Mutant of L1 protein of human papillomavirus type 39
11427618 · 2022-08-30
Assignee
Inventors
- Shaowei Li (Fujian, CN)
- Daning Wang (Fujian, CN)
- Zhiping Wang (Fujian, CN)
- Xinlin Liu (Fujian, CN)
- Jun Zhang (Fujian, CN)
- Ningshao Xia (Fujian, CN)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2710/20022
CHEMISTRY; METALLURGY
C07K14/025
CHEMISTRY; METALLURGY
C12N2710/20034
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
International classification
C07K14/025
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
Abstract
The invention relates to a mutated HPV39 L1 protein (or a variant thereof), a sequence encoding the same, a method for preparing the same, and a virus-like particle comprising the same, wherein the protein (or a variant thereof) and the virus-like particle can induce the generation of neutralizing antibodies against at least two HPV types (e.g. HPV39 and HPV68, or HPV39, HPV68 and HPV70), and therefore can be used to prevent infection by said at least two HPV types, and a disease caused by said infection, such as cervical cancer and condyloma acuminatum. The invention further relates to the use of the protein and the virus-like particle in the manufacture of a pharmaceutical composition or a vaccine for preventing infection by said at least two HPV types, and a disease caused by said infection, such as cervical cancer and condyloma acuminatum.
Claims
1. A mutated HPV39 L1 protein, wherein as compared with a wild type HPV39 L1 protein, (I) the mutated HPV39 L1 protein has the following mutations: (1) N-terminal truncation of any number of amino acids from 1 to 25; and (2) substitution of amino acid residues at positions of the wild type HPV39 L1 protein which correspond to positions 269-288 of SEQ ID NO: 1 with amino acid residues at the corresponding positions of a L1 protein of a second type of wild-type HPV; or, (II) the mutated HPV39 L1 protein has the mutations as defined in (1) and (2), and further has the following mutation: (3)(a) substitution of amino acid residues at positions of the wild type HPV39 L1 protein which correspond to positions 117-140 of SEQ ID NO: 1 with amino acid residues at the corresponding positions of a L1 protein of a third type of wild-type HPV; or (b) substitution of amino acid residues at positions of the wild type HPV39 L1 protein which correspond to positions 169-181 of SEQ ID NO: 1 with amino acid residues at the corresponding positions of a L1 protein of a third type of wild-type HPV; or (c) substitution of amino acid residues at positions of the wild type HPV39 L1 protein which correspond to positions 347-358 of SEQ ID NO: 1 with amino acid residues at the corresponding positions of a L1 protein of a third type of wild-type HPV, wherein said corresponding positions are determined by optimal alignment of the sequences being compared, and wherein the L1 proteins of the second type of wild-type HPV comprises different amino acid sequences at a region corresponding to positions 269-288 of SEQ ID. NO.1, and the L1 protein of the third type of wild-type HPV comprises different amino acid sequences at regions correspondence to positions 117-140, 169-181, or 347-358 of SEQ ID NO.1.
2. An isolated nucleic acid, encoding the mutated HPV39 L1 protein according to claim 1.
3. A vector comprising the isolated nucleic acid according to claim 2.
4. An isolated host cell comprising the isolated nucleic acid according to claim 2 and/or a vector comprising the isolated nucleic acid according to claim 2.
5. An HPV virus-like particle, comprising or consisting of the mutated HPV39 L1 protein according to claim 1.
6. A composition, comprising: (i) the mutated HPV39 L1 protein according to claim 1, or (ii) an isolated nucleic acid encoding the mutated HPV39 L1 protein as described in (i), or (iii) a vector comprising the isolated nucleic acid as described in (ii), or (iv) an isolated host cell comprising the isolated nucleic acid as described in (ii) and/or the vector comprising the isolated nucleic acid as described in (iii), or (v) an HPV virus-like particle comprising or consisting of the mutated HPV39 L1 protein as described in (i).
7. A pharmaceutical composition or vaccine, comprising the HPV virus-like particle according to claim 5, and optionally a pharmaceutically acceptable carrier and/or excipient.
8. A method for preparing the mutated HPV39 L1 protein according to claim 1, comprising expressing the mutated HPV39 L1 protein in a host cell, and then recovering the mutated HPV39 L1 protein from a culture of the host cell.
9. A method for preparing a vaccine, comprising combining the HPV virus-like particle according to claim 5 with a pharmaceutically acceptable carrier and/or excipient.
10. A method for preventing HPV infection or a disease caused by HPV infection, comprising administering to a subject a prophylactically effective amount of the HPV virus-like particle according to claim 5 or a pharmaceutical composition or vaccine comprising the HPV virus-like particle according to claim 5 and optionally a pharmaceutically acceptable carrier and/or excipient.
11. The mutated HPV39 L1 protein according to claim 1, wherein the mutated HPV39 L1 protein is characterized by one or more of the following items: (i) the mutated HPV39 L1 protein has 3, 5, 8, 10, 12, 15, 18, 20 or 22 amino acids truncated at N-terminal, as compared with the wild type HPV39 L1 protein; (ii) the second type of wild-type HPV is HPV68; (iii) the amino acid residues at the corresponding positions as described in (2) are amino acid residues at positions 270-289 of a wild type HPV68 L1 protein; (iv) the third type of wild-type HPV is HPV70; (v) the amino acid residues at the corresponding positions as described in (3) (a) are amino acid residues at positions 117-141 of a wild type HPV70 L1 protein; (vi) the amino acid residues at the corresponding positions as described in (3) (b) are amino acid residues at positions 170-182 of a wild type HPV70 L1 protein; (vii) the amino acid residues at the corresponding positions as described in (3) (c) are amino acid residues at positions 348-359 of a wild type HPV70 L1 protein; (viii) the wild type HPV39 L1 protein has an amino acid sequence as set forth in SEQ ID NO: 1; (ix) the wild type HPV68 L1 protein has an amino acid sequence as set forth in SEQ ID NO: 2; (x) the wild type HPV70 L1 protein has an amino acid sequence as set forth in SEQ ID NO: 3.
12. The mutated HPV39 L1 protein according to claim 1, wherein the mutated HPV39 L1 protein has an amino acid sequence selected from the group consisting of: SEQ ID NO: 7, 10, 11 and 12.
13. The isolated nucleic acid according to claim 2, wherein the isolated nucleic acid has a nucleotide sequence selected from the group consisting of: SEQ ID NO: 19, 22, 23 and 24.
14. The pharmaceutical composition or vaccine according to claim 7, wherein the HPV virus-like particle is present in an amount effective for preventing HPV infection or a disease caused by HPV infection.
15. The pharmaceutical composition or vaccine according to claim 14, wherein the HPV infection is infection by one or more HPV types, and/or, the disease caused by HPV infection is selected from the group consisting of cervical cancer and condyloma acuminatum.
16. The pharmaceutical composition or vaccine according to claim 15, wherein the HPV infection is selected from: HPV39 infection, HPV68 infection, HPV70 infection and any combination thereof.
17. The method according to claim 8, wherein the host cell is E. coli.
18. The method according to claim 17, wherein the method comprises the steps of: expressing the mutated HPV39 L1 protein in E. coli, and then obtaining the mutated HPV39 L1 protein by purifying a lysate supernatant of the E. coli.
19. The method according to claim 10, wherein the HPV infection is infection by one or more HPV types, and/or, the disease caused by HPV infection is selected from the group consisting of cervical cancer and condyloma acuminatum.
20. The method according to claim 19, wherein the HPV infection is selected from: HPV39 infection, HPV68 infection, HPV70 infection and any combination thereof.
Description
DESCRIPTION OF DRAWINGS
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SEQUENCE INFORMATION
(11) Some of the sequences involved in the invention are provided in the following Table 1.
(12) TABLE-US-00001 TABLE 1 Description of sequences SEQ ID NO: Description 1 wild type HPV39 L1 protein 2 wild type HPV68 L1 protein, HPV68NO 3 wild type HPV70 L1 protein 4 the mutated HPV39 L1 protein comprising Segment 1 of HPV68 L1 protein, H39N15-68T1 5 the mutated HPV39 L1 protein comprising Segment 2 of HPV68 L1 protein, H39N15-68T2 6 the mutated HPV39 L1 protein comprising Segment 3 of HPV68 L1 protein, H39N15-68T3 7 the mutated HPV39 L1 protein comprising Segment 4 of HPV68 L1 protein, H39N15-68T4 8 the mutated HPV39 L1 protein comprising Segment 5 of HPV68 L1 protein, H39N15-68T5 9 the mutated HPV39 L1 protein comprising Segment 4 of HPV68 L1 protein and Segment 1 of HPV70 L1 protein, H39N15-68T4-7051 10 the mutated HPV39 L1 protein comprising Segment 4 of HPV68 L1 protein and Segment 2 of HPV70 L1 protein, H39N15-68T4-7052 11 the mutated HPV39 L1 protein comprising Segment 4 of HPV68 L1 protein and Segment 3 of HPV70 L1 protein, H39N15-68T4-7053 12 the mutated HPV39 L1 protein comprising Segment 4 of HPV68 L1 protein and Segment 5 of HPV70 L1 protein, H39N15-68T4-7055 13 the DNA sequence encoding SEQ ID NO: 1 14 the DNA sequence encoding SEQ ID NO: 2 15 the DNA sequence encoding SEQ ID NO: 3 16 the DNA sequence encoding SEQ ID NO: 4 17 the DNA sequence encoding SEQ ID NO: 5 18 the DNA sequence encoding SEQ ID NO: 6 19 the DNA sequence encoding SEQ ID NO: 7 20 the DNA sequence encoding SEQ ID NO: 8 21 the DNA sequence encoding SEQ ID NO: 9 22 the DNA sequence encoding SEQ ID NO: 10 23 the DNA sequence encoding SEQ ID NO: 11 24 the DNA sequence encoding SEQ ID NO: 12 25 the sequence of the amino acid residues at positions 270-289 of wild type HPV68 L1 protein, i.e., Segment 4 of HPV68 L1 protein 26 the sequence of the amino acid residues at positions 117-141 of wild type HPV70 L1 protein, i.e., Segment 2 of HPV70 L1 protein 27 the sequence of the amino acid residues at positions 170-182 of wild type