Patent application title: Receptor Binding Polypeptides
Inventors:
Pele Choi Sing Chong (Richmond Hill, CA)
Shie-Liang Hsieh (Taipei, TW)
Assignees:
NATIONAL HEALTH RESEARCH INSTITUTES
IPC8 Class: AA61K3942FI
USPC Class:
4241591
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds virus or component thereof
Publication date: 2009-01-22
Patent application number: 20090022735
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Patent application title: Receptor Binding Polypeptides
Inventors:
Pele Choi Sing Chong
Shie-Liang Hsieh
Agents:
OCCHIUTI ROHLICEK & TSAO, LLP
Assignees:
National Health Research Institutes
Origin: CAMBRIDGE, MA US
IPC8 Class: AA61K3942FI
USPC Class:
4241591
Abstract:
Isolated polypeptides containing fragments of SARS CoV S protein and
functional equivalents thereof. Also disclosed are isolated nucleic acids
encoding the polypeptides, related expression vectors, related host
cells, related antibodies, and related compositions. Methods of producing
the polypeptide, diagnosing infection with a coronavirus, and identifying
a test compound for treating infection with a coronavirus are also
disclosed.Claims:
1-11. (canceled)
12. An isolated nucleic acid comprising a sequence encoding a polypeptide or a complement thereof, wherein the polypeptide comprises SEQ ID NO: 4 or an immunogenic fragment thereof, and the immunogenic fragment is at least 10 amino acid residues in length.
13. The nucleic acid of claim 12, wherein the sequence contains SEQ ID NO: 3, 5, 6, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27; or encodes one of the peptide sequences listed in Table 2.
14. The nucleic acid of claim 13, wherein the sequence contains SEQ ID NO: 23 or 25.
15. An expression vector comprising a nucleic acid of claim 12.
16. A host cell comprising a nucleic acid of claim 12.
17. A method of producing a polypeptide, comprising culturing the host cell of claim 16 in a medium under conditions permitting expression of a polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cell or the medium of the cell, wherein the polypeptide comprises SEQ ID NO: 4 or an immunogenic fragment thereof, and the immunogenic fragment is at least 10 amino acid residues in length.
18. (canceled)
19. A method of generating an antibody against a polypeptide, the method comprising administering to a non-human animal a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises SEQ ID NO: 4 or an immunogenic fragment thereof, and the immunogenic fragment is at least 10 amino acid residues in length.
20. A method of inducing an immune response in a subject against a coronavirus, the method comprising administering to the subject a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises SEQ ID NO: 4 or an immunogenic fragment thereof, and the immunogenic fragment is at least 10 amino acid residues in length.
21-24. (canceled)
25. A method of treating an infection with a coronavirus, the method comprising administering to a subject in need thereof an effective amount of a polypeptide, wherein the polypeptide comprises SEQ ID NO: 4 or an immunogenic fragment thereof, and the immunogenic fragment is at least 10 amino acid residues in length.
26-29. (canceled)
Description:
RELATED APPLICATION
[0001]This application is a divisional of U.S. application Ser. No. 11/033,455, filed on Jan. 10, 2005, which_claims priority to U.S. Provisional Application Ser. No. 60/535,641, filed on Jan. 9, 2004, the contents of which are incorporated by reference in its entirety.
BACKGROUND
[0002]Virus is the cause of various disorders. For example, members of the coronavirus family cause hepatitis in mice, gastroenteritis in pigs, and respiratory infections in birds and humans. Among the more than 30 strains isolated so far, three or four infect humans. The severe acute respiratory syndrome (SARS), a newly found infectious disease, is associated with a novel coronavirus. This life-threatening respiratory coronavirus touched off worldwide outbreaks in 2003. Vaccines and drugs against SARS coronavirus (CoV) are being vigorously sought. Nevertheless, the progress has been rather slow due to safety concerns.
SUMMARY
[0003]This invention is based, at least in part, on the discovery of receptor binding domains of the SARS CoV Spike (S) protein. Genomic sequences of a number of SARS CoV strains can be found in GenBank. GenBank Accession No. AY278741 (SEQ ID NO: 1) represents the genomic sequence of the SARS CoV Urbani strain, which contains an open reading frame encoding a polypeptide that is 7,073 amino acid residues (aa.) in length (SEQ ID NO: 2). The nucleic acid encoding the S protein of this strain corresponds to nucleotides (nt) 21,492-25,259 of GenBank Accession No. AY278741. Listed below are the nucleic acid and amino acid sequences of the S protein:
TABLE-US-00001 (SEQ ID NO: 3) 21492 atgtttatt ttcttattat ttcttactct cactagtggt agtgaccttg 21541 accggtgcac cacttttgat gatgttcaag ctcctaatta cactcaacat acttcatcta 21601 tgaggggggt ttactatcct gatgaaattt ttagatcaga cactctttat ttaactcagg 21661 atttatttct tccattttat tctaatgtta cagggtttca tactattaat catacgtttg 21721 gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg 21781 ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgattatta 21841 ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt 21901 tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc gataatgcat 21961 ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag 22021 gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt 22081 ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt aacactttga 22141 aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc attcttacag 22201 ccttttcacc tgctcaagac atttggggca cgtcagctgc agcctatttt gttggctatt 22261 taaagccaac tacatttatg ctcaagtatg atgaaaatgg tacaatcaca gatgctgttg 22321 attgttctca aaatccactt gctgaactca aatgctctgt taagagcttt gagattgaca 22381 aaggaattta ccagacctct aatttcaggg ttgttccctc aggagatgtt gtgagattcc 22441 ctaatattac aaacttgtgt ccttttggag aggtttttaa tgctactaaa ttcccttctg 22501 tctacgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct gtgctctaca 22561 actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc 22621 tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa 22681 tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca 22741 tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact ggtaattata 22801 attataaata taggtatctt agacatggca agcttaggcc ccttgagaga gacatatcta 22861 atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc 22921 cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct tacagagttg 22981 tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaattatcca 23041 ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg 23101 tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgtttctg 23161 atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgct 23221 cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc 23281 tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac 23341 cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta 23401 taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt 23461 gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt 23521 atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac 23581 ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct 23641 ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc 23701 aatatggtag cttttgcaca caactaaatc gtgcactctc aggtattgct gctgaacagg 23761 atcgcaacac acgtgaagtg ttcgctcaag tcaaacaaat gtacaaaacc ccaactttga 23821 aatattttgg tggttttaat ttttcacaaa tattacctga ccctctaaag ccaactaaga 23881 ggtcttttat tgaggacttg ctctttaata aggtgacact cgctgatgct ggcttcatga 23941 agcaatatgg cgaatgccta ggtgatatta atgctagaga tctcatttgt gcgcagaagt 24001 tcaatggact tacagtgttg ccacctctgc tcactgatga tatgattgct gcctacactg 24061 ctgctctagt tagtggtact gccactgctg gatggacatt tggtgctggc gctgctcttc 24121 aaataccttt tgctatgcaa atggcatata ggttcaatgg cattggagtt acccaaaatg 24181 ttctctatga gaaccaaaaa caaatcgcca accaatttaa caaggcgatt agtcaaattc 24241 aagaatcact tacaacaaca tcaactgcat tgggcaagct gcaagacgtt gttaaccaga 24301 atgctcaagc attaaacaca cttgttaaac aacttagctc taattttggt gcaatttcaa 24361 gtgtgctaaa tgatatcctt tcgcgacttg ataaagtcga ggcggaggta caaattgaca 24421 ggttaattac aggcagactt caaagccttc aaacctatgt aacacaacaa ctaatcaggg 24481 ctgctgaaat cagggcttct gctaatcttg ctgctactaa aatgtctgag tgtgttcttg 24541 gacaatcaaa aagagttgac ttttgtggaa agggctacca ccttatgtcc ttcccacaag 24601 cagccccgca tggtgttgtc ttcctacatg tcacgtatgt gccatcccag gagaggaact 24661 tcaccacagc gccagcaatt tgtcatgaag gcaaagcata cttccctcgt gaaggtgttt 24721 ttgtgtttaa tggcacttct tggtttatta cacagaggaa cttcttttct ccacaaataa 24781 ttactacaga caatacattt gtctcaggaa attgtgatgt cgttattggc atcattaaca 24841 acacagttta tgatcctctg caacctgagc tcgactcatt caaagaagag ctggacaagt 24901 acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt 24961 ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg 25021 aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt 25081 atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt 25141 gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca 25201 agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacacataa 25259 MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSD TLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTM IFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQP IDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAYF VGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSN FRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVL YNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVI ADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDI SNVPFSPDGKPCTPPALNCYWPLNDYGEYTTTGIGYQPYRVVVLSFELLN APATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDV SDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTD VSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGI CASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTE VMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDR NTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLENKVT LADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALV SGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNK AISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDI LSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAIC HEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGII NNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEI DRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLC CMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT (SEQ ID NO: 4; the two underlines segments represent two receptor binding domains.) MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSD TLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTM IFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQP IDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAYF VGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSN FRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVL YNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVI ADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDI SNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLN APATVCGPTLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDV SDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTD VSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGI CASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTE VMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDR NTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVT LADAGFMKQYGECLCDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALV SGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNK AISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDI LSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAIC HEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGII NNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEI DRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLC CMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT (SEQ ID NO: 4; the two underlines segments represent two receptor binding domains.)
[0004]One aspect of the invention features an isolated polypeptide containing SEQ ID NO: 4 or an immunogenic fragment derived from SEQ ID NO: 4. The immunogenic fragment is at least 10 amino acid residues in length, i.e., any number between 10 and 1255 (the length of SEQ ID NO: 4), inclusive. Examples of such an immunogenic fragment include the domains listed below:
TABLE-US-00002 Corresponding aa. position within Domain Name SEQ ID NO: 4 SEQ ID NO Receptor binding domain 1 80-227 SEQ ID NO: 6 (RBD1) Receptor binding domain 2 284-735 SEQ ID NO: 8 (RBD2) S1 1-333 SEQ ID NO: 18 S2 334-666 SEQ ID NO: 20 S3 667-1000 SEQ ID NO: 22 RBD2-consensus (RBD2-C) 434-467 SEQ ID NO: 24 RBD-55 564-613 SEQ ID NO: 26 Transmembrane domain (TM) 1128-1255 SEQ ID NO: 28
Examples also include the fusions of two or more of the above-listed domains, e.g., RBD1-(Gly)8-TM (SEQ ID NO: 10), RBD2-(Gly)8-TM (SEQ ID NO: 12), RBD1-(Gly)8-RBD2 (SEQ ID NO: 14), and RBD1-(Gly)8-RBD2-(Gly)8-TM (SEQ ID NO: 16). In these fusions, different S protein fragments are joined by a linker of 8 glycines. Additional examples include those listed in Table 2 shown in Example 6 below. Preferably, the polypeptide of this invention contains SEQ ID NO: 24 or 26. In one embodiment, the polypeptide is a glycoprotein containing a polysaccharide, e.g., a polysaccharide from S. pneumococcal. In another embodiment, the polypeptide is a fusion protein including a heterologous polypeptide that contains an Fc portion of an immunoglobin, e.g., an IgG. Preferably, the immunoglobin is IgG1, and more preferably, a human IgG1. The fusion protein can also include a heterologous polypeptide that contains a surface portion of a protein of a pathogen, such as the HA or NA of an influenza virus.
[0005]An "isolated polypeptide" refers to a polypeptide substantially free from naturally associated molecules, i.e., it is at least 75% (i.e., any number between 75% and 100%, inclusive) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide of the invention can be purified from a natural source, produced by recombinant DNA techniques, or by chemical methods. A "heterologous" protein or nucleic acid is one that originates from a foreign species, or, if from the same species, is substantially modified from its original form.
[0006]The invention also features an isolated nucleic acid that contains a sequence encoding one of the above-mentioned polypeptides. Examples of the sequence include (1) those encoding S, RBD1, RBD2, S1, S2, S3, RBD-2C, RBD-55, and TM, which, respectively, correspond to nt. 21492-25259, 21729-22172,--22341-23696, 21492-22490, 22491-23489, 23490-24491, 22791-22892, 23181-23330, and 24873-25256 of GenBank Accession No. AY278741 (SEQ ID NOs: 3, 5, 7, 17, 19, 21, 23, 25, and 27, respectively)
[0007]Listed below are exemplary sequences that encode fusion proteins RBD1-(Gly)8-TM, RBD2-(Gly)8-TM, RBD1-(Gly)8-RBD2, and RBD1-(Gly)8-RBD2-(Gly)8-TM (SEQ ID NOs: 9, 11, 13, and 15, respectively; linkers shown in the upper case):
TABLE-US-00003 SEQ ID NO: 9 catacgtttg gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgattatta ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc gataatgcat ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt ataagggcta tcaacctata gacgtagttc gtgatctacc ttctggtttt aacactttga aacctatttt taagttgcct ctcggtatta acattacaaa ttttagagcc GAATTCGGGG GCGGGGGTGG AGGTGGTGGC tcatt caaagaagag ctggacaagt acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacaca SEQ ID NO: 11 gagattgaca aaggaattta ccagacctct aatttcaggg ttgttccctc aggagatgtt gtgagattcc ctaatattac aaacttgtgt ccttttggag aggtttttaa tgctactaaa ttcccttctg tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct gtgctctaca actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact ggtaattata attataaata taggtatctt agacatggca agcttaggcc ctttgagaga gacatatcta atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc cattaaatga ttatggtttt tacaccacta ccggcattgg ctaccaacct tacagagttg tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaatcatcca ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgcttctg atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgct cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc aatatggGCG GCCGCCTGGG GGCGGGGGTG GAGGTGGTGG Ctcatt caaagaagag ctggacaagt acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacaca SEQ ID NO: 13 catacgtttg gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgattatta ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc gataatgcat ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt aacactttga aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc GAATTCGGGG GCGGGGGTGG AGGTGGTGGC gagattgaca aaggaattta ccagacctct aatttcaggg ttgttccctc aggagatgtt gtgagattcc ctaatattac aaacttgtgt ccttttggag aggtttttaa tgccactaaa ttcccttctg tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgactactct gtgctctaca actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca tgggttgtgt ccttgcttgg aatactagga acattgatgc tacctcaact ggtaattata attataaata taggtatctt agacatggca agcttaggcc ctttgagaga gacatatcta atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct tacagagttg tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaattatcca ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgtttctg atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgct cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc aatatgg SEQ ID NO: 15 catacgtttg gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgactatta ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc gataatgcat ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt aacactttga aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc GAATTCGGGG GCGGGGGTGG AGGTGGTGGC gagattgaca aaggaattta ccagacctct aattccaggg ttgttccctc aggagatgtt gtgagattcc ctaatattac aaacttgtgt ccttttggag aggtttttaa tgctactaaa ttcccttctg tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct gtgctctaca actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact ggtaattata attataaata taggtatctt agacatggca agcttaggcc ctttgagaga gacatatcta atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct tacagagttg tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaattatcca ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgtttctg atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgct cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc aatatggGCG GCCGCCTGGG GGCGGGGGTG GAGGTGGTGG Ctcatt caaagaagag ctggacaagt acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacaca
[0008]Additional examples of the nucleic acid of this invention include nucleic acids encoding the peptides listed in Table 2. In a preferred embodiment, the nucleic acid contains SEQ ID NO: 23 or 25.
[0009]A "nucleic acid" refers to a DNA molecule (e.g., a cDNA or genomic DNA), an RNA molecule (e.g., an mRNA), or a DNA or RNA analog. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated nucleic acid" is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The nucleic acid described above can be used to express the polypeptide of this invention. For this purpose, one can operatively linked the nucleic acid to suitable regulatory sequences to generate an expression vector.
[0010]A "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The vector can be capable of autonomous replication or integrate into a host DNA. Examples of the vector include a plasmid, cosmid, or viral vector. The vector of this invention includes a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. A "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vector can be introduced into host cells to produce the polypeptide of this invention. Also within the scope of this invention is a host cell that contains the above-described nucleic acid. Examples include E. coli cells, insect cells (e.g., using baculovirus expression vectors), yeast "effective amount" is an amount of the composition that is capable of producing a medically desirable result, e.g., as described above, in a treated subject.
[0011]The details of one or more embodiments of the invention are set forth in the accompanying description below. Other advantages, features, and objects of the invention will be apparent from the detailed description and the claims.
DETAILED DESCRIPTION
[0012]This invention relates to receptor binding domains or immunogenic fragments of the S protein of a coronavirus, such as SARS. Since these domains mediate target cell binding and entry of the coronavirus or induce immune response, they can be targeted for diagnosing or treating an infection with the coronavirus.
[0013]A polypeptide of this invention contains the sequence of the S protein, such as SEQ ID NO: 4 or an immunogenic fragment thereof. It can also contain the sequence of the S protein of SARS CoV TW1, Tor-2, SIN2500, SIN2774, SIN2748, SIN2677, SIN2679, CUHK-W1, HKU39849, GZO1, BJO1, BJO2, BJO3 BJO4, and other strains. In a particular embodiment, the polypeptide contains a receptor-binding domain of the S protein or a functional equivalent. A functional equivalent of the a protein receptor binding domain refers to a polypeptide derived from the coronavirus S protein, e.g., a fusion polypeptide or a polypeptide having one or more point mutations, insertions, deletions, truncations, or a combination thereof. In particular, such functional equivalents include polypeptides, whose sequences differ from the S protein by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions. Such a functional equivalent can be encoded by a nucleic acid that hybridizes under high stringency conditions to a probe the sequence of which consists of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25. The term "hybridizes under stringent conditions" refers to conditions for hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. All of the above-described functional equivalents retain substantially the receptor binding activity of coronavirus, e.g., SRAS CoV S protein, i.e., binding to target cells including VERO E6, NIH3T3. This activity can be determined by the assays described in the examples presented below.
[0014]A polypeptide of the invention can be obtained as a synthetic polypeptide or a recombinant polypeptide. To prepare a recombinant polypeptide, a nucleic acid encoding it can be linked to another nucleic acid encoding a fusion partner, e.g., Glutathione-S-Transferase (GST), 6×-His epitope tag, or M13 Gene 3 protein. The resultant fusion nucleic acid expresses in suitable host cells a fusion protein that can be isolated by methods known in the art. The isolated fusion protein can be further treated, e.g., by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of this invention.
[0015]A polypeptide of the invention can be used to generate antibodies in animals (for production of antibodies) or humans (for treatment of diseases). Methods of making monoclonal and polyclonal antibodies and fragments thereof in animals are known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term "antibody" includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, Fv, scFv (single chain antibody), and dAb (domain antibody; Ward, et. al. (1989) Nature, 341, 544). These antibodies can be used for detecting the S polypeptide, e.g., in determining whether a test sample from a subject contains coronavirus or in identifying a compound that binds to the polypeptide. As these antibodies interfere with the cell binding and entry of the coronavirus, they are also useful for treating a coronavirus infection.
[0016]In general, to produce antibodies against a polypeptide, the polypeptide is coupled to a carrier protein, such as KLH, mixed with an adjuvant, and injected into a host animal. Antibodies produced in the animal can then be purified by peptide affinity chromatography. Commonly employed host animals include rabbits, mice, guinea pigs, and rats. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, CpG, surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Useful human adjuvants include BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
[0017]Polyclonal antibodies, heterogeneous populations of antibody molecules, are present in the sera of the immunized subjects. Monoclonal antibodies, homogeneous populations of antibodies to a polypeptide of this invention, can be prepared using standard hybridoma technology (see, for example, Kohler et al. (1975) Nature 256, 495; Kohler et al. (1976) Eur. J. Immunol. 6, 511; Kohler et al. (1976) Eur J Immunol 6, 292; and Hammerling et al. (1981) Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y.). In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described in Kohler et al. (1975) Nature 256, 495 and U.S. Pat. No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al. (1983) Immunol Today 4, 72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2026, and the EBV-hybridoma technique (Cole et al. (1983) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the monoclonal antibodies of the invention may be cultivated in vitro or in vivo. The ability to produce high titers of monoclonal antibodies in vivo makes it a particularly useful method of production.
[0018]In addition, techniques developed for the production of "chimeric antibodies" can be used. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage library of single chain Fv antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge. Moreover, antibody fragments can be generated by known techniques. For example, such fragments include, but are not limited to, F(ab')2 fragments that can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments. Antibodies can also be humanized by methods known in the art. For example, monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; and Oxford Molecular, Palo Alto, Calif.). Fully human antibodies, such as those expressed in transgenic animals are also features of the invention (see, e.g., Green et al. (1994) Nature Genetics 7, 13; and U.S. Pat. Nos. 5,545,806 and 5,569,825).
[0019]A polypeptide of the invention can also be used to prepare an immunogenic composition (e.g., a vaccine) for generating antibodies against coronavirus (e.g., SRAS CoV) in a subject susceptible to the coronavirus. Such compositions can be prepared, e.g., according to the method described in the examples below, or by any other equivalent methods known in the art. The composition contains an effective amount of a polypeptide of the invention, and a pharmaceutically acceptable carrier such as phosphate buffered saline or a bicarbonate solution. The carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. An adjuvant, e.g., a cholera toxin, Escherichia coli heat-labile enterotoxin (LT), liposome, immune-stimulating complex (ISCOM), or immunostimulatory sequences oligodeoxynucleotides (ISS-ODN), can also be included in a composition of the invention, if necessary. The S protein, fragments or analogs thereof or peptides may be components of a multivalent composition of vaccine against respiratory diseases. This multivalent composition contains at least one immunogenic fragment of S protein described above, along with at least one protective antigen isolated from influenza virus, para-influenza virus 3, Strentococcus pneumoniae, Branhamella (Moroxella) gatarhalis, Staphylococcus aureus, or respiratory syncytial virus, in the presence or absence of adjuvant.
[0020]Methods for preparing vaccines are generally well known in the art, as exemplified by U.S. Pat. Nos. 4,601,903; 4,599,231; 4,599,230; and 4,596,792. Vaccines may be prepared as injectables, as liquid solutions or emulsions. The S protein, fragments or analogs thereof or peptides corresponding to portions of S protein may be mixed with physiologically acceptable and excipients compatible. Excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof. The vaccine may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness of the vaccines. Methods of achieving adjuvant effect for the vaccine includes use of agents, such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solutions in phosphate buffered saline. Vaccines may be administered parenterally, by injection subcutaneously or intramuscularly. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides. Oral formulations may include normally employed incipients such as, for example, pharmaceutical grades of saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of the S protein, fragment analogs, or peptides.
[0021]The vaccines are administered in a manner compatible with the dosage formulation, and in an amount that is therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms of the polypeptide of this invention. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage of the vaccine may also depend on the route of administration and varies according to the size of the host.
[0022]Use of polypeptide in vivo may first require chemical modification of the peptides since they may not have a sufficiently long half-life. A chemically modified peptide or a peptide analog includes any functional chemical equivalent of the peptide characterized by its increased stability and/or efficacy in vivo or in vitro in respect of the practice of the invention. The term peptide analog also refers to any amino acid derivative of a peptide as described herein. A peptide analog can be produced by procedures that include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of cross-linkers and other methods that impose conformational constraint on the peptides or their analogs. Examples of side chain modifications include modification of amino groups, such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with methylacetimidate; acetylation with acetic anhydride; carbamylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzene sulfonic acid (TNBS); alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxa-5'-phosphate followed by reduction with NABH4. The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via o-acylisourea formation followed by subsequent derivatization, for example, to a corresponding amide. Sulfhydryl groups may be modified by methods, such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide; maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuric-4-nitrophenol and other mercurials; carbamylation with cyanate at alkaline pH. Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tryosine residues may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
[0023]A nucleic acid molecule of this invention may also be used directly for immunization by administration of the nucleic acid directly to a subject via a live vector, such as Salmonella, BCG, adenovirus, poxvirus or vaccinia. Immunization methods based on nucleic acids are well known in the art.
[0024]A subject susceptible to coronavirus infection can be identified and administered a polypeptide-containing composition of the invention. The dose of the composition depends, for example, on the particular polypeptide, whether an adjuvant is co-administered with the polypeptide, the type of adjuvant co-administered, the mode and frequency of administration, as can be determined by one skilled in the art. Administration is repeated as necessary, as can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly intervals. A booster shot can be given at 4 to 8 weeks after the first immunization, and a second booster can be given at 8 to 12 weeks, using the same formulation. Sera or T-cells can be taken from the subject for testing the immune response elicited by the composition against the coronavirus S protein or infection. Methods of assaying antibodies or cytotoxic T cells against a protein or infection are well known in the art. Additional boosters can be given as needed. By varying the amount of polypeptide, the dose of the composition, and frequency of administration, the immunization protocol can be optimized for eliciting a maximal immune response. Before a large scale administering, efficacy testing is desirable. In an efficacy testing, a non-human subject can be administered via an oral or parenteral route with a composition of the invention. After the initial administration or after optional booster administration, both the test subject and the control subject (receiving mock administration) are challenged with an LD95 dose of a coronavirus. End points other than lethality can also be used. Efficacy is determined if subjects receiving the composition dies at a rate lower than control subjects. The difference in death rates should be statistically significant.
[0025]The above-described S protein and its fragment can be used as a carrier and linked to other antigens of interest to generate antibodies against the antigens. The S protein or its fragment can be generally utilized to prepare chimeric molecules and conjugate compositions against pathogenic bacteria, including encapsulated bacteria. For example, the glycoconjugates of the present inventions may be applied to immunize a subject to generate antibodies against the bacteria and confer protection against infection with any bacteria having polysaccharide antigens, e.g., Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli, Neisseria meningitidis, Salmonella typhi, Streptococcus mutans, Cryptococcus neoformans, Klebsiella, Staphylococcus aureus, and Pseudomonas aeruginosa. In addition, as a carrier, the S protein or fragment may be used to induce immunity toward abnormal polysaccharides of tumor cells, thereby to produce anti-tumor antibodies for chemotherapy or diagnosis.
[0026]Also within the scope of this invention is a diagnosing method using the above-described polypeptides or antibodies. Presence of the polypeptides or antibodies in a subject indicates that the subject is infected with a coronavirus. To detect the antibodies or polypeptides, one can obtain a test sample from a subject and detect the presence or absence of the antibodies or polypeptides using standard techniques, including ELISAs, immunoprecipitations, immunofluorescence, EIA, RIA, and Western blotting analysis.
[0027]The nucleic acid of this invention is useful as a hybridization probe for identifying coronavirus, e.g., SARS CoV, in a sample. The sample can be a clinical sample, including exudates, body fluids (e.g., serum, amniotic fluid, middle ear effusion, sputum, bronchoalveolar lavage fluid) and tissues. A variety of hybridization conditions may be employed to achieve varying degrees of selectivity of the probe toward the target sequences. A high degree of selectivity requires stringent conditions, such as that described in the Summary section
[0028]A hybridization reaction can be performed both in a solution or on a solid phrase. In a solid phase, a test sequence from a sample is affixed to a selected matrix or surface. The fixed nucleic acid is then subjected to specific hybridization with selected probes comprising the nucleic acid of the present invention under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required depending on, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe etc. Following washing of the hybridization surface to remove non-specifically bound probe molecules, specific hybridization is detected or quantified, by means of the label. The selected probe should be at least 18 bp and may be in the range of 30 bp to 90 bp long.
[0029]In addition, A small interference RNA (SiRNA) corresponding to the nucleotide sequences of the present invention comprising the sequence of the S protein receptor binding domains such as RBD1 and RBD2, can be useful to block SARS CoV replication in vivo.
[0030]A polypeptide of this invention can also be used in a screening method of identifying a compound for treating an infection with a coronavirus, e.g., SARS CoV. The method includes (1) contacting a polypeptide of this invention with a suitable cell, to which the coronavirus binds to; and (2) determining a binding level between the polypeptide and the cell the presence or absence of a test compound. The binding level in the presence of the test compound, if lower than that in the absence of the test compound, indicates that the test compound can be used to treat an infection with the coronavirus. Examples of the cell include VERO E6 cells, NIH3T3 cells, HeLa cells, BHK-21 cells, and COS-7 cells. One can also use other cells that are capable of binding to a coronavirus.
[0031]The above-described polypeptides and antibodies can be used for treating an infection with a coronavirus, e.g., SARS. The invention therefore features a method of treating SARS, e.g., by administering to a subject in need thereof an effective amount of a polypeptide, an antibody, or a compound of the invention. Subjects to be treated can be identified as having, or being at risk for acquiring, a condition characterized by SARS. This method can be performed alone or in conjunction with other drugs or therapy.
[0032]Thus, also within the scope of this invention is a pharmaceutical composition that contains a pharmaceutically acceptable carrier and an effective amount of a polypeptide, an antibody, or a compound of the invention. The pharmaceutical composition can be used to treat coronavirus infection, such as SARS. The pharmaceutically acceptable carrier includes a solvent, a dispersion medium, a coating, an antibacterial and antifungal agent, and an isotonic and absorption delaying agent.
[0033]In one in vivo approach, a composition of this invention (e.g., a composition containing a polypeptide, an antibody, or a compound of the invention) is administered to a subject. Generally, the antibody or the compound is suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
[0034]The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compositions available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the composition in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
[0035]A pharmaceutical composition of the invention can be formulated into dosage forms for different administration routes utilizing conventional methods. For example, it can be formulated in a capsule, a gel seal, or a tablet for oral administration. Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the composition with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. The composition can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, conventional filler, and a tableting agent. The pharmaceutical composition can be administered via the parenteral route. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipient. Cyclodextrins, or other solubilizing agents well known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic agent.
[0036]The efficacy of a composition of this invention can be evaluated both in vitro and in vivo. Briefly, the composition can be tested for its ability to inhibit the binding between a coronavirus and its target cell in vitro. For in vivo studies, the composition can be injected into an animal (e.g., a mouse model) and its therapeutic effects are then accessed. Based on the results, an appropriate dosage range and administration route can be determined.
[0037]The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.
EXAMPLE 1
[0038]In this example, the gene encoding S protein of SARS CoV was cloned. A SARS CoV, designated as "SARS-CoV TW1," was isolated from a SARS patient in Taiwan. Seven pairs of PCR primers were designed based on the sequence of the Urbani strain (SEQ ID NO: 1) or the SARS CoV TOR2 strain. The positions of the primers' 5' ends within the Urbani genome were summarized below:
TABLE-US-00004 5' primer 3' primer Pair 1 21,492 22,000 Pair 2 22,000 22,600 Pair 3 22,600 23,100 Pair 4 23,075 23,780 Pair 5 23,765 24,320 Pair 6 24,300 24,875 Pair 7 24,850 25,244
[0039]Seven products were generated by PCR reactions respectively and ligated together to form a sequence that encoded the S protein. The sequence was then subcloned into pUC19 to produce pUC19/S and used to transform E. coli HB101. Plasmid DNA was prepared from two E. coli HB101 colonies and sequenced on an ABI 370A DNA sequencer. Subsequent sequence analysis revealed that the sequence differed from that of the TOR2 strain by 3 base pairs and that it is about 30.1% identical to that of human coronavirus 229E.
[0040]SARS CoV M and E proteins (GenBank Accession Nos. AAP13443 and 13444) were also cloned and expressed. The E-M fusion protein corresponds to residues 8751 to 9057 of the first open reading frame of SEQ ID NO: 1. Construction of DNA plasmids containing genes for E and M proteins was performed by standard molecular biology methods (Sambrook et al (1989) Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.). The constructs utilized a pUC-based expression vector, which was shown to result in optimal expression of reporter genes. Each vector employed the human cytomegalovirus promoter, enhancer, intron A, and the bovine growth hormone termination and polyasenylation sequences. The tissue plasminogen activator signal sequence was use to enhance the level of expression. The M and E proteins were further expressed in host cells to generated virus like particles.
EXAMPLE 2
[0041]It is known that, in SARS CoV, the S native protein is expressed in small quantities. To obtain a large amount of the S protein, there is a need to either express it in a heterologous system, such as E. coli, or to modify SARS CoV to increase the native S protein expression.
[0042]The above-described PUC19/S was transformed into E. coli. to express the S protein. It was found that the full-length recombinant S (rS) protein was not expressed in E. coli. Vectors encoding different S protein fragments fused to Myc-His tag were then constructed and transformed in E. coli. The fragments include the N-terminal amino acids 80-228 of the S protein (receptor binding domain 1; RBD1); the middle region encompassing amino acids 284-735 of the S protein (receptor binding domain 2; RBD2), the transmembrane domain (TM), and fusions of them.
