Mutant of L1 protein of human papillomavirus type 16
11213580 · 2022-01-04
Assignee
Inventors
- Ying Gu (Xiamen, CN)
- Shaowei Li (Xiamen, CN)
- Shuo Song (Xiamen, CN)
- Maozhou He (Xiamen, CN)
- Zhihai Li (Xiamen, CN)
- Ningshao Xia (Xiamen, CN)
Cpc classification
C12N2710/20022
CHEMISTRY; METALLURGY
C12N2710/20034
CHEMISTRY; METALLURGY
A61P17/12
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
C12N7/00
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
Abstract
Provided are a mutated HPV16 L1 protein (or a variant thereof), a sequence encoding the same and a method for preparing the same, as well as a virus-like particle comprising the same. The protein (or variant thereof) and the virus-like particle are capable of inducing neutralizing antibodies against at least two types of HPV (e.g., HPV16 and HPV35, or HPV16, HPV35, and HPV31), 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. Also provided are a use of the above protein and virus-like particle in the manufacture of a pharmaceutical composition or 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 HPV16 L1 protein or a variant thereof, wherein the mutated HPV16 L1 protein has the following mutations compared to a wild-type HPV16 L1 protein: (1) a N-terminal truncation of 4-50 amino acids; and (2) a substitution of amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein with amino acid residues at the corresponding positions of a L1 protein of a second type of wild-type HPV; optionally, the mutated HPV16 L1 protein further has the following mutation: (3) a substitution of amino acid residues at positions 76-87 of the wild-type HPV16 L1 protein with amino acid residues at the corresponding positions of a L1 protein of a third type of wild-type HPV; (4) a substitution of amino acid residues at positions 152-167 of the wild-type HPV16 L1 protein with amino acid residues at the corresponding positions of a L1 protein of a third type of wild-type HPV; or, (5) a substitution of amino acid residues at positions 202-207 of the wild-type HPV16 L1 protein with amino acid residues at the corresponding positions of a L1 protein of a third type of wild-type HPV; and, the variant differs from the mutated HPV16 L1 protein only by substitution, addition or deletion of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, and maintains the function of the mutated HPV16 L1 protein, including an ability of inducing neutralizing antibodies against at least two types of HPV.
2. An isolated nucleic acid, which encodes the mutated HPV16 L1 protein or variant thereof according to claim 1.
3. A vector comprising the isolated nucleic acid according to claim 2.
4. A host cell comprising the isolated nucleic acid according to claim 2 and/or a vector comprising the isolated nucleic acid.
5. A HPV virus-like particle, which comprises or consists of the mutated HPV16 L1 protein or variant thereof according to claim 1.
6. A composition, which comprises: (i) the mutated HPV16 L1 protein or a variant thereof according to claim 1, or (ii) an isolated nucleic acid encoding the mutated HPV16 L1 protein or variant thereof as described in (i), or (iii) a vector comprising the isolated nucleic acid as described in (ii), or (iv) a host cell comprising the isolated nucleic acid as described in (ii) or the vector as described in (iii), or (v) a HPV virus-like particle comprising or consisting of the mutated HPV16 L1 protein or variant thereof as described in (i).
7. A pharmaceutical composition or vaccine, which comprises the HPV virus-like particle according to claim 5, and optionally further comprises a pharmaceutically acceptable carrier and/or excipient.
8. A method for preparing the mutated HPV16 L1 protein or variant thereof according to claim 1, which comprises expressing the mutated HPV16 L1 protein or variant thereof in a host cell, and then recovering the mutated HPV16 L1 protein or a variant thereof from a culture of the host cell.
9. A method for preparing a vaccine, which comprises 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, which comprises 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 and optionally a pharmaceutically acceptable carrier and/or excipient.
11. The mutated HPV16 L1 protein or variant thereof according to claim 1, wherein the mutated HPV16 L1 protein is characterized by one or more of the following items: (i) the mutated HPV16 L1 protein has a N-terminal truncation of 4, 6, 8, 10, 20, 30 or 40 amino acids compared with a wild-type HPV16 L1 protein; (ii) the second type of wild-type HPV is HPV35; (iii) the third type of wild-type HPV is HPV31; (iv) the amino acid residues at the corresponding positions described in (2) are the amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein; (v) the amino acid residues at the corresponding positions described in (3) are the amino acid residues at positions 50-62 of the wild-type HPV31 L1 protein; (vi) the amino acid residues at the corresponding positions described in (4) are the amino acid residues at positions 127-142 of the wild-type HPV31 L1 protein; (vii) the amino acid residues at the corresponding positions described in (5) are the amino acid residues at positions 177-182 of the wild-type HPV31 L1 protein; (viii) the wild-type HPV16 L1 protein has an amino acid sequence set forth in SEQ ID NO: 1; (ix) the wild-type HPV35 L1 protein has an amino acid sequence set forth in SEQ ID NO: 2; and (x) the wild-type HPV31 L1 protein has an amino acid sequence set forth in SEQ ID NO: 3.
12. The mutated HPV16 L1 protein or variant thereof according to claim 1, wherein the mutated HPV16 L1 protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, 10, and 11.
13. 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.
14. The pharmaceutical composition or vaccine according to claim 13, 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 a cervical cancer and a condyloma acuminatum.
15. The pharmaceutical composition or vaccine according to claim 13, wherein the HPV infection is one or more selected from the group consisting of the following: HPV 16 infection, HPV 35 infection, and HPV 31 infection.
16. The method according to claim 8, wherein the host cell is E. coli.
17. The method according to claim 8, wherein the method comprises: expressing the mutated HPV16 L1 protein or a variant thereof in E. coli, and then obtaining the mutated HPV16 L1 protein or a variant thereof by purifying a lysate supernatant of the E. coli.
18. 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.
19. The method according to claim 10, wherein the HPV infection is one or more selected from the group consisting of the following: HPV 16 infection, HPV 35 infection, and HPV 31 infection.
Description
DESCRIPTION OF THE DRAWINGS
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SEQUENCE INFORMATION
(11) Some of the sequences involved in the present invention are provided in Table 1 below.
(12) TABLE-US-00001 TABLE 1 Description of sequences SEQ ID NO: Description 1 wild-type HPV16 L1 protein 2 wild-type HPV35 L1 protein 3 wild-type HPV31 L1 protein 4 The mutated HPV16 L1 protein containing Segment 1 of HPV35 L1 protein, H16N30-35T1 5 The mutated HPV16 L1 protein containing Segment 2 of HPV35 L1 protein, H16N30-35T2 6 The mutated HPV16 L1 protein containing Segment 3 of HPV35 L1 protein, H16N30-35T3 7 The mutated HPV16 L1 protein containing Segment 4 of HPV35 L1 protein, H16N30-35T4 8 The mutated HPV16 L1 protein containing Segment 5 of HPV35 L1 protein, H16N30-35T5 9 The mutated HPV16 L1 protein containing Segment 4 of HPV35 L1 protein and Segment 1 of HPV31 L1 protein, H16N30-35T4-31S1 10 The mutated HPV16 L1 protein containing Segment 4 of HPV35 L1 protein and Segment 2 of HPV31 L1 protein, H16N30-35T4-31S2 11 The mutated HPV16 L1 protein containing Segment 4 of HPV35 L1 protein and Segment 3 of HPV31 L1 protein, H16N30-35T4-31S3 12 The mutated HPV16 L1 protein containing Segment 4 of HPV35 L1 protein and Segment 5 of HPV31 L1 protein, H16N30-35T4-31S5 13 DNA sequence encoding SEQ ID NO: 1 14 DNA sequence encoding SEQ ID NO: 2 15 DNA sequence encoding SEQ ID NO: 3 16 DNA sequence encoding SEQ ID NO: 4 17 DNA sequence encoding SEQ ID NO: 5 18 DNA sequence encoding SEQ ID NO: 6 19 DNA sequence encoding SEQ ID NO: 7 20 DNA sequence encoding SEQ ID NO: 8 21 DNA sequence encoding SEQ ID NO: 9 22 DNA sequence encoding SEQ ID NO: 10 23 DNA sequence encoding SEQ ID NO: 11 24 DNA sequence encoding SEQ ID NO: 12 25 Sequence of amino acid residues at positions 266-288 of wild-type HPV35 L1 protein 26 Sequence of amino acid residues at positions 50-62 of wild-type HPV31 L1 protein 27 Sequence of amino acid residues at positions 127-142 of wild-type HPV31 L1 protein 28 Sequence of amino acid residues at positions 177-182 of wild-type HPV31 L1 protein
