Patent application title: TICK CHITINASE
Inventors:
Kozo Fujisaki (Hokkaido, JP)
Hideyuki Nagasawa (Hokkaido, JP)
Ikuo Igarashi (Hokkaido, JP)
Hiroshi Suzuki (Hokkaido, JP)
Chihiro Sugimoto (Hokkaido, JP)
Gakunan Gen (Hokkaido, JP)
Myonjo Yu (Hokkaido, JP)
Naotoshi Tsuji (Ibaraki, JP)
Assignees:
MEIJI SEIKA KAISHA, LTD.
IPC8 Class: AA61K3900FI
USPC Class:
4241851
Class name: Drug, bio-affecting and body treating compositions antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same
Publication date: 2009-11-05
Patent application number: 20090274715
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Patent application title: TICK CHITINASE
Inventors:
Chihiro Sugimoto
Kozo Fujisaki
Hideyuki Nagasawa
Ikuo Igarashi
Hiroshi Suzuki
Gakunan Gen
Myonjo Yu
Naotoshi Tsuji
Agents:
SUGHRUE MION, PLLC
Assignees:
Origin: WASHINGTON, DC US
IPC8 Class: AA61K3900FI
USPC Class:
4241851
Patent application number: 20090274715
Abstract:
A novel chitinase, a polynucleotide encoding the same, a vector and a
transformant comprising the polynucleotide, an antibody against the
chitinase, and a screening method for screening a substance capable of
modifying the chitinase, are disclosed. According to the chitinase,
polynucleotide, or vector, it is possible, for example, to exterminate
ticks, or to treat or prevent tick-borne infections such as
piroplasmosis, Q fever, or viral encephalitis.Claims:
1. A method for treating or preventing a tick-borne infection, comprising
administering to a subject in need thereof a polypeptide selected from
the group consisting of:(1) a polypeptide consisting of the amino acid
sequence of SEQ ID NO: 2;(2) a polypeptide comprising the amino acid
sequence of SEQ ID NO: 2 and exhibiting a chitinase activity;(3) a
polypeptide exhibiting a chitinase activity and comprising an amino acid
sequence in which one or plural amino acids are substituted, deleted,
and/or inserted at one or plural positions in the amino acid sequence of
SEQ ID NO: 2; and(4) a polypeptide comprising an amino acid sequence
having a 60% or more homology with the amino acid sequence of SEQ ID NO:
2, and exhibiting a chitinase activity,or a fragment of the polypeptide
in an amount effective therefor.
2. The method according to claim 1, wherein the fragment of the polypeptide is an immunogenic fragment of the polypeptide.
3. The method according to claim 1, wherein the polypeptide is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.
4. The method according to claim 1, wherein the polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and exhibiting a chitinase activity.
5. The method according to claim 1, wherein the polypeptide is a polypeptide comprising an amino acid sequence having a 70% or more homology with the amino acid sequence of SEQ ID NO: 2, and exhibiting a chitinase activity.
6. The method according to claim 1, wherein the polypeptide is a polypeptide comprising an amino acid sequence having a 80% or more homology with the amino acid sequence of SEQ ID NO: 2, and exhibiting a chitinase activity.
7. The method according to claim 1, wherein the polypeptide is a polypeptide comprising an amino acid sequence having a 90% or more homology with the amino acid sequence of SEQ ID NO: 2, and exhibiting a chitinase activity.
8. The method according to claim 1, wherein the polypeptide is a polypeptide comprising an amino acid sequence having a 95% or more homology with the amino acid sequence of SEQ ID NO: 2, and exhibiting a chitinase activity.
Description:
CROSS-REFERENCE TO RELATED APPLICATION
[0001]This application is a Divisional of U.S. application Ser. No. 10/506,010, filed Nov. 19, 2004 (now allowed); which is a 371 of PCT/JP03/02335, filed Feb. 28, 2003; the disclosure of each of which is incorporated herein by reference.
TECHNICAL FIELD
[0002]This invention relates to a tick chitinase.
BACKGROUND ART
[0003]Ticks are the cause, directly or indirectly, of extensive damage to animals or humans. Examples of the direct damage are pruritus or bleeding caused by biting or blood-sucking, or tick paralysis or allergic diseases caused by saliva secreted in blood-sucking or regurgitation of midgut contents. Examples of the indirect damage are various diseases in livestock, caused by viruses, rickettsiae, bacterium, spirochaeta, protozoa, nematoda, or the like. This damage causes enormous losses at home and abroad, and threat of emerging and re-emerging zoonotic diseases by ticks is becoming a serious problem.
[0004]Under these circumstances, various methods to exterminate ticks are used in many countries. Among these methods, the major one is the use of agents such as organic phosphorus agents, carbamate agents, pyrethroid or macrolide antibiotics, or the like. However, in any agent, a drug resistance is established by using the agent successively or heavily, and thus many agents lose their miticidal activity. Further, when using such agents, it is necessary to take side effects to animals into consideration. In addition, there is a problem of a remnant agent which may threaten the safety of foods and the environment, and people tend to avoid the use of such agents. Furthermore, the use of agents is approaching limitation, with respect to the enormous development cost, in addition to the effectiveness thereof and an applicable area. As described above, it is considered difficult to prevent the parasitism of ticks to humans or animals, and the damage caused by ticks-borne infection in the 21st century, by means of the use of agents.
[0005]In hematophagous arthropods including ticks, acquisition of protective immune response against reinfection in a host against a viral or bacterial infection is known and has been confirmed in the laboratory stage [Fujisaki, Nat. Inst. Anim. Hlth. Quart. (Tokyo), 18, 27-38 (1978)]. Due to the recent progress in gene recombination techniques, genes encoding protective antigens, enzymes related to metamorphosis specific to hematophagous arthropods, or the like are being intensively cloned in many countries, and an attempt to manufacture safe vaccine proteins or chemotherapeutic agents has been made.
[0006]However, such an agent in practical use is only that against Boophilus microplus, which was developed by Willadesen [Willadesen and Jogejan, Prasitology Today. 15, 258-262 (1999)]. There is now a search for a vaccine against Haemaphysalis longicornis, which is widely distributed over Asian countries including Japan and the Eurasia continent and mediates zoonotic diseases such as piroplasmosis, Q fever, or viral encephaliti, and thus the rapid development and practical application of such a vaccine is greatly desired.
DISCLOSURE OF INVENTION
[0007]The present inventors have conducted intensive studies into obtaining a novel polypeptide useful as a candidate for a vaccine against ticks, particularly Haemaphysalis longicornis, and a polynucleotide encoding the polypeptide and, as a result, found a novel chitinase and a polynucleotide encoding the same. Further, the present inventors inoculated the chitinase into mice, to observe the induction of an antibody production, and confirmed that the chitinase is useful as a tick vaccine. The present invention is based on the above findings.
[0008]The object of the present invention is to provide a novel chitinase useful as a tick vaccine, and a polynucleotide encoding the chitinase.
[0009]The object can be solved by a polypeptide of the present invention, i.e., (1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2;
(2) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and exhibiting a chitinase activity;(3) a polypeptide exhibiting a chitinase activity and comprising an amino acid sequence in which one or plural amino acids are substituted, deleted, and/or inserted at one or plural positions in the amino acid sequence of SEQ ID NO: 2; or(4) a polypeptide comprising an amino acid sequence having a 60% or more homology with the amino acid sequence of SEQ ID NO: 2, and exhibiting a chitinase activity.
[0010]The present invention relates to a polynucleotide encoding the polypeptide.
[0011]The present invention relates to a vector comprising the polynucleotide.
[0012]The present invention relates to a transformant comprising the polynucleotide.
[0013]The present invention relates to a process for producing the polypeptide, comprising the step of culturing the transformant.
[0014]The present invention relates to a medicament comprising the polypeptide or a fragment thereof, the polynucleotide, or the vector.
[0015]The present invention relates to a pharmaceutical composition comprising the polypeptide or a fragment thereof, the polynucleotide, or the vector, and a pharmaceutically or veterinary acceptable carrier or diluent.
[0016]The present invention relates to a method for exterminating ticks, comprising administering to a subject in need thereof the polypeptide or a fragment thereof, the polynucleotide, or the vector in an amount effective therefor.
[0017]The present invention relates to a method for treating or preventing a tick-borne infection, comprising administering to a subject in need thereof the polypeptide or a fragment thereof, the polynucleotide, or the vector in an amount effective therefor.
[0018]The present invention relates to an antibody or a fragment thereof, which binds to the polypeptide.
[0019]The present invention relates to a method for screening a substance capable of modifying a chitinase activity of the polypeptide, comprising the steps of:
bringing the polypeptide into contact with a substance to be tested; andanalyzing the chitinase activity of the polypeptide.