[0043]To examine the expressed protein, antisera against various SARS CoV proteins were generated The following SARS CoV polypeptides were synthesized by standard techniques:
TABLE-US-00005 RBD1-specific peptide (SEQ ID NO:29) KSGNFKHLREFVFKNKDGFLYVYKGQPIDV RBD2-specific peptide (SEQ ID NO:24) GNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPC TM-specific peptide (SEQ ID NO:30) DSFKEELDRY FKNHTSPDVD LGDISGINAS VV E-specific peptide (SEQ ID NO:31) ALRLCAYCCN IVNVSLVKPT VYVYSRVKNL NSSEG M-specific peptide (SEQ ID NO:32) MADNGTITVE ELKQLLEQWN LVICFLFLAW IML
[0044]More specifically, 200 μg of each peptide was mixed with the completed Fruend's adjuvant and injected at day 0 and injected into rabbits by standard techniques At day 14 and 56, the rabbits were boosted with half of the amount of the peptide in the incomplete Fruend's adjuvant At day 78, the rabbits were bleed, and the blood were tested for antiserum titer by ELISA The results are shown in Table 1
TABLE-US-00006 TABLE 1 Rabbit immunogenicity of SARS CoV peptides Reactivity of Anti-peptide sera to target peptide Peptides Pre-Immune Post-booster Final Bleed RBD1-specific 0 5120 10240 RBD2-specific 0 10240 41440 TM-specific 0 5120 10240 E-specific 0 1280 5120 M-specific 0 5120 10240
[0045]To generate a vector encoding RBD1, the following two primers were used for PCR: 5' primer: GGATCCGCCACC ATG catacgtttg g (SEQ ID NO:33); and 3' primer: aa ttttagagcc GAATTC (SEQ ID NO:34) The two primers contained EcoRI and BamHI sites to facilitate subsequent cloning of PCR products A ˜500 base pair (bp) fragment was obtained and subcloned into pcDNA-A4 to generate pcDNA-A4-D1 plasmid, which encoded a fusion protein of Myc-His-RBD This plasmid was transformed into E coli HB101 to express recombinant RBD1 (rRBD1) It was found that, upon induction, the transformed clones expressed a 20 kDa protein This protein was expressed at high levels in inclusion bodies and was recognized by anti-RBD-1 antisera and anti-His tag antibody on Western blot analysis It was also found that protein was highly immunogenic, but not able to elicit protective antibodies against live virus challenge.
[0046]A vector encoding RBD2 was also generated More specifically, PCR was conducted using the following two primers 5' primer: GGATCCGCCACCATG gagattgaca (SEQ ID NO:35) and 3' primer: aatatgg GCGGCCGC (SEQ ID NO:36) to generate a 14 kb fragment After being digested by BamH1H-Not1, the resulting fragment was also subcloned into pcDNA-A4 The resultant vector was used to express RBD2 in the same manner described above It was found that a 50-kDa protein was expressed at high levels in both soluble form and in inclusion bodies Western blot analysis revealed that this protein was recognized by the S-specific antisera This rRBD2 fragment was highly immunogenic too and elicited even stronger neutralizing antibodies that could block SARS CoV binding to Vero cell (see Example 9 below).
[0047]The above-described recombinant proteins were isolated from E. coli. More specifically, E. coli pellet from a 250 mL culture was resuspended in 40 mL of 50 mM Tris, pH 8.0, and disrupted by sonication (3×10 minutes, 70% duty circle). The resultant mixture was centrifuged at 20,000×g. The pellet was re-extracted with 40 mL of 50 mM Tris, 0.5% Triton X-100, 10 mM EDTA, pH 8.0. The suspension was then sonicated for 10 minutes at 70% duty circle and centrifuged at 300×g for 5 minutes. The resulting supernatant was centrifuged again at 20,000×g for 30 minutes. The pellet was resuspended in 50 mM Tris, 0.5% Triton X-100, 10 mM EDTA, pH 8.0 and mixed with PBS/8 M urea to a final urea concentration of 6 M urea. The mixture was then dialyzed against PBS to remove urea and centrifuged at 300×g for 10 minutes. The supernatant was saved and stored at 4° C.
[0048]Ni-affinity chromatography was used to isolate rRBD1 and rRBD2 fusion proteins from inclusion body. The just described supernatant was loaded onto a Ni affinity column (2 mL) equilibrated with PBS containing 1% Triton X-100. The run-through of the column was discarded. After washing the column with 20 mL of PBS, the affinity column was eluted with 50 mM Tris-HCl buffer, pH 8.0, containing 5 mM EDTA. The protein-containing factions were collected and the purity was analyzed by SDS-PAGE.
[0049]It was estimated that about 10 mg of rRBD2 was recovered from 1 L of E. coli bacterial culture. The identity of rRBD2 was confirmed by both immunoblotting and protein sequencing. The N-terminal sequence of this polypeptide was found to be Met-Ala-Glu-Leu-Lys-Cys, which corresponds to residues 284 to 288 of the sequence of S protein.
EXAMPLE 3
[0050]In this example, additional fragments of the SARS coronavirus S protein were expressed in baculovirus and SF21 insect cell.
[0051]Nucleic acids encoding 1-333, 334-666, and 667-999 amino acid of the S protein (spike1, spike2, and spike3; S1, S2, and S3, respectively) were obtained by PCR with primer sets listed below, respectively, in the manner similar to that described in Example 1
TABLE-US-00007 Amplified Primer fragment name Sequence (5' to 3') Sense Spike1 S1F AGGGGATCCATGTTTATTTTCT S (1-333 aa) TATTATTTCTTACTC S1R CCTGGATCCTTTAGTAGCATTA AS AAAACCTCTCCA Spike2 S2F AGGGGATCCTTCCCTTCTGTCT S (334-666 aa) ATGCATGGGAGA S2R CCTGGATCCTAATAAAGAAACT AS GTATGGTAACTA Spike3 S3F AGGGGATCCCGTAGTACTAGCC S (667-999 aa) AAAAATCTATTG S3R CCTGGATCCTTCAGCAGCCCTG AS ATTAGTTGTTGT RBD1 RBD1F CATACGTTTGGCAACCCTGTC S (74-253 aa) RBD1R AACATTACAAATTTTAGAGCC AS RBD2 RBD2F GAGATTGACAAAGGAATTTAC S (294-739 aa) RBD2R CTAATTTGCTTCTCCAATATGG AS RBD3 RBD3F ATGGCTAAAACCTCCGTAGAT S (713-1113 aa) RBD3R AATTGTGATGTCGTTATTGGC AS TM TM1F ACTTCAAAAATCATACATCA S (1130-1255 aa) TM1R GGTGTCAAATTACATTACACAT AS AA SEQ ID NOs 37-50, respectively
[0052]The PCR products were inserted into the pCR2.1 vector by TA cloning. The coding sequences were than released by BamHI digestions and ligated to BamHI-cutted pSecTagb/hIgG1.Fc vector, thereby in-frame fusing the S protein-encoding sequence to that encoding the human IgG1 Fc, The resultant vectors encodes fusion proteins spike1-Fc, spike2-Fc, and spike3-Fc. To generate corresponding baculovirus transfer vectors, the three fusion genes were released by NheI/XhoI digestion and ligated to XbaI/XhoI-cutted pBacPAK9 vectors.
[0053]The just-described pBacPAK9 vectors were co-transfected into Sf21 cells with Bsu36 I-digested BacPAK6 viral DNA by Bacfectin (Clontech 6144-1). Each resulting viral plaque was picked by performing plaque assays on the co-transfection supernatant. The recombinant viruses were confirmed by PCR. Sf21 cells were then infected with virus at a small scale to characterize gene expression and to determine the optimum harvest time and infection ratio by standard methods. Recombinant viruses were amplified to high virus titer to obtain working stocks for large-scale infection.
[0054]To purify recombinant proteins, Sf21 cells were cultured in spinner flask at a starting concentration of 2×105/ml in the first 3-5 days. After reaching 1-2×106 cells/ml, the cells were infected with the above-described recombinant baculoviruses at M.O.I. of 5-10 and cultured for 4-5 days. The supernatants were then collected and cell debris was removed by centrifugation. The supernatant was loaded onto protein A Sepharose® 4 Fast Flow beads (Amersham Biosciences 17-0974). Finally, the bound Fc-fusion protein was eluted with a 0.1 M glycine buffer (pH 3.0), followed by dialysis against PBS. The purity and the concentration of purified proteins were assessed by a standard silver staining method.
[0055]Five milligrams of S1-Fc fusion protein crude extract prepared in the manner described above were dissolved in 5 mL of phosphate buffer saline (PBS) containing 1% Triton X-100. The solution was then loaded onto a Protein A-Sepharose 4B column (2 mL) equilibrated with PBS containing 1% Triton X-100. The run-through of the column was discarded. The column was washed with 20 mL of PBS and the S1-FC fusion protein was eluted with 50 mM Gly-HCl buffer, pH 3.0. Elution was monitored by absorbance at 280 nm. Protein-containing fractions (2 mL/fraction) were collected and pooled. The purity of the protein was assessed by SDS-PAGE.
[0056]Certain plasmids described above was deposited with the American Type Culture Collection (ATCC) located at 10801 University Boulevard, Manassas, Va. 20110-2209 U.S.A. pursuant to the Budapest Treaty and prior to the filing of this application. Samples of the deposited plasmids will become available to the public upon grant of a patent based upon this United States patent application.
EXAMPLE 4
[0057]The above-described recombinant RBD1, RBD2, S1-FC, and S2-Fc were used to produce of S-specific antisera. The purified recombinant proteins were emulsified in the Freund's complete adjuvant (Difco) and injected intramuscularly (IM) into New Zealand White rabbits (Maple Lane) or guinea pigs (Charles River) at a dose of 10 to 100 μg/injection. The animals were boosted on day 28 with another half of dose of the corresponding S fragment emulsified in Freund's incomplete adjuvant. On day 42, a blood sample was taken from each animal via the marginal ear vein for titer determination by standard methods. Animals that generated specific antibodies were bled to obtain more antisera.
[0058]To examine the immunogenicity of the RBD1 or 2 fusion protein, guinea pigs or mice were immunized with RBD1 or 2 of various amounts. The doses between 10 to 100 μg/injection RBD1 induced high IgG titers in guinea pigs when administered in the presence of either Freund's adjuvant or AlPO4. In the mice, RBD1 or 2 appeared to be immunogenic at a dose as low as 5 μg/injection in either Freund's adjuvant.
[0059]A ferret model was used to examine the protective ability of anti-RBD1 or 2 sera against a SARS CoV infection. It was found that ferrate passively immunized with guinea pig anti-RBD2 antisera, but not anti-RBD1 sera, were significantly protected than controls injected with pre-immune sera.
[0060]The above-described S1-Fc or S2-Fc fusion protein was used to purify S protein-specific polyclonal antibodies by affinity chromatography. The recombinant S1-Fc or S2-Fc fusion protein was conjugated to cyanogen bromide-activated Sepharose to form an affinity column. The affinity column was then used to purify antibodies from a rabbit hyperimmune anti-inactivated SARS CoV antiserum. The affinity purified-antibodies were shown by immunoblotting to react with a 200-kDa component present in the lysates of SARS Cov isolates.
[0061]Similarly, antisera raised against the recombinant fusion protein or the purified RBD1, RBD2, S1 and S2 can also be purified in the same manner.
EXAMPLE 5
[0062]Purified recombinant RBD2 were conjugated with S. pneumococcal oligosaccharides 14 (14F) by periodate oxidation in the manner described in U.S. Pat. No. 4,356,170. S. pneumococcal oligosaccharides 14 was prepared by controlled acid hydrolysis. The mean molecular size of the 14F molecules used for conjugation was determined as approximately 20,000 Daltons. The conjugation was carried out with or without a linker molecule. A 14/RBD2 molar ratio of approximately 7 was used to provide an excess of 14F hapten.
[0063]To prepare 14-BSA conjugates, 0.5 mL of periodate-oxidized 14 (25 mg in 1 mL of 0.1 M sodium phosphate buffer, pH 6.0), prepared from native 14F treated with aqueous periodic acid (Carlone et al, 1986 J. Clin. Microbiol. 24:330-331.), was added to bovine serum albumin (BSA) (1.32 mg; 0.02 μmol) in 0.5 mL of 0.2 M sodium phosphate buffer, pH 8.0, followed by the addition of sodium cyanoborohydride (14 μg; 0.22 μmol; 10 eqv. to BSA). After incubation at 37° C. for 5 days, the reaction mixture was dialyzed against 4 L of 0.1 M phosphate buffer, pH 7.5. The resulting solution was applied onto an analytical Superose 12 column (15×0.300 mm, Pharmacia) equilibrated with 0.2 M sodium phosphate buffer, pH 7.2, and eluted with the same buffer. Fractions were monitored for absorbance at 230 nm. The first major protein peak was pooled and concentrated in a Centriprep 30 to 2.2 mL. The amount of protein was found, by the Bio Rad protein assay, to be 300 ug/mL. The presence of 14 oligosccharides in the protein conjugate fraction was confirmed by the Orcinol test.
[0064]The above-described RBD2-14 S. pneumococcal polysaccharide conjugate was then used to produce anti-14 S. pneumococcal polysaccharide antisera in animals. Rabbits were immunized intramuscularly with 14-RBD2 conjugates (5 to 50 μg 14 equivalent) mixed with 3 mg AlPO4 per mL, followed by two booster doses (half amount of the same immunogen) at 2-week intervals. Antisera were collected every 2 weeks after the first injection, heat-inactivated at 56° C. for 30 minutes and stored at -20° C. It was found that the immunization elicited both primary and secondary immune responses against PRP-IgG and S protein. Rabbit anti-RBD2-14F antisera also strongly reacted with both native S and rS as determined by immunoblot analysis. These results indicate that RBD2 can be used as a carrier protein in a conjugate vaccine. Since RBD2-14 S. pneumococcal polysaccharide conjugate elicited antibodies against both 14F and S, it can be used to d thus should enhance the level of protection against S. pneumococcal-related diseases, especially in infants.
EXAMPLE 6
[0065]To map the linear B-cell epitopes of the SARS S protein, overlapping synthetic peptides covering the entire S protein were synthesized. These peptides were listed in Table 2 below.
TABLE-US-00008 TABLE 2 Synthetic SARS CoV S peptides Peptide SEQ ID ID No MW Sequence NO: RBD1-related fragments 1 1,6812 VIPFKDGIYFAATEK 51 2 1,6520 DGIYFAATEKSNVVR 52 3 1,6029 AATEKSNVVRGWVFG 53 4 1,6499 SNVVRGWVFGSTMNN 54 5 1,6238 GWVEGSTMNNKSQSV 55 6 1,6449 STMNNKSQSVIIINN 56 7 1,5978 KSQSVIIINNSTNVV 57 8 1,6261 IIINNSTNVVIRACN 58 9 1,6661 STNVVIRACNFELCD 59 10 1,7423 IRACNFELCDNPFFA 60 11 1,7272 FELCDNPFFAVSKPM 61 12 1,6439 NPFFAVSKPMGTQTH 62 13 1,6750 VSKPMGTQTHTMIFD 63 14 1,6820 GTQTHTMIFDNAFNC 64 15 1,8113 TMIFDNAFNCTFEYI 65 16 1,7111 NAFNCTFEYISDAFS 66 17 1,7050 TFEYISDAFSLDVSE 67 18 1,5849 SDAFSLDVSEKSGNF 68 19 1,7412 LDVSEKSGNFKHLRE 69 20 1,8334 KSGNFKHLREFVFKN 70 21 1,8605 KHLREFVFKNKDGFL 71 22 1,8074 FVFKNKDGFLYVYKG 72 23 1,7882 KDGFLYVYKGYQPID 73 24 1,8101 YVYKGYQPIDVVRDL 74 25 1,7019 YQPIDVVRDLPSGFN 75 26 1,6381 VVRDLPSGFNTLKPI 76 27 1,6543 PSGFNTLKPIFKLPL 77 RBD2-related fragments 28 1,6362 AELKCSVKSFEIDKG 78 29 1,6840 SVKSFEIDKGIYQTS 79 30 1,7510 EIDKGIYQTSNFRVV 80 31 1,6638 IYQTSNFRVVPSGDV 81 32 1,6849 NFRVVPSGDVVRFPN 82 33 1,6140 PSGDVVRFPNITNLC 83 34 1,6881 VRFPNITNLCPFGEV 84 35 1,6361 ITNLCPFGEVFNATK 85 36 1,6850 PFGEVFNATKFPSVY 86 37 1,8262 FNATKFPSVYAWERK 87 38 1,8103 FPSVYAWERKKISNC 88 39 1,7522 AWERKKISNCVADYS 89 40 1,6581 KISNCVADYSVLYNS 90 41 1,6960 VADYSVLYNSTFFST 91 42 1,7593 VLYNSTFFSTFKCYG 92 43 1,6692 TFFSTFKCYGVSATK 93 44 1,6443 FKCYGVSATKLNDLC 94 45 1,6561 VSATKLNDLCFSNVY 95 46 1,6891 LNDLCFSNVYADSFV 96 47 1,6449 FSNVYADSFVVKGDD 97 48 1,6018 ADSFVVKGDDVRQIA 98 49 1,5226 VKGDDVRQIAPGQTG 99 50 1,5697 VRQIAPGQTGVIADY 100 51 1,6179 PGQTGVIADYNYKLP 101 52 1,7432 VIADYNYKLPDDFMG 102 53 1,7543 NYKLPDDFMGCVLAW 103 54 1,7372 DDFMGCVLAWNTRNI 104 55 1,6470 CVLAWNTRNIDATST 105 56 1,6859 NTRNIDATSTGNYNY 106 57 1,8112 DATSTGNYNYKYRYL 107 58 1,9276 GNYNYKYRYLRHGKL 108 59 2,0017 KYRYLRHGKLRPFER 109 60 1,8063 RHGKLRPFERDISNV 110 61 1,7580 RPFERDISNVPFSPD 111 62 1,5589 DISNVPFSPDGKPCT 112 63 1,5229 PFSPDGKPCTPPALN 113 64 1,6422 GKPCTPPALNCYWPL 114 65 1,7522 PPALNCYWPLNDYGF 115 66 1,7832 CYWPLNDYGFYTTTG 116 67 1,6789 NDYGFYTTTGTGYQP 117 68 1,6989 YTTTGIGYQPYRVVV 118 69 1,7651 IGYQPYRVVVLSFEL 119 70 1,6731 YRVVVLSFELLNAPA 120 71 1,5140 LSFELLNAPATVCGP 121 72 1,4689 LNAPATVCGPKLSTD 122 73 1,5990 TVCGPKLSTDLIKNQ 123 74 1,7191 KLSTDLIKNQCVNFN 124 75 1,7071 LIKNQCVNFNFNGLT 125 76 1,5380 CVNFNFNGLTGTGVL 126 77 1,4609 FNGLTGTGVLTPSSK 127 78 1,6039 GTGVLTPSSKRFQPF 128 79 1,7928 TPSSKRFQPFQQFGR 129 80 1,8558 RFQPFQQFGRDVSDF 130 81 1,7387 QQFGRDVSDFTDSVR 131 82 1,6508 DVSDFTDSVRDPKTS 132 83 1,6710 TDSVRDPKTSEILDI 133 84 1,6181 DPKTSEILDISPCAF 134 85 1,4890 EILDISPCAFGGVSV 135 86 1,3748 SPCAFGGVSVITPGT 136 87 1,3576 GGVSVITPGTNASSE 137 88 1,5038 ITPGTNASSEVAVLY 138 89 1,5937 NASSEVAVLYQDVNC 139 90 1,6087 VAVLYQDVNCTDVST 140 91 1,5707 QDVNCTDVSTAIHAD 141 92 1,5217 TDVSTAIHADQLTPA 142 93 1,7241 AIHADQLTPAWRIYS 143 94 1,7019 QLTPAWRIYSTGNNV 144 95 1,7668 WRIYSTGNNVFQTQA 145 96 1,5047 TGNNVFQTQAGCLIG 146 97 1,5708 FQTQAGCLIGAEHVD 147 98 1,5791 GCLIGAEHVDTSYEC 148 99 1,6310 AEHVDTSYECDIPIG 149 100 1,4951 TSYECDIPIGAGICA 150 101 1,4991 DIPIGAGICASYHTV 151 102 1,5602 AGICASYHTVSLLRS 152 103 1,6760 SYHTVSLLRSTSQKS 153 104 1,6360 SLLRSTSQKSIVAYT 154 105 1,5389 TSQKSIVAYTMSLGA 155 106 1,4810 IVAYTMSLGADSSIA 156 107 1,5129 MSLGADSSIAYSNNT 157 108 1,5489 DSSIAYSNNTIAIPT 158 109 1,6240 YSNNTIAIPTNFSIS 159 110 1,5880 IAIPTNFSISITTEV 160 111 1,6380 NFSISITTEVMPVSM 161 112 1,5759 ITTEVMPVSMAKTSV 162 113 1,6591 MPVSMAKTSVDCNMY 163 114 1,5891 AKTSVDCNMYICGDS 164 115 1,6211 DCNMYICGDSTECAN 165 TM-related Fragments 116 1,8833 DSFKEELDKYFKNHT 166 117 1,7901 ELDKYFKNHTSPDVD 167 118 1,6270 FKNHTSPDVDLGDIS 168 119 1,4418 SPDVDLGDISGINAS 169 120 1,4818 LGDISGINASVVNIQ 170 121 1,6379 GINASVVNIQKEIDR 171
122 1,7219 VVNIQKEIDRLNEVA 172 123 1,7671 KEIDRLNEVAKNLNE 173 124 1,6671 LNEVAKNLNESLIDL 174 125 1,6961 KNLNESLIDLQELGK 175 126 1,7941 SLIDLQELGKYEQYI 176 127 2,0132 QELGKYEQYIKWPWY 177 128 2,0604 YEQYIKWPWYVWLGF 178 129 1,8315 KWPWYVWLGFIAGLI 179 130 1,5843 VWLGFIAGLIAIVMV 180 131 1,5254 IAGLIAIVMVTILLC 181 132 1,5834 AIVMVTILLCCMTSC 182 133 1,6046 TILLCCMTSCCSCLK 183 134 1,4824 CMTSCCSCLKGACSC 184 135 1,4354 CSCLKGACSCGSCCK 185 136 1,5221 GACSCGSCCKFDEDD 186 137 1,6260 GSCCKFDEDDSEPVL 187 138 1,6730 FDEDDSEPVLKGVKL 188 S3 Fragments S3-1 GDSTECANLLLQYGS 189 S3-2 LQYGSFCTQLNRALS 190 S3-3 NRALSGIAAEQDRNT 191 S3-4 QDRNTREVFAQVKQM 192 S3-5 QVKQMYKTPTLKYFG 193 S3-6 LKYFGGFNFSQILPD 194 S3-7 QILPDPLKPTKRSFI 195 S3-8 KRSFIEDLLFNKVTL 196 S3-9 KVTLLADAGFMKQYG 197 S3-10 MKQYGECLGDINARD 198 S3-11 INARDLICAQKFNGL 199 S3-12 KFNGLTVLPPLLTDD 200 S3-13 LLTDDMIAAYTAALV 201 S3-14 TAALVSGTATAGWTF 202 S3-15 AGWTFGAGAALQIPF 203 S3-16 LQIPFAMQMAYRFNG 204 S3-17 YRFNGIGVTQNVLYE 205 S3-18 NVLYENQKQTANQFN 206 S3-19 ANQFNKAISQIQESL 207 S3-20 IQESLTTTSTALGKL 208 S3-21 ALGKLQDVVNQNAQA 209 S3-22 QNAQALNTLVKQLSS 210 S3-23 KQLSSNFGAISSVLN 211 S3-24 SSVLNDILSRLDKVEA 212 S3-25 LDKVEAEVQIDRLITG 213 S3-26 RLITGRLQSLQTYVTQQLIRA 214 (SEQ ID NO:24) RBD2 GNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPC (SEQ ID NO:26) RBD55 DPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQD VNCTDVSTAIHAD Note: RBD-55 includes the amino acids covering S84 to S91
[0066]The peptides were synthesized by an ABI 433A peptide synthesizer and optimized F-Moc chemistry according to the manufacturer's manual. The synthesized peptides were cleaved from the resin by Trifluoroacetic acid (TFA). They were then purified by reversed-phase high performance liquid chromatography (RP-HPLC) on a Vydac C4 semi-preparative column (1×30 cm) using a 15 to 55% acetonitrile gradient in 0.1% trifluoryl acetic acid (TFA) developed over 40 minutes at a flow rate of 2 mL/min. All synthetic peptides used in subsequent biochemical and immunological studies were >95% pure as determined by analytical HPLC. Amino acid compositions of these peptides were determined on a Waters Pico-Tag system. The results indicated a good agreement with their expected compositions.
[0067]ELISA was used to map B-cell epitopes. Microtiter wells (Nunc-Immunoplate, Nunc, Denmark) were coated with 50 μL of a coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) containing 200 ng of purified recombinant S fragments or 500 ng of individual peptides (listed in Table 3 below) for 16 hours at room temperature. The plates were then blocked in 0.1% (w/v) BSA in phosphate buffer saline (PBS) for 30 minutes at room temperature. Serially diluted antisera were added to the wells and incubated for 1 hour at room temperature. After removal of the antisera, the plates were washed five times with PBS containing 0.1% (w/v) Tween-20 and 0.1% (w/v) BSA. Fab'2 fragments from goat anti-rabbit, -guinea pig, -mouse, or -human IgG antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch Labs Inc., PA) were diluted (1/8,000) with a washing buffer, and added to the microtiter wells. After incubating for 1 hour at room temperature, the wells were washed five times with the washing buffer and then developed using the substrates tetramethylbenzidine (TMB) and H2O2 (ADI, Toronto). The reaction was stopped by adding 1N H2 SO4 and the optical density was measured at 450 nm by a Titretek Multiskan TI (Flow Labs., Virginia). Two irrelevant peptides were used as negative controls. All assays were performed in triplicate, and the reactive titer of each antiserum was defined as the dilution consistently showing 2-fold increase absorbance value over those obtained from the negative controls. Immunodomiant B-cell epitopes were identified to residues 125-146, 334-348, 409-423, 449-468, 589-603, and 1232-1246. These results indicate that these regions contain the linear B-cell epitope sequences and that they can be used as target antigens in, e.g., diagnostic kits to detect the presence of anti-S and anti-SARS CoV antibodies in samples.
EXAMPLE 7
[0068]It is known that SRAS CoV binds to VERO E6 cells. The above-described S protein fragments were tests for their ability to bind to VERO E6 cells. Vero E6 cells (1×104 cells per mL) were incubated with S1-Fc, S2-Fc, S3-Fc, or human IgG1 at various concentrations in a volume of 1 mL for 2 hours at room temperature. The cells were then washed in PBS containing 0.5% BSA and 0.1% NaN3, incubated with FITC-labeled goat anti-human IgG Fc (Sigma), and analyzed by flow cytometry. It was found that S1-Fc and S2-Fc bound to VERO E6 cells at 1 μg/ml and 0.1 g/ml, respectively. In contrast, S3-fc and human IgG1 did not bind to VERO E6 cell even at 10 μg/ml.
[0069]The just-described VERO E6 cell model was used to examine the ability of anti-S1-Fc or anti-S2-Fc serum to inhibit the binding of SARS CoV to VERO E6 cells. VERO E6 cells were cultured on a 24-well plate until they reached approximately 50% confluent. The cells were then incubated with SARS-CoV Tw1 strain (MOI 1:10) and human sera that had a 1/128 virus neutralization titer in the presence or absence of 0.1 to 10 μg/mL corresponding S fusion proteins. After 24-48 hours, the cells were examined under a microscope. The presence of mltinucleated giant cells indicated infected cells. The results indicated that human sera blocked the viral infection, and that this blocking activity was repressed by the recombinant S fusion proteins.
EXAMPLE 8
[0070]Since S2-Fc fusion protein strongly bound to VERO E6 cell and inhibited human neutralizing antibody activity against SARS CoV, it was of interest to identify the protective epitope(s) of this S2 fragment. Eighty-eight peptides from S2 (shown in Table 2 above) were synthesized based upon the sequence of the SARS CoV TW1 S protein.
[0071]Five convalescent sera were obtained from patients infected with SARS CoV and three sera were obtained from guinea pigs immunized with RBD2 in the manner described in Examples 5 and 6 above. These antisera were mixed with the peptides shown in Table 3. These peptides covered residues 522 to 600 of the S protein. The reactive titer of each antiserum was determined. The results are summarized in Table 3
TABLE-US-00009 TABLE 3 Reactivity of human or guinea pig anti-RBD2 antisera with synthetic peptides Reactive Titers Peptide ID No. Synthetic Guinea (SEQ ID NO:) peptides Human pig 76 (SEQ ID NO: 126) CVNFNFNGLTGTGVL 1/5 0/3 77 (SEQ ID NO: 127) FNGLTGTGVLTPSSK 1/5 0/3 78 (SEQ ID NO: 128) GTGVLTPSSKRFQPF 0/5 0/3 79 (SEQ ID NO: 129) TPSSKRFQPFQQFGR 0/5 0/3 80 (SEQ ID NO: 130) RFQPFQQFGRDVSDF 0/5 0/3 81 (SEQ ID NO: 131) QQFGRDVSDFTDSVR 1/5 0/3 82 (SEQ ID NO: 132) DVSDFTDSVRDPKTS 2/5 1/3 83 (SEQ ID NO: 133) TDSVRDPKTSEILDI 15 1/3 84 (SEQ ID NO: 134) DPKTSEILDISPCAF 0/5 0/3 85 (SEQ ID NO: 135) EILDISPCAFGGVSV 1/5 0/3 86 (SEQ ID NO: 136) SPCAFGGVSVITPGT 0/5 0/3 87 (SEQ ID NO: 137) GGVSVITPGTNASSE 0/5 0/3 88 (SEQ ID NO: 138) ITPGTNASSEVAVLY 5/5 3/3 89 (SEQ ID NO: 139) NASSEVAVLYQDVNC 5/5 3/3 90 (SEQ ID NO: 140) VAVLYQDVNCTDVST 4/5 1/3 91 (SEQ ID NO: 141) QDVNCTDVSTAIHAD 1/5 0/3 RBD-55 5/5 3/3 (SEQ ID NO: 26)
[0072]As shown in Table 3, most of the peptides successfully detected the presence of anti-S protein antibody in the samples.
EXAMPLE 9
[0073]Further studies were performed to determine whether the binding of S2-Fc to VERO E6 cells could be neutralized by S protein or its fragments.
[0074]Recombinant RBD2 was tested first. 104 of VERO E6 cells were incubated with 330 ng/mL of S2-Fc protein in the presence or absence of know amount of RBD2 protein solution. It was found that 1 μg of RBD2 significantly reduced the S2-Fc binding to VERO E6 cells.
[0075]The inhibition assays were repeated with 11 cocktails, each containing nine RBD2 fragment and covering(S28 to S115). More specifically, the VERO E6 cells were harvested and washed twice with a FACS staining/washing buffer. 2×105 cells were incubated with various peptides and then stained in a final volume of 100 ml with recombinant S-Fc protein (1 mg), S2-Fc protein (0.2-0.3 mg), or hIgG1 as isotype control for 30 minutes at 4° C. Cells were washed twice and stained with the RPE-conjugated anti-hIg Abs for 30 minutes at 4° C. After washing, cells were fixed with fixation buffer for 30 minutes at 4° C., and then the fluorescence was detected with FACS Calibur (Becton Dickinson). The results are summarized in Table 4 below. The inhibition level by RBD2 was designated as 100%.