(13) TABLE-US-00002 Sequence 1 (SEQ ID NO: 1): MQVTFIYILVITCYENDVNVYHIFFQMSLWLPSEATVYLPPVPVSKVV STDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNNKILVPKVSGLQY RVFRIFILPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGI SGHPLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPI GEHWGKGSPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTL QANKSEVPLDICTSICKYPDYIKMVSEPYGDSLFFYLRREQMFVRHLE NRAGAVGDNVPDDLYIKGSGSTANLASSNYFPTPSGSMVTSDAQIFNK PYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNIVISLCAAISTSETTY KNTNFKEYLRHGEEYDLQFIFQLCKITLTADIVITYIHSMNSTILEDW NFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVN LKEKFSADLDQFPLGRKFLLQAGLEAKPKFTLGKRKATPTTSSTSTTA KRKKRKL Sequence 2 (SEQ ID NO: 2): MSLWRSNEATVYLPPVSVSKVVSTDEYVTRTNIYYHAGSSRLLAVGHP YYAIKKQDSNKIAVPKVSGLQYRVFRVKLPDPNKFGFPDTSFYDPASQ RLVWACTGVEVGRGQPLGVGISGHPLLNKLDDTENSNKYVGNSGTDNR ECISMDYKQTQLCLIGCRPPIGEHWGKGTPCNANQVKAGECPPLELLN TVLQDGDMVDTGFGAMDFTTLQANKSDVPLDICSSICKYPDYLKMVSE PYGDMLFFYLRREQMFVRITLFNRAGTVGETVPADLYIKGTTGTLPST SYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWSNQLFVTVVDTT RSTNMSVCSAVSSSDSTYKNDNFICEYLRHGEEYDLQFIFQLCKITLT PADVMTYIHSMNPSILEDWNFGLTPPSGTLEDTYRYVISQAVTCQKPS APKPKDDPLKNYTFWEVDLKEKFSADLDQFPLGRKFLLQAGLKARPNF RLGKRAAPASTSKKSSTKRRKVKS Sequence 3 (SEQ ID NO: 3): MSLWRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHP YYSIPKSDNPKKIVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPET QRLVWACVGLEVGRGQPLGVGISGHPLLNKFDDTENSNRYAGGPGTDN RECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITPGDCPPLELK NSVIQDGDIVIVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKM VAEPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPTDLYIKOSGSTAT LANSTYFPTPSGSMVTSDAQTFNKPYWMQRAQGHNNGICWGNQLFVTV VDTTRSTNMSVCAAIANSDTTFKSSNFKEYLRHGEEFDLQFIFQLCKI TLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVTSQAITCQ KTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGYRAR PKFKAGKRSAPSASTTTPAKRKKTKK Sequence 4 (SEQ ID NO: 4): MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYYA IKKQDSNKIAVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLV WACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRECI SMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVI QDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYG DSLFFYLRREQMYVRIELFNRAGAVGDNVPDDLYIKGSGSTANLASSN YFPTPSGSMVTSDAWNICPYWLQRAQGHNNGICWGNQLFVTVVDTTRS TNIVISLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTA DIMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTP PAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLEAKPKFT LGKRKATPTTSSTSTTAKRKKRKL Sequence 5 (SEQ ID NO: 5): MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHIPYF PIKKPNNNKILVPKVSGLQYRVFRITILPDPNKFGFPDTSFYNPDTQR LVWACVGVEVGRGQPLGVGISGHPLLNKLDDTENSNKYVGNSGTDNRE CISMDYKQTQLCLIGCKPPIGEHVVGKGSPCTNVAVNPGDCPPLELIN TVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSE PYGDSLFFYLRREQMFVRHLFNRAGAVGDNVPDDLYIKGSGSTANLAS SNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDT TRSTNIVISLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKIT LTADIMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQK HTPPAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLEAKP KFTLGKRKATPTTSSTSTTAKRKKRKL Sequence 6 (SEQ ID NO: 6): MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFP IKKPNNNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLV WACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAVAANAGVDNRECI SMDYKQTQLCLIGCKPPIGEHWGKGTPCNANQVKAGECPPLELINTVI QDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYG DSLFFYLRREQMFVRHLFNRAGAVGDNVPDDLY1KGSGSTANLASSNY FPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRS TNIVISLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTA DIMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTP PAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLEAKPKFT LGKRKATPTTSSTSTTAKRKKRKL Sequence 7 (SEQ ID NO: 7): MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFP IKKPNNNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLV WACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRECI SMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVI QDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYG DSLFFYLRREQMFVRHLENRAGTVGETVPADLYIKGTTGTLPSTSYFP TPSGSMVTSDAQWNKPYWLQRAQGHNNGICWSNQLFVTVVDTTRSTNM SLCAAISTSETTYKNTNFKEYLRHGEEYDLQFWQLCKITLTADIIVIT YWISMNSTILEDWNEGLQPPPGGTLEDTYREVTSQAIACQICHTPPAP KEDPLKKYTFWEVNLKEKESADLDQFPLGRKFLLQAGLEAKPKFTLGK RKATPTTSSTSTTAKRKKRKL Sequence 8 (SEQ ID NO: 8): MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFP IKKPNJKILVPKVSGLQYRVERIHLPDPNKFGFPDTSFYNPDTQRLVW ACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRECIS MDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVIQ DGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYGD SLFFYLRREQMFVRHLENRAGAVGDNVPDDLYIKGSGSTANLASSNYF PTPSGSMVTSDAQWNKPYWLQRAQGHNNGICWSNQLFVTVVDTTRSTN MSLCAAVSSSDSTYKNDNFKEYLRHGEEYDLQFIFQLCKITLTADWIT YIHSMNSTILEDWNEGLQPPPGGTLEDTYREVTSQAIACQKHTPPAPK EDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLEAKPKFTLGKR KATPTTSSTSTTAKRKKRKL Sequence 9 (SEQ ID NO: 9): MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGITPYY STPKSDNPKKIVVPKVSGLQYRVERIHLPDPNKFGFPDTSFYNPDTQR LVWACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRE CISMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINT VIQDGDMVDTGEGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEP YGDSLFFYLRREQMFVRHLENRAGTVGETVPADLYIKGTTGTLPSTSY FPTPSGSMVTSDAQIFNICPYWLQRAQGHNNGICWSNQLFVTVVDTTR STNIVISLCAAISTSETTYKNTNEKEYLRHGEEYDLQFIFQLCKITLT ADIMTYIHSMNSTILEDWNEGLQPPPGGTLEDTYRFVTSQAIACQKHT PPAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLEAKPKF TLGKRKATPTTSSTSTTAKRKKRKL Sequence 10 (SEQ ID NO: 10): MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFP IKKPNNNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQREV WACVGVEVGRGQPLGVGISGHPLLNKFDDTENSNRYAGGPGTDNRECI SMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVI QDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYG DSLFFYLRREQMFVRHLFNRAGTVGETVPADEYIKGTTGTLPSTSYFP TPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWSNQLFVTVVDTTRSTN MSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADIMT YIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPK EDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLEAKPKFTLGKR KATPTTSSTSTTAKRKKRKL Sequence 11 (SEQ ID NO: 11): MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGFIPYF PIKKPNNNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRL VWACVGVEVGRGQPLGVGISGFIPLLNKLDDTENASAYAANAGVDNRE CISMDYKQTQLCLIGCKPPIGEHWGKGSPCSNNAITPGDCPPLELINT VIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEP YGDSLFFYLRREQMFVRHLFNRAGTVGETVPADLYIKGTTGTLPSTSY FPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWSNQLFVTVVDTTRS TNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADI MTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPA PKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLEAKPKFTLG KRKATPTTSSTSTTAKRKKRKL Sequence 12 (SEQ ID NO: 12): MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFP IKKPNNNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLV WACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRECI SMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVI QDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYG DSLFFYLRREQMFVRHLFNRAGTVGETVPADLYIKGTTGTLPSTSYFP TPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWSNQLFVTVVDTTRSTN MSLCAAIANSDTTFKSSNFKEYLRHGEEYDLQHFQLCKITLTADIMTY IFISMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPK EDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLEAKPKFTLGKR KATPTTSSTSTTAKRKKRKL Sequence 13 (SEQ ID NO: 13): ATGCAGGTGACTTTTATTTACATCCTAGTTATTACATGTTACGAAAAC GACGTAAACGTTTACCATATTTTTTTTCAGATGTCTCTTTGGCTTCCT AGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCTAAGGTTGTA AGCACGGATGAATATGTTGCACGCACAAACATATATTATCATGCAGGA ACATCCAGACTACTTGCAGTTGGACATCCCTATTTTCCTATTAAAAAA CCTAACAATAACAAAATATTAGTTCCTAAAGTATCAGGATTACAATAC AGGGTATTTAGAATACATTTACCTGACCCCAATAAGTTTGGTTTTCCT GACACCTCATTTTATAATCCAGATACACAGCGGCTGGTTTGGGCCTGT GTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTGGGCATTAGT GGCCATCCTTTATTAAATAAATTGGATGACACAGAAAATGCTAGTGCT TATGCAGCAAATGCAGGTGTGGATAATAGAGAATGTATATCTATGGAT TACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCACCTATAGGG GAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCAGTAAATCCA GGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATTCAGGATGGT GATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACTACATTACAG GCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCTATTTGCAAA TATCCAGATTATATTAAAATGGTGTCAGAACCATATGGCGACAGCTTA TTTTTTTATCTACGAAGGGAACAAATGTTTGTTAGACATTTATTTAAT AGGGCTGGTGCTGTTGGTGATAATGTACCAGACGATTTATACATTAAA GGCTCTGGGTCTACTGCAAATTTAGCCAGTTCAAATTATTTTCCTACA CCTAGTGGTTCTATGGTTACCTCTGATGCCCAAATATTCAATAAACCT TACTGGTTACAACGAGCACAGGGCCACAATAATGGCATTTGTTGGGGT AACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACAAATATG TCATTATGTGCTGCCATATCTACTTCAGAAACTACATATAAAAATACT AACTTTAAGGAGTACCTACGACATGGGGAGGAATATGATTTACAGTTT ATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACATTATGACATAC ATACATTCTATGAATTCCACTATTTTGGAGGACTGGAATTTTGGTCTA CAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGGTTTGTAACA TCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCACCTAAAGAA GATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTAAAGGAAAAG TTTTCTGCAGACCTAGATCAGTTTCCTTTAGGACGCAAATTTTTACTA CAAGCAGGATTGGAGGCCAAACCAAAATTTACATTAGGAAAACGAAAA GCTACACCCACCACCTCATCTACCTCTACAACTGCTAAACGCAAAAAA CGTAAGCTGTAA Sequence 14 (SEQ ID NO: 14): ATGAGCCTGTGGAGGAGCAACGAGGCCACCGTGTACCTGCCCCCCGTG AGCGTGAGCAAGGTGGTGAGCACCGACGAGTACGTGACCAGGACCAAC ATCTACTACCACGCCGGCAGCAGCAGGCTGCTGGCCGTGGGCCACCCC TACTACGCCATCAAGAAGCAGGACAGCAACAAGATCGCCGTGCCCAAG GTGAGCGGCCTGCAGTACAGGGTGTTCAGGGTGAAGCTGCCCGACCCC AACAAGTTCGGCTTCCCCGACACCAGCTTCTACGACCCCGCCAGCCAG AGGCTGGTGTGGGCCTGCACCGGCGTGGAGGTGGGCAGGGGCCAGCCC CTGGGCGTGGGCATCAGCGGCCACCCCCTGCTGAACAAGCTGGACGAC ACCGAGAACAGCAACAAGTACGTGGGCAACAGCGGCACCGACAACAGG GAGTGCATCAGCATGGACTACAAGCAGACCCAGCTGTGCCTGATCGGC TGCAGGCCCCCCATCGGCGAGCACTGGGGCAAGGGCACCCCCTGCAAC GCCAACCAGGTGAAGGCCGGCGAGTGCCCCCCCCTGGAGCTGCTGAAC ACCGTGCTGCAGGACGGCGACATGGTGGACACCGGCTTCGGCGCCATG GACTTCACCACCCTGCAGGCCAACAAGAGCGACGTGCCCCTGGACATC TGCAGCAGCATCTGCAAGTACCCCGACTACCTGAAGATGGTGAGCGAG CCCTACGGCGACATGCTGTTCTTCTACCTGAGGAGGGAGCAGATGTTC GTGAGGCACCTGTTCAACAGGGCCGGCACCGTGGGCGAGACCGTGCCC GCCGACCTGTACATCAAGGGCACCACCGGCACCCTGCCCAGCACCAGC TACTTCCCCACCCCCAGCGGCAGCATGGTGACCAGCGACGCCCAGATC TTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGC ATCTGCTGGAGCAACCAGCTGTTCGTGACCGTGGTGGACACCACCAGG AGCACCAACATGAGCGTGTGCAGCGCCGTGAGCAGCAGCGACAGCACC TACAAGAACGACAACTTCAAGGAGTACCTGAGGCACGGCGAGGAGTAC GACCTGCAGTTCATCTTCCAGCTGTGCAAGATCACCCTGACCGCCGAC GTGATGACCTACATCCACAGCATGAACCCCAGCATCCTGGAGGACTGG AACTTCGGCCTGACCCCCCCCCCCAGCGGCACCCTGGAGGACACCTAC AGGTACGTGACCAGCCAGGCCGTGACCTGCCAGAAGCCCAGCGCCCCC AAGCCCAAGGACGACCCCCTGAAGAACTACACCTTCTGGGAGGTGGAC CTGAAGGAGAAGTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGG AAGTTCCTGCTGCAGGCCGGCCTGAAGGCCAGGCCCAACTTCAGGCTG GGCAAGAGGGCCGCCCCCGCCAGCACCAGCAAGAAGAGCAGCACCAAG AGGAGGAAGGTGAAGAGCTGA Sequence 15 (SEQ ID NO: 15): ATGAGCCTGTGGAGGCCCAGCGAGGCCACCGTGTACCTGCCCCCCGTG CCCGTGAGCAAGGTGGTGAGCACCGACGAGTACGTGACCAGGACCAAC ATCTACTACCACGCCGGCAGCGCCAGGCTGCTGACCGTGGGCCACCCC