antibody (Molecular Probes) was used against MCM3. Counter-staining was carried out using TOTO-3 iodide (Molecular Probes), followed by observation with a confocal laser scanning microscope (FV500; Olympus).

2.3. cDNA Constructs for ExpressionpSRα-MCM3-HA is described in the literature (Kimura, H. et al., (1995) Nucl. Acids Res. 23, 2097-2104). A vector pECFP-Nuc carrying the three nuclear localization signals (NLSs) of SV40 T-Ag was purchased from Clontech. PCR fragments obtained by using the following combinations of 3' and 5' primers were introduced into pGEX-4T-1 (Amersham). Using the resultant plasmids, different forms of mouse ganp cDNAs were expressed as fusion proteins with glutathione-S-transferase (GST).

TABLE-US-00006 (SEQ ID NO: 11) GANP1-5': 5'-GGGGATCCATACCCGG TGAACCCCTT-3' (SEQ ID NO: 12) GANP1-3': 5'-GGGTCGACGCGCACAGACTTTCCCCTGA-3' (SEQ ID NO: 13) GANP2-5': 5'-GGGAATTCTCCCGCCTTCCAGCTGTGAC-3' (SEQ ID NO: 14) GANP2-3': 5'-GGGTCGACGTGCTGCTGTGTTATGTCCT-3' (SEQ ID NO: 15) GANP3-5': 5'-GGGAATTCCATGAGCT GAGACCCTCAGC-3' (SEQ ID NO: 16) GANP3-3': 5'-GGGTCGACTGAGGATGCAGGAGGCGGCT-3' (SEQ ID NO: 17) GANP4-5': 5'-GGGAATTCTACGTTGGAGAGAGCCTGGC-3' (SEQ ID NO: 18) GANP4-3': 5'-GGGTCGACCATGCTGTCATCTCCTGTGA-3' (SEQ ID NO: 19) GANP5-5': 5'-GGGAATTCGAGAA CCTGGCCAAGGGTCT-3' (SEQ ID NO: 20) GANP5-3': 5'-GGGTCGACGAAAAACCGACGGCTGAACT-3' (SEQ ID NO: 21) GANP6-5': 5'-GGGAATTCAAGCCCTTCCAGCCTGCCCT-3' (SEQ ID NO: 22) GANP6-3': 5'-GGGTCGACCGAGGGAACGTGGTATTTTC-3' (SEQ ID NO: 23) GANP7-5': 5'-GGCCCGGGCC CGTGGGATGACATCATCA-3' (SEQ ID NO: 24) GANP7-3': 5'-GGCTCGAGCATGTCCACCATCTC CAGCA-3'

[0307]cDNA constructs were prepared by introducing PCR fragments into pSVEGFP pA to thereby obtain green fluorescence protein (GFP)-tagged Ganp mutants (Kuwata, N. et al., (1999) J. Immunol. 163, 6355-6359). Subsequently, these constructs were introduced into a mammalian expression vector pCXN2 (Niwa, H. et al., (1991) Gene 108, 193-200). Primer sequences were designed as described below so that they encode Ganp.

TABLE-US-00007 Gp-gfp-5': (SEQ ID NO: 25) 5'-GGGGATCCGAATTCCACCATGGCAGTCTTCAAACCGATACC-3' Gp-gfp-3': (SEQ ID NO: 26) 5'-GCAGGGGCTCCTCCTGATCT-3' Gsac-gfp-5': (SEQ ID NO: 27) 5'-GGGGATC CGAATTCCACCATGTCCGAGGGCCTTGGTTCTTG-3' Gsac-gfp-3': (SEQ ID NO: 28) 5'-CTGTCTT GTTTCTAAGCCGC-3' Gmap80-gfp-5': (SEQ ID NO: 29) 5'-GGGGATCCGAATTCCACCATGGAGA ACCTGGCCAAGGGTCT-3' Gmap80-gfp-3': (SEQ ID NO: 30) 5'-GAGGACTTGTAGATGTTTTCAC CATGG-3'

[0308]FLAG-tagged Ganp mutants were prepared by introducing into pCXN2 the cDNA fragments obtained by PCR using the following primers.

TABLE-US-00008 FLAG-Gp-5': (SEQ ID NO: 31) 5'-GGGAATTCCACCATGGATTACAAGGATGACGACGATAAGGCAGTCTT CAA CCGATACC-3' FL4G-Gp-3': (SEQ ID NO: 32) 5'-GGGAATTCCTCCGGGTCTCCCTCAAGTA-3' FL4G-Gsac-5': (SEQ ID NO: 33) 5'-GGGAATTCCACCATGGATTACAAGGATGACGACGATAAGTCCGAGGG CCTTGGTTCTTG-3' FLAGGsac-3': (SEQ ID NO: 34) 5'-GGGAATTCGCTGTCTTGTTTCTAAGCCG-3' FL4G-Gmap-5': (SEQ ID NO: 35) 5'-GGGAATTCCACCATGGATTACAAGGATGACGACGATAAGGAGAACCT GGCCAAGGGTCT-3' FLAG-Gmap-3': (SEQ ID NO: 36) 5'-GGGAATTCTGAGGACTTG TAGATGTTTT-3'

[0309]Internal deletion mutant GpΔNLS-GFP and 13 mutant (MCMΔNLS-HA) were prepared as described in the literature (Imai, Y et al., (1991) Nucl. Acids Res. 19, 2785-2785). All of the constructs were sequenced to confirm that they have the proper orientation and that the reading frame of codons will be correct when they are expressed as tagged fusion proteins. Thus, their quality was controlled. Expression vectors comprising a mutant RNA/DNA primase domain (PD) are described in the literature (Gp mutant from Ser⁵⁰² to Ala [GpS502A] or Glu [GpS502E]) (Kuwahara, K. et al., (2001). Proc. Natl. Acad. Sci. USA 98, 10279-10283).

2.4. Detection of Transgene Product with Confocal Microscope

[0310]NIH-3T3 cells were transfected with pCXN2-ganp-gfp and/or pSRα-MCM3-HA using FuGENE 6 (Roche Diagnostics). Sixteen hours before fixation, leptomycin B (LMB) (Kudo, N. et al., (1999) Proc. Natl. Acad. Sci. USA 96, 9112-9117) was added to the medium. In the co-transfection experiment, rabbit anti-HA antibody (Santa Cruz) and Alexa 546-conjugated goat anti-rabbit IgG antibody were used. In the single transfection experiment, Alexa 488-conjugated goat anti-rabbit IgG antibody (Molecular Probes) was used. Thus, exogenous MCM3 protein was stained. Nuclear acid was counter-stained with TOTO-3 iodide in the co-transfection experiment and with propidium iodide (PI; Sigma) in the single transfection experiment.

2.5. GST Pull Down Assay

[0311]GST fusion proteins were purified as described in the literature (Kuwahara, K. et al., (2000) Blood 95, 2321-2328). Various GST fusion proteins (5 μg each) immobilized on glutathione-Sepharose beads (Amersham) were incubated with BAL17 lysate prepared with TNE buffer (10 mM Tris-HCl [pH 7.8], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10 μg of aprotinin, 1 mM phenylmethyl-sulfonylfluoride [PMSF]). Bound proteins were separated by 8% SDS-PAGE, transferred onto a nitrocellulose filter and blocked. Subsequently, the filter was incubated with rabbit anti-mouse MCM3 antibody (Kimura, H. et al., (1994) EMBO J. 13, 4311-4320) and then with peroxidase-labeled protein A (Amersham) serially. Finally, signals were visualized with ECL detection kit (Amersham). For direct binding assay, radio-labeled MCM3 was prepared with ³⁵S-methionin (Amersham) using In vitro Transcription and Translation Binding System (Novagen) according to the manufacturer's instructions. Thus, [³⁵S]-labeled MCM3 was detected by autoradiography.

2.6. Immunoprecipitation and Western Blotting of Transgene Product

[0312]COS7 cells were transfected with pCXN2-FLAG-ganp and/or pSRα-MCM3-HA using FuGENE 6. After 26 hrs, cells were lysed in TNE buffer. The resultant lysate was incubated with a combination of protein A-Sepharose (Amersham) and anti-HA antibody. The resultant immunoprecipitates were separated by 8% SDS-PAGE, transferred on a nitrocellulose filter, and blocked. Subsequently, the filter was incubated with anti-mouse FLAG M2 antibody (Stratagene) and then with peroxidase-labeled goat anti-mouse IgG (H+L) antibody (Zymed). For the detection of Gp-GFP and mutants thereof, the blotted filter was probed with rabbit anti-GFP antibody (Santa Cruz) and peroxidase-conjugated protein A (Zymed).

2.7. Heterokaryon Assay

[0313]HeLa cells were transfected with pSRα-MCM3-HA using FuGENE 6. After 20 hrs, transfected HeLa cells and untransfected mouse Swiss-3T3 cells were treated with trypsin and seeded in culture dishes at a ratio of 1:1. After 24 hrs, cells were fused using polyethylene glycol 1500 (Roche Diagnostics) at room temperature for 2 min (Schmidt-Zachmann, M. S. et al., (1993) Cell 74, 493-504). The culture dishes were washed with the medium 4 times. Then, cycloheximide-containing medium was added thereto (at a final concentration of 20 μg/ml), and the cells were incubated in CO₂ incubator at 37° C. for 5 hrs. Subsequently, the cells were fixed with 4% paraformaldehyde in 250 mM HEPES-NaOH (pH 7.4) for 20 min, made transparent using 0.5% Triton X-100 in PBS for 30 min, and washed with PBS. The cells were stained using anti-HA antibody (12CA5; Covence Research Products) and Cy3-conjugated donkey anti-mouse Ig antibody (Jackson). Also, DNA was counter-stained with 100 ng/ml of Hoechst 33342 (Sigma) in PBS for 20 min. Images were collected using Zeiss Axioplan equipped with 100× PlanNeofluar phase-contrast objective lens (NA 1.3) and SpotII CCD.

3. Results and Observations

[0314]3.1. Association of GANP with MCM3

[0315]The interaction between GANP and MCM3 in B cell lineage has been already demonstrated by immunoprecipitation (Kuwahara, K. et al., (2000) Blood 95, 2321-2328). Since a C-terminal domain of GANP is identical with total Map80 protein, it is predicted that GANP associates with MCM3 at this domain. In order to determine which domain of GANP associates with MCM3, the present inventor performed a pull down assay using GST fusion proteins containing the various truncated GANP proteins as shown in FIG. 32. Briefly, GST was fused to the N-terminus of each of the truncated GANP proteins designated 1 to 7 and 5-7' in FIG. 32. In the lower panel of FIG. 33, Map80 domain (designated GANP5-7') pulled down MCM3 from cell extract as described previously (Kimura, H. et al., (1994) EMBO J. 13, 4311-4320).

[0316]Surprisingly, GANP1 and GANP2 (which are partial fragments of GANP) also pulled down MCM3 (FIG. 33: upper and lower panels).

[0317]Subsequently, this binding was examined using MCM3 synthesized in vitro in a reticulocyte lysate system (FIG. 34). GST-GANP1 and GST-GANP2 also pulled down [³⁵S]-MCM3 from the in vitro translation cocktail.

[0318]GST alone (negative control: first lane) or GST fused with an irrelevant protein did not show any signal. Further, the binding to GST-GANP1 was stronger than the binding to Map80 domain (GST-GANP5-7'). This binding was also confirmed in cells by a DNA transfection experiment using FLAG-tagged constructs (FIG. 35).

[0319]Briefly, COS7 cells were co-transfected with pCXN2-FLAG-Ganp, pCXN2-FLAG-Gp and pCXN2-FLAG-Gmap80 in combination with pSRα-MCM3-HA or pSRa-I3-HA. After immunoprecipitation with anti-HA antibody, Western blotting was performed using anti-FLAG monoclonal antibody. The predicted sizes of FLAG-labeled proteins are shown in individual lanes with triangle marks. In the left and right panels, the migration of bands is similar, but the light exposure for ECL detection was 1 min for the left panel and 3 min for the right panel (FIG. 35).

[0320]FLAG-Ganp, FLAG-Gp and FLAG-Gmap80 bound to wild-type MCM3-HA (HA epitope-tagged MCM3) (FIG. 35: left panel). Only FLAG-Gsac did not bind thereto. With respect to the binding with 13 mutant MCM3 (MCM3ΔNLS), only FLAG-Gmap80 showed a positive result (FIG. 35: right panel). Gp domain carrying the N-terminal NLS associates with MCM3 consistently in cells containing a large quantity of MCM3 (FIG. 35: left panel). These results suggest that GANP associates with the NLS domain of MCM3 through Gp domain.

[0321]The present inventor further examined whether the state of phosphorylation of Ser⁵⁰² in Gp domain influences the binding GANP to MCM3 or not. A GANP mutant lacking primase site (GanpΔPD-GFP) and other GANP mutants prepared as GanpS502A and GanpS502E having a mutation at Ser⁵⁰² were fused with GFP (FIG. 36A). Cells were co-transfected with pCXN2-Ganp-gfp and pSRα-MCM3-HA, and cell lysate was used in immunoprecipitation with anti-HA antibody.

[0322]GFP signals were detected with anti-GFP antibody (FIG. 36B: upper panel). This means that GANP has bound to MCM.

[0323]Co-transfection using Ganp-GFP mutants was also performed in the same manner. In order to determine the predicted position of each protein, lysates were separated by SDS-PAGE and blotted with anti-GFP antibody in the same manner (FIG. 36B: lower panel).

[0324]The non-phosphorylated mutant (GanpS502A-GFP) bound to MCM3 as wild-type Ganp-GFP and GanpS502E-GFP (a mutant much resembling phosphoserine) did (FIG. 36B: upper panel). Interestingly, GanpΔPD-GFP does not co-precipitate with MCM3-HA (FIG. 36B: upper panel).

[0325]Regardless of the latent binding activity of Map80 domain, GANP molecule as a whole needs RNA primase domain (PD) for its binding to MCM3. Open triangle in FIG. 36B indicates the position of GanpΔPD-GFP. The size of Ganp-GFP, which is equal to the sizes of Ganp S502A-GFP and Ganp S502E-GFP, is indicated with filled triangle (FIG. 36B: lower panel). These results suggest that the binding of GANP to MCM3 is mediated by its PD domain, but phosphorylation at Ser⁵⁰² does not influence this binding.

[0326]The experiment using truncated constructs revealed the association of GANP with MCM3 in a wide region. However, the association of the entire GANP (involving its N-terminal 600 amino acid region) with MCM3 requires the NLS of MCM3. NLS-deficient MCM3 mutant was unable to effectively associate with entire GANP molecule in cells. Map80 domain bound to NLS-negative MCM3, suggesting that GANP mainly binds to a domain of MCM3 other than the domain required for the interaction with Map80. Although Map80 is considered to be an MCM3 import factor, GANP may play a different role in cooperation with MCM3. It seems that GANP has many potential phosphorylation sites and has many association components in cells (Kuwahara, K. et al., (2000) Blood 95, 2321-2328). Therefore, it will be necessary to specify a domain whose state of phosphorylation influences the GANP/MCM3 association and transport between the cytoplasm and the nuclear compartment.

3.2. Intracellular Localization of Map80 and Ganp Mutants Shown by Transfection

[0327]GANP has two potential NLSs. One is located in the N-terminal primase domain and the other in the C-terminal Map80 domain. GANP also has two nuclear export signal (NES)-like motifs. On is located between SAC3 homologous domain and Map80 domain and the other within Map80 domain.

[0328]NIH-3T3 cells were transfected with pCXN2-Ganp-gfp or pCXN2-Gmap80-gfp, followed by fixation 48 hrs later. LMB was added 16 hrs before the fixation. Nuclei were pre-stained with PI, and images were collected with a confocal microscope. Representative expression properties are shown in FIG. 37. The numbers of cells were counted by property and expressed in % (FIG. 37).

[0329]It was found that Ganp-GFP (almost full-length GANP tagged with GFP) is present in both the cytoplasm and the nuclear compartment, though the ratio of cells showing nuclear dominant expression (N and N>C: 38%) or cells showing cytoplasm dominant expression (C and C>N: 31%) was varied (% from total 500 cells) (FIG. 37: Ganp-GFP, LMB-). In contrast, Gmap80-GFP was found in the cytoplasm for the most part, showing no nuclear dominant expression according to the inventor's classification (N>C, 0%; N═C, 35%; C and C>N, 65%) (FIG. 37: Gmap80-GFP, LMB-). The localization of Ganp-GFP is different from the localization of Gmap80-GFP.

[0330]In order to examine whether the N-terminal NLS motif is functional or not, 5' 1-kb DNA fragment comprising RNA/DNA primase domain and the N-terminal NLS (but not NES-like motif) was fused with GFP (FIG. 38: Gp-GFP). Although this Gp-GFP product was present in the nucleus alone (N and N>C: 94%) (FIG. 38), NLS-deficient mutant GpGFP (GpΔNLS-GFP; as shown in FIG. 38, amino acids from position 497 to 500 are deleted) was found to be cytoplasmic. Thus, it was confirmed that the N-terminal NLS is involved in the nuclear localization.