TABLE-US-00010 TABLE 4 Inhibition S2-Fc/VERO E6 cell Binding by S Peptides Percent of Inhibition Concentration of Synthetic peptides (μg/mL) Blocking agents 1 10 100 Negative control (SEQ ID NOs:) 0 0 0 Gp(28-35) (SEQ ID NOs: 78-85) 0 0 0 Gp(36-43) (SEQ ID NOs: 86-93) 0 0 0 Gp(44-51) (SEQ ID NOs: 94-101) 0 0 0 Gp(52-59) (SEQ ID NOs: 102-109) 0 0 0 Gp(60-67) (SEQ ID NOs: 110-117) 0 0 0 Gp(68-75) (SEQ ID NOs: 118-125) 0 0 0 Gp(76-83) (SEQ ID NOs: 126-133) 0 0 0 Gp(84-91) (SEQ ID NOs: 134-141) 0 10% 30% Gp(92-99) (SEQ ID NOs: 142-149) 0 0 0 Gp(100-107) (SEQ ID NOs: 150-157) 0 0 0 Gp(108-115) (SEQ ID NOs: 158-165) 0 0 0 RRBD2 (SEQ ID NO: 24) 100% 100% NA
[0076]As shown in Table 4, the peptide cocktail containing S peptides 84 to 91 (group #8) strongly inhibited the binding between S2-Fc and VERO-6 cells by 30% as compared with those in the RBD2. These results indicate that the major B-cell epitopes of S2 were located within the region covering these 9 peptides, i.e., residues 540 to 600 of S protein.
[0077]To more clearly define the protective epitope(s) of the S2 fragment, individual peptides S84-91 were also tested. 104 of VERO E6 cells were incubated with 330 ng per mL of S2-Fc protein in the presence or absence of the peptides, respectively. The inhibitions of the binding of S-Fc to VERO E6 cells were determined in the same manner described above. The same experiment was repeated using a polypeptide containing with 50 amino acids covering S84 to S91 ("RBD-55" shown in Table 2 above). The results are summarized in Table 5 below.
TABLE-US-00011 TABLE 5 Inhibition Activity of S Synthetic Peptides against S2-Fc/VERO E6 cell Binding Percent of Inhibition Concentration of Synthetic peptides (μg/mL) Blocking agents 1 10 100 Negative control (SEQ ID NOs:) 0 0 0 Gp(76-83) (SEQ ID NOs: 126-133) 0 0 0 Gp(84-91) (SEQ ID NO: 134-141) 0 10 30 S84 (SEQ ID NO: 134) 0 0 0 S85 (SEQ ID NO: 135) 0 0 0 S86 (SEQ ID NO: 136) 0 0 10 S87 (SEQ ID NO: 137) 0 0 0 S88 (SEQ ID NO: 138) 0 0 0 S89 (SEQ ID NO: 139) 0 0 10 S90 (SEQ ID NO: 140) 0 0 0 S91 (SEQ ID NO: 141) 0 0 0 S86 + S87 (SEQ ID NOs: 136 and 137) 0 20 40 S86 + S88 (SEQ ID NOs: 136 and 138) 0 0 0 S86 + S89 (SEQ ID NOs: 136 and 139) 0 20 40 S86 + S90 (SEQ ID NOs: 136 and 140) 0 0 0 S86 + S91 (SEQ ID NOs: 136 and 141) 0 0 0 RBD-55 (SEQ ID NO: 26) 10 30 60 rRBD2 (SEQ ID NOs: 24) 100 100 Not test
[0078]As shown in Table 5, both S86 and S89 statistically significantly inhibited the S2-Fc/VERO cell binding. Furthermore, S86 and S87, or S86 and S89 exhibited synergetic effect and could inhibit 30% of S2-Fc/Vero cell binding. Each of S86 and S89 contains two cysteine residues on both termini, which could form a disulfide bridge and might lead to strong inhibition. RBD-55 inhibited the S2-FC/VERO E6 cell binding more significantly than S86 or S89 peptide (60% inhibition vs 10% inhibition). These results indicate that RBD-55 could be used as an immunogen to induce protective antibodies against SARS CoV.
EXAMPLE 10
[0079]The above-described peptides were used to generate S peptide-specific antisera. Guinea pigs and rabbits were immunized with peptides cocktail (50 to 200 μg) emulsified with the Freund's complete adjuvant and injected intramuscularly. The animals were boosted with the same amount of peptide cocktails in the incomplete Freund's adjuvant at days 14 and 28. Antisera were collected on day 42 and tested by ELISAs and immunoblotting. Both rabbit and guinea pig antisera were shown to be monospecific for their respective immunizing peptides by the peptide-specific ELISAs. In addition, both guinea pig and rabbit antisera raised against S peptides reacted with SARS CoV on immunoblot analyses. Since most S peptides induced strong anti-peptide antibody responses in at least one animal species, they are appropriate immunogens to be included in immunogenic compositions, e.g., vaccines.
EXAMPLE 11
[0080]Infant ferrets were used to examine the protective activity of S-specific antisera against SARS CoV challenge as described by NIH (Yang et al., Nature (2004) 428:561-564.). Five-day old infant ferrates were inoculated subcutaneously (SC) on the dorsum with 0.15 mL of two different rabbit anti-S fragments. Pre-immune sera were used as negative controls. One day after this passive immunization, the infant ferrets were injected intraperitoneally (IP) with 4000 plaque-forming units (cfu) of SARS CoV Tor2 strain (0.1 ml) freshly grown and isolated from a Vero cell culture medium supplemented with cofactors and diluted in PBS containing 0.5 mM MgCl2 and 0.15 mM CaCl2. One day later, blood samples were collected via cardiac puncture under methoxyflurane anaesthesia and cultured in the Vero cell media. The number of virus per mL of blood was determined after 24 hours. The Student's t-test was used to analyze differences observed in the levels of viramia relative to controls. The results indicate that the antibodies protect against SARS CoV challenge The protective ability of anti-RBD1 sera against SARS CoV infection was examined in the ferret model. It was that ferret passively immunized with guinea pig anti-RBD1 antisera were not more protective than pre-bleed serum control.
EXAMPLE 12
[0081]Little is known about the cellular immune response to SARS CoV and its role in protecting against SARS CoV infection. To examine the cellular response elicited by SARS CoV, T-cell lines' proliferative responses to S peptides were determined by conventional cytokine assays as described below.
[0082]S-specific T-cell lines were generated. BALB/c (H-2d) mice (Charles River Animal Farm, Montreal, Canada) were primed subcutaneously with 20 μg of recombinant S adsorbed to 1.5 mg of aluminium phosphate (alum) in presence of 100 μg of CpG. The mice were boosted twice with the same dose of immunogen at 3-week intervals.
[0083]Ten days after the final boost, the spleen of each immunized mouse was removed. Splenocytes were isolated and cultured in 200 μL of RPMI 1640 medium (Flow Lab) at 5.75×105 cells per well of a microtiter plate. The medium was supplemented with 10% heat-inactivated fetal calf serum (Gibson), 2 mM L-glutamine, 100 U/mL) penicillin, and 5×0.10-5 M 2-mercaptoethanol and contained varying concentrations (1, 10 and 100 μg per mL) of individual S peptides. The cultures were kept in a humidified incubator in the presence of 5% CO2/air. Triplicate cultures were performed for each concentration of each peptide. Five days later, 150 μL of 10% rat concanavalin A culture supernatant diluted in the culture medium was added to the microtiter plate wells. The supernatant contained Interleukin-2 (IL-2), which expand peptide-specific T-cells.
[0084]Six days later, 150 μL of the supernatant were removed from each microculture, and 150 μL of a fresh IL-2 containing culture supernatant added to further expand and maintain the viability of the peptide-specific T-cells. After another 6 day-incubation, the cells were washed with 200 μL culture medium for three times. Each set of cultures were then stimulated with a peptide at concentrations of 1, 10, and 100 μg/mL, respectively in the presence of 2×0.105 irradiated (1,500 rad) BALB/c spleen cells in a final volume of 200 μL culture medium. Sixty microliters of the supernatant were then removed from each triplicate culture and pooled. All supernatants were then assayed for IL-2, IL-4, and Interferon-gamma (IFN-gamma) using murine IL-2 and IL-4 ELISA kits (Endogen Inc, MA, U.S.A.) and a mouse IFN-gamma ELISA kit (Genzyme Corporation. MA, U.S.A.). Test culture supernatants were assayed at 1 in 5 dilution according to the manufacturers' instructions.
[0085]The results indicated that peptides corresponding to residues 120-134, 649-688, and 699-713 elicited proliferative responses and the release of specific cytokines. Because of this strong ability to induce cellular immune response, these immunodominant T-cell epitopes can be used as carriers for pneumococcal polysaccharides and/or S B-cell epitopes to enhance the immunogenicity. The Th1 cell epitopes identified above can be used in SARS CoV vaccine formulations to induce SARS-specific cellular immune responses.
EXAMPLE 13
[0086]In this example, murine anti-S monoclonal antibodies were generated. BALB/c mice were immunized intraperitoneally with 20 to 50 μg of RBD2 emulsified in the Freund's complete adjuvant. Two weeks later, the mice were injected with the same amount of immunogen in the incomplete Freund's adjuvant. The anti-S titers were examined. Positive mice were selected for making hybridomas by standard cell fusion techniques. Three days before the fusion, the mice were boosted again with the same amount of immunogen in the incomplete Freund's adjuvant. Hybridomas were produced by fusion of splenic lymphocytes from immunized mice with non-secreting Sp2/0 myeloma cells in the manner described in Hamel et al. 1987, J. Med. Microbiol. 23:163-170. S-specific hybridomas were cloned by sequential limiting dilutions and screened for anti-S monoclonal antibody production. Eight S-specific hybridoma cell lines were identified, expanded, and frozen in liquid nitrogen by standard techniques.
EXAMPLE 14
[0087]The mechanism of SARS CoV infection is unclear although it was reported that infection took place through enteric route, respiratory tract, and skin. As discussed above, S1-Fc and S2-Fc, but not S3-Fc, bind to VERO cells. To test whether S3-Fc binds to any other cells, a panel of cell lines were tested. About 1×104 cells/mL were incubated with 0.1, 0.3, and 1 μg of S3-Fc or the same amount of S1-Fc or S2-Fc in a volume of 1 mL for 2 hours at room temperature. The cells were washed in PBS with 0.5% BSA and 0.1% NaN3, incubated with FITC-labeled goat anti-human IgG Fc (Sigma), and analyzed by flow cytometry.
[0088]It was unexpected that S3-Fc bound strongly to NIH 3T3 cells but not to Jarket cells. S3-Fc showed strong binding to NIH 3T3 cells even at a concentration as low as 0.1 μg/mL. In contrast, S1-Fc did not bind to NIH3T3 cells even at 10 g/mL, and S2-Fc showed some binding to NIH 3T3 cell at 1 μg/mL. These results indicate that S3-Fc had specificity toward receptors in NIH 3T3 cells.
[0089]It was also unexpected that S protein also binds to HeLa, BHK-21, and COS-7 cells. Three separated receptor-binding domains of S protein were identified: (1) the low affinity mapped to the N-terminal 333 residues, (2) a intermediate affinity receptor-binding domain (with 1 μM avidity) mapped to residues 334 to 666, and (3) a high affinity domain within residues 667 to 999. Beside VERO E6 cells, all these cell lines had not been reported before to be the hosts for SARS CoV replication. This explained why SARS CoV could infect patient via skin contact with infected solutions.
OTHER EMBODIMENTS
[0090]All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
[0091]From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claim.
Sequence CWU
1
214129727DNASARS coronavirus Urbani 1atattaggtt tttacctacc caggaaaagc
caaccaacct cgatctcttg tagatctgtt 60ctctaaacga actttaaaat ctgtgtagct
gtcgctcggc tgcatgccta gtgcacctac 120gcagtataaa caataataaa ttttactgtc
gttgacaaga aacgagtaac tcgtccctct 180tctgcagact gcttacggtt tcgtccgtgt
tgcagtcgat catcagcata cctaggtttc 240gtccgggtgt gaccgaaagg taagatggag
agccttgttc ttggtgtcaa cgagaaaaca 300cacgtccaac tcagtttgcc tgtccttcag
gttagagacg tgctagtgcg tggcttcggg 360gactctgtgg aagaggccct atcggaggca
cgtgaacacc tcaaaaatgg cacttgtggt 420ctagtagagc tggaaaaagg cgtactgccc
cagcttgaac agccctatgt gttcattaaa 480cgttctgatg ccttaagcac caatcacggc
cacaaggtcg ttgagctggt tgcagaaatg 540gacggcattc agtacggtcg tagcggtata
acactgggag tactcgtgcc acatgtgggc 600gaaaccccaa ttgcataccg caatgttctt
cttcgtaaga acggtaataa gggagccggt 660ggtcatagct atggcatcga tctaaagtct
tatgacttag gtgacgagct tggcactgat 720cccattgaag attatgaaca aaactggaac
actaagcatg gcagtggtgc actccgtgaa 780ctcactcgtg agctcaatgg aggtgcagtc
actcgctatg tcgacaacaa tttctgtggc 840ccagatgggt accctcttga ttgcatcaaa
gattttctcg cacgcgcggg caagtcaatg 900tgcactcttt ccgaacaact tgattacatc
gagtcgaaga gaggtgtcta ctgctgccgt 960gaccatgagc atgaaattgc ctggttcact
gagcgctctg ataagagcta cgagcaccag 1020acacccttcg aaattaagag tgccaagaaa
tttgacactt tcaaagggga atgcccaaag 1080tttgtgtttc ctcttaactc aaaagtcaaa
gtcattcaac cacgtgttga aaagaaaaag 1140actgagggtt tcatggggcg tatacgctct
gtgtaccctg ttgcatctcc acaggagtgt 1200aacaatatgc acttgtctac cttgatgaaa
tgtaatcatt gcgatgaagt ttcatggcag 1260acgtgcgact ttctgaaagc cacttgtgaa
cattgtggca ctgaaaattt agttattgaa 1320ggacctacta catgtgggta cctacctact
aatgctgtag tgaaaatgcc atgtcctgcc 1380tgtcaagacc cagagattgg acctgagcat
agtgttgcag attatcacaa ccactcaaac 1440attgaaactc gactccgcaa gggaggtagg
actagatgtt ttggaggctg tgtgtttgcc 1500tatgttggct gctataataa gcgtgcctac
tgggttcctc gtgctagtgc tgatattggc 1560tcaggccata ctggcattac tggtgacaat
gtggagacct tgaatgagga tctccttgag 1620atactgagtc gtgaacgtgt taacattaac
attgttggcg attttcattt gaatgaagag 1680gttgccatca ttttggcatc tttctctgct
tctacaagtg cctttattga cactataaag 1740agtcttgatt acaagtcttt caaaaccatt
gttgagtcct gcggtaacta taaagttacc 1800aagggaaagc ccgtaaaagg tgcttggaac
attggacaac agagatcagt tttaacacca 1860ctgtgtggtt ttccctcaca ggctgctggt
gttatcagat caatttttgc gcgcacactt 1920gatgcagcaa accactcaat tcctgatttg
caaagagcag ctgtcaccat acttgatggt 1980atttctgaac agtcattacg tcttgtcgac
gccatggttt atacttcaga cctgctcacc 2040aacagtgtca ttattatggc atatgtaact
ggtggtcttg tacaacagac ttctcagtgg 2100ttgtctaatc ttttgggcac tactgttgaa
aaactcaggc ctatctttga atggattgag 2160gcgaaactta gtgcaggagt tgaatttctc
aaggatgctt gggagattct caaatttctc 2220attacaggtg tttttgacat cgtcaagggt
caaatacagg ttgcttcaga taacatcaag 2280gattgtgtaa aatgcttcat tgatgttgtt
aacaaggcac tcgaaatgtg cattgatcaa 2340gtcactatcg ctggcgcaaa gttgcgatca
ctcaacttag gtgaagtctt catcgctcaa 2400agcaagggac tttaccgtca gtgtatacgt
ggcaaggagc agctgcaact actcatgcct 2460cttaaggcac caaaagaagt aacctttctt
gaaggtgatt cacatgacac agtacttacc 2520tctgaggagg ttgttctcaa gaacggtgaa
ctcgaagcac tcgagacgcc cgttgatagc 2580ttcacaaatg gagctatcgt tggcacacca
gtctgtgtaa atggcctcat gctcttagag 2640attaaggaca aagaacaata ctgcgcattg
tctcctggtt tactggctac aaacaatgtc 2700tttcgcttaa aagggggtgc accaattaaa
ggtgtaacct ttggagaaga tactgtttgg 2760gaagttcaag gttacaagaa tgtgagaatc
acatttgagc ttgatgaacg tgttgacaaa 2820gtgcttaatg aaaagtgctc tgtctacact
gttgaatccg gtaccgaagt tactgagttt 2880gcatgtgttg tagcagaggc tgttgtgaag
actttacaac cagtttctga tctccttacc 2940aacatgggta ttgatcttga tgagtggagt
gtagctacat tctacttatt tgatgatgct 3000ggtgaagaaa acttttcatc acgtatgtat
tgttcctttt accctccaga tgaggaagaa 3060gaggacgatg cagagtgtga ggaagaagaa
attgatgaaa cctgtgaaca tgagtacggt 3120acagaggatg attatcaagg tctccctctg
gaatttggtg cctcagctga aacagttcga 3180gttgaggaag aagaagagga agactggctg
gatgatacta ctgagcaatc agagattgag 3240ccagaaccag aacctacacc tgaagaacca
gttaatcagt ttactggtta tttaaaactt 3300actgacaatg ttgccattaa atgtgttgac
atcgttaagg aggcacaaag tgctaatcct 3360atggtgattg taaatgctgc taacatacac
ctgaaacatg gtggtggtgt agcaggtgca 3420ctcaacaagg caaccaatgg tgccatgcaa
aaggagagtg atgattacat taagctaaat 3480ggccctctta cagtaggagg gtcttgtttg
ctttctggac ataatcttgc taagaagtgt 3540ctgcatgttg ttggacctaa cctaaatgca
ggtgaggaca tccagcttct taaggcagca 3600tatgaaaatt tcaattcaca ggacatctta
cttgcaccat tgttgtcagc aggcatattt 3660ggtgctaaac cacttcagtc tttacaagtg
tgcgtgcaga cggttcgtac acaggtttat 3720attgcagtca atgacaaagc tctttatgag
caggttgtca tggattatct tgataacctg 3780aagcctagag tggaagcacc taaacaagag
gagccaccaa acacagaaga ttccaaaact 3840gaggagaaat ctgtcgtaca gaagcctgtc
gatgtgaagc caaaaattaa ggcctgcatt 3900gatgaggtta ccacaacact ggaagaaact
aagtttctta ccaataagtt actcttgttt 3960gctgatatca atggtaagct ttaccatgat
tctcagaaca tgcttagagg tgaagatatg 4020tctttccttg agaaggatgc accttacatg
gtaggtgatg ttatcactag tggtgatatc 4080acttgtgttg taataccctc caaaaaggct
ggtggcacta ctgagatgct ctcaagagct 4140ttgaagaaag tgccagttga tgagtatata
accacgtacc ctggacaagg atgtgctggt 4200tatacacttg aggaagctaa gactgctctt
aagaaatgca aatctgcatt ttatgtacta 4260ccttcagaag cacctaatgc taaggaagag
attctaggaa ctgtatcctg gaatttgaga 4320gaaatgcttg ctcatgctga agagacaaga
aaattaatgc ctatatgcat ggatgttaga 4380gccataatgg caaccatcca acgtaagtat
aaaggaatta aaattcaaga gggcatcgtt 4440gactatggtg tccgattctt cttttatact
agtaaagagc ctgtagcttc tattattacg 4500aagctgaact ctctaaatga gccgcttgtc
acaatgccaa ttggttatgt gacacatggt 4560tttaatcttg aagaggctgc gcgctgtatg
cgttctctta aagctcctgc cgtagtgtca 4620gtatcatcac cagatgctgt tactacatat
aatggatacc tcacttcgtc atcaaagaca 4680tctgaggagc actttgtaga aacagtttct
ttggctggct cttacagaga ttggtcctat 4740tcaggacagc gtacagagtt aggtgttgaa
tttcttaagc gtggtgacaa aattgtgtac 4800cacactctgg agagccccgt cgagtttcat
cttgacggtg aggttctttc acttgacaaa 4860ctaaagagtc tcttatccct gcgggaggtt
aagactataa aagtgttcac aactgtggac 4920aacactaatc tccacacaca gcttgtggat
atgtctatga catatggaca gcagtttggt 4980ccaacatact tggatggtgc tgatgttaca
aaaattaaac ctcatgtaaa tcatgagggt 5040aagactttct ttgtactacc tagtgatgac
acactacgta gtgaagcttt cgagtactac 5100catactcttg atgagagttt tcttggtagg
tacatgtctg ctttaaacca cacaaagaaa 5160tggaaatttc ctcaagttgg tggtttaact
tcaattaaat gggctgataa caattgttat 5220ttgtctagtg ttttattagc acttcaacag
cttgaagtca aattcaatgc accagcactt 5280caagaggctt attatagagc ccgtgctggt
gatgctgcta acttttgtgc actcatactc 5340gcttacagta ataaaactgt tggcgagctt
ggtgatgtca gagaaactat gacccatctt 5400ctacagcatg ctaatttgga atctgcaaag
cgagttctta atgtggtgtg taaacattgt 5460ggtcagaaaa ctactacctt aacgggtgta
gaagctgtga tgtatatggg tactctatct 5520tatgataatc ttaagacagg tgtttccatt
ccatgtgtgt gtggtcgtga tgctacacaa 5580tatctagtac aacaagagtc ttcttttgtt
atgatgtctg caccacctgc tgagtataaa 5640ttacagcaag gtacattctt atgtgcgaat
gagtacactg gtaactatca gtgtggtcat 5700tacactcata taactgctaa ggagaccctc
tatcgtattg acggagctca ccttacaaag 5760atgtcagagt acaaaggacc agtgactgat
gttttctaca aggaaacatc ttacactaca 5820accatcaagc ctgtgtcgta taaactcgat
ggagttactt acacagagat tgaaccaaaa 5880ttggatgggt attataaaaa ggataatgct
tactatacag agcagcctat agaccttgta 5940ccaactcaac cattaccaaa tgcgagtttt
gataatttca aactcacatg ttctaacaca 6000aaatttgctg atgatttaaa tcaaatgaca
ggcttcacaa agccagcttc acgagagcta 6060tctgtcacat tcttcccaga cttgaatggc
gatgtagtgg ctattgacta tagacactat 6120tcagcgagtt tcaagaaagg tgctaaatta
ctgcataagc caattgtttg gcacattaac 6180caggctacaa ccaagacaac gttcaaacca
aacacttggt gtttacgttg tctttggagt 6240acaaagccag tagatacttc aaattcattt
gaagttctgg cagtagaaga cacacaagga 6300atggacaatc ttgcttgtga aagtcaacaa
cccacctctg aagaagtagt ggaaaatcct 6360accatacaga aggaagtcat agagtgtgac
gtgaaaacta ccgaagttgt aggcaatgtc 6420atacttaaac catcagatga aggtgttaaa
gtaacacaag agttaggtca tgaggatctt 6480atggctgctt atgtggaaaa cacaagcatt
accattaaga aacctaatga gctttcacta 6540gccttaggtt taaaaacaat tgccactcat
ggtattgctg caattaatag tgttccttgg 6600agtaaaattt tggcttatgt caaaccattc
ttaggacaag cagcaattac aacatcaaat 6660tgcgctaaga gattagcaca acgtgtgttt
aacaattata tgccttatgt gtttacatta 6720ttgttccaat tgtgtacttt tactaaaagt
accaattcta gaattagagc ttcactacct 6780acaactattg ctaaaaatag tgttaagagt
gttgctaaat tatgtttgga tgccggcatt 6840aattatgtga agtcacccaa attttctaaa
ttgttcacaa tcgctatgtg gctattgttg 6900ttaagtattt gcttaggttc tctaatctgt
gtaactgctg cttttggtgt actcttatct 6960aattttggtg ctccttctta ttgtaatggc
gttagagaat tgtatcttaa ttcgtctaac 7020gttactacta tggatttctg tgaaggttct
tttccttgca gcatttgttt aagtggatta 7080gactcccttg attcttatcc agctcttgaa
accattcagg tgacgatttc atcgtacaag 7140ctagacttga caattttagg tctggccgct
gagtgggttt tggcatatat gttgttcaca 7200aaattctttt atttattagg tctttcagct
ataatgcagg tgttctttgg ctattttgct 7260agtcatttca tcagcaattc ttggctcatg
tggtttatca ttagtattgt acaaatggca 7320cccgtttctg caatggttag gatgtacatc
ttctttgctt ctttctacta catatggaag 7380agctatgttc atatcatgga tggttgcacc
tcttcgactt gcatgatgtg ctataagcgc 7440aatcgtgcca cacgcgttga gtgtacaact
attgttaatg gcatgaagag atctttctat 7500gtctatgcaa atggaggccg tggcttctgc
aagactcaca attggaattg tctcaattgt 7560gacacatttt gcactggtag tacattcatt
agtgatgaag ttgctcgtga tttgtcactc 7620cagtttaaaa gaccaatcaa ccctactgac
cagtcatcgt atattgttga tagtgttgct 7680gtgaaaaatg gcgcgcttca cctctacttt
gacaaggctg gtcaaaagac ctatgagaga 7740catccgctct cccattttgt caatttagac
aatttgagag ctaacaacac taaaggttca 7800ctgcctatta atgtcatagt ttttgatggc
aagtccaaat gcgacgagtc tgcttctaag 7860tctgcttctg tgtactacag tcagctgatg
tgccaaccta ttctgttgct tgaccaagtt 7920cttgtatcag acgttggaga tagtactgaa
gtttccgtta agatgtttga tgcttatgtc 7980gacacctttt cagcaacttt tagtgttcct
atggaaaaac ttaaggcact tgttgctaca 8040gctcacagcg agttagcaaa gggtgtagct
ttagatggtg tcctttctac attcgtgtca 8100gctgcccgac aaggtgttgt tgataccgat
gttgacacaa aggatgttat tgaatgtctc 8160aaactttcac atcactctga cttagaagtg
acaggtgaca gttgtaacaa tttcatgctc 8220acctataata aggttgaaaa catgacgccc
agagatcttg gcgcatgtat tgactgtaat 8280gcaaggcata tcaatgccca agtagcaaaa
agtcacaatg tttcactcat ctggaatgta 8340aaagactaca tgtctttatc tgaacagctg
cgtaaacaaa ttcgtagtgc tgccaagaag 8400aacaacatac cttttagact aacttgtgct
acaactagac aggttgtcaa tgtcataact 8460actaaaatct cactcaaggg tggtaagatt
gttagtactt gttttaaact tatgcttaag 8520gccacattat tgtgcgttct tgctgcattg
gtttgttata tcgttatgcc agtacataca 8580ttgtcaatcc atgatggtta cacaaatgaa
atcattggtt acaaagccat tcaggatggt 8640gtcactcgtg acatcatttc tactgatgat
tgttttgcaa ataaacatgc tggttttgac 8700gcatggttta gccagcgtgg tggttcatac
aaaaatgaca aaagctgccc tgtagtagct 8760gctatcatta caagagagat tggtttcata
gtgcctggct taccgggtac tgtgctgaga 8820gcaatcaatg gtgacttctt gcattttcta
cctcgtgttt ttagtgctgt tggcaacatt 8880tgctacacac cttccaaact cattgagtat
agtgattttg ctacctctgc ttgcgttctt 8940gctgctgagt gtacaatttt taaggatgct
atgggcaaac ctgtgccata ttgttatgac 9000actaatttgc tagagggttc tatttcttat
agtgagcttc gtccagacac tcgttatgtg 9060cttatggatg gttccatcat acagtttcct
aacacttacc tggagggttc tgttagagta 9120gtaacaactt ttgatgctga gtactgtaga
catggtacat gcgaaaggtc agaagtaggt 9180atttgcctat ctaccagtgg tagatgggtt
cttaataatg agcattacag agctctatca 9240ggagttttct gtggtgttga tgcgatgaat
ctcatagcta acatctttac tcctcttgtg 9300caacctgtgg gtgctttaga tgtgtctgct
tcagtagtgg ctggtggtat tattgccata 9360ttggtgactt gtgctgccta ctactttatg
aaattcagac gtgtttttgg tgagtacaac 9420catgttgttg ctgctaatgc acttttgttt
ttgatgtctt tcactatact ctgtctggta 9480ccagcttaca gctttctgcc gggagtctac
tcagtctttt acttgtactt gacattctat 9540ttcaccaatg atgtttcatt cttggctcac
cttcaatggt ttgccatgtt ttctcctatt 9600gtgccttttt ggataacagc aatctatgta
ttctgtattt ctctgaagca ctgccattgg 9660ttctttaaca actatcttag gaaaagagtc
atgtttaatg gagttacatt tagtaccttc 9720gaggaggctg ctttgtgtac ctttttgctc
aacaaggaaa tgtacctaaa attgcgtagc 9780gagacactgt tgccacttac acagtataac
aggtatcttg ctctatataa caagtacaag 9840tatttcagtg gagccttaga tactaccagc
tatcgtgaag cagcttgctg ccacttagca 9900aaggctctaa atgactttag caactcaggt
gctgatgttc tctaccaacc accacagaca 9960tcaatcactt ctgctgttct gcagagtggt
tttaggaaaa tggcattccc gtcaggcaaa 10020gttgaagggt gcatggtaca agtaacctgt
ggaactacaa ctcttaatgg attgtggttg 10080gatgacacag tatactgtcc aagacatgtc
atttgcacag cagaagacat gcttaatcct 10140aactatgaag atctgctcat tcgcaaatcc
aaccatagct ttcttgttca ggctggcaat 10200gttcaacttc gtgttattgg ccattctatg
caaaattgtc tgcttaggct taaagttgat 10260acttctaacc ctaagacacc caagtataaa
tttgtccgta tccaacctgg tcaaacattt 10320tcagttctag catgctacaa tggttcacca
tctggtgttt atcagtgtgc catgagacct 10380aatcatacca ttaaaggttc tttccttaat
ggatcatgtg gtagtgttgg ttttaacatt 10440gattatgatt gcgtgtcttt ctgctatatg
catcatatgg agcttccaac aggagtacac 10500gctggtactg acttagaagg taaattctat
ggtccatttg ttgacagaca aactgcacag 10560gctgcaggta cagacacaac cataacatta
aatgttttgg catggctgta tgctgctgtt 10620atcaatggtg ataggtggtt tcttaataga
ttcaccacta ctttgaatga ctttaacctt 10680gtggcaatga agtacaacta tgaacctttg
acacaagatc atgttgacat attgggacct 10740ctttctgctc aaacaggaat tgccgtctta
gatatgtgtg ctgctttgaa agagctgctg 10800cagaatggta tgaatggtcg tactatcctt
ggtagcacta ttttagaaga tgagtttaca 10860ccatttgatg ttgttagaca atgctctggt
gttaccttcc aaggtaagtt caagaaaatt 10920gttaagggca ctcatcattg gatgctttta
actttcttga catcactatt gattcttgtt 10980caaagtacac agtggtcact gtttttcttt
gtttacgaga atgctttctt gccatttact 11040cttggtatta tggcaattgc tgcatgtgct
atgctgcttg ttaagcataa gcacgcattc 11100ttgtgcttgt ttctgttacc ttctcttgca
acagttgctt actttaatat ggtctacatg 11160cctgctagct gggtgatgcg tatcatgaca
tggcttgaat tggctgacac tagcttgtct 11220ggttataggc ttaaggattg tgttatgtat
gcttcagctt tagttttgct tattctcatg 11280acagctcgca ctgtttatga tgatgctgct
agacgtgttt ggacactgat gaatgtcatt 11340acacttgttt acaaagtcta ctatggtaat
gctttagatc aagctatttc catgtgggcc 11400ttagttattt ctgtaacctc taactattct
ggtgtcgtta cgactatcat gtttttagct 11460agagctatag tgtttgtgtg tgttgagtat
tacccattgt tatttattac tggcaacacc 11520ttacagtgta tcatgcttgt ttattgtttc
ttaggctatt gttgctgctg ctactttggc 11580cttttctgtt tactcaaccg ttacttcagg
cttactcttg gtgtttatga ctacttggtc 11640tctacacaag aatttaggta tatgaactcc
caggggcttt tgcctcctaa gagtagtatt 11700gatgctttca agcttaacat taagttgttg
ggtattggag gtaaaccatg tatcaaggtt 11760gctactgtac agtctaaaat gtctgacgta
aagtgcacat ctgtggtact gctctcggtt 11820cttcaacaac ttagagtaga gtcatcttct
aaattgtggg cacaatgtgt acaactccac 11880aatgatattc ttcttgcaaa agacacaact
gaagctttcg agaagatggt ttctcttttg 11940tctgttttgc tatccatgca gggtgctgta
gacattaata ggttgtgcga ggaaatgctc 12000gataaccgtg ctactcttca ggctattgct
tcagaattta gttctttacc atcatatgcc 12060gcttatgcca ctgcccagga ggcctatgag
caggctgtag ctaatggtga ttctgaagtc 12120gttctcaaaa agttaaagaa atctttgaat