HPV70 L1 protein, i.e., Segment 3 of HPV70 L1 protein 28 the sequence of the amino acid residues at positions 348-359 of wild type HPV70 L1 protein, i.e., Segment 5 of HPV70 L1 protein 29 the HPV39 L1 protein having 15 amino acids truncated at N-terminal, HPV39N15 30 the DNA sequence encoding SEQ ID NO:29 31 the HPV70 L1 protein having 10 amino acids truncated at N-terminal, HPV70N10 32 the DNA sequence encoding SEQ ID NO:31 33 the sequence of the amino acid residues at positions 53-61 of wild type HPV68 L1 protein, i.e., Segment 1 of HPV68 L1 protein 34 the sequence of the amino acid residues at positions 117-151 of wild type HPV68 L1 protein, i.e., Segment 2 of HPV68 L1 protein 35 the sequence of the amino acid residues at positions 170-182 of wild type HPV68 L1 protein, i.e., Segment 3 of HPV68 L1 protein 36 the sequence of the amino acid residues at positions 348-359 of wild type HPV68 L1 protein, i.e., Segment 5 of HPV68 L1 protein 37 the sequence of the amino acid residues at positions 53-61 of wild type HPV70 L1 protein, i.e., Segment 1 of HPV70 L1 protein Sequence 1 (SEQ ID NO: 1): MALWRSSDSMVYLPPPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVS AYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSP FSSTTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTMEDGDMID TGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSNIFFCLRREQLFARHFWNRGGMVG DAIPAQLYIKGTDIRANPGSSVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVD TTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNF AVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQ ARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK Sequence 2 (SEQ ID NO: 2): MALWRASDNMVYLPPPSVAKVVNTDDYVTRTGMYYYAGTSRLLTVGHPYFKVPMSGGRKQGIPKV SAYQYRVFRVTLPDPNKFSVPESTLYNPDTQRMVWACVGVEIGRGQPLGVGLSGHPLYNRLDDTENS PFSSNKNPKDSRDNVAVDCKQTQLCIIGCVPAIGEHWAKGKSCKPTNVQQGDCPPLELVNTMEDGD MIDTGYGAMDFGTLQETKSEVPLDICQSVCKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGG MVGDTIPTDMYIKGTDIRETPSSYVYAPSPSGSMVSSDSQLFNKPYWLHKAQGHNNGICWHNQLFLT VVDTTRSTNFTLSTTTDSTVPAVYDSNKFKEYVRHVEEYDLQFIFQLCTITLSTDVMSYIHTMNPAILD DWNFGVAPPPSASLVDTYRYLQSAAITCQKDAPAPVKKDPYDGLNFWNVDLKEKFSSELDQFPLGRK FLLQAGVRRRPTIGPRKRTATAATTSTSKHKRKRVSK Sequence 3 (SEQ ID NO: 3): MALWRSSDNTVYLPPPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVPVNGGRKQEIPKVSA YQYRVFRVSLPDPNKFGLPDPSLYNPDTQRLVWACIGVEIGRGQPLGVGVSGHPLYNRLDDTENSHFS SAVNTQDSRDNVSVDYKQTQLCIIGCVPAMGEHWAKGKACKSTTVQQGDCPPLELVNTAIEDGDMI DTGYGAMDFRTLQETKSEVPLDICQSVCKYPDYLQMSADVYGDSMFFCLRKEQLFARHFWNRGGM VGDTIPSELYIKGTDIRDRPGTHVYSPSPSGSMVSSDSQLFNKPYWLHKAQGHNNGICWHNQLFITVV DTTRSTNFTLSACTETAIPAVYSPTKFKEYTRHVEEYDLQFIFQLCTITLTADVMAYIHTMNPAILDNW NIGVTPPPSASLVDTYRYLQSAAIACQKDAPAPEKKDPYDDLKFWNVDLKEKFSTELDQFPLGRKFLL QVGARRRPTIGPRKRPASAKSSSSASKHKRKRVSK Sequence 4 (SEQ ID NO: 4): NIPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVPMSGGRKQGIPKVSAYQYRVFRVTLPDP NKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSSTTNKDSRDNVS VDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQE TKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDAIPAQLYIKGTDI RANPGSSVYCPSPSGSMVTSDSQLFNKPYWLIIKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIES SIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTY RYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRP AASTSSSSATKHKRKRVSK Sequence 5 (SEQ ID NO: 5): NIPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDP NKFSIPDASLYNPETQRL VWACVGVEVGRGQPLGVGLSGHPLYNRLDDTENSPFSSNKNPKDSRDNV AVDCKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQ ETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDAIPAQLYIKGTD IRANPGSSVYCPSPSGSMVTSDSQLFNKPYWLIIKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIE SSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDT YRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRK RPAASTSSSSATKHKRKRVSK Sequence 6 (SEQ ID NO: 6): NIP SVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDP NKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSSTTNKDSRDNVS VDYKQTQLCIIGCVPAIGEHWAKGKSCKPTNVQQGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQE TKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDAIPAQLYIKGTDI RANPGSSVYCPSPSGSMVTSDSQLFNKPYWLIIKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIES SIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTY RYLQ SAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRP AASTSSSSATKHKRKRVSK Sequence 7 (SEQ ID NO: 7): NIPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDP NKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSSTTNKDSRDNVS VDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQE TKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDTIPTDMYIKGTDI RETPSSYVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIES SIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTY RYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRP AASTSSSSATKHKRKRVSK Sequence 8 (SEQ ID NO: 8): NIPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDP NKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSSTTNKDSRDNVS VDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQE TKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDAIPAQLYIKGTDI RANPGSSVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSTD STVPAVYDSNKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVD TYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRK RPAASTSSSSATKHKRKRVSK Sequence 9 (SEQ ID NO: 9): NIPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVPVNGGRKQEIPKVSAYQYRVFRVTLPDPN KFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSSTTNKDSRDNVSV DYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQET KSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDTIPTDMYIKGTDIR ETPSSYVYCPSPSGSMVTSDSQLFNKPYWLIIKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSI PSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYR YLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPA ASTSSSSATKHKRKRVSK Sequence 10 (SEQ ID NO: 10): NIPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDP NKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGVSGHPLYNRLDDTENSHFSSAVNTQDSRDN VSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGAL QETKSEVPLDICQSICKYPDYLQMSADVYGDSNIFFCLRREQLFARHFWNRGGMVGDTIPTDMYIKGT DIRETPSSYVYCPSPSGSMVTSDSQLFNKPYWLIIKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSI ESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVD TYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRK RPAASTSSSSATKHKRKRVSK Sequence 11 (SEQ ID NO: 11): NIPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDP NKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSSTTNKDSRDNVS VDYKQTQLCIIGCVPAIGEHWAKGKACKSTTVQQGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQE TKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDTIPTDMYIKGTDI RETPSSYVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTSTSIES SIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTY RYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRP AASTSSSSATKHKRKRVSK Sequence 12 (SEQ ID NO: 12): NIPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDP NKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSSTTNKDSRDNVS VDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQE TKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDTIPTDMYIKGTDI RETPSSYVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSTET AIPAVYSPTKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTY RYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRP AASTSSSSATKHKRKRVSK Sequence 13 (SEQ ID NO: 13): ATGGCCCTCTGGCGCAGCTCCGATTCCATGGTCTACCTCCCCCCCCCCAGCGTCGCCAAGGTCGT GAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTACGCCGGGTCCAGCCGCCTGCTGA CCGTGGGCCACCCCTACTTCAAGGTCGGCATGAACGGCGGGCGCAAGCAGGATATCCCCAAGGT CAGCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGACCCCAACAAGTTCTCCATCCCCG ACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGGCCTGCGTGGGCGTCGAAGTCGG GCGCGGGCAGCCCCTCGGCGTCGGCATCTCCGGCCACCCCCTGTACAACCGCCAGGACGACACC GAGAATAGCCCCTTCAGCAGCACAACAAACAAGGATTCCCGCGACAACGTCAGCGTCGACTACA AGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCGGCGAACACTGGGGCAAGGGCAA GGCCTGTAAGCCAAACAACGTGAGCACCGGCGATTGCCCCCCCCTGGAGCTGGTGAATACACCC ATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATGGATTTCGGCGCCCTCCAGGAGA CAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAAGTACCCCGACTACCTCCAGATG AGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCCGCGAGCAGCTCTTCGCCCGCCA CTTCTGGAACCGCGGCGGCATGGTCGGCGATGCAATCCCCGCACAGCTCTACATCAAGGGGACC GACATCCGCGCCAATCCAGGCTCCAGCGTGTATTGTCCAAGCCCATCCGGCAGCATGGTGACAAG CGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGCACAAGGCACAGGGGCATAATAACGGCATC TGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGATACCACACGCTCCACAAATTTCACCCTGAG CACAAGCATCGAAAGCAGCATCCCCAGCACCTACGACCCCAGCAAGTTCAAGGAGTACACACGC CACGTCGAAGAATACGACCTGCAGTTCATCTTCCAGCTCTGCACCGTGACCCTGACCACCGACGT CATGAGCTACATCCACACCATGAACAGCAGCATCCTCGATAACTGGAACTTCGCCGTGGCCCCCC CCCCCAGCGCATCCCTCGTGGATACCTATCGCTATCTGCAGAGCGCCGCAATCACCTGCCAGAAG GACGCCCCCGCCCCCGAGAAGAAGGACCCCTACGATGGCCTGAAGTTCTGGAACGTCGATCTGC GCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCCCTCGGCCGCAAGTTCCTCCTCCAGGCACGC GTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCGCCCCGCCGCCAGCACCAGCAGCAGCAGCG CCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 14 (SEQ ID NO: 14): ATGGCACTGTGGAGAGCCAGCGACAACATGGTGTACCTGCCCCCTCCCAGCGTGGCCAAGGTGG TCAACACCGACGACTACGTGACCCGGACCGGCATGTACTACTACGCCGGCACCTCTCGGCTCCTG ACCGTGGGCCACCCCTACTTCAAGGTGCCCATGAGCGGCGGCAGAAAGCAGGGCATCCCCAAGG TGTCCGCCTACCAGTACCGGGTGTTCAGAGTGACCCTGCCCGACCCCAACAAGTTCAGCGTGCCC GAGAGCACCCTGTACAACCCCGACACCCAGCGGATGGTCTGGGCCTGCGTGGGCGTGGAGATCG GCAGAGGCCAGCCCCTGGGCGTGGGCCTGAGCGGCCACCCCCTGTACAATCGGCTGGACGACAC CGAGAACAGCCCCTTCAGCAGCAACAAGAACCCCAAGGACAGCCGGGACAACGTGGCCGTGGA CTGCAAGCAGACCCAGCTGTGCATCATCGGCTGCGTGCCTGCCATTGGCGAGCACTGGGCCAAG GGCAAGAGCTGCAAGCCCACCAACGTGCAGCAGGGCGACTGCCCCCCTCTGGAACTGGTCAACA CACCCATCGAGGACGGCGACATGATCGACACCGGCTACGGCGCCATGGACTTCGGCACCCTGCA GGAAACCAAGAGCGAGGTCCCCCTGGACATCTGCCAGAGCGTGTGCAAGTACCCCGACTACCTG CAGATGAGCGCCGACGTGTACGGCGACAGCATGTTCTTTTGCCTGCGGCGGGAGCAGCTGTTCGC CCGGCACTTCTGGAACAGAGGCGGCATGGTCGGCGACACCATCCCCACCGACATGTACATCAAG GGCACCGACATCAGAGAGACACCCAGCAGCTACGTGTACGCCCCCAGCCCCAGCGGCAGCATGG TGTCCAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGCACAAGGCCCAGGGCCACAACAA CGGCATCTGCTGGCACAACCAGCTGTTTCTGACCGTGGTGGACACCACCAGAAGCACCAACTTCA