TACTACAGCATCCCCAAGAGCGACAACCCCAAGAAGATCGTGGTGCCC AAGGTGAGCGGCCTGCAGTACAGGGTGTTCAGGGTGAGGCTGCCCGAC CCCAACAAGTTCGGCTTCCCCGACACCAGCTTCTACAACCCCGAGACC CAGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGGTGGGCAGGGGCCAG CCCCTGGGCGTGGGCATCAGCGGCCACCCCCTGCTGAACAAGTTCGAC GACACCGAGAACAGCAACAGGTACGCCGGCGGCCCCGGCACCGACAAC AGGGAGTGCATCAGCATGGACTACAAGCAGACCCAGCTGTGCCTGCTG GGCTGCAAGCCCCCCATCGGCGAGCACTGGGGCAAGGGCAGCCCCTGC AGCAACAACGCCATCACCCCCGGCGACTGCCCCCCCCTGGAGCTGAAG AACAGCGTGATCCAGGACGGCGACATGGTGGACACCGGCTTCGGCGCC ATGGACTTCACCGCCCTGCAGGACACCAAGAGCAACGTGCCCCTGGAC ATCTGCAACAGCATCTGCAAGTACCCCGACTACCTGAAGATGGTGGCC GAGCCCTACGGCGACACCCTGTTCTTCTACCTGAGGAGGGAGCAGATG TTCGTGAGGCACTTCTTCAACAGGAGCGGCACCGTGGGCGAGAGCGTG CCCACCGACCTGTACATCAAGGGCAGCGGCAGCACCGCCACCCTGGCC AACAGCACCTACTTCCCCACCCCCAGCGGCAGCATGGTGACCAGCGAC GCCCAGATCTTCAACAAGCCCTACTGGATGCAGAGGGCCCAGGGCCAC AACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTGGTGGAC ACCACCAGGAGCACCAACATGAGCGTGTGCGCCGCCATCGCCAACAGC GACACCACCTTCAAGAGCAGCAACTTCAAGGAGTACCTGAGGCACGGC GAGGAGTTCGACCTGCAGTTCATCTTCCAGCTGTGCAAGATCACCCTG AGCGCCGACATCATGACCTACATCCACAGCATGAACCCCGCCATCCTG GAGGACTGGAACTTCGGCCTGACCACCCCCCCCAGCGGCAGCCTGGAG GACACCTACAGGTTCGTGACCAGCCAGGCCATCACCTGCCAGAAGACC GCCCCCCAGAAGCCCAAGGAGGACCCCTTCAAGGACTACGTGTTCTGG GAGGTGAACCTGAAGGAGAAGTTCAGCGCCGACCTGGACCAGTTCCCC CTGCTGCAGGAAGTTCCTGCTGCAGGCCGGCTACAGGGCCAGGCCCAA GTTCAAGGCCGGCAAGAGGAGCGCCCCCAGCGCCAGCACCACCACCCC CGCCAAGAGGAAGAAGACCAAGAAGTAA Sequence 16 (SEQ ID NO: 16): ATGCTTCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCT AAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTAT CATGCAGGAACAAGCAGGCTGCTGGCCGTGGGCCACCCCTACTACGCC ATCAAGAAGCAGGACAGCAACAAGATCGCCGTGCCCAAGGTGAGCGGC CTGCAGTACAGGGTGTTCAGGATACATTTACCTGACCCCAATAAGTTT GGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTGGTT TGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTG GGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGACACAGAAAAT GCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGAGAATGTATA TCTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCA CCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCA GTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATT CAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACT ACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCT ATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATACGGC GACAGCTTATTTTTTTATCTACGAAGGGAACAAATGTTTGTTAGACAT TTATTTAATAGGGCTGGTGCTGTTGGTGATAATGTACCAGACGATTTA TACATTAAAGGCTCTGGGTCTACTGCAAATTTAGCCAGTTCAAATTAT TTTCCTACACCTAGTGGTTCTATGGTTACCTCTGATGCCCAAATATTC AATAAACCTTACTGGTTACAACGAGCACAGGGCCACAATAATGGCATT TGTTGGGGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGT ACAAATATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATAT AAAAATACTAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGAT TTACAGTTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACATT ATGACATACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAAT TTTGGTCTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGG TTTGTAACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCA CCTAAAGAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTA AAGGAAAAGTTTTCTGCAGACCTAGATCAGTTTCCTTTAGGACGCAAA TTTTTACTACAAGCAGGATTGGAGGCCAAACCAAAATTTACATTAGGA AAACGAAAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAA CGCAAAAAACGTAAGCTGTAA Sequence 17 (SEQ ID NO: 17): ATGCTTCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCT AAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTAT CATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCCTATTTTCCT ATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAAGTATCAGGA TTACAATACAGGGTATTTAGAATACATTTACCTGACCCCAATAAGTTT GGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTGGTT TGGGCCTGTGTAGGCGTGGAGGTGGGCAGCTGGCCAGCCCCTGGGCGT GGGCATCAGCGGCCACCCCCTGCTGAACAAGCTGGACGACACCGAGAA CAGCAACAAGTACGTGGGCAACAGCGGCACCGACAACAGGGAGTGCAT CAGCATGGACTACAAGCAGACCCAGCTGTGCCTGATCGGCTGCAAACC ACCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGC AGTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTAT TCAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTAC TACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATC TATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATACGG CGACAGCTTATTTTTTTATCTACGAAGGGAACAAATGTTTGTTAGACA TTTATTTAATAGGGCTGGTGCTGTTGGTGATAATGTACCAGACGATTT ATACATTAAAGGCTCTGGGTCTACTGCAAATTTAGCCAGTTCAAATTA TTTTCCTACACCTAGTGGTTCTATGGTTACCTCTGATGCCCAAATATT CAATAAACCTTACTGGTTACAACGAGCACAGGGCCACAATAATGGCAT TTGTTGGGGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAG TACAAATATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATA TAAAAATACTAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGA TTTACAGTTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACAT TATGACATACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAA TTTTGGTCTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAG GTTTGTAACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGC ACCTAAAGAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTT AAAGGAAAAGTTTTCTGCAGACCTAGATCAGTTTCCTTTAGGACGCAA ATTTTTACTACAAGCAGGATTGGAGGCCAAACCAAAATTTACATTAGG AAAACGAAAAGCTACACCCACCACCTCATCTACCTCTACAACTGrCTA AACGCAAAAAACGTAAGCTGTAA Sequence 18 (SEQ ID NO: 18): ATGCTTCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCT AAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTAT CATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCCTATTTTCCT ATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAAGTATCAGGA TTACAATACAGGGTATTTAGAATACATTTACCTGACCCCAATAAGTTT CGGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGTGGTT TGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTG GGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGACACAGAAAAT GCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGAGAATGTATA TCTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCA CCTATAGGGGAACACTGGGGCAAAGGAACCCCATGTAACGCTAATCAA GTAAAGGCAGGTGAGTGTCCACCATTAGAGTTAATAAACACAGTTATT CAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACT ACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCT ATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATACGGC GACAGCTTATTTTTTTATCTACGAAGGGAACAAATGTTTGTTAGACAT TTATTTAATAGGGCTGGTGCTGTTGGTGATAATGTACCAGACGATTTA TACATTAAAGGCTCTGGGTCTACTGCAAATTTAGCCAGTTCAAATTAT TTTCCTACACCTAGTGGTTCTATGGTTACCTCTGATGCCCAAATATTC AATAAACCTTACTGGTTACAACGAGCACAGGGCCACAATAATGGCATT TGTTGGGGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGT ACAAATATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATAT AAAAATACTAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGAT TTACAGTTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACATT ATGACATACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAAT TTTGGTCTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGG TTTGTAACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCA CCTAAAGAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTA AAGGAAAAGTTTTCTGCAGACCTAGATCAGTTTCCTTTAGGACGCAAA TTTTTACTACAAGCAGGATTGGAGGCCAAACCAAAATTTACATTAGGA AAACGAAAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAA CGCAAAAAACGTAAGCTGTAA Sequence 19 (SEQ ID NO: 19): ATGCTTCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCT AAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTAT CATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCCTATTTTCCT ATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAAGTATCAGGA TTACAATACAGGGTATTTAGAATACATTTACCTGACCCCAATAAGTTT GGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTGGTT TGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTG GGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGACACAGAAAAT GCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGAGAATGTATA TCTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCA CCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCA GTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATT CAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACT ACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCT ATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATATGGC GACAGCTTATTCTTCTACCTGAGGAGGGAGCAGATGTTCGTGAGGCAC CTGTTCAACAGGGCCGGCACCGTGGGCGAGACCGTGCCCGCCGACCTG TACATCAAGGGCACCACCGGCACCCTGCCCAGCACCAGCTACTTCCCC ACCCCCAGCGGCAGCATGGTGACCAGCGACGCCCAGATCTTCAACAAG CCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGG AGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACAAAT ATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATATAAAAAT ACTAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGATTTACAG TTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACATTATGACA TACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAATTTTGGT CTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGGTTTGTA ACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCACCTAAA CGAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTAAAGGA CCAAAGTTTTCTGCAGACTAGATCAGTTTCCTTTAGGACGCAAATTTT TACTACAAGCAGGATTGGAGGCCAAAAAAATTTACATTAGGAAAACGA AAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAACGCAAA AAACGTAAGCTGTAA Sequence 20 (SEQ ID NO: 20): ATGCTTCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCT AAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTAT CATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCCTATTTTCCT ATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAAGTATCAGGA TTACAATACAGGGTATTTAGAATACATTTACCTGACCCCAATAAGTTT GGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTGGTT TGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTG GGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGACACAGAAAAT GCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGAGAATGTATA TCTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCA CCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCA GTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATT CAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACT ACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCT ATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATACGGC GACAGCTTATTTTTTTATCTACGAAGGGAACAAATGTTTGTTAGACAT TTATTTAATAGGGCTGGTGCTGTTGGTGATAATGTACCAGACGATTTA TACATTAAAGGCTCTGGGTCTACTGCAAATTTAGCCAGTTCAAATTAT