gtggctaaat ctgagtttga ccgtgatgct 12180gccatgcaac gcaagttgga aaagatggca
gatcaggcta tgacccaaat gtacaaacag 12240gcaagatctg aggacaagag ggcaaaagta
actagtgcta tgcaaacaat gctcttcact 12300atgcttagga agcttgataa tgatgcactt
aacaacatta tcaacaatgc gcgtgatggt 12360tgtgttccac tcaacatcat accattgact
acagcagcca aactcatggt tgttgtccct 12420gattatggta cctacaagaa cacttgtgat
ggtaacacct ttacatatgc atctgcactc 12480tgggaaatcc agcaagttgt tgatgcggat
agcaagattg ttcaacttag tgaaattaac 12540atggacaatt caccaaattt ggcttggcct
cttattgtta cagctctaag agccaactca 12600gctgttaaac tacagaataa tgaactgagt
ccagtagcac tacgacagat gtcctgtgcg 12660gctggtacca cacaaacagc ttgtactgat
gacaatgcac ttgcctacta taacaattcg 12720aagggaggta ggtttgtgct ggcattacta
tcagaccacc aagatctcaa atgggctaga 12780ttccctaaga gtgatggtac aggtacaatt
tacacagaac tggaaccacc ttgtaggttt 12840gttacagaca caccaaaagg gcctaaagtg
aaatacttgt acttcatcaa aggcttaaac 12900aacctaaata gaggtatggt gctgggcagt
ttagctgcta cagtacgtct tcaggctgga 12960aatgctacag aagtacctgc caattcaact
gtgctttcct tctgtgcttt tgcagtagac 13020cctgctaaag catataagga ttacctagca
agtggaggac aaccaatcac caactgtgtg 13080aagatgttgt gtacacacac tggtacagga
caggcaatta ctgtaacacc agaagctaac 13140atggaccaag agtcctttgg tggtgcttca
tgttgtctgt attgtagatg ccacattgac 13200catccaaatc ctaaaggatt ctgtgacttg
aaaggtaagt acgtccaaat acctaccact 13260tgtgctaatg acccagtggg ttttacactt
agaaacacag tctgtaccgt ctgcggaatg 13320tggaaaggtt atggctgtag ttgtgaccaa
ctccgcgaac ccttgatgca gtctgcggat 13380gcatcaacgt ttttaaacgg gtttgcggtg
taagtgcagc ccgtcttaca ccgtgcggca 13440caggcactag tactgatgtc gtctacaggg
cttttgatat ttacaacgaa aaagttgctg 13500gttttgcaaa gttcctaaaa actaattgct
gtcgcttcca ggagaaggat gaggaaggca 13560atttattaga ctcttacttt gtagttaaga
ggcatactat gtctaactac caacatgaag 13620agactattta taacttggtt aaagattgtc
cagcggttgc tgtccatgac tttttcaagt 13680ttagagtaga tggtgacatg gtaccacata
tatcacgtca gcgtctaact aaatacacaa 13740tggctgattt agtctatgct ctacgtcatt
ttgatgaggg taattgtgat acattaaaag 13800aaatactcgt cacatacaat tgctgtgatg
atgattattt caataagaag gattggtatg 13860acttcgtaga gaatcctgac atcttacgcg
tatatgctaa cttaggtgag cgtgtacgcc 13920aatcattatt aaagactgta caattctgcg
atgctatgcg tgatgcaggc attgtaggcg 13980tactgacatt agataatcag gatcttaatg
ggaactggta cgatttcggt gatttcgtac 14040aagtagcacc aggctgcgga gttcctattg
tggattcata ttactcattg ctgatgccca 14100tcctcacttt gactagggca ttggctgctg
agtcccatat ggatgctgat ctcgcaaaac 14160cacttattaa gtgggatttg ctgaaatatg
attttacgga agagagactt tgtctcttcg 14220accgttattt taaatattgg gaccagacat
accatcccaa ttgtattaac tgtttggatg 14280ataggtgtat ccttcattgt gcaaacttta
atgtgttatt ttctactgtg tttccaccta 14340caagttttgg accactagta agaaaaatat
ttgtagatgg tgttcctttt gttgtttcaa 14400ctggatacca ttttcgtgag ttaggagtcg
tacataatca ggatgtaaac ttacatagct 14460cgcgtctcag tttcaaggaa cttttagtgt
atgctgctga tccagctatg catgcagctt 14520ctggcaattt attgctagat aaacgcacta
catgcttttc agtagctgca ctaacaaaca 14580atgttgcttt tcaaactgtc aaacccggta
attttaataa agacttttat gactttgctg 14640tgtctaaagg tttctttaag gaaggaagtt
ctgttgaact aaaacacttc ttctttgctc 14700aggatggcaa cgctgctatc agtgattatg
actattatcg ttataatctg ccaacaatgt 14760gtgatatcag acaactccta ttcgtagttg
aagttgttga taaatacttt gattgttacg 14820atggtggctg tattaatgcc aaccaagtaa
tcgttaacaa tctggataaa tcagctggtt 14880tcccatttaa taaatggggt aaggctagac
tttattatga ctcaatgagt tatgaggatc 14940aagatgcact tttcgcgtat actaagcgta
atgtcatccc tactataact caaatgaatc 15000ttaagtatgc cattagtgca aagaatagag
ctcgcaccgt agctggtgtc tctatctgta 15060gtactatgac aaatagacag tttcatcaga
aattattgaa gtcaatagcc gccactagag 15120gagctactgt ggtaattgga acaagcaagt
tttacggtgg ctggcataat atgttaaaaa 15180ctgtttacag tgatgtagaa actccacacc
ttatgggttg ggattatcca aaatgtgaca 15240gagccatgcc taacatgctt aggataatgg
cctctcttgt tcttgctcgc aaacataaca 15300cttgctgtaa cttatcacac cgtttctaca
ggttagctaa cgagtgtgcg caagtattaa 15360gtgagatggt catgtgtggc ggctcactat
atgttaaacc aggtggaaca tcatccggtg 15420atgctacaac tgcttatgct aatagtgtct
ttaacatttg tcaagctgtt acagccaatg 15480taaatgcact tctttcaact gatggtaata
agatagctga caagtatgtc cgcaatctac 15540aacacaggct ctatgagtgt ctctatagaa
atagggatgt tgatcatgaa ttcgtggatg 15600agttttacgc ttacctgcgt aaacatttct
ccatgatgat tctttctgat gatgccgttg 15660tgtgctataa cagtaactat gcggctcaag
gtttagtagc tagcattaag aactttaagg 15720cagttcttta ttatcaaaat aatgtgttca
tgtctgaggc aaaatgttgg actgagactg 15780accttactaa aggacctcac gaattttgct
cacagcatac aatgctagtt aaacaaggag 15840atgattacgt gtacctgcct tacccagatc
catcaagaat attaggcgca ggctgttttg 15900tcgatgatat tgtcaaaaca gatggtacac
ttatgattga aaggttcgtg tcactggcta 15960ttgatgctta cccacttaca aaacatccta
atcaggagta tgctgatgtc tttcacttgt 16020atttacaata cattagaaag ttacatgatg
agcttactgg ccacatgttg gacatgtatt 16080ccgtaatgct aactaatgat aacacctcac
ggtactggga acctgagttt tatgaggcta 16140tgtacacacc acatacagtc ttgcaggctg
taggtgcttg tgtattgtgc aattcacaga 16200cttcacttcg ttgcggtgcc tgtattagga
gaccattcct atgttgcaag tgctgctatg 16260accatgtcat ttcaacatca cacaaattag
tgttgtctgt taatccctat gtttgcaatg 16320ccccaggttg tgatgtcact gatgtgacac
aactgtatct aggaggtatg agctattatt 16380gcaagtcaca taagcctccc attagttttc
cattatgtgc taatggtcag gtttttggtt 16440tatacaaaaa cacatgtgta ggcagtgaca
atgtcactga cttcaatgcg atagcaacat 16500gtgattggac taatgctggc gattacatac
ttgccaacac ttgtactgag agactcaagc 16560ttttcgcagc agaaacgctc aaagccactg
aggaaacatt taagctgtca tatggtattg 16620ctactgtacg cgaagtactc tctgacagag
aattgcatct ttcatgggag gttggaaaac 16680ctagaccacc attgaacaga aactatgtct
ttactggtta ccgtgtaact aaaaatagta 16740aagtacagat tggagagtac acctttgaaa
aaggtgacta tggtgatgct gttgtgtaca 16800gaggtactac gacatacaag ttgaatgttg
gtgattactt tgtgttgaca tctcacactg 16860taatgccact tagtgcacct actctagtgc
cacaagagca ctatgtgaga attactggct 16920tgtacccaac actcaacatc tcagatgagt
tttctagcaa tgttgcaaat tatcaaaagg 16980tcggcatgca aaagtactct acactccaag
gaccacctgg tactggtaag agtcattttg 17040ccatcggact tgctctctat tacccatctg
ctcgcatagt gtatacggca tgctctcatg 17100cagctgttga tgccctatgt gaaaaggcat
taaaatattt gcccatagat aaatgtagta 17160gaatcatacc tgcgcgtgcg cgcgtagagt
gttttgataa attcaaagtg aattcaacac 17220tagaacagta tgttttctgc actgtaaatg
cattgccaga aacaactgct gacattgtag 17280tctttgatga aatctctatg gctactaatt
atgacttgag tgttgtcaat gctagacttc 17340gtgcaaaaca ctacgtctat attggcgatc
ctgctcaatt accagccccc cgcacattgc 17400tgactaaagg cacactagaa ccagaatatt
ttaattcagt gtgcagactt atgaaaacaa 17460taggtccaga catgttcctt ggaacttgtc
gccgttgtcc tgctgaaatt gttgacactg 17520tgagtgcttt agtttatgac aataagctaa
aagcacacaa ggataagtca gctcaatgct 17580tcaaaatgtt ctacaaaggt gttattacac
atgatgtttc atctgcaatc aacagacctc 17640aaataggcgt tgtaagagaa tttcttacac
gcaatcctgc ttggagaaaa gctgttttta 17700tctcacctta taattcacag aacgctgtag
cttcaaaaat cttaggattg cctacgcaga 17760ctgttgattc atcacagggt tctgaatatg
actatgtcat attcacacaa actactgaaa 17820cagcacactc ttgtaatgtc aaccgcttca
atgtggctat cacaagggca aaaattggca 17880ttttgtgcat aatgtctgat agagatcttt
atgacaaact gcaatttaca agtctagaaa 17940taccacgtcg caatgtggct acattacaag
cagaaaatgt aactggactt tttaaggact 18000gtagtaagat cattactggt cttcatccta
cacaggcacc tacacacctc agcgttgata 18060taaagttcaa gactgaagga ttatgtgttg
acataccagg cataccaaag gacatgacct 18120accgtagact catctctatg atgggtttca
aaatgaatta ccaagtcaat ggttacccta 18180atatgtttat cacccgcgaa gaagctattc
gtcacgttcg tgcgtggatt ggctttgatg 18240tagagggctg tcatgcaact agagatgctg
tgggtactaa cctacctctc cagctaggat 18300tttctacagg tgttaactta gtagctgtac
cgactggtta tgttgacact gaaaataaca 18360cagaattcac cagagttaat gcaaaacctc
caccaggtga ccagtttaaa catcttatac 18420cactcatgta taaaggcttg ccctggaatg
tagtgcgtat taagatagta caaatgctca 18480gtgatacact gaaaggattg tcagacagag
tcgtgttcgt cctttgggcg catggctttg 18540agcttacatc aatgaagtac tttgtcaaga
ttggacctga aagaacgtgt tgtctgtgtg 18600acaaacgtgc aacttgcttt tctacttcat
cagatactta tgcctgctgg aatcattctg 18660tgggttttga ctatgtctat aacccattta
tgattgatgt tcagcagtgg ggctttacgg 18720gtaaccttca gagtaaccat gaccaacatt
gccaggtaca tggaaatgca catgtggcta 18780gttgtgatgc tatcatgact agatgtttag
cagtccatga gtgctttgtt aagcgcgttg 18840attggtctgt tgaataccct attataggag
atgaactgag ggttaattct gcttgcagaa 18900aagtacaaca catggttgtg aagtctgcat
tgcttgctga taagtttcca gttcttcatg 18960acattggaaa tccaaaggct atcaagtgtg
tgcctcaggc tgaagtagaa tggaagttct 19020acgatgctca gccatgtagt gacaaagctt
acaaaataga ggagctcttc tattcttatg 19080ctacacatca cgataaattc actgatggtg
tttgtttgtt ttggaattgt aacgttgatc 19140gttacccagc caatgcaatt gtgtgtaggt
ttgacacaag agtcttgtca aacttgaact 19200taccaggctg tgatggtggt agtttgtatg
tgaataagca tgcattccac actccagctt 19260tcgataaaag tgcatttact aatttaaagc
aattgccttt cttttactat tctgatagtc 19320cttgtgagtc tcatggcaaa caagtagtgt
cggatattga ttatgttcca ctcaaatctg 19380ctacgtgtat tacacgatgc aatttaggtg
gtgctgtttg cagacaccat gcaaatgagt 19440accgacagta cttggatgca tataatatga
tgatttctgc tggatttagc ctatggattt 19500acaaacaatt tgatacttat aacctgtgga
atacatttac caggttacag agtttagaaa 19560atgtggctta taatgttgtt aataaaggac
actttgatgg acacgccggc gaagcacctg 19620tttccatcat taataatgct gtttacacaa
aggtagatgg tattgatgtg gagatctttg 19680aaaataagac aacacttcct gttaatgttg
catttgagct ttgggctaag cgtaacatta 19740aaccagtgcc agagattaag atactcaata
atttgggtgt tgatatcgct gctaatactg 19800taatctggga ctacaaaaga gaagccccag
cacatgtatc tacaataggt gtctgcacaa 19860tgactgacat tgccaagaaa cctactgaga
gtgcttgttc ttcacttact gtcttgtttg 19920atggtagagt ggaaggacag gtagaccttt
ttagaaacgc ccgtaatggt gttttaataa 19980cagaaggttc agtcaaaggt ctaacacctt
caaagggacc agcacaagct agcgtcaatg 20040gagtcacatt aattggagaa tcagtaaaaa
cacagtttaa ctactttaag aaagtagacg 20100gcattattca acagttgcct gaaacctact
ttactcagag cagagactta gaggatttta 20160agcccagatc acaaatggaa actgactttc
tcgagctcgc tatggatgaa ttcatacagc 20220gatataagct cgagggctat gccttcgaac
acatcgttta tggagatttc agtcatggac 20280aacttggcgg tcttcattta atgataggct
tagccaagcg ctcacaagat tcaccactta 20340aattagagga ttttatccct atggacagca
cagtgaaaaa ttacttcata acagatgcgc 20400aaacaggttc atcaaaatgt gtgtgttctg
tgattgatct tttacttgat gactttgtcg 20460agataataaa gtcacaagat ttgtcagtga
tttcaaaagt ggtcaaggtt acaattgact 20520atgctgaaat ttcattcatg ctttggtgta
aggatggaca tgttgaaacc ttctacccaa 20580aactacaagc aagtcaagcg tggcaaccag
gtgttgcgat gcctaacttg tacaagatgc 20640aaagaatgct tcttgaaaag tgtgaccttc
agaattatgg tgaaaatgct gttataccaa 20700aaggaataat gatgaatgtc gcaaagtata
ctcaactgtg tcaatactta aatacactta 20760ctttagctgt accctacaac atgagagtta
ttcactttgg tgctggctct gataaaggag 20820ttgcaccagg tacagctgtg ctcagacaat
ggttgccaac tggcacacta cttgtcgatt 20880cagatcttaa tgacttcgtc tccgacgcag
attctacttt aattggagac tgtgcaacag 20940tacatacggc taataaatgg gaccttatta
ttagcgatat gtatgaccct aggaccaaac 21000atgtgacaaa agagaatgac tctaaagaag
ggtttttcac ttatctgtgt ggatttataa 21060agcaaaaact agccctgggt ggttctatag
ctgtaaagat aacagagcat tcttggaatg 21120ctgaccttta caagcttatg ggccatttct
catggtggac agcttttgtt acaaatgtaa 21180atgcatcatc atcggaagca tttttaattg
gggctaacta tcttggcaag ccgaaggaac 21240aaattgatgg ctataccatg catgctaact
acattttctg gaggaacaca aatcctatcc 21300agttgtcttc ctattcactc tttgacatga
gcaaatttcc tcttaaatta agaggaactg 21360ctgtaatgtc tcttaaggag aatcaaatca
atgatatgat ttattctctt ctggaaaaag 21420gtaggcttat cattagagaa aacaacagag
ttgtggtttc aagtgatatt cttgttaaca 21480actaaacgaa catgtttatt ttcttattat
ttcttactct cactagtggt agtgaccttg 21540accggtgcac cacttttgat gatgttcaag
ctcctaatta cactcaacat acttcatcta 21600tgaggggggt ttactatcct gatgaaattt
ttagatcaga cactctttat ttaactcagg 21660atttatttct tccattttat tctaatgtta
cagggtttca tactattaat catacgtttg 21720gcaaccctgt catacctttt aaggatggta
tttattttgc tgccacagag aaatcaaatg 21780ttgtccgtgg ttgggttttt ggttctacca
tgaacaacaa gtcacagtcg gtgattatta 21840ttaacaattc tactaatgtt gttatacgag
catgtaactt tgaattgtgt gacaaccctt 21900tctttgctgt ttctaaaccc atgggtacac
agacacatac tatgatattc gataatgcat 21960ttaattgcac tttcgagtac atatctgatg
ccttttcgct tgatgtttca gaaaagtcag 22020gtaattttaa acacttacga gagtttgtgt
ttaaaaataa agatgggttt ctctatgttt 22080ataagggcta tcaacctata gatgtagttc
gtgatctacc ttctggtttt aacactttga 22140aacctatttt taagttgcct cttggtatta
acattacaaa ttttagagcc attcttacag 22200ccttttcacc tgctcaagac atttggggca
cgtcagctgc agcctatttt gttggctatt 22260taaagccaac tacatttatg ctcaagtatg
atgaaaatgg tacaatcaca gatgctgttg 22320attgttctca aaatccactt gctgaactca
aatgctctgt taagagcttt gagattgaca 22380aaggaattta ccagacctct aatttcaggg
ttgttccctc aggagatgtt gtgagattcc 22440ctaatattac aaacttgtgt ccttttggag
aggtttttaa tgctactaaa ttcccttctg 22500tctatgcatg ggagagaaaa aaaatttcta
attgtgttgc tgattactct gtgctctaca 22560actcaacatt tttttcaacc tttaagtgct
atggcgtttc tgccactaag ttgaatgatc 22620tttgcttctc caatgtctat gcagattctt
ttgtagtcaa gggagatgat gtaagacaaa 22680tagcgccagg acaaactggt gttattgctg
attataatta taaattgcca gatgatttca 22740tgggttgtgt ccttgcttgg aatactagga
acattgatgc tacttcaact ggtaattata 22800attataaata taggtatctt agacatggca
agcttaggcc ctttgagaga gacatatcta 22860atgtgccttt ctcccctgat ggcaaacctt
gcaccccacc tgctcttaat tgttattggc 22920cattaaatga ttatggtttt tacaccacta
ctggcattgg ctaccaacct tacagagttg 22980tagtactttc ttttgaactt ttaaatgcac
cggccacggt ttgtggacca aaattatcca 23040ctgaccttat taagaaccag tgtgtcaatt
ttaattttaa tggactcact ggtactggtg 23100tgttaactcc ttcttcaaag agatttcaac
catttcaaca atttggccgt gatgtttctg 23160atttcactga ttccgttcga gatcctaaaa
catctgaaat attagacatt tcaccttgct 23220cttttggggg tgtaagtgta attacacctg
gaacaaatgc ttcatctgaa gttgctgttc 23280tatatcaaga tgttaactgc actgatgttt
ctacagcaat tcatgcagat caactcacac 23340cagcttggcg catatattct actggaaaca
atgtattcca gactcaagca ggctgtctta 23400taggagctga gcatgtcgac acttcttatg
agtgcgacat tcctattgga gctggcattt 23460gtgctagtta ccatacagtt tctttattac
gtagtactag ccaaaaatct attgtggctt 23520atactatgtc tttaggtgct gatagttcaa
ttgcttactc taataacacc attgctatac 23580ctactaactt ttcaattagc attactacag
aagtaatgcc tgtttctatg gctaaaacct 23640ccgtagattg taatatgtac atctgcggag
attctactga atgtgctaat ttgcttctcc 23700aatatggtag cttttgcaca caactaaatc
gtgcactctc aggtattgct gctgaacagg 23760atcgcaacac acgtgaagtg ttcgctcaag
tcaaacaaat gtacaaaacc ccaactttga 23820aatattttgg tggttttaat ttttcacaaa
tattacctga ccctctaaag ccaactaaga 23880ggtcttttat tgaggacttg ctctttaata
aggtgacact cgctgatgct ggcttcatga 23940agcaatatgg cgaatgccta ggtgatatta
atgctagaga tctcatttgt gcgcagaagt 24000tcaatggact tacagtgttg ccacctctgc
tcactgatga tatgattgct gcctacactg 24060ctgctctagt tagtggtact gccactgctg
gatggacatt tggtgctggc gctgctcttc 24120aaataccttt tgctatgcaa atggcatata
ggttcaatgg cattggagtt acccaaaatg 24180ttctctatga gaaccaaaaa caaatcgcca
accaatttaa caaggcgatt agtcaaattc 24240aagaatcact tacaacaaca tcaactgcat
tgggcaagct gcaagacgtt gttaaccaga 24300atgctcaagc attaaacaca cttgttaaac
aacttagctc taattttggt gcaatttcaa 24360gtgtgctaaa tgatatcctt tcgcgacttg
ataaagtcga ggcggaggta caaattgaca 24420ggttaattac aggcagactt caaagccttc
aaacctatgt aacacaacaa ctaatcaggg 24480ctgctgaaat cagggcttct gctaatcttg
ctgctactaa aatgtctgag tgtgttcttg 24540gacaatcaaa aagagttgac ttttgtggaa
agggctacca ccttatgtcc ttcccacaag 24600cagccccgca tggtgttgtc ttcctacatg
tcacgtatgt gccatcccag gagaggaact 24660tcaccacagc gccagcaatt tgtcatgaag
gcaaagcata cttccctcgt gaaggtgttt 24720ttgtgtttaa tggcacttct tggtttatta
cacagaggaa cttcttttct ccacaaataa 24780ttactacaga caatacattt gtctcaggaa
attgtgatgt cgttattggc atcattaaca 24840acacagttta tgatcctctg caacctgagc
tcgactcatt caaagaagag ctggacaagt 24900acttcaaaaa tcatacatca ccagatgttg
atcttggcga catttcaggc attaacgctt 24960ctgtcgtcaa cattcaaaaa gaaattgacc
gcctcaatga ggtcgctaaa aatttaaatg 25020aatcactcat tgaccttcaa gaattgggaa
aatatgagca atatattaaa tggccttggt 25080atgtttggct cggcttcatt gctggactaa
ttgccatcgt catggttaca atcttgcttt 25140gttgcatgac tagttgttgc agttgcctca
agggtgcatg ctcttgtggt tcttgctgca 25200agtttgatga ggatgactct gagccagttc
tcaagggtgt caaattacat tacacataaa 25260cgaacttatg gatttgttta tgagattttt
tactcttgga tcaattactg cacagccagt 25320aaaaattgac aatgcttctc ctgcaagtac
tgttcatgct acagcaacga taccgctaca 25380agcctcactc cctttcggat ggcttgttat
tggcgttgca tttcttgctg tttttcagag 25440cgctaccaaa ataattgcgc tcaataaaag
atggcagcta gccctttata agggcttcca 25500gttcatttgc aatttactgc tgctatttgt
taccatctat tcacatcttt tgcttgtcgc 25560tgcaggtatg gaggcgcaat ttttgtacct
ctatgccttg atatattttc tacaatgcat 25620caacgcatgt agaattatta tgagatgttg
gctttgttgg aagtgcaaat ccaagaaccc 25680attactttat gatgccaact actttgtttg
ctggcacaca cataactatg actactgtat 25740accatataac agtgtcacag atacaattgt
cgttactgaa ggtgacggca tttcaacacc 25800aaaactcaaa gaagactacc aaattggtgg
ttattctgag gataggcact caggtgttaa 25860agactatgtc gttgtacatg gctatttcac
cgaagtttac taccagcttg agtctacaca 25920aattactaca gacactggta ttgaaaatgc
tacattcttc atctttaaca agcttgttaa 25980agacccaccg aatgtgcaaa tacacacaat
cgacggctct tcaggagttg ctaatccagc 26040aatggatcca atttatgatg agccgacgac
gactactagc gtgcctttgt aagcacaaga 26100aagtgagtac gaacttatgt actcattcgt
ttcggaagaa acaggtacgt taatagttaa 26160tagcgtactt ctttttcttg ctttcgtggt
attcttgcta gtcacactag ccatccttac 26220tgcgcttcga ttgtgtgcgt actgctgcaa
tattgttaac gtgagtttag taaaaccaac 26280ggtttacgtc tactcgcgtg ttaaaaatct
gaactcttct gaaggagttc ctgatcttct 26340ggtctaaacg aactaactat tattattatt
ctgtttggaa ctttaacatt gcttatcatg 26400gcagacaacg gtactattac cgttgaggag
cttaaacaac tcctggaaca atggaaccta 26460gtaataggtt tcctattcct agcctggatt
atgttactac aatttgccta ttctaatcgg 26520aacaggtttt tgtacataat aaagcttgtt
ttcctctggc tcttgtggcc agtaacactt 26580gcttgttttg tgcttgctgc tgtctacaga
attaattggg tgactggcgg gattgcgatt 26640gcaatggctt gtattgtagg cttgatgtgg
cttagctact tcgttgcttc cttcaggctg 26700tttgctcgta cccgctcaat gtggtcattc
aacccagaaa caaacattct tctcaatgtg 26760cctctccggg ggacaattgt gaccagaccg
ctcatggaaa gtgaacttgt cattggtgct 26820gtgatcattc gtggtcactt gcgaatggcc
ggacaccccc tagggcgctg tgacattaag 26880gacctgccaa aagagatcac tgtggctaca
tcacgaacgc tttcttatta caaattagga 26940gcgtcgcagc gtgtaggcac tgattcaggt
tttgctgcat acaaccgcta ccgtattgga 27000aactataaat taaatacaga ccacgccggt
agcaacgaca atattgcttt gctagtacag 27060taagtgacaa cagatgtttc atcttgttga
cttccaggtt acaatagcag agatattgat 27120tatcattatg aggactttca ggattgctat
ttggaatctt gacgttataa taagttcaat 27180agtgagacaa ttatttaagc ctctaactaa
gaagaattat tcggagttag atgatgaaga 27240acctatggag ttagattatc cataaaacga
acatgaaaat tattctcttc ctgacattga 27300ttgtatttac atcttgcgag ctatatcact
atcaggagtg tgttagaggt acgactgtac 27360tactaaaaga accttgccca tcaggaacat
acgagggcaa ttcaccattt caccctcttg 27420ctgacaataa atttgcacta acttgcacta
gcacacactt tgcttttgct tgtgctgacg 27480gtactcgaca tacctatcag ctgcgtgcaa
gatcagtttc accaaaactt ttcatcagac 27540aagaggaggt tcaacaagag ctctactcgc
cactttttct cattgttgct gctctagtat 27600ttttaatact ttgcttcacc attaagagaa
agacagaatg aatgagctca ctttaattga 27660cttctatttg tgctttttag cctttctgct
attccttgtt ttaataatgc ttattatatt 27720ttggttttca ctcgaaatcc aggatctaga
agaaccttgt accaaagtct aaacgaacat 27780gaaacttctc attgttttga cttgtatttc
tctatgcagt tgcatatgca ctgtagtaca 27840gcgctgtgca tctaataaac ctcatgtgct
tgaagatcct tgtaaggtac aacactaggg 27900gtaatactta tagcactgct tggctttgtg
ctctaggaaa ggttttacct tttcatagat 27960ggcacactat ggttcaaaca tgcacaccta
atgttactat caactgtcaa gatccagctg 28020gtggtgcgct tatagctagg tgttggtacc
ttcatgaagg tcaccaaact gctgcattta 28080gagacgtact tgttgtttta aataaacgaa
caaattaaaa tgtctgataa tggaccccaa 28140tcaaaccaac gtagtgcccc ccgcattaca
tttggtggac ccacagattc aactgacaat 28200aaccagaatg gaggacgcaa tggggcaagg
ccaaaacagc gccgacccca aggtttaccc 28260aataatactg cgtcttggtt cacagctctc
actcagcatg gcaaggagga acttagattc 28320cctcgaggcc agggcgttcc aatcaacacc
aatagtggtc cagatgacca aattggctac 28380taccgaagag ctacccgacg agttcgtggt
ggtgacggca aaatgaaaga gctcagcccc 28440agatggtact tctattacct aggaactggc
ccagaagctt cacttcccta cggcgctaac 28500aaagaaggca tcgtatgggt tgcaactgag
ggagccttga atacacccaa agaccacatt 28560ggcacccgca atcctaataa caatgctgcc
accgtgctac aacttcctca aggaacaaca 28620ttgccaaaag gcttctacgc agagggaagc
agaggcggca gtcaagcctc ttctcgctcc 28680tcatcacgta gtcgcggtaa ttcaagaaat
tcaactcctg gcagcagtag gggaaattct 28740cctgctcgaa tggctagcgg aggtggtgaa
actgccctcg cgctattgct gctagacaga 28800ttgaaccagc ttgagagcaa agtttctggt
aaaggccaac aacaacaagg ccaaactgtc 28860actaagaaat ctgctgctga ggcatctaaa
aagcctcgcc aaaaacgtac tgccacaaaa 28920cagtacaacg tcactcaagc atttgggaga
cgtggtccag aacaaaccca aggaaatttc 28980ggggaccaag acctaatcag acaaggaact
gattacaaac attggccgca aattgcacaa 29040tttgctccaa gtgcctctgc attctttgga
atgtcacgca ttggcatgga agtcacacct 29100tcgggaacat ggctgactta tcatggagcc
attaaattgg atgacaaaga tccacaattc 29160aaagacaacg tcatactgct gaacaagcac
attgacgcat acaaaacatt cccaccaaca 29220gagcctaaaa aggacaaaaa gaaaaagact
gatgaagctc agcctttgcc gcagagacaa 29280aagaagcagc ccactgtgac tcttcttcct
gcggctgaca tggatgattt ctccagacaa 29340cttcaaaatt ccatgagtgg agcttctgct
gattcaactc aggcataaac actcatgatg 29400accacacaag gcagatgggc tatgtaaacg
ttttcgcaat tccgtttacg atacatagtc 29460tactcttgtg cagaatgaat tctcgtaact
aaacagcaca agtaggttta gttaacttta 29520atctcacata gcaatcttta atcaatgtgt
aacattaggg aggacttgaa agagccacca 29580cattttcatc gaggccacgc ggagtacgat
cgagggtaca gtgaataatg ctagggagag 29640ctgcctatat ggaagagccc taatgtgtaa
aattaatttt agtagtgcta tccccatgtg 29700attttaatag cttcttagga gaatgac
2972727073PRTSARS coronavirus Urbani 2Met
Glu Ser Leu Val Leu Gly Val Asn Glu Lys Thr His Val Gln Leu1
5 10 15Ser Leu Pro Val Leu Gln Val Arg
Asp Val Leu Val Arg Gly Phe Gly20 25
30Asp Ser Val Glu Glu Ala Leu Ser Glu Ala Arg Glu His Leu Lys Asn35
40 45Gly Thr Cys Gly Leu Val Glu Leu Glu Lys
Gly Val Leu Pro Gln Leu50 55 60Glu Gln
Pro Tyr Val Phe Ile Lys Arg Ser Asp Ala Leu Ser Thr Asn65
70 75 80His Gly His Lys Val Val Glu
Leu Val Ala Glu Met Asp Gly Ile Gln85 90
95Tyr Gly Arg Ser Gly Ile Thr Leu Gly Val Leu Val Pro His Val Gly100
105 110Glu Thr Pro Ile Ala Tyr Arg Asn Val
Leu Leu Arg Lys Asn Gly Asn115 120 125Lys
Gly Ala Gly Gly His Ser Tyr Gly Ile Asp Leu Lys Ser Tyr Asp130
135 140Leu Gly Asp Glu Leu Gly Thr Asp Pro Ile Glu
Asp Tyr Glu Gln Asn145 150 155
160Trp Asn Thr Lys His Gly Ser Gly Ala Leu Arg Glu Leu Thr Arg
Glu165 170 175Leu Asn Gly Gly Ala Val Thr
Arg Tyr Val Asp Asn Asn Phe Cys Gly180 185
190Pro Asp Gly Tyr Pro Leu Asp Cys Ile Lys Asp Phe Leu Ala Arg Ala195
200 205Gly Lys Ser Met Cys Thr Leu Ser Glu
Gln Leu Asp Tyr Ile Glu Ser210 215 220Lys
Arg Gly Val Tyr Cys Cys Arg Asp His Glu His Glu Ile Ala Trp225
230 235 240Phe Thr Glu Arg Ser Asp
Lys Ser Tyr Glu His Gln Thr Pro Phe Glu245 250
255Ile Lys Ser Ala Lys Lys Phe Asp Thr Phe Lys Gly Glu Cys Pro
Lys260 265 270Phe Val Phe Pro Leu Asn Ser
Lys Val Lys Val Ile Gln Pro Arg Val275 280
285Glu Lys Lys Lys Thr Glu Gly Phe Met Gly Arg Ile Arg Ser Val Tyr290
295 300Pro Val Ala Ser Pro Gln Glu Cys Asn
Asn Met His Leu Ser Thr Leu305 310 315
320Met Lys Cys Asn His Cys Asp Glu Val Ser Trp Gln Thr Cys
Asp Phe325 330 335Leu Lys Ala Thr Cys Glu
His Cys Gly Thr Glu Asn Leu Val Ile Glu340 345
350Gly Pro Thr Thr Cys Gly Tyr Leu Pro Thr Asn Ala Val Val Lys
Met355 360 365Pro Cys Pro Ala Cys Gln Asp
Pro Glu Ile Gly Pro Glu His Ser Val370 375
380Ala Asp Tyr His Asn His Ser Asn Ile Glu Thr Arg Leu Arg Lys Gly385
390 395 400Gly Arg Thr Arg
Cys Phe Gly Gly Cys Val Phe Ala Tyr Val Gly Cys405 410
415Tyr Asn Lys Arg Ala Tyr Trp Val Pro Arg Ala Ser Ala Asp
Ile Gly420 425 430Ser Gly His Thr Gly Ile
Thr Gly Asp Asn Val Glu Thr Leu Asn Glu435 440
445Asp Leu Leu Glu Ile Leu Ser Arg Glu Arg Val Asn Ile Asn Ile
Val450 455 460Gly Asp Phe His Leu Asn Glu
Glu Val Ala Ile Ile Leu Ala Ser Phe465 470
475 480Ser Ala Ser Thr Ser Ala Phe Ile Asp Thr Ile Lys
Ser Leu Asp Tyr485 490 495Lys Ser Phe Lys
Thr Ile Val Glu Ser Cys Gly Asn Tyr Lys Val Thr500 505
510Lys Gly Lys Pro Val Lys Gly Ala Trp Asn Ile Gly Gln Gln
Arg Ser515 520 525Val Leu Thr Pro Leu Cys
Gly Phe Pro Ser Gln Ala Ala Gly Val Ile530 535
540Arg Ser Ile Phe Ala Arg Thr Leu Asp Ala Ala Asn His Ser Ile
Pro545 550 555 560Asp Leu
Gln Arg Ala Ala Val Thr Ile Leu Asp Gly Ile Ser Glu Gln565
570 575Ser Leu Arg Leu Val Asp Ala Met Val Tyr Thr Ser
Asp Leu Leu Thr580 585 590Asn Ser Val Ile
Ile Met Ala Tyr Val Thr Gly Gly Leu Val Gln Gln595 600
605Thr Ser Gln Trp Leu Ser Asn Leu Leu Gly Thr Thr Val Glu
Lys Leu610 615 620Arg Pro Ile Phe Glu Trp
Ile Glu Ala Lys Leu Ser Ala Gly Val Glu625 630
635 640Phe Leu Lys Asp Ala Trp Glu Ile Leu Lys Phe
Leu Ile Thr Gly Val645 650 655Phe Asp Ile
Val Lys Gly Gln Ile Gln Val Ala Ser Asp Asn Ile Lys660
665 670Asp Cys Val Lys Cys Phe Ile Asp Val Val Asn Lys
Ala Leu Glu Met675 680 685Cys Ile Asp Gln
Val Thr Ile Ala Gly Ala Lys Leu Arg Ser Leu Asn690 695
700Leu Gly Glu Val Phe Ile Ala Gln Ser Lys Gly Leu Tyr Arg
Gln Cys705 710 715 720Ile
Arg Gly Lys Glu Gln Leu Gln Leu Leu Met Pro Leu Lys Ala Pro725
730 735Lys Glu Val Thr Phe Leu Glu Gly Asp Ser His
Asp Thr Val Leu Thr740 745 750Ser Glu Glu
Val Val Leu Lys Asn Gly Glu Leu Glu Ala Leu Glu Thr755
760 765Pro Val Asp Ser Phe Thr Asn Gly Ala Ile Val Gly
Thr Pro Val Cys770 775 780Val Asn Gly Leu
Met Leu Leu Glu Ile Lys Asp Lys Glu Gln Tyr Cys785 790
795 800Ala Leu Ser Pro Gly Leu Leu Ala Thr
Asn Asn Val Phe Arg Leu Lys805 810 815Gly
Gly Ala Pro Ile Lys Gly Val Thr Phe Gly Glu Asp Thr Val Trp820
825 830Glu Val Gln Gly Tyr Lys Asn Val Arg Ile Thr
Phe Glu Leu Asp Glu835 840 845Arg Val Asp
Lys Val Leu Asn Glu Lys Cys Ser Val Tyr Thr Val Glu850
855 860Ser Gly Thr Glu Val Thr Glu Phe Ala Cys Val Val
Ala Glu Ala Val865 870 875
880Val Lys Thr Leu Gln Pro Val Ser Asp Leu Leu Thr Asn Met Gly Ile885
890 895Asp Leu Asp Glu Trp Ser Val Ala Thr
Phe Tyr Leu Phe Asp Asp Ala900 905 910Gly
Glu Glu Asn Phe Ser Ser Arg Met Tyr Cys Ser Phe Tyr Pro Pro915
920 925Asp Glu Glu Glu Glu Asp Asp Ala Glu Cys Glu