BRIEF DESCRIPTION OF DRAWINGS
[0020]FIG. 1 shows the result of SDS-polyacrylamide gel electrophoresis of a recombinant chitinase fusion protein.
[0021]FIG. 2 shows the result of electrophoresis of native (wild type) chitinase in a tick egg lysate by immunoblotting.
BEST MODE FOR CARRYING OUT THE INVENTION
[0022]The present invention will be explained in detail hereinafter.
[1] Polypeptide of the Present Invention
[0023]The polypeptides of the present invention includes
(1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2;(2) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and exhibiting a chitinase activity;(3) a polypeptide comprising an amino acid sequence in which one or plural amino acids are substituted, deleted, and/or inserted at one or plural positions in the amino acid sequence of SEQ ID NO: 2, and exhibiting a chitinase activity (hereinafter referred to as a variation functionally equivalent); and(4) a polypeptide comprising an amino acid sequence having a 60% or more homology with the amino acid sequence of SEQ ID NO: 2, and exhibiting a chitinase activity (hereinafter referred to as a homologous polypeptide).
[0024]The term "chitinase activity" as used herein means an enzyme activity in which chitin [poly(β-1,4-N-acetyl D-glucosamine)] is digested to generate oligosaccharides and N-acetylglucosamine. Whether or not a polypeptide to be tested exhibits the chitinase activity may be easily confirmed, for example, by a known method for measuring the chitinase activity, in which the polypeptide to be tested is brought into contact with a substrate of chitinase, and then the digestion and/or a degree thereof of the chitinase substrate is analyzed [for example, Johannes et al., Infect. Immun., 69, 4041-4047 (2001)]. The method is not particularly limited, but is preferably confirmed by a method described in Example 6.
[0025]More particularly, for example, a polypeptide to be tested is added to a well of an agarose gel containing an appropriate substrate of chitinase (for example, glycol chitin or chitin), and incubated for a predetermined period (for example, at 37° C. for 12 hours). The gel is stained with an appropriate dye [for example, Fluorescent Brightener 28 (Sigma)] and observed under an ultraviolet ray. The portion in which chitin is digested by chitinase does not react with the dye, and becomes black. In this case, it may be judged that the polypeptide to be tested exhibits the chitinase activity. Conversely, when the chitinase reaction does not occur, the gel is brightened by the reaction with the dye. In this case, it may be judged that the polypeptide to be tested does not exhibit the chitinase activity.
[0026]The "polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and exhibiting the chitinase activity" as the polypeptide of the present invention includes, for example, a fusion polypeptide consisting of an amino acid sequence in which an appropriate marker sequence or the like is added to the N-terminus and/or the C-terminus of the amino acid sequence of SEQ ID NO: 2, and exhibiting the chitinase activity; or a fusion polypeptide of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and a partner for fusion, and exhibiting the chitinase activity.
[0027]As the marker sequence, for example, a sequence for easily carrying out a confirmation of polypeptide expression, a confirmation of intracellular localization thereof, a purification thereof, or the like may be used. As the sequence, there may be mentioned, for example, a FLAG tag, a hexa-histidine tag, a hemagglutinin tag, a myc epitope, or the like.
[0028]As the partner for fusion, there may be mentioned, for example, a polypeptide for purification [for example, glutathione S-transferase (GST) or a fragment thereof], a polypeptide for detection [for example, hemagglutinin or β-galactosidase αpeptide (LacZ α), or a fragment thereof], a polypeptide for expression (for example, a signal sequence), or the like.
[0029]In the above fusion polypeptide, an amino acid sequence which can be specifically digested with a protease such as thrombin or factor Xa may be optionally inserted between the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and the marker sequence or the partner for fusion.
[0030]The variation functionally equivalent of the present invention is not particularly limited, so long as it is a polypeptide comprising an amino acid sequence in which one or plural (preferably 1 to 10, more preferably 1 to 7, most preferably 1 to 5) amino acids in total (for example, one to several amino acids in total) are deleted, substituted, and/or inserted at one or plural positions in the amino acid sequence of SEQ ID NO: 2, and exhibiting the chitinase activity. Further, an origin of the variation functionally equivalent is not limited to Haemaphysalis longicornis.
[0031]The variation functionally equivalent of the present invention includes not only Haemaphysalis longicornis variations of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, but also variations functionally equivalent derived from organisms other than Haemaphysalis longicornis [for example, Ixodids (hard ticks) other than Haemaphysalis longicornis, or Argasids (soft ticks)]. Further, it includes polypeptides prepared using polynucleotides obtained by artificially modifying their amino acid sequences encoded thereby by genetic engineering techniques, on the basis of polynucleotides encoding these native polypeptides (i.e., Haemaphysalis longicornis variations or variations functionally equivalent derived from organisms other than Haemaphysalis longicornis), or on the basis of polynucleotides encoding the amino acid sequence of SEQ ID NO: 2. The term "variation" as used herein means an individual difference between the same polypeptides in the same species or a difference between homologous polypeptides in several species.
[0032]Haemaphysalis longicornis variations of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or variations functionally equivalent derived from organisms other than Haemaphysalis longicornis may be obtained by those skilled in the art in accordance with the information of a base sequence (for example, the base sequence of the 571st to 3360th bases in the base sequence of SEQ ID NO: 1) of a polynucleotide encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. In this connection, genetic engineering techniques may be generally performed in accordance with known methods (for example, Sambrook et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1989), unless otherwise specified.
[0033]For example, an appropriate probe or appropriate primers are designed in accordance with the information of a base sequence of a polynucleotide encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. A polymerase chain reaction (PCR) method (Saiki, R. K. et al., Science, 239, 487-491, 1988) or a hybridization method is carried out using a sample (for example, total RNA or an mRNA fraction, a cDNA library, or a phage library) derived prepared from an organism [for example, Ixodids (hard ticks) other than Haemaphysalis longicornis, or Argasids (soft ticks)] of interest and the primers or the probe to obtain a polynucleotide encoding the polypeptide. A desired polypeptide may be obtained by expressing the resulting polynucleotide in an appropriate expression system and confirming that the expressed polypeptide exhibits the chitinase activity by, for example, the method described in Example 6.
[0034]Further, the polypeptide artificially modified by genetic engineering techniques may be obtained by, for example, the following procedure. A gene encoding the polypeptide is obtained by a conventional method such as site-specific directed mutagenesis (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA, 81, 5662-5666, 1984). A desired polypeptide may be obtained by expressing the resulting polynucleotide in an appropriate expression system and confirming that the expressed polypeptide exhibits the chitinase activity by, for example, the method described in Example 6.
[0035]The homologous polypeptide of the present invention is not particularly limited, so long as it is a polypeptide having an amino acid sequence having a 60% or more homology with the amino acid sequence of SEQ ID NO: 2, and exhibiting the chitinase activity. The homologous polypeptide of the present invention may have an amino acid sequence having preferably a 70% or more homology, more preferably a 80% or more homology, more preferably a 90% or more homology, more preferably a 95% or more homology, most preferably a 98% or more homology, with respect to the amino acid sequence of SEQ ID NO: 2. The term "homology" as used herein means a value obtained by a Clustal program (Higgins and Sharp, Gene, 73, 237-244, 1988; and Thompson et al., Nucleic Acid Res., 22, 4673-4680, 1994) in accordance with a default parameter.
[0036]The above-mentioned novel polypeptide of the present invention may be manufactured by various known methods, for example, known genetic engineering techniques using the polynucleotide of the present invention which encodes the polypeptide of the present invention. More particularly, the polypeptide of the present invention may be prepared by culturing the transformant of the present invention described below (i.e., the transformant comprising the polynucleotide of the present invention) under conditions in which the novel polypeptide of the present invention can be expressed, and then separating and purifying the desired polypeptide from the resulting culture in accordance with a conventional method for a polypeptide separation and purification. As the separation and purification method, there may be mentioned, for example, salting-out with ammonium sulfate, an ion exchange column chromatography using ion exchange cellulose, a molecular sieve column chromatography using molecular sieve gel, an affinity column chromatography using protein A agarose, dialysis, lyophilization, or the like.
[0037]The present invention includes a fragment of the polypeptide of the present invention. The fragment of the present invention is useful as an active ingredient for the medicament of the present invention or as an antigen for preparing the antibody of the present invention.
[2] Polynucleotide of the Present Invention
[0038]The polynucleotide of the present invention is not particularly limited, so long as it encodes the polypeptide of the present invention. As the polynucleotide of the present invention, there may be mentioned, for example, a polynucleotide consisting of the 571st to 3360th bases in the base sequence of the SEQ ID NO: 1. In this connection, the term "polynucleotide" as used herein includes both DNA and RNA.