TTTCCTACACCTAGTGGTTCTATGGTTACCTCTGATGCCCAAATATTC AATAAACCTTACTGGTTACAACGAGCACAGGGCCACAATAATGGCATT TGTTGGAGCAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGT ACAAATATGTCATTATGTGCTGCCGTATCTAGTTCAGACAGTACATAT AAAAATGATAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGAT TTACAGTTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACATT ATGACATACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAAT TTTGGTCTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGG TTTGTAACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCA CCTAAAGAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTA AAGGAAAAGTTTTCTGCAGACCTAGATCAGTTTCCTTTAGGACGCAAA TTTTTACTACAAGCAGGATTGGAGGCCAAACCAAAATTTACATTAGGA AAACGAAAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAA CGCAAAAAACGTAAGCTGTAA Sequence 21 (SEQ ID NO: 21): ATGCTTCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCT AAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTAT CATGCAGGAACATCCAGACTACTTGCAGTGGGCCACCCCTACTACAGC ATCCCCAAGAGCGACAACCCCAAGAAGATCGTGGTGCCCAAGGTGAGC GGCCTGCAGTACAGGGTGTTCAGGATACATTTACCTGACCCCAATAAG TTTGGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTG GTTTGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGT GTGGGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGACACAGAA AATGCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGAGAATGT ATATCTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAA CCACCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTT GCAGTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTT ATTCAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTT ACTACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACA TTCTATTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATAT GGCGACAGCTTATTCTTCTACCTGAGGAGGGAGCAGATGTTCGTGAGG CACCTGTTCAACAGGGCCGGCACCGTGGGCGAGACCGTGCCCGCCGAC CTGTACATCAAGGGCACCACCGGCACCCTGCCCAGCACCAGCTACTTC CCCACCCCCAGCGGCAGCATGGTGACCAGCGACGCCCAGATCTTCAAC CAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTG TGGAGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACA GAATATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATATAA AAATACTAACTTTAAGGATACCTACGACATGGGGAGGAATATGATTTA CAGTTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACATTATG ACATACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAATTTT GGTCTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGGTTT GTAACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCACCT AAAGAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTAAAG GAAAAGTTTTCTGCAGACCTAGATCAGTTTCCTTTAGGACGCAAATTT TTACTACAAGCAGGATTGGAGGCCAAACCAAAATTTACATTAGGAAAA CGAAAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAACGC AAAAAACGTAAGCTGTAA Sequence 22 (SEQ ID NO: 22): ATGCTTCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCT AAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTAT CATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCCTATTTTCCT ATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAAGTATCAGGA TTACAATACAGGGTATTTAGAATACATTTACCTGACCCCAATAAGTTT GGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTGGTT TGGGCCTGTGTAGGTGTTGAGGTGGGCAGGGGCCAGCCCCTGGGCGTG GGCATCAGCGGCCACCCCCTGCTGAACAAGTTCGACGACACCGAGAAC AAGCAACAGGTACGCCGGCGGCCCCGGCACCGACAACAGGGAGTGCAT CAGCATGGACTACAAGCAGCCCAGCTGTGCCTGATTGGTTGCAAACCA CCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCA GTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATT CAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACT ACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCT ATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATATGGC GACAGCTTATTCTTCTACCTGAGGAGGGAGCAGATGTTCGTGAGGCAC CTGTTCAACAGGGCCGGCACCGTGGGCGAGACCGTGCCCGCCGACCTG TACATCAAGGGCACCACCGGCACCCTGCCCAGCACCAGCTACTTCCCC ACCCCCAGCGGCAGCATGGTGACCAGCGACGCCCAGATCTTCAACAAG CCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGG AGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACAAAT ATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATATAAAAAT ACTAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGATTTACAG TTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACATTATGACA TACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAATTTTGGT CTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGGTTTGTA AACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCACCTAA AGAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTAAAGGA AAAGTTTTCTGCAGCCTAGATCAGTTTCCTTTAGGACGCAAATTTTTA CTACAAGCAGGATTGGAGGCCAAACCAAAATTTACATTAGGAAAACGA AAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAACGCAAA AAACGTAAGCTGTAA Sequence 23 (SEQ ID NO: 23): ATGCTTCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCT AAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTAT CATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCCTATTTTCCT ATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAAGTATCAGGA TTACAATACAGGGTATTTAGAATACATTTACCTGACCCCAATAAGTTT GGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTGGTT TGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTG GGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGACACAGAAAAT GCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGAGAATGTATA GTCTATGGATTACAAACAAACACAATTGTGTTTAATTGGCTGCAAGCC CCCCATCGGCGAGCACTGGGGCAAGGGCACCCCTGCAGCAACAACGCC ACATCACCCCCGGCGACTGCCCCCCCCTGGAGCTGATAAACACAGTTA TTCAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTT ACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCT ATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATATGGC GACAGCTTATTCTTCTACCTGAGGAGGGAGCAGATGTTCGTGAGGCAC CTGTTCAACAGGGCCGGCACCGTGGGCGAGACCGTGCCCGCCGACCTG TACATCAAGGGCACCACCGGCACCCTGCCCAGCACCAGCTACTTCCCC ACCCCCAGCGGCAGCATGGTGACCAGCGACGCCCAGATCTTCAACAAG GCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTG AGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACAAAT ATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATATAAAAAT CACTAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGATTTACA GTTTATTTTTCAACTGTGCAAAATAACTTAACTGCAGACATTATGACA TACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAATTTTGGT ACTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGGTTTGT AACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCACCTAA AGAAGTCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTAAAGGAA AAGTTTTCTGCAGACCTAGATCAGTTTCCTTTAGGACGCAAATTTTTA CTACAAGCAGGATTGGAGGCCAAACCAAAATTTACATTAGGAAAACGA AAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAACGCAAA AAACGTAAGCTGTAA Sequence 24 (SEQ ID NO: 24): ATGCTTCCTAGTGAGGCCACTGTCTACTTGCCTCCTGTCCCAGTATCT AAGGTTGTAAGCACGGATGAATATGTTGCACGCACAAACATATATTAT CATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCCTATTTTCCT ATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAAGTATCAGGA TTACAATACAGGGTATTTAGAATACATTTACCTGACCCCAATAAGTTT GGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTGGTT TGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTG GGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGACACAGAAAAT GCTAGTGCTTATGCAGCAAATGCAGGTGTGGATAATAGAGAATGTATA TCTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCA CCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCA GTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATT CAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACT ACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCT ATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATATGGC GACAGCTTATTCTTCTACCTGAGGAGGGAGCAGATGTTCGTGAGGCAC CTGTTCAACAGGGCCGGCACCGTGGGCGAGACCGTGCCCGCCGACCTG TACATCAAGGGCACCACCGGCACCCTGCCCAGCACCAGCTACTTCCCC ACCCCCAGCGGCAGCATGGTGACCAGCGACGCCCAGATCTTCAACAAG CCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGG AGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACAAAT ATGTCATTATGCGCCGCCATCGCCAACAGCGACACCACCTTCAAGAGC AGCAACTTCAAGGAGTACCTGAGGCACGGCGAGGAGTATGATTTACAG TTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACATTATGACA TACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAATTTTGGT CTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGGTTTGTA ACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCACCTAAA GAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTAAAGGAA AAGTTTTCTGCAGACCTAGATCAGTTTCCTTTAGGACGCAAATTTTTA CTACAAGCAGGATTGGAGGCCAAACCAAAATTTACATTAGGAAAACGA AAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAACGCAAA AAACGTAAGCTGTAA Sequence 25 (SEQ ID NO: 25): TVGETVPADLYIKGTTGTLPSTS Sequence 26 (SEQ ID NO: 26): YSIPKSDNPKKIV Sequence 27 (SEQ ID NO: 27): FDDTENSNRYAGGPGT Sequence 28 (SEQ ID NO: 28): SNNAIT
Specific Models for Carrying Out the Invention
(14) The invention is described with reference to the following examples which are intended to illustrate, but not limit the invention.
(15) Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention were carried out substantially by referring to the procedures of J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd 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; restriction endonucleases were used under the conditions recommended by the manufacturers. It will be understood by those skilled in the art that the present invention is described by way of examples, and the examples are not intended to limit the scope of the invention.
Example 1. Expression and Purification of Mutated HPV16 L1 Protein
(16) Construction of Expression Vector
(17) An expression vector encoding the mutated HPV16 L1 protein containing Segment 3 or Segment 5 derived from HPV35 L1 protein was constructed by PCR for multi-site mutagenesis, in which the initial template used was pTO-T7-HPV16L1N30C plasmid (which encoded the HPV16 L1 protein with a N-terminal truncation of 30 amino acids (this protein was named as HPV16N30); abbreviated as 16L1N30 in Table 2). The templates and primers used for each PCR reaction were shown in Table 2, and the amplification conditions of the PCR reaction were set as: denaturating at 94° C. for 10 minutes; 25 cycles of (denaturating at 94° C. for 50 seconds, annealing at a specified temperature for a certain time, extending at 72° C. for 7 minutes and 30 seconds); finally extending at 72° C. for 7 minutes. The sequences of the used PCR primers were listed in Table 3.