[0331]The present inventor examined whether or not the mutation of the adjacent Ser⁵⁰² to alanine (GpS502A-GFP; non-phosphorylated type) or to glutamic acid (GpS502E-GFP; phosphoserine-mimic type) influences this localization (FIG. 38). Then, the present inventor observed that these mutations do not alter the localization of Gp. This suggests that the N-terminal NLS is functional regardless of the state of phosphorylation of Ser⁵⁰² (FIG. 38). In contrast, it seems that Gac-GFP having neither N-terminal NLS nor C-terminal NLS is present in the cytoplasm for the most part (N and N>C: 0%; N═C: 3%; C and C>N: 97%) (FIG. 38).

[0332]These results suggest that the N-terminal NLS plays a functional role for Ganp to enter into the nucleus. However, the NLS may not be so strong to maintain GANP expression within the nucleus, because Ganp-GFP is also present in the cytoplasm (FIG. 37). In order to examine this issue further, cells were treated with leptomycin B (LMB) after cDNA transfection in order to inhibit the Crm1-mediated export to the nucleus (Kudo, N. et al., (1999) Proc. Natl. Acad. Sci. USA 96, 9112-9117).

[0333]In LMB-treated cells, Ganp-GFP localized in the nucleus for most of the transfectants (FIG. 37). The cell fraction showing cytoplasm dominant expression decreased from 31% to 4%, while the cell fraction showing nuclear dominant expression increased from 38% to 81%. Therefore, it appears that the movement of Ganp to the cytoplasm is inhibited by LMB.

[0334]The localization of Gmap80-GFP has also changed dramatically after LMB treatment (FIG. 37). The cell fraction showing cytoplasm dominant expression decreased from 65% to 37%, and the cell fraction showing nuclear dominant expression increased from 0% to 41%. These findings were reproduced in other cell systems including COS7 and Ltk.sup.- cells, suggesting that the export of GANP from the nucleus to the cytoplasm is regulated by Crm1-dependent passway. Therefore, GANP and Map80 seem to shuttle between the nucleus and the cytoplasm, and their localization seem to depend on the balance between nuclear import and export mechanisms maintained in cooperation with other molecules.

3.3. Localization of MCM3 and GANP in Cotransfected Cells

[0335]Subsequently, the present inventor examined whether or not the movement of GANP is related to MCM3 expression. Mammal MCM3 alters the state of binding with chromatin during cell cycle, but it is present only in the nucleus throughout the interphase (Kimura, H. et al., (1994) EMBO J. 13, 4311-4320). NIH-3T3 cells were transfected with pSRα-MCM3-HA or pSRα-I3-HA, fixed, immunolabeled with anti-HA antibody (Alexa 488) and stained with PI.

[0336]MCM3-HA in transfected cells agreed with the representative presence of NLS (Kimura, H. et al., (1994) EMBO J. 13, 4311-4320, Takei, Y. et al., (1998) J. Biol. Chem. 273, 22177-22180) and localized in the nucleus (FIG. 39). This nuclear localization was dependent on the NLS of MCM3, because an MCM3 mutant lacking this NLS (13; MCM3ΔNLS-HA) was expressed only in the cytoplasm (FIG. 39: right panel).

[0337]Cells were cotransfected with pCXN2-Ganp-gfp or pCXN2-Gmap80-gfp and pSRα-MCM3-HA, fixed and immunolabeled with anti-HA antibody (Alexa 546), and nuclei were pre-stained with TOTO-3 (FIG. 40). Cell counts are shown below the panel (FIG. 40).

[0338]Interestingly, when cells were cotransfected with Ganp-GFP, cytoplasmic localization of MCM3 was induced in 17% of the cells (FIG. 40: marked with white arrows). When cells were cotransfected with Gmap80-GFP or Gp-GFP, such a result was not observed.

[0339]In order to prove that the effect of Ganp on MCM3 is specific, expression of ECFP-Nuc which appears in the nucleus was examined before and after transfection using different ganp-gfp constructs. Representative images obtained from transfection with Ganp-GFP are shown in FIG. 41. The localization of ECFP-Nuc in the nucleus was not influenced by any cotransfection using Ganp-GFP (FIG. 41) or Gmap80-GFP or Gp-GFP.

[0340]Coexpression of Ganp and MCM3 has also altered the localization of GANP. Compared to the transfection with Ganp-GFP alone (38%) or with Gmap80-GFP alone (0%) (FIG. 37), cotransfection using MCM3 raised the nuclear expression levels of Ganp-GFP (74%) and Gmap80-GFP (64%) (FIG. 40). MCM3 retained GANP and Map80 within the nucleus, but overexpression of Ganp alone enhanced the expression of MCM3 in the cytoplasm (FIG. 40: 17% by Ganp-GFP expression). On the other hand, Gmap80 did not enhance the expression of MCM3 in the cytoplasm (4% by Gmap80-GFP expression). The mutation of MDM3 at its NLS (13; MCM3ΔNLS-HA) (as a result, MCM3 is present in the cytoplasm) did not induce the accumulation of Ganp-GFP or Gmap80-GFP in the nucleus (FIG. 42).

[0341]Unlike wild-type MCM3, 13 mutant (MCM3ΔNLS-HA) does not associate with Ganp or Ga (FIG. 35). Considering this fact together, it is suggested that the NLS motif of MCM3 is necessary for the functional association with GANP and for the transport of GANP between the nucleus and the cytoplasm.

[0342]DNA transfection experiments demonstrated that Ganp-GFP is accumulated in the nuclear compartment when co-introduced with MCM3, suggesting the formation of GANP/MCM3 complex in the nucleus. MCM3 does not contain a definite common NES-like motif recognizable by Crm1. Therefore, the export of MCM3 from the nucleus probably depends on other binding molecules having an NES-like motif or a different export mechanism. The two NES-like motifs on GANP seems to be involved in an LMB sensitive, Crm1 dependent export passway (FIG. 37). The two NES-like motifs carried by GANP (these might be recognized by Crm1) might possibly be involved in the transport of the complex.

[0343]Recently, it was shown that yeast SAC3 carrying a GANP homologous domain is involved in the export of a specific protein from the nucleus and associates with a component of nuclear pore complex (Jones, A. L. et al., (2000) Proc. Natl. Acad. Sci. USA 97, 3224-3229). Coexpression with GANP altered the localization of MCM3 in the cytoplasmic compartment.

[0344]The nuclear-cytoplasmic shuttling of MCM3 was examined using cell fusion techniques (Schmidt-Zachmann, M. S. et al., (1993) Cell 74, 493-504). HeLa cells were transfected with MCM3-HA and then fused with untransfected mouse Swiss-3T3 cells. After a 5-hour incubation in the presence of cycloheximide to inhibit protein synthesis, cells were fixed and immunolabeled with MCM3-HA. Heterokaryons were examined by Hoechst staining. This staining discriminates mouse nuclei (marked with arrows) with "mottled" heterochromatins from human HeLa nuclei.

[0345]As representative images are shown in FIG. 43, MCM3-HA was found in both human nuclei and mouse nuclei in heterokaryons. Unfused mouse cells do not exhibit such staining. This suggests that MCM3-HA has been exported from the HeLa nucleus to the cytoplasm, and then imported into the mouse nucleus.

[0346]From these results, it is concluded that MCM3-HA is a shuttling protein. It should be noted here that proving the movement of an endogenous protein from the nucleus to the cytoplasm with a sensitivity similar to the sensitivity achieved when transgene products are handled is often difficult (Kimura, H., Ohtomo, T. et al., (1996) Genes Cells 1, 977-993; Mizuno, T. et al., (1999) Mol. Cell. Biol. 19, 7886-7896). That was the case with the results shown in the present Example. It is also difficult to prove the movement of endogenous MCM protein from the nucleus to the cytoplasm in mammal cells with a sensitivity achieved in more primitive cells such as yeast.

[0347]However, the results of the present inventor suggest that the nuclear-cytoplasmic shuttling of MCM protein is probably important in untreated cells (though experiments were performed by DNA transfection). To facilitate definite understanding of the nuclear-cytoplasmic shuttling of the MCM complex during cell cycle, discovery of a further component may be necessary.

3.4. Localization of GANP During Cell Cycle

[0348]Using a monoclonal antibody specific to the epitope of RNA/DNA primase domain (pSer⁵⁰² GANP) peculiar to GANP, the localization of GANP in NIH-3T3 cells was examined under a confocal laser scanning microscope (Kuwahara, K. et al., (2001) Proc. Natl. Acad. Sci. USA 98, 10279-10283). NIH-3T3 cells at different stages of cell cycle were immunostained with anti-pSer⁵⁰² GANP (Alexa 488; green) and anti-MCM3 (Alexa 546; red) antibodies. Nuclei were pre-stained with TOTO-3 iodide (blue). During the interphase, the above-described monoclonal antibody reacted everything within the nucleus except for the nucleolus (FIG. 44).

[0349]By the triple labeling with anti-MCM3 antibody and TOTO-3 for staining nucleic acid, the localization of GANP during mitosis was analyzed in detail. As cells proceed from the prometaphase to the metaphase, GANP seems to be dissociated from concentrated chromatin (FIG. 44). The yellow signal in the superimposed image indicates colocalization of GANP and MCM3, but some blue staining shows that GANP alone is also observed in the central part of the prometaphase image. At this stage, GANP and MCM3 are not superimposed with the concentrated chromosome. In metaphase cells, GANP is detected in the spindle region. This signal decreases when chromosomes are separated into two daughter cells in the anaphase.

[0350]In the anaphase of mitosis, most of GANP molecules are found in the cytoplasmic compartment until nuclei are formed (telophase). These results suggest that the behaviors of GANP and MCM3 are similar and that they are almost colocalized in the nucleus throughout the interphase. This is consistent with the interassociation of these two molecules. However, as shown by the confocal microscopic examination during mitosis, GANP and MCM3 may be present separately (FIG. 44).

[0351]The biological meaning of the nuclear-cytoplasmic shuttling of GANP with respect to the second type RNA/DNA primase remains to be investigated. The shuttling may be associated with the generation of RNA primer at the final stage of DNA repairing. Although the expression level of GANP is low in normal cells, GANP expression is up-regulated in the germinal center where cells rapidly proliferate (Kuwahara, K. et al., (2000) Blood 95, 2321-2328; Kuwahara, K. et al., (2001). Proc. Natl. Acad. Sci. USA 98, 10279-10283). Further, GANP is expressed at higher levels in certain types of cells having rapid cell cycle. This suggests the possibility that association into MCM complex may stimulate DNA replication (Kuwahara, K. et al., (2001). Proc. Natl. Acad. Sci. USA 98, 10279-10283). The expression of GANP having RNA/DNA primase activity, MCM3 binding ability and an acetyltransferase domain (Takei, Y. et al., (2001) EMBO Rep. 2, 119-123) may be involved in the regulation of cell cycle progress.

Sequence Listing Free Text

[0352]SEQ ID NO: 5: primer

[0353]SEQ ID NO: 6: primer

[0354]SEQ ID NO: 7: primer

[0355]SEQ ID NO: 8: primer

[0356]SEQ ID NO: 9: primer

[0357]SEQ ID NO: 10: primer

[0358]SEQ ID NO: 11: primer

[0359]SEQ ID NO: 12: primer

[0360]SEQ ID NO: 13: primer

[0361]SEQ ID NO: 14: primer

[0362]SEQ ID NO: 15: primer

[0363]SEQ ID NO: 16: primer

[0364]SEQ ID NO: 17: primer

[0365]SEQ ID NO: 18: primer

[0366]SEQ ID NO: 19: primer

[0367]SEQ ID NO: 20: primer

[0368]SEQ ID NO: 21: primer

[0369]SEQ ID NO: 22: primer

[0370]SEQ ID NO: 23: primer

[0371]SEQ ID NO: 24: primer

[0372]SEQ ID NO: 25: primer

[0373]SEQ ID NO: 26: primer

[0374]SEQ ID NO: 27: primer

[0375]SEQ ID NO: 28: primer

[0376]SEQ ID NO: 29: primer

[0377]SEQ ID NO: 30: primer

[0378]SEQ ID NO: 31: primer

[0379]SEQ ID NO: 32: primer

[0380]SEQ ID NO: 33: primer

[0381]SEQ ID NO: 34: primer

[0382]SEQ ID NO: 35: primer

[0383]SEQ ID NO: 36: primer

INDUSTRIAL APPLICABILITY

[0384]By using the GANP overexpressing mouse of the invention, it is possible to rapidly prepare antibodies specific to viral antigens and having high affinity therefor, which could not be obtained by conventional methods. Therefore, it is expected that specific and potent antibodies can be obtained rapidly enough to keep up with the mutations of viral antigens in order to prevent the worsening of conditions caused by prolonged infection such as AIDS or hepatitis C. Further, with the transgenic animal of the invention, it is possible to prepare tailored, specific antibodies corresponding to the mutations of viral antigens from infected patients. The period of immunization necessary for antibody preparation is about only 10 days, and the efficiency of producing antibodies with high affinity mutations reaches almost 60%. High affinity antibody production protocol using bed side patients' samples is expected to become a new immunotherapy that will take the place of vaccine therapy.