CCCTGAGCACCACCACCGACAGCACCGTGCCCGCCGTGTACGACAGCAATAAGTTCAAAGAATA CGTGCGGCACGTGGAGGAATACGACCTGCAGTTCATCTTCCAGCTGTGTACCATCACCCTGTCCA CCGACGTGATGAGCTACATCCACACCATGAACCCCGCCATCCTGGACGACTGGAACTTCGGCGTG GCCCCTCCCCCTAGCGCCAGCCTGGTGGATACCTACAGATACCTGCAGAGCGCCGCCATCACCTG CCAGAAGGACGCCCCTGCCCCCGTGAAGAAGGACCCCTACGACGGCCTGAACTTCTGGAATGTG GACCTGAAAGAGAAGTTCAGCAGCGAGCTGGACCAGTTCCCCCTGGGCCGGAAGTTCCTGCTGC AAGCCGGCGTGCGGAGAAGGCCCACCATCGGCCCCAGAAAGCGGACCGCCACCGCAGCCACAA CCTCCACCTCCAAGCACAAGCGGAAGCGGGTGTCCAAGTGA Sequence 15 (SEQ ID NO: 15): ATGGCTTTGTGGCGGTCTAGTGACAACACGGTGTATTTGCCACCCCCTTCTGTGGCGAAGGTTGT CAATACAGATGATTATGTAACACGTACAGGCATATATTATTATGCTGGAAGCTCTCGCTTATTAA CAGTAGGGCATCCTTATTTTAAGGTACCTGTAAATGGTGGCCGCAAGCAGGAAATACCTAAGGTG TCTGCATATCAGTATAGGGTATTTAGGGTATCCCTACCTGATCCTAATAAGTTTGGCCTTCCGGAT CCTTCCCTTTATAATCCTGACACACAACGCCTGGTATGGGCCTGTATAGGTGTGGAAATTGGTAG AGGCCAGCCATTGGGCGTTGGTGTTAGTGGACATCCTTTATATAATAGATTGGATGATACTGAAA ATTCACATTTTTCCTCTGCTGTTAATACACAGGACAGTAGGGACAATGTGTCTGTGGACTATAAG CAGACACAGTTATGTATTATAGGCTGTGTTCCTGCTATGGGAGAGCACTGGGCAAAGGGCAAGG CCTGTAAGTCCACTACTGTACAACAGGGCGATTGTCCACCATTAGAATTAGTTAATACTGCAATT GAGGATGGCGATATGATAGATACAGGCTATGGAGCCATGGACTTTCGTACATTGCAGGAAACCA AAAGTGAGGTACCACTAGATATTTGCCAATCCGTGTGTAAATATCCTGATTATTTGCAGATGTCT GCTGATGTATATGGGGACAGTATGTTTTTTTGTTTGCGCAAGGAACAGTTATTTGCCAGACACTTT TGGAATAGAGGTGGCATGGTGGGCGACACAATACCTTCAGAGTTATATATTAAAGGCACGGATA TACGTGATCGTCCTGGTACTCATGTATATTCCCCTTCCCCAAGTGGCTCTATGGTTTCTTCTGATTC CCAGTTGTTTAATAAGCCCTATTGGTTGCATAAGGCCCAGGGACACAATAATGGCATTTGTTGGC ATAACCAGTTGTTTATTACTGTGGTGGACACTACACGTAGTACTAATTTTACATTGTCTGCCTGCA CCGAAACAGCCATACCTGCTGTATATAGCCCTACAAAGTTTAAGGAATATACTAGGCATGTGGAG GAATATGATTTACAATTTATATTTCAGTTGTGTACTATCACATTAACTGCAGACGTTATGGCCTAC ATCCATACTATGAATCCTGCAATTTTGGACAATTGGAATATAGGCGTTACCCCTCCACCATCTGC AAGCTTGGTGGACACGTATAGGTATTTACAATCAGCAGCTATAGCATGTCAGAAGGATGCTCCTG CACCTGAAAAAAAGGATCCCTATGACGATTTAAAATTTTGGAATGTTGATTTAAAGGAAAAGTTT AGTACAGAACTAGATCAGTTTCCTTTGGGGCGCAAATTTTTACTACAGGTAGGGGCTCGCAGACG TCCTACTATAGGCCCTCGCAAACGCCCTGCATCAGCTAAATCGTCTTCCTCAGCCTCTAAACACA AACGGAAACGTGTGTCCAAGTAA Sequence 16 (SEQ ID NO: 16): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTCAAGGTGCCCATGAGCGGCGGCA GAAAGCAGGGCATCCCCAAGGTGTCCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGAC CCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGGC CTGCGTGGGCGTCGAAGTCGGGCGCGGGCAGCCCCTCGGCGTCGGCATCTCCGGCCACCCCCTGT ACAACCGCCAGGACGACACCGAGAATAGCCCCTTCAGCAGCACAACAAACAAGGATTCCCGCGA CAACGTCAGCGTCGACTACAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCGGCG AACACTGGGGCAAGGGCAAGGCCTGTAAGCCAAACAACGTGAGCACCGGCGATTGCCCCCCCCT GGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATGGAT TTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAAGTA CCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCCGCG AGCAGCTCTTCGCCCGCCACTTCTGGAACCGCGGCGGCATGGTCGGCGATGCAATCCCCGCACAG CTCTACATCAAGGGGACCGACATCCGCGCCAATCCAGGCTCCAGCGTGTATTGTCCAAGCCCATC CGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGCACAAGGCACAG GGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGATACCACACGCTC CACAAATTTCACCCTGAGCACAAGCATCGAAAGCAGCATCCCCAGCACCTACGACCCCAGCAAG TTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTTCCAGCTCTGCACCGT GACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCATCCTCGATAACTGG AACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCTATCTGCAGAGCGC CGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTACGATGGCCTGAAG TTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCCCTCGGCCGCAA GTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCGCCCCGCCGCCA GCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 17 (SEQ ID NO: 17): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTCAAGGTCGGCATGAACGGCGGG CGCAAGCAGGATATCCCCAAGGTCAGCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGA CCCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGG CCTGCGTGGGCGTCGAAGTCGGCAGAGGCCAGCCCCTGGGCGTGGGCCTGAGCGGCCACCCCCT GTACAATCGGCTGGACGACACCGAGAACAGCCCCTTCAGCAGCAACAAGAACCCCAAGGACAGC CGGGACAACGTGGCCGTGGACTGCAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAA TCGGCGAACACTGGGGCAAGGGCAAGGCCTGTAAGCCAAACAACGTGAGCACCGGCGATTGCCC CCCCCTGGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCC ATGGATTTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTG CAAGTACCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGC GCCGCGAGCAGCTCTTCGCCCGCCACTTCTGGAACCGCGGCGGCATGGTCGGCGATGCAATCCCC GCACAGCTCTACATCAAGGGGACCGACATCCGCGCCAATCCAGGCTCCAGCGTGTATTGTCCAAG CCCATCCGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGCACAAG GCACAGGGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGATACCAC ACGCTCCACAAATTTCACCCTGAGCACAAGCATCGAAAGCAGCATCCCCAGCACCTACGACCCC AGCAAGTTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTTCCAGCTCTG CACCGTGACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCATCCTCGATA ACTGGAACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCTATCTGCAG AGCGCCGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTACGATGGCC TGAAGTTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCCCTCGGC CGCAAGTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCGCCCCGC CGCCAGCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 18 (SEQ ID NO: 18): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTCAAGGTCGGCATGAACGGCGGG CGCAAGCAGGATATCCCCAAGGTCAGCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGA CCCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGG CCTGCGTGGGCGTCGAAGTCGGGCGCGGGCAGCCCCTCGGCGTCGGCATCTCCGGCCACCCCCTG TACAACCGCCAGGACGACACCGAGAATAGCCCCTTCAGCAGCACAACAAACAAGGATTCCCGCG ACAACGTCAGCGTCGACTACAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCGGC GAACACTGGGCCAAGGGCAAGAGCTGCAAGCCCACCAACGTGCAGCAGGGCGACTGCCCCCCTC TGGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATGGA TTTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAAGT ACCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCCGC GAGCAGCTCTTCGCCCGCCACTTCTGGAACCGCGGCGGCATGGTCGGCGATGCAATCCCCGCACA GCTCTACATCAAGGGGACCGACATCCGCGCCAATCCAGGCTCCAGCGTGTATTGTCCAAGCCCAT CCGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGCACAAGGCACA GGGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGATACCACACGCT CCACAAATTTCACCCTGAGCACAAGCATCGAAAGCAGCATCCCCAGCACCTACGACCCCAGCAA GTTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTTCCAGCTCTGCACCG TGACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCATCCTCGATAACTGG AACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCTATCTGCAGAGCGC CGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTACGATGGCCTGAAG TTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCCCTCGGCCGCAA GTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCGCCCCGCCGCCA GCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 19 (SEQ ID NO: 19): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTCAAGGTCGGCATGAACGGCGGG CGCAAGCAGGATATCCCCAAGGTCAGCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGA CCCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGG CCTGCGTGGGCGTCGAAGTCGGGCGCGGGCAGCCCCTCGGCGTCGGCATCTCCGGCCACCCCCTG TACAACCGCCAGGACGACACCGAGAATAGCCCCTTCAGCAGCACAACAAACAAGGATTCCCGCG ACAACGTCAGCGTCGACTACAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCGGC GAACACTGGGGCAAGGGCAAGGCCTGTAAGCCAAACAACGTGAGCACCGGCGATTGCCCCCCCC TGGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATGGA TTTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAAGT ACCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCCGC GAGCAGCTCTTCGCCCGCCACTTCTGGAACAGAGGCGGCATGGTCGGCGACACCATCCCCACCG ACATGTACATCAAGGGCACCGACATCAGAGAGACACCCAGCAGCTACGTGTACTGT CCAAGCCCATCCGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGC ACAAGGCACAGGGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGA TACCACACGCTCCACAAATTTCACCCTGAGCACAAGCATCGAAAGCAGCATCCCCAGCACCTACG ACCCCAGCAAGTTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTTCCA GCTCTGCACCGTGACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCATCC TCGATAACTGGAACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCTAT CTGCAGAGCGCCGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTACG ATGGCCTGAAGTTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCC CTCGGCCGCAAGTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCG CCCCGCCGCCAGCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 20 (SEQ ID NO: 20): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTCAAGGTCGGCATGAACGGCGGG CGCAAGCAGGATATCCCCAAGGTCAGCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGA CCCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGG CCTGCGTGGGCGTCGAAGTCGGGCGCGGGCAGCCCCTCGGCGTCGGCATCTCCGGCCACCCCCTG TACAACCGCCAGGACGACACCGAGAATAGCCCCTTCAGCAGCACAACAAACAAGGATTCCCGCG ACAACGTCAGCGTCGACTACAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCGGC GAACACTGGGGCAAGGGCAAGGCCTGTAAGCCAAACAACGTGAGCACCGGCGATTGCCCCCCCC TGGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATGGA TTTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAAGT ACCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCCGC GAGCAGCTCTTCGCCCGCCACTTCTGGAACCGCGGCGGCATGGTCGGCGATGCAATCCCCGCACA GCTCTACATCAAGGGGACCGACATCCGCGCCAATCCAGGCTCCAGCGTGTATTGTCCAAGCCCAT CCGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGCACAAGGCACA GGGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGATACCACACGCT CCACAAATTTCACCCTGAGCACAAGCACCGACAGCACCGTGCCCGCCGTGTACGACAGCAATAA GTTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTTCCAGCTCTGCACCG TGACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCATCCTCGATAACTGG AACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCTATCTGCAGAGCGC CGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTACGATGGCCTGAAG TTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCCCTCGGCCGCAA GTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCGCCCCGCCGCCA GCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 21 (SEQ ID NO: 21): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTTAAGGTACCTGTAAATGGTGGCC GCAAGCAGGAAATACCTAAGGTGTCTGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGAC CCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGGC CTGCGTGGGCGTCGAAGTCGGGCGCGGGCAGCCCCTCGGCGTCGGCATCTCCGGCCACCCCCTGT ACAACCGCCAGGACGACACCGAGAATAGCCCCTTCAGCAGCACAACAAACAAGGATTCCCGCGA CAACGTCAGCGTCGACTACAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCGGCG AACACTGGGGCAAGGGCAAGGCCTGTAAGCCAAACAACGTGAGCACCGGCGATTGCCCCCCCCT GGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATGGAT TTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAAGTA CCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCCGCG AGCAGCTCTTCGCCCGCCACTTCTGGAACAGAGGCGGCATGGTCGGCGACACCATCCCCACCGAC ATGTACATCAAGGGCACCGACATCAGAGAGACACCCAGCAGCTACGTGTACTGT CCAAGCCCATCCGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGC ACAAGGCACAGGGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGA TACCACACGCTCCACAAATTTCACCCTGAGCACAAGCATCGAAAGCAGCATCCCCAGCACCTACG ACCCCAGCAAGTTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTTCCA GCTCTGCACCGTGACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCATCC TCGATAACTGGAACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCTAT CTGCAGAGCGCCGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTACG ATGGCCTGAAGTTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCC CTCGGCCGCAAGTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCG CCCCGCCGCCAGCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 22 (SEQ ID NO: 22): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTCAAGGTCGGCATGAACGGCGGG CGCAAGCAGGATATCCCCAAGGTCAGCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGA CCCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGG CCTGCGTGGGCGTCGAAGTCGGTAGAGGCCAGCCATTGGGCGTTGGTGTTAGTGGACATCCTTTA TATAATAGATTGGATGATACTGAAAATTCACATTTTTCCTCTGCTGTTAATACACAGGACAGTAG GGACAATGTGTCTGTGGACTATAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCG GCGAACACTGGGGCAAGGGCAAGGCCTGTAAGCCAAACAACGTGAGCACCGGCGATTGCCCCCC CCTGGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATG GATTTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAA GTACCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCC GCGAGCAGCTCTTCGCCCGCCACTTCTGGAACAGAGGCGGCATGGTCGGCGACACCATCCCCACC GACATGTACATCAAGGGCACCGACATCAGAGAGACACCCAGCAGCTACGTGTAC TGTCCAAGCCCATCCGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCT GCACAAGGCACAGGGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTC GATACCACACGCTCCACAAATTTCACCCTGAGCACAAGCATCGAAAGCAGCATCCCCAGCACCT ACGACCCCAGCAAGTTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTT CCAGCTCTGCACCGTGACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCA TCCTCGATAACTGGAACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCT ATCTGCAGAGCGCCGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTA CGATGGCCTGAAGTTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCC CCCTCGGCCGCAAGTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAG CGCCCCGCCGCCAGCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGT GA Sequence 23 (SEQ ID NO: 23): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTCAAGGTCGGCATGAACGGCGGG CGCAAGCAGGATATCCCCAAGGTCAGCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGA CCCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGG CCTGCGTGGGCGTCGAAGTCGGGCGCGGGCAGCCCCTCGGCGTCGGCATCTCCGGCCACCCCCTG TACAACCGCCAGGACGACACCGAGAATAGCCCCTTCAGCAGCACAACAAACAAGGATTCCCGCG ACAACGTCAGCGTCGACTACAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCGGC GAACACTGGGCAAAGGGCAAGGCCTGTAAGTCCACTACTGTACAACAGGGCGATTGTCCACCAC TGGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATGGA TTTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAAGT ACCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCCGC GAGCAGCTCTTCGCCCGCCACTTCTGGAACAGAGGCGGCATGGTCGGCGACACCATCCCCACCG ACATGTACATCAAGGGCACCGACATCAGAGAGACACCCAGCAGCTACGTGTACTGT CCAAGCCCATCCGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGC ACAAGGCACAGGGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGA TACCACACGCTCCACAAATTTCACCCTGAGCACAAGCATCGAAAGCAGCATCCCCAGCACCTACG ACCCCAGCAAGTTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTTCCA GCTCTGCACCGTGACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCATCC TCGATAACTGGAACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCTAT CTGCAGAGCGCCGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTACG ATGGCCTGAAGTTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCC CTCGGCCGCAAGTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCG CCCCGCCGCCAGCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 24 (SEQ ID NO: 24): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTCAAGGTCGGCATGAACGGCGGG CGCAAGCAGGATATCCCCAAGGTCAGCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGA CCCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGG CCTGCGTGGGCGTCGAAGTCGGGCGCGGGCAGCCCCTCGGCGTCGGCATCTCCGGCCACCCCCTG TACAACCGCCAGGACGACACCGAGAATAGCCCCTTCAGCAGCACAACAAACAAGGATTCCCGCG ACAACGTCAGCGTCGACTACAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCGGC GAACACTGGGGCAAGGGCAAGGCCTGTAAGCCAAACAACGTGAGCACCGGCGATTGCCCCCCCC TGGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATGGA TTTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAAGT ACCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCCGC GAGCAGCTCTTCGCCCGCCACTTCTGGAACAGAGGCGGCATGGTCGGCGACACCATCCCCACCG ACATGTACATCAAGGGCACCGACATCAGAGAGACACCCAGCAGCTACGTGTACTGT CCAAGCCCATCCGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGC ACAAGGCACAGGGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGA TACCACACGCTCCACAAATTTCACCCTGAGCACAAGCACCGAAACAGCCATACCTGCTGTATATA GCCCTACAAAGTTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTTCCAG CTCTGCACCGTGACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCATCCT CGATAACTGGAACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCTATC TGCAGAGCGCCGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTACGA TGGCCTGAAGTTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCCC TCGGCCGCAAGTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCGC CCCGCCGCCAGCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 25 (SEQ ID NO: 25): TIPTDMYIKGTDIRETPSSY Sequence 26 (SEQ ID NO: 26): VSGHPLYNRLDDTENSHFSSAVNTQ Sequence 27 (SEQ ID NO: 27): AKGKACKSTTVQQ Sequence 28 (SEQ ID NO: 28): TETAIPAVYSPT Sequence 29 (SEQ ID NO: 29): NIPSVAKVVNTDDYVTRTGIYYYAGSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDP NKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSSTTNKDSRDNVS VDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQE TKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDAIPAQLYIKGTDI RANPGSSVYCPSPSGSMVTSDSQLFNKPYWLIIKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIES SIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTY RYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRP AASTSSSSATKHKRKRVSK Sequence 30 (SEQ ID NO: 30): ATGCCCAGCGTCGCCAAGGTCGTGAACACCGACGACTACGTCACCCGCACCGGGATCTACTACTA CGCCGGGTCCAGCCGCCTGCTGACCGTGGGCCACCCCTACTTCAAGGTCGGCATGAACGGCGGG CGCAAGCAGGATATCCCCAAGGTCAGCGCCTACCAGTACCGCGTGTTCCGCGTCACCCTCCCAGA CCCCAACAAGTTCTCCATCCCCGACGCCAGCCTGTACAACCCCGAGACCCAGCGCCTGGTGTGGG CCTGCGTGGGCGTCGAAGTCGGGCGCGGGCAGCCCCTCGGCGTCGGCATCTCCGGCCACCCCCTG TACAACCGCCAGGACGACACCGAGAATAGCCCCTTCAGCAGCACAACAAACAAGGATTCCCGCG ACAACGTCAGCGTCGACTACAAGCAGACCCAGCTCTGTATCATCGGGTGCGTCCCAGCAATCGGC GAACACTGGGGCAAGGGCAAGGCCTGTAAGCCAAACAACGTGAGCACCGGCGATTGCCCCCCCC TGGAGCTGGTGAATACACCCATCGAAGACGGCGACATGATCGACACCGGGTACGGCGCCATGGA TTTCGGCGCCCTCCAGGAGACAAAGTCCGAAGTCCCCCTGGACATCTGCCAGAGCATCTGCAAGT ACCCCGACTACCTCCAGATGAGCGCCGACGTCTACGGCGATTCCATGTTCTTCTGCCTGCGCCGC GAGCAGCTCTTCGCCCGCCACTTCTGGAACCGCGGCGGCATGGTCGGCGATGCAATCCCCGCACA GCTCTACATCAAGGGGACCGACATCCGCGCCAATCCAGGCTCCAGCGTGTATTGTCCAAGCCCAT CCGGCAGCATGGTGACAAGCGACAGCCAGCTGTTCAACAAGCCCTACTGGCTGCACAAGGCACA GGGGCATAATAACGGCATCTGCTGGCACAACCAGCTGTTCCTGACCGTCGTCGATACCACACGCT CCACAAATTTCACCCTGAGCACAAGCATCGAAAGCAGCATCCCCAGCACCTACGACCCCAGCAA GTTCAAGGAGTACACACGCCACGTCGAAGAATACGACCTGCAGTTCATCTTCCAGCTCTGCACCG TGACCCTGACCACCGACGTCATGAGCTACATCCACACCATGAACAGCAGCATCCTCGATAACTGG AACTTCGCCGTGGCCCCCCCCCCCAGCGCATCCCTCGTGGATACCTATCGCTATCTGCAGAGCGC CGCAATCACCTGCCAGAAGGACGCCCCCGCCCCCGAGAAGAAGGACCCCTACGATGGCCTGAAG TTCTGGAACGTCGATCTGCGCGAGAAGTTCTCCCTGGAGCTGGACCAGTTCCCCCTCGGCCGCAA GTTCCTCCTCCAGGCACGCGTGCGCCGCCGCCCCACCATCGGCCCACGCAAGCGCCCCGCCGCCA GCACCAGCAGCAGCAGCGCCACCAAGCACAAGCGCAAGCGCGTCAGCAAGTGA Sequence 31 (SEQ ID NO: 31): MVYLPPPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVPVNGGRKQEIPKVSAYQYRVFRVS LPDPNKFGLPDPSLYNPDTQRLVWACIGVEIGRGQPLGVGVSGHPLYNRLDDTENSHFSSAVNTQDSR DNVSVDYKQTQLCIIGCVPAMGEHWAKGKACKSTTVQQGDCPPLELVNTAIEDGDMIDTGYGAMDF RTLQETKSEVPLDICQSVCKYPDYLQMSADVYGDSNIFFCLRKEQLFARHFWNRGGMVGDTIPSELYI KGTD1RDRPGTHVYSPSPSGSMVSSDSQLFNKPYWLHKAQGHNNGICWHNQLFITVVDTTRSTNFTLS ACTETAIPAVYSPTKFKEYTRHVEEYDLQFIFQLCTITLTADVMAYIHTMNPAILDNWNIGVTPPPSAS LVDTYRYLQSAAIACQKDAPAPEKKDPYDDLKFWNVDLKEKFSTELDQFPLGRKFLLQVGARRRPTI GPRKRPASAKSSSSASKHKRKRVSK Sequence 32 (SEQ ID NO: 32): ATGGTGTATTTGCCACCCCCTTCTGTGGCGAAGGTTGTCAATACAGATGATTATGTAACACGTAC AGGCATATATTATTATGCTGGAAGCTCTCGCTTATTAACAGTAGGGCATCCTTATTTTAAGGTACC TGTAAATGGTGGCCGCAAGCAGGAAATACCTAAGGTGTCTGCATATCAGTATAGGGTATTTAGG GTATCCCTACCTGATCCTAATAAGTTTGGCCTTCCGGATCCTTCCCTTTATAATCCTGACACACAA CGCCTGGTATGGGCCTGTATAGGTGTGGAAATTGGTAGAGGCCAGCCATTGGGCGTTGGTGTTAG TGGACATCCTTTATATAATAGATTGGATGATACTGAAAATTCACATTTTTCCTCTGCTGTTAATAC ACAGGACAGTAGGGACAATGTGTCTGTGGACTATAAGCAGACACAGTTATGTATTATAGGCTGT GTTCCTGCTATGGGAGAGCACTGGGCAAAGGGCAAGGCCTGTAAGTCCACTACTGTACAACAGG GCGATTGTCCACCATTAGAATTAGTTAATACTGCAATTGAGGATGGCGATATGATAGATACAGGC TATGGAGCCATGGACTTTCGTACATTGCAGGAAACCAAAAGTGAGGTACCACTAGATATTTGCCA ATCCGTGTGTAAATATCCTGATTATTTGCAGATGTCTGCTGATGTATATGGGGACAGTATGTTTTT TTGTTTGCGCAAGGAACAGTTATTTGCCAGACACTTTTGGAATAGAGGTGGCATGGTGGGCGACA CAATACCTTCAGAGTTATATATTAAAGGCACGGATATACGTGATCGTCCTGGTACTCATGTATAT TCCCCTTCCCCAAGTGGCTCTATGGTTTCTTCTGATTCCCAGTTGTTTAATAAGCCCTATTGGTTGC ATAAGGCCCAGGGACACAATAATGGCATTTGTTGGCATAACCAGTTGTTTATTACTGTGGTGGAC ACTACACGTAGTACTAATTTTACATTGTCTGCCTGCACCGAAACAGCCATACCTGCTGTATATAG CCCTACAAAGTTTAAGGAATATACTAGGCATGTGGAGGAATATGATTTACAATTTATATTTCAGT TGTGTACTATCACATTAACTGCAGACGTTATGGCCTACATCCATACTATGAATCCTGCAATTTTGG ACAATTGGAATATAGGCGTTACCCCTCCACCATCTGCAAGCTTGGTGGACACGTATAGGTATTTA CAATCAGCAGCTATAGCATGTCAGAAGGATGCTCCTGCACCTGAAAAAAAGGATCCCTATGACG ATTTAAAATTTTGGAATGTTGATTTAAAGGAAAAGTTTAGTACAGAACTAGATCAGTTTCCTTTG GGGCGCAAATTTTTACTACAGGTAGGGGCTCGCAGACGTCCTACTATAGGCCCTCGCAAACGCCC TGCATCAGCTAAATCGTCTTCCTCAGCCTCTAAACACAAACGGAAACGTGTGTCCAAGTAA Sequence 33 (SEQ ID NO: 33): PMSGGRKQG Sequence 34 (SEQ ID NO: 34): LSGIIPLYNRLDDTENSPFSSNKNPKDSRDNVAVDC Sequence 35 (SEQ ID NO: 35): AKGKSCKPTNVQQ Sequence 36 (SEQ ID NO: 36): TDSTVPAVYDSN Sequence 37 (SEQ ID NO: 37): PVNGGRKQE
SPECIFIC MODES FOR CARRYING OUT THE INVENTION
(13) The present invention is further described by reference to the examples as follows, wherein the examples are used only for the purpose of illustrating the present invention, rather than limiting the present invention.