(18) 2 μL of restriction endonuclease DpnI was added to the amplification product (50 μL), and incubated at 37° C. for 60 minutes. 10 μL of the enzyme-digested product was used for transformation of 40 μL of competent E. coli ER2566 (purchased from New England Biolabs) prepared by the calcium chloride method. The transformed E. coli was spread onto a solid LB medium (LB medium components: 10 g/L peptone, 5 g/L yeast powder, 10 g/L sodium chloride, the same hereinafter) containing kanamycin (final concentration: 25 mg/mL, the same hereinafter), and was subjected to static culture at 37° C. for 10-12 hours until single colonies were observed clearly. Single colonies were picked and inoculated into a test tube containing 4 mL of liquid LB medium (containing kanamycin), and cultured with shaking at 220 rpm for 10 hours at 37° C. Subsequently, 1 mL of the bacterial solution was taken and stored at −70° C. Plasmid was extracted from the E. coli, and the nucleotide sequences of the target fragments inserted into the plasmid were sequenced using T7 primer. The sequencing results showed that the nucleotide sequences of the target fragments inserted in each of the constructed plasmids (expression vectors) were SEQ ID NOs: 18 and 20, respectively, and the amino acid sequences encoded thereby were SEQ ID NOs: 6 and 8 (the corresponding proteins were named as H16N30-35T3 and H16N30-35T5, respectively).
(19) The mutated protein H16N30-35T3 differed from HPV16N30 in that the amino acid residues at positions 199-210 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 173-184 of the wild-type HPV35 L1 protein. The mutated protein H16N30-35T5 differed from HPV16N30 in that the amino acid residues at positions 374-384 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 346-356 of the wild-type HPV35 L1 protein.
(20) An expression vector of the mutated HPV16 L1 protein containing Segment 1, Segment 2 or Segment 4 derived from HPV35 L1 protein was constructed by using 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). Briefly, a short fragment containing a mutation and a long fragment containing no mutation were first obtained by PCR reaction, and then the two fragments were ligated into a loop using a Gibson assembly system. The initial template used was pTO-T7-HPV16L1N30C plasmid and pTO-T7-HPV35L1 plasmid (which encoded HPV35 L1 protein; abbreviated as 35L1 in Table 2). The templates and primers used for the respective PCR reactions were shown in Table 2, and the amplification conditions for the PCR reaction for amplifying the short fragment were set as: denaturating at 94° C. for 10 minutes; 25 cycles of (denaturating at 94° C. for 50 seconds, annealing at a specified temperature for a certain time, extending at 72° C. for 1 minute); finally extending at 72° C. for 10 minutes. The amplification conditions for the PCR reaction for amplifying the long fragment were set as: denaturating at 94° C. for 10 minutes; 25 cycles of (denaturating at 94° C. for 50 seconds, annealing at a specified temperature for a certain time, extending at 72° C. for 7 minutes and 30 seconds); finally extending at 72° C. for 10 minutes. The sequences of the PCR primers used were listed in Table 3. The amplified product was subjected to electrophoresis, and then the target fragments were recovered using a DNA recovery kit and their concentrations were determined. The amplified short and long fragments were mixed at a molar ratio of 2:1 (total volume of 3 μL), followed by the addition of 3 μL of 2× Gibson Assembly premixes (2× Gibson Assembly Master Mix, purchased from NEB, containing T5 exonuclease, Phusion DNA polymerase, Taq DNA ligase), and reacted at 50° C. for 1 hour.
(21) 40 μL of competent E. coli ER2566 (purchased from New England Biolabs) prepared by the calcium chloride method was transformed with the assembled product (6 μL). The transformed E. coli was spread on a solid LB medium containing kanamycin and were subjected to static culture at 37° C. for 10-12 hours until the single colonies were observed clearly. Single colonies were picked and inoculated into a test tube containing 4 mL of a liquid LB medium (containing kanamycin), and cultured with shaking at 220 rpm for 10 hours at 37° C. Subsequently, 1 mL of the bacterial solution was taken and stored at −70° C. The plasmid was extracted from E. coli, and the nucleotide sequences of the target fragments inserted into the plasmid were sequenced using T7 primer. The sequencing results showed that the nucleotide sequences of the target fragments inserted in each of the constructed plasmids (expression vectors) were SEQ ID NOs: 16, 17, and 19, respectively, and the amino acid sequences encoded thereby were SEQ ID NOs: 4, 5, and 7 (the corresponding proteins were named as H16N30-35T1, H16N30-35T2 and H16N30-35T4, respectively).
(22) The mutated protein H16N30-35T1 differed from HPV in that the amino acid residues at positions 76-87 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 50-61 of the wild-type HPV35 L1 protein. The mutated protein H16N30-35T2 differed from HPV16N30 in that the amino acid residues at positions 158-167 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 132-141 of the wild-type HPV35 L1 protein. The mutated protein H16N30-35T4 differed from HPV16N30 in that the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein.
(23) An expression vector encoding the mutated HPV16 L1 protein with double-substitution which contains a segment derived from HPV35 L1 and a segment derived from HPV31 L1, was constructed by using Gibson assembly. Briefly, a short fragment containing a mutation and a long fragment containing no mutation were firstly obtained by PCR reaction, and then the two fragments were ligated into a loop using a Gibson assembly system. The initial template used included pTO-T7-H16N30-35T4 plasmid (which encoded the mutated protein H16N30-35T4; abbreviated as H16N30-35T4 in Table 2), and p TO-T7-HPV31L1 plasmid (which encoded HPV31 L1 protein; abbreviated as 31L1 in Table 2). The templates and primers used for the respective PCR reactions were shown in Table 2, and the amplification conditions for the PCR reaction for amplifying the short fragment were set as: denaturating at 94° C. for 10 minutes; 25 cycles of (denaturating at 94° C. for 50 seconds, annealing at a specified temperature for a certain time, extending at 72° C. for 1 minute); finally extending at 72° C. for 10 minutes. The amplification conditions for the PCR reaction for amplifying the long fragment were set as: denaturating at 94° C. for 10 minutes; 25 cycles of (denaturating at 94° C. for 50 seconds, annealing at a specified temperature for a certain time, extending at 72° C. for 7 minutes and 30 seconds); finally extending at 72° C. for 10 minutes. The sequences of the PCR primers used are listed in Table 3. The amplified product was subjected to electrophoresis, and then the target fragments were recovered using a DNA recovery kit and its concentration was determined. The amplified short and long fragments were mixed at a molar ratio of 2:1 (total volume of 3 μL), followed by the addition of 3 μL of 2× Gibson Assembly premixes (2× Gibson Assembly Master Mix, purchased from NEB, containing T5 exonuclease, Phusion DNA polymerase, Taq DNA ligase), and reacted at 50° C. for 1 hour.
(24) 40 μL of competent E. coli ER2566 (purchased from New England Biolabs) prepared by the calcium chloride method was transformed with the assembled product (6 μL). The transformed E. coli was spread on a solid LB medium containing kanamycin and subjected to static culture at 37° C. for 10-12 hours until the single colonies were observed clearly. Single colonies were picked and inoculated into a test tube containing 4 mL of a liquid LB medium (containing kanamycin), and cultured with shaking at 220 rpm for 10 hours at 37° C. Subsequently, 1 mL of the bacterial solution was taken and stored at −70° C. The plasmid was extracted from E. coli, and the nucleotide sequences of the target fragments inserted into the plasmid was sequenced using T7 primer. The sequencing results showed that the nucleotide sequences of the desired fragments inserted in each of the constructed plasmids (expression vectors) were SEQ ID NOs: 21, 22, 23, and 24, respectively, and the amino acid sequences encoded thereby were SEQ ID NOs: 9, 10, 11, 12 (the corresponding proteins were named as H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, and H16N30-35T4-31S5).
(25) The mutated protein H16N30-35T4-31S1 differed from HPV16N30 in that the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and the amino acid residues at positions 76-87 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 50-62 of the wild-type HPV31 L1 protein. The mutated protein H16N30-35T4-31S2 differed from HPV16N30 in that the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and the amino acid residues at positions 152-167 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 127-142 of the wild-type HPV31 L1 protein. The mutated protein H16N30-35T4-31S3 differed from HPV16N30 in that the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and the amino acid residues at positions 202-207 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 177-182 of the wild type HPV31 L1 protein. The mutated protein H16N30-35T4-31S5 differed from HPV16N30 in that the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and the amino acid residues at positions 375-384 of the wild-type HPV16 L1 protein were replaced with the amino acid residues at positions 350-359 of the wild-type HPV31 L1 protein.