[0039]The present invention includes a polynucleotide comprising a base sequence which can hybridize with the polynucleotide of the present invention, preferably a polynucleotide consisting of a base sequence which can hybridize with the polynucleotide of the present invention. The base sequence capable of hybridizing with the polynucleotide of the present invention is preferably a base sequence complementary to the base sequence (or a partial sequence thereof) of the polynucleotide of the present invention, more preferably a base sequence complementary to the base sequence (or a partial sequence thereof) consisting of the 571st to 3360th bases in the base sequence of the SEQ ID NO: 1.
[3] Vector and Transformant of the Present Invention
[0040]The vector of the present invention is not particularly limited, so long as it comprises the polynucleotide of the present invention. As the vector, there may be mentioned, for example, a vector obtained by introducing the polynucleotide of the present invention into a known expression vector appropriately selected in accordance with a host cell to be used.
[0041]The transformant of the present invention is not particularly limited, so long as it comprises the polynucleotide of the present invention. The transformant of the present invention may be, for example, a cell in which the polynucleotide is integrated into a chromosome of a host cell, or a transformant containing the polynucleotide as a vector comprising polynucleotide. Further, the transformant of the present invention may be a transformant expressing the polypeptide of the present invention, or a transformant not expressing the polypeptide of the present invention. The transformant of the present invention may be obtained by, for example, transfecting a desired host cell with the vector of the present invention or the polynucleotide of the present invention per se.
[0042]The host cell may be, for example, a known microorganism usually used, for example, an Escherichia coli or yeast (Saccharomyces cerevisiae), or a known cultivated cell, such as an animal cell, such as a CHO cell, an HEK-293 cell, or a COS cell, or an insect cell such as a BmN4 cell.
[0043]The known expression vector may be, for example, pUC, pTV, pGEX, pKK, or pTrcHis for an Escherichia coli; pEMBLY or pYES2 for the yeast; pcDNA3 or pMAMneo for the CHO cell; pcDNA3 for the HEK-293 cell; pcDNA3 for the COS cell; a vector (such as pBK283) containing a polyhedrin promoter of a silkworm nucleopolyhederovirus (BmNPV) for the BmN4 cell. Further, the expression vector includes a virus vector which can be used as a vector for a gene therapy, such as retrovirus, adenovirus, or Sendai virus.
[4] Medicament of the Present Invention
[0044]The medicament of the present invention (preferably a tick vaccine) comprises, as a active ingredient, the polypeptide of the present invention or a fragment thereof, the polynucleotide of the present invention, or the vector of the present invention. In the present invention, the polypeptide of the present invention or a fragment thereof, the polynucleotide of the present invention, or the vector of the present invention can be orally or parenterally administered alone, or preferably together with a pharmaceutically or veterinarily acceptable carrier or diluent, to an animal (preferably a mammal, particularly a human) in need of an extermination of ticks.
[0045]When the active ingredient in the medicament of the present invention (i.e., the polypeptide of the present invention or a fragment thereof, the polynucleotide of the present invention, or the vector of the present invention) is administered to an animal as a tick vaccine, an antibody production may be induced and then ticks may be terminated by protective immune response against reinfection in the host animal. Further, as a result, it is possible to treat or prevent tick-borne infections such as piroplasmosis, Q fever, or viral encephalitis.
[0046]In other words, the pharmaceutical composition (preferably pharmaceutical composition for exterminating ticks or pharmaceutical composition for treating or preventing a tick-borne infection) of the present invention comprises the polypeptide of the present invention or a fragment thereof, the polynucleotide of the present invention, or the vector of the present invention as the active ingredient, and a pharmaceutically or veterinary acceptable carrier or diluent. The active ingredient in the present invention (i.e., the polypeptide of the present invention or a fragment thereof, the polynucleotide of the present invention, or the vector of the present invention) can be used in the manufacture of the above medicament (preferably medicament for exterminating ticks or medicament for treating or preventing a tick-borne infection).
[0047]When the medicament of the present invention is used as a tick vaccine, the fragment of the polypeptide of the present invention is not particularly limited, so long as the fragment administered to a subject can induce immunity thereagainst. The fragment can be appropriately selected by those skilled in the art.
[0048]The medicament (particularly the tick vaccine) of the present invention can be used, for example, by mixing the polypeptide of the present invention with an adjuvant or the like and inoculating the resulting mixture into an animal (for example, livestock) at an appropriate interval as a tick vaccine. Further, it can be used by dissolving or suspending the polypeptide of the present invention directly in an appropriate solvent, or by enclosing it in liposomes or integrating a DNA encoding it in an appropriate vector. Furthermore, it can be used in an appropriate formulation such as injections, tablets, capsules, eye drops, creams, suppositories, sprays, poultices, or the like, optionally by adding a pharmaceutical acceptable carrier to the polypeptide of the present invention.
[0049]As the pharmaceutical acceptable carrier, well-known solvents, bases, stabilizing agents, antiseptics, solubilizing agents, fillers, buffers, and the like may be used. When the polypeptide of the present invention contained in the medicament of the present invention is used in the above formulation, the administration method and the dose may be determined in accordance with, for example, the age or sex of each subject, or the kind or degree of each disease.
[0050]The oral administration includes a sublingual administration. As the parenteral administration, for example, inhalation, percutaneous administration, ophthalmic administration, vaginal administration, intra-articular administration, rectal administration, intra-arterial administration, intravenous administration, local administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, or the like may be selected.
[5] Antibody and the Fragment Thereof of the Present Invention
[0051]An antibody, such as a polyclonal antibody or a monoclonal antibody, which reacts with the polypeptide of the present invention may be obtained by directly administering the polypeptide of the present invention or a fragment thereof to various animals. Alternatively, it may be obtained by a DNA vaccine method (Raz, E. et al., Proc. Natl. Acad. Sci. USA, 91, 9519-9523, 1994; or Donnelly, J. J. et al., J. Infect. Dis., 173, 314-320, 1996), using a plasmid into which a polynucleotide encoding the polypeptide of the present invention is inserted.
[0052]The polyclonal antibody may be produced from a serum or eggs of an animal such as a rabbit, a rat, a goat, or a chicken, in which the animal is immunized and sensitized by the polypeptide of the present invention or a fragment thereof emulsified in an appropriate adjuvant (for example, Freund's complete adjuvant) by intraperitoneal, subcutaneous, or intravenous administration. The polyclonal antibody may be separated and purified from the resulting serum or eggs in accordance with conventional methods for polypeptide isolation and purification. Examples of the separation and purification methods include, for example, centrifugal separation, dialysis, salting-out with ammonium sulfate, or a chromatographic technique using such as DEAE-cellulose, hydroxyapatite, protein A agarose, and the like.
[0053]The monoclonal antibody may be easily produced by those skilled in the art, according to, for example, a cell fusion method of Kohler and Milstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975).
[0054]A mouse is immunized intraperitoneally, subcutaneously, or intravenously several times at an interval of a few weeks by a repeated inoculation of emulsions in which the polypeptide of the present invention or a fragment thereof is emulsified into a suitable adjuvant such as Freund's complete adjuvant. Spleen cells are removed after the final immunization, and then fused with myeloma cells to prepare hybridomas.
[0055]As a myeloma cell for obtaining a hybridoma, a myeloma cell having a marker such as a deficiency in hypoxanthine-guanine phosphoribosyltransferase or thymidine kinase (for example, mouse myeloma cell line P3X63Ag8.U1) may be used. As a fusing agent, polyethylene glycol may be used. As a medium for preparation of hybridomas, for example, a commonly used medium such as an Eagle's minimum essential medium, a Dulbecco's modified minimum essential medium, or an RPMI-1640 medium may be used by adding properly 10 to 30% of a fetal bovine serum. The fused strains may be selected by a HAT selection method. A culture supernatant of the hybridomas is screened by a well-known method such as an ELISA method or an immunohistological method, to select hybridoma clones secreting the antibody of interest. The monoclonality of the selected hybridoma is guaranteed by repeating subcloning by a limiting dilution method. Antibodies in an amount which may be purified are produced by culturing the resulting hybridomas in a medium for 2 to 4 days, or in the peritoneal cavity of a pristane-pretreated BALB/c strain mouse for 10 to 20 days.
[0056]The resulting monoclonal antibodies in the culture supernatant or the ascites may be separated and purified by conventional polypeptide isolation and purification methods. Examples of the separation and purification methods include, for example, centrifugal separation, dialysis, salting-out with ammonium sulfate, or chromatographic technique using such as DEAE-cellulose, hydroxyapatite, protein A agarose, and the like.
[0057]Further, the monoclonal antibodies or the antibody fragments containing a part thereof may be produced by inserting the whole or a part of a gene encoding the monoclonal antibody into an expression vector and introducing the resulting expression vector into appropriate host cells (such as E. coli, yeast, or animal cells).
[0058]Antibody fragments comprising an active part of the antibody such as F(ab')2, Fab, Fab', or Fv may be obtained by a conventional method, for example, by digesting the separated and purified antibodies (including polyclonal antibodies and monoclonal antibodies) with a protease such as pepsin, papain, and the like, and separating and purifying the resulting fragments by standard polypeptide isolation and purification methods.