(14) Unless indicated otherwise, the molecular biological experimental methods and immunological assays used in the present invention are carried out substantially in accordance with the methods as described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; and restriction enzymes are used under the conditions recommended by the manufacturers. Those skilled in the art understand that the examples are used for illustrating the present invention, but not intended to limit the protection scope of the present invention.
Example 1. Expression and Purification of the Mutated HPV39 L1 Proteins Construction of Expression Vectors
(15) An expression vector encoding the mutated HPV39 L1 protein comprising a segment from HPV68 L1 protein was constructed by PCR for multi-site mutagenesis, wherein the initial template used was the plasmid pTO-T7-HPV39N15C (encoding the HPV39 L1 protein having 15 amino acids truncated at N-terminal; abbreviated as 39L1N15 in Table 2). The templates and primers for each PCR were shown in Table 2, and the amplification conditions for PCR were as followed: denaturation at 94° C. for 10 min; 25 cycles (denaturation at 94° C. for 50 sec, annealing at a given temperature for a certain period of time, and extension at 72° C. for 7.5 min); and final extension at 72° C. for 10 min. The temperature and time of annealing were listed in Table 2. The sequences of the PCR primers used were listed in Table 3.
(16) To the amplification product (50 μL), 2 μL restriction endonuclease DpnI (Fermentas (MBI), Cat. No. FD1704, 2500U/tube) was added, and the resultant mixture was incubated at 37° C. for 60 min. 10 μL of the product of digestion was used to transform 40 μL competent E. coli ER2566 (purchased from New England Biolabs) prepared by the Calcium chloride method. The transformed E. coli was spread onto solid LB medium (the components of the LB medium: 10 g/L peptone, 5 g/L yeast powder, 10 g/L NaCl, the same hereinafter) containing kanamycin (at a final concentration of 25 μg/mL, the same hereinafter), and was subjected to static culture at 37° C. for 10-12 h until single colonies could be observed clearly. Single colony was picked and inoculated into a tube containing 4 mL liquid LB medium (containing kanamycin), and cultured with shaking at 220 rpm for 10 h at 37° C., and then 1 ml bacterial solution was taken and stored at −70° C. Plasmids were extracted from E. coli, and T7 primer was used to sequence the nucleotide sequences of the fragments of interest inserted into the plasmids. The sequencing result showed that the nucleotide sequence of the fragments of interest inserted into the constructed plasmids (expression vectors) was SEQ ID NO: 16, and their encoded amino acid sequences was SEQ ID NO: 4 (the corresponding protein was designated as H39N15-68T1). The mutated protein H39N15-68T1 differs from HPV39N15 by: the substitution of the amino acid residues from positions 53-61 of wild type HPV39 L1 protein with the amino acid residues from positions 53-61 of wild type HPV68 L1 protein.
(17) Gibson assembly (Gibson D G, Young L, Chuang R Y, Venter J C, Hutchison C A, Smith H O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009; 6:343-5. doi: 10.1038/nmeth.1318) was used to construct the expression vector encoding the other mutated HPV39 L1 protein, wherein the mutated HPV39 L1 protein comprised a specific segment from HPV68 L1 and a specific segment from HPV70L1. In brief, a short fragment comprising mutations and a long fragment comprising no mutation were obtained by PCR, and Gibson assembly system was then used to ligate the two fragments to form a ring. The initial template used comprised the plasmid pTO-T7-HPV39N15 (encoding the HPV39 L1 protein having 15 amino acids truncated at N-terminal; abbreviated as 39L1N15 in Table 2), the plasmid pTO-T7-HPV68L1 (encoding the HPV68 L1 protein; abbreviated as 68L1N0 in Table 2), the plasmid pTO-T7-H39N15-68T4 (encoding the mutated protein H39N15-68T4; abbreviated as H39N15-68T4 in Table 2), and the plasmid pTO-T7-HPV70N10 (encoding the HPV70 L1 protein having 10 amino acids truncated at N-terminal; abbreviated as 70L1N10 in Table 2). The templates and primers for each PCR were shown in Table 2, and, the amplification conditions for PCR for amplifying the short fragment were as followed: denaturation at 94° C. for 10 min; 25 cycles (denaturation at 94° C. for 50 sec, annealing at a given temperature for a certain period of time, and extension at 72° C. for 1 min); and final extension at 72° C. for 10 min. The amplification conditions for PCR for amplifying the long fragment were as followed: denaturation at 94° C. for 10 min; 25 cycles (denaturation at 94° C. for 50 sec, annealing at a given temperature for a certain period of time, and extension at 72° C. for 7.5 min); and final extension at 72° C. for 10 min. The sequences of the PCR primers used were listed in Table 3. The amplification product was subjected to electrophoresis, the fragment of interest was then recovered by using DNA Extraction Kit (BEYOTIME, Cat. No. D0033), and its concentration was determined. The short fragment and long fragment obtained by amplification were mixed at a molar ratio of 2:1 (a total volume of 3 μL), and 3 μL of 2× Gibson Assembly Master Mix (purchased from NEB, containing T5 exonuclease, Phusion DNA polymerase, Taq DNA ligase) was then added, and reacted at 50° C. for 1 h.
(18) The assembled product (6 μL) was used to transform 40 μL competent E. coli ER2566 (purchased from New England Biolabs) prepared by the Calcium chloride method. The transformed E. coli were spread onto solid LB medium containing kanamycin, and were subjected to static culture at 37° C. for 10-12 h until single colonies could be observed clearly. Single colony was picked and inoculated into a tube containing 4 mL liquid LB medium (containing kanamycin), and cultured with shaking at 220 rpm for 10h at 37° C., and then 1 ml bacterial solution was taken and stored at −70° C. Plasmids were extracted from E. coli, and T7 primer was used to sequence the nucleotide sequences of the fragments of interest inserted into the plasmids. The sequencing result showed that the nucleotide sequences of the fragments of interest inserted into the constructed plasmids (expression vectors) were SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, and 24, respectively, and their encoded amino acid sequences were SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, and 12, respectively (the corresponding proteins were designated as H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-7051, H39N15- 68T4-7052, H39N15-68T4-7053, and H39N15-68T4-7055, respectively).
(19) The mutated protein H39N15-68T2 differs from HPV39N15 by: the substitution of the amino acid residues from positions 117-150 of wild type HPV39 L1 protein with the amino acid residues from positions 117-151 of wild type HPV68 L1 protein. The mutated protein H39N15-68T3 differs from HPV39N15 by: the substitution of the amino acid residues from positions 169-181 of wild type HPV39 L1 protein with the amino acid residues from positions 170-182 of wild type HPV68 L1 protein. The mutated protein H39N15-68T4 differs from HPV39N15 by: the substitution of the amino acid residues from positions 269-288 of wild type HPV39 L1 protein with the amino acid residues from positions 270-289 of wild type HPV68 L1 protein. The mutated protein H39N15-68T5 differs from HPV39N15 by: the substitution of the amino acid residues from positions 347-358 of wild type HPV39 L1 protein with the amino acid residues from positions 348-359 of wild type HPV68 L1 protein.
(20) The mutated protein H39N15-68T4-7051 differs from HPV39N15 by: the substitution of the amino acid residues from positions 269-288 of wild type HPV39 L1 protein with the amino acid residues from positions 270-289 of wild type HPV68 L1 protein, and the substitution of the amino acid residues from positions 53-61 of wild type HPV39 L1 protein with the amino acid residues from positions 53-61 of wild type HPV70 L1 protein. The mutated protein H39N15-68T4-7052 differs from HPV39N15 by: the substitution of the amino acid residues from positions 269-288 of wild type HPV39 L1 protein with the amino acid residues from positions 270-289 of wild type HPV68 L1 protein, and the substitution of the amino acid residues from positions 117-140 of wild type HPV39 L1 protein with the amino acid residues from positions 117-141 of wild type HPV70 L1 protein. The mutated protein H39N15-68T4-7053 differs from HPV39N15 by: the substitution of the amino acid residues from positions 269-288 of wild type HPV39 L1 protein with the amino acid residues from positions 270-289 of wild type HPV68 L1 protein, and the substitution of the amino acid residues from positions 169-181 of wild type HPV39 L1 protein with the amino acid residues from positions 170-182 of wild type HPV70 L1 protein. The mutated protein H39N15-68T4-7055 differs from HPV39N15 by: the substitution of the amino acid residues from positions 269-288 of wild type HPV39 L1 protein with the amino acid residues from positions 270-289 of wild type HPV68 L1 protein, and the substitution of the amino acid residues from positions 347-358 of wild type HPV39 L1 protein with the amino acid residues from positions 348-359 of wild type HPV70 L1 protein.