(26) TABLE-US-00003 TABLE 2 Templates and primers for PCR reactions used to construct expression vectors Upstream Downstream Template primer primer Product 16L1N30 G-V-H16N30- G-V-H16N30- H16N30-35T1 long 35T1-F 35T1-R fragment 35L1 G-H16N30- G-H16N30- H16N30-35T1 short 35T1-F 35T1-R fragment 16L1N30 G-V-H16N30- G-V-H16N30- H16N30-35T2 long 35T2-F 35T2-R fragment 35L1 G-H16N30- G-H16N30- H16N30-35T2 short 35T2-F 35T2-R fragment 16L1N30 H16N30-35T3-F H16N30-35T3-R H16N30-35T3 16L1N30 G-V-H16N30- G-V-H16N30- H16N30-35T4 long 35T4-F 35T4-R fragment 35L1 G-H16N30- G-H16N30- H16N30-35T4 short 35T4-F 35T4-R fragment 16L1N30 H16N30-35T5-F H16N30-35T5-R H16N30-35T5 H16N30- G-V-H16N30- G-V-H16N30- H16N30-35T4-31S1 35T4 35T4-31S1-F 35T4-31S1-R long fragment 31L1 G-H16N30- G-H16N30- H16N30-35T4-31S1 35T4-31S1-F 35T4-31S1-R short fragment H16N30- G-V-H16N30- G-V-H16N30- H16N30-35T4-31S2 35T4 35T4-31S2-F 35T4-31S2-R long fragment 31L1 G-H16N30- G-H16N30- H16N30-35T4-31S2 35T4-31S2-F 35T4-31S2-R short fragment H16N30- G-V-H16N30- G-V-H16N30- H16N30-35T4-31S3 35T4 35T4-31S3-F 35T4-31S3-R long fragment 31L1 G-H16N30- G-H16N30- H16N30-35T4-31S3 35T4-31S3-F 35T4-31S3-R short fragment H16N30- G-V-H16N30- G-V-H16N30- H16N30-35T4-31S5 35T4 35T4-31S5-F 35T4-31S5-R long fragment 31L1 G-H16N30- G-H16N30- H16N30-35T4-31S5 35T4-31S5-F 35T4-31S5-R short fragment
(27) TABLE-US-00004 TABLE 3 Sequences of the used primers (SEQ ID NO: 29-60) SEQ ID Primer sequence NO: Primer name (5′-3′) 29 G-V-H16N30- ATACATTTACCTGACC 35T1-F CCAATAAG 30 G-V-H16N30- TGTTCCTGCATGATAA 35T1-R TATATGTTTG 31 G-H16N30- AACATATATTATCATGC 35T1-F AGGAACAAGCAGGCTGC TGGCCGTGGGC 32 G-H16N30- CTTATTGGGGTCAGGTA 35T1-R AATGTATCCTGAACACC CTGTACTGCAGGC 33 G-V-H16N30- AAACCACCTATAGGGGA 35T2-F ACACTG 34 G-V-H16N30- TACACAGGCCCAAACCA 35T2-R GCCGC 35 G-1-116N30- GCGGCTGGTTTGGGCCTGT 35T2-F GTAGGCGTGGAGGTGGGCA GGGGCC 36 G-H16N30- CAGTGTTCCCCTATAGG 35T2-R TGGTTTGCAGCCGATCA GGCACAGCTGGG 37 H16N30- GGCAAAGGAACCCCATGT 35T3-F AACGCTAATCAAGTAAAG GCAGGTGAGTGTCCACCAT 38 H16N30- ATGGTGGACACTCACCT 35T3-R GCCTTTACTTGATTAGC GTTACATGGGGTTCCTT TGCC 39 G-V-H16N30- TGGGGTAACCAACTATT 35T4-F TGTTACTG 40 G-V-H16N30- TAAGCTGTCGCCATATG 35T4-R GTTCTG 41 G-H16N30- CAGAACCATATGGCGACA 35T4-F GCTTATTCTTCTACCTGA GGAGGGAGC 42 G-H16N30- CAGTAACAAATAGTTGGTT 35T4-R ACCCCAGCAGATGCCGTTG TTGTGG 43 H16N30- ATGTGCTGCCGTATCTAGT 35T5-F TCAGACAGTACATATAAAA ATGATAACTTTAAGGAG 44 H16N30- CTCCTTAAAGTTATCATTT 35T5-R TTATATGTACTGTCTGAA CTAGATACGGCAGCACAT 45 G-V-H16N30- ATACATTTACCTGACCC 35T4-31S1-F CAATAAGTT 46 G-V-H16N30- TGCAAGTAGTCTGGAT 35T4-31S1-R GTTCCTGC 47 G-H16N30- CAGGAACATCCAGACTACT 335T4-1S1-F TGCAGTGGGCCACCCCTAC TACAGCAT 48 G-H16N30- CTTATTGGCTGTCAGGTAA 35T4-31S1-R ATGTATCCTGAACACCCTG TACTGCAGGC 49 G-V-H16N30- ATTGGTTGCAAACC 35T4-31S2-F ACCTATAGGGG 50 G-V-H16N30- AACACCTACACAGGC 35T4-31S2-R CCAAACCAGC 51 G-H16N30- TGGTTTGGGCCTGTGTAGG 35T4-31S2-F TGTTGAGGTGGGCAGGGGC CAGCC 52 G-H16N30- CCTATAGGTGGTTTGCAAC 35T4-3152-R CAATCAGGCACAGCTGGGT CTGCTTG 53 G-V-H16N30- ATAAACACAGTTATTCAG 35T4-31S3-F GATGG 54 G-V-H16N30- AATTAAACACAATTGTG 35T4-31S3-R TTTGTTTGT 55 G-H16N30- AACAAACACAATTGTGTTT 35T4-31S3-F AATTGGCTGCAAGCCCCCC ATCGGCG 56 G-H16N30- CCATCCTGAATAACTGTGT 35T4-31S3-R TTATCAGCTCCAGGGGGGG GCAGTCGC 57 G-V-H16N30- TATGATTTACAGTTTATT 35T4-31S5-F TTTC 58 G-V-H16N30- TAATGACATATTTGTACT 35T4-31S5-R GCGTG 59 G-H16N30- CACGCAGTACAAATATGTC 35T4-31S5-F ATTATGCGCCGCCATCGCC AACAGCG 60 G-H16N30- TGAAAAATAAACTGTAA 35T4-31S5-R ATCATACTCCTCGCCGT GCCTCAGGTACT
Expression of Mutated Proteins on a Large Scale
(28) The bacteria liquids of E. coli carrying recombinant plasmids pTO-T7-H16N30-35T1, pTO-T7-H16N30-35T2, pTO-T7-H16N30-35T3, pTO-T7-H16N30-35T4, pTO-T7-H16N30-35T5, pTO-T7-H16N30-35T4-31S1, pTO-T7-H16N30-35T4-31 S2, pTO-T7-H16N30-35T4-31S3, pTO-T7-H16N30-35T4-31S5 were taken out from the −70° C. refrigerator, separately inoculated into 100 ml of kanamycin-containing LB liquid medium, and cultured at 200 rpm and 37° C. for about 8 hours; then transferred and inoculated into 500 ml of kanamycin-containing LB medium (1 ml of bacterial liquid was inoculated), and cultured continuously. When the bacterial concentration reached an OD.sub.600 of about 0.6, the culture temperature was lowered to 25° C., and 500 μL, of IPTG was added to each culture flask, and the culturing was continued for 8 hours. At the end of culturing, the bacterial cells were collected by centrifugation. The bacterial cells separately expressing H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31 S2, H16N30-35T4-31 S3, H16N30-35T4-31 S5 proteins were obtained.
(29) Disruption of Bacterial Cells Expressing Mutated Proteins
(30) The obtained cells were resuspended in a ratio of 1 g of bacterial cells to 10 mL of a lysate (20 mM Tris buffer, pH 7.2, 300 mM NaCl). The cells were disrupted by a ultrasonicator for 30 minutes. The lysate containing the disrupted cells was centrifuged at 13500 rpm (30000 g) for 15 minutes, and the supernatant (i.e., the supernatant of the disrupted bacterial cells) was taken.
(31) Chromatographic Purification of Mutated Proteins
(32) Instrument System: AKTA explorer 100 preparative liquid chromatography system, manufactured by GE Healthcare (formerly, Amershan Pharmacia).
(33) Chromatographic media: SP Sepharose 4 Fast Flow (GE Healthcare), CHT-II (purchased from Bio-RAD), and Butyl Sepharose 4 Fast Flow (GE Healthcare).
(34) Buffer solutions: 20 mM phosphate buffer, pH 8.0, 20 mM DTT; and, 20 mM phosphate buffer, pH 8.0, 20 mM DTT, 2 M NaCl.
(35) Samples: the obtained supernatants of the disrupted bacterial cells containing H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31 S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5, respectively.
(36) Elution Procedure:
(37) (1) Cation exchange purification of the supernatants of the disrupted bacterial cells were carried out by using SP Sepharose 4 Fast Flow: the sample was loaded to a column, and then a buffer containing 400 mM NaCl was used to elute undesired proteins, and a buffer containing 800 mM NaCl was used to elute target protein, the fraction eluted by the buffer containing 800 mM NaCl was collected;
(38) (2) Chromatographic purification of the eluted fraction obtained in the previous step was carried out by using CHT II (hydroxyapatite chromatography): the eluted fraction obtained in the previous step was diluted to reduce the concentration of NaCl to 0.5 M; the sample was loaded to a column, and then a buffer containing 500 mM NaCl was used to elute undesired proteins, and a buffer containing 1000 mM NaCl was used to elute target protein, and the fraction eluted by the buffer containing 1000 mM NaCl was collected;
(39) (3) Chromatographic purification of the eluted fraction obtained in the previous step was carried out by using HIC (hydrophobic interaction chromatography): the sample was loaded to a column, and then a buffer containing 1000 mM NaCl was used to elute undesired proteins, and a buffer containing 200 mM NaCl was used to elute target protein, and the fraction eluted by the buffer containing 200 mM NaCl was collected.
(40) 150 μL of the eluted fraction obtained in step (3) was taken, added to 30 μL of 6× Loading Buffer, mixed, and incubated in a water bath at 80° C. for 10 min. Then, 10 μl of the sample was electrophoresed in a 10% SDS-polyacrylamide gel at 120 V for 120 min; and the electrophoresis band was visualized by Coomassie blue staining. The results of electrophoresis were shown in
(41) By similar methods, HPV16N30 protein was prepared and purified using E. coli and pTO-T7-HPV16L1N30C plasmid; HPV35 L1 protein (SEQ ID NO: 2) was prepared and purified using E. coli and pTO-T7-HPV35L1 plasmid; HPV31 L1 protein (SEQ ID NO: 3) was prepared and purified using E. coli and pTO-T7-HPV31L1 plasmid.
(42) Western Blotting of Mutated Proteins
(43) The purified proteins H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 were subjected to electrophoresis by the above methods. After electrophoresis, Western Blot detection was carried out using a broad-spectrum antibody 4B3 against HPV L1 protein, and the results were shown in
Example 2: Assembly and Particle Morphology Detection of HPV Virus-Like Particles
(44) Assembly of HPV Virus-Like Particles
(45) A given volume (about 2m1) of the protein H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, or H16N30-35T4-31S5 was dialyzed to (1) 2 L storage buffer (20 mM sodium phosphate buffer, pH 6.5, 0.5M 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, 0.5M NaCl, successively. The dialysis was carried out for 12 h in each of the three buffers.
(46) By similar method, the proteins HPV16N30, HPV35 L1 and HPV31 L1 were assembled into HPV16N30 VLP, HPV35 VLP and HPV31 VLP, respectively.