[0059]Further, an antibody which reacts to the polypeptide of the present invention may be obtained in a form of single chain Fv or Fab in accordance with a method of Clackson et al. or a method of Zebedee et al. (Clackson, T. et al., Nature, 352, 624-628, 1991; or Zebedee, S. et al., Proc. Natl. Acad. Sci. USA, 89, 3175-3179, 1992). Furthermore, a humanized antibody may be obtained by immunizing a transgenic mouse in which mouse antibody genes are substituted with human antibody genes (Lonberg, N. et al., Nature, 368, 856-859, 1994).
[6] Screening Method of the Present Invention
[0060]It is possible to determine whether or not a substance to be tested modifies (for example, suppresses or promotes) the chitinase activity of the polypeptide according to the present invention, using the polypeptide of the present invention.
[0061]Substances to be tested to which may be applied the screening method of the present invention are not particularly limited, but there may be mentioned, for example, various known compounds (including peptides) registered in chemical files, compounds obtained by combinatorial chemistry techniques (Terrett, N. K. et al., Tetrahedron, 51, 8135-8137, 1995), or random peptides prepared by employing a phage display method (Felici, F. et al., J. Mol. Biol., 222, 301-310, 1991) or the like. In addition, culture supernatants of microorganisms, natural components derived from plants or marine organisms, or animal tissue extracts may be used as the test substances for screening. Further, compounds (including peptides) obtained by chemically or biologically modifying compounds (including peptides) selected by the screening method of the present invention may be used.
[0062]The screening method of the present invention may be carried out by a method similar to the above-mentioned method for confirming the chitinase activity, except that the polypeptide of the present invention, a substrate of chitinase, and the test substance are brought into contact with each other instead of bringing the test polypeptide into contact with a substrate of chitinase.
[0063]Namely, in the screening method of the present invention, it is confirmed whether or not the test substance modifies the chitinase activity of the polypeptide of the present invention by bringing into contact the polypeptide of the present invention, a substrate of chitinase, and the test substance, and then analyzing whether or not the substrate of chitinase is digested (or a degree of the digestion) by the chitinase activity of the polypeptide of the present invention in the presence of the test substance. When the substrate of chitinase is not degraded by the chitinase activity of the polypeptide of the present invention, or the degree of the digestion is decreased, it is possible to confirm that the test substance suppresses the chitinase activity of the polypeptide of the present invention. Alternatively, when the degree of the digestion of the substrate of chitinase by the chitinase activity of the polypeptide of the present invention is increased, it is possible to confirm that the test substance promotes the chitinase activity of the polypeptide of the present invention.
EXAMPLES
[0064]The present invention now will be further illustrated by, but is by no means limited to, the following Examples. The procedures described in the following Examples were performed in accordance with various techniques commonly used in molecular biology, acarology, arthropodology, immunology, or biochemistry, described in, for example, Sambrook et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1989 or similar books. As a software for analyzing DNA, MacVector® (Oxford Molecular) was used.
Example 1
Isolation of a Gene Encoding a Novel Tick Chitinase
[0065]Total RNA was extracted from eggs of a Haemaphysalis longicornis Okayama strain [Fujisaki et al., Nat. Inst. Anim. Hlth Quart. (Tokyo), 16, 122-128 (1976)] by an Acid Guanidinium-phenol-chloroform method [Chomczynski et al., Anal. Biochem., 162, 156-159 (1987)]. From the resulting total RNA, poly A.sup.+ RNA was purified using an mRNA isolation kit [Oligotex-dT30 (Super), code W9021B; Takara] in accordance with a protocol attached to the kit.
[0066]The following procedures, i.e., construction of a cDNA library, screening, and insertion into plasmid of a cDNA clone (in vivo Excision) were performed using commercially available reagent kits (Stratagen) in accordance with protocols attached thereto.
[0067]More particularly, cDNA was synthesized using 5 μg of tick mRNA as a template and a cDNA synthesis kit (ZAP-cDNA Synthesis Kit, Cat. No. 200401-5; Stratagen). The resulting cDNA was fractionated by a size fractionation with a Sepharose CL-2B gel column, inserted into a vector (Uni-ZAP XR Vector, Cat. No. 237211; Stratagen), and packaged using a packaging reagent (GigapackIII Gold packaging extract; Stratagen). Escherichia coli (E. coli XL1-Blue MRF' strain) was transfected with the packaged product to obtain a library containing approximately 500,000 cDNA clones.
[0068]The cDNA library was plaque-screened using a polymerase chain reaction (PCR) method, to obtain four overlapping positive clones. More particularly, a fragment of a tick chitinase gene obtained by PCR, as a probe, was reacted with the cDNA library. A positive colony was observed as a black dot. Screening was performed by confirming the black dots. In this connection, the PCR was carried out using two primers having the base sequences of SEQ ID NOS: 7 and 8 designed from the amino acid sequences of SEQ ID NOS: 5 and 6, respectively. In the PCR, 50 μL of a reaction liquid [template DNA 1 μg, 0.1 μmol/L primer, 10 mmol/L Tris-HCl (pH8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, and 2.5 U Taq Gold DNA polymerase (Part No. N808-0244; Perkin Elmer)] was used, and a cycle composed of treatments at 94° C. for 1 minute, 50° C. for 1 minute, and 72° C. for 2 minutes was repeated 40 times.
[0069]These positive clones were inserted into plasmid (i.e., converted into a pBluescript) by an in vivo Excision method.
[0070]Each plasmid containing a cDNA fragment was purified using a plasmid purification kit (Cat no. 12125; Qiagen), and then PCR was carried out using a sequencing kit (Dye Primer Cycle Sequencing Kit, Part No. 4303153; Perkin Elmer) in accordance with a protocol attached to the kit. Each resulting PCR product was analyzed with a DNA sequencer (ABI PRISM 3100 Genetic Analyzer; Perkin Elmer) to determine a base sequence of each cDNA fragment.
[0071]As a result, it was found that all four clones were derived from a single gene. The longest clone was used in the following analyses.
[0072]The full length of the cDNA was 6439 bp, and the base sequence thereof was that of SEQ ID NO: 1. It was confirmed that the base sequence contains an open reading frame consisting of 2790 bp (a base sequence consisting of the 571st to 3360th bases in the base sequence of SEQ ID NO: 1). The amino acid sequence of a protein deduced from the open reading frame was the amino acid sequence of SEQ ID NO: 2 consisting of 929 amino acid residues, and the deduced molecular weight was 104 kDa.
[0073]The homology search of the deduced amino acid sequence was carried out by a BLAST method (Basic local alignment search tool; Altschul, S. F. et al., J. Mol. Biol., 215, 403-410, 1990; obtained from the National Center for Biotechnology Information). As a result, it was confirmed that the amino acid sequence had a high homology with known chitinase proteins derived from other organisms. For example, it had an approximately 30% homology with yellow fever mosquito (Aedes aegypti) chitinase [Insect Mol. Biol., 7(3), 233-239 (1998)].
Example 2
Construction of Vector for Expressing Tick Chitinase Fusion Protein
[0074]For cloning the ORF region of the tick chitinase gene, a DNA amplification was carried out by PCR using the cDNA clone obtained in Example 1 as a template, and a sense primer (consisting of the base sequence of SEQ ID NO: 3 and containing the EcoRI recognition site "gaattc" at the 5' terminus) and an antisense primer (consisting of the base sequence of SEQ ID NO: 4 and containing the XhoI recognition site "ctcgag" at the 5' terminus). In the PCR, 50 μL of a reaction liquid [template DNA 1 μg, 0.1 μmol/L primer, 10 mmol/L Tris-HCl (pH8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, and 2.5 U Taq Gold DNA polymerase (Part No. N808-0244; Perkin Elmer)] was used, and a cycle composed of treatments at 94° C. for 1 minute, 50° C. for 1 minute, and 72° C. for 2 minutes was repeated 40 times.
[0075]The PCR product was treated with phenol/chloroform, collected by an ethanol precipitation method, and dissolved in distilled water. The resulting DNA solution was digested with the restriction enzyme EcoRI, and then the DNA fragment was separated by electrophoresis, purified using a DNA purification kit (Cat no. 1001-400; Biotechnologies), and collected in distilled water.
[0076]Meanwhile, a vector pGEMEX-4T-3 (Product no. 27-4583; Pharmacia Biotech) for expression in Escherichia coli was digested with the restriction enzyme EcoRI, dephosphorylated with alkaline phosphatase, and purified in the manner similar to that used in the purification of the PCR product.