Sequence CWU 1

10516429DNAMus musculusCDS(384)..(6299) 1gttgcggtgc ggtgggcccg gtagaggctg cacgcagact gtgggcgagc acaagcgctg 60gcgacagtgg ccgtatctgg cggacttgct cctccctccg cggcctccgc tgtcccttgt 120gtctttgccg agttgctgaa ggccttcact agtcttcgct cgaaggcgtc tgttaaccta 180gcggccggct tccggagtgt taagcatcgg ggataaaaag ctattatttc tagaccaggg 240catcgcaagt tcgagttacc gggagaaaaa tgagatggtc atcctgagga tgaaggagag 300cttcccctgg caacagataa tttaaagagg agagctactt gtgtatagtc catatttatt 360gccttcagat aattggcttg aag atg cac ccg gtg aac ccc ttc gga ggc agc 413 Met His Pro Val Asn Pro Phe Gly Gly Ser 1 5 10agc cca agt gct ttt gcg gta tct tcc agc acc acg gga aca tat cag 461Ser Pro Ser Ala Phe Ala Val Ser Ser Ser Thr Thr Gly Thr Tyr Gln 15 20 25act aaa tca cca ttt cga ttt ggc cag cct tcc ctt ttt gga cag aac 509Thr Lys Ser Pro Phe Arg Phe Gly Gln Pro Ser Leu Phe Gly Gln Asn 30 35 40agc aca ccc agc aag agc ctg gcg ttt tca caa gta cca agc ttt gca 557Ser Thr Pro Ser Lys Ser Leu Ala Phe Ser Gln Val Pro Ser Phe Ala 45 50 55aca ccc tct gga gga agc cat tct tcc tcc ttg cca gca ttt gga ctc 605Thr Pro Ser Gly Gly Ser His Ser Ser Ser Leu Pro Ala Phe Gly Leu 60 65 70acc caa acc tca agt gtg gga ctc ttc tct agt ctc gaa tcc aca cct 653Thr Gln Thr Ser Ser Val Gly Leu Phe Ser Ser Leu Glu Ser Thr Pro75 80 85 90tct ttc gca gct act tcg agt tcc tct gtg ccc ggc aat acg gca ttc 701Ser Phe Ala Ala Thr Ser Ser Ser Ser Val Pro Gly Asn Thr Ala Phe 95 100 105agc ttt aag tca acc tct agc gtt ggg gtt ttc cca agt ggc gct act 749Ser Phe Lys Ser Thr Ser Ser Val Gly Val Phe Pro Ser Gly Ala Thr 110 115 120ttt ggg cca gaa acc gga gaa gta gca ggt tct ggc ttt cgg aag acg 797Phe Gly Pro Glu Thr Gly Glu Val Ala Gly Ser Gly Phe Arg Lys Thr 125 130 135gaa ttc aag ttt aaa cct ctg gaa aat gca gtc ttc aaa ccg ata ccg 845Glu Phe Lys Phe Lys Pro Leu Glu Asn Ala Val Phe Lys Pro Ile Pro 140 145 150ggg cct gag tca gag cca gaa aaa acc cag agc cag att tct tct gga 893Gly Pro Glu Ser Glu Pro Glu Lys Thr Gln Ser Gln Ile Ser Ser Gly155 160 165 170ttt ttt aca ttt tcc cat ccc gtt ggt agc ggg tct gga ggc ctg acc 941Phe Phe Thr Phe Ser His Pro Val Gly Ser Gly Ser Gly Gly Leu Thr 175 180 185cct ttt tct ttc cca cag gtg aca aat agt tcg gtg act agc tca agt 989Pro Phe Ser Phe Pro Gln Val Thr Asn Ser Ser Val Thr Ser Ser Ser 190 195 200ttt atc ttt tcg aaa cca gtt act agt aat act cct gcc ttt gcc tct 1037Phe Ile Phe Ser Lys Pro Val Thr Ser Asn Thr Pro Ala Phe Ala Ser 205 210 215cct ttg tct aac caa aat gta gaa gaa gag aag agg gtt tct acg tca 1085Pro Leu Ser Asn Gln Asn Val Glu Glu Glu Lys Arg Val Ser Thr Ser 220 225 230gcg ttt gga agc tca aac agt agc ttc agt act ttc ccc aca gcg tca 1133Ala Phe Gly Ser Ser Asn Ser Ser Phe Ser Thr Phe Pro Thr Ala Ser235 240 245 250cca gga tct ttg ggg gag ccc ttc cca gct aac aaa cca agc ctc cgc 1181Pro Gly Ser Leu Gly Glu Pro Phe Pro Ala Asn Lys Pro Ser Leu Arg 255 260 265caa gga tgt gag gaa gcc atc tcc cag gtg gag cca ctt ccc acc ctc 1229Gln Gly Cys Glu Glu Ala Ile Ser Gln Val Glu Pro Leu Pro Thr Leu 270 275 280atg aag gga tta aag agg aaa gag gac cag gat cgc tcc ccg agg aga 1277Met Lys Gly Leu Lys Arg Lys Glu Asp Gln Asp Arg Ser Pro Arg Arg 285 290 295cat tgc cac gag gca gca gaa gac cct gat ccc ctg tcc agg ggc gac 1325His Cys His Glu Ala Ala Glu Asp Pro Asp Pro Leu Ser Arg Gly Asp 300 305 310cat ccc cca gat aaa cgg cca gtc cgc ctc aac aga ccc cgg gga ggt 1373His Pro Pro Asp Lys Arg Pro Val Arg Leu Asn Arg Pro Arg Gly Gly315 320 325 330act ttg ttt ggc cgg aca ata cag gag gtc ttc aaa agc aat aaa gag 1421Thr Leu Phe Gly Arg Thr Ile Gln Glu Val Phe Lys Ser Asn Lys Glu 335 340 345gca ggc cgc ctg ggc agc aag gaa tcc aag gag agt ggc ttt gcg gaa 1469Ala Gly Arg Leu Gly Ser Lys Glu Ser Lys Glu Ser Gly Phe Ala Glu 350 355 360cct ggg gaa agt gac cac gcg gcc gtc cca gga ggg agt cag tcc acc 1517Pro Gly Glu Ser Asp His Ala Ala Val Pro Gly Gly Ser Gln Ser Thr 365 370 375atg gta cct tcc cgc ctt cca gct gtg act aaa gag gaa gaa gaa agt 1565Met Val Pro Ser Arg Leu Pro Ala Val Thr Lys Glu Glu Glu Glu Ser 380 385 390aga gat gag aaa gaa gat tct ctc agg gga aag tct gtg cgc cag agt 1613Arg Asp Glu Lys Glu Asp Ser Leu Arg Gly Lys Ser Val Arg Gln Ser395 400 405 410aag cga agg gaa gag tgg atc tac agc ctc ggg ggc gtg tct tct tta 1661Lys Arg Arg Glu Glu Trp Ile Tyr Ser Leu Gly Gly Val Ser Ser Leu 415 420 425gag ctc aca gcc atc cag tgc aag aac atc ccc gac tac ctc aac gac 1709Glu Leu Thr Ala Ile Gln Cys Lys Asn Ile Pro Asp Tyr Leu Asn Asp 430 435 440aga gcc atc ctg gag aaa cac ttc agc aaa atc gct aaa gtc cag cgg 1757Arg Ala Ile Leu Glu Lys His Phe Ser Lys Ile Ala Lys Val Gln Arg 445 450 455gtc ttc acc aga cgc agc aag aag ctc gcc gtg att cat ttt ttc gac 1805Val Phe Thr Arg Arg Ser Lys Lys Leu Ala Val Ile His Phe Phe Asp 460 465 470cac gca tcg gca gcc ctg gct agg aag aag ggg aaa ggt ctg cat aag 1853His Ala Ser Ala Ala Leu Ala Arg Lys Lys Gly Lys Gly Leu His Lys475 480 485 490gac gtg gtt atc ttt tgg cac aag aag aaa ata agt ccc agc aag aaa 1901Asp Val Val Ile Phe Trp His Lys Lys Lys Ile Ser Pro Ser Lys Lys 495 500 505ctc ttt ccc ctg aag gag aag ctt ggt gag agt gaa gcc agc cag ggc 1949Leu Phe Pro Leu Lys Glu Lys Leu Gly Glu Ser Glu Ala Ser Gln Gly 510 515 520atc gag gac tcc ccc ttt cag cac tcg cct ctc agc aag ccc atc gtg 1997Ile Glu Asp Ser Pro Phe Gln His Ser Pro Leu Ser Lys Pro Ile Val 525 530 535agg cct gca gcc ggc agc ctc ctc agc aaa agc tct cca gtg aag aag 2045Arg Pro Ala Ala Gly Ser Leu Leu Ser Lys Ser Ser Pro Val Lys Lys 540 545 550ccg agt ctt ctg aag atg cac cag ttt gag gcg gat cct ttt gac tct 2093Pro Ser Leu Leu Lys Met His Gln Phe Glu Ala Asp Pro Phe Asp Ser555 560 565 570gga tct gag ggc tcc gag ggc ctt ggt tct tgc gtg tca tct ctt agc 2141Gly Ser Glu Gly Ser Glu Gly Leu Gly Ser Cys Val Ser Ser Leu Ser 575 580 585acc ctg ata ggg act gtg gca gac aca tct gag gag aag tac cgc ctt 2189Thr Leu Ile Gly Thr Val Ala Asp Thr Ser Glu Glu Lys Tyr Arg Leu 590 595 600ctg gac cag aga gac cgc atc atg cgg caa gct cga gtg aag agg acg 2237Leu Asp Gln Arg Asp Arg Ile Met Arg Gln Ala Arg Val Lys Arg Thr 605 610 615gac ctg gac aaa gcc agg gca ttt gtt ggg act tgc cct gac atg tgt 2285Asp Leu Asp Lys Ala Arg Ala Phe Val Gly Thr Cys Pro Asp Met Cys 620 625 630ccc gag aag gag cgg tac ttg agg gag acc cgg agc cag ctg agc gtg 2333Pro Glu Lys Glu Arg Tyr Leu Arg Glu Thr Arg Ser Gln Leu Ser Val635 640 645 650ttt gaa gtt gtc cca ggg act gac cag gtg gac cat gca gca gcc gtg 2381Phe Glu Val Val Pro Gly Thr Asp Gln Val Asp His Ala Ala Ala Val 655 660 665aag gag tac agc cgg tcc tct gca gat cag gag gag ccc ctg cca cat 2429Lys Glu Tyr Ser Arg Ser Ser Ala Asp Gln Glu Glu Pro Leu Pro His 670 675 680gag ctg aga ccc tca gca gtt ctc agc agg acc atg gac tac ctg gtg 2477Glu Leu Arg Pro Ser Ala Val Leu Ser Arg Thr Met Asp Tyr Leu Val 685 690 695acc cag atc atg gac caa aag gaa ggc agc ctt cgg gat tgg tat gac 2525Thr Gln Ile Met Asp Gln Lys Glu Gly Ser Leu Arg Asp Trp Tyr Asp 700 705 710ttc gtg tgg aac cgc acc cgg ggt ata cgg aag gac ata aca cag cag 2573Phe Val Trp Asn Arg Thr Arg Gly Ile Arg Lys Asp Ile Thr Gln Gln715 720 725 730cac ctc tgt gat ccc ctg acg gtg tct ctg atc gag aag tgt acc cga 2621His Leu Cys Asp Pro Leu Thr Val Ser Leu Ile Glu Lys Cys Thr Arg 735 740 745ttt cac att cac tgt gcc cac ttt atg tgt gag gag cct atg tct tcc 2669Phe His Ile His Cys Ala His Phe Met Cys Glu Glu Pro Met Ser Ser 750 755 760ttt gat gcc aag atc aac aat gag aac atg acc aag tgt cta cag agt 2717Phe Asp Ala Lys Ile Asn Asn Glu Asn Met Thr Lys Cys Leu Gln Ser 765 770 775ctg aag gag atg tac cag gac ctg agg aac aag ggt gtt ttt tgt gcc 2765Leu Lys Glu Met Tyr Gln Asp Leu Arg Asn Lys Gly Val Phe Cys Ala 780 785 790agt gaa gca gag ttt cag ggc tac aat gtc ctg ctt aat ctc aac aaa 2813Ser Glu Ala Glu Phe Gln Gly Tyr Asn Val Leu Leu Asn Leu Asn Lys795 800 805 810gga gac att ttg aga gaa gtg cag cag ttc cac cct gac gtt agg aac 2861Gly Asp Ile Leu Arg Glu Val Gln Gln Phe His Pro Asp Val Arg Asn 815 820 825tcc cca gag gtg aac ttc gct gtc cag gct ttt gct gca ttg aac agc 2909Ser Pro Glu Val Asn Phe Ala Val Gln Ala Phe Ala Ala Leu Asn Ser 830 835 840aat aat ttt gtg aga ttt ttc aaa ctg gtt cag tca gct tct tac ctg 2957Asn Asn Phe Val Arg Phe Phe Lys Leu Val Gln Ser Ala Ser Tyr Leu 845 850 855aat gcg tgc ctg tta cac tgt tac ttt aat cag atc cgc aag gat gcc 3005Asn Ala Cys Leu Leu His Cys Tyr Phe Asn Gln Ile Arg Lys Asp Ala 860 865 870ctc cgg gca ctc aat gtt gct tat act gta agc aca cag cgc tct acc 3053Leu Arg Ala Leu Asn Val Ala Tyr Thr Val Ser Thr Gln Arg Ser Thr875 880 885 890gtc ttc ccc ctg gat ggt gtc gtc cgc atg ctg ctg ttc aga gat agt 3101Val Phe Pro Leu Asp Gly Val Val Arg Met Leu Leu Phe Arg Asp Ser 895 900 905gaa gag gcg aca aac ttc ctc aat tac cat ggc ctc act gta gct gat 3149Glu Glu Ala Thr Asn Phe Leu Asn Tyr His Gly Leu Thr Val Ala Asp 910 915 920ggc tgt gtt gag ctg aat cgg tcg gca ttc ttg gaa ccg gag gga tta 3197Gly Cys Val Glu Leu Asn Arg Ser Ala Phe Leu Glu Pro Glu Gly Leu 925 930 935tgc aag gcc agg aag tca gtg ttt att ggc cgg aag ctg acg gtg tca 3245Cys Lys Ala Arg Lys Ser Val Phe Ile Gly Arg Lys Leu Thr Val Ser 940 945 950gtt ggg gaa gtt gtg aat gga ggg ccg ttg ccc cct gtt cct cgc cat 3293Val Gly Glu Val Val Asn Gly Gly Pro Leu Pro Pro Val Pro Arg His955 960 965 970aca cct gtg tgc agc ttc aac tcc cag aat aag tac gtt gga gag agc 3341Thr Pro Val Cys Ser Phe Asn Ser Gln Asn Lys Tyr Val Gly Glu Ser 975 980 985ctg gct acg gag ctg ccc atc agc act cag aga gct ggt gga gac cca 3389Leu Ala Thr Glu Leu Pro Ile Ser Thr Gln Arg Ala Gly Gly Asp Pro 990 995 1000gca ggt ggt ggc aga gga gag gac tgt gag gca gag gtg gac ttg 3434Ala Gly Gly Gly Arg Gly Glu Asp Cys Glu Ala Glu Val Asp Leu 1005 1010 1015cca aca ttg gcg gtc ctc cca cag ccg cct cct gca tcc tca gcc 3479Pro Thr Leu Ala Val Leu Pro Gln Pro Pro Pro Ala Ser Ser Ala 1020 1025 1030acg ccg gcg ctt cat gtc cag cca ctg gcc cca gcc gca gca ccc 3524Thr Pro Ala Leu His Val Gln Pro Leu Ala Pro Ala Ala Ala Pro 1035 1040 1045agc ctt ctc cag gcc tcc acg cag cct gag gtg ctg ctt cca aag 3569Ser Leu Leu Gln Ala Ser Thr Gln Pro Glu Val Leu Leu Pro Lys 1050 1055 1060cct gcg cct gtg tac tct gac tcg gac ctg gta cag gtg gtg gac 3614Pro Ala Pro Val Tyr Ser Asp Ser Asp Leu Val Gln Val Val Asp 1065 1070 1075gag ctc atc cag gag gct ctg caa gtg gac tgt gag gaa gtc agc 3659Glu Leu Ile Gln Glu Ala Leu Gln Val Asp Cys Glu Glu Val Ser 1080 1085 1090tcc gct ggg gca gcc tac gta gcc gca gct ctg ggc gtt tcc aat 3704Ser Ala Gly Ala Ala Tyr Val Ala Ala Ala Leu Gly Val Ser Asn 1095 1100 1105gct gct gtg gag gat ctg att act gct gcg acc acg ggc att ctg 3749Ala Ala Val Glu Asp Leu Ile Thr Ala Ala Thr Thr Gly Ile Leu 1110 1115 1120agg cac gtt gcc gct gag gaa gtt tcc atg gaa agg cag aga cta 3794Arg His Val Ala Ala Glu Glu Val Ser Met Glu Arg Gln Arg Leu 1125 1130 1135gag gaa gag aag caa cga gct gag gag gaa cgg ttg aag caa gag 3839Glu Glu Glu Lys Gln Arg Ala Glu Glu Glu Arg Leu Lys Gln Glu 1140 1145 1150aga gaa ctg atg tta act cag ctg agc gag ggt ctg gcc gca gag 3884Arg Glu Leu Met Leu Thr Gln Leu Ser Glu Gly Leu Ala Ala Glu 1155 1160 1165ctg aca gaa ctc acg gtg aca gag tgt gtg tgg gaa acc tgc tct 3929Leu Thr Glu Leu Thr Val Thr Glu Cys Val Trp Glu Thr Cys Ser 1170 1175 1180cag gag cta cag agt gca gta aaa ata gac cag aag gtc cgt gtg 3974Gln Glu Leu Gln Ser Ala Val Lys Ile Asp Gln Lys Val Arg Val 1185 1190 1195gcc cgc tgt tgt gaa gcc gtc tgt gca cac ctg gtg gat ttg ttt 4019Ala Arg Cys Cys Glu Ala Val Cys Ala His Leu Val Asp Leu Phe 1200 1205 1210ctt gct gag gaa att ttc cag act gca aaa gag aca ctc cag gaa 4064Leu Ala Glu Glu Ile Phe Gln Thr Ala Lys Glu Thr Leu Gln Glu 1215 1220 1225ctc cag tgt ttc tgc aag tat cta caa cgg tgg agg gag gct gtt 4109Leu Gln Cys Phe Cys Lys Tyr Leu Gln Arg Trp Arg Glu Ala Val 1230 1235 1240gca gct cgg aag aaa ttc cgg cgt cag atg cgg gcc ttc cct gca 4154Ala Ala Arg Lys Lys Phe Arg Arg Gln Met Arg Ala Phe Pro Ala 1245 1250 1255gcg cca tgc tgt gtg gat gtg aat gac cgg ctg cag gca cta gtg 4199Ala Pro Cys Cys Val Asp Val Asn Asp Arg Leu Gln Ala Leu Val 1260 1265 1270ccc agc gca gag tgc ccc att act gag gag aac ctg gcc aag ggt 4244Pro Ser Ala Glu Cys Pro Ile Thr Glu Glu Asn Leu Ala Lys Gly 1275 1280 1285ctt ttg gac ctg ggc cac gca ggc aaa gta ggc gtc tcc tgt acc 4289Leu Leu Asp Leu Gly His Ala Gly Lys Val Gly Val Ser Cys Thr 1290 1295 1300agg ttg agg cgg ctt aga aac aag aca gct cac cag ata aag gtc 4334Arg Leu Arg Arg Leu Arg Asn Lys Thr Ala His Gln Ile Lys Val 1305 1310 1315cag cac ttc cac cag cag ctg ctg agg aat gct gca tgg gca cct 4379Gln His Phe His Gln Gln Leu Leu Arg Asn Ala Ala Trp Ala Pro 1320 1325 1330ctg gac ctg cca tcc att gtg tct gag cac ctc ccc atg aag cag 4424Leu Asp Leu Pro Ser Ile Val Ser Glu His Leu Pro Met Lys Gln 1335 1340 1345aag cga agg ttt tgg aaa ctg gtg ctg gtg ttg cct gat gtg gaa 4469Lys Arg Arg Phe Trp Lys Leu Val Leu Val Leu Pro Asp Val Glu 1350 1355 1360gag cag act cca gag agt cct ggc aga ata cta gaa aac tgg cta 4514Glu Gln Thr Pro Glu Ser Pro Gly Arg Ile Leu Glu Asn Trp Leu 1365 1370 1375aag gtc aaa ttc aca gga gat gac agc atg gtg ggt gac ata gga 4559Lys Val Lys Phe Thr Gly Asp Asp Ser Met Val Gly Asp Ile Gly 1380 1385 1390gat aat gct ggt gat atc cag acc ctc tca gtc ttt aat aca ctt 4604Asp Asn Ala Gly Asp Ile Gln Thr Leu Ser Val Phe Asn Thr Leu 1395 1400 1405agt agt aaa ggg gat caa aca gtt tct gtc aac gtg tgt ata aag 4649Ser Ser Lys Gly Asp Gln Thr Val Ser Val Asn Val Cys Ile Lys 1410 1415 1420gtg gct cat ggc acc ctt agt gac agt gcc ctt gat gct gtg gag 4694Val Ala His Gly Thr Leu Ser Asp Ser Ala Leu Asp Ala Val Glu 1425 1430 1435acc cag aag gac ctg ttg gga acc agt ggg ctc atg ctg ctg ctt 4739Thr Gln Lys Asp Leu Leu Gly Thr Ser Gly Leu Met Leu Leu Leu 1440 1445 1450ccc ccg aaa gtg aag agt gag gag gtg gca gag gag gaa ctg tcc 4784Pro Pro Lys Val Lys Ser Glu Glu Val Ala Glu Glu Glu Leu Ser 1455 1460