(47) Molecular Sieve Chromatography Analysis
(48) The dialyzed samples were analyzed by molecular sieve chromatography using a 1120 Compact LC high performance liquid chromatography system from Agilent, USA, using an analytical column of TSK Gel PW5000xl 7.8×300 mm. The results of the analysis were shown in
(49) Sedimentation Velocity Analysis
(50) The instrument used for sedimentation velocity analysis was a Beckman XL-A analytical type ultracentrifuge equipped with optical inspection system, and An-50Ti and An-60Ti rotors. Sedimentation velocity method was used to analyze the sedimentation coefficients of HPV16N30 VLP, HPV35 VLP, HPV31 VLP, H16N30-35T1 VLP, H16N30-35T2 VLP, H16N30-35T3 VLP, H16N30-35T4 VLP, H16N30-35T5 VLP, H16N30-35T4-31S1 VLP, H16N30-35T4-31S2 VLP, H16N30-35T4-31S3 VLP, H16N30-35T4-31S5 VLP. The results were shown in
(51) Morphological Detection of Virus-Like Particles
(52) 100 μL of the sample containing VLP was taken for observation of transmission electron microscopy. The instrument used was a 100 kV transmission electron microscope manufactured by JEOL, at a magnification of 100,000 times. Briefly, 13.5 μL of the sample was taken, negatively stained with 2% phosphotungstic acid at pH 7.0, and fixed on a carbon-coated copper mesh, and then observed by the transmission electron microscopy. The results of the observation were shown in
Example 3: Evaluation of Thermal Stability of Virus-Like Particles
(53) The thermal stability of the VLPs formed by H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 were evaluated using a differential temperature calorimeter VP Capillary DSC purchased from GE Corporation (formerly, MicroCal Corporation), in which the storage buffer of the proteins was used as a control, and each of the proteins was scanned under a heating rate of 1.5° C./min within a range from 10° C. to 90° C. The test results were shown in
Example 4: Evaluation 1 of Immunoprotection of Virus-Like Particles in Animals
(54) The immunoprotective properties of VLPs formed by H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 were evaluated using mice. The animals used for immunization were 5-6 week old BalB/c mice (ordinary grade) (purchased from Shanghai SLAC Laboratory Animal Co., Ltd.).
(55) The above-prepared H16N30-35T1 VLP, H16N30-35T2 VLP, H16N30-35T3 VLP, H16N30-35T4 VLP, H16N30-35T5 VLP, HPV16N30 VLP, HPV35 VLP, and the mixed HPV16/HPV35 VLP (i.e., a mixture of HPV16N30 VLP and HPV35 VLP) were adsorbed onto aluminum adjuvant, respectively. The mice were divided into 8 groups according to different immunogens, and each group contained 5 mice. The immunization procedure was: primary immunization was carried out at the 0.sup.th week; and booster immunizations were carried out at the 2.sup.nd and 4.sup.th weeks respectively. The immunization method was intraperitoneal injection, and the immunogens and doses used were as shown in Table 4. At the 8.sup.th week after the primary immunization, venous blood was collected from eyeball, and the serum was separated, and then the titers of the neutralizing antibodies in the serum were measured. The test results were shown in
(56) TABLE-US-00005 TABLE 4 Immunization protocols Immunization Immunization Immunization antigen Adjuvant dose Number protocol (week) H16N30-35T1 Aluminum 5 μg 5 0, 2, 4 VLP adjuvant H16N30-35T2 Aluminum 5 μg 5 0, 2, 4 VLP adjuvant H16N30-35T3 Aluminum 5 μg 5 0, 2, 4 VLP adjuvant H16N30-35T4 Aluminum 5 μg 5 0, 2, 4 VLP adjuvant H16N30-35T5 Aluminum 5 μg 5 0, 2, 4 VLP adjuvant HPV16N30 Aluminum 5 μg 5 0, 2, 4 VLP adjuvant HPV35 VLP Aluminum 5 μg 5 0, 2, 4 adjuvant HPV16/HPV35 Aluminum 5 μg for 5 0, 2, 4 VLP adjuvant each VLP
(57) In addition, the above-prepared H16N30-35T4-31S1 VLP, H16N30-35T4-31S2 VLP, H16N30-35T4-31S3 VLP, H16N30-35T4-31S5 VLP, HPV16N30 VLP, HPV31 VLP, HPV35 VLP, as well as the mixed HPV16/HPV35/HPV31 (i.e., a mixture of HPV16N30 VLP, HPV35 VLP and HPV31 VLP) were adsorbed onto aluminum adjuvant, respectively. Mice were divided into 8 groups according to different immunogens, and each group contained 5 mice. The immunization procedure was: primary immunization was carried out at the 0.sup.th week; booster immunizations were carried out at the 2.sup.nd and 4.sup.th weeks respectively. The immunization method was intraperitoneal injection, and the immunogens and doses used were as shown in Table 5. At the 8.sup.th week after the primary immunization, venous blood was collected from eyeball, and the serum was separated, and then the titers of the neutralizing antibodies in the serum were measured. The test results were shown in
(58) TABLE-US-00006 TABLE 5 Immunization protocols Immuni- Immuni- Immuni- zation zation Adju- zation Num- protocol antigen vant dose ber (week) H16N30-35T4-31S1 aluminum 5 μg 5 0, 2, 4 VLP adjuvant H16N30-35T4-31S2 aluminum 5 μg 5 0, 2, 4 VLP adjuvant H16N30-35T4-31S3 aluminum 5 μg 5 0, 2, 4 VLP adjuvant H16N30-35T4-31S5 aluminum 5 μg 5 0, 2, 4 VLP adjuvant HPV16N30 aluminum 5 μg 5 0, 2, 4 VLP adjuvant HPV31 aluminum 5 μg 5 0, 2, 4 VLP adjuvant HPV35 aluminum 5 μg 5 0, 2, 4 VLP adjuvant HPV16/HPV35/ aluminum 5 μg 5 0, 2, 4 HPV31 VLP adjuvant for each VLP
Example 5: Evaluation 2 of Immunoprotection of Virus-Like Particles in Animals
(59) ED50 of H16N30-35T4 VLP
(60) Six weeks old BalB/c female mice (8 mice) were immunized with aluminum adjuvant by single intraperitoneal injection, in which the experimental groups were administered with the H16N30-35T4 VLP (immunization doses were 0.300 μg, 0.100 μg, 0.033 μg, 0.011 μg, and 0.004 μg), the control groups were administered with the HPV16N30 VLP alone and the HPV35 VLP alone (immunization doses were 0.300 μg, 0.100 μg, 0.033 μg, 0.011 μg, 0.004 μg), or the mixed HPV16/HPV35 VLP (immunization doses for each VLP were 0.300 μg, 0.100 μg, 0.033 μg, 0.011 μg, 0.004 μg); and the immunization volume was 1 mL. In the 5.sup.th week after immunization, venous blood was collected from eyeball and the HPV antibodies in the blood were detected. ED50 for inducing seroconversion (i.e. inducing the generation of antibodies in mice) was calculated for each sample, by Reed-Muench method (Reed L J M H. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938; 27:493-7). The results were shown in Tables 6-9.
(61) TABLE-US-00007 TABLE 6 ED.sub.50 values of HPV16N30 VLP for inducing the generation of anti-HPV16 and anti-HPV35 antibodies in mice Number of Seroconversion Dose mice number Seroconversion ED50 Type (μg) (mice) (mice) rate (μg) HPV16 0.300 8 8 100.00% 0.019 0.100 8 8 100.00% 0.033 8 5 72.73% 0.011 8 3 27.27% 0.004 8 0 0.00% HPV35 0.300 8 0 0.00% >0.3 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% 0.004 8 0 0.00%
(62) TABLE-US-00008 TABLE 7 ED.sub.50 values of HPV35 VLP for inducing the generation of anti-HPV16 and anti-HPV35 antibodies in mice Number of Seroconversion Dose mice number Seroconversion ED50 Type (μg) (mice) (mice) rate (μg) HPV16 0.300 8 0 0.00% >0.3 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% 0.004 8 0 0.00% HPV35 0.300 8 5 70.00% 0.196 0.100 8 2 18.18% 0.033 8 0 0.00% 0.011 8 0 0.00% 0.004 8 0 0.00%
(63) TABLE-US-00009 TABLE 8 ED.sub.50 values of the mixed HPV16/HPV35 VLP for inducing the generation of anti-HPV16 and anti-HPV35 antibodies in mice Number Seroconversion Dose of mice number Seroconversion ED50 Type (μg) (mice) (mice) rate (μg) HPV16 0.300 8 7 95.65% 0.023 0.100 8 8 93.75% 0.033 8 6 70.00% 0.011 8 1 9.09% 0.004 8 0 0.00% HPV35 0.300 8 8 100.00% 0.042 0.100 8 8 100.00% 0.033 8 3 37.50% 0.011 8 0 0.00% 0.004 8 0 0.00%
(64) TABLE-US-00010 TABLE 9 ED.sub.50 values of H16N30-35T4 VLP for inducing the generation of anti-HPV16 and anti-HPV35 antibodies in mice Number Seroconversion Dose of mice number Seroconversion ED50 Type (μg) (mice) (mice) rate (μg) HPV16 0.300 8 7 92.86% 0.081 0.100 8 5 60.00% 0.033 8 1 8.33% 0.011 8 0 0.00% 0.004 8 0 0.00% HPV35 0.300 8 5 100.00% 0.264 0.100 8 1 100.00% 0.033 8 0 22.22% 0.011 8 0 6.67% 0.004 8 0 0.00%
(65) The results showed that after 5 weeks of immunization in mice, the ED.sub.50 of the H16N30-35T4 VLP for inducing the generation of anti-HPV16 antibody in mice was comparable to those of the HPV16N30 VLP alone and the mixed HPV16/HPV35 VLP, and was significantly better than that of the HPV35 VLP alone; and the ED.sub.50 thereof for inducing the generation of anti-HPV35 antibody in mice was comparable to those of the HPV35 VLP alone and the mixed HPV16/HPV35 VLP, and was significantly better than that of the HPV16N30 VLP alone. This indicated that the H16N30-35T4 VLP had good cross-immunogenicity and cross-protection against HPV16 and HPV35.
(66) ED50 of H16N30-35T4-31S3 VLP
(67) Six weeks old BalB/c female mice (8 mice) were immunized with aluminum adjuvant by single intraperitoneal injection. The experimental groups were administered with the H16N30-35T4-31S3 VL (immunization doses were 0.300 μg, 0.100 μg, 0.033 μg, 0.011 μg, 0.004 μg); the control groups were administered with the HPV16N30 VLP alone, the HPV35 VLP alone, the HPV31 VLP alone (immunization doses were 0.300 μg, 0.100 μg, 0.033 μg, 0.011 μg, 0.004 μg), and the mixed HPV16/HPV35/HPV31 VLP (immunization doses of each VLP were 0.300 μg, 0.100 μg, 0.033 μg, 0.011 μg, 0.004 μg); and the immunological volume was 1 mL. In the 5.sup.th week after immunization, venous blood was collected from eyeball and the HPV antibodies in the blood were detected. ED50 for inducing seroconversion (i.e. inducing the generation of antibodies in mice) was calculated for each sample, by Reed-Muench method (Reed L J M H. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938; 27:493-7). The results were shown in Tables 10-14.