[0077]The purified PCR product and vector were reacted using a DNA ligation kit (Cat no. 6022; Takara) in accordance with a protocol attached to the kit. Escherichia coli DH5α was transformed with the ligation reaction product, and then recombinant clones in which the chitinase ORF fragment was inserted in the same direction as that of glutathione S-transferase (GST) in the vector were selected. A recombinant plasmid was purified using a plasmid purification kit (Cat no. 12125; Qiagen).
Example 3
Expression of Tick Chitinase Recombinant Protein in Escherichia coli
[0078]Escherichia coli JM109 (DE3) (Promega) was transformed with the recombinant plasmid prepared in Example 2, and then the transformants were cultured at 37° C. in an LB medium containing ampicillin. When OD.sub.600nm of the culture became 0.3˜0.5, isopropyl-thio-galactoside (IPTG) was added to the culture so that the final concentration became 0.5 mmol/L, and then the transformants were further cultured at 37° C. for 4 hours.
[0079]The expression of tick chitinase recombinant protein was confirmed by carrying out 10% sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis [Laemmli et al., Nature, 227, 680-685 (1970)] followed by Coomassie staining.
[0080]As a result, the expression of the recombinant protein having a molecular weight of approximately 130 kDa was observed, and it was confirmed that the recombinant protein was a fusion protein of a GST leader protein (26 kDa) and the tick chitinase protein (104 kDa).
Example 4
Purification of Tick Chitinase Recombinant Protein and Preparation of Antiserum
[0081]The recombinant chitinase fusion protein expressed in Escherichia coli by the method described in Example 3 was purified in accordance with a protocol attached to a commercially available kit (Bulk GST Purification Module; Amersham Bioscience).
[0082]The result of electrophoresis of the purified recombinant chitinase fusion protein is shown in FIG. 1. In this connection, the electrophoresis and staining were performed in the manner similar to that described in Example 3. In FIG. 1, lane 1 is the result of molecular weight markers, lane 2 is the result of the purified recombinant chitinase fusion protein, and lane 3 is the result of the purified GST protein. The arrow at the right side of lane 3 indicates the recombinant chitinase fusion protein, and the numbers at the right side of lane 3 mean the molecular weights of the recombinant chitinase fusion protein (130 kDa), and the tick chitinase protein (104 kDa) and the GST leader protein (26 kDa) which are composed of the recombinant chitinase fusion protein.
[0083]An emulsion was prepared by mixing 200 μL of a solution containing 100 μg of the purified recombinant chitinase fusion protein with 200 μL of a complete Freund's adjuvant (Adjuvant Complete Freund; Difco). The emulsion was intraperitoneally inoculated into a 8-week-old female BALB/c mouse. After 2 and 4 weeks from the intraperitoneal inoculation, 100 μg of the recombinant chitinase fusion protein was mixed with an incomplete Freund's adjuvant (Difco), and each booster inoculation was carried out. After 2 weeks from the final inoculation, blood was collected and the resulting serum was kept at -20° C.
Example 5
Identification of Native (Wild Type) Chitinase by Immunoblotting
[0084]The wild type chitinase protein was identified by immunoblotting [Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350-4354 (1979)] using the anti-recombinant chitinase fusion protein mouse serum obtained in Example 4. As samples, a lysate of tick eggs, i.e., a supernatant prepared in accordance with a method described in You et al., Insect Biochem. Mol. Biol., 32, 67-73 (2000), was used. More particularly, the lasate was prepared by homogenizing tick eggs in 20 mmol/L Tris-HCl and centrifuging the homogenate at 1000 rpm for 30 minutes.
[0085]The result is shown in FIG. 2. In FIG. 2, lane 1 is the result when the anti-recombinant chitinase fusion protein immune serum was used, and lane 2 is the result when the anti-GST protein immune serum was used (negative control). The arrow at the right side of lane 2 indicates the wild type chitinase protein, and the numbers at the right side of lane 2 mean molecular weights.
[0086]As shown in lane 1 in FIG. 2, the specific band of the wild type chitinase protein (114 kDa) was detected in the egg lysate. The measured molecular weight was approximately 10 kDa higher than the molecular weight (104 kDa) deduced from the amino acid sequence of SEQ ID NO: 2. The difference seems to be due to glycosylation.
Example 6
Confirmation of Chitinase Activity in Recombinant Chitinase Fusion Protein
[0087]In this Example, the chitinase activity in the recombinant chitinase fusion protein was examined on a 1% agarose gel containing 0.01 glycol chitin, in accordance with a known method for measuring the chitinase activity [Johannes et al., Infect. Immun., 69, 4041-4047 (2001)]. For comparison, the chitinase activity in Serratia marcescens chitinase (Sigma) or β-galactosidase was examined.
[0088]More particularly, 10 μL of each protein solution obtained by dissolving each protein in PBS (phosphate buffer) was added to each well in the agarose gel. After an incubation at 37° C. for 12 hours, the gel was stained in a 0.01% staining liquid (Fluorescent Brightener 28; Sigma), washed with distilled water, and observed under an ultraviolet ray.
[0089]The recombinant chitinase fusion protein or Serratia marcescens chitinase reacted with chitin more strongly than did the β-galactosidase or PBS (control). As a result, it was confirmed that the recombinant chitinase fusion protein or Serratia marcescens chitinase exhibits a property of digesting chitin.
INDUSTRIAL APPLICABILITY
[0090]According to the polypeptide, polynucleotide, vector, transformant, and antibody of the present invention, the medicament of the present invention, particularly a tick vaccine, can be provided.
[0091]Further, according to the medicament of the present invention, particularly a tick vaccine, it is possible, for example, to exterminate ticks, or to treat or prevent tick-borne infections such as piroplasmosis, Q fever, or viral encephalitis.