1465tgg ctg tcg gct tta ctg cag ctc aag cag ctt ctg cag gcc aag 4829Trp Leu Ser Ala Leu Leu Gln Leu Lys Gln Leu Leu Gln Ala Lys 1470 1475 1480ccc ttc cag cct gcc ctg ccg ctg gtg gtc ctc gtg ccc agc tcc 4874Pro Phe Gln Pro Ala Leu Pro Leu Val Val Leu Val Pro Ser Ser 1485 1490 1495aga ggg gac tcc gcg ggg agg gca gta gag gac ggt ctg atg tta 4919Arg Gly Asp Ser Ala Gly Arg Ala Val Glu Asp Gly Leu Met Leu 1500 1505 1510cag gat ttg gtt tca gcc aag ctg att tcc gat tac att gtt gtt 4964Gln Asp Leu Val Ser Ala Lys Leu Ile Ser Asp Tyr Ile Val Val 1515 1520 1525gag att cct gac tct gtt aat gat tta caa ggc aca gtg aag gtt 5009Glu Ile Pro Asp Ser Val Asn Asp Leu Gln Gly Thr Val Lys Val 1530 1535 1540tct gga gca gtc cag tgg ctg atc tcc gga tgt cct caa gcc cta 5054Ser Gly Ala Val Gln Trp Leu Ile Ser Gly Cys Pro Gln Ala Leu 1545 1550 1555gac ctt tgc tgc cag acc ctt gtt cag tat gtt gag gat ggg atc 5099Asp Leu Cys Cys Gln Thr Leu Val Gln Tyr Val Glu Asp Gly Ile 1560 1565 1570agc cgc gag ttc agc cgt cgg ttt ttc cac gac agg aga gag agg 5144Ser Arg Glu Phe Ser Arg Arg Phe Phe His Asp Arg Arg Glu Arg 1575 1580 1585cgc ctg gct agc ctg ccc tcc cag gag cct agc acc att att gag 5189Arg Leu Ala Ser Leu Pro Ser Gln Glu Pro Ser Thr Ile Ile Glu 1590 1595 1600ttg ttc aac agt gtg ctg cag ttc ctg gcc tct gtg gta tcc tct 5234Leu Phe Asn Ser Val Leu Gln Phe Leu Ala Ser Val Val Ser Ser 1605 1610 1615gag cag ctg tgt gac atc tcc tgg cct gtc atg gaa ttt gcc gaa 5279Glu Gln Leu Cys Asp Ile Ser Trp Pro Val Met Glu Phe Ala Glu 1620 1625 1630gtg gga ggc agc cag ctg ctt cct cac ctg cac tgg aac tca cca 5324Val Gly Gly Ser Gln Leu Leu Pro His Leu His Trp Asn Ser Pro 1635 1640 1645gag cat cta gcg tgg ctg aaa caa gct gtg ctt ggg ttc cag ctt 5369Glu His Leu Ala Trp Leu Lys Gln Ala Val Leu Gly Phe Gln Leu 1650 1655 1660cca cag atg gac ctt cca ccc cca ggg gcc ccc tgg ctc cct gtg 5414Pro Gln Met Asp Leu Pro Pro Pro Gly Ala Pro Trp Leu Pro Val 1665 1670 1675tgt tcc atg gtc att cag tac acc tcc cag att ccc agc tca agc 5459Cys Ser Met Val Ile Gln Tyr Thr Ser Gln Ile Pro Ser Ser Ser 1680 1685 1690cag aca cag cct gtc ctc cag tcc cag gcg gag aac ctg ctg tgc 5504Gln Thr Gln Pro Val Leu Gln Ser Gln Ala Glu Asn Leu Leu Cys 1695 1700 1705aga aca tac cag aag tgg aag aac aag agc ctc tct cca ggc cag 5549Arg Thr Tyr Gln Lys Trp Lys Asn Lys Ser Leu Ser Pro Gly Gln 1710 1715 1720gag ttg ggg cct tct gtt gcc gag atc ccg tgg gat gac atc atc 5594Glu Leu Gly Pro Ser Val Ala Glu Ile Pro Trp Asp Asp Ile Ile 1725 1730 1735acc tta tgc atc aat cat aag ctg agg gac tgg aca ccc ccc agg 5639Thr Leu Cys Ile Asn His Lys Leu Arg Asp Trp Thr Pro Pro Arg 1740 1745 1750ctc cct gtc aca tta gag gcg ctg agt gaa gat ggt caa ata tgt 5684Leu Pro Val Thr Leu Glu Ala Leu Ser Glu Asp Gly Gln Ile Cys 1755 1760 1765gtg tat ttt ttc aaa aac ctt tta aga aaa tac cac gtt ccc tcg 5729Val Tyr Phe Phe Lys Asn Leu Leu Arg Lys Tyr His Val Pro Ser 1770 1775 1780tca tgg gaa cag gcc aga atg cag acg cag cgg gaa ctg cag ctg 5774Ser Trp Glu Gln Ala Arg Met Gln Thr Gln Arg Glu Leu Gln Leu 1785 1790 1795agt cat gga cgt tcg ggg atg agg tcc atc cat cct cct aca agc 5819Ser His Gly Arg Ser Gly Met Arg Ser Ile His Pro Pro Thr Ser 1800 1805 1810act ttt cct act cca ttg ctt cat gta cac cag aaa ggg aag aaa 5864Thr Phe Pro Thr Pro Leu Leu His Val His Gln Lys Gly Lys Lys 1815 1820 1825aag gaa gag agt ggc cga gag ggg agc ctc agt aca gag gac ctc 5909Lys Glu Glu Ser Gly Arg Glu Gly Ser Leu Ser Thr Glu Asp Leu 1830 1835 1840ctg cgg ggg gct tct gca gaa gag ctc ctg gca cag agt ctg tcc 5954Leu Arg Gly Ala Ser Ala Glu Glu Leu Leu Ala Gln Ser Leu Ser 1845 1850 1855agc agt ctt ctg gaa gag aag gaa gag aac aag agg ttt gaa gat 5999Ser Ser Leu Leu Glu Glu Lys Glu Glu Asn Lys Arg Phe Glu Asp 1860 1865 1870caa ctt cag cag tgg tta tcg caa gac tca cag gca ttc aca gag 6044Gln Leu Gln Gln Trp Leu Ser Gln Asp Ser Gln Ala Phe Thr Glu 1875 1880 1885tca act cgg ctt cct ctc tac ctc cct cag acg cta gtg tcc ttt 6089Ser Thr Arg Leu Pro Leu Tyr Leu Pro Gln Thr Leu Val Ser Phe 1890 1895 1900cct gat tct atc aaa act cag acc atg gtg aaa aca tct aca agt 6134Pro Asp Ser Ile Lys Thr Gln Thr Met Val Lys Thr Ser Thr Ser 1905 1910 1915cct cag aat tca gga aca gga aag cag ttg agg ttc tca gag gca 6179Pro Gln Asn Ser Gly Thr Gly Lys Gln Leu Arg Phe Ser Glu Ala 1920 1925 1930tcc ggt tca tcc ctg acg gaa aag ctg aag ctc ctg gaa agg ctg 6224Ser Gly Ser Ser Leu Thr Glu Lys Leu Lys Leu Leu Glu Arg Leu 1935 1940 1945atc cag agc tca agg gcg gaa gaa gca gcc tcc gag ctg cac ctc 6269Ile Gln Ser Ser Arg Ala Glu Glu Ala Ala Ser Glu Leu His Leu 1950 1955 1960tct gca ctg ctg gag atg gtg gac atg tag ctgtctgacg ggagacggat 6319Ser Ala Leu Leu Glu Met Val Asp Met 1965 1970ctctaattca taatgctttg tctgtattca attgtgttat agatgctgtt ggaaatgtga 6379ctattaatta tgcaaataaa ctttttgaat cattccaaaa aaaaaaccat 642921971PRTMus musclus 2Met His Pro Val Asn Pro Phe Gly Gly Ser Ser Pro Ser Ala Phe Ala1 5 10 15Val Ser Ser Ser Thr Thr Gly Thr Tyr Gln Thr Lys Ser Pro Phe Arg 20 25 30Phe Gly Gln Pro Ser Leu Phe Gly Gln Asn Ser Thr Pro Ser Lys Ser 35 40 45Leu Ala Phe Ser Gln Val Pro Ser Phe Ala Thr Pro Ser Gly Gly Ser 50 55 60His Ser Ser Ser Leu Pro Ala Phe Gly Leu Thr Gln Thr Ser Ser Val65 70 75 80Gly Leu Phe Ser Ser Leu Glu Ser Thr Pro Ser Phe Ala Ala Thr Ser 85 90 95Ser Ser Ser Val Pro Gly Asn Thr Ala Phe Ser Phe Lys Ser Thr Ser 100 105 110Ser Val Gly Val Phe Pro Ser Gly Ala Thr Phe Gly Pro Glu Thr Gly 115 120 125Glu Val Ala Gly Ser Gly Phe Arg Lys Thr Glu Phe Lys Phe Lys Pro 130 135 140Leu Glu Asn Ala Val Phe Lys Pro Ile Pro Gly Pro Glu Ser Glu Pro145 150 155 160Glu Lys Thr Gln Ser Gln Ile Ser Ser Gly Phe Phe Thr Phe Ser His 165 170 175Pro Val Gly Ser Gly Ser Gly Gly Leu Thr Pro Phe Ser Phe Pro Gln 180 185 190Val Thr Asn Ser Ser Val Thr Ser Ser Ser Phe Ile Phe Ser Lys Pro 195 200 205Val Thr Ser Asn Thr Pro Ala Phe Ala Ser Pro Leu Ser Asn Gln Asn 210 215 220Val Glu Glu Glu Lys Arg Val Ser Thr Ser Ala Phe Gly Ser Ser Asn225 230 235 240Ser Ser Phe Ser Thr Phe Pro Thr Ala Ser Pro Gly Ser Leu Gly Glu 245 250 255Pro Phe Pro Ala Asn Lys Pro Ser Leu Arg Gln Gly Cys Glu Glu Ala 260 265 270Ile Ser Gln Val Glu Pro Leu Pro Thr Leu Met Lys Gly Leu Lys Arg 275 280 285Lys Glu Asp Gln Asp Arg Ser Pro Arg Arg His Cys His Glu Ala Ala 290 295 300Glu Asp Pro Asp Pro Leu Ser Arg Gly Asp His Pro Pro Asp Lys Arg305 310 315 320Pro Val Arg Leu Asn Arg Pro Arg Gly Gly Thr Leu Phe Gly Arg Thr 325 330 335Ile Gln Glu Val Phe Lys Ser Asn Lys Glu Ala Gly Arg Leu Gly Ser 340 345 350Lys Glu Ser Lys Glu Ser Gly Phe Ala Glu Pro Gly Glu Ser Asp His 355 360 365Ala Ala Val Pro Gly Gly Ser Gln Ser Thr Met Val Pro Ser Arg Leu 370 375 380Pro Ala Val Thr Lys Glu Glu Glu Glu Ser Arg Asp Glu Lys Glu Asp385 390 395 400Ser Leu Arg Gly Lys Ser Val Arg Gln Ser Lys Arg Arg Glu Glu Trp 405 410 415Ile Tyr Ser Leu Gly Gly Val Ser Ser Leu Glu Leu Thr Ala Ile Gln 420 425 430Cys Lys Asn Ile Pro Asp Tyr Leu Asn Asp Arg Ala Ile Leu Glu Lys 435 440 445His Phe Ser Lys Ile Ala Lys Val Gln Arg Val Phe Thr Arg Arg Ser 450 455 460Lys Lys Leu Ala Val Ile His Phe Phe Asp His Ala Ser Ala Ala Leu465 470 475 480Ala Arg Lys Lys Gly Lys Gly Leu His Lys Asp Val Val Ile Phe Trp 485 490 495His Lys Lys Lys Ile Ser Pro Ser Lys Lys Leu Phe Pro Leu Lys Glu 500 505 510Lys Leu Gly Glu Ser Glu Ala Ser Gln Gly Ile Glu Asp Ser Pro Phe 515 520 525Gln His Ser Pro Leu Ser Lys Pro Ile Val Arg Pro Ala Ala Gly Ser 530 535 540Leu Leu Ser Lys Ser Ser Pro Val Lys Lys Pro Ser Leu Leu Lys Met545 550 555 560His Gln Phe Glu Ala Asp Pro Phe Asp Ser Gly Ser Glu Gly Ser Glu 565 570 575Gly Leu Gly Ser Cys Val Ser Ser Leu Ser Thr Leu Ile Gly Thr Val 580 585 590Ala Asp Thr Ser Glu Glu Lys Tyr Arg Leu Leu Asp Gln Arg Asp Arg 595 600 605Ile Met Arg Gln Ala Arg Val Lys Arg Thr Asp Leu Asp Lys Ala Arg 610 615 620Ala Phe Val Gly Thr Cys Pro Asp Met Cys Pro Glu Lys Glu Arg Tyr625 630 635 640Leu Arg Glu Thr Arg Ser Gln Leu Ser Val Phe Glu Val Val Pro Gly 645 650 655Thr Asp Gln Val Asp His Ala Ala Ala Val Lys Glu Tyr Ser Arg Ser 660 665 670Ser Ala Asp Gln Glu Glu Pro Leu Pro His Glu Leu Arg Pro Ser Ala 675 680 685Val Leu Ser Arg Thr Met Asp Tyr Leu Val Thr Gln Ile Met Asp Gln 690 695 700Lys Glu Gly Ser Leu Arg Asp Trp Tyr Asp Phe Val Trp Asn Arg Thr705 710 715 720Arg Gly Ile Arg Lys Asp Ile Thr Gln Gln His Leu Cys Asp Pro Leu 725 730 735Thr Val Ser Leu Ile Glu Lys Cys Thr Arg Phe His Ile His Cys Ala 740 745 750His Phe Met Cys Glu Glu Pro Met Ser Ser Phe Asp Ala Lys Ile Asn 755 760 765Asn Glu Asn Met Thr Lys Cys Leu Gln Ser Leu Lys Glu Met Tyr Gln 770 775 780Asp Leu Arg Asn Lys Gly Val Phe Cys Ala Ser Glu Ala Glu Phe Gln785 790 795 800Gly Tyr Asn Val Leu Leu Asn Leu Asn Lys Gly Asp Ile Leu Arg Glu 805 810 815Val Gln Gln Phe His Pro Asp Val Arg Asn Ser Pro Glu Val Asn Phe 820 825 830Ala Val Gln Ala Phe Ala Ala Leu Asn Ser Asn Asn Phe Val Arg Phe 835 840 845Phe Lys Leu Val Gln Ser Ala Ser Tyr Leu Asn Ala Cys Leu Leu His 850 855 860Cys Tyr Phe Asn Gln Ile Arg Lys Asp Ala Leu Arg Ala Leu Asn Val865 870 875 880Ala Tyr Thr Val Ser Thr Gln Arg Ser Thr Val Phe Pro Leu Asp Gly 885 890 895Val Val Arg Met Leu Leu Phe Arg Asp Ser Glu Glu Ala Thr Asn Phe 900 905 910Leu Asn Tyr His Gly Leu Thr Val Ala Asp Gly Cys Val Glu Leu Asn 915 920 925Arg Ser Ala Phe Leu Glu Pro Glu Gly Leu Cys Lys Ala Arg Lys Ser 930 935 940Val Phe Ile Gly Arg Lys Leu Thr Val Ser Val Gly Glu Val Val Asn945 950 955 960Gly Gly Pro Leu Pro Pro Val Pro Arg His Thr Pro Val Cys Ser Phe 965 970 975Asn Ser Gln Asn Lys Tyr Val Gly Glu Ser Leu Ala Thr Glu Leu Pro 980 985 990Ile Ser Thr Gln Arg Ala Gly Gly Asp Pro Ala Gly Gly Gly Arg Gly 995 1000 1005Glu Asp Cys Glu Ala Glu Val Asp Leu Pro Thr Leu Ala Val Leu 1010 1015 1020Pro Gln Pro Pro Pro Ala Ser Ser Ala Thr Pro Ala Leu His Val 1025 1030 1035Gln Pro Leu Ala Pro Ala Ala Ala Pro Ser Leu Leu Gln Ala Ser 1040 1045 1050Thr Gln Pro Glu Val Leu Leu Pro Lys Pro Ala Pro Val Tyr Ser 1055 1060 1065Asp Ser Asp Leu Val Gln Val Val Asp Glu Leu Ile Gln Glu Ala 1070 1075 1080Leu Gln Val Asp Cys Glu Glu Val Ser Ser Ala Gly Ala Ala Tyr 1085 1090 1095Val Ala Ala Ala Leu Gly Val Ser Asn Ala Ala Val Glu Asp Leu 1100 1105 1110Ile Thr Ala Ala Thr Thr Gly Ile Leu Arg His Val Ala Ala Glu 1115 1120 1125Glu Val Ser Met Glu Arg Gln Arg Leu Glu Glu Glu Lys Gln Arg 1130 1135 1140Ala Glu Glu Glu Arg Leu Lys Gln Glu Arg Glu Leu Met Leu Thr 1145 1150 1155Gln Leu Ser Glu Gly Leu Ala Ala Glu Leu Thr Glu Leu Thr Val 1160 1165 1170Thr Glu Cys Val Trp Glu Thr Cys Ser Gln Glu Leu Gln Ser Ala 1175 1180 1185Val Lys Ile Asp Gln Lys Val Arg Val Ala Arg Cys Cys Glu Ala 1190 1195 1200Val Cys Ala His Leu Val Asp Leu Phe Leu Ala Glu Glu Ile Phe 1205 1210 1215Gln Thr Ala Lys Glu Thr Leu Gln Glu Leu Gln Cys Phe Cys Lys 1220 1225 1230Tyr Leu Gln Arg Trp Arg Glu Ala Val Ala Ala Arg Lys Lys Phe 1235 1240 1245Arg Arg Gln Met Arg Ala Phe Pro Ala Ala Pro Cys Cys Val Asp 1250 1255 1260Val Asn Asp Arg Leu Gln Ala Leu Val Pro Ser Ala Glu Cys Pro 1265 1270 1275Ile Thr Glu Glu Asn Leu Ala Lys Gly Leu Leu Asp Leu Gly His 1280 1285 1290Ala Gly Lys Val Gly Val Ser Cys Thr Arg Leu Arg Arg Leu Arg 1295 1300 1305Asn Lys Thr Ala His Gln Ile Lys Val Gln His Phe His Gln Gln 1310 1315 1320Leu Leu Arg Asn Ala Ala Trp Ala Pro Leu Asp Leu Pro Ser Ile 1325 1330 1335Val Ser Glu His Leu Pro Met Lys Gln Lys Arg Arg Phe Trp Lys 1340 1345 1350Leu Val Leu Val Leu Pro Asp Val Glu Glu Gln Thr Pro Glu Ser 1355 1360 1365Pro Gly Arg Ile Leu Glu Asn Trp Leu Lys Val Lys Phe Thr Gly 1370 1375 1380Asp Asp Ser Met Val Gly Asp Ile Gly Asp Asn Ala Gly Asp Ile 1385 1390 1395Gln Thr Leu Ser Val Phe Asn Thr Leu Ser Ser Lys Gly Asp Gln 1400 1405 1410Thr Val Ser Val Asn Val Cys Ile Lys Val Ala His Gly Thr Leu 1415 1420 1425Ser Asp Ser Ala Leu Asp Ala Val Glu Thr Gln Lys Asp Leu Leu 1430 1435 1440Gly Thr Ser Gly Leu Met Leu Leu Leu Pro Pro Lys Val Lys Ser 1445 1450 1455Glu Glu Val Ala Glu Glu Glu Leu Ser Trp Leu Ser Ala Leu Leu 1460 1465 1470Gln Leu Lys Gln Leu Leu Gln Ala Lys Pro Phe Gln Pro Ala Leu 1475 1480 1485Pro Leu Val Val Leu Val Pro Ser Ser Arg Gly Asp Ser Ala Gly 1490 1495 1500Arg Ala Val Glu Asp Gly Leu Met Leu Gln Asp Leu Val Ser Ala 1505 1510 1515Lys Leu Ile Ser Asp Tyr Ile Val Val Glu Ile Pro Asp Ser Val 1520 1525 1530Asn Asp Leu Gln Gly Thr Val Lys Val Ser Gly Ala Val Gln Trp 1535 1540 1545Leu Ile Ser Gly Cys Pro Gln Ala Leu Asp Leu Cys Cys Gln Thr 1550 1555 1560Leu Val Gln Tyr Val Glu Asp Gly Ile Ser Arg Glu Phe Ser Arg 1565