Glu Glu Glu Ile Asp930 935 940Glu Thr Cys
Glu His Glu Tyr Gly Thr Glu Asp Asp Tyr Gln Gly Leu945
950 955 960Pro Leu Glu Phe Gly Ala Ser
Ala Glu Thr Val Arg Val Glu Glu Glu965 970
975Glu Glu Glu Asp Trp Leu Asp Asp Thr Thr Glu Gln Ser Glu Ile Glu980
985 990Pro Glu Pro Glu Pro Thr Pro Glu Glu
Pro Val Asn Gln Phe Thr Gly995 1000
1005Tyr Leu Lys Leu Thr Asp Asn Val Ala Ile Lys Cys Val Asp Ile Val1010
1015 1020Lys Glu Ala Gln Ser Ala Asn Pro Met
Val Ile Val Asn Ala Ala Asn1025 1030 1035
1040Ile His Leu Lys His Gly Gly Gly Val Ala Gly Ala Leu Asn
Lys Ala1045 1050 1055Thr Asn Gly Ala Met
Gln Lys Glu Ser Asp Asp Tyr Ile Lys Leu Asn1060 1065
1070Gly Pro Leu Thr Val Gly Gly Ser Cys Leu Leu Ser Gly His Asn
Leu1075 1080 1085Ala Lys Lys Cys Leu His
Val Val Gly Pro Asn Leu Asn Ala Gly Glu1090 1095
1100Asp Ile Gln Leu Leu Lys Ala Ala Tyr Glu Asn Phe Asn Ser Gln
Asp1105 1110 1115 1120Ile
Leu Leu Ala Pro Leu Leu Ser Ala Gly Ile Phe Gly Ala Lys Pro1125
1130 1135Leu Gln Ser Leu Gln Val Cys Val Gln Thr Val
Arg Thr Gln Val Tyr1140 1145 1150Ile Ala
Val Asn Asp Lys Ala Leu Tyr Glu Gln Val Val Met Asp Tyr1155
1160 1165Leu Asp Asn Leu Lys Pro Arg Val Glu Ala Pro Lys
Gln Glu Glu Pro1170 1175 1180Pro Asn Thr
Glu Asp Ser Lys Thr Glu Glu Lys Ser Val Val Gln Lys1185
1190 1195 1200Pro Val Asp Val Lys Pro Lys
Ile Lys Ala Cys Ile Asp Glu Val Thr1205 1210
1215Thr Thr Leu Glu Glu Thr Lys Phe Leu Thr Asn Lys Leu Leu Leu Phe1220
1225 1230Ala Asp Ile Asn Gly Lys Leu Tyr His
Asp Ser Gln Asn Met Leu Arg1235 1240
1245Gly Glu Asp Met Ser Phe Leu Glu Lys Asp Ala Pro Tyr Met Val Gly1250
1255 1260Asp Val Ile Thr Ser Gly Asp Ile Thr
Cys Val Val Ile Pro Ser Lys1265 1270 1275
1280Lys Ala Gly Gly Thr Thr Glu Met Leu Ser Arg Ala Leu Lys
Lys Val1285 1290 1295Pro Val Asp Glu Tyr
Ile Thr Thr Tyr Pro Gly Gln Gly Cys Ala Gly1300 1305
1310Tyr Thr Leu Glu Glu Ala Lys Thr Ala Leu Lys Lys Cys Lys Ser
Ala1315 1320 1325Phe Tyr Val Leu Pro Ser
Glu Ala Pro Asn Ala Lys Glu Glu Ile Leu1330 1335
1340Gly Thr Val Ser Trp Asn Leu Arg Glu Met Leu Ala His Ala Glu
Glu1345 1350 1355 1360Thr
Arg Lys Leu Met Pro Ile Cys Met Asp Val Arg Ala Ile Met Ala1365
1370 1375Thr Ile Gln Arg Lys Tyr Lys Gly Ile Lys Ile
Gln Glu Gly Ile Val1380 1385 1390Asp Tyr
Gly Val Arg Phe Phe Phe Tyr Thr Ser Lys Glu Pro Val Ala1395
1400 1405Ser Ile Ile Thr Lys Leu Asn Ser Leu Asn Glu Pro
Leu Val Thr Met1410 1415 1420Pro Ile Gly
Tyr Val Thr His Gly Phe Asn Leu Glu Glu Ala Ala Arg1425
1430 1435 1440Cys Met Arg Ser Leu Lys Ala
Pro Ala Val Val Ser Val Ser Ser Pro1445 1450
1455Asp Ala Val Thr Thr Tyr Asn Gly Tyr Leu Thr Ser Ser Ser Lys Thr1460
1465 1470Ser Glu Glu His Phe Val Glu Thr Val
Ser Leu Ala Gly Ser Tyr Arg1475 1480
1485Asp Trp Ser Tyr Ser Gly Gln Arg Thr Glu Leu Gly Val Glu Phe Leu1490
1495 1500Lys Arg Gly Asp Lys Ile Val Tyr His
Thr Leu Glu Ser Pro Val Glu1505 1510 1515
1520Phe His Leu Asp Gly Glu Val Leu Ser Leu Asp Lys Leu Lys
Ser Leu1525 1530 1535Leu Ser Leu Arg Glu
Val Lys Thr Ile Lys Val Phe Thr Thr Val Asp1540 1545
1550Asn Thr Asn Leu His Thr Gln Leu Val Asp Met Ser Met Thr Tyr
Gly1555 1560 1565Gln Gln Phe Gly Pro Thr
Tyr Leu Asp Gly Ala Asp Val Thr Lys Ile1570 1575
1580Lys Pro His Val Asn His Glu Gly Lys Thr Phe Phe Val Leu Pro
Ser1585 1590 1595 1600Asp
Asp Thr Leu Arg Ser Glu Ala Phe Glu Tyr Tyr His Thr Leu Asp1605
1610 1615Glu Ser Phe Leu Gly Arg Tyr Met Ser Ala Leu
Asn His Thr Lys Lys1620 1625 1630Trp Lys
Phe Pro Gln Val Gly Gly Leu Thr Ser Ile Lys Trp Ala Asp1635
1640 1645Asn Asn Cys Tyr Leu Ser Ser Val Leu Leu Ala Leu
Gln Gln Leu Glu1650 1655 1660Val Lys Phe
Asn Ala Pro Ala Leu Gln Glu Ala Tyr Tyr Arg Ala Arg1665
1670 1675 1680Ala Gly Asp Ala Ala Asn Phe
Cys Ala Leu Ile Leu Ala Tyr Ser Asn1685 1690
1695Lys Thr Val Gly Glu Leu Gly Asp Val Arg Glu Thr Met Thr His Leu1700
1705 1710Leu Gln His Ala Asn Leu Glu Ser Ala
Lys Arg Val Leu Asn Val Val1715 1720
1725Cys Lys His Cys Gly Gln Lys Thr Thr Thr Leu Thr Gly Val Glu Ala1730
1735 1740Val Met Tyr Met Gly Thr Leu Ser Tyr
Asp Asn Leu Lys Thr Gly Val1745 1750 1755
1760Ser Ile Pro Cys Val Cys Gly Arg Asp Ala Thr Gln Tyr Leu
Val Gln1765 1770 1775Gln Glu Ser Ser Phe
Val Met Met Ser Ala Pro Pro Ala Glu Tyr Lys1780 1785
1790Leu Gln Gln Gly Thr Phe Leu Cys Ala Asn Glu Tyr Thr Gly Asn
Tyr1795 1800 1805Gln Cys Gly His Tyr Thr
His Ile Thr Ala Lys Glu Thr Leu Tyr Arg1810 1815
1820Ile Asp Gly Ala His Leu Thr Lys Met Ser Glu Tyr Lys Gly Pro
Val1825 1830 1835 1840Thr
Asp Val Phe Tyr Lys Glu Thr Ser Tyr Thr Thr Thr Ile Lys Pro1845
1850 1855Val Ser Tyr Lys Leu Asp Gly Val Thr Tyr Thr
Glu Ile Glu Pro Lys1860 1865 1870Leu Asp
Gly Tyr Tyr Lys Lys Asp Asn Ala Tyr Tyr Thr Glu Gln Pro1875
1880 1885Ile Asp Leu Val Pro Thr Gln Pro Leu Pro Asn Ala
Ser Phe Asp Asn1890 1895 1900Phe Lys Leu
Thr Cys Ser Asn Thr Lys Phe Ala Asp Asp Leu Asn Gln1905
1910 1915 1920Met Thr Gly Phe Thr Lys Pro
Ala Ser Arg Glu Leu Ser Val Thr Phe1925 1930
1935Phe Pro Asp Leu Asn Gly Asp Val Val Ala Ile Asp Tyr Arg His Tyr1940
1945 1950Ser Ala Ser Phe Lys Lys Gly Ala Lys
Leu Leu His Lys Pro Ile Val1955 1960
1965Trp His Ile Asn Gln Ala Thr Thr Lys Thr Thr Phe Lys Pro Asn Thr1970
1975 1980Trp Cys Leu Arg Cys Leu Trp Ser Thr
Lys Pro Val Asp Thr Ser Asn1985 1990 1995
2000Ser Phe Glu Val Leu Ala Val Glu Asp Thr Gln Gly Met Asp
Asn Leu2005 2010 2015Ala Cys Glu Ser Gln
Gln Pro Thr Ser Glu Glu Val Val Glu Asn Pro2020 2025
2030Thr Ile Gln Lys Glu Val Ile Glu Cys Asp Val Lys Thr Thr Glu
Val2035 2040 2045Val Gly Asn Val Ile Leu
Lys Pro Ser Asp Glu Gly Val Lys Val Thr2050 2055
2060Gln Glu Leu Gly His Glu Asp Leu Met Ala Ala Tyr Val Glu Asn
Thr2065 2070 2075 2080Ser
Ile Thr Ile Lys Lys Pro Asn Glu Leu Ser Leu Ala Leu Gly Leu2085
2090 2095Lys Thr Ile Ala Thr His Gly Ile Ala Ala Ile
Asn Ser Val Pro Trp2100 2105 2110Ser Lys
Ile Leu Ala Tyr Val Lys Pro Phe Leu Gly Gln Ala Ala Ile2115
2120 2125Thr Thr Ser Asn Cys Ala Lys Arg Leu Ala Gln Arg
Val Phe Asn Asn2130 2135 2140Tyr Met Pro
Tyr Val Phe Thr Leu Leu Phe Gln Leu Cys Thr Phe Thr2145
2150 2155 2160Lys Ser Thr Asn Ser Arg Ile
Arg Ala Ser Leu Pro Thr Thr Ile Ala2165 2170
2175Lys Asn Ser Val Lys Ser Val Ala Lys Leu Cys Leu Asp Ala Gly Ile2180
2185 2190Asn Tyr Val Lys Ser Pro Lys Phe Ser
Lys Leu Phe Thr Ile Ala Met2195 2200
2205Trp Leu Leu Leu Leu Ser Ile Cys Leu Gly Ser Leu Ile Cys Val Thr2210
2215 2220Ala Ala Phe Gly Val Leu Leu Ser Asn
Phe Gly Ala Pro Ser Tyr Cys2225 2230 2235
2240Asn Gly Val Arg Glu Leu Tyr Leu Asn Ser Ser Asn Val Thr
Thr Met2245 2250 2255Asp Phe Cys Glu Gly
Ser Phe Pro Cys Ser Ile Cys Leu Ser Gly Leu2260 2265
2270Asp Ser Leu Asp Ser Tyr Pro Ala Leu Glu Thr Ile Gln Val Thr
Ile2275 2280 2285Ser Ser Tyr Lys Leu Asp
Leu Thr Ile Leu Gly Leu Ala Ala Glu Trp2290 2295
2300Val Leu Ala Tyr Met Leu Phe Thr Lys Phe Phe Tyr Leu Leu Gly
Leu2305 2310 2315 2320Ser
Ala Ile Met Gln Val Phe Phe Gly Tyr Phe Ala Ser His Phe Ile2325
2330 2335Ser Asn Ser Trp Leu Met Trp Phe Ile Ile Ser
Ile Val Gln Met Ala2340 2345 2350Pro Val
Ser Ala Met Val Arg Met Tyr Ile Phe Phe Ala Ser Phe Tyr2355
2360 2365Tyr Ile Trp Lys Ser Tyr Val His Ile Met Asp Gly
Cys Thr Ser Ser2370 2375 2380Thr Cys Met
Met Cys Tyr Lys Arg Asn Arg Ala Thr Arg Val Glu Cys2385
2390 2395 2400Thr Thr Ile Val Asn Gly Met
Lys Arg Ser Phe Tyr Val Tyr Ala Asn2405 2410
2415Gly Gly Arg Gly Phe Cys Lys Thr His Asn Trp Asn Cys Leu Asn Cys2420
2425 2430Asp Thr Phe Cys Thr Gly Ser Thr Phe
Ile Ser Asp Glu Val Ala Arg2435 2440
2445Asp Leu Ser Leu Gln Phe Lys Arg Pro Ile Asn Pro Thr Asp Gln Ser2450
2455 2460Ser Tyr Ile Val Asp Ser Val Ala Val
Lys Asn Gly Ala Leu His Leu2465 2470 2475
2480Tyr Phe Asp Lys Ala Gly Gln Lys Thr Tyr Glu Arg His Pro
Leu Ser2485 2490 2495His Phe Val Asn Leu
Asp Asn Leu Arg Ala Asn Asn Thr Lys Gly Ser2500 2505
2510Leu Pro Ile Asn Val Ile Val Phe Asp Gly Lys Ser Lys Cys Asp
Glu2515 2520 2525Ser Ala Ser Lys Ser Ala
Ser Val Tyr Tyr Ser Gln Leu Met Cys Gln2530 2535
2540Pro Ile Leu Leu Leu Asp Gln Val Leu Val Ser Asp Val Gly Asp
Ser2545 2550 2555 2560Thr
Glu Val Ser Val Lys Met Phe Asp Ala Tyr Val Asp Thr Phe Ser2565
2570 2575Ala Thr Phe Ser Val Pro Met Glu Lys Leu Lys
Ala Leu Val Ala Thr2580 2585 2590Ala His
Ser Glu Leu Ala Lys Gly Val Ala Leu Asp Gly Val Leu Ser2595
2600 2605Thr Phe Val Ser Ala Ala Arg Gln Gly Val Val Asp
Thr Asp Val Asp2610 2615 2620Thr Lys Asp
Val Ile Glu Cys Leu Lys Leu Ser His His Ser Asp Leu2625
2630 2635 2640Glu Val Thr Gly Asp Ser Cys
Asn Asn Phe Met Leu Thr Tyr Asn Lys2645 2650
2655Val Glu Asn Met Thr Pro Arg Asp Leu Gly Ala Cys Ile Asp Cys Asn2660
2665 2670Ala Arg His Ile Asn Ala Gln Val Ala
Lys Ser His Asn Val Ser Leu2675 2680
2685Ile Trp Asn Val Lys Asp Tyr Met Ser Leu Ser Glu Gln Leu Arg Lys2690
2695 2700Gln Ile Arg Ser Ala Ala Lys Lys Asn
Asn Ile Pro Phe Arg Leu Thr2705 2710 2715
2720Cys Ala Thr Thr Arg Gln Val Val Asn Val Ile Thr Thr Lys
Ile Ser2725 2730 2735Leu Lys Gly Gly Lys
Ile Val Ser Thr Cys Phe Lys Leu Met Leu Lys2740 2745
2750Ala Thr Leu Leu Cys Val Leu Ala Ala Leu Val Cys Tyr Ile Val
Met2755 2760 2765Pro Val His Thr Leu Ser
Ile His Asp Gly Tyr Thr Asn Glu Ile Ile2770 2775
2780Gly Tyr Lys Ala Ile Gln Asp Gly Val Thr Arg Asp Ile Ile Ser
Thr2785 2790 2795 2800Asp
Asp Cys Phe Ala Asn Lys His Ala Gly Phe Asp Ala Trp Phe Ser2805
2810 2815Gln Arg Gly Gly Ser Tyr Lys Asn Asp Lys Ser
Cys Pro Val Val Ala2820 2825 2830Ala Ile
Ile Thr Arg Glu Ile Gly Phe Ile Val Pro Gly Leu Pro Gly2835
2840 2845Thr Val Leu Arg Ala Ile Asn Gly Asp Phe Leu His
Phe Leu Pro Arg2850 2855 2860Val Phe Ser
Ala Val Gly Asn Ile Cys Tyr Thr Pro Ser Lys Leu Ile2865
2870 2875 2880Glu Tyr Ser Asp Phe Ala Thr
Ser Ala Cys Val Leu Ala Ala Glu Cys2885 2890
2895Thr Ile Phe Lys Asp Ala Met Gly Lys Pro Val Pro Tyr Cys Tyr Asp2900
2905 2910Thr Asn Leu Leu Glu Gly Ser Ile Ser
Tyr Ser Glu Leu Arg Pro Asp2915 2920
2925Thr Arg Tyr Val Leu Met Asp Gly Ser Ile Ile Gln Phe Pro Asn Thr2930
2935 2940Tyr Leu Glu Gly Ser Val Arg Val Val
Thr Thr Phe Asp Ala Glu Tyr2945 2950 2955
2960Cys Arg His Gly Thr Cys Glu Arg Ser Glu Val Gly Ile Cys
Leu Ser2965 2970 2975Thr Ser Gly Arg Trp
Val Leu Asn Asn Glu His Tyr Arg Ala Leu Ser2980 2985
2990Gly Val Phe Cys Gly Val Asp Ala Met Asn Leu Ile Ala Asn Ile
Phe2995 3000 3005Thr Pro Leu Val Gln Pro
Val Gly Ala Leu Asp Val Ser Ala Ser Val3010 3015
3020Val Ala Gly Gly Ile Ile Ala Ile Leu Val Thr Cys Ala Ala Tyr
Tyr3025 3030 3035 3040Phe
Met Lys Phe Arg Arg Val Phe Gly Glu Tyr Asn His Val Val Ala3045
3050 3055Ala Asn Ala Leu Leu Phe Leu Met Ser Phe Thr
Ile Leu Cys Leu Val3060 3065 3070Pro Ala
Tyr Ser Phe Leu Pro Gly Val Tyr Ser Val Phe Tyr Leu Tyr3075
3080 3085Leu Thr Phe Tyr Phe Thr Asn Asp Val Ser Phe Leu
Ala His Leu Gln3090 3095 3100Trp Phe Ala
Met Phe Ser Pro Ile Val Pro Phe Trp Ile Thr Ala Ile3105
3110 3115 3120Tyr Val Phe Cys Ile Ser Leu
Lys His Cys His Trp Phe Phe Asn Asn3125 3130
3135Tyr Leu Arg Lys Arg Val Met Phe Asn Gly Val Thr Phe Ser Thr Phe3140
3145 3150Glu Glu Ala Ala Leu Cys Thr Phe Leu
Leu Asn Lys Glu Met Tyr Leu3155 3160
3165Lys Leu Arg Ser Glu Thr Leu Leu Pro Leu Thr Gln Tyr Asn Arg Tyr3170
3175 3180Leu Ala Leu Tyr Asn Lys Tyr Lys Tyr
Phe Ser Gly Ala Leu Asp Thr3185 3190 3195
3200Thr Ser Tyr Arg Glu Ala Ala Cys Cys His Leu Ala Lys Ala
Leu Asn3205 3210 3215Asp Phe Ser Asn Ser
Gly Ala Asp Val Leu Tyr Gln Pro Pro Gln Thr3220 3225
3230Ser Ile Thr Ser Ala Val Leu Gln Ser Gly Phe Arg Lys Met Ala
Phe3235 3240 3245Pro Ser Gly Lys Val Glu
Gly Cys Met Val Gln Val Thr Cys Gly Thr3250 3255
3260Thr Thr Leu Asn Gly Leu Trp Leu Asp Asp Thr Val Tyr Cys Pro
Arg3265 3270 3275 3280His
Val Ile Cys Thr Ala Glu Asp Met Leu Asn Pro Asn Tyr Glu Asp3285
3290 3295Leu Leu Ile Arg Lys Ser Asn His Ser Phe Leu
Val Gln Ala Gly Asn3300 3305 3310Val Gln
Leu Arg Val Ile Gly His Ser Met Gln Asn Cys Leu Leu Arg3315
3320 3325Leu Lys Val Asp Thr Ser Asn Pro Lys Thr Pro Lys
Tyr Lys Phe Val3330 3335 3340Arg Ile Gln
Pro Gly Gln Thr Phe Ser Val Leu Ala Cys Tyr Asn Gly3345
3350 3355 3360Ser Pro Ser Gly Val Tyr Gln
Cys Ala Met Arg Pro Asn His Thr Ile3365 3370
3375Lys Gly Ser Phe Leu Asn Gly Ser Cys Gly Ser Val Gly Phe Asn Ile3380
3385 3390Asp Tyr Asp Cys Val Ser Phe Cys Tyr
Met His His Met Glu Leu Pro3395 3400
3405Thr Gly Val His Ala Gly Thr Asp Leu Glu Gly Lys Phe Tyr Gly Pro3410
3415 3420Phe Val Asp Arg Gln Thr Ala Gln Ala
Ala Gly Thr Asp Thr Thr Ile3425 3430 3435
3440Thr Leu Asn Val Leu Ala Trp Leu Tyr Ala Ala Val Ile Asn
Gly Asp3445 3450 3455Arg Trp Phe Leu Asn
Arg Phe Thr Thr Thr Leu Asn Asp Phe Asn Leu3460 3465
3470Val Ala Met Lys Tyr Asn Tyr Glu Pro Leu Thr Gln Asp His Val
Asp3475 3480 3485Ile Leu Gly Pro Leu Ser
Ala Gln Thr Gly Ile Ala Val Leu Asp Met3490 3495
3500Cys Ala Ala Leu Lys Glu Leu Leu Gln Asn Gly Met Asn Gly Arg
Thr3505 3510 3515 3520Ile
Leu Gly Ser Thr Ile Leu Glu Asp Glu Phe Thr Pro Phe Asp Val3525
3530 3535Val Arg Gln Cys Ser Gly Val Thr Phe Gln Gly
Lys Phe Lys Lys Ile3540 3545 3550Val Lys
Gly Thr His His Trp Met Leu Leu Thr Phe Leu Thr Ser Leu3555
3560 3565Leu Ile Leu Val Gln Ser Thr Gln Trp Ser Leu Phe
Phe Phe Val Tyr3570 3575 3580Glu Asn Ala
Phe Leu Pro Phe Thr Leu Gly Ile Met Ala Ile Ala Ala3585
3590 3595 3600Cys Ala Met Leu Leu Val Lys
His Lys His Ala Phe Leu Cys Leu Phe3605 3610
3615Leu Leu Pro Ser Leu Ala Thr Val Ala Tyr Phe Asn Met Val Tyr Met3620
3625 3630Pro Ala Ser Trp Val Met Arg Ile Met
Thr Trp Leu Glu Leu Ala Asp3635 3640
3645Thr Ser Leu Ser Gly Tyr Arg Leu Lys Asp Cys Val Met Tyr Ala Ser3650
3655 3660Ala Leu Val Leu Leu Ile Leu Met Thr
Ala Arg Thr Val Tyr Asp Asp3665 3670 3675
3680Ala Ala Arg Arg Val Trp Thr Leu Met Asn Val Ile Thr Leu
Val Tyr3685 3690 3695Lys Val Tyr Tyr Gly
Asn Ala Leu Asp Gln Ala Ile Ser Met Trp Ala3700 3705
3710Leu Val Ile Ser Val Thr Ser Asn Tyr Ser Gly Val Val Thr Thr
Ile3715 3720 3725Met Phe Leu Ala Arg Ala
Ile Val Phe Val Cys Val Glu Tyr Tyr Pro3730 3735
3740Leu Leu Phe Ile Thr Gly Asn Thr Leu Gln Cys Ile Met Leu Val
Tyr3745 3750 3755 3760Cys
Phe Leu Gly Tyr Cys Cys Cys Cys Tyr Phe Gly Leu Phe Cys Leu3765
3770 3775Leu Asn Arg Tyr Phe Arg Leu Thr Leu Gly Val
Tyr Asp Tyr Leu Val3780 3785 3790Ser Thr
Gln Glu Phe Arg Tyr Met Asn Ser Gln Gly Leu Leu Pro Pro3795
3800 3805Lys Ser Ser Ile Asp Ala Phe Lys Leu Asn Ile Lys
Leu Leu Gly Ile3810 3815 3820Gly Gly Lys
Pro Cys Ile Lys Val Ala Thr Val Gln Ser Lys Met Ser3825
3830 3835 3840Asp Val Lys Cys Thr Ser Val
Val Leu Leu Ser Val Leu Gln Gln Leu3845 3850
3855Arg Val Glu Ser Ser Ser Lys Leu Trp Ala Gln Cys Val Gln Leu His3860
3865 3870Asn Asp Ile Leu Leu Ala Lys Asp Thr
Thr Glu Ala Phe Glu Lys Met3875 3880
3885Val Ser Leu Leu Ser Val Leu Leu Ser Met Gln Gly Ala Val Asp Ile3890
3895 3900Asn Arg Leu Cys Glu Glu Met Leu Asp
Asn Arg Ala Thr Leu Gln Ala3905 3910 3915
3920Ile Ala Ser Glu Phe Ser Ser Leu Pro Ser Tyr Ala Ala Tyr
Ala Thr3925 3930 3935Ala Gln Glu Ala Tyr
Glu Gln Ala Val Ala Asn Gly Asp Ser Glu Val3940 3945
3950Val Leu Lys Lys Leu Lys Lys Ser Leu Asn Val Ala Lys Ser Glu
Phe3955 3960 3965Asp Arg Asp Ala Ala Met
Gln Arg Lys Leu Glu Lys Met Ala Asp Gln3970 3975
3980Ala Met Thr Gln Met Tyr Lys Gln Ala Arg Ser Glu Asp Lys Arg
Ala3985 3990 3995 4000Lys
Val Thr Ser Ala Met Gln Thr Met Leu Phe Thr Met Leu Arg Lys4005
4010 4015Leu Asp Asn Asp Ala Leu Asn Asn Ile Ile Asn
Asn Ala Arg Asp Gly4020 4025 4030Cys Val
Pro Leu Asn Ile Ile Pro Leu Thr Thr Ala Ala Lys Leu Met4035
4040 4045Val Val Val Pro Asp Tyr Gly Thr Tyr Lys Asn Thr
Cys Asp Gly Asn4050 4055 4060Thr Phe Thr
Tyr Ala Ser Ala Leu Trp Glu Ile Gln Gln Val Val Asp4065
4070 4075 4080Ala Asp Ser Lys Ile Val Gln
Leu Ser Glu Ile Asn Met Asp Asn Ser4085 4090
4095Pro Asn Leu Ala Trp Pro Leu Ile Val Thr Ala Leu Arg Ala Asn Ser4100
4105 4110Ala Val Lys Leu Gln Asn Asn Glu Leu
Ser Pro Val Ala Leu Arg Gln4115 4120
4125Met Ser Cys Ala Ala Gly Thr Thr Gln Thr Ala Cys Thr Asp Asp Asn4130
4135 4140Ala Leu Ala Tyr Tyr Asn Asn Ser Lys
Gly Gly Arg Phe Val Leu Ala4145 4150 4155
4160Leu Leu Ser Asp His Gln Asp Leu Lys Trp Ala Arg Phe Pro
Lys Ser4165 4170 4175Asp Gly Thr Gly Thr
Ile Tyr Thr Glu Leu Glu Pro Pro Cys Arg Phe4180 4185
4190Val Thr Asp Thr Pro Lys Gly Pro Lys Val Lys Tyr Leu Tyr Phe
Ile4195 4200 4205Lys Gly Leu Asn Asn Leu
Asn Arg Gly Met Val Leu Gly Ser Leu Ala4210 4215
4220Ala Thr Val Arg Leu Gln Ala Gly Asn Ala Thr Glu Val Pro Ala
Asn4225 4230 4235 4240Ser
Thr Val Leu Ser Phe Cys Ala Phe Ala Val Asp Pro Ala Lys Ala4245
4250 4255Tyr Lys Asp Tyr Leu Ala Ser Gly Gly Gln Pro
Ile Thr Asn Cys Val4260 4265 4270Lys Met
Leu Cys Thr His Thr Gly Thr Gly Gln Ala Ile Thr Val Thr4275
4280 4285Pro Glu Ala Asn Met Asp Gln Glu Ser Phe Gly Gly
Ala Ser Cys Cys4290 4295 4300Leu Tyr Cys
Arg Cys His Ile Asp His Pro Asn Pro Lys Gly Phe Cys4305
4310 4315 4320Asp Leu Lys Gly Lys Tyr Val
Gln Ile Pro Thr Thr Cys Ala Asn Asp4325 4330
4335Pro Val Gly Phe Thr Leu Arg Asn Thr Val Cys Thr Val Cys Gly Met4340
4345 4350Trp Lys Gly Tyr Gly Cys Ser Cys Asp
Gln Leu Arg Glu Pro Leu Met4355 4360
4365Gln Ser Ala Asp Ala Ser Thr Phe Leu Asn Arg Val Cys Gly Val Ser4370
4375 4380Ala Ala Arg Leu Thr Pro Cys Gly Thr
Gly Thr Ser Thr Asp Val Val4385 4390 4395
4400Tyr Arg Ala Phe Asp Ile Tyr Asn Glu Lys Val Ala Gly Phe
Ala Lys4405 4410 4415Phe Leu Lys Thr Asn
Cys Cys Arg Phe Gln Glu Lys Asp Glu Glu Gly4420 4425
4430Asn Leu Leu Asp Ser Tyr Phe Val Val Lys Arg His Thr Met Ser
Asn4435 4440 4445Tyr Gln His Glu Glu Thr
Ile Tyr Asn Leu Val Lys Asp Cys Pro Ala4450 4455
4460Val Ala Val His Asp Phe Phe Lys Phe Arg Val Asp Gly Asp Met
Val4465 4470 4475 4480Pro
His Ile Ser Arg Gln Arg Leu Thr Lys Tyr Thr Met Ala Asp Leu4485
4490 4495Val Tyr Ala Leu Arg His Phe Asp Glu Gly Asn
Cys Asp Thr Leu Lys4500 4505 4510Glu Ile
Leu Val Thr Tyr Asn Cys Cys Asp Asp Asp Tyr Phe Asn Lys4515
4520 4525Lys Asp Trp Tyr Asp Phe Val Glu Asn Pro Asp Ile
Leu Arg Val Tyr4530 4535 4540Ala Asn Leu
Gly Glu Arg Val Arg Gln Ser Leu Leu Lys Thr Val Gln4545
4550 4555 4560Phe Cys Asp Ala Met Arg Asp
Ala Gly Ile Val Gly Val Leu Thr Leu4565 4570
4575Asp Asn Gln Asp Leu Asn Gly Asn Trp Tyr Asp Phe Gly Asp Phe Val4580
4585 4590Gln Val Ala Pro Gly Cys Gly Val Pro
Ile Val Asp Ser Tyr Tyr Ser4595 4600
4605Leu Leu Met Pro Ile Leu Thr Leu Thr Arg Ala Leu Ala Ala Glu Ser4610
4615 4620His Met Asp Ala Asp Leu Ala Lys Pro
Leu Ile Lys Trp Asp Leu Leu4625 4630 4635
4640Lys Tyr Asp Phe Thr Glu Glu Arg Leu Cys Leu Phe Asp Arg
Tyr Phe4645 4650 4655Lys Tyr Trp Asp Gln
Thr Tyr His Pro Asn Cys Ile Asn Cys Leu Asp4660 4665
4670Asp Arg Cys Ile Leu His Cys Ala Asn Phe Asn Val Leu Phe Ser
Thr4675 4680 4685Val Phe Pro Pro Thr Ser
Phe Gly Pro Leu Val Arg Lys Ile Phe Val4690 4695
4700Asp Gly Val Pro Phe Val Val Ser Thr Gly Tyr His Phe Arg Glu
Leu4705 4710 4715 4720Gly
Val Val His Asn Gln Asp Val Asn Leu His Ser Ser Arg Leu Ser4725
4730 4735Phe Lys Glu Leu Leu Val Tyr Ala Ala Asp Pro
Ala Met His Ala Ala4740 4745 4750Ser Gly
Asn Leu Leu Leu Asp Lys Arg Thr Thr Cys Phe Ser Val Ala4755
4760 4765Ala Leu Thr Asn Asn Val Ala Phe Gln Thr Val Lys
Pro Gly Asn Phe4770 4775 4780Asn Lys Asp
Phe Tyr Asp Phe Ala Val Ser Lys Gly Phe Phe Lys Glu4785
4790 4795 4800Gly Ser Ser Val Glu Leu Lys
His Phe Phe Phe Ala Gln Asp Gly Asn4805 4810
4815Ala Ala Ile Ser Asp Tyr Asp Tyr Tyr Arg Tyr Asn Leu Pro Thr Met4820
4825 4830Cys Asp Ile Arg Gln Leu Leu Phe Val
Val Glu Val Val Asp Lys Tyr4835 4840
4845Phe Asp Cys Tyr Asp Gly Gly Cys Ile Asn Ala Asn Gln Val Ile Val4850
4855 4860Asn Asn Leu Asp Lys Ser Ala Gly Phe
Pro Phe Asn Lys Trp Gly Lys4865 4870 4875
4880Ala Arg Leu Tyr Tyr Asp Ser Met Ser Tyr Glu Asp Gln Asp
Ala Leu4885 4890 4895Phe Ala Tyr Thr Lys
Arg Asn Val Ile Pro Thr Ile Thr Gln Met Asn4900 4905
4910Leu Lys Tyr Ala Ile Ser Ala Lys Asn Arg Ala Arg Thr Val Ala
Gly4915 4920 4925Val Ser Ile Cys Ser Thr
Met Thr Asn Arg Gln Phe His Gln Lys Leu4930 4935
4940Leu Lys Ser Ile Ala Ala Thr Arg Gly Ala Thr Val Val Ile Gly
Thr4945 4950 4955 4960Ser
Lys Phe Tyr Gly Gly Trp His Asn Met Leu Lys Thr Val Tyr Ser4965
4970 4975Asp Val Glu Thr Pro His Leu Met Gly Trp Asp
Tyr Pro Lys Cys Asp4980 4985 4990Arg Ala
Met Pro Asn Met Leu Arg Ile Met Ala Ser Leu Val Leu Ala4995
5000 5005Arg Lys His Asn Thr Cys Cys Asn Leu Ser His Arg
Phe Tyr Arg Leu5010 5015 5020Ala Asn Glu
Cys Ala Gln Val Leu Ser Glu Met Val Met Cys Gly Gly5025
5030 5035 5040Ser Leu Tyr Val Lys Pro Gly
Gly Thr Ser Ser Gly Asp Ala Thr Thr5045 5050
5055Ala Tyr Ala Asn Ser Val Phe Asn Ile Cys Gln Ala Val Thr Ala Asn5060
5065 5070Val Asn Ala Leu Leu Ser Thr Asp Gly
Asn Lys Ile Ala Asp Lys Tyr5075 5080
5085Val Arg Asn Leu Gln His Arg Leu Tyr Glu Cys Leu Tyr Arg Asn Arg5090
5095 5100Asp Val Asp His Glu Phe Val Asp Glu
Phe Tyr Ala Tyr Leu Arg Lys5105 5110 5115
5120His Phe Ser Met Met Ile Leu Ser Asp Asp Ala Val Val Cys
Tyr Asn5125 5130 5135Ser Asn Tyr Ala Ala
Gln Gly Leu Val Ala Ser Ile Lys Asn Phe Lys5140 5145
5150Ala Val Leu Tyr Tyr Gln Asn Asn Val Phe Met Ser Glu Ala Lys
Cys5155 5160 5165Trp Thr Glu Thr Asp Leu
Thr Lys Gly Pro His Glu Phe Cys Ser Gln5170 5175
5180His Thr Met Leu Val Lys Gln Gly Asp Asp Tyr Val Tyr Leu Pro
Tyr5185 5190 5195 5200Pro
Asp Pro Ser Arg Ile Leu Gly Ala Gly Cys Phe Val Asp Asp Ile5205
5210 5215Val Lys Thr Asp Gly Thr Leu Met Ile Glu Arg
Phe Val Ser Leu Ala5220 5225 5230Ile Asp
Ala Tyr Pro Leu Thr Lys His Pro Asn Gln Glu Tyr Ala Asp5235
5240 5245Val Phe His Leu Tyr Leu Gln Tyr Ile Arg Lys Leu
His Asp Glu Leu5250 5255 5260Thr Gly His
Met Leu Asp Met Tyr Ser Val Met Leu Thr Asn Asp Asn5265
5270 5275 5280Thr Ser Arg Tyr Trp Glu Pro
Glu Phe Tyr Glu Ala Met Tyr Thr Pro5285 5290
5295His Thr Val Leu Gln Ala Val Gly Ala Cys Val Leu Cys Asn Ser Gln5300
5305 5310Thr Ser Leu Arg Cys Gly Ala Cys Ile
Arg Arg Pro Phe Leu Cys Cys5315 5320
5325Lys Cys Cys Tyr Asp His Val Ile Ser Thr Ser His Lys Leu Val Leu5330
5335 5340Ser Val Asn Pro Tyr Val Cys Asn Ala
Pro Gly Cys Asp Val Thr Asp5345 5350 5355
5360Val Thr Gln Leu Tyr Leu Gly Gly Met Ser Tyr Tyr Cys Lys
Ser His5365 5370 5375Lys Pro Pro Ile Ser
Phe Pro Leu Cys Ala Asn Gly Gln Val Phe Gly5380 5385
5390Leu Tyr Lys Asn Thr Cys Val Gly Ser Asp Asn Val Thr Asp Phe
Asn5395 5400 5405Ala Ile Ala Thr Cys Asp
Trp Thr Asn Ala Gly Asp Tyr Ile Leu Ala5410 5415
5420Asn Thr Cys Thr Glu Arg Leu Lys Leu Phe Ala Ala Glu Thr Leu
Lys5425 5430 5435 5440Ala
Thr Glu Glu Thr Phe Lys Leu Ser Tyr Gly Ile Ala Thr Val Arg5445
5450 5455Glu Val Leu Ser Asp Arg Glu Leu His Leu Ser
Trp Glu Val Gly Lys5460 5465 5470Pro Arg
Pro Pro Leu Asn Arg Asn Tyr Val Phe Thr Gly Tyr Arg Val5475
5480 5485Thr Lys Asn Ser Lys Val Gln Ile Gly Glu Tyr Thr
Phe Glu Lys Gly5490 5495 5500Asp Tyr Gly
Asp Ala Val Val Tyr Arg Gly Thr Thr Thr Tyr Lys Leu5505
5510 5515 5520Asn Val Gly Asp Tyr Phe Val
Leu Thr Ser His Thr Val Met Pro Leu5525 5530
5535Ser Ala Pro Thr Leu Val Pro Gln Glu His Tyr Val Arg Ile Thr Gly5540
5545 5550Leu Tyr Pro Thr Leu Asn Ile Ser Asp
Glu Phe Ser Ser Asn Val Ala5555 5560
5565Asn Tyr Gln Lys Val Gly Met Gln Lys Tyr Ser Thr Leu Gln Gly Pro5570
5575 5580Pro Gly Thr Gly Lys Ser His Phe Ala
Ile Gly Leu Ala Leu Tyr Tyr5585 5590 5595
5600Pro Ser Ala Arg Ile Val Tyr Thr Ala Cys Ser His Ala Ala
Val Asp5605 5610 5615Ala Leu Cys Glu Lys
Ala Leu Lys Tyr Leu Pro Ile Asp Lys Cys Ser5620 5625
5630Arg Ile Ile Pro Ala Arg Ala Arg Val Glu Cys Phe Asp Lys Phe
Lys5635 5640 5645Val Asn Ser Thr Leu Glu
Gln Tyr Val Phe Cys Thr Val Asn Ala Leu5650 5655
5660Pro Glu Thr Thr Ala Asp Ile Val Val Phe Asp Glu Ile Ser Met
Ala5665 5670 5675 5680Thr
Asn Tyr Asp Leu Ser Val Val Asn Ala Arg Leu Arg Ala Lys His5685
5690 5695Tyr Val Tyr Ile Gly Asp Pro Ala Gln Leu Pro
Ala Pro Arg Thr Leu5700 5705 5710Leu Thr
Lys Gly Thr Leu Glu Pro Glu Tyr Phe Asn Ser Val Cys Arg5715
5720 5725Leu Met Lys Thr Ile Gly Pro Asp Met Phe Leu Gly
Thr Cys Arg Arg5730 5735 5740Cys Pro Ala
Glu Ile Val Asp Thr Val Ser Ala Leu Val Tyr Asp Asn5745
5750 5755 5760Lys Leu Lys Ala His Lys Asp
Lys Ser Ala Gln Cys Phe Lys Met Phe5765 5770
5775Tyr Lys Gly Val Ile Thr His Asp Val Ser Ser Ala Ile Asn Arg Pro5780
5785 5790Gln Ile Gly Val Val Arg Glu Phe Leu
Thr Arg Asn Pro Ala Trp Arg5795 5800
5805Lys Ala Val Phe Ile Ser Pro Tyr Asn Ser Gln Asn Ala Val Ala Ser5810
5815 5820Lys Ile Leu Gly Leu Pro Thr Gln Thr
Val Asp Ser Ser Gln Gly Ser5825 5830 5835
5840Glu Tyr Asp Tyr Val Ile Phe Thr Gln Thr Thr Glu Thr Ala
His Ser5845 5850 5855Cys Asn Val Asn Arg
Phe Asn Val Ala Ile Thr Arg Ala Lys Ile Gly5860 5865
5870Ile Leu Cys Ile Met Ser Asp Arg Asp Leu Tyr Asp Lys Leu Gln
Phe5875 5880 5885Thr Ser Leu Glu Ile Pro
Arg Arg Asn Val Ala Thr Leu Gln Ala Glu5890 5895
5900Asn Val Thr Gly Leu Phe Lys Asp Cys Ser Lys Ile Ile Thr Gly
Leu5905 5910 5915 5920His
Pro Thr Gln Ala Pro Thr His Leu Ser Val Asp Ile Lys Phe Lys5925
5930 5935Thr Glu Gly Leu Cys Val Asp Ile Pro Gly Ile
Pro Lys Asp Met Thr5940 5945 5950Tyr Arg
Arg Leu Ile Ser Met Met Gly Phe Lys Met Asn Tyr Gln Val5955
5960 5965Asn Gly Tyr Pro Asn Met Phe Ile Thr Arg Glu Glu
Ala Ile Arg His5970 5975 5980Val Arg Ala
Trp Ile Gly Phe Asp Val Glu Gly Cys His Ala Thr Arg5985
5990 5995 6000Asp Ala Val Gly Thr Asn Leu
Pro Leu Gln Leu Gly Phe Ser Thr Gly6005 6010
6015Val Asn Leu Val Ala Val Pro Thr Gly Tyr Val Asp Thr Glu Asn Asn6020
6025 6030Thr Glu Phe Thr Arg Val Asn Ala Lys
Pro Pro Pro Gly Asp Gln Phe6035 6040
6045Lys His Leu Ile Pro Leu Met Tyr Lys Gly Leu Pro Trp Asn Val Val6050
6055 6060Arg Ile Lys Ile Val Gln Met Leu Ser
Asp Thr Leu Lys Gly Leu Ser6065 6070 6075
6080Asp Arg Val Val Phe Val Leu Trp Ala His Gly Phe Glu Leu
Thr Ser6085 6090 6095Met Lys Tyr Phe Val