Free Text in Sequence Listing
[0092]Each of the base sequences of SEQ ID NOS: 3, 4, 7, and 8 in the Sequence Listing is an artificially synthesized primer sequence.
[0093]In the base sequence of SEQ ID NO: 8 in the Sequence Listing, the alphabet "n" means A (i.e., adenine), C (i.e., cytosine), G (i.e., guanine), or T (i.e., thymine).
[0094]Although the present invention has been described with reference to specific embodiments, various changes and modifications obvious to those skilled in the art are possible without departing from the scope of the appended claims.
Sequence CWU
1
816439DNAHaemaphysalis longicornisCDS(571)..(3360) 1ggtcaggcca gtcgccatac
cgctccgtct ggtgcacgag caagtgcatt agagtgtgtg 60ctccttcgag aaggcgccag
caaccggaac tatatccgtc cgggtcctgc agcgtgacta 120gcgcccaacg tgcgcgaaga
ctttttgggt agcggagttg ggttgagtgt atcgtctgct 180tcaagtgggg tcggaggaaa
ccgaaaccgt ctgcgcggct cttttgagag cgcgcgccat 240catcgtctgc agccgagcgt
gtctacgacg cgtcttgtcg ccgggtgcgc ttaggtggat 300accgttcgtc tccctgttcg
aaaaaaactt cttgtgcccg tggcctgacc tgcggattga 360aggcgcttcg cggggactcc
gtaggctgat ttttttcccc atcgtgcgga aaaaaaggac 420accagcgttg gagcaagtgg
ttcttttcta gcatcgtgtg tggtaccttg tgagagcgct 480gtcttcctcg ggttaccgat
tctctttccg agcaaggact acgcaccctt cactggattc 540gcgagccttt ctgcgtagag
gacatacatc atg aag ccc agg tgg ttc ttc ctg 594
Met Lys Pro Arg Trp Phe Phe Leu
1 5gcc ttc gtg gcc ctt ttg agc ttc aca aat gtc gtc gac gca
agg aga 642Ala Phe Val Ala Leu Leu Ser Phe Thr Asn Val Val Asp Ala
Arg Arg 10 15 20cca ctc cgg aaa gtc
agc ctc aca agt ccg tcg gcg ccg agc ggc ggc 690Pro Leu Arg Lys Val
Ser Leu Thr Ser Pro Ser Ala Pro Ser Gly Gly25 30
35 40aac acg ggc aag aag atc gtg tgc tac ttc
acc aac tgg gcc cag tat 738Asn Thr Gly Lys Lys Ile Val Cys Tyr Phe
Thr Asn Trp Ala Gln Tyr 45 50
55cga cag ggt gat ggc aag ttt ctg ccg gag gac atc gac ccg acg cta
786Arg Gln Gly Asp Gly Lys Phe Leu Pro Glu Asp Ile Asp Pro Thr Leu
60 65 70tgc aca cac atc atc tac
gcc ttc ggc tgg atg aag aag cac aag ctg 834Cys Thr His Ile Ile Tyr
Ala Phe Gly Trp Met Lys Lys His Lys Leu 75 80
85tcg tcc ttc gac gcc gcc gac gac acc aag aac ggc aag aag
ggc ctc 882Ser Ser Phe Asp Ala Ala Asp Asp Thr Lys Asn Gly Lys Lys
Gly Leu 90 95 100tac gag agg gtc att
gac ctc aag aag aag aac ccc agt ctc aag gtc 930Tyr Glu Arg Val Ile
Asp Leu Lys Lys Lys Asn Pro Ser Leu Lys Val105 110
115 120ctg ctg gcc gtg ggc ggt tgg tcg ttc ggc
acg cag cgc ttc aag gag 978Leu Leu Ala Val Gly Gly Trp Ser Phe Gly
Thr Gln Arg Phe Lys Glu 125 130
135atg gcc tcg aac agc tac aac cgg cgg ctg ttc atc ttc agc gcg ctc
1026Met Ala Ser Asn Ser Tyr Asn Arg Arg Leu Phe Ile Phe Ser Ala Leu
140 145 150aac ttc ctg cgc agg cgc
aag ttc gac ggt ctc gat ctc gac tgg gag 1074Asn Phe Leu Arg Arg Arg
Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu 155 160
165ttc ccc cgc gga aac gag gac aag aag aac ttc gtc gaa cta
gtc agg 1122Phe Pro Arg Gly Asn Glu Asp Lys Lys Asn Phe Val Glu Leu
Val Arg 170 175 180gaa ctt cgc gag gca
ttc gag gcc gag gcc aaa gag aag aag ctg cca 1170Glu Leu Arg Glu Ala
Phe Glu Ala Glu Ala Lys Glu Lys Lys Leu Pro185 190
195 200cga ctg ctg ctc acg gcg gcc gtg tcc gcc
ggc gcg gaa acc atc cgg 1218Arg Leu Leu Leu Thr Ala Ala Val Ser Ala
Gly Ala Glu Thr Ile Arg 205 210
215gga ggc tac gac gtg ccc gcc gtg gcg gcc tac gtg gac ttc ctg aac
1266Gly Gly Tyr Asp Val Pro Ala Val Ala Ala Tyr Val Asp Phe Leu Asn
220 225 230gtc atg tcg tac gac ttc
cac ggc aag tgg gag agc atg act gga cac 1314Val Met Ser Tyr Asp Phe
His Gly Lys Trp Glu Ser Met Thr Gly His 235 240
245aac agc ccg ctc tac gcg cag gcc aat gag acc acg tgg cgg
aag cag 1362Asn Ser Pro Leu Tyr Ala Gln Ala Asn Glu Thr Thr Trp Arg
Lys Gln 250 255 260ctg tgc atg gac ttt
ggt gtg aaa acc tgg gag cgt ctt ggt gcg ccg 1410Leu Cys Met Asp Phe
Gly Val Lys Thr Trp Glu Arg Leu Gly Ala Pro265 270
275 280aag gaa aag atc gtt gtc ggc acg ggc act
tac ggc aga acc ttc acc 1458Lys Glu Lys Ile Val Val Gly Thr Gly Thr
Tyr Gly Arg Thr Phe Thr 285 290
295ttg gcg aat ccc aac aac aat ggc atg aac gcg cca tcg tct ggt ggt
1506Leu Ala Asn Pro Asn Asn Asn Gly Met Asn Ala Pro Ser Ser Gly Gly
300 305 310ggt gac gcc gga cag ttc
aca aag gaa gct ggc ttc ctt gct tac tac 1554Gly Asp Ala Gly Gln Phe
Thr Lys Glu Ala Gly Phe Leu Ala Tyr Tyr 315 320
325gag att tgt gat atg ctg aaa aag ggt gcc gac tac gtg tgg
gac gag 1602Glu Ile Cys Asp Met Leu Lys Lys Gly Ala Asp Tyr Val Trp
Asp Glu 330 335 340gaa cag ctg gtc ccg
tac gcc tac ctc ggc aac caa tgg gtt ggc ttc 1650Glu Gln Leu Val Pro
Tyr Ala Tyr Leu Gly Asn Gln Trp Val Gly Phe345 350
355 360gac gac gag aga agc att agg gcc aag atg
caa tgg att aag atg aac 1698Asp Asp Glu Arg Ser Ile Arg Ala Lys Met
Gln Trp Ile Lys Met Asn 365 370
375ggc tac gct gga gcc atg gtc tgg acc gta gac atg gat gat ttt cga
1746Gly Tyr Ala Gly Ala Met Val Trp Thr Val Asp Met Asp Asp Phe Arg
380 385 390gga cgc tgc acg acg aaa
acg tgg cct ctg att ggc gct atg gcc gag 1794Gly Arg Cys Thr Thr Lys
Thr Trp Pro Leu Ile Gly Ala Met Ala Glu 395 400
405gaa ttg ctg aac agg cca agt cgg gga cct aaa aat ctg ctg
cct ttc 1842Glu Leu Leu Asn Arg Pro Ser Arg Gly Pro Lys Asn Leu Leu
Pro Phe 410 415 420gtc aag aag cag cgg
acg tct agc gcc aag tca acc ggc acc act gaa 1890Val Lys Lys Gln Arg
Thr Ser Ser Ala Lys Ser Thr Gly Thr Thr Glu425 430
435 440cca cca aac tcc atc aag gga tcg gac ccg
ttg aag gcg gcg cca tcc 1938Pro Pro Asn Ser Ile Lys Gly Ser Asp Pro
Leu Lys Ala Ala Pro Ser 445 450
455aac ctt ccg gtc aag gac gag gcc cca gaa agt gac acc aac gct cgc
1986Asn Leu Pro Val Lys Asp Glu Ala Pro Glu Ser Asp Thr Asn Ala Arg
460 465 470gtc gtc tgc tac ttc acc
aac tgg tcc gcc aag cgg aag ggt aag ggc 2034Val Val Cys Tyr Phe Thr
Asn Trp Ser Ala Lys Arg Lys Gly Lys Gly 475 480
485cac tac gag cca gag gac atc gat gcc acc ctc tgc acg cac
gtc atc 2082His Tyr Glu Pro Glu Asp Ile Asp Ala Thr Leu Cys Thr His
Val Ile 490 495 500tac gcg ttc gcc aac
atc aaa gag ttc aag atc gtt ccc acc gag ccc 2130Tyr Ala Phe Ala Asn
Ile Lys Glu Phe Lys Ile Val Pro Thr Glu Pro505 510
515 520gtg gat gaa ggc gac ggc agc gcg aag aag