(21) TABLE-US-00002 TABLE 2 PCR templates and primers for constructing expression vectors Temperature/ Time of Template Upstream primer Downstream primer Product annealing 39L1N15 H39N15-68T1-F H39N15-68T1-R H39N15-68T1 56° C./50 s 39L1N15 G-V-H39N15-68T2-F G-V-H39N15-68T2-R H39N15-68T2 56° C./50 s long fragment 39L1N15 G-V-H39N15-68T3-F G-V-H39N15-68T3-R H39N15-68T3 56° C./50 s long fragment 39L1N15 G-V-H39N15-68T4-F G-V-H39N15-68T4-R H39N15-68T4 56° C./50 s long fragment 39L1N15 G-V-H39N15-68T5-F G-V-H39N15-68T5-R H39N15-68T5 56° C./50 s long fragment 68L1N0 G-H39N15-68T2-F G-H39N15-68T2-R H39N15-68T2 56° C./30 s short fragment 68L1N0 G-H39N15-68T3-F G-H39N15-68T3-R H39N15-68T3 56° C./30 s short fragment 68L1N0 G-H39N15-68T4-F G-H39N15-68T4-R H39N15-68T4 56° C./30 s short fragment 68L1N0 G-H39N15-68T5-F G-H39N15-68T5-R H39N15-68T5 56° C./30 s short fragment H39N15- G-V-H39N15-68T4-70S1-F G-V-H39N15-68T4-70S1-R H39N15-68T4-70S1 56° C./50 s 68T4 long fragment H39N15- G-V-H39N15-68T4-70S2-F G-V-H39N15-68T4-70S2-R H39N15-68T4-70S2 56° C./50 s 68T4 long fragment H39N15- G-V-H39N15-68T4-70S3-F G-V-H39N15-68T4-70S3-R H39N15-68T4-70S3 56° C./50 s 68T4 long fragment H39N15- G-V-H39N15-68T4-70S5-F G-V-H39N15-68T4-70S5-R H39N15-68T4-70S5 56° C./50 s 68T4 long fragment 70L1N10 G-H39N15-68T4-70S1-F G-H39N15-68T4-70S1-R H39N15-68T4-70S1 56° C./30 s short fragment 70L1N10 G-H39N15-68T4-70S2-F G-H39N15-68T4-70S2-R H39N15-68T4-70S2 56° C./30 s short fragment 70L1N10 G-H39N15-68T4-70S3-F G-H39N15-68T4-70S3-R H39N15-68T4-70S3 56° C./30 s short fragment 70L1N10 G-H39N15-68T4-70S5-F G-H39N15-68T4-70S5-R H39N15-68T4-70S5 56° C./30 s short fragment
(22) TABLE-US-00003 TABLE 3 Sequences of the primers used (SEQ ID NOs: 38-71) SEQ ID NO: Primer name Primer sequence (5′-3′) 38 H39N15-68T1-F TTCAAGGTCCCCATGAGCGGCGGGCGCAAGCAGGAT ATCCCCAAGGTC 39 H39N15-68T1-R CCCGCCGCTCATGGGGACCTTGAAGTAGGGGTGGCC CACGGTCAGCAG 40 G-V-H39N15-68T2-F GACTTCGACGCCCACGCAGGCCCA 41 G-V-H39N15-68T2-R AAGCAGACCCAGCTCTGTATCATC 42 G-V-H39N15-68T3-F TTCGCCGATTGCTGGGACGCACCC 43 G-V-H39N15-68T3-R CTGGAGCTGGTGAATACACCCATC 44 G-V-H39N15-68T4-F GTGGCGGGCGAAGAGCTGCTCGCG 45 G-V-H39N15-68T4-R TGTCCAAGCCCATCCGGCAGCATG 46 G-V-H39N15-68T5-F GCTTGTGCTCAGGGTGAAATTTGTGGAGCG 47 G-V-H39N15-68T5-F TTCAAGGAGTACACACGCCACGTCGAAGAA 48 G-H39N15-68T2-F TGGGCCTGCGTGGGCGTCGAAGTCGGCAGAGGCCAG CCCCTGGGC 49 G-H39N15-68T2-R GATGATACAGAGCTGGGTCTGCTTGCAGTCCACGGC CACGTTGTC 50 G-H39N15-68T3-F GGGTGCGTCCCAGCAATCGGCGAACACTGGGCCAAG GGCAAGAGC 51 G-H39N15-68T3-R GATGGGTGTATTCACCAGCTCCAGAGGGGGGCAGTC GCCCTGCTG 52 G-H39N15-68T4-F CGCGAGCAGCTCTTCGCCCGCCACTTCTGGAACAGA GGCGGCATG 53 G-H39N15-68T4-R CATGCTGCCGGATGGGCTTGGACAGTACACGTAGCT GCTGGGTGT 54 G-H39N15-68T5-F CGCTCCACAAATTTCACCCTGAGCACAAGCACCGAC AGCACCGTGCCCGCC 55 G-H39N15-68T5-R TTCTTCGACGTGGCGTGTGTACTCCTTGAACTTATTG CTGTCGTACACGGC 56 G-V-H39N15-68T4-70S1-F GTAGGGGTGGCCCACGGTCAG 57 G-V-H39N15-68T4-70S1-R GCCTACCAGTACCGCGTGTTC 58 G-V-H39N15-68T4-70S2-F GACTTCGACGCCCACGCAGGC 59 G-V-H39N15-68T4-70S2-R AAGCAGACCCAGCTCTGTATC 60 G-V-H39N15-68T4-70S3-F TTCGCCGATTGCTGGGACGCA 61 G-V-H39N15-68T4-70S3-R CTGGAGCTGGTGAATACACCC 62 G-V-H39N15-68T4-70S5-F GCTTGTGCTCAGGGTGAAATT 63 G-V-H39N15-68T4-70S5-R TTCAAGGAGTACACACGCCAC 64 G-H39N15-68T4-70S1-F CTGACCGTGGGCCACCCCTACTTTAAGGTACCTGTAA ATGGT 65 G-H39N15-68T4-70S1-R GAACACGCGGTACTGGTAGGCAGACACCTTAGGTAT TTCCTG 66 G-H39N15-68T4-70S2-F GCCTGCGTGGGCGTCGAAGTCGGTAGAGGCCAGCCA TTGGGC 67 G-H39N15-68T4-70S2-R GATACAGAGCTGGGTCTGCTTATAGTCCACAGACAC ATTGTC 68 G-H39N15-68T4-70S3-F TGCGTCCCAGCAATCGGCGAACACTGGGCAAAGGGC AAGGCC 69 G-H39N15-68T4-70S3-R GGGTGTATTCACCAGCTCCAGTGGTGGACAATCGCC CTGTTGTAC 70 G-H39N15-68T4-70S5-F AATTTCACCCTGAGCACAAGCACCGAAACAGCCATA CCTGCT 71 G-H39N15-68T4-70S5-R GTGGCGTGTGTACTCCTTGAACTTTGTAGGGCTATAT ACAGC
(23) Expression of the Mutated Proteins on a Large Scale
(24) The E. coli solutions comprising the recombinant plasmid pTO-T7-H39N15-68T1, pTO-T7-H39N15-68T2, pTO-T7-H39N15-68T3, pTO-T7-H39N15-68T4, pTO-T7-H39N15-68T5, pTO-T7-H39N15-68T4-70S1, pTO-T7-H39N15-68T4-70S2, pTO-T7-H39N15-68T4-70S3, and pTO-T7-H39N15-68T4-70S5, respectively, were taken from −70° C. refrigerator, were inoculated in 100 mL LB liquid medium containing kanamycin, and incubated at 200 rpm and 37° C. for about 8 h. Then, the culture was transferred to 500 mL LB medium containing kanamycin (1 ml bacterial solution was transferred), and was further incubated. When the bacterial concentration reached an OD600 of about 0.6, the culturing temperature was lowered to 25° C. and 500 μL IPTG was added to each culture bottle. The incubation was further performed for 8 h. After the incubation was finished, the bacteria were collected by centrifugation. The bacteria expressing H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3 and H39N15-68T4-70S5 protein were obtained, respectively.
(25) Disruption of Bacteria Expressing the Mutated Proteins
(26) The bacteria obtained were re-suspended at a ratio of 1 g bacteria to 10 mL lysis buffer (20 mM Tris buffer, pH7.2, 300 mM NaCl). The bacteria were disrupted by using an ultrasonic apparatus for 30 min. The lysis solution containing the disrupted bacteria were centrifuged at 13500 rpm (30000 g) for 15 min, and the supernatant (i.e. the supernatant of disrupted bacteria) was obtained.
(27) Chromatographic Purification of the Mutated Protein
(28) Equipment: AKTA Explorer 100 preparative liquid chromatography system produced by GE Healthcare (i.e. the original Amershan Pharmacia Co.)
(29) Chromatographic media: SP Sepharose 4 Fast Flow (GE Healthcare Co.), CHT-II (purchased from Bio-RAD) and Butyl Sepharose 4 Fast Flow (GE Healthcare Co.)
(30) Buffer: Buffer A (20 mM phosphate buffer, pH8.0, 20 mM DTT); and Buffer B (20 mM phosphate buffer, pH8.0, 20 mM DTT, 2 M NaCl). The buffers containing different concentrations of NaCl used in the following elution protocol were prepared by mixing Buffer A and Buffer B at a certain ratio.
(31) Sample: the supernatants of disrupted bacteria containing H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5, respectively, as obtained above.
(32) Elution Protocol:
(33) (1) Cation exchange purification of the supernatant of disrupted bacteria by SP Sepharose 4 Fast Flow: the sample was loaded on the column, undesired proteins were then eluted with a buffer containing 400 mM NaCl (80% Buffer A+20% Buffer B), followed by the elution of the protein of interest with a buffer containing 800 mM NaCl (60% Buffer A+40% Buffer B), and the fraction eluted with the buffer containing 800 mM NaCl was collected;
(34) (2) Chromatographic purification of the elution fraction obtained in the step (1) by CHTII (hydroxyapatite chromatography): the elution fraction obtained in the step (1) was diluted so that the NaCl concentration was decreased to 0.5 M; the sample was loaded on the column, undesired proteins were then eluted with a buffer containing 500 mM NaCl (75% Buffer A+25% Buffer B), followed by the elution of the protein of interest with a buffer containing 1000 mM NaCl (50% Buffer A+50% Buffer B), and the fraction eluted with the buffer containing 1000 mM NaCl was collected;
(35) (3) Chromatographic purification of the elution fraction obtained in the step (2) by HIC (hydrophobic interaction chromatography): the sample was loaded on the column, undesired proteins were then eluted with a buffer containing 1000 mM NaCl, followed by the elution of the protein of interest with a buffer containing 200 mM NaCl (90% Buffer A+10% Buffer B), and the fraction eluted with the buffer containing 200 mM NaCl was collected.
(36) 150 μL of elution fraction obtained in the step (3) was added to 30 μL of 6× Loading Buffer (1 L of which contained 300 ml of 1 M TB 6.8, 600 ml of 100% glycerol, 120 g of SDS, 6 g of bromophenol blue, and 50 ml of β-mercaptoethanol). The resultant solution was mixed well and incubated in 80° C. water bath for 10 min. 10 μl of the resultant sample was then subjected to 10% SDS-PAGE at 120V for 120 min; and the electrophoretic bands were stained by Coomassie brilliant blue. The electrophoretic result was shown in
(37) By similar methods, HPV39N15 protein was prepared and purified by using E. coli and the plasmid pTO-T7-HPV39N15; HPV68N0 protein was prepared and purified by using E. coli and the plasmid pTO-T7-HPV68L1N0; and HPV70N10 protein was prepared and purified by using E. coli and the plasmid pTO-T7-HPV70N10.