1570 1575Arg Phe Phe His Asp Arg Arg Glu Arg Arg Leu Ala Ser Leu Pro 1580 1585 1590Ser Gln Glu Pro Ser Thr Ile Ile Glu Leu Phe Asn Ser Val Leu 1595 1600 1605Gln Phe Leu Ala Ser Val Val Ser Ser Glu Gln Leu Cys Asp Ile 1610 1615 1620Ser Trp Pro Val Met Glu Phe Ala Glu Val Gly Gly Ser Gln Leu 1625 1630 1635Leu Pro His Leu His Trp Asn Ser Pro Glu His Leu Ala Trp Leu 1640 1645 1650Lys Gln Ala Val Leu Gly Phe Gln Leu Pro Gln Met Asp Leu Pro 1655 1660 1665Pro Pro Gly Ala Pro Trp Leu Pro Val Cys Ser Met Val Ile Gln 1670 1675 1680Tyr Thr Ser Gln Ile Pro Ser Ser Ser Gln Thr Gln Pro Val Leu 1685 1690 1695Gln Ser Gln Ala Glu Asn Leu Leu Cys Arg Thr Tyr Gln Lys Trp 1700 1705 1710Lys Asn Lys Ser Leu Ser Pro Gly Gln Glu Leu Gly Pro Ser Val 1715 1720 1725Ala Glu Ile Pro Trp Asp Asp Ile Ile Thr Leu Cys Ile Asn His 1730 1735 1740Lys Leu Arg Asp Trp Thr Pro Pro Arg Leu Pro Val Thr Leu Glu 1745 1750 1755Ala Leu Ser Glu Asp Gly Gln Ile Cys Val Tyr Phe Phe Lys Asn 1760 1765 1770Leu Leu Arg Lys Tyr His Val Pro Ser Ser Trp Glu Gln Ala Arg 1775 1780 1785Met Gln Thr Gln Arg Glu Leu Gln Leu Ser His Gly Arg Ser Gly 1790 1795 1800Met Arg Ser Ile His Pro Pro Thr Ser Thr Phe Pro Thr Pro Leu 1805 1810 1815Leu His Val His Gln Lys Gly Lys Lys Lys Glu Glu Ser Gly Arg 1820 1825 1830Glu Gly Ser Leu Ser Thr Glu Asp Leu Leu Arg Gly Ala Ser Ala 1835 1840 1845Glu Glu Leu Leu Ala Gln Ser Leu Ser Ser Ser Leu Leu Glu Glu 1850 1855 1860Lys Glu Glu Asn Lys Arg Phe Glu Asp Gln Leu Gln Gln Trp Leu 1865 1870 1875Ser Gln Asp Ser Gln Ala Phe Thr Glu Ser Thr Arg Leu Pro Leu 1880 1885 1890Tyr Leu Pro Gln Thr Leu Val Ser Phe Pro Asp Ser Ile Lys Thr 1895 1900 1905Gln Thr Met Val Lys Thr Ser Thr Ser Pro Gln Asn Ser Gly Thr 1910 1915 1920Gly Lys Gln Leu Arg Phe Ser Glu Ala Ser Gly Ser Ser Leu Thr 1925 1930 1935Glu Lys Leu Lys Leu Leu Glu Arg Leu Ile Gln Ser Ser Arg Ala 1940 1945 1950Glu Glu Ala Ala Ser Glu Leu His Leu Ser Ala Leu Leu Glu Met 1955 1960 1965Val Asp Met 197036114DNAHomo sapiensCDS(38)..(5977) 3gtaatactta attaccttct aataattgga gcagaag atg aac cca act aat cct 55 Met Asn Pro Thr Asn Pro 1 5ttc agt ggg cag cag cct agt gct ttt tcg gcg tct tct agt aat gta 103Phe Ser Gly Gln Gln Pro Ser Ala Phe Ser Ala Ser Ser Ser Asn Val 10 15 20gga aca ctt cca tct aag ccg cca ttt cga ttt ggt caa cct tct ctt 151Gly Thr Leu Pro Ser Lys Pro Pro Phe Arg Phe Gly Gln Pro Ser Leu 25 30 35ttt gga caa aac agt acc tta tct ggg aag agc tcg gga ttt tca cag 199Phe Gly Gln Asn Ser Thr Leu Ser Gly Lys Ser Ser Gly Phe Ser Gln 40 45 50gta tcc agc ttt cca gcg tct tct gga gta agt cat tcc tct tca gtg 247Val Ser Ser Phe Pro Ala Ser Ser Gly Val Ser His Ser Ser Ser Val55 60 65 70caa aca tta ggg ttc acc caa acc tca agt gtt gga ccc ttt tct gga 295Gln Thr Leu Gly Phe Thr Gln Thr Ser Ser Val Gly Pro Phe Ser Gly 75 80 85ctt gag cac act tcc acc ttt gtg gct acc tct ggg cct tca agt tca 343Leu Glu His Thr Ser Thr Phe Val Ala Thr Ser Gly Pro Ser Ser Ser 90 95 100tct gtg ctg gga aac aca gga ttt agt ttt aaa tca ccc acc agt gtt 391Ser Val Leu Gly Asn Thr Gly Phe Ser Phe Lys Ser Pro Thr Ser Val 105 110 115ggg gct ttc cca agc act tct gct ttt gga caa gaa gct gga gaa ata 439Gly Ala Phe Pro Ser Thr Ser Ala Phe Gly Gln Glu Ala Gly Glu Ile 120 125 130gtg aac tct ggt ttt ggg aaa aca gaa ttc agc ttt aaa cct ctg gaa 487Val Asn Ser Gly Phe Gly Lys Thr Glu Phe Ser Phe Lys Pro Leu Glu135 140 145 150aat gca gtg ttc aaa cca ata ctg ggg gct gaa tct gag cca gag aaa 535Asn Ala Val Phe Lys Pro Ile Leu Gly Ala Glu Ser Glu Pro Glu Lys 155 160 165acc cag agc caa att gct tct ggg ttt ttt aca ttt tcc cac cca att 583Thr Gln Ser Gln Ile Ala Ser Gly Phe Phe Thr Phe Ser His Pro Ile 170 175 180agt agt gca cct gga ggc ctg gcc cct ttc tct ttt cct caa gta aca 631Ser Ser Ala Pro Gly Gly Leu Ala Pro Phe Ser Phe Pro Gln Val Thr 185 190 195agt agt tca gct acc act tca aat ttt acc ttt tca aaa cct gtt agt 679Ser Ser Ser Ala Thr Thr Ser Asn Phe Thr Phe Ser Lys Pro Val Ser 200 205 210agt aat aat tca tta tct gcc ttt acc cct gct ttg tca aac caa aat 727Ser Asn Asn Ser Leu Ser Ala Phe Thr Pro Ala Leu Ser Asn Gln Asn215 220 225 230gta gag gaa gag aag aga gga cct aag tca ata ttt gga agt tct aat 775Val Glu Glu Glu Lys Arg Gly Pro Lys Ser Ile Phe Gly Ser Ser Asn 235 240 245aat agc ttc agt agc ttc cct gta tca tct gcg gtt ttg ggc gaa cct 823Asn Ser Phe Ser Ser Phe Pro Val Ser Ser Ala Val Leu Gly Glu Pro 250 255 260ttc cag gct agc aaa gca ggt gtc agg cag ggg tgt gaa gaa gct gtt 871Phe Gln Ala Ser Lys Ala Gly Val Arg Gln Gly Cys Glu Glu Ala Val 265 270 275tcc cag gtg gaa cca ctt ccc agc cta atg aaa gga ctg aaa agg aag 919Ser Gln Val Glu Pro Leu Pro Ser Leu Met Lys Gly Leu Lys Arg Lys 280 285 290gag gac cag gat cgc tcc cca agg aga cat ggc cac gag cca gca gaa 967Glu Asp Gln Asp Arg Ser Pro Arg Arg His Gly His Glu Pro Ala Glu295 300 305 310gat tcg gat cct ctg tcc cgg ggc gat cat cct cca gac aaa cga cct 1015Asp Ser Asp Pro Leu Ser Arg Gly Asp His Pro Pro Asp Lys Arg Pro 315 320 325gtc cgc ctg aat cga ccc cgg gga ggt act tta ttt ggt cgg acg ata 1063Val Arg Leu Asn Arg Pro Arg Gly Gly Thr Leu Phe Gly Arg Thr Ile 330 335 340cag gat gtt ttc aaa agc aat aag gaa gta ggt cgt ctg ggc aac aag 1111Gln Asp Val Phe Lys Ser Asn Lys Glu Val Gly Arg Leu Gly Asn Lys 345 350 355gag gcc aaa aag gaa act ggc ttt gtt gag tct gca gaa agt gac cac 1159Glu Ala Lys Lys Glu Thr Gly Phe Val Glu Ser Ala Glu Ser Asp His 360 365 370atg gct atc cca gga ggg aat cag tct gtc ctg gca cct tcc cgg att 1207Met Ala Ile Pro Gly Gly Asn Gln Ser Val Leu Ala Pro Ser Arg Ile375 380 385 390cca ggt gtg aat aaa gag gaa gaa act gaa agt aga gag aag aaa gaa 1255Pro Gly Val Asn Lys Glu Glu Glu Thr Glu Ser Arg Glu Lys Lys Glu 395 400 405gat tct cta aga gga act ccg gcg cgt cag agt aac aga agc gag agc 1303Asp Ser Leu Arg Gly Thr Pro Ala Arg Gln Ser Asn Arg Ser Glu Ser 410 415 420aca gac agt ctt ggg ggc ttg tct ccc tct gaa gtc aca gcc atc cag 1351Thr Asp Ser Leu Gly Gly Leu Ser Pro Ser Glu Val Thr Ala Ile Gln 425 430 435tgc aag aac atc cct gac tac ctc aac gac agg acc att ctg gag aac 1399Cys Lys Asn Ile Pro Asp Tyr Leu Asn Asp Arg Thr Ile Leu Glu Asn 440 445 450cat ttt ggc aaa att gct aaa gtg cag cgc atc ttt acc agg cgc agc 1447His Phe Gly Lys Ile Ala Lys Val Gln Arg Ile Phe Thr Arg Arg Ser455 460 465 470aaa aag ctt gca gtg gta cat ttc ttt gat cat gca tct gca gcc ctg 1495Lys Lys Leu Ala Val Val His Phe Phe Asp His Ala Ser Ala Ala Leu 475 480 485gct aga aag aag ggg aaa agt ttg cat aaa gac atg gct atc ttt tgg 1543Ala Arg Lys Lys Gly Lys Ser Leu His Lys Asp Met Ala Ile Phe Trp 490 495 500cac agg aag aaa ata agc ccc aat aag aaa ccc ttt tcc ctg aag gag 1591His Arg Lys Lys Ile Ser Pro Asn Lys Lys Pro Phe Ser Leu Lys Glu 505 510 515aag aaa cca ggt gac ggt gaa gtc agc ccg agc aca gag gat gca ccc 1639Lys Lys Pro Gly Asp Gly Glu Val Ser Pro Ser Thr Glu Asp Ala Pro 520 525 530ttt cag cac tct cct ctt ggc aag gcc gca ggg agg act ggt gct agc 1687Phe Gln His Ser Pro Leu Gly Lys Ala Ala Gly Arg Thr Gly Ala Ser535 540 545 550agc ctc ctg aat aaa agc tct cca gtg aag aag cca agt ctt cta aag 1735Ser Leu Leu Asn Lys Ser Ser Pro Val Lys Lys Pro Ser Leu Leu Lys 555 560 565gcc cac caa ttc gag gga gac tct ttt gac tca gcc tcc gag ggc tcc 1783Ala His Gln Phe Glu Gly Asp Ser Phe Asp Ser Ala Ser Glu Gly Ser 570 575 580gag ggc ctc ggg cca tgt gtg ctc tcc ctc agt acc ctg ata ggc act 1831Glu Gly Leu Gly Pro Cys Val Leu Ser Leu Ser Thr Leu Ile Gly Thr 585 590 595gtg gct gag aca tcc aag gag aag tac cgc ctg ctt gac cag aga gac 1879Val Ala Glu Thr Ser Lys Glu Lys Tyr Arg Leu Leu Asp Gln Arg Asp 600 605 610agg atc atg cgg caa gct cgg gtg aag aga acc gat ctg gac aaa gcg 1927Arg Ile Met Arg Gln Ala Arg Val Lys Arg Thr Asp Leu Asp Lys Ala615 620 625 630agg act ttt gtt ggc acc tgc ctg gat atg tgt cct gag aag gag agg 1975Arg Thr Phe Val Gly Thr Cys Leu Asp Met Cys Pro Glu Lys Glu Arg 635 640 645tac atg cgg gag acc cgt agc cag ctg agc gtg ttc gaa gtg gtc cca 2023Tyr Met Arg Glu Thr Arg Ser Gln Leu Ser Val Phe Glu Val Val Pro 650 655 660ggg act gac cag gtg gac cac gca gca gct gtg aaa gag tac agt cgg 2071Gly Thr Asp Gln Val Asp His Ala Ala Ala Val Lys Glu Tyr Ser Arg 665 670 675tcc tcg gcg gat cag gag gag ccc ctg ccc cac gag ctg cgg ccc ttg 2119Ser Ser Ala Asp Gln Glu Glu Pro Leu Pro His Glu Leu Arg Pro Leu 680 685 690cca gtg ctc agc agg acc atg gac tac ctg gtg acc cag atc atg gac 2167Pro Val Leu Ser Arg Thr Met Asp Tyr Leu Val Thr Gln Ile Met Asp695 700 705 710cag aag gag ggc agc ctg cgg gat tgg tat gac ttc gtg tgg aac cgc 2215Gln Lys Glu Gly Ser Leu Arg Asp Trp Tyr Asp Phe Val Trp Asn Arg 715 720 725acg cgt ggc ata cgg aag gat atc acg cag cag cac ctc tgt gac ccc 2263Thr Arg Gly Ile Arg Lys Asp Ile Thr Gln Gln His Leu Cys Asp Pro 730 735 740ctg acg gtg tcc ctg att gag aag tgc acc cgg ttt cac atc cac tgt 2311Leu Thr Val Ser Leu Ile Glu Lys Cys Thr Arg Phe His Ile His Cys 745 750 755gcc cac ttc atg tgt gag gag ccc atg tcc tcc ttt gat gcc aag atc 2359Ala His Phe Met Cys Glu Glu Pro Met Ser Ser Phe Asp Ala Lys Ile 760 765 770aat aat gag aac atg acc aag tgc ctg cag agc ctg aag gag atg tac 2407Asn Asn Glu Asn Met Thr Lys Cys Leu Gln Ser Leu Lys Glu Met Tyr775 780 785 790cag gac ctg aga aac aag ggt gtc ttc tgt gcc agc gaa gcg gag ttc 2455Gln Asp Leu Arg Asn Lys Gly Val Phe Cys Ala Ser Glu Ala Glu Phe 795 800 805cag ggc tac aat gtt ctg ctc agt ctc aac aag gga gac atc cta aga 2503Gln Gly Tyr Asn Val Leu Leu Ser Leu Asn Lys Gly Asp Ile Leu Arg 810 815 820gaa gta caa cag ttc cat cct gct gtt aga aac tca tct gag gtg aaa 2551Glu Val Gln Gln Phe His Pro Ala Val Arg Asn Ser Ser Glu Val Lys 825 830 835ttt gct gtt cag gct ttt gct gca ttg aac agt aat aat ttt gtg aga 2599Phe Ala Val Gln Ala Phe Ala Ala Leu Asn Ser Asn Asn Phe Val Arg 840 845 850ttt ttc aaa ctg gtc cag tca gct tct tac ctg aac gct tgt ctt tta 2647Phe Phe Lys Leu Val Gln Ser Ala Ser Tyr Leu Asn Ala Cys Leu Leu855 860 865 870cac tgt tac ttc agt cag atc cgc aag gat gct ctc cgg gcg ctc aac 2695His Cys Tyr Phe Ser Gln Ile Arg Lys Asp Ala Leu Arg Ala Leu Asn 875 880 885ttt gcg tac acg gtg agc aca cag cga tct acc atc ttt ccc ctg gat 2743Phe Ala Tyr Thr Val Ser Thr Gln Arg Ser Thr Ile Phe Pro Leu Asp 890 895 900ggt gtg gtg cgc atg ctg ctg ttc aga gac tgt gaa gag gcc acc gac 2791Gly Val Val Arg Met Leu Leu Phe Arg Asp Cys Glu Glu Ala Thr Asp 905 910 915ttc ctc acc tgc cac ggc ctc acc gtt tcc gac ggc tgt gtg gag ctg 2839Phe Leu Thr Cys His Gly Leu Thr Val Ser Asp Gly Cys Val Glu Leu 920 925 930aac cgg tct gca ttc ctg gaa cca gag gga tta tcc aag acc agg aag 2887Asn Arg Ser Ala Phe Leu Glu Pro Glu Gly Leu Ser Lys Thr Arg Lys935 940 945 950tcg gtg ttt att act agg aag ctg acg gtg tca gtc ggg gaa att gtg 2935Ser Val Phe Ile Thr Arg Lys Leu Thr Val Ser Val Gly Glu Ile Val 955 960 965aac gga ggg cca ttg ccc ccc gtc cct cgt cac acc cct gtg tgc agc 2983Asn Gly Gly Pro Leu Pro Pro Val Pro Arg His Thr Pro Val Cys Ser 970 975 980ttc aac tcc cag aac aag tac atc ggg gag agc ctg gcc gcg gag ctg 3031Phe Asn Ser Gln Asn Lys Tyr Ile Gly Glu Ser Leu Ala Ala Glu Leu 985 990 995ccc gtc agc acc cag aga ccc ggc tcc gac aca gtg ggc gga ggg 3076Pro Val Ser Thr Gln Arg Pro Gly Ser Asp Thr Val Gly Gly Gly 1000 1005 1010aga gga gag gag tgt ggt gta gag ccg gat gca ccc ctg tcc agt 3121Arg Gly Glu Glu Cys Gly Val Glu Pro Asp Ala Pro Leu Ser Ser 1015 1020 1025ctc cca cag tct cta cca gcc cct gcg ccc tca cca gtg cct ctg 3166Leu Pro Gln Ser Leu Pro Ala Pro Ala Pro Ser Pro Val Pro Leu 1030 1035 1040cct cct gtc ctg gca ctg acc ccg tct gtg gcg ccc agc ctc ttc 3211Pro Pro Val Leu Ala Leu Thr Pro Ser Val Ala Pro Ser Leu Phe 1045 1050 1055cag ctg tct gtg cag cct gaa cca ccg cct cca gag ccc gtg ccc 3256Gln Leu Ser Val Gln Pro Glu Pro Pro Pro Pro Glu Pro Val Pro 1060 1065 1070atg tac tct gac gag gac ctg gcg cag gtg gtg gac gag ctc atc 3301Met Tyr Ser Asp Glu Asp Leu Ala Gln Val Val Asp Glu Leu Ile 1075 1080 1085cag gag gcc ctg cag agg gac tgt gag gaa gtt ggc tct gcg ggt 3346Gln Glu Ala Leu Gln Arg Asp Cys Glu Glu Val Gly Ser Ala Gly 1090 1095 1100gct gcc tac gca gct gcc gcc ctg ggt gtt tct aat gct gct atg 3391Ala Ala Tyr Ala Ala Ala Ala Leu Gly Val Ser Asn Ala Ala Met 1105 1110 1115gag gat ttg tta aca gct gca acc acg ggc att ttg agg cac att 3436Glu Asp Leu Leu Thr Ala Ala Thr Thr Gly Ile Leu Arg His Ile 1120 1125 1130gca gct gaa gaa gtg tct aag gaa aga gag cga agg gag cag gag 3481Ala Ala Glu Glu Val Ser Lys Glu Arg Glu Arg Arg Glu Gln Glu 1135 1140 1145agg cag cgg gct gaa gag gaa agg ttg aaa caa gag aga gag ctg 3526Arg Gln Arg Ala Glu Glu Glu Arg Leu Lys Gln Glu Arg Glu Leu 1150 1155 1160gtg tta agt gag ctg agc cag ggc ctg gcc gtg gag ctg atg gaa 3571Val Leu Ser Glu Leu Ser Gln Gly Leu Ala Val Glu Leu Met Glu 1165 1170 1175cgc gtg atg atg gag ttt gtg agg gaa acc tgc tcc cag gag ttg 3616Arg Val Met Met Glu Phe Val Arg Glu Thr Cys Ser Gln Glu Leu 1180 1185 1190aag aat gca gta gag aca gac cag agg gtc cgt gtg gcc cgt tgc 3661Lys Asn Ala Val Glu Thr Asp Gln Arg Val Arg Val Ala Arg Cys 1195 1200 1205tgt gag gat gtc tgt gcc cac tta gtg gac ttg ttt ctc gtg gag 3706Cys Glu Asp Val Cys Ala His Leu Val Asp Leu Phe Leu Val Glu 1210 1215 1220gaa atc ttc cag act gca aag gag acc ctc cag gag ctt cag tgc 3751Glu Ile Phe Gln Thr Ala Lys Glu Thr Leu Gln Glu Leu Gln Cys 1225 1230 1235ttc tgc aag tat cta cag cgg tgg agg gaa gct gtc aca gcc cgc 3796Phe Cys Lys Tyr Leu Gln Arg Trp Arg Glu Ala Val Thr Ala Arg 1240 1245 1250aag aaa ctg agg cgc caa atg cgg gct ttc cct gct gcg ccc tgc 3841Lys Lys Leu Arg Arg Gln Met Arg Ala Phe Pro Ala Ala Pro Cys 1255 1260