ggc ttc tgg gag aga atc 2178Val Asp Glu Gly Asp Gly Ser Ala Lys Lys
Gly Phe Trp Glu Arg Ile 525 530
535gtg gcc ctg aag act aag aac ccg cag cta aag gtg atg ctc gcc gtc
2226Val Ala Leu Lys Thr Lys Asn Pro Gln Leu Lys Val Met Leu Ala Val
540 545 550gga gga tgg atg ctc gga
tcc gct ccc ttc agg gag gtc acc gag aac 2274Gly Gly Trp Met Leu Gly
Ser Ala Pro Phe Arg Glu Val Thr Glu Asn 555 560
565tcc tac agg caa tct ttg ttc gtg ttc aac gcc atc gac ttc
ttg agg 2322Ser Tyr Arg Gln Ser Leu Phe Val Phe Asn Ala Ile Asp Phe
Leu Arg 570 575 580gaa aag ggt ttc gat
ggt ctc gac gtc gac tgg gag ttt ccc cga ggt 2370Glu Lys Gly Phe Asp
Gly Leu Asp Val Asp Trp Glu Phe Pro Arg Gly585 590
595 600gct gag gac aag aag aag ctg gct ggt ctc
atc aag gaa ctg aga gtg 2418Ala Glu Asp Lys Lys Lys Leu Ala Gly Leu
Ile Lys Glu Leu Arg Val 605 610
615gcg ttc gat ggc gag ggc ttg gcg tcc aag aag ccc agg ctc atc ctc
2466Ala Phe Asp Gly Glu Gly Leu Ala Ser Lys Lys Pro Arg Leu Ile Leu
620 625 630tcc atg gcg gca ccg gcc
agc ttc gag gcc atc tct gcc ggc tac gac 2514Ser Met Ala Ala Pro Ala
Ser Phe Glu Ala Ile Ser Ala Gly Tyr Asp 635 640
645gtc gag gag ctc aac aag cat gtt gat atg atc aat atg atg
acg tac 2562Val Glu Glu Leu Asn Lys His Val Asp Met Ile Asn Met Met
Thr Tyr 650 655 660gac ttc cac gga gac
tgg gaa cgt caa gta gga cac cac agc ccc ctg 2610Asp Phe His Gly Asp
Trp Glu Arg Gln Val Gly His His Ser Pro Leu665 670
675 680ttc ccg ctg tac aca gcc agc agt ttc cag
aag aag ctt aca gtg gac 2658Phe Pro Leu Tyr Thr Ala Ser Ser Phe Gln
Lys Lys Leu Thr Val Asp 685 690
695tac agc gcg ggt gag tgg gtt cgc aag ggg gcc agt aag gaa aag ctg
2706Tyr Ser Ala Gly Glu Trp Val Arg Lys Gly Ala Ser Lys Glu Lys Leu
700 705 710ctg gtt ggc att ccc acg
tac ggg cgt acc ttc act ctc ggt gac aac 2754Leu Val Gly Ile Pro Thr
Tyr Gly Arg Thr Phe Thr Leu Gly Asp Asn 715 720
725aac ctg acc gac gtc ggt gct ccg gcc aca gct ggc ggc agg
cct ggc 2802Asn Leu Thr Asp Val Gly Ala Pro Ala Thr Ala Gly Gly Arg
Pro Gly 730 735 740aac tat act ggc gaa
act ggg ttc ctg tcc ttt ttc gag atc tgc gac 2850Asn Tyr Thr Gly Glu
Thr Gly Phe Leu Ser Phe Phe Glu Ile Cys Asp745 750
755 760ctt ctg cgt tct gga gca acc ctc gta tgg
gac aat gag cag atg gtt 2898Leu Leu Arg Ser Gly Ala Thr Leu Val Trp
Asp Asn Glu Gln Met Val 765 770
775ccg tac gcc tac aag gac gac cag tgg gtt ggc ttc gac gac cag aga
2946Pro Tyr Ala Tyr Lys Asp Asp Gln Trp Val Gly Phe Asp Asp Gln Arg
780 785 790agt ctc aag ctt aag gtc
cag tgg ctg aaa cag gcc ggc tac ggc ggg 2994Ser Leu Lys Leu Lys Val
Gln Trp Leu Lys Gln Ala Gly Tyr Gly Gly 795 800
805gtg atg gtc tgg tcc gta gac ttg gac gac ttc aag ggc acg
tgc acg 3042Val Met Val Trp Ser Val Asp Leu Asp Asp Phe Lys Gly Thr
Cys Thr 810 815 820ggc cac agc tac ccg
ctg ctt acg gca atc aag gag gag ctc aag gga 3090Gly His Ser Tyr Pro
Leu Leu Thr Ala Ile Lys Glu Glu Leu Lys Gly825 830
835 840tac aag gtg gcc aac ctc gaa gtt gcc tct
tcc aac ata ctc aac tcg 3138Tyr Lys Val Ala Asn Leu Glu Val Ala Ser
Ser Asn Ile Leu Asn Ser 845 850
855tac gga cag ctc gtc gat ccg aat gaa gtg gtg tgc gac gaa gag gac
3186Tyr Gly Gln Leu Val Asp Pro Asn Glu Val Val Cys Asp Glu Glu Asp
860 865 870ggc cac atc agc tat cac
ttg gac aag aag gac tgc acc atg tac tac 3234Gly His Ile Ser Tyr His
Leu Asp Lys Lys Asp Cys Thr Met Tyr Tyr 875 880
885atg tgc gag gga aag cga cgc cac cac atg ccg tgt ccc act
aac ctc 3282Met Cys Glu Gly Lys Arg Arg His His Met Pro Cys Pro Thr
Asn Leu 890 895 900gta ttt aac tta aac
gag agc gtc tgc gac tgg cca gag aat gtg gac 3330Val Phe Asn Leu Asn
Glu Ser Val Cys Asp Trp Pro Glu Asn Val Asp905 910
915 920gac tgc aag cat atc gct gct aag acg tag
tgtttctgta caacgtttcg 3380Asp Cys Lys His Ile Ala Ala Lys Thr
925ctaacccatt tgccgtcgtc gtgccgtcag cttgccagcg gctgccaggg
atcggtttca 3440ggaaggggct aagttgtggc gataaagata tttgcttcgc gattccctta
ggtggctcgc 3500actttcaaaa atatcgcttc acccctgcga tctgtgtggg gcattcacta
tggagaagtg 3560tctgtgtcgc atgccgcctt agaaaaacgc gtaattcaac tgacttgaga
atttgcttga 3620aacagtaaag agggcgatat gagcctaaaa agtgccccaa aacaagtggc
gctctatcac 3680ggtagctcat gttgcttaac tgttggtgat acgttcccga tcaaaataaa
gtcacagcaa 3740aataatcgcg attatcaggt ctctgtatga ccgcacaatt aggcgcctaa
atattagccg 3800tgaaaagtta cgacgacggc gaattcgtaa gcggcaattt tgttgttcgg
ttctgagtat 3860tctcttcaca agtgctctca tgtctaaatg cgctcctcac gtgacagaag
ccacggagaa 3920gacggggtct ccttcagcgt accccgcact ggctttggta acatgcctct
ttgtcacaaa 3980gctcaatgat gctatatacc caaaacgctg ccaaaaaatc gaaacattac
ttgacgctac 4040ccagtttatt tttacgtact ttgacctcaa aggtacccgt gaagtgccag
aaaaaaaaat 4100tctacgccta cgtttgtggg atccagaaat tagtacctca tgagaagcca
cacactgtag 4160tagattttta tatatttggg atacaataaa tgcaatgggg cgttgggctg
gtgtcaacgt 4220tttggggggc gccagagtcc gtcagcatac cttcatcttt tcttagtttt
ctgaaatcgt 4280tcgtttgtgt tatgaagagc tcggcttggt agaaatggtc gggctgcaaa
tttttaccat 4340ttgcttcaga gcaatatcgt tgttcttttt gttttcgaga aatactacgt
gatgttcttt 4400atttcatcgc agggatgaaa cctttgggaa gagtgtttat ttgtatgaaa
agaaaggaag 4460atgtttgata tgttgcttcc atgttttgtt gcaagtattg tattgtgtac
gtcttcagca 4520ttgttatcac gctcgatcgt ttttaagtag agtagattta tcctggtctt
tccagatgtc 4580aatcataaat atgcgtgttt ttcgacctta taaaaagtgg tgctatatat
atattagagc 4640aataatagag agggagggta attaggctaa atattcttgc gcgtctatct
caaatatttt 4700ctatcgcctt aaacgaatac cttttctttc aactgaaatg ctctttcttt
taaattattg 4760caaaattcgg tagctctcaa aagtgccttt gcttgtgact aacataacac
acacaccgca 4820agactcgctt tgttaatttc tactttgctg cgcagcacat caaaaacaac
aaaatatttt 4880aaacgtagag aacaactgaa tgcagaacat gtatcacagc tccgcctcct
tgtcgtatgg 4940cgcactcaag tcagttttaa aatattttac gaatgcgaca gcgacattat
aatacctatt 5000acattgcaat gaaagctaaa gtaaatgcaa tatatacttc aaaacgcaag
tcctttgttt 5060tattaggcac tcgtttacac actataaact gataattttt tgttatatcg
aggaatactg 5120taagcaatgc ttgtgtagaa gacaccgatt ttgaacagga actttgaact
ctctaggtta 5180tagtagatga attttatccg taggaaaacg taatacctat cgagtaaata
tacaagataa 5240gaggaaggca cagcagaagc cgtgaaaatg tcaatgtgcc gtgccacacg
tttataacct 5300cctatttgta agttgttctc caatttggta aacttctaaa ctaccgtggc
aactctgcaa 5360aaaacgtgct cattagcatg cgttaacttc attctaaaca ctcagggttg
gctcgtattc 5420gcaacataag gtttcagatc tgatttctgc atgtcaaaag cgccagcaca
attttttcgc 5480caagtgactg cttgtcatgc atatattcgg tgcacacatt tatacagcga