Lys Ile Gly Pro Glu Arg Thr Cys Cys Leu Cys6100 6105
6110Asp Lys Arg Ala Thr Cys Phe Ser Thr Ser Ser Asp Thr Tyr Ala
Cys6115 6120 6125Trp Asn His Ser Val Gly
Phe Asp Tyr Val Tyr Asn Pro Phe Met Ile6130 6135
6140Asp Val Gln Gln Trp Gly Phe Thr Gly Asn Leu Gln Ser Asn His
Asp6145 6150 6155 6160Gln
His Cys Gln Val His Gly Asn Ala His Val Ala Ser Cys Asp Ala6165
6170 6175Ile Met Thr Arg Cys Leu Ala Val His Glu Cys
Phe Val Lys Arg Val6180 6185 6190Asp Trp
Ser Val Glu Tyr Pro Ile Ile Gly Asp Glu Leu Arg Val Asn6195
6200 6205Ser Ala Cys Arg Lys Val Gln His Met Val Val Lys
Ser Ala Leu Leu6210 6215 6220Ala Asp Lys
Phe Pro Val Leu His Asp Ile Gly Asn Pro Lys Ala Ile6225
6230 6235 6240Lys Cys Val Pro Gln Ala Glu
Val Glu Trp Lys Phe Tyr Asp Ala Gln6245 6250
6255Pro Cys Ser Asp Lys Ala Tyr Lys Ile Glu Glu Leu Phe Tyr Ser Tyr6260
6265 6270Ala Thr His His Asp Lys Phe Thr Asp
Gly Val Cys Leu Phe Trp Asn6275 6280
6285Cys Asn Val Asp Arg Tyr Pro Ala Asn Ala Ile Val Cys Arg Phe Asp6290
6295 6300Thr Arg Val Leu Ser Asn Leu Asn Leu
Pro Gly Cys Asp Gly Gly Ser6305 6310 6315
6320Leu Tyr Val Asn Lys His Ala Phe His Thr Pro Ala Phe Asp
Lys Ser6325 6330 6335Ala Phe Thr Asn Leu
Lys Gln Leu Pro Phe Phe Tyr Tyr Ser Asp Ser6340 6345
6350Pro Cys Glu Ser His Gly Lys Gln Val Val Ser Asp Ile Asp Tyr
Val6355 6360 6365Pro Leu Lys Ser Ala Thr
Cys Ile Thr Arg Cys Asn Leu Gly Gly Ala6370 6375
6380Val Cys Arg His His Ala Asn Glu Tyr Arg Gln Tyr Leu Asp Ala
Tyr6385 6390 6395 6400Asn
Met Met Ile Ser Ala Gly Phe Ser Leu Trp Ile Tyr Lys Gln Phe6405
6410 6415Asp Thr Tyr Asn Leu Trp Asn Thr Phe Thr Arg
Leu Gln Ser Leu Glu6420 6425 6430Asn Val
Ala Tyr Asn Val Val Asn Lys Gly His Phe Asp Gly His Ala6435
6440 6445Gly Glu Ala Pro Val Ser Ile Ile Asn Asn Ala Val
Tyr Thr Lys Val6450 6455 6460Asp Gly Ile
Asp Val Glu Ile Phe Glu Asn Lys Thr Thr Leu Pro Val6465
6470 6475 6480Asn Val Ala Phe Glu Leu Trp
Ala Lys Arg Asn Ile Lys Pro Val Pro6485 6490
6495Glu Ile Lys Ile Leu Asn Asn Leu Gly Val Asp Ile Ala Ala Asn Thr6500
6505 6510Val Ile Trp Asp Tyr Lys Arg Glu Ala
Pro Ala His Val Ser Thr Ile6515 6520
6525Gly Val Cys Thr Met Thr Asp Ile Ala Lys Lys Pro Thr Glu Ser Ala6530
6535 6540Cys Ser Ser Leu Thr Val Leu Phe Asp
Gly Arg Val Glu Gly Gln Val6545 6550 6555
6560Asp Leu Phe Arg Asn Ala Arg Asn Gly Val Leu Ile Thr Glu
Gly Ser6565 6570 6575Val Lys Gly Leu Thr
Pro Ser Lys Gly Pro Ala Gln Ala Ser Val Asn6580 6585
6590Gly Val Thr Leu Ile Gly Glu Ser Val Lys Thr Gln Phe Asn Tyr
Phe6595 6600 6605Lys Lys Val Asp Gly Ile
Ile Gln Gln Leu Pro Glu Thr Tyr Phe Thr6610 6615
6620Gln Ser Arg Asp Leu Glu Asp Phe Lys Pro Arg Ser Gln Met Glu
Thr6625 6630 6635 6640Asp
Phe Leu Glu Leu Ala Met Asp Glu Phe Ile Gln Arg Tyr Lys Leu6645
6650 6655Glu Gly Tyr Ala Phe Glu His Ile Val Tyr Gly
Asp Phe Ser His Gly6660 6665 6670Gln Leu
Gly Gly Leu His Leu Met Ile Gly Leu Ala Lys Arg Ser Gln6675
6680 6685Asp Ser Pro Leu Lys Leu Glu Asp Phe Ile Pro Met
Asp Ser Thr Val6690 6695 6700Lys Asn Tyr
Phe Ile Thr Asp Ala Gln Thr Gly Ser Ser Lys Cys Val6705
6710 6715 6720Cys Ser Val Ile Asp Leu Leu
Leu Asp Asp Phe Val Glu Ile Ile Lys6725 6730
6735Ser Gln Asp Leu Ser Val Ile Ser Lys Val Val Lys Val Thr Ile Asp6740
6745 6750Tyr Ala Glu Ile Ser Phe Met Leu Trp
Cys Lys Asp Gly His Val Glu6755 6760
6765Thr Phe Tyr Pro Lys Leu Gln Ala Ser Gln Ala Trp Gln Pro Gly Val6770
6775 6780Ala Met Pro Asn Leu Tyr Lys Met Gln
Arg Met Leu Leu Glu Lys Cys6785 6790 6795
6800Asp Leu Gln Asn Tyr Gly Glu Asn Ala Val Ile Pro Lys Gly
Ile Met6805 6810 6815Met Asn Val Ala Lys
Tyr Thr Gln Leu Cys Gln Tyr Leu Asn Thr Leu6820 6825
6830Thr Leu Ala Val Pro Tyr Asn Met Arg Val Ile His Phe Gly Ala
Gly6835 6840 6845Ser Asp Lys Gly Val Ala
Pro Gly Thr Ala Val Leu Arg Gln Trp Leu6850 6855
6860Pro Thr Gly Thr Leu Leu Val Asp Ser Asp Leu Asn Asp Phe Val
Ser6865 6870 6875 6880Asp
Ala Asp Ser Thr Leu Ile Gly Asp Cys Ala Thr Val His Thr Ala6885
6890 6895Asn Lys Trp Asp Leu Ile Ile Ser Asp Met Tyr
Asp Pro Arg Thr Lys6900 6905 6910His Val
Thr Lys Glu Asn Asp Ser Lys Glu Gly Phe Phe Thr Tyr Leu6915
6920 6925Cys Gly Phe Ile Lys Gln Lys Leu Ala Leu Gly Gly
Ser Ile Ala Val6930 6935 6940Lys Ile Thr
Glu His Ser Trp Asn Ala Asp Leu Tyr Lys Leu Met Gly6945
6950 6955 6960His Phe Ser Trp Trp Thr Ala
Phe Val Thr Asn Val Asn Ala Ser Ser6965 6970
6975Ser Glu Ala Phe Leu Ile Gly Ala Asn Tyr Leu Gly Lys Pro Lys Glu6980
6985 6990Gln Ile Asp Gly Tyr Thr Met His Ala
Asn Tyr Ile Phe Trp Arg Asn6995 7000
7005Thr Asn Pro Ile Gln Leu Ser Ser Tyr Ser Leu Phe Asp Met Ser Lys7010
7015 7020Phe Pro Leu Lys Leu Arg Gly Thr Ala
Val Met Ser Leu Lys Glu Asn7025 7030 7035
7040Gln Ile Asn Asp Met Ile Tyr Ser Leu Leu Glu Lys Gly Arg
Leu Ile7045 7050 7055Ile Arg Glu Asn Asn
Arg Val Val Val Ser Ser Asp Ile Leu Val Asn7060 7065
7070Asn33768DNASARS coronavirus Urbani 3atgtttattt tcttattatt
tcttactctc actagtggta gtgaccttga ccggtgcacc 60acttttgatg atgttcaagc
tcctaattac actcaacata cttcatctat gaggggggtt 120tactatcctg atgaaatttt
tagatcagac actctttatt taactcagga tttatttctt 180ccattttatt ctaatgttac
agggtttcat actattaatc atacgtttgg caaccctgtc 240atacctttta aggatggtat
ttattttgct gccacagaga aatcaaatgt tgtccgtggt 300tgggtttttg gttctaccat
gaacaacaag tcacagtcgg tgattattat taacaattct 360actaatgttg ttatacgagc
atgtaacttt gaattgtgtg acaacccttt ctttgctgtt 420tctaaaccca tgggtacaca
gacacatact atgatattcg ataatgcatt taattgcact 480ttcgagtaca tatctgatgc
cttttcgctt gatgtttcag aaaagtcagg taattttaaa 540cacttacgag agtttgtgtt
taaaaataaa gatgggtttc tctatgttta taagggctat 600caacctatag atgtagttcg
tgatctacct tctggtttta acactttgaa acctattttt 660aagttgcctc ttggtattaa
cattacaaat tttagagcca ttcttacagc cttttcacct 720gctcaagaca tttggggcac
gtcagctgca gcctattttg ttggctattt aaagccaact 780acatttatgc tcaagtatga
tgaaaatggt acaatcacag atgctgttga ttgttctcaa 840aatccacttg ctgaactcaa
atgctctgtt aagagctttg agattgacaa aggaatttac 900cagacctcta atttcagggt
tgttccctca ggagatgttg tgagattccc taatattaca 960aacttgtgtc cttttggaga
ggtttttaat gctactaaat tcccttctgt ctatgcatgg 1020gagagaaaaa aaatttctaa
ttgtgttgct gattactctg tgctctacaa ctcaacattt 1080ttttcaacct ttaagtgcta
tggcgtttct gccactaagt tgaatgatct ttgcttctcc 1140aatgtctatg cagattcttt
tgtagtcaag ggagatgatg taagacaaat agcgccagga 1200caaactggtg ttattgctga
ttataattat aaattgccag atgatttcat gggttgtgtc 1260cttgcttgga atactaggaa
cattgatgct acttcaactg gtaattataa ttataaatat 1320aggtatctta gacatggcaa
gcttaggccc tttgagagag acatatctaa tgtgcctttc 1380tcccctgatg gcaaaccttg
caccccacct gctcttaatt gttattggcc attaaatgat 1440tatggttttt acaccactac
tggcattggc taccaacctt acagagttgt agtactttct 1500tttgaacttt taaatgcacc
ggccacggtt tgtggaccaa aattatccac tgaccttatt 1560aagaaccagt gtgtcaattt
taattttaat ggactcactg gtactggtgt gttaactcct 1620tcttcaaaga gatttcaacc
atttcaacaa tttggccgtg atgtttctga tttcactgat 1680tccgttcgag atcctaaaac
atctgaaata ttagacattt caccttgctc ttttgggggt 1740gtaagtgtaa ttacacctgg
aacaaatgct tcatctgaag ttgctgttct atatcaagat 1800gttaactgca ctgatgtttc
tacagcaatt catgcagatc aactcacacc agcttggcgc 1860atatattcta ctggaaacaa
tgtattccag actcaagcag gctgtcttat aggagctgag 1920catgtcgaca cttcttatga
gtgcgacatt cctattggag ctggcatttg tgctagttac 1980catacagttt ctttattacg
tagtactagc caaaaatcta ttgtggctta tactatgtct 2040ttaggtgctg atagttcaat
tgcttactct aataacacca ttgctatacc tactaacttt 2100tcaattagca ttactacaga
agtaatgcct gtttctatgg ctaaaacctc cgtagattgt 2160aatatgtaca tctgcggaga
ttctactgaa tgtgctaatt tgcttctcca atatggtagc 2220ttttgcacac aactaaatcg
tgcactctca ggtattgctg ctgaacagga tcgcaacaca 2280cgtgaagtgt tcgctcaagt
caaacaaatg tacaaaaccc caactttgaa atattttggt 2340ggttttaatt tttcacaaat
attacctgac cctctaaagc caactaagag gtcttttatt 2400gaggacttgc tctttaataa
ggtgacactc gctgatgctg gcttcatgaa gcaatatggc 2460gaatgcctag gtgatattaa
tgctagagat ctcatttgtg cgcagaagtt caatggactt 2520acagtgttgc cacctctgct
cactgatgat atgattgctg cctacactgc tgctctagtt 2580agtggtactg ccactgctgg
atggacattt ggtgctggcg ctgctcttca aatacctttt 2640gctatgcaaa tggcatatag
gttcaatggc attggagtta cccaaaatgt tctctatgag 2700aaccaaaaac aaatcgccaa
ccaatttaac aaggcgatta gtcaaattca agaatcactt 2760acaacaacat caactgcatt
gggcaagctg caagacgttg ttaaccagaa tgctcaagca 2820ttaaacacac ttgttaaaca
acttagctct aattttggtg caatttcaag tgtgctaaat 2880gatatccttt cgcgacttga
taaagtcgag gcggaggtac aaattgacag gttaattaca 2940ggcagacttc aaagccttca
aacctatgta acacaacaac taatcagggc tgctgaaatc 3000agggcttctg ctaatcttgc
tgctactaaa atgtctgagt gtgttcttgg acaatcaaaa 3060agagttgact tttgtggaaa
gggctaccac cttatgtcct tcccacaagc agccccgcat 3120ggtgttgtct tcctacatgt
cacgtatgtg ccatcccagg agaggaactt caccacagcg 3180ccagcaattt gtcatgaagg
caaagcatac ttccctcgtg aaggtgtttt tgtgtttaat 3240ggcacttctt ggtttattac
acagaggaac ttcttttctc cacaaataat tactacagac 3300aatacatttg tctcaggaaa
ttgtgatgtc gttattggca tcattaacaa cacagtttat 3360gatcctctgc aacctgagct
cgactcattc aaagaagagc tggacaagta cttcaaaaat 3420catacatcac cagatgttga
tcttggcgac atttcaggca ttaacgcttc tgtcgtcaac 3480attcaaaaag aaattgaccg
cctcaatgag gtcgctaaaa atttaaatga atcactcatt 3540gaccttcaag aattgggaaa
atatgagcaa tatattaaat ggccttggta tgtttggctc 3600ggcttcattg ctggactaat
tgccatcgtc atggttacaa tcttgctttg ttgcatgact 3660agttgttgca gttgcctcaa
gggtgcatgc tcttgtggtt cttgctgcaa gtttgatgag 3720gatgactctg agccagttct
caagggtgtc aaattacatt acacataa 376841255PRTSARS
coronavirus Urbani 4Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly
Ser Asp Leu1 5 10 15Asp
Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln20
25 30His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro
Asp Glu Ile Phe Arg35 40 45Ser Asp Thr
Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser50 55
60Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly
Asn Pro Val65 70 75
80Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn85
90 95Val Val Arg Gly Trp Val Phe Gly Ser Thr
Met Asn Asn Lys Ser Gln100 105 110Ser Val
Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys115
120 125Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val
Ser Lys Pro Met130 135 140Gly Thr Gln Thr
His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr145 150
155 160Phe Glu Tyr Ile Ser Asp Ala Phe Ser
Leu Asp Val Ser Glu Lys Ser165 170 175Gly
Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly180
185 190Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile
Asp Val Val Arg Asp195 200 205Leu Pro Ser
Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu210
215 220Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr
Ala Phe Ser Pro225 230 235
240Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr245
250 255Leu Lys Pro Thr Thr Phe Met Leu Lys
Tyr Asp Glu Asn Gly Thr Ile260 265 270Thr
Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys275
280 285Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile
Tyr Gln Thr Ser Asn290 295 300Phe Arg Val
Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr305
310 315 320Asn Leu Cys Pro Phe Gly Glu
Val Phe Asn Ala Thr Lys Phe Pro Ser325 330
335Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr340
345 350Ser Val Leu Tyr Asn Ser Thr Phe Phe
Ser Thr Phe Lys Cys Tyr Gly355 360 365Val
Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala370
375 380Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg
Gln Ile Ala Pro Gly385 390 395
400Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
Phe405 410 415Met Gly Cys Val Leu Ala Trp
Asn Thr Arg Asn Ile Asp Ala Thr Ser420 425
430Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu435
440 445Arg Pro Phe Glu Arg Asp Ile Ser Asn
Val Pro Phe Ser Pro Asp Gly450 455 460Lys
Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp465
470 475 480Tyr Gly Phe Tyr Thr Thr
Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val485 490
495Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys
Gly500 505 510Pro Lys Leu Ser Thr Asp Leu
Ile Lys Asn Gln Cys Val Asn Phe Asn515 520
525Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg530
535 540Phe Gln Pro Phe Gln Gln Phe Gly Arg
Asp Val Ser Asp Phe Thr Asp545 550 555
560Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser
Pro Cys565 570 575Ala Phe Gly Gly Val Ser
Val Ile Thr Pro Gly Thr Asn Ala Ser Ser580 585
590Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser
Thr595 600 605Ala Ile His Ala Asp Gln Leu
Thr Pro Ala Trp Arg Ile Tyr Ser Thr610 615
620Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu625
630 635 640His Val Asp Thr
Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile645 650
655Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser
Gln Lys660 665 670Ser Ile Val Ala Tyr Thr
Met Ser Leu Gly Ala Asp Ser Ser Ile Ala675 680
685Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser
Ile690 695 700Thr Thr Glu Val Met Pro Val
Ser Met Ala Lys Thr Ser Val Asp Cys705 710
715 720Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala
Asn Leu Leu Leu725 730 735Gln Tyr Gly Ser
Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile740 745
750Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln
Val Lys755 760 765Gln Met Tyr Lys Thr Pro
Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe770 775
780Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe
Ile785 790 795 800Glu Asp
Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met805
810 815Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala
Arg Asp Leu Ile820 825 830Cys Ala Gln Lys
Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr835 840
845Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly
Thr Ala850 855 860Thr Ala Gly Trp Thr Phe
Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe865 870
875 880Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
Gly Val Thr Gln Asn885 890 895Val Leu Tyr
Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala900
905 910Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser
Thr Ala Leu Gly915 920 925Lys Leu Gln Asp
Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu930 935
940Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
Leu Asn945 950 955 960Asp
Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp965
970 975Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
Thr Tyr Val Thr Gln980 985 990Gln Leu Ile
Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala995
1000 1005Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys
Arg Val Asp Phe1010 1015 1020Cys Gly Lys
Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His1025
1030 1035 1040Gly Val Val Phe Leu His Val
Thr Tyr Val Pro Ser Gln Glu Arg Asn1045 1050
1055Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro1060
1065 1070Arg Glu Gly Val Phe Val Phe Asn Gly
Thr Ser Trp Phe Ile Thr Gln1075 1080
1085Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val1090
1095 1100Ser Gly Asn Cys Asp Val Val Ile Gly
Ile Ile Asn Asn Thr Val Tyr1105 1110 1115
1120Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu
Asp Lys1125 1130 1135Tyr Phe Lys Asn His
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser1140 1145
1150Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
Leu1155 1160 1165Asn Glu Val Ala Lys Asn
Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu1170 1175
1180Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp
Leu1185 1190 1195 1200Gly
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu1205
1210 1215Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
Gly Ala Cys Ser Cys1220 1225 1230Gly Ser
Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys1235
1240 1245Gly Val Lys Leu His Tyr Thr1250
12555444DNASARS coronavirus UrbaniCDS(1)...(444) 5gtc ata cct ttt aag gat
ggt att tat ttt gct gcc aca gag aaa tca 48Val Ile Pro Phe Lys Asp
Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser1 5
10 15aat gtt gtc cgt ggt tgg gtt ttt ggt tct acc atg
aac aac aag tca 96Asn Val Val Arg Gly Trp Val Phe Gly Ser Thr Met
Asn Asn Lys Ser20 25 30cag tcg gtg att
att att aac aat tct act aat gtt gtt ata cga gca 144Gln Ser Val Ile
Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala35 40
45tgt aac ttt gaa ttg tgt gac aac cct ttc ttt gct gtt tct
aaa ccc 192Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser
Lys Pro50 55 60atg ggt aca cag aca cat
act atg ata ttc gat aat gca ttt aat tgc 240Met Gly Thr Gln Thr His
Thr Met Ile Phe Asp Asn Ala Phe Asn Cys65 70
75 80act ttc gag tac ata tct gat gcc ttt tcg ctt
gat gtt tca gaa aag 288Thr Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu
Asp Val Ser Glu Lys85 90 95tca ggt aat
ttt aaa cac tta cga gag ttt gtg ttt aaa aat aaa gat 336Ser Gly Asn
Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp100
105 110ggg ttt ctc tat gtt tat aag ggc tat caa cct ata
gat gta gtt cgt 384Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile
Asp Val Val Arg115 120 125gat cta cct tct
ggt ttt aac act ttg aaa cct att ttt aag ttg cct 432Asp Leu Pro Ser
Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro130 135
140ctt ggt att aac
444Leu Gly Ile Asn1456148PRTSARS coronavirus Urbani 6Val Ile
Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser1 5
10 15Asn Val Val Arg Gly Trp Val Phe Gly
Ser Thr Met Asn Asn Lys Ser20 25 30Gln
Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala35
40 45Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe
Ala Val Ser Lys Pro50 55 60Met Gly Thr
Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys65 70
75 80Thr Phe Glu Tyr Ile Ser Asp Ala
Phe Ser Leu Asp Val Ser Glu Lys85 90
95Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp100
105 110Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln
Pro Ile Asp Val Val Arg115 120 125Asp Leu
Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro130
135 140Leu Gly Ile Asn14571356DNASARS coronavirus Urbani
7gctgaactca aatgctctgt taagagcttt gagattgaca aaggaattta ccagacctct
60aatttcaggg ttgttccctc aggagatgtt gtgagattcc ctaatattac aaacttgtgt
120ccttttggag aggtttttaa tgctactaaa ttcccttctg tctatgcatg ggagagaaaa
180aaaatttcta attgtgttgc tgattactct gtgctctaca actcaacatt tttttcaacc
240tttaagtgct atggcgtttc tgccactaag ttgaatgatc tttgcttctc caatgtctat
300gcagattctt ttgtagtcaa gggagatgat gtaagacaaa tagcgccagg acaaactggt
360gttattgctg attataatta taaattgcca gatgatttca tgggttgtgt ccttgcttgg
420aatactagga acattgatgc tacttcaact ggtaattata attataaata taggtatctt
480agacatggca agcttaggcc ctttgagaga gacatatcta atgtgccttt ctcccctgat
540ggcaaacctt gcaccccacc tgctcttaat tgttattggc cattaaatga ttatggtttt
600tacaccacta ctggcattgg ctaccaacct tacagagttg tagtactttc ttttgaactt
660ttaaatgcac cggccacggt ttgtggacca aaattatcca ctgaccttat taagaaccag
720tgtgtcaatt ttaattttaa tggactcact ggtactggtg tgttaactcc ttcttcaaag
780agatttcaac catttcaaca atttggccgt gatgtttctg atttcactga ttccgttcga
840gatcctaaaa catctgaaat attagacatt tcaccttgct cttttggggg tgtaagtgta
900attacacctg gaacaaatgc ttcatctgaa gttgctgttc tatatcaaga tgttaactgc
960actgatgttt ctacagcaat tcatgcagat caactcacac cagcttggcg catatattct
1020actggaaaca atgtattcca gactcaagca ggctgtctta taggagctga gcatgtcgac
1080acttcttatg agtgcgacat tcctattgga gctggcattt gtgctagtta ccatacagtt
1140tctttattac gtagtactag ccaaaaatct attgtggctt atactatgtc tttaggtgct
1200gatagttcaa ttgcttactc taataacacc attgctatac ctactaactt ttcaattagc
1260attactacag aagtaatgcc tgtttctatg gctaaaacct ccgtagattg taatatgtac
1320atctgcggag attctactga atgtgctaat ttgctt
13568452PRTSARS coronavirus Urbani 8Ala Glu Leu Lys Cys Ser Val Lys Ser
Phe Glu Ile Asp Lys Gly Ile1 5 10
15Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val
Arg20 25 30Phe Pro Asn Ile Thr Asn Leu
Cys Pro Phe Gly Glu Val Phe Asn Ala35 40
45Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn50
55 60Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
Ser Thr Phe Phe Ser Thr65 70 75
80Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys
Phe85 90 95Ser Asn Val Tyr Ala Asp Ser
Phe Val Val Lys Gly Asp Asp Val Arg100 105
110Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys115
120 125Leu Pro Asp Asp Phe Met Gly Cys Val
Leu Ala Trp Asn Thr Arg Asn130 135 140Ile
Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu145
150 155 160Arg His Gly Lys Leu Arg
Pro Phe Glu Arg Asp Ile Ser Asn Val Pro165 170
175Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys
Tyr180 185 190Trp Pro Leu Asn Asp Tyr Gly
Phe Tyr Thr Thr Thr Gly Ile Gly Tyr195 200
205Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro210
215 220Ala Thr Val Cys Gly Pro Lys Leu Ser
Thr Asp Leu Ile Lys Asn Gln225 230 235
240Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val
Leu Thr245 250 255Pro Ser Ser Lys Arg Phe
Gln Pro Phe Gln Gln Phe Gly Arg Asp Val260 265
270Ser Asp Phe Thr Asp Ser Val Arg Asp Pro Lys Thr Ser Glu Ile
Leu275 280 285Asp Ile Ser Pro Cys Ala Phe
Gly Gly Val Ser Val Ile Thr Pro Gly290 295
300Thr Asn Ala Ser Ser Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys305
310 315 320Thr Asp Val Ser
Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp325 330
335Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln Ala
Gly Cys340 345 350Leu Ile Gly Ala Glu His
Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro355 360
365Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser Leu Leu
Arg370 375 380Ser Thr Ser Gln Lys Ser Ile
Val Ala Tyr Thr Met Ser Leu Gly Ala385 390
395 400Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala
Ile Pro Thr Asn405 410 415Phe Ser Ile Ser
Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys420 425
430Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr
Glu Cys435 440 445Ala Asn Leu
Leu4509891DNAArtificial SequenceSynthetically generated oligonucleotide
9cat acg ttt ggc aac cct gtc ata cct ttt aag gat ggt att tat ttt
48gct gcc aca gag aaa tca aat gtt gtc cgt ggt tgg gtt ttt ggt tct
96acc atg aac aac aag tca cag tcg gtg att att att aac aat tct act
144aat gtt gtt ata cga gca tgt aac ttt gaa ttg tgt gac aac cct ttc
192ttt gct gtt tct aaa ccc atg ggt aca cag aca cat act atg ata ttc
240gat aat gca ttt aat tgc act ttc gag tac ata tct gat gcc ttt tcg
288ctt gat gtt tca gaa aag tca ggt aat ttt aaa cac tta cga gag ttt
336gtg ttt aaa aat aaa gat ggg ttt ctc tat gtt tat aag ggc tat caa
384cct ata gat gta gtt cgt gat cta cct tct ggt ttt aac act ttg aaa
432cct att ttt aag ttg cct ctt ggt att aac att aca aat ttt aga gcc
480gaa ttc ggg ggc ggg ggt gga ggt ggt ggc tca ttc aaa gaa gag ctg
528gac aag tac ttc aaa aat cat aca tca cca gat gtt gat ctt ggc gac
576att tca ggc att aac gct tct gtc gtc aac att caa aaa gaa att gac
624cgc ctc aat gag gtc gct aaa aat tta aat gaa tca ctc att gac ctt
672caa gaa ttg gga aaa tat gag caa tat att aaa tgg cct tgg tat gtt
720tgg ctc ggc ttc att gct gga cta att gcc atc gtc atg gtt aca atc
768ttg ctt tgt tgc atg act agt tgt tgc agt tgc ctc aag ggt gca tgc
816tct tgt ggt tct tgc tgc aag ttt gat gag gat gac tct gag cca gtt
864ctc aag ggt gtc aaa tta cat tac aca
89110284PRTArtificial SequenceSynthetically generated peptide 10Val Ile
Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser1 5
10 15Asn Val Val Arg Gly Trp Val Phe Gly
Ser Thr Met Asn Asn Lys Ser20 25 30Gln
Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala35
40 45Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe
Ala Val Ser Lys Pro50 55 60Met Gly Thr
Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys65 70
75 80Thr Phe Glu Tyr Ile Ser Asp Ala
Phe Ser Leu Asp Val Ser Glu Lys85 90
95Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp100
105 110Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln
Pro Ile Asp Val Val Arg115 120 125Asp Leu
Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro130
135 140Leu Gly Ile Asn Gly Gly Gly Gly Gly Gly Gly Gly
Asp Ser Phe Lys145 150 155
160Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp165
170 175Leu Gly Asp Ile Ser Gly Ile Asn Ala
Ser Val Val Asn Ile Gln Lys180 185 190Glu
Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu195
200 205Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln
Tyr Ile Lys Trp Pro210 215 220Trp Tyr Val
Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met225
230 235 240Val Thr Ile Leu Leu Cys Cys
Met Thr Ser Cys Cys Ser Cys Leu Lys245 250
255Gly Ala Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser260
265 270Glu Pro Val Leu Lys Gly Val Lys Leu
His Tyr Thr275 280111752DNAArtificial
SequenceSynthetically generated oligonucleotide 11gagattgaca aaggaattta
ccagacctct aatttcaggg ttgttccctc aggagatgtt 60gtgagattcc ctaatattac
aaacttgtgt ccttttggag aggtttttaa tgctactaaa 120ttcccttctg tctatgcatg
ggagagaaaa aaaatttcta attgtgttgc tgattactct 180gtgctctaca actcaacatt
tttttcaacc tttaagtgct atggcgtttc tgccactaag 240ttgaatgatc tttgcttctc
caatgtctat gcagattctt ttgtagtcaa gggagatgat 300gtaagacaaa tagcgccagg
acaaactggt gttattgctg attataatta taaattgcca 360gatgatttca tgggttgtgt
ccttgcttgg aatactagga acattgatgc tacttcaact 420ggtaattata attataaata
taggtatctt agacatggca agcttaggcc ctttgagaga 480gacatatcta atgtgccttt
ctcccctgat ggcaaacctt gcaccccacc tgctcttaat 540tgttattggc cattaaatga
ttatggtttt tacaccacta ctggcattgg ctaccaacct 600tacagagttg tagtactttc
ttttgaactt ttaaatgcac cggccacggt ttgtggacca 660aaattatcca ctgaccttat
taagaaccag tgtgtcaatt ttaattttaa tggactcact 720ggtactggtg tgttaactcc
ttcttcaaag agatttcaac catttcaaca atttggccgt 780gatgtttctg atttcactga
ttccgttcga gatcctaaaa catctgaaat attagacatt 840tcaccttgct cttttggggg
tgtaagtgta attacacctg gaacaaatgc ttcatctgaa 900gttgctgttc tatatcaaga
tgttaactgc actgatgttt ctacagcaat tcatgcagat 960caactcacac cagcttggcg
catatattct actggaaaca atgtattcca gactcaagca 1020ggctgtctta taggagctga
gcatgtcgac acttcttatg agtgcgacat tcctattgga 1080gctggcattt gtgctagtta
ccatacagtt tctttattac gtagtactag ccaaaaatct 1140attgtggctt atactatgtc