1265tgc gtg gac gtg agc gac cgg ctg agg gcg ctg gcg ccc agc gca 3886Cys Val Asp Val Ser Asp Arg Leu Arg Ala Leu Ala Pro Ser Ala 1270 1275 1280gag tgc ccc att gct gaa gag aac ctg gcc agg ggc ctc ctg gac 3931Glu Cys Pro Ile Ala Glu Glu Asn Leu Ala Arg Gly Leu Leu Asp 1285 1290 1295ctg ggc cat gca ggg aga ttg ggc atc tct tgc acc agg tta agg 3976Leu Gly His Ala Gly Arg Leu Gly Ile Ser Cys Thr Arg Leu Arg 1300 1305 1310cgg ctc aga aac aag aca gct cac cag atg aag gtt cag cac ttc 4021Arg Leu Arg Asn Lys Thr Ala His Gln Met Lys Val Gln His Phe 1315 1320 1325tac cag cag ctg ctg agt gat gtg gca tgg gcg tct ctg gac ctg 4066Tyr Gln Gln Leu Leu Ser Asp Val Ala Trp Ala Ser Leu Asp Leu 1330 1335 1340cca tcc ctc gtg gct gag cac ctc cct ggg agg cag gag cat gtg 4111Pro Ser Leu Val Ala Glu His Leu Pro Gly Arg Gln Glu His Val 1345 1350 1355ttt tgg aag ctg gtg ctg gtg ttg ccg gat gta gag gag cag tcc 4156Phe Trp Lys Leu Val Leu Val Leu Pro Asp Val Glu Glu Gln Ser 1360 1365 1370cca gag agt tgt ggc aga att cta gca aat tgg tta aaa gtc aag 4201Pro Glu Ser Cys Gly Arg Ile Leu Ala Asn Trp Leu Lys Val Lys 1375 1380 1385ttc atg gga gat gaa ggc tca gtg gat gac aca tcc agc gat gct 4246Phe Met Gly Asp Glu Gly Ser Val Asp Asp Thr Ser Ser Asp Ala 1390 1395 1400ggt ggg att cag acg ctt tcg ctt ttc aac tca ctt agc agc aaa 4291Gly Gly Ile Gln Thr Leu Ser Leu Phe Asn Ser Leu Ser Ser Lys 1405 1410 1415ggg gat cag atg att tct gtt aac gtg tgt ata aag gtg gcc cat 4336Gly Asp Gln Met Ile Ser Val Asn Val Cys Ile Lys Val Ala His 1420 1425 1430ggc gcc ctc agt gat ggt gcc att gat gct gtg gag aca cag aag 4381Gly Ala Leu Ser Asp Gly Ala Ile Asp Ala Val Glu Thr Gln Lys 1435 1440 1445gac ctc ctg gga gcc agt ggg ctc atg ctg ctg ctt ccc ccc aaa 4426Asp Leu Leu Gly Ala Ser Gly Leu Met Leu Leu Leu Pro Pro Lys 1450 1455 1460atg aag agt gag gac atg gca gag gag gac gtg tac tgg ctg tcg 4471Met Lys Ser Glu Asp Met Ala Glu Glu Asp Val Tyr Trp Leu Ser 1465 1470 1475gcc ttg ctg cag ctc aag cag ctc ctg cag gct aag ccc ttc cag 4516Ala Leu Leu Gln Leu Lys Gln Leu Leu Gln Ala Lys Pro Phe Gln 1480 1485 1490cct gcg ctt cct ctg gtg gtt ctt gtg cct agc cca gga ggg gac 4561Pro Ala Leu Pro Leu Val Val Leu Val Pro Ser Pro Gly Gly Asp 1495 1500 1505gcc gtt gag aag gaa gta gaa gat ggt ctg atg cta cag gac ttg 4606Ala Val Glu Lys Glu Val Glu Asp Gly Leu Met Leu Gln Asp Leu 1510 1515 1520gtt tca gct aag ctg att tca gat tac act gtt acc gag atc cct 4651Val Ser Ala Lys Leu Ile Ser Asp Tyr Thr Val Thr Glu Ile Pro 1525 1530 1535gat acc att aat gat cta caa ggt tca act aag gtt ttg caa gca 4696Asp Thr Ile Asn Asp Leu Gln Gly Ser Thr Lys Val Leu Gln Ala 1540 1545 1550gtg cag tgg ctg gtt tcc cac tgc ccc cat tcc ctt gac ctc tgc 4741Val Gln Trp Leu Val Ser His Cys Pro His Ser Leu Asp Leu Cys 1555 1560 1565tgc cag act ctc att cag tac gtc gaa gac ggg att ggc cat gag 4786Cys Gln Thr Leu Ile Gln Tyr Val Glu Asp Gly Ile Gly His Glu 1570 1575 1580ttt agt ggc cgc ttt ttc cat gac aga aga gag agg cgt ctg ggc 4831Phe Ser Gly Arg Phe Phe His Asp Arg Arg Glu Arg Arg Leu Gly 1585 1590 1595ggt ctt gct tct cag gag cct ggc gcc atc att gag ctg ttt aac 4876Gly Leu Ala Ser Gln Glu Pro Gly Ala Ile Ile Glu Leu Phe Asn 1600 1605 1610agt gtg ctg cag ttc ctg gct tct gtg gtg tcc tct gaa cag ctg 4921Ser Val Leu Gln Phe Leu Ala Ser Val Val Ser Ser Glu Gln Leu 1615 1620 1625tgt gac ctg tcc tgg cct gtc act gag ttt gct gag gca ggg ggc 4966Cys Asp Leu Ser Trp Pro Val Thr Glu Phe Ala Glu Ala Gly Gly 1630 1635 1640agc cgg ctg ctt cct cac ctg cac tgg aat gcc cca gag cac ctg 5011Ser Arg Leu Leu Pro His Leu His Trp Asn Ala Pro Glu His Leu 1645 1650 1655gcc tgg ctg aag cag gct gtg ctc ggg ttc cag ctt ccg cag atg 5056Ala Trp Leu Lys Gln Ala Val Leu Gly Phe Gln Leu Pro Gln Met 1660 1665 1670gac ctt cca ccc ctg ggg gcc ccc tgg ctc ccc gtg tgc tcc atg 5101Asp Leu Pro Pro Leu Gly Ala Pro Trp Leu Pro Val Cys Ser Met 1675 1680 1685gtt gtc cag tac gcc tcc cag atc ccc agc tca cgc cag aca cag 5146Val Val Gln Tyr Ala Ser Gln Ile Pro Ser Ser Arg Gln Thr Gln 1690 1695 1700cct gtc ctc cag tcc cag gtg gag aac ctg ctc cac aga acc tac 5191Pro Val Leu Gln Ser Gln Val Glu Asn Leu Leu His Arg Thr Tyr 1705 1710 1715tgt agg tgg aag agc aag agt ccc tcc cca gtc cat ggg gca ggc 5236Cys Arg Trp Lys Ser Lys Ser Pro Ser Pro Val His Gly Ala Gly 1720 1725 1730ccc tcg gtc atg gag atc cca tgg gat gat ctt atc gcc ttg tgt 5281Pro Ser Val Met Glu Ile Pro Trp Asp Asp Leu Ile Ala Leu Cys 1735 1740 1745atc aac cac aag ctg aga gac tgg acg ccc ccc cgg ctt cct gtt 5326Ile Asn His Lys Leu Arg Asp Trp Thr Pro Pro Arg Leu Pro Val 1750 1755 1760aca tca gag gcg ctg agt gaa gat ggt cag ata tgt gtg tat ttt 5371Thr Ser Glu Ala Leu Ser Glu Asp Gly Gln Ile Cys Val Tyr Phe 1765 1770 1775ttt aaa aac gat ttg aaa aaa tat gat gtt cct ttg tcg tgg gaa 5416Phe Lys Asn Asp Leu Lys Lys Tyr Asp Val Pro Leu Ser Trp Glu 1780 1785 1790caa gcc agg ttg cag acg cag aag gag cta cag ctg aga gag gga 5461Gln Ala Arg Leu Gln Thr Gln Lys Glu Leu Gln Leu Arg Glu Gly 1795 1800 1805cgt ttg gca ata aag cct ttt cat cct tct gca aac aat ttt ccc 5506Arg Leu Ala Ile Lys Pro Phe His Pro Ser Ala Asn Asn Phe Pro 1810 1815 1820ata cca ttg ctt cac atg cac cgt aac tgg aag agg agc aca gag 5551Ile Pro Leu Leu His Met His Arg Asn Trp Lys Arg Ser Thr Glu 1825 1830 1835tgt gct caa gag ggg agg att ccc agc aca gag gat ctg atg cga 5596Cys Ala Gln Glu Gly Arg Ile Pro Ser Thr Glu Asp Leu Met Arg 1840 1845 1850gga gct tct gct gag gag ctc ttg gcg cag tgt ttg tcg agc agt 5641Gly Ala Ser Ala Glu Glu Leu Leu Ala Gln Cys Leu Ser Ser Ser 1855 1860 1865ctg ctg ctg gag aaa gaa gag aac aag agg ttt gaa gat cag ctt 5686Leu Leu Leu Glu Lys Glu Glu Asn Lys Arg Phe Glu Asp Gln Leu 1870 1875 1880cag caa tgg ttg tct gaa gac tca gga gca ttt acg gat tta act 5731Gln Gln Trp Leu Ser Glu Asp Ser Gly Ala Phe Thr Asp Leu Thr 1885 1890 1895tcc ctt ccc ctc tat ctt cct cag act cta gtg tct ctt tct cac 5776Ser Leu Pro Leu Tyr Leu Pro Gln Thr Leu Val Ser Leu Ser His 1900 1905 1910act att gaa cct gtg atg aaa aca tct gta act act agc cca cag 5821Thr Ile Glu Pro Val Met Lys Thr Ser Val Thr Thr Ser Pro Gln 1915 1920 1925agt gac atg atg agg gag caa ctg cag ctg tca gag gcg aca gga 5866Ser Asp Met Met Arg Glu Gln Leu Gln Leu Ser Glu Ala Thr Gly 1930 1935 1940acg tgt cta ggc gaa cga cta aag cac ctg gaa agg ctg atc cgg 5911Thr Cys Leu Gly Glu Arg Leu Lys His Leu Glu Arg Leu Ile Arg 1945 1950 1955agt tca agg gaa gag gaa gtt gcc tct gag ctc cat ctc tct gcg 5956Ser Ser Arg Glu Glu Glu Val Ala Ser Glu Leu His Leu Ser Ala 1960 1965 1970ctg cta gac atg gtg gac att tgagcagcct gacctgtggg gagggggtct 6007Leu Leu Asp Met Val Asp Ile 1975 1980ctcccgaaga gtttctgttt ttactcaaaa taatgttatt ctcagatgct tgatgcactg 6067ttggaaatgt gattaattta atcatgcaga taaaccattt aaatgtc 611441980PRTHomo sapiens 4Met Asn Pro Thr Asn Pro Phe Ser Gly Gln Gln Pro Ser Ala Phe Ser1 5 10 15Ala Ser Ser Ser Asn Val Gly Thr Leu Pro Ser Lys Pro Pro Phe Arg 20 25 30Phe Gly Gln Pro Ser Leu Phe Gly Gln Asn Ser Thr Leu Ser Gly Lys 35 40 45Ser Ser Gly Phe Ser Gln Val Ser Ser Phe Pro Ala Ser Ser Gly Val 50 55 60Ser His Ser Ser Ser Val Gln Thr Leu Gly Phe Thr Gln Thr Ser Ser65 70 75 80Val Gly Pro Phe Ser Gly Leu Glu His Thr Ser Thr Phe Val Ala Thr 85 90 95Ser Gly Pro Ser Ser Ser Ser Val Leu Gly Asn Thr Gly Phe Ser Phe 100 105 110Lys Ser Pro Thr Ser Val Gly Ala Phe Pro Ser Thr Ser Ala Phe Gly 115 120 125Gln Glu Ala Gly Glu Ile Val Asn Ser Gly Phe Gly Lys Thr Glu Phe 130 135 140Ser Phe Lys Pro Leu Glu Asn Ala Val Phe Lys Pro Ile Leu Gly Ala145 150 155 160Glu Ser Glu Pro Glu Lys Thr Gln Ser Gln Ile Ala Ser Gly Phe Phe 165 170 175Thr Phe Ser His Pro Ile Ser Ser Ala Pro Gly Gly Leu Ala Pro Phe 180 185 190Ser Phe Pro Gln Val Thr Ser Ser Ser Ala Thr Thr Ser Asn Phe Thr 195 200 205Phe Ser Lys Pro Val Ser Ser Asn Asn Ser Leu Ser Ala Phe Thr Pro 210 215 220Ala Leu Ser Asn Gln Asn Val Glu Glu Glu Lys Arg Gly Pro Lys Ser225 230 235 240Ile Phe Gly Ser Ser Asn Asn Ser Phe Ser Ser Phe Pro Val Ser Ser 245 250 255Ala Val Leu Gly Glu Pro Phe Gln Ala Ser Lys Ala Gly Val Arg Gln 260 265 270Gly Cys Glu Glu Ala Val Ser Gln Val Glu Pro Leu Pro Ser Leu Met 275 280 285Lys Gly Leu Lys Arg Lys Glu Asp Gln Asp Arg Ser Pro Arg Arg His 290 295 300Gly His Glu Pro Ala Glu Asp Ser Asp Pro Leu Ser Arg Gly Asp His305 310 315 320Pro Pro Asp Lys Arg Pro Val Arg Leu Asn Arg Pro Arg Gly Gly Thr 325 330 335Leu Phe Gly Arg Thr Ile Gln Asp Val Phe Lys Ser Asn Lys Glu Val 340 345 350Gly Arg Leu Gly Asn Lys Glu Ala Lys Lys Glu Thr Gly Phe Val Glu 355 360 365Ser Ala Glu Ser Asp His Met Ala Ile Pro Gly Gly Asn Gln Ser Val 370 375 380Leu Ala Pro Ser Arg Ile Pro Gly Val Asn Lys Glu Glu Glu Thr Glu385 390 395 400Ser Arg Glu Lys Lys Glu Asp Ser Leu Arg Gly Thr Pro Ala Arg Gln 405 410 415Ser Asn Arg Ser Glu Ser Thr Asp Ser Leu Gly Gly Leu Ser Pro Ser 420 425 430Glu Val Thr Ala Ile Gln Cys Lys Asn Ile Pro Asp Tyr Leu Asn Asp 435 440 445Arg Thr Ile Leu Glu Asn His Phe Gly Lys Ile Ala Lys Val Gln Arg 450 455 460Ile Phe Thr Arg Arg Ser Lys Lys Leu Ala Val Val His Phe Phe Asp465 470 475 480His Ala Ser Ala Ala Leu Ala Arg Lys Lys Gly Lys Ser Leu His Lys 485 490 495Asp Met Ala Ile Phe Trp His Arg Lys Lys Ile Ser Pro Asn Lys Lys 500 505 510Pro Phe Ser Leu Lys Glu Lys Lys Pro Gly Asp Gly Glu Val Ser Pro 515 520 525Ser Thr Glu Asp Ala Pro Phe Gln His Ser Pro Leu Gly Lys Ala Ala 530 535 540Gly Arg Thr Gly Ala Ser Ser Leu Leu Asn Lys Ser Ser Pro Val Lys545 550 555 560Lys Pro Ser Leu Leu Lys Ala His Gln Phe Glu Gly Asp Ser Phe Asp 565 570 575Ser Ala Ser Glu Gly Ser Glu Gly Leu Gly Pro Cys Val Leu Ser Leu 580 585 590Ser Thr Leu Ile Gly Thr Val Ala Glu Thr Ser Lys Glu Lys Tyr Arg 595 600 605Leu Leu Asp Gln Arg Asp Arg Ile Met Arg Gln Ala Arg Val Lys Arg 610 615 620Thr Asp Leu Asp Lys Ala Arg Thr Phe Val Gly Thr Cys Leu Asp Met625 630 635 640Cys Pro Glu Lys Glu Arg Tyr Met Arg Glu Thr Arg Ser Gln Leu Ser 645 650 655Val Phe Glu Val Val Pro Gly Thr Asp Gln Val Asp His Ala Ala Ala 660 665 670Val Lys Glu Tyr Ser Arg Ser Ser Ala Asp Gln Glu Glu Pro Leu Pro 675 680 685His Glu Leu Arg Pro Leu Pro Val Leu Ser Arg Thr Met Asp Tyr Leu 690 695 700Val Thr Gln Ile Met Asp Gln Lys Glu Gly Ser Leu Arg Asp Trp Tyr705 710 715 720Asp Phe Val Trp Asn Arg Thr Arg Gly Ile Arg Lys Asp Ile Thr Gln 725 730 735Gln His Leu Cys Asp Pro Leu Thr Val Ser Leu Ile Glu Lys Cys Thr 740 745 750Arg Phe His Ile His Cys Ala His Phe Met Cys Glu Glu Pro Met Ser 755 760 765Ser Phe Asp Ala Lys Ile Asn Asn Glu Asn Met Thr Lys Cys Leu Gln 770 775 780Ser Leu Lys Glu Met Tyr Gln Asp Leu Arg Asn Lys Gly Val Phe Cys785 790 795 800Ala Ser Glu Ala Glu Phe Gln Gly Tyr Asn Val Leu Leu Ser Leu Asn 805 810 815Lys Gly Asp Ile Leu Arg Glu Val Gln Gln Phe His Pro Ala Val Arg 820 825 830Asn Ser Ser Glu Val Lys Phe Ala Val Gln Ala Phe Ala Ala Leu Asn 835 840 845Ser Asn Asn Phe Val Arg Phe Phe Lys Leu Val Gln Ser Ala Ser Tyr 850 855 860Leu Asn Ala Cys Leu Leu His Cys Tyr Phe Ser Gln Ile Arg Lys Asp865 870 875 880Ala Leu Arg Ala Leu Asn Phe Ala Tyr Thr Val Ser Thr Gln Arg Ser 885 890 895Thr Ile Phe Pro Leu Asp Gly Val Val Arg Met Leu Leu Phe Arg Asp 900 905 910Cys Glu Glu Ala Thr Asp Phe Leu Thr Cys His Gly Leu Thr Val Ser 915 920 925Asp Gly Cys Val Glu Leu Asn Arg Ser Ala Phe Leu Glu Pro Glu Gly 930 935 940Leu Ser Lys Thr Arg Lys Ser Val Phe Ile Thr Arg Lys Leu Thr Val945 950 955 960Ser Val Gly Glu Ile Val Asn Gly Gly Pro Leu Pro Pro Val Pro Arg 965 970 975His Thr Pro Val Cys Ser Phe Asn Ser Gln Asn Lys Tyr Ile Gly Glu 980 985 990Ser Leu Ala Ala Glu Leu Pro Val Ser Thr Gln Arg Pro Gly Ser Asp 995 1000 1005Thr Val Gly Gly Gly Arg Gly Glu Glu Cys Gly Val Glu Pro Asp 1010 1015 1020Ala Pro Leu Ser Ser Leu Pro Gln Ser Leu Pro Ala Pro Ala Pro 1025 1030 1035Ser Pro Val Pro Leu Pro Pro Val Leu Ala Leu Thr Pro Ser Val 1040 1045 1050Ala Pro Ser Leu Phe Gln Leu Ser Val Gln Pro Glu Pro Pro Pro 1055 1060 1065Pro Glu Pro Val Pro Met Tyr Ser Asp Glu Asp Leu Ala Gln Val 1070 1075 1080Val Asp Glu Leu Ile Gln Glu Ala Leu Gln Arg Asp Cys Glu Glu 1085 1090 1095Val Gly Ser Ala Gly Ala Ala Tyr Ala Ala Ala Ala Leu Gly Val 1100 1105 1110Ser Asn Ala Ala Met Glu Asp Leu Leu Thr Ala Ala Thr Thr Gly 1115 1120 1125Ile Leu Arg His Ile Ala Ala Glu Glu Val Ser Lys Glu Arg Glu 1130 1135 1140Arg Arg Glu Gln Glu Arg Gln Arg Ala Glu Glu Glu Arg Leu Lys 1145 1150 1155Gln Glu Arg Glu Leu Val Leu Ser Glu Leu Ser Gln Gly Leu Ala 1160 1165 1170Val Glu Leu Met Glu Arg Val Met Met Glu Phe Val Arg Glu Thr 1175 1180 1185Cys Ser Gln Glu Leu Lys Asn Ala Val Glu Thr Asp Gln Arg Val 1190 1195 1200Arg Val Ala Arg Cys Cys Glu Asp Val Cys Ala His Leu Val Asp 1205 1210 1215Leu Phe Leu Val Glu Glu Ile Phe Gln Thr Ala Lys Glu Thr Leu 1220 1225 1230Gln Glu Leu Gln Cys Phe Cys Lys Tyr Leu Gln Arg Trp Arg Glu 1235 1240 1245Ala Val Thr Ala