tctgctacac 5540tgcaccgaca agttttattt cgctgtgttt tctgcgcttt tctgtccatg
tgtacggcgc 5600acttcaaata agcaaagcta gcacaaaaac ccgtgcagat gagaaatgta
ttcgggcatc 5660taacattcca aaaatcatcc ggtgacttcg tggtacgctt acgtggttta
gacaaatacg 5720tctcccctat tttgtcgtag cttggagctc agctacacta agaataaaaa
agaagcgagc 5780gttatatttt ccgacagtta cactttcgta cagttgtacg tttgctgcaa
cctccttaat 5840ggctttcaag gccagcacta aacgagcttt tttattatcc tctgtttaaa
aaaaatttcg 5900caaaaaatgg gctcttacaa aaaccaatgc cagaagcatg aagagacaac
ggggttatgt 5960gcataagtgg ttttgcatgg cgggccacat atcttaaata tcacatagcg
ccttctttgt 6020cgtacgatgc ctcctgcagt aagacagcgc ttctctttgc atagcagcga
aggcggcgcg 6080cacatctcga gtattcaaac ggtcttccgc aggtacgctg cgcttgtttt
aaggtaacgt 6140ctctctaact ttcttattta ttatcacgca taccgcctat gacacttcca
gatcgggtgt 6200gcttatgctt ttattccttg tttgtgtttt ttctctcctt tcttctctac
gctgtaccta 6260ttgcatttga ctgtgtgctt cgagccgtgt gtgcaggtcc cccccacaaa
taggagtgga 6320gagccggcca ctcgtgcttc tgtacaaagt cgcctgccat ctaggagaaa
aagaaagcga 6380ataaatacaa acaaaaatta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaa 64392929PRTHaemaphysalis longicornis 2Met Lys Pro Arg Trp
Phe Phe Leu Ala Phe Val Ala Leu Leu Ser Phe1 5
10 15Thr Asn Val Val Asp Ala Arg Arg Pro Leu Arg
Lys Val Ser Leu Thr 20 25
30Ser Pro Ser Ala Pro Ser Gly Gly Asn Thr Gly Lys Lys Ile Val Cys
35 40 45Tyr Phe Thr Asn Trp Ala Gln Tyr
Arg Gln Gly Asp Gly Lys Phe Leu 50 55
60Pro Glu Asp Ile Asp Pro Thr Leu Cys Thr His Ile Ile Tyr Ala Phe65
70 75 80Gly Trp Met Lys Lys
His Lys Leu Ser Ser Phe Asp Ala Ala Asp Asp 85
90 95Thr Lys Asn Gly Lys Lys Gly Leu Tyr Glu Arg
Val Ile Asp Leu Lys 100 105
110Lys Lys Asn Pro Ser Leu Lys Val Leu Leu Ala Val Gly Gly Trp Ser
115 120 125Phe Gly Thr Gln Arg Phe Lys
Glu Met Ala Ser Asn Ser Tyr Asn Arg 130 135
140Arg Leu Phe Ile Phe Ser Ala Leu Asn Phe Leu Arg Arg Arg Lys
Phe145 150 155 160Asp Gly
Leu Asp Leu Asp Trp Glu Phe Pro Arg Gly Asn Glu Asp Lys
165 170 175Lys Asn Phe Val Glu Leu Val
Arg Glu Leu Arg Glu Ala Phe Glu Ala 180 185
190Glu Ala Lys Glu Lys Lys Leu Pro Arg Leu Leu Leu Thr Ala
Ala Val 195 200 205Ser Ala Gly Ala
Glu Thr Ile Arg Gly Gly Tyr Asp Val Pro Ala Val 210
215 220Ala Ala Tyr Val Asp Phe Leu Asn Val Met Ser Tyr
Asp Phe His Gly225 230 235
240Lys Trp Glu Ser Met Thr Gly His Asn Ser Pro Leu Tyr Ala Gln Ala
245 250 255Asn Glu Thr Thr Trp
Arg Lys Gln Leu Cys Met Asp Phe Gly Val Lys 260
265 270Thr Trp Glu Arg Leu Gly Ala Pro Lys Glu Lys Ile
Val Val Gly Thr 275 280 285Gly Thr
Tyr Gly Arg Thr Phe Thr Leu Ala Asn Pro Asn Asn Asn Gly 290
295 300Met Asn Ala Pro Ser Ser Gly Gly Gly Asp Ala
Gly Gln Phe Thr Lys305 310 315
320Glu Ala Gly Phe Leu Ala Tyr Tyr Glu Ile Cys Asp Met Leu Lys Lys
325 330 335Gly Ala Asp Tyr
Val Trp Asp Glu Glu Gln Leu Val Pro Tyr Ala Tyr 340
345 350Leu Gly Asn Gln Trp Val Gly Phe Asp Asp Glu
Arg Ser Ile Arg Ala 355 360 365Lys
Met Gln Trp Ile Lys Met Asn Gly Tyr Ala Gly Ala Met Val Trp 370
375 380Thr Val Asp Met Asp Asp Phe Arg Gly Arg
Cys Thr Thr Lys Thr Trp385 390 395
400Pro Leu Ile Gly Ala Met Ala Glu Glu Leu Leu Asn Arg Pro Ser
Arg 405 410 415Gly Pro Lys
Asn Leu Leu Pro Phe Val Lys Lys Gln Arg Thr Ser Ser 420
425 430Ala Lys Ser Thr Gly Thr Thr Glu Pro Pro
Asn Ser Ile Lys Gly Ser 435 440
445Asp Pro Leu Lys Ala Ala Pro Ser Asn Leu Pro Val Lys Asp Glu Ala 450
455 460Pro Glu Ser Asp Thr Asn Ala Arg
Val Val Cys Tyr Phe Thr Asn Trp465 470
475 480Ser Ala Lys Arg Lys Gly Lys Gly His Tyr Glu Pro
Glu Asp Ile Asp 485 490
495Ala Thr Leu Cys Thr His Val Ile Tyr Ala Phe Ala Asn Ile Lys Glu
500 505 510Phe Lys Ile Val Pro Thr
Glu Pro Val Asp Glu Gly Asp Gly Ser Ala 515 520
525Lys Lys Gly Phe Trp Glu Arg Ile Val Ala Leu Lys Thr Lys
Asn Pro 530 535 540Gln Leu Lys Val Met
Leu Ala Val Gly Gly Trp Met Leu Gly Ser Ala545 550
555 560Pro Phe Arg Glu Val Thr Glu Asn Ser Tyr
Arg Gln Ser Leu Phe Val 565 570
575Phe Asn Ala Ile Asp Phe Leu Arg Glu Lys Gly Phe Asp Gly Leu Asp
580 585 590Val Asp Trp Glu Phe
Pro Arg Gly Ala Glu Asp Lys Lys Lys Leu Ala 595
600 605Gly Leu Ile Lys Glu Leu Arg Val Ala Phe Asp Gly
Glu Gly Leu Ala 610 615 620Ser Lys Lys
Pro Arg Leu Ile Leu Ser Met Ala Ala Pro Ala Ser Phe625
630 635 640Glu Ala Ile Ser Ala Gly Tyr
Asp Val Glu Glu Leu Asn Lys His Val 645
650 655Asp Met Ile Asn Met Met Thr Tyr Asp Phe His Gly
Asp Trp Glu Arg 660 665 670Gln
Val Gly His His Ser Pro Leu Phe Pro Leu Tyr Thr Ala Ser Ser 675
680 685Phe Gln Lys Lys Leu Thr Val Asp Tyr
Ser Ala Gly Glu Trp Val Arg 690 695
700Lys Gly Ala Ser Lys Glu Lys Leu Leu Val Gly Ile Pro Thr Tyr Gly705
710 715 720Arg Thr Phe Thr
Leu Gly Asp Asn Asn Leu Thr Asp Val Gly Ala Pro 725
730 735Ala Thr Ala Gly Gly Arg Pro Gly Asn Tyr
Thr Gly Glu Thr Gly Phe 740 745
750Leu Ser Phe Phe Glu Ile Cys Asp Leu Leu Arg Ser Gly Ala Thr Leu
755 760 765Val Trp Asp Asn Glu Gln Met
Val Pro Tyr Ala Tyr Lys Asp Asp Gln 770 775
780Trp Val Gly Phe Asp Asp Gln Arg Ser Leu Lys Leu Lys Val Gln
Trp785 790 795 800Leu Lys
Gln Ala Gly Tyr Gly Gly Val Met Val Trp Ser Val Asp Leu
805 810 815Asp Asp Phe Lys Gly Thr Cys
Thr Gly His Ser Tyr Pro Leu Leu Thr 820 825
830Ala Ile Lys Glu Glu Leu Lys Gly Tyr Lys Val Ala Asn Leu
Glu Val 835 840 845Ala Ser Ser Asn
Ile Leu Asn Ser Tyr Gly Gln Leu Val Asp Pro Asn 850
855 860Glu Val Val Cys Asp Glu Glu Asp Gly His Ile Ser
Tyr His Leu Asp865 870 875
880Lys Lys Asp Cys Thr Met Tyr Tyr Met Cys Glu Gly Lys Arg Arg His
885 890 895His Met Pro Cys Pro
Thr Asn Leu Val Phe Asn Leu Asn Glu Ser Val 900
905 910Cys Asp Trp Pro Glu Asn Val Asp Asp Cys Lys His
Ile Ala Ala Lys 915 920
925Thr324DNAArtificial SequenceAn artificially synthesized primer
sequence 3acgaattcca tcatgaagcc cagg
24424DNAArtificial SequenceAn artificially synthesized primer
sequence 4acctcgagct acgtcttagc agcg
24513PRTAedes aegypti 5Lys Val Thr Leu Ala Ile Gly Gly Trp Asn Asp
Ser Ala1 5 10613PRTAedes aegypti 6Tyr Gly
Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro1 5
10716DNAArtificial SequenceAn artificially synthesized primer
sequence 7tggkcvrtst aycgdc
16820DNAArtificial SequenceAn artificially synthesized primer
sequence 8ccartcrakr tcnadnccvt
20
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