(38) Western Blot Assay of the Mutated Proteins
(39) The H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4- 70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4-70S5 protein purified by the method above were subjected to electrophoresis. After electrophoresis, Western Blot assay was carried out by using a broad-spectrum antibody 4B3 against HPV L1 protein, and the result was shown in
Example 2: Assembly of HPV Virus-Like Particles and Morphological Detection of Particles Assembly of HPV Virus-Like Particles
(40) A given volume (about 2 ml) of the protein H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3 or H39N15-68T4-70S5 was dialyzed to (1) 2 L storage buffer (20 mM sodium phosphate buffer pH 6.5, 0.5 M NaCl); (2) 2 L renaturation buffer (50 mM sodium phosphate buffer pH 6.0, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2, 0.5 M NaCl); and (3) 20 mM sodium phosphate buffer pH 7.0, 0.5 M NaCl, successively. The dialysis was performed in each of the three buffers for 12 h.
(41) By similar methods, the HPV39N15, HPV68N0 and HPV70N10 protein were assembled into HPV39N15 VLP, HPV68N0 VLP and HPV70N10 VLP, respectively.
(42) Molecular Sieve Chromatographic Analysis
(43) The dialyzed sample was subjected to molecular sieve chromatographic analysis by 1120 Compact LC High Performance Liquid Chromatographic System (Agilent Technologies), wherein the analytical column used was TSK Gel PW5000×17.8×300 mm. The analysis results were shown in
(44) Sedimentation Velocity Analysis
(45) The apparatus for sedimentation velocity analysis was Beckman XL-A Analytical Ultracentrifuge, equipped with optical inspection system and An-50Ti and An-60Ti rotor. The sedimentation coefficients of HPV39N15 VLP, HPV68N0 VLP, HPV70N10 VLP, H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, H39N15-68T5 VLP, H39N15-68T4-7051 VLP, H39N15-68T4-7052 VLP, H39N15-68T4-7053 VLP and H39N15-68T4-7055 VLP were analyzed by sedimentation velocity method. The results were shown in
(46) Morphological Test of Virus-Like Particles
(47) A 100 μL sample comprising VLP was observed by transmission electron microscope (TEM). The apparatus used was a 100 kV Transmission Electron Microscope supplied by JEOL Ltd. (100,000× magnification). In brief, a 13.5 μL of sample was negatively stained with 2% phosphotungstic acid (pH 7.0), fixed on a carbon-coated copper grid, and then observed by TEM. The results were shown in
Example 3: Evaluation 1 of Neutralizing Antibody Titer in Serum of Mice Vaccinated with Virus-Like Particles
(48) In this experiment, virus-like particles used were H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP and H39N15-68T5 VLP.
(49) In this experiment, vaccination schedule was shown in Table 4. All the mice (6-week old BalB/c female mice) were divided into 3 groups: Aluminum adjuvant group 1 (at an immunizing dose of 5 μg, using aluminum adjuvant), Aluminum adjuvant group 2 (at an immunizing dose of 1 μg, using aluminum adjuvant), and Aluminum adjuvant group 3 (at an immunizing dose of 0.2 μg, using aluminum adjuvant). Each group was further divided into 8 subgroups. The Control subgroups 1 and 2 were vaccinated with HPV39N15 VLP alone and HPV68N0 VLP alone, respectively, the Control subgroup 3 was vaccinated with the mixed HPV39/HPV68 VLP (i.e. a mixture of HPV39N15 VLP and HPV68N0 VLP, at a given immunizing dose for each VLP). The Experimental subgroups 1, 2, 3, 4 and 5 were vaccinated with H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP and H39N15-68T5 VLP, respectively.
(50) In Aluminum adjuvant groups 1-3, 5 mice/subgroup were vaccinated by intraperitoneal injection, at an immunizing dose 5 μg, 1 μg, and 0.2 μg, respectively, and an injection volume of 1 mL. All the mice were subjected to the first vaccination at Week 0, and then subjected to the booster vaccination at Weeks 2 and 4, respectively. At Week 8, blood sample was collected via orbital bleeding, and the titers of antibodies against HPV39 and HPV68 in serum were analyzed. The analysis results were shown in
(51) TABLE-US-00004 TABLE 4 Vaccination schedule Vaccination Immunizing procedure Group Antigen for vaccination Adjuvant dose Number (week) Aluminum HPV39N15 VLP aluminum 5 μg 5 0, 2, 4 adjuvant adjuvant group 1 HPV68N0 VLP aluminum 5 μg 5 0, 2, 4 adjuvant The mixed aluminum 5 μg for 5 0, 2, 4 HPV39/HPV68 VLP adjuvant each VLP H39N15-68T1 VLP aluminum 5 μg 5 0, 2, 4 adjuvant H39N15-68T2 VLP aluminum 5 μg 5 0, 2, 4 adjuvant H39N15-68T3 VLP aluminum 5 μg 5 0, 2, 4 adjuvant H39N15-68T4 VLP aluminum 5 μg 5 0, 2, 4 adjuvant H39N15-68T5 VLP aluminum 5 μg 5 0, 2, 4 adjuvant Aluminum HPV39N15 VLP aluminum 1 μg 5 0, 2, 4 adjuvant adjuvant group 2 HPV68N0 VLP aluminum 1 μg 5 0, 2, 4 adjuvant The mixed aluminum 1 μg for 5 0, 2, 4 HPV39/HPV68 VLP adjuvant each VLP H39N15-68T1 VLP aluminum 1 μg 5 0, 2, 4 adjuvant H39N15-68T2 VLP aluminum 1 μg 5 0, 2, 4 adjuvant H39N15-68T3 VLP aluminum 1 μg 5 0, 2, 4 adjuvant H39N15-68T4 VLP aluminum 1 μg 5 0, 2, 4 adjuvant H39N15-68T5 VLP aluminum 1 μg 5 0, 2, 4 adjuvant Aluminum HPV39N15 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant adjuvant group 3 HPV68N0 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant The mixed aluminum 0.2 μg for 5 0, 2, 4 HPV39/HPV68 VLP adjuvant each VLP H39N15-68T1 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant H39N15-68T2 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant H39N15-68T3 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant H39N15-68T4 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant aluminum H39N15-68T5 VLP adjuvant 0.2 μg 5 0, 2, 4
Example 4: Evaluation 1 of ED.SUB.50 .of Virus-Like Particles for Inducing Seroconversion
(52) In this experiment, the virus-like particle used was H39N15-68T4 VLP. 6-Week old BalB/c female mice (8 mice) were vaccinated with aluminum adjuvant by single intraperitoneal injection, wherein H39N15-68T4 VLP (at an immunizing dose of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg or 0.011 μg) was used in the Experimental groups, and HPV68N0 VLP alone (at an immunizing dose of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg or 0.011 μg), HPV39N15 VLP alone (at an immunizing dose of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg or 0.011 μg) or the mixed HPV39/HPV68 VLP (i.e. a mixture of HPV39N15 VLP and HPV68N0 VLP, at an immunizing dose of 0.900 μg, 0.300 μg, 0.100 μg, 0.033 μg or 0.011 μg for each VLP); the immunizing volume was 1 mL. In addition, the diluent used to dilute the vaccine was used as a blank control. 8 Mice were vaccinated in each group, and at Week 5 after vaccination, venous blood was collected from eyeball. Antibodies against HPV in the serum were detected, and by Reed-Muench method (Reed L J M H. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938; 27:493-7), ED.sub.50 for inducing seroconversion (i.e. inducing the generation of antibodies in mice) was calculated for each sample. The results were shown in Tables 5-8.
(53) TABLE-US-00005 TABLE 5 ED.sub.50 of HPV39N15 VLP for inducing the generation of antibodies against HPV39 and HPV68 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 8 100.00% 0.019 0.300 8 8 100.00% 0.100 8 6 88.89% 0.033 8 8 83.33% 0.011 8 2 20.00% HPV68 0.900 8 0 0.00% >0.9 0.300 8 0 0.00% 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 8 0.00%
(54) TABLE-US-00006 TABLE 6 ED.sub.50 of HPV68N0 VLP for inducing the generation of antibodies against HPV39 and HPV68 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 0 0.00% >0.9 0.300 8 0 0.00% 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% HPV68 0.900 8 8 100.00% 0.021 0.300 8 8 100.00% 0.100 8 7 93.75% 0.033 8 7 80.00% 0.011 8 1 10.00%
(55) TABLE-US-00007 TABLE 7 ED.sub.50 of H39N15-68T4 VLP for inducing the generation of antibodies against HPV39 and HPV68 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 6 90.48% 0.091 0.300 8 6 76.47% 0.100 8 6 53.85% 0.033 8 1 7.14% 0.11 8 0 0.00% HPV68 0.900 8 4 75.00% 0.300 0.300 8 4 50.00% 0.100 8 2 22.22% 0.033 8 2 9.09% 0.011 8 0 0.00%
(56) TABLE-US-00008 TABLE 8 ED.sub.50 of the mixed HPV39/HPV68 VLP for inducing the generation of antibodies against HPV39 and HPV68 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 7 97.06% 0.017 0.300 8 8 96.30% 0.100 8 8 94.74% 0.033 8 8 90.91% 0.011 8 2 22.22% HPV68 0.900 8 7 96.88% 0.021 0.300 8 8 96.00% 0.100 8 8 94.12% 0.033 8 7 80.00% 0.011 8 1 10.00%
(57) The results showed that 5 weeks after vaccination of mice, ED.sub.50 of H39N15-68T4 VLP for inducing the generation of antibodies against HPV39 in mice was comparable to that of HPV39N15 VLP alone, and was significantly superior to that of HPV68N0 VLP alone; and its ED.sub.50 for inducing the generation of antibodies against HPV68 was slightly weaker than that of HPV68N0 VLP alone, but was significantly superior to that of HPV39N15 VLP alone. This showed that H39N15-68T4 VLP had good cross-immunogenicity and cross-protection against HPV68 and HPV39.
Example 5: Evaluation 2 of Neutralizing Antibody Titer in Serum of Mice Vaccinated with Virus-Like Particles
(58) In this experiment, virus-like particles used were H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP and H39N15-68T4-70S5 VLP.
(59) In this experiment, vaccination schedule was shown in Table 9. All the mice (6-week old BalB/c female mice) were divided into 3 groups: Aluminum adjuvant group 1 (at an immunizing dose of 5 μg, using aluminum adjuvant), Aluminum adjuvant group 2 (at an immunizing dose of 1 μg, using aluminum adjuvant), and Aluminum adjuvant group 3 (at an immunizing dose of 0.2 μg, using aluminum adjuvant). Each group was further divided into 8 subgroups. The Control subgroups 1, 2 and 3 were vaccinated with HPV39N15 VLP alone, HPV68N0 VLP alone and HPV70N10 VLP alone, respectively, the Control subgroup 4 was vaccinated with the mixed HPV39/HPV68/HPV70 VLP (i.e. a mixture of HPV39N15 VLP, HPV68N0 VLP and HPV70N10 VLP, at a given immunizing dose for each VLP). The Experimental subgroups 1, 2, 3 and 4 were vaccinated with H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP and H39N15-68T4-70S5 VLP, respectively.