Arg Lys Lys Leu Arg Arg Gln Met Arg Ala Phe 1250 1255 1260Pro Ala Ala Pro Cys Cys Val Asp Val Ser Asp Arg Leu Arg Ala 1265 1270 1275Leu Ala Pro Ser Ala Glu Cys Pro Ile Ala Glu Glu Asn Leu Ala 1280 1285 1290Arg Gly Leu Leu Asp Leu Gly His Ala Gly Arg Leu Gly Ile Ser 1295 1300 1305Cys Thr Arg Leu Arg Arg Leu Arg Asn Lys Thr Ala His Gln Met 1310 1315 1320Lys Val Gln His Phe Tyr Gln Gln Leu Leu Ser Asp Val Ala Trp 1325 1330 1335Ala Ser Leu Asp Leu Pro Ser Leu Val Ala Glu His Leu Pro Gly 1340 1345 1350Arg Gln Glu His Val Phe Trp Lys Leu Val Leu Val Leu Pro Asp 1355 1360 1365Val Glu Glu Gln Ser Pro Glu Ser Cys Gly Arg Ile Leu Ala Asn 1370 1375 1380Trp Leu Lys Val Lys Phe Met Gly Asp Glu Gly Ser Val Asp Asp 1385 1390 1395Thr Ser Ser Asp Ala Gly Gly Ile Gln Thr Leu Ser Leu Phe Asn 1400 1405 1410Ser Leu Ser Ser Lys Gly Asp Gln Met Ile Ser Val Asn Val Cys 1415 1420 1425Ile Lys Val Ala His Gly Ala Leu Ser Asp Gly Ala Ile Asp Ala 1430 1435 1440Val Glu Thr Gln Lys Asp Leu Leu Gly Ala Ser Gly Leu Met Leu 1445 1450 1455Leu Leu Pro Pro Lys Met Lys Ser Glu Asp Met Ala Glu Glu Asp 1460 1465 1470Val Tyr Trp Leu Ser Ala Leu Leu Gln Leu Lys Gln Leu Leu Gln 1475 1480 1485Ala Lys Pro Phe Gln Pro Ala Leu Pro Leu Val Val Leu Val Pro 1490 1495 1500Ser Pro Gly Gly Asp Ala Val Glu Lys Glu Val Glu Asp Gly Leu 1505 1510 1515Met Leu Gln Asp Leu Val Ser Ala Lys Leu Ile Ser Asp Tyr Thr 1520 1525 1530Val Thr Glu Ile Pro Asp Thr Ile Asn Asp Leu Gln Gly Ser Thr 1535 1540 1545Lys Val Leu Gln Ala Val Gln Trp Leu Val Ser His Cys Pro His 1550 1555 1560Ser Leu Asp Leu Cys Cys Gln Thr Leu Ile Gln Tyr Val Glu Asp 1565 1570 1575Gly Ile Gly His Glu Phe Ser Gly Arg Phe Phe His Asp Arg Arg 1580 1585 1590Glu Arg Arg Leu Gly Gly Leu Ala Ser Gln Glu Pro Gly Ala Ile 1595 1600 1605Ile Glu Leu Phe Asn Ser Val Leu Gln Phe Leu Ala Ser Val Val 1610 1615 1620Ser Ser Glu Gln Leu Cys Asp Leu Ser Trp Pro Val Thr Glu Phe 1625 1630 1635Ala Glu Ala Gly Gly Ser Arg Leu Leu Pro His Leu His Trp Asn 1640 1645 1650Ala Pro Glu His Leu Ala Trp Leu Lys Gln Ala Val Leu Gly Phe 1655 1660 1665Gln Leu Pro Gln Met Asp Leu Pro Pro Leu Gly Ala Pro Trp Leu 1670 1675 1680Pro Val Cys Ser Met Val Val Gln Tyr Ala Ser Gln Ile Pro Ser 1685 1690 1695Ser Arg Gln Thr Gln Pro Val Leu Gln Ser Gln Val Glu Asn Leu 1700 1705 1710Leu His Arg Thr Tyr Cys Arg Trp Lys Ser Lys Ser Pro Ser Pro 1715 1720 1725Val His Gly Ala Gly Pro Ser Val Met Glu Ile Pro Trp Asp Asp 1730 1735 1740Leu Ile Ala Leu Cys Ile Asn His Lys Leu Arg Asp Trp Thr Pro 1745 1750 1755Pro Arg Leu Pro Val Thr Ser Glu Ala Leu Ser Glu Asp Gly Gln 1760 1765 1770Ile Cys Val Tyr Phe Phe Lys Asn Asp Leu Lys Lys Tyr Asp Val 1775 1780 1785Pro Leu Ser Trp Glu Gln Ala Arg Leu Gln Thr Gln Lys Glu Leu 1790 1795 1800Gln Leu Arg Glu Gly Arg Leu Ala Ile Lys Pro Phe His Pro Ser 1805 1810 1815Ala Asn Asn Phe Pro Ile Pro Leu Leu His Met His Arg Asn Trp 1820 1825 1830Lys Arg Ser Thr Glu Cys Ala Gln Glu Gly Arg Ile Pro Ser Thr 1835 1840 1845Glu Asp Leu Met Arg Gly Ala Ser Ala Glu Glu Leu Leu Ala Gln 1850 1855 1860Cys Leu Ser Ser Ser Leu Leu Leu Glu Lys Glu Glu Asn Lys Arg 1865 1870 1875Phe Glu Asp Gln Leu Gln Gln Trp Leu Ser Glu Asp Ser Gly Ala 1880 1885 1890Phe Thr Asp Leu Thr Ser Leu Pro Leu Tyr Leu Pro Gln Thr Leu 1895 1900 1905Val Ser Leu Ser His Thr Ile Glu Pro Val Met Lys Thr Ser Val 1910 1915 1920Thr Thr Ser Pro Gln Ser Asp Met Met Arg Glu Gln Leu Gln Leu 1925 1930 1935Ser Glu Ala Thr Gly Thr Cys Leu Gly Glu Arg Leu Lys His Leu 1940 1945 1950Glu Arg Leu Ile Arg Ser Ser Arg Glu Glu Glu Val Ala Ser Glu 1955 1960 1965Leu His Leu Ser Ala Leu Leu Asp Met Val Asp Ile 1970 1975 1980533DNAArtificial sequenceLVH3 Primer 5ctataaccat ggaccatgga catactttgt tcc 33636DNAArtificial sequenceXbaI-CH1-Cu Primer 6tgcatgcatt ctagagttgc cgttggggtg ctggac 36720DNAArtificial sequencePrimer used in the preparation of GANP-Transgenic (Tg) Mouse 7tcccgccttc cagctgtgac 20820DNAArtificial sequencePrimer used in the preparation of GANP-Transgenic (Tg) Mouse 8gtgctgctgt gttatgtcct 20930DNAArtificial sequenceneo2 Primer 9gcctgcttgc cgaatatcat ggtggaaaat 301028DNAArtificial sequenceCGK3'-2 Primer 10ggcaccaagc atgcacggag tacacaga 281126DNAArtificial sequenceGANP1-5' Primer 11ggggatccat acccggtgaa cccctt 261228DNAArtificial sequenceGANP1-3' Primer 12gggtcgacgc gcacagactt tcccctga 281328DNAArtificial sequenceGANP2-5' Primer 13gggaattctc ccgccttcca gctgtgac 281428DNAArtificial sequenceGANP2-3' Primer 14gggtcgacgt gctgctgtgt tatgtcct 281528DNAArtificial sequenceGANP3-5' Primer 15gggaattcca tgagctgaga ccctcagc 281628DNAArtificial sequenceGANP3-3' Primer 16gggtcgactg aggatgcagg aggcggct 281728DNAArtificial sequenceGANP4-5' Primer 17gggaattcta cgttggagag agcctggc 281828DNAArtificial sequenceGANP4-3' Primer 18gggtcgacca tgctgtcatc tcctgtga 281928DNAArtificial sequenceGANP5-5'Primer 19gggaattcga gaacctggcc aagggtct 282028DNAArtificial sequenceGANP5-3'Primer 20gggtcgacga aaaaccgacg gctgaact 282128DNAArtificial sequenceGANP6-5' Primer 21gggaattcaa gcccttccag cctgccct 282228DNAArtificial sequenceGANP6-3' Primer 22gggtcgaccg agggaacgtg gtattttc 282328DNAArtificial sequenceGANP7-5' Primer 23ggcccgggcc cgtgggatga catcatca 282428DNAArtificial sequenceGANP7-3' Primer 24ggctcgagca tgtccaccat ctccagca 282541DNAArtificial sequenceGp-gfp-5' Primer 25ggggatccga attccaccat ggcagtcttc aaaccgatac c 412620DNAArtificial sequenceGp-gfp-3' Primer 26gcaggggctc ctcctgatct 202741DNAArtificial sequenceGsac-gfp-5' Primer 27ggggatccga attccaccat gtccgagggc cttggttctt g 412820DNAArtificial sequenceGsac-gfp-3' Primer 28ctgtcttgtt tctaagccgc 202941DNAArtificial sequenceGmap80-gfp-5' Primer 29ggggatccga attccaccat ggagaacctg gccaagggtc t 413027DNAArtificial sequenceGmap80-gfp-3' Primer 30gaggacttgt agatgttttc accatgg 273158DNAArtificial sequenceFLAG-Gp-5' Primer 31gggaattcca ccatggatta caaggatgac gacgataagg cagtcttcaa ccgatacc 583228DNAArtificial sequenceFLAG-Gp-3' Primer 32gggaattcct ccgggtctcc ctcaagta 283359DNAArtificial sequenceFLAG-Gsac-5' Primer 33gggaattcca ccatggatta caaggatgac gacgataagt ccgagggcct tggttcttg 593428DNAArtificial sequenceFLAGGsac-3' Primer 34gggaattcgc tgtcttgttt ctaagccg 283559DNAArtificial sequenceFLAG-Gmap-5' Primer 35gggaattcca ccatggatta caaggatgac gacgataagg agaacctggc caagggtct 593628DNAArtificial sequenceFLAG-Gmap-3' Primer 36gggaattctg aggacttgta gatgtttt 283798PRTMus musculusmisc_feature(1)..(98)Fig. 10 WT 37Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg38294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT 38caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29439294DNAMus musculusmisc_feature(1)..(39)Fig. 10 WT-4 39caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc gagt 29440294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-5 40caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc atctacttaa tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaact tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29441294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-6 41caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agttacttga tgcactgggt gaagcagggg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc gagt 29442294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-9 42caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaaga cttctggcta ctccttcacc agctacttta tacactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaaat tcaagagcag ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29443294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-10 43caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactggat gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaggtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29444294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-11 44caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtaggca aaccctccag cacagcctac 240atgcggctca gcagcctgac atctgaggac tctgcggtct attattgtgc acgt 29445294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-14 45caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgttgaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29446294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-16 46caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc aactacttga tgcactgggt gaagcagagg 120cctggacgag gccttgggtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc gagt 29447294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-17 47caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg ttctaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcacctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29448294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-18 48caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcgatct attattgtgc aaga 29449294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-19 49caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgttccta atagtggtga tactaagtac 180aatgagaagt tcaagaacaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29450294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-20 50caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtta tactaggtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctcc 240atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aaga 29451294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-21 51caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aagg 29452294DNAMus musculusmisc_feature(1)..(294)Fig. 10 WT-22 52caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaggg attgatccta atagtggtta tactaggtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctcc 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 2945398PRTMus musculusmisc_feature(1)..(98)Fig. 10 TG 53Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg54294DNAMus musculusmisc_feature(1)..(294)Fig. 10 TG 54caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29455294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-3 55caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctacctga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atcgtggtgg tactaagtac 180aatgagaagt

tcattaacaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29456294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-4 56caggtccaac tgcagcagcc tggggctgaa cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc aactacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29457294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-5 57caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac acctgaggac tctgcggtct attattgtgc aaga 29458294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-7 58caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcaccgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca gaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29459294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-8 59caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctgggtcttc agtgaagctg 60tcctgcaagc cttctggcta caccttcacc acctactgga tacactgggt gaggcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29460294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-9 60caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29461294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-10 61caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcaac agttactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aatcctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29462294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-11 62caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccttccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29463294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-12 63caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29464294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-13 64caggtccaac tgcagcagcc tggggctgag cttgtgaagt ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctacttga tgcactgggt gaagcagtgg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagaacaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgtc atctgaggac tctgcggtct attattgtgc aaga 29465294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-14 65caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaaag cttctggcta caccttcacc agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccca atagtggtgg tactaagtac 180aatgagaagt tcaggagcag ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29466294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-15 66caggtccgac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc ggctactgga tggactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg atcgatccta atagtggtgg cactaagtac 180aaagagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29467294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-16 67caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcaat agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagaacaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcacctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29468294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-17 68caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta attctggtgg tactaagtac 180aatgagaagt tcaagaccaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29469294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-18 69caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta cattttcacc agctacctga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atcgtggtgg tactaagtac 180aatgagaagt tcattaacaa ggccacactg actgtagaca aaccctccac cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29470294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-20 70caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaggtac 180aatgagaggt tcaagagcaa ggccacactg tctgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29471294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-21 71caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29472294DNAMus musculusmisc_feature(1)..(294)Fig. 10 Tg-23 72caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atactggtgg tactaagtac 180gatgagaagt tcaagaccaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagtctgac atctgaggac tctgcggtct attattgtgc aaga 2947398PRTMus musculusmisc_feature(1)..(98)Fig. 20A-20F Cre-flox/+ 73Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg74294DNAMus musculusmisc_feature(1)..(294)Fig. 20A-20F Cre-flox/+ 74caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29475294DNAMus musculusmisc_feature(1)..(294)Fig. 20 1-5 75caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgattctg 60tcctgcaagg cttctgccta caccttcacc agttactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29476294DNAMus musculusmisc_feature(1)..(294)Fig. 20 1-6 76caggtccaac tgcagcagcc tggggctgaa cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29477294DNAMus musculusmisc_feature(1)..(294)Fig. 20 3-1 77caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctatagga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtga tacaaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29478294DNAMus musculusmisc_feature(1)..(294)Fig. 20 3-2 78caggtccaac tgcagcagcc tggggctgag cttgtgaggc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc acctacttga ttcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta tgagtggtgg cagtaggtac 180aatgagtact tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac actgcggtcg attattgtgc aaga 29479294DNAMus musculusmisc_feature(1)..(294)Fig. 20 3-3 79caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaacctg 60tcctgcaagg cttctggcta aattttcacc agctagtgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacattg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29480294DNAMus musculusmisc_feature(1)..(294)Fig. 20 4-2 80caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctgggggttc agtgaagctg 60tcctgcaagg cttctggtta caccctcacc acctacttaa tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actatagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aagg 29481294DNAMus musculusmisc_feature(1)..(294)Fig. 20 4-4 81caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctgggggttc agtgaagctg 60tcctgcaagg cttctggcta caccctcacc acctacttaa tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actatagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aagg 29482294DNAMus musculusmisc_feature(1)..(294)Fig. 20 4-6 82caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccctcacc acctacttaa tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actatagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aagg 29483294DNAMus musculusmisc_feature(1)..(294)Fig. 20 1-8 83caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc acctacttga tacactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta aaagtggtgg tactaagtac 180agtgagaagt tcaagagcaa ggccacactg actgtagacc aaccctccag cacagcctac 240atgcagttca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29484294DNAMus musculusmisc_feature(1)..(294)Fig. 20 1-10 84caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc acctacttga ttcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg gttgatccta atactggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29485294DNAMus musculusmisc_feature(1)..(294)Fig. 20 4-7 85caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctgggggttc agtgaagctg 60tcctgcaagg cttctggcta caccctcacc acctacttaa tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtga tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca acagcctgac atctgaggac tctgcggtct attattgtgc aaga 29486294DNAMus musculusmisc_feature(1)..(294)Fig. 20 6-1 86caggtccaac tgcagcagcc tgggactgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggctt caccttcacc agctacttga tgcactgggt gaaacagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagacgt tcaagaacaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29487294DNAMus musculusmisc_feature(1)..(294)Fig. 20 6-2 87caggtccaac tgcagcagcc tggggctgag ctagtgaagc ctggggcttc agtgaaggtg 60tcctgcaagg cttctggcta caccttcacc agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtag tactaagtac 180aatgagaagt tcaagaccaa ggccacactg actgtagaca aaccctccag tacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29488294DNAMus musculusmisc_feature(1)..(294)Fig. 20 7-1 88caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc aactacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagacgt tcaagaacaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 2948998PRTMus musculusmisc_feature(1)..(98)Fig.20G-20L B-Ganp-/- 89Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg90294DNAMus musculusmisc_feature(1)..(98)Fig.20G-20L B-Ganp-/- 90caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29491294DNAMus musculusmisc_feature(1)..(294)Fig. 20 1-1 91caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagtaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc acga 29492294DNAMus musculusmisc_feature(1)..(294)Fig. 20 1-5 92caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgattctg 60tcctgcaagg cttctgccta caccttcacc agttactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29493294DNAMus musculusmisc_feature(1)..(294)Fig. 20 1-6 93caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcaactca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29494294DNAMus musculusmisc_feature(1)..(294)Fig. 20 2-2 94caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttcgggcta caccttcacc aactattgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg ttctaagtac 180aatgagaagt tcaagagcaa ggccacactg actgcagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aaga 29495294DNAMus musculusmisc_feature(1)..(294)Fig. 20 2-3 95caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagt attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagaacaa ggccacacta actgtggaca aaccctccag cacagcctac 240atgcagctca gcagcctgac

atctgaggac tctgcggtct attattgtgc aaga 29496294DNAMus musculusmisc_feature(1)..(294)Fig. 20 4-3 96caggtccaac tgcagcagcc tgggactgaa ctggtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaaat attaatccta atagtggtgg tactaactac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aatcctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aaga 29497294DNAMus musculusmisc_feature(1)..(294)Fig. 20 4-4 97caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atggtggtgg tactaagtac 180aatgagaagt tcaagaccaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 29498294DNAMus musculusmisc_feature(1)..(294)Fig. 20 6-1 98caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta cactttaacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtat 180aatgaggagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct actattgtgc aaga 29499294DNAMus musculusmisc_feature(1)..(294)Fig. 20 6-2 99caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta cactttaacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtat 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct actattgtgc aaga 294100294DNAMus musculusmisc_feature(1)..(294)Fig. 20 7-1 100caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctgctgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atggtggtgg tactaagttc 180gatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctat 240atgcaactca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 294101294DNAMus musculusmisc_feature(1)..(294)Fig. 20 8-1 101caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc aactacttga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 294102294DNAMus musculusmisc_feature(1)..(294)Fig. 20 8-2 102caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atggtggtgg tactaaatac 180aatgagaggt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagttca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 294103294DNAMus musculusmisc_feature(1)..(294)Fig. 20 9-1 103caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc aactactgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tgccaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccttccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 294104294DNAMus musculusmisc_feature(1)..(294)Fig. 20 9-3 104caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaaga cttctggcta caccttcacc acctactggc tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattgggagg attgatccta atagtggcgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 294105294DNAMus musculusmisc_feature(1)..(294)Fig. 20 9-4 105caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc agctattgga tgcactgggt gaagcagagg 120cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca aatcctccag cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aaga 294

Patents by BIRCH STEWART KOLASCH & BIRCH

Patents in class IMMUNOGLOBULIN, ANTISERUM, ANTIBODY, OR ANTIBODY FRAGMENT, EXCEPT CONJUGATE OR COMPLEX OF THE SAME WITH NONIMMUNOGLOBULIN MATERIAL

Patents in all subclasses IMMUNOGLOBULIN, ANTISERUM, ANTIBODY, OR ANTIBODY FRAGMENT, EXCEPT CONJUGATE OR COMPLEX OF THE SAME WITH NONIMMUNOGLOBULIN MATERIAL

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Patent application title: Transgenic mammal carrying GANP gene transferred thereinto and utilization thereof

Inventors: Nobuo Sakaguchi
Agents: BIRCH STEWART KOLASCH & BIRCH
Assignees: Immunokick Incorporation
Origin: FALLS CHURCH, VA US
IPC8 Class: AA61K39395FI
USPC Class: 4241301

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Patent application title: Transgenic mammal carrying GANP gene transferred thereinto and utilization thereof

Inventors: Nobuo Sakaguchi Agents: BIRCH STEWART KOLASCH & BIRCH Assignees: Immunokick Incorporation Origin: FALLS CHURCH, VA US IPC8 Class: AA61K39395FI USPC Class: 4241301

Abstract:

Claims:

Description:

Inventors: Nobuo Sakaguchi
Agents: BIRCH STEWART KOLASCH & BIRCH
Assignees: Immunokick Incorporation
Origin: FALLS CHURCH, VA US
IPC8 Class: AA61K39395FI
USPC Class: 4241301