tttaggtgct gatagttcaa ttgcttactc taataacacc 1200attgctatac ctactaactt
ttcaattagc attactacag aagtaatgcc tgtttctatg 1260gctaaaacct ccgtagattg
taatatgtac atctgcggag attctactga atgtgctaat 1320ttgcttctcc aatatgggcg
gccgcctggg ggcgggggtg gaggtggtgg ctcattcaaa 1380gaagagctgg acaagtactt
caaaaatcat acatcaccag atgttgatct tggcgacatt 1440tcaggcatta acgcttctgt
cgtcaacatt caaaaagaaa ttgaccgcct caatgaggtc 1500gctaaaaatt taaatgaatc
actcattgac cttcaagaat tgggaaaata tgagcaatat 1560attaaatggc cttggtatgt
ttggctcggc ttcattgctg gactaattgc catcgtcatg 1620gttacaatct tgctttgttg
catgactagt tgttgcagtt gcctcaaggg tgcatgctct 1680tgtggttctt gctgcaagtt
tgatgaggat gactctgagc cagttctcaa gggtgtcaaa 1740ttacattaca ca
175212588PRTArtificial
SequenceSynthetically generated peptide 12Ala Glu Leu Lys Cys Ser Val Lys
Ser Phe Glu Ile Asp Lys Gly Ile1 5 10
15Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val
Arg20 25 30Phe Pro Asn Ile Thr Asn Leu
Cys Pro Phe Gly Glu Val Phe Asn Ala35 40
45Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn50
55 60Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
Ser Thr Phe Phe Ser Thr65 70 75
80Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys
Phe85 90 95Ser Asn Val Tyr Ala Asp Ser
Phe Val Val Lys Gly Asp Asp Val Arg100 105
110Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys115
120 125Leu Pro Asp Asp Phe Met Gly Cys Val
Leu Ala Trp Asn Thr Arg Asn130 135 140Ile
Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu145
150 155 160Arg His Gly Lys Leu Arg
Pro Phe Glu Arg Asp Ile Ser Asn Val Pro165 170
175Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys
Tyr180 185 190Trp Pro Leu Asn Asp Tyr Gly
Phe Tyr Thr Thr Thr Gly Ile Gly Tyr195 200
205Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro210
215 220Ala Thr Val Cys Gly Pro Lys Leu Ser
Thr Asp Leu Ile Lys Asn Gln225 230 235
240Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val
Leu Thr245 250 255Pro Ser Ser Lys Arg Phe
Gln Pro Phe Gln Gln Phe Gly Arg Asp Val260 265
270Ser Asp Phe Thr Asp Ser Val Arg Asp Pro Lys Thr Ser Glu Ile
Leu275 280 285Asp Ile Ser Pro Cys Ala Phe
Gly Gly Val Ser Val Ile Thr Pro Gly290 295
300Thr Asn Ala Ser Ser Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys305
310 315 320Thr Asp Val Ser
Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp325 330
335Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln Ala
Gly Cys340 345 350Leu Ile Gly Ala Glu His
Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro355 360
365Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser Leu Leu
Arg370 375 380Ser Thr Ser Gln Lys Ser Ile
Val Ala Tyr Thr Met Ser Leu Gly Ala385 390
395 400Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala
Ile Pro Thr Asn405 410 415Phe Ser Ile Ser
Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys420 425
430Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr
Glu Cys435 440 445Ala Asn Leu Leu Gly Gly
Gly Gly Gly Gly Gly Gly Asp Ser Phe Lys450 455
460Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val
Asp465 470 475 480Leu Gly
Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys485
490 495Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu
Asn Glu Ser Leu500 505 510Ile Asp Leu Gln
Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro515 520
525Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile
Val Met530 535 540Val Thr Ile Leu Leu Cys
Cys Met Thr Ser Cys Cys Ser Cys Leu Lys545 550
555 560Gly Ala Cys Ser Cys Gly Ser Cys Cys Lys Phe
Asp Glu Asp Asp Ser565 570 575Glu Pro Val
Leu Lys Gly Val Lys Leu His Tyr Thr580
585131847DNAArtificial SequenceSynthetically generated oligonucleotide
13catacgtttg gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag
60aaatcaaatg ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg
120gtgattatta ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt
180gacaaccctt tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc
240gataatgcat ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca
300gaaaagtcag gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt
360ctctatgttt ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt
420aacactttga aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc
480gaattcgggg gcgggggtgg aggtggtggc gagattgaca aaggaattta ccagacctct
540aatttcaggg ttgttccctc aggagatgtt gtgagattcc ctaatattac aaacttgtgt
600ccttttggag aggtttttaa tgctactaaa ttcccttctg tctatgcatg ggagagaaaa
660aaaatttcta attgtgttgc tgattactct gtgctctaca actcaacatt tttttcaacc
720tttaagtgct atggcgtttc tgccactaag ttgaatgatc tttgcttctc caatgtctat
780gcagattctt ttgtagtcaa gggagatgat gtaagacaaa tagcgccagg acaaactggt
840gttattgctg attataatta taaattgcca gatgatttca tgggttgtgt ccttgcttgg
900aatactagga acattgatgc tacttcaact ggtaattata attataaata taggtatctt
960agacatggca agcttaggcc ctttgagaga gacatatcta atgtgccttt ctcccctgat
1020ggcaaacctt gcaccccacc tgctcttaat tgttattggc cattaaatga ttatggtttt
1080tacaccacta ctggcattgg ctaccaacct tacagagttg tagtactttc ttttgaactt
1140ttaaatgcac cggccacggt ttgtggacca aaattatcca ctgaccttat taagaaccag
1200tgtgtcaatt ttaattttaa tggactcact ggtactggtg tgttaactcc ttcttcaaag
1260agatttcaac catttcaaca atttggccgt gatgtttctg atttcactga ttccgttcga
1320gatcctaaaa catctgaaat attagacatt tcaccttgct cttttggggg tgtaagtgta
1380attacacctg gaacaaatgc ttcatctgaa gttgctgttc tatatcaaga tgttaactgc
1440actgatgttt ctacagcaat tcatgcagat caactcacac cagcttggcg catatattct
1500actggaaaca atgtattcca gactcaagca ggctgtctta taggagctga gcatgtcgac
1560acttcttatg agtgcgacat tcctattgga gctggcattt gtgctagtta ccatacagtt
1620tctttattac gtagtactag ccaaaaatct attgtggctt atactatgtc tttaggtgct
1680gatagttcaa ttgcttactc taataacacc attgctatac ctactaactt ttcaattagc
1740attactacag aagtaatgcc tgtttctatg gctaaaacct ccgtagattg taatatgtac
1800atctgcggag attctactga atgtgctaat ttgcttctcc aatatgg
184714608PRTArtificial SequenceSynthetically generated peptide 14Val Ile
Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser1 5
10 15Asn Val Val Arg Gly Trp Val Phe Gly
Ser Thr Met Asn Asn Lys Ser20 25 30Gln
Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala35
40 45Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe
Ala Val Ser Lys Pro50 55 60Met Gly Thr
Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys65 70
75 80Thr Phe Glu Tyr Ile Ser Asp Ala
Phe Ser Leu Asp Val Ser Glu Lys85 90
95Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp100
105 110Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln
Pro Ile Asp Val Val Arg115 120 125Asp Leu
Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro130
135 140Leu Gly Ile Asn Gly Gly Gly Gly Gly Gly Gly Gly
Ala Glu Leu Lys145 150 155
160Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser165
170 175Asn Phe Arg Val Val Pro Ser Gly Asp
Val Val Arg Phe Pro Asn Ile180 185 190Thr
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro195
200 205Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser
Asn Cys Val Ala Asp210 215 220Tyr Ser Val
Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr225
230 235 240Gly Val Ser Ala Thr Lys Leu
Asn Asp Leu Cys Phe Ser Asn Val Tyr245 250
255Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro260
265 270Gly Gln Thr Gly Val Ile Ala Asp Tyr
Asn Tyr Lys Leu Pro Asp Asp275 280 285Phe
Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr290
295 300Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr
Leu Arg His Gly Lys305 310 315
320Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro
Asp325 330 335Gly Lys Pro Cys Thr Pro Pro
Ala Leu Asn Cys Tyr Trp Pro Leu Asn340 345
350Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg355
360 365Val Val Val Leu Ser Phe Glu Leu Leu
Asn Ala Pro Ala Thr Val Cys370 375 380Gly
Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe385
390 395 400Asn Phe Asn Gly Leu Thr
Gly Thr Gly Val Leu Thr Pro Ser Ser Lys405 410
415Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe
Thr420 425 430Asp Ser Val Arg Asp Pro Lys
Thr Ser Glu Ile Leu Asp Ile Ser Pro435 440
445Cys Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser450
455 460Ser Glu Val Ala Val Leu Tyr Gln Asp
Val Asn Cys Thr Asp Val Ser465 470 475
480Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile
Tyr Ser485 490 495Thr Gly Asn Asn Val Phe
Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala500 505
510Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
Gly515 520 525Ile Cys Ala Ser Tyr His Thr
Val Ser Leu Leu Arg Ser Thr Ser Gln530 535
540Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile545
550 555 560Ala Tyr Ser Asn
Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser565 570
575Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser
Val Asp580 585 590Cys Asn Met Tyr Ile Cys
Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu595 600
605152262DNAArtificial SequenceSynthetically generated
oligonucletide 15catacgtttg gcaaccctgt catacctttt aaggatggta tttattttgc
tgccacagag 60aaatcaaatg ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa
gtcacagtcg 120gtgattatta ttaacaattc tactaatgtt gttatacgag catgtaactt
tgaattgtgt 180gacaaccctt tctttgctgt ttctaaaccc atgggtacac agacacatac
tatgatattc 240gataatgcat ttaattgcac tttcgagtac atatctgatg ccttttcgct
tgatgtttca 300gaaaagtcag gtaattttaa acacttacga gagtttgtgt ttaaaaataa
agatgggttt 360ctctatgttt ataagggcta tcaacctata gatgtagttc gtgatctacc
ttctggtttt 420aacactttga aacctatttt taagttgcct cttggtatta acattacaaa
ttttagagcc 480gaattcgggg gcgggggtgg aggtggtggc gagattgaca aaggaattta
ccagacctct 540aatttcaggg ttgttccctc aggagatgtt gtgagattcc ctaatattac
aaacttgtgt 600ccttttggag aggtttttaa tgctactaaa ttcccttctg tctatgcatg
ggagagaaaa 660aaaatttcta attgtgttgc tgattactct gtgctctaca actcaacatt
tttttcaacc 720tttaagtgct atggcgtttc tgccactaag ttgaatgatc tttgcttctc
caatgtctat 780gcagattctt ttgtagtcaa gggagatgat gtaagacaaa tagcgccagg
acaaactggt 840gttattgctg attataatta taaattgcca gatgatttca tgggttgtgt
ccttgcttgg 900aatactagga acattgatgc tacttcaact ggtaattata attataaata
taggtatctt 960agacatggca agcttaggcc ctttgagaga gacatatcta atgtgccttt
ctcccctgat 1020ggcaaacctt gcaccccacc tgctcttaat tgttattggc cattaaatga
ttatggtttt 1080tacaccacta ctggcattgg ctaccaacct tacagagttg tagtactttc
ttttgaactt 1140ttaaatgcac cggccacggt ttgtggacca aaattatcca ctgaccttat
taagaaccag 1200tgtgtcaatt ttaattttaa tggactcact ggtactggtg tgttaactcc
ttcttcaaag 1260agatttcaac catttcaaca atttggccgt gatgtttctg atttcactga
ttccgttcga 1320gatcctaaaa catctgaaat attagacatt tcaccttgct cttttggggg
tgtaagtgta 1380attacacctg gaacaaatgc ttcatctgaa gttgctgttc tatatcaaga
tgttaactgc 1440actgatgttt ctacagcaat tcatgcagat caactcacac cagcttggcg
catatattct 1500actggaaaca atgtattcca gactcaagca ggctgtctta taggagctga
gcatgtcgac 1560acttcttatg agtgcgacat tcctattgga gctggcattt gtgctagtta
ccatacagtt 1620tctttattac gtagtactag ccaaaaatct attgtggctt atactatgtc
tttaggtgct 1680gatagttcaa ttgcttactc taataacacc attgctatac ctactaactt
ttcaattagc 1740attactacag aagtaatgcc tgtttctatg gctaaaacct ccgtagattg
taatatgtac 1800atctgcggag attctactga atgtgctaat ttgcttctcc aatatgggcg
gccgcctggg 1860ggcgggggtg gaggtggtgg ctcattcaaa gaagagctgg acaagtactt
caaaaatcat 1920acatcaccag atgttgatct tggcgacatt tcaggcatta acgcttctgt
cgtcaacatt 1980caaaaagaaa ttgaccgcct caatgaggtc gctaaaaatt taaatgaatc
actcattgac 2040cttcaagaat tgggaaaata tgagcaatat attaaatggc cttggtatgt
ttggctcggc 2100ttcattgctg gactaattgc catcgtcatg gttacaatct tgctttgttg
catgactagt 2160tgttgcagtt gcctcaaggg tgcatgctct tgtggttctt gctgcaagtt
tgatgaggat 2220gactctgagc cagttctcaa gggtgtcaaa ttacattaca ca
226216744PRTArtificial SequenceSynthetically generated peptide
16Val Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser1
5 10 15Asn Val Val Arg Gly Trp
Val Phe Gly Ser Thr Met Asn Asn Lys Ser20 25
30Gln Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala35
40 45Cys Asn Phe Glu Leu Cys Asp Asn Pro
Phe Phe Ala Val Ser Lys Pro50 55 60Met
Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys65
70 75 80Thr Phe Glu Tyr Ile Ser
Asp Ala Phe Ser Leu Asp Val Ser Glu Lys85 90
95Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp100
105 110Gly Phe Leu Tyr Val Tyr Lys Gly
Tyr Gln Pro Ile Asp Val Val Arg115 120
125Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro130
135 140Leu Gly Ile Asn Gly Gly Gly Gly Gly
Gly Gly Gly Ala Glu Leu Lys145 150 155
160Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln
Thr Ser165 170 175Asn Phe Arg Val Val Pro
Ser Gly Asp Val Val Arg Phe Pro Asn Ile180 185
190Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe
Pro195 200 205Ser Val Tyr Ala Trp Glu Arg
Lys Lys Ile Ser Asn Cys Val Ala Asp210 215
220Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr225
230 235 240Gly Val Ser Ala
Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr245 250
255Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile
Ala Pro260 265 270Gly Gln Thr Gly Val Ile
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp275 280
285Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala
Thr290 295 300Ser Thr Gly Asn Tyr Asn Tyr
Lys Tyr Arg Tyr Leu Arg His Gly Lys305 310
315 320Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro
Phe Ser Pro Asp325 330 335Gly Lys Pro Cys
Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn340 345
350Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro
Tyr Arg355 360 365Val Val Val Leu Ser Phe
Glu Leu Leu Asn Ala Pro Ala Thr Val Cys370 375
380Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn
Phe385 390 395 400Asn Phe
Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys405
410 415Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val
Ser Asp Phe Thr420 425 430Asp Ser Val Arg
Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro435 440
445Cys Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn
Ala Ser450 455 460Ser Glu Val Ala Val Leu
Tyr Gln Asp Val Asn Cys Thr Asp Val Ser465 470
475 480Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala
Trp Arg Ile Tyr Ser485 490 495Thr Gly Asn
Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala500
505 510Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro
Ile Gly Ala Gly515 520 525Ile Cys Ala Ser
Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln530 535
540Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser
Ser Ile545 550 555 560Ala
Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser565
570 575Ile Thr Thr Glu Val Met Pro Val Ser Met Ala
Lys Thr Ser Val Asp580 585 590Cys Asn Met
Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu595
600 605Gly Gly Gly Gly Gly Gly Gly Gly Asp Ser Phe Lys
Glu Glu Leu Asp610 615 620Lys Tyr Phe Lys
Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile625 630
635 640Ser Gly Ile Asn Ala Ser Val Val Asn
Ile Gln Lys Glu Ile Asp Arg645 650 655Leu
Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln660
665 670Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp
Pro Trp Tyr Val Trp675 680 685Leu Gly Phe
Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu690
695 700Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
Gly Ala Cys Ser705 710 715
720Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu725
730 735Lys Gly Val Lys Leu His Tyr
Thr74017999DNASARS coronavirus Urbani 17atgtttattt tcttattatt tcttactctc
actagtggta gtgaccttga ccggtgcacc 60acttttgatg atgttcaagc tcctaattac
actcaacata cttcatctat gaggggggtt 120tactatcctg atgaaatttt tagatcagac
actctttatt taactcagga tttatttctt 180ccattttatt ctaatgttac agggtttcat
actattaatc atacgtttgg caaccctgtc 240atacctttta aggatggtat ttattttgct
gccacagaga aatcaaatgt tgtccgtggt 300tgggtttttg gttctaccat gaacaacaag
tcacagtcgg tgattattat taacaattct 360actaatgttg ttatacgagc atgtaacttt
gaattgtgtg acaacccttt ctttgctgtt 420tctaaaccca tgggtacaca gacacatact
atgatattcg ataatgcatt taattgcact 480ttcgagtaca tatctgatgc cttttcgctt
gatgtttcag aaaagtcagg taattttaaa 540cacttacgag agtttgtgtt taaaaataaa
gatgggtttc tctatgttta taagggctat 600caacctatag atgtagttcg tgatctacct
tctggtttta acactttgaa acctattttt 660aagttgcctc ttggtattaa cattacaaat
tttagagcca ttcttacagc cttttcacct 720gctcaagaca tttggggcac gtcagctgca
gcctattttg ttggctattt aaagccaact 780acatttatgc tcaagtatga tgaaaatggt
acaatcacag atgctgttga ttgttctcaa 840aatccacttg ctgaactcaa atgctctgtt
aagagctttg agattgacaa aggaatttac 900cagacctcta atttcagggt tgttccctca
ggagatgttg tgagattccc taatattaca 960aacttgtgtc cttttggaga ggtttttaat
gctactaaa 99918333PRTSARS coronavirus Urbani
18Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu1
5 10 15Asp Arg Cys Thr Thr Phe
Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln20 25
30His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg35
40 45Ser Asp Thr Leu Tyr Leu Thr Gln Asp
Leu Phe Leu Pro Phe Tyr Ser50 55 60Asn
Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val65
70 75 80Ile Pro Phe Lys Asp Gly
Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn85 90
95Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln100
105 110Ser Val Ile Ile Ile Asn Asn Ser
Thr Asn Val Val Ile Arg Ala Cys115 120
125Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met130
135 140Gly Thr Gln Thr His Thr Met Ile Phe
Asp Asn Ala Phe Asn Cys Thr145 150 155
160Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu
Lys Ser165 170 175Gly Asn Phe Lys His Leu
Arg Glu Phe Val Phe Lys Asn Lys Asp Gly180 185
190Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg
Asp195 200 205Leu Pro Ser Gly Phe Asn Thr
Leu Lys Pro Ile Phe Lys Leu Pro Leu210 215
220Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro225
230 235 240Ala Gln Asp Ile
Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr245 250
255Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly
Thr Ile260 265 270Thr Asp Ala Val Asp Cys
Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys275 280
285Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser
Asn290 295 300Phe Arg Val Val Pro Ser Gly
Asp Val Val Arg Phe Pro Asn Ile Thr305 310
315 320Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
Lys325 33019999DNASARS coronavirus Urbani 19ttcccttctg
tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct 60gtgctctaca
actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag 120ttgaatgatc
tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat 180gtaagacaaa
tagcgccagg acaaactggt gttattgctg attataatta taaattgcca 240gatgatttca
tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact 300ggtaattata
attataaata taggtatctt agacatggca agcttaggcc ctttgagaga 360gacatatcta
atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat 420tgttattggc
cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct 480tacagagttg
tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca 540aaattatcca
ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact 600ggtactggtg
tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt 660gatgtttctg
atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt 720tcaccttgct
cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa 780gttgctgttc
tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat 840caactcacac
cagcttggcg catatattct actggaaaca atgtattcca gactcaagca 900ggctgtctta
taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga 960gctggcattt
gtgctagtta ccatacagtt tctttatta 99920333PRTSARS
coronavirus Urbani 20Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser
Asn Cys Val1 5 10 15Ala
Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys20
25 30Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp
Leu Cys Phe Ser Asn35 40 45Val Tyr Ala
Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile50 55
60Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr
Lys Leu Pro65 70 75
80Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp85
90 95Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys
Tyr Arg Tyr Leu Arg His100 105 110Gly Lys
Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser115
120 125Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn
Cys Tyr Trp Pro130 135 140Leu Asn Asp Tyr
Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro145 150
155 160Tyr Arg Val Val Val Leu Ser Phe Glu
Leu Leu Asn Ala Pro Ala Thr165 170 175Val
Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val180
185 190Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly
Val Leu Thr Pro Ser195 200 205Ser Lys Arg
Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp210
215 220Phe Thr Asp Ser Val Arg Asp Pro Lys Thr Ser Glu
Ile Leu Asp Ile225 230 235
240Ser Pro Cys Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn245
250 255Ala Ser Ser Glu Val Ala Val Leu Tyr
Gln Asp Val Asn Cys Thr Asp260 265 270Val
Ser Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile275
280 285Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln
Ala Gly Cys Leu Ile290 295 300Gly Ala Glu
His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly305
310 315 320Ala Gly Ile Cys Ala Ser Tyr
His Thr Val Ser Leu Leu325 330211002DNASARS coronavirus
Urbani 21cgtagtacta gccaaaaatc tattgtggct tatactatgt ctttaggtgc
tgatagttca 60attgcttact ctaataacac cattgctata cctactaact tttcaattag
cattactaca 120gaagtaatgc ctgtttctat ggctaaaacc tccgtagatt gtaatatgta
catctgcgga 180gattctactg aatgtgctaa tttgcttctc caatatggta gcttttgcac
acaactaaat 240cgtgcactct caggtattgc tgctgaacag gatcgcaaca cacgtgaagt
gttcgctcaa 300gtcaaacaaa tgtacaaaac cccaactttg aaatattttg gtggttttaa
tttttcacaa 360atattacctg accctctaaa gccaactaag aggtctttta ttgaggactt
gctctttaat 420aaggtgacac tcgctgatgc tggcttcatg aagcaatatg gcgaatgcct
aggtgatatt 480aatgctagag atctcatttg tgcgcagaag ttcaatggac ttacagtgtt
gccacctctg 540ctcactgatg atatgattgc tgcctacact gctgctctag ttagtggtac
tgccactgct 600ggatggacat ttggtgctgg cgctgctctt caaatacctt ttgctatgca
aatggcatat 660aggttcaatg gcattggagt tacccaaaat gttctctatg agaaccaaaa
acaaatcgcc 720aaccaattta acaaggcgat tagtcaaatt caagaatcac ttacaacaac
atcaactgca 780ttgggcaagc tgcaagacgt tgttaaccag aatgctcaag cattaaacac
acttgttaaa 840caacttagct ctaattttgg tgcaatttca agtgtgctaa atgatatcct
ttcgcgactt 900gataaagtcg aggcggaggt acaaattgac aggttaatta caggcagact
tcaaagcctt 960caaacctatg taacacaaca actaatcagg gctgctgaaa tc
100222334PRTSARS coronavirus Urbani 22Arg Ser Thr Ser Gln Lys
Ser Ile Val Ala Tyr Thr Met Ser Leu Gly1 5
10 15Ala Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala
Ile Pro Thr20 25 30Asn Phe Ser Ile Ser
Ile Thr Thr Glu Val Met Pro Val Ser Met Ala35 40
45Lys Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr
Glu50 55 60Cys Ala Asn Leu Leu Leu Gln
Tyr Gly Ser Phe Cys Thr Gln Leu Asn65 70
75 80Arg Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg
Asn Thr Arg Glu85 90 95Val Phe Ala Gln
Val Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr100 105
110Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Leu
Lys Pro115 120 125Thr Lys Arg Ser Phe Ile
Glu Asp Leu Leu Phe Asn Lys Val Thr Leu130 135
140Ala Asp Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp
Ile145 150 155 160Asn Ala
Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val165
170 175Leu Pro Pro Leu Leu Thr Asp Asp Met Ile Ala Ala
Tyr Thr Ala Ala180 185 190Leu Val Ser Gly
Thr Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala195 200
205Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe
Asn Gly210 215 220Ile Gly Val Thr Gln Asn
Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala225 230
235 240Asn Gln Phe Asn Lys Ala Ile Ser Gln Ile Gln
Glu Ser Leu Thr Thr245 250 255Thr Ser Thr
Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala260
265 270Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser
Asn Phe Gly Ala275 280 285Ile Ser Ser Val
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu290 295
300Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln
Ser Leu305 310 315 320Gln
Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile325
33023102DNASARS coronavirus Urbani 23ggtaattata attataaata taggtatctt
agacatggca agcttaggcc ctttgagaga 60gacatatcta atgtgccttt ctcccctgat
ggcaaacctt gc 1022434PRTSARS coronavirus Urbani
24Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg1
5 10 15Pro Phe Glu Arg Asp Ile
Ser Asn Val Pro Phe Ser Pro Asp Gly Lys20 25
30Pro Cys25150DNASARS coronavirus Urbani 25gatcctaaaa catctgaaat
attagacatt tcaccttgct cttttggggg tgtaagtgta 60attacacctg gaacaaatgc
ttcatctgaa gttgctgttc tatatcaaga tgttaactgc 120actgatgttt ctacagcaat
tcatgcagat 1502650PRTSARS coronavirus
Urbani 26Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys Ala Phe Gly1
5 10 15Gly Val Ser Val
Ile Thr Pro Gly Thr Asn Ala Ser Ser Glu Val Ala20 25
30Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr Ala
Ile His35 40 45Ala Asp5027384DNASARS
coronavirus Urbani 27gactcattca aagaagagct ggacaagtac ttcaaaaatc
atacatcacc agatgttgat 60cttggcgaca tttcaggcat taacgcttct gtcgtcaaca
ttcaaaaaga aattgaccgc 120ctcaatgagg tcgctaaaaa tttaaatgaa tcactcattg
accttcaaga attgggaaaa 180tatgagcaat atattaaatg gccttggtat gtttggctcg
gcttcattgc tggactaatt 240gccatcgtca tggttacaat cttgctttgt tgcatgacta
gttgttgcag ttgcctcaag 300ggtgcatgct cttgtggttc ttgctgcaag tttgatgagg
atgactctga gccagttctc 360aagggtgtca aattacatta caca
38428128PRTSARS coronavirus Urbani 28Asp Ser Phe
Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser1 5
10 15Pro Asp Val Asp Leu Gly Asp Ile Ser Gly
Ile Asn Ala Ser Val Val20 25 30Asn Ile
Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu35
40 45Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys
Tyr Glu Gln Tyr50 55 60Ile Lys Trp Pro
Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile65 70
75 80Ala Ile Val Met Val Thr Ile Leu Leu
Cys Cys Met Thr Ser Cys Cys85 90 95Ser