(60) In Aluminum adjuvant groups 1-3, 5 mice/subgroup were vaccinated by intraperitoneal injection, at an immunizing dose 5 μg, 1 μg, and 0.2 μg, respectively, and an injection volume of 1 mL. All the mice were subjected to the first vaccination at Week 0, and then subjected to the booster vaccination at Weeks 2 and 4, respectively. At Week 8, blood sample was collected via orbital bleeding, and the titers of antibodies against HPV39, and HPV68 and HPV70 in serum were analyzed. The analysis results were shown in
(61) TABLE-US-00009 TABLE 9 Vaccination schedule Vaccination Immunizing procedure Group Antigen for vaccination Adjuvant dose Number (week) Aluminum HPV39N15 VLP aluminum 5 μg 5 0, 2, 4 adjuvant adjuvant group 1 HPV68N0 VLP aluminum 5 μg 5 0, 2, 4 adjuvant HPV70N10 VLP aluminum 5 μg 5 0, 2, 4 adjuvant The mixed aluminum 5 μg for 5 0, 2, 4 HPV39/HPV68/HPV70 VLP adjuvant each VLP H39N15-68T4-70S1 VLP aluminum 5 μg 5 0, 2, 4 adjuvant H39N15-68T4-70S2 VLP aluminum 5 μg 5 0, 2, 4 adjuvant H39N15-68T4-70S3 VLP aluminum 5 μg 5 0, 2, 4 adjuvant H39N15-68T4-70S5 VLP aluminum 5 μg 5 0, 2, 4 adjuvant HPV39N15 VLP aluminum 1 μg 5 0, 2, 4 adjuvant HPV68N0 VLP aluminum 1 μg 5 0, 2, 4 adjuvant HPV70N10 VLP aluminum 1 μg 5 0, 2, 4 adjuvant Aluminum The mixed aluminum 1 μg for 5 0, 2, 4 adjuvant HPV39/HPV68/HPV70 VLP adjuvant each VLP group 2 H39N15-68T4-70S1 VLP aluminum 1 μg 5 0, 2, 4 adjuvant H39N15-68T4-70S2 VLP aluminum 1 μg 5 0, 2, 4 adjuvant H39N15-68T4-70S3 VLP aluminum 1 μg 5 0, 2, 4 adjuvant H39N15-68T4-70S5 VLP aluminum 1 μg 5 0, 2, 4 adjuvant HPV39N15 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant HPV68N0 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant HPV70N10 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant Aluminum The mixed aluminum 0.2 μg for 5 0, 2, 4 adjuvant HPV39/HPV68/HPV70 VLP adjuvant each VLP group 3 H39N15-68T4-70S1 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant H39N15-68T4-70S2 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant H39N15-68T4-70S3 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant H39N15-68T4-70S5 VLP aluminum 0.2 μg 5 0, 2, 4 adjuvant
Example 6: Evaluation 2 of ED.SUB.50 .of Virus-Like Particles for Inducing Seroconversion
(62) In this experiment, virus-like particles used were H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP and H39N15-68T4-70S5 VLP.
(63) 6-Week old BalB/c female mice (8 mice) were vaccinated with aluminum adjuvant by single intraperitoneal injection, wherein H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP and H39N15-68T4-70S5 VLP (at an immunizing dose of 0.900m, 0.300m, 0.100m, 0.033m or 0.011m) were used in the Experimental groups, and HPV39N15 VLP alone, HPV68N0 VLP alone, HPV70N10 VLP alone (at an immunizing dose of 0.900m, 0.300m, 0.100m, 0.033m or 0.011 μg) or the mixed HPV39/HPV68/HPV70 VLP (i.e. a mixture of HPV39N15 VLP, HPV68N0 VLP and HPV70N10 VLP, at an immunizing dose of 0.900m, 0.300m, 0.100m, 0.033m or 0.011m for each VLP); the immunizing volume was 1 mL. In addition, the diluent used to dilute the vaccine was used as a blank control. 8 Mice were vaccinated in each group, and at Week 5 after vaccination, venous blood was collected from eyeball. Antibodies against HPV in the serum were detected, and by Reed-Muench method (Reed L J M H. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938; 27:493-7), ED.sub.50 for inducing seroconversion (i.e. inducing the generation of antibodies in mice) was calculated for each sample. The results were shown in Tables 10-16.
(64) TABLE-US-00010 TABLE 10 ED.sub.50 of HPV39N15 VLP for inducing the generation of antibodies against HPV39, HPV68 and HPV70 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 8 100.00% 0.019 0.300 8 8 100.00% 0.100 8 8 100.00% 0.033 8 7 88.89% 0.011 8 1 11.11% HPV68 0.900 8 0 11.11% >0.9 0.300 8 0 5.88% 0.100 8 0 4.00% 0.033 8 1 3.13% 0.011 8 0 0.00% HPV70 0.900 8 0 11.11% >0.9 0.300 8 1 6.25% 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00%
(65) TABLE-US-00011 TABLE 11 ED.sub.50 of HPV68N0 VLP for inducing the generation of antibodies against HPV39, HPV68 and HPV70 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 0 20.00% >0.9 0.300 8 0 11.11% 0.100 8 1 8.00% 0.033 8 1 3.23% 0.011 8 0 0.00% HPV68 0.900 8 8 100.00% 0.021 0.300 8 8 100.00% 0.100 8 8 100.00% 0.033 8 6 77.78% 0.011 8 1 10.00% HPV70 0.900 8 2 50.00% 0.9 0.300 8 2 25.00% 0.100 8 0 9.09% 0.033 8 2 7.14% 0.011 8 0 0.00%
(66) TABLE-US-00012 TABLE 12 ED.sub.50 of HP70N10 VLP for inducing the generation of antibodies against HPV39, HPV68 and HPV70 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 2 40.00% >0.9 0.300 8 0 12.50% 0.100 8 1 8.70% 0.033 8 1 3.45% 0.011 8 0 0.00% HPV68 0.900 8 0 0.00% >0.9 0.300 8 0 0.00% 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% HPV70 0.900 8 8 100.00% 0.017 0.300 8 8 100.00% 0.100 8 8 100.00% 0.033 8 7 90.00% 0.011 8 2 22.22%
(67) TABLE-US-00013 TABLE 13 ED.sub.50 of the mixed HPV39/HPV68/HPV70 VLP for inducing the generation of antibodies against HPV39, HPV68 and HPV70 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 μg for 8 8 100.00% 0.021 each VLP 0.300 μg for 8 8 100.00% each VLP 0.100 μg for 8 7 93.75% each VLP 0.033 μg for 8 7 80.00% each VLP 0.011 μg for 8 1 10.00% each VLP HPV68 0.900 μg for 8 8 100.00% 0.019 each VLP 0.300 μg for 8 8 100.00% each VLP 0.100 μg for 8 7 94.12% each VLP 0.033 μg for 8 7 81.82% each VLP 0.011 μg for 8 2 20.00% each VLP HPV70 0.900 μg for 8 8 100.00% 0.021 each VLP 0.300 μg for 8 8 100.00% each VLP 0.100 μg for 8 7 93.75% each VLP 0.033 μg for 8 7 80.00% each VLP 0.011 μg for 8 1 10.00% each VLP
(68) TABLE-US-00014 TABLE 14 ED.sub.50 of H39N15-68T4-70S2 VLP for inducing the generation of antibodies against HPV39, HPV68 and HPV70 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 8 100.00% 0.017 0.300 8 7 96.15% 0.100 8 8 94.74% 0.033 8 8 90.91% 0.011 8 2 22.22% HPV68 0.900 8 6 100.00% 0.028 0.300 8 8 91.67% 0.100 8 7 82.35% 0.033 8 6 58.33% 0.011 8 1 7.69% HPV70 0.900 8 7 96.30% 0.033 0.300 8 7 90.48% 0.100 8 5 70.59% 0.033 8 6 50.00% 0.011 8 1 6.67%
(69) TABLE-US-00015 TABLE 15 ED.sub.50 of H39N15-68T4-70S3 VLP for inducing the generation of antibodies against HPV39, HPV68 and HPV70 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 3 50.00% 0.9 0.300 8 1 14.29% 0.100 8 0 4.76% 0.033 8 1 3.57% 0.011 8 0 0.00% HPV68 0.900 8 7 96.88% 0.020 0.300 8 7 92.31% 0.100 8 7 85.00% 0.033 8 8 76.92% 0.011 8 2 18.18% HPV70 0.900 8 4 63.64% 0.611 0.300 8 3 25.00% 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00%
(70) TABLE-US-00016 TABLE 16 ED.sub.50 of H39N15-68T4-70S5 VLP for inducing the generation of antibodies against HPV39, HPV68 and HPV70 (seroconversion) in mice Number of Total mice with Positive Immunizing number positive conversion ED.sub.50 Type dose (μg) of mice conversion rate (μg) HPV39 0.900 8 7 96.97% 0.019 0.300 8 7 92.59% 0.100 8 8 90.00% 0.033 8 8 83.33% 0.011 8 2 20.00% HPV68 0.900 8 8 100.00% 0.148 0.300 8 8 100.00% 0.100 8 1 22.22% 0.033 8 1 6.67% 0.011 8 0 0.00% HPV70 0.900 8 7 96.30% 0.042 0.300 8 6 86.36% 0.100 8 8 81.25% 0.033 8 4 41.67% 0.011 8 1 6.67%
(71) The results showed that 5 weeks after vaccination of mice, ED.sub.50 of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP for inducing the generation of antibodies against HPV39 in mice was comparable to that of HPV39N15 VLP alone and that of the mixed of HPV39/HPV68/HPV70 VLP, and was significantly superior to that of HPV68N0 VLP alone and that of HPV70N10 VLP alone; and their ED.sub.50 for inducing the generation of antibodies against HPV68 in mice was comparable to that of HPV68N0 VLP alone and that of the mixed of HPV39/HPV68/HPV70 VLP, and was significantly superior to that of HPV39N15 VLP alone and that of HPV70N10 VLP alone; and their ED.sub.50 for inducing the generation of antibodies against HPV70 in mice was comparable to that of HPV70N10 VLP alone and that of the mixed of HPV39/HPV68/HPV70 VLP, and was significantly superior to that of HPV39N15 VLP alone and that of HPV68N0 VLP alone. This showed that H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP had good cross-immunogenicity and cross-protection against HPV39, HPV68 and HPV70.
Example 7: Evaluation of Thermostability of Virus-Like Particles
(72) The VLPs formed by HPV39N15 protein, HPV68N0 protein, HPV70N10 protein, H39N15-68T4 protein, H39N15-68T4-70S2 protein and H39N15-68T4-70S5 protein were evaluated for their thermostability by using a differential scanning calorimeter VP Capillary DSC purchased from GE Company (i.e. the original MicroCal Co.), wherein the storage buffer for the protein was used as control, and the proteins were scanned at a heating rate of 1.5° C./min within a temperature range of 10° C.-90° C. The detection results were shown in
Example 8: Reconstruction of Three-Dimensional Structures of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP
(73) The three-dimensional structures of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP were reconstructed by three-dimensional structure reconstruction experiment using cryo-electron microscopy (cryo-EM) (Wolf M, Garcea R L, Grigorieff N. et al. Proc Natl Acad Sci USA. (2010), 107(14): 6298-303). In brief, in the cryo-electron microscopy (cryo-EM) photograph of H39N15-68T4-70S2 VLP (A of
(74) Although the specific embodiments of the present invention have been described in details, those skilled in the art would understand that, according to the teachings disclosed in the specification, various modifications and changes can be made thereto, and that such modifications and changes are within the scope of the present invention. The scope of the present invention is given by the appended claims and any equivalents thereof.