Cys Leu Lys Gly Ala Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp100
105 110Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val
Lys Leu His Tyr Thr115 120
1252930PRTArtificial SequenceSynthetically generated SARS CoV peptide
29Lys Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys1
5 10 15Asp Gly Phe Leu Tyr Val
Tyr Lys Gly Gln Pro Ile Asp Val20 25
303032PRTArtificial SequenceSynthetically generated SARS CoV peptide
30Asp Ser Phe Lys Glu Glu Leu Asp Arg Tyr Phe Lys Asn His Thr Ser1
5 10 15Pro Asp Val Asp Leu Gly
Asp Ile Ser Gly Ile Asn Ala Ser Val Val20 25
303135PRTArtificial SequenceSynthetically generated SARS CoV
peptide 31Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn Val Ser
Leu1 5 10 15Val Lys Pro
Thr Val Tyr Val Tyr Ser Arg Val Lys Asn Leu Asn Ser20 25
30Ser Glu Gly353233PRTArtificial SequenceSynthetically
generated SARS CoV peptide 32Met Ala Asp Asn Gly Thr Ile Thr Val Glu Glu
Leu Lys Gln Leu Leu1 5 10
15Glu Gln Trp Asn Leu Val Ile Cys Phe Leu Phe Leu Ala Trp Ile Met20
25 30Leu3326DNAArtificial SequencePrimer
33ggatccgcca ccatgcatac gtttgg
263418DNAArtificial SequencePrimer 34cttaagccga gattttaa
183525DNAArtificial
SequenceSynthetically generated SARS CoV peptide 35ggatccgcca ccatggagat
tgaca 253615DNAArtificial
SequencePrimer 36cgccggcggg tataa
153737DNAArtificial SequencePrimer 37aggggatcca tgtttatttt
cttattattt cttactc 373834DNAArtificial
SequencePrimer 38cctggatcct ttagtagcat taaaaacctc tcca
343934DNAArtificial SequencePrimer 39aggggatcct tcccttctgt
ctatgcatgg gaga 344034DNAArtificial
SequenceSynthetically generated SARS CoV peptide 40cctggatcct aataaagaaa
ctgtatggta acta 344134DNAArtificial
SequencePrimer 41aggggatccc gtagtactag ccaaaaatct attg
344234DNAArtificial SequencePrimer 42cctggatcct tcagcagccc
tgattagttg ttgt 344321DNAArtificial
SequencePrimer 43catacgtttg gcaaccctgt c
214421DNAArtificial SequencePrimer 44aacattacaa attttagagc c
214521DNAArtificial
SequencePrimer 45gagattgaca aaggaattta c
214622DNAArtificial SequencePrimer 46ctaatttgct tctccaatat
gg 224721DNAArtificial
SequencePrimer 47atggctaaaa cctccgtaga t
214821DNAArtificial SequencePrimer 48aattgtgatg tcgttattgg c
214920DNAArtificial
SequencePrimer 49acttcaaaaa tcatacatca
205024DNAArtificial SequencePrimer 50ggtgtcaaat tacattacac
ataa 245115PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 51Val Ile Pro Phe Lys
Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys1 5
10 155215PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 52Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn Val
Val Arg1 5 10
155315PRTArtificial SequenceSynthetically generated SARS CoV S peptide
53Ala Ala Thr Glu Lys Ser Asn Val Val Arg Gly Trp Val Phe Gly1
5 10 155415PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 54Ser Asn Val Val Arg
Gly Trp Val Phe Gly Ser Thr Met Asn Asn1 5
10 155515PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 55Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln
Ser Val1 5 10
155615PRTArtificial SequenceSynthetically generated SARS CoV S peptide
56Ser Thr Met Asn Asn Lys Ser Gln Ser Val Ile Ile Ile Asn Asn1
5 10 155715PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 57Lys Ser Gln Ser Val
Ile Ile Ile Asn Asn Ser Thr Asn Val Val1 5
10 155815PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 58Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala
Cys Asn1 5 10
155915PRTArtificial SequenceSynthetically generated SARS CoV S peptide
59Ser Thr Asn Val Val Ile Arg Ala Cys Asn Phe Glu Leu Cys Asp1
5 10 156015PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 60Ile Arg Ala Cys Asn
Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala1 5
10 156115PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 61Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys
Pro Met1 5 10
156215PRTArtificial SequenceSynthetically generated SARS CoV S peptide
62Asn Pro Phe Phe Ala Val Ser Lys Pro Met Gly Thr Gln Thr His1
5 10 156315PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 63Val Ser Lys Pro Met
Gly Thr Gln Thr His Thr Met Ile Phe Asp1 5
10 156415PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 64Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe
Asn Cys1 5 10
156515PRTArtificial SequenceSynthetically generated SARS CoV S peptide
65Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr Phe Glu Tyr Ile1
5 10 156615PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 66Asn Ala Phe Asn Cys
Thr Phe Glu Tyr Ile Ser Asp Ala Phe Ser1 5
10 156715PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 67Thr Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val
Ser Glu1 5 10
156815PRTArtificial SequenceSynthetically generated SARS CoV S peptide
68Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe1
5 10 156915PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 69Leu Asp Val Ser Glu
Lys Ser Gly Asn Phe Lys His Leu Arg Glu1 5
10 157015PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 70Lys Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe
Lys Asn1 5 10
157115PRTArtificial SequenceSynthetically generated SARS CoV S peptide
71Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu1
5 10 157215PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 72Phe Val Phe Lys Asn
Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly1 5
10 157315PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 73Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro
Ile Asp1 5 10
157415PRTArtificial SequenceSynthetically generated SARS CoV S peptide
74Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp Leu1
5 10 157515PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 75Tyr Gln Pro Ile Asp
Val Val Arg Asp Leu Pro Ser Gly Phe Asn1 5
10 157615PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 76Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys
Pro Ile1 5 10
157715PRTArtificial SequenceSynthetically generated SARS CoV S peptide
77Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu1
5 10 157815PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 78Ala Glu Leu Lys Cys
Ser Val Lys Ser Phe Glu Ile Asp Lys Gly1 5
10 157915PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 79Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln
Thr Ser1 5 10
158015PRTArtificial SequenceSynthetically generated SARS CoV S peptide
80Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Val1
5 10 158115PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 81Ile Tyr Gln Thr Ser
Asn Phe Arg Val Val Pro Ser Gly Asp Val1 5
10 158215PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 82Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe
Pro Asn1 5 10
158315PRTArtificial SequenceSynthetically generated SARS CoV S peptide
83Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn Leu Cys1
5 10 158415PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 84Val Arg Phe Pro Asn
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val1 5
10 158515PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 85Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
Thr Lys1 5 10
158615PRTArtificial SequenceSynthetically generated SARS CoV S peptide
86Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr1
5 10 158715PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 87Phe Asn Ala Thr Lys
Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys1 5
10 158815PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 88Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser
Asn Cys1 5 10
158915PRTArtificial SequenceSynthetically generated SARS CoV S peptide
89Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser1
5 10 159015PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 90Lys Ile Ser Asn Cys
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser1 5
10 159115PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 91Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe
Ser Thr1 5 10
159215PRTArtificial SequenceSynthetically generated SARS CoV S peptide
92Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly1
5 10 159315PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 93Thr Phe Phe Ser Thr
Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys1 5
10 159415PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 94Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp
Leu Cys1 5 10
159515PRTArtificial SequenceSynthetically generated SARS CoV S peptide
95Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr1
5 10 159615PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 96Leu Asn Asp Leu Cys
Phe Ser Asn Val Tyr Ala Asp Ser Phe Val1 5
10 159715PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 97Phe Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly
Asp Asp1 5 10
159815PRTArtificial SequenceSynthetically generated SARS CoV S peptide
98Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala1
5 10 159915PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 99Val Lys Gly Asp Asp
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly1 5
10 1510015PRTArtificial SequenceSynthetically generated
SARS CoV S peptide 100Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Val Ile
Ala Asp Tyr1 5 10
1510115PRTArtificial SequenceSynthetically generated SARS CoV S peptide
101Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro1
5 10 1510215PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 102Val Ile Ala Asp
Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met Gly1 5
10 1510315PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 103Asn Tyr Lys Leu Pro Asp Asp Phe Met Gly
Cys Val Leu Ala Trp1 5 10
1510415PRTArtificial SequenceSynthetically generated SARS CoV S peptide
104Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile1
5 10 1510515PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 105Cys Val Leu Ala
Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr1 5
10 1510615PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 106Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr
Gly Asn Tyr Asn Tyr1 5 10
1510715PRTArtificial SequenceSynthetically generated SARS CoV S peptide
107Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu1
5 10 1510815PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 108Gly Asn Tyr Asn
Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu1 5
10 1510915PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 109Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu
Arg Pro Phe Glu Arg1 5 10
1511015PRTArtificial SequenceSynthetically generated SARS CoV S peptide
110Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val1
5 10 1511115PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 111Arg Pro Phe Glu
Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp1 5
10 1511215PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 112Asp Ile Ser Asn Val Pro Phe Ser Pro Asp
Gly Lys Pro Cys Thr1 5 10
1511315PRTArtificial SequenceSynthetically generated SARS CoV S peptide
113Pro Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn1
5 10 1511415PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 114Gly Lys Pro Cys
Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu1 5
10 1511515PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 115Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu
Asn Asp Tyr Gly Phe1 5 10
1511615PRTArtificial SequenceSynthetically generated SARS CoV S peptide
116Cys Tyr Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly1
5 10 1511715PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 117Asn Asp Tyr Gly
Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro1 5
10 1511815PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 118Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro
Tyr Arg Val Val Val1 5 10
1511915PRTArtificial SequenceSynthetically generated SARS CoV S peptide
119Ile Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu1
5 10 1512015PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 120Tyr Arg Val Val
Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala1 5
10 1512115PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 121Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala
Thr Val Cys Gly Pro1 5 10
1512215PRTArtificial SequenceSynthetically generated SARS CoV S peptide
122Leu Asn Ala Pro Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Asp1
5 10 1512315PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 123Thr Val Cys Gly
Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln1 5
10 1512415PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 124Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln
Cys Val Asn Phe Asn1 5 10
1512515PRTArtificial SequenceSynthetically generated SARS CoV S peptide
125Leu Ile Lys Asn Gln Cys Val Asn Phe Asn Phe Asn Gly Leu Thr1
5 10 1512615PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 126Cys Val Asn Phe
Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu1 5
10 1512715PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 127Phe Asn Gly Leu Thr Gly Thr Gly Val Leu
Thr Pro Ser Ser Lys1 5 10
1512815PRTArtificial SequenceSynthetically generated SARS CoV S peptide
128Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg Phe Gln Pro Phe1
5 10 1512915PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 129Thr Pro Ser Ser
Lys Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg1 5
10 1513015PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 130Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg
Asp Val Ser Asp Phe1 5 10
1513115PRTArtificial SequenceSynthetically generated SARS CoV S peptide
131Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp Ser Val Arg1
5 10 1513215PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 132Asp Val Ser Asp
Phe Thr Asp Ser Val Arg Asp Pro Lys Thr Ser1 5
10 1513315PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 133Thr Asp Ser Val Arg Asp Pro Lys Thr Ser
Glu Ile Leu Asp Ile1 5 10
1513415PRTArtificial SequenceSynthetically generated SARS CoV S peptide
134Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys Ala Phe1
5 10 1513515PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 135Glu Ile Leu Asp
Ile Ser Pro Cys Ala Phe Gly Gly Val Ser Val1 5
10 1513615PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 136Ser Pro Cys Ala Phe Gly Gly Val Ser Val
Ile Thr Pro Gly Thr1 5 10
1513715PRTArtificial SequenceSynthetically generated SARS CoV S peptide
137Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser Glu1
5 10 1513815PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 138Ile Thr Pro Gly
Thr Asn Ala Ser Ser Glu Val Ala Val Leu Tyr1 5
10 1513915PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 139Asn Ala Ser Ser Glu Val Ala Val Leu Tyr
Gln Asp Val Asn Cys1 5 10
1514015PRTArtificial SequenceSynthetically generated SARS CoV S peptide
140Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr1
5 10 1514115PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 141Gln Asp Val Asn
Cys Thr Asp Val Ser Thr Ala Ile His Ala Asp1 5
10 1514215PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 142Thr Asp Val Ser Thr Ala Ile His Ala Asp
Gln Leu Thr Pro Ala1 5 10
1514315PRTArtificial SequenceSynthetically generated SARS CoV S peptide
143Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser1
5 10 1514415PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 144Gln Leu Thr Pro
Ala Trp Arg Ile Tyr Ser Thr Gly Asn Asn Val1 5
10 1514515PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 145Trp Arg Ile Tyr Ser Thr Gly Asn Asn Val
Phe Gln Thr Gln Ala1 5 10
1514615PRTArtificial SequenceSynthetically generated SARS CoV S peptide
146Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly1
5 10 1514715PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 147Phe Gln Thr Gln
Ala Gly Cys Leu Ile Gly Ala Glu His Val Asp1 5
10 1514815PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 148Gly Cys Leu Ile Gly Ala Glu His Val Asp
Thr Ser Tyr Glu Cys1 5 10
1514915PRTArtificial SequenceSynthetically generated SARS CoV S peptide
149Ala Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly1
5 10 1515015PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 150Thr Ser Tyr Glu
Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala1 5
10 1515115PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 151Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
Ser Tyr His Thr Val1 5 10
1515215PRTArtificial SequenceSynthetically generated SARS CoV S peptide
152Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser1
5 10 1515315PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 153Ser Tyr His Thr
Val Ser Leu Leu Arg Ser Thr Ser Gln Lys Ser1 5
10 1515415PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 154Ser Leu Leu Arg Ser Thr Ser Gln Lys Ser
Ile Val Ala Tyr Thr1 5 10
1515515PRTArtificial SequenceSynthetically generated SARS CoV S peptide
155Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala1
5 10 1515615PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 156Ile Val Ala Tyr
Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala1 5
10 1515715PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 157Met Ser Leu Gly Ala Asp Ser Ser Ile Ala
Tyr Ser Asn Asn Thr1 5 10
1515815PRTArtificial SequenceSynthetically generated SARS CoV S peptide
158Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr1
5 10 1515915PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 159Tyr Ser Asn Asn
Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser1 5
10 1516015PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 160Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser
Ile Thr Thr Glu Val1 5 10
1516115PRTArtificial SequenceSynthetically generated SARS CoV S peptide
161Asn Phe Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val Ser Met1
5 10 1516215PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 162Ile Thr Thr Glu
Val Met Pro Val Ser Met Ala Lys Thr Ser Val1 5
10 1516315PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 163Met Pro Val Ser Met Ala Lys Thr Ser Val
Asp Cys Asn Met Tyr1 5 10
1516415PRTArtificial SequenceSynthetically generated SARS CoV S peptide
164Ala Lys Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser1
5 10 1516515PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 165Asp Cys Asn Met
Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn1 5
10 1516615PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 166Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
Phe Lys Asn His Thr1 5 10
1516715PRTArtificial SequenceSynthetically generated SARS CoV S peptide
167Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp1
5 10 1516815PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 168Phe Lys Asn His
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser1 5
10 1516915PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 169Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
Gly Ile Asn Ala Ser1 5 10
1517015PRTArtificial SequenceSynthetically generated SARS CoV S peptide
170Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln1
5 10 1517115PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 171Gly Ile Asn Ala
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg1 5
10 1517215PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 172Val Val Asn Ile Gln Lys Glu Ile Asp Arg
Leu Asn Glu Val Ala1 5 10
1517315PRTArtificial SequenceSynthetically generated SARS CoV S peptide
173Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu1
5 10 1517415PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 174Leu Asn Glu Val
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu1 5
10 1517515PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 175Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
Gln Glu Leu Gly Lys1 5 10
1517615PRTArtificial SequenceSynthetically generated SARS CoV S peptide
176Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile1
5 10 1517715PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 177Gln Glu Leu Gly
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr1 5
10 1517815PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 178Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
Val Trp Leu Gly Phe1 5 10
1517915PRTArtificial SequenceSynthetically generated SARS CoV S peptide
179Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile1
5 10 1518015PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 180Val Trp Leu Gly
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val1 5
10 1518115PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 181Ile Ala Gly Leu Ile Ala Ile Val Met Val
Thr Ile Leu Leu Cys1 5 10
1518215PRTArtificial SequenceSynthetically generated SARS CoV S peptide
182Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys Met Thr Ser Cys1
5 10 1518315PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 183Thr Ile Leu Leu
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys1 5
10 1518415PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 184Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
Gly Ala Cys Ser Cys1 5 10
1518515PRTArtificial SequenceSynthetically generated SARS CoV S peptide
185Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys Gly Ser Cys Cys Lys1
5 10 1518615PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 186Gly Ala Cys Ser
Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp1 5
10 1518715PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 187Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp
Ser Glu Pro Val Leu1 5 10
1518815PRTArtificial SequenceSynthetically generated SARS CoV S peptide
188Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu1
5 10 1518915PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 189Gly Asp Ser Thr
Glu Cys Ala Asn Leu Leu Leu Gln Tyr Gly Ser1 5
10 1519015PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 190Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu
Asn Arg Ala Leu Ser1 5 10
1519115PRTArtificial SequenceSynthetically generated SARS CoV S peptide
191Asn Arg Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg Asn Thr1
5 10 1519215PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 192Gln Asp Arg Asn
Thr Arg Glu Val Phe Ala Gln Val Lys Gln Met1 5
10 1519315PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 193Gln Val Lys Gln Met Tyr Lys Thr Pro Thr
Leu Lys Tyr Phe Gly1 5 10
1519415PRTArtificial SequenceSynthetically generated SARS CoV S peptide
194Leu Lys Tyr Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp1
5 10 1519515PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 195Gln Ile Leu Pro
Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile1 5
10 1519615PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 196Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe
Asn Lys Val Thr Leu1 5 10
1519715PRTArtificial SequenceSynthetically generated SARS CoV S peptide
197Lys Val Thr Leu Leu Ala Asp Ala Gly Phe Met Lys Gln Tyr Gly1
5 10 1519815PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 198Met Lys Gln Tyr
Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp1 5
10 1519915PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 199Ile Asn Ala Arg Asp Leu Ile Cys Ala Gln
Lys Phe Asn Gly Leu1 5 10
1520015PRTArtificial SequenceSynthetically generated SARS CoV S peptide
200Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Asp1
5 10 1520115PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 201Leu Leu Thr Asp
Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val1 5
10 1520215PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 202Thr Ala Ala Leu Val Ser Gly Thr Ala Thr
Ala Gly Trp Thr Phe1 5 10
1520315PRTArtificial SequenceSynthetically generated SARS CoV S peptide
203Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe1
5 10 1520415PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 204Leu Gln Ile Pro
Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly1 5
10 1520515PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 205Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln
Asn Val Leu Tyr Glu1 5 10
1520615PRTArtificial SequenceSynthetically generated SARS CoV S peptide
206Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn1
5 10 1520715PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 207Ala Asn Gln Phe
Asn Lys Ala Ile Ser Gln Ile Gln Glu Ser Leu1 5
10 1520815PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 208Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr
Ala Leu Gly Lys Leu1 5 10
1520915PRTArtificial SequenceSynthetically generated SARS CoV S peptide
209Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala1
5 10 1521015PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 210Gln Asn Ala Gln
Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser1 5
10 1521115PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 211Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile
Ser Ser Val Leu Asn1 5 10
1521216PRTArtificial SequenceSynthetically generated SARS CoV S peptide
212Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala1
5 10 1521316PRTArtificial
SequenceSynthetically generated SARS CoV S peptide 213Leu Asp Lys Val
Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly1 5
10 1521421PRTArtificial SequenceSynthetically
generated SARS CoV S peptide 214Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
Gln Thr Tyr Val Thr Gln1 5 10
15Gln Leu Ile Arg Ala20
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