(68) TABLE-US-00011 TABLE 10 ED.sub.50 values of HPV16N30 VLP for inducing the generation of anti-HPV16, anti-HPV35 and anti-HPV31 antibodies in mice Number Seroconversion Dose of mice number Seroconversion ED50 Type (μg) (mice) (mice) rate (μg) HPV16 0.300 8 7 96.67% 0.008 0.100 8 8 95.65% 0.033 8 8 93.33% 0.011 8 6 66.67% 0.004 8 0 0.00% HPV35 0.300 8 0 0.00% >0.3 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% 0.004 8 0 0.00% HPV31 0.300 8 0 0.00% >0.3 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% 0.004 8 0 0.00%
(69) TABLE-US-00012 TABLE 11 ED.sub.50 values of HPV35 VLP for inducing the generation of anti-HPV16, anti-HPV35 and anti-HPV31 antibodies in mice Number Seroconversion Dose of mice number Seroconversion ED50 Type (μg) (mice) (mice) rate (μg) HPV16 0.300 8 0 0.00% >0.3 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% 0.004 8 0 0.00% HPV35 0.300 8 8 100.00% 0.017 0.100 8 8 100.00% 0.033 8 7 90.00% 0.011 8 2 22.22% 0.004 8 0 0.00% HPV31 0.300 8 0 0.00% >0.3 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% 0.004 8 0 0.00%
(70) TABLE-US-00013 TABLE 12 ED.sub.50 values of HPV31 VLP for inducing the generation of anti-HPV16, anti-HPV35 and anti-HPV31 antibodies in mice Number Seroconversion Dose of mice number Seroconversion ED50 Type (μg) (mice) (mice) rate (μg) HPV16 0.300 8 0 0.00% >0.3 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% 0.004 8 0 0.00% HPV35 0.300 8 0 0.00% >0.3 0.100 8 0 0.00% 0.033 8 0 0.00% 0.011 8 0 0.00% 0.004 8 0 0.00% HPV31 0.300 8 8 100.00% 0.014 0.100 8 8 100.00% 0.033 8 7 91.67% 0.011 8 3 40.00% 0.004 8 1 7.14%
(71) TABLE-US-00014 TABLE 13 ED.sub.50 values of the mixed HPV16/HPV35/HPV31 VLP for inducing the generation of anti-HPV16, anti-HPV35 and anti-HPV31 antibodies in mice Num- Serocon- ber version Serocon- Dose of mice number version ED50 Type (μg) (mice) (mice) rate (μg) HPV16 0.300 for each VLP 8 7 95.24% 0.043 0 100 for each VLP 8 5 76.47% 0.033 for each VLP 8 1 42.11% 0.011 for each VLP 8 5 33.33% 0.004 for each VLP 8 2 9.09% HPV35 0.300 for each VLP 8 8 100.00% 0.011 0.100 for each VLP 8 8 100.00% 0.033 for each VLP 8 8 100.00% 0.011 for each VLP 8 4 50.00% 0.004 for each VLP 8 0 0.00% HPV31 0 300 for each VLP 8 8 100.00% 0.006 0.100 for each VLP 8 8 100.00% 0.033 for each VLP 8 8 100.00% 0.011 for each VLP 8 7 88.89% 0.004 for each VLP 8 1 11.11%
(72) TABLE-US-00015 TABLE 14 ED.sub.50 values of H16N30-35T4-31S3 VLP for inducing the generation of anti-HPV16, anti-HPV35 and anti-HPV31 antibodies in mice Number Seroconversion Dose of mice number Seroconversion ED50 Type (μg) (mice) (mice) rate (μg) HPV16 0.300 8 8 100.00% 0.017 0.100 8 7 94.44% 0.033 8 7 83.33% 0.011 8 3 30.00% 0.004 8 0 0.00% HPV35 0.900 8 6 86.67% 0.100 0.300 8 3 50.00% 0.033 8 1 22.22% 0.011 8 3 13.64% 0.004 8 0 0.00% HPV31 0.900 8 5 80.00% 0.121 0.100 8 2 43.75% 0.033 8 5 29.41% 0.011 8 0 0.00% 0.004 8 0 0.00% The results showed that after 5 weeks of immunization in mice, the ED.sub.50 of H16N30-35T4-31S3 VLP for inducing the generation of anti-HPV16 antibody in mice was comparable to those of the HPV16N30 VLP alone and the mixed HPV16/HPV35/HPV31 VLP, and was significantly better than those of the HPV35 VLP alone and the HPV31 VLP alone; and the ED.sub.50 thereof for inducing the generation of anti-HPV35 antibody in mice was comparable to those of the HPV35 VLP alone and the mixed HPV16/HPV35/HPV31 VLP, and was significantly better than those of the HPV16N30 VLP alone and the HPV31 VLP alone; the ED.sub.50 thereof for inducing the generation of anti-HPV31 antibody in mice was comparable to those of the HPV31 VLP alone and the mixed HPV16/HPV35/HPV31 VLP, and was significantly better than those of the HPV16N30 VLP alone and the HPV35 VLP alone. This indicated that the H16N30-35T4-31S3 VLP had good cross-immunogenicity and cross-protection against HPV16, HPV35 and HPV31.
Evaluation of Titers of Neutralizing Antibodies in Serum After Immunization with H16N30-35T4 VLP in Mice
(73) In this experiment, the immunization protocols were shown in Table 15. All mice (6-weeks old BalB/c female mice) were divided into 3 groups: 10 μg dose group (immunization dose was 10 μg, using aluminum adjuvant), 1 μg dose group (immunization dose was 1 μg, using aluminum adjuvant), and 0.1 μg dose group (immunization dose was 0.1 μg, using aluminum adjuvant). Each group was subdivided into 4 subgroups, in which the control subgroups 1 and 2 were immunized with the HPV16N30 VLP alone and the HPV35 VLP alone respectively, the control subgroup 3 was immunized with the mixed HPV16/HPV35 VLP, and the experimental subgroup was immunized with H16N30-35T4 VLP.
(74) Six mice per subgroup were immunized by intraperitoneal injection, and the immunization doses were 10 μg, 1 μg, and 0.1 μg, respectively, and the injection volume was 1 ml. All mice were immunized initially at the 0.sup.th week, and boosted at the 2.sup.nd and 4.sup.th weeks, respectively. Blood samples were collected from the mice via orbital bleeding at the 8.sup.th week, and the titers of antibodies against HPV16 and HPV35 in the serum were analyzed. The results of the analysis were shown in
(75) TABLE-US-00016 TABLE 15 Immunization protocols Immunization Group Immunization antigen Adjuvant Immunization dose Number protocol (week) 10 μg HPV16N30 VLP Aluminum 10 μg 6 0, 2, 4 dose adjuvant group HPV35 VLP Aluminum 10 μg 6 0, 2, 4 adjuvant HPV16/HPV35 VLP Aluminum 10 μg for 6 0, 2, 4 adjuvant each VLP H16N30-35T4 VLP Aluminum 10 μg 6 0, 2, 4 adjuvant 1 μg HPV16N30 VLP Aluminum 1 μg 6 0, 2, 4 dose adjuvant group HPV35 VLP Aluminum 1 μg 6 0, 2, 4 adjuvant HPV16/HPV35 VLP Aluminum 1 μg for 6 0, 2, 4 adjuvant each VLP H16N30-35T4 VLP Aluminum 1 μg 6 0, 2, 4 adjuvant 0.1 μg HPV16N30 VLP Aluminum 0.1 μg 6 0, 2, 4 dose adjuvant group HPV35 VLP Aluminum 0.1 μg 6 0, 2, 4 adjuvant HPV16/HPV35 VLP Aluminum 0.1 μg for 6 0, 2, 4 adjuvant each VLP H16N30-35T4 VLP Aluminum 0.1 μg 6 0, 2, 4 adjuvant
Evaluation of Titers of Neutralizing Antibodies in Serum After Immunization with H16N30-35T4-31S3 VLP in Mice
(76) In this experiment, the immunization protocols were shown in Table 16. All mice (6-weeks old BalB/c female mice) were divided into 3 groups: 10 μg dose group (immunization dose was 10 μg, using aluminum adjuvant), 1 μg dose group (immunization dose was 1 μg, using aluminum adjuvant), and 0.1 μg dose group (immunization dose was 0.1 μg, using aluminum adjuvant). Each group was subdivided into 6 subgroups, in which the control subgroups 1, 2, and 3 were immunized with the HPV16N30 VLP alone, the HPV35 VLP alone and the HPV31 VLP alone, respectively, the control subgroup 4 was immunized with the mixed HPV16/HPV35/HPV31 VLP, and the experimental subgroup was immunized with H16N30-35T4-31S3 VLP alone.
(77) Six mice per subgroup were immunized by intraperitoneal injection, and the immunization doses were 10 μg, 1 μg, 0.1 μg, and the injection volume was 1 ml. All mice were immunized initially at the 0.sup.th week, and boosted at the 2.sup.nd and 4.sup.th weeks, respectively. Blood samples were collected from the mice via orbital bleeding at the 8.sup.th week, and the titers of antibodies against HPV16, HPV35 and HPV31 in the serum were analyzed. The results of the analysis were shown in
(78) TABLE-US-00017 TABLE 16 Immunization protocols Immunization Group Immunization antigen Adjuvant Immunization dose Number protocol (week) 10 μg HPV16N30 VLP aluminum 10 μg 6 0, 2, 4 dose adjuvant group HPV35 VLP aluminum 10 μg 6 0, 2, 4 adjuvant HPV31 VLP aluminum 10 μg 6 0, 2, 4 adjuvant HPV16/HPV35/HPV31 VLP aluminum 10 μg for 6 0, 2, 4 adjuvant each VLP H16N30-35T4-31S3 VLP aluminum 10 μg 6 0, 2, 4 adjuvant 1 μg HPV16N30 VLP aluminum 1 μg 6 0, 2, 4 dose adjuvant group HPV35 VLP aluminum 1 μg 6 0, 2, 4 adjuvant HPV31 VLP aluminum 1 μg 6 0, 2, 4 adjuvant HPV16/HPV35/HPV31 VLP aluminum 1 μg for 6 0, 2, 4 adjuvant each VLP H16N30-35T4-31S3 VLP aluminum 1 μg 6 0, 2, 4 adjuvant 0.1 μg HPV16N30 VLP aluminum 0.1 μg 6 0, 2, 4 dose adjuvant group HPV35 VLP aluminum 0.1 μg 6 0, 2, 4 adjuvant HPV31 VLP aluminum 0.1 μg 6 0, 2, 4 adjuvant HPV16/HPV35/HPV31 VLP aluminum 0.1 μg for 6 0, 2, 4 adjuvant each VLP H16N30-35T4-31S3 VLP aluminum 0.1 μg 6 0, 2, 4 adjuvant
(79) Although specific embodiments of the invention have been described in detail, a person skilled in the art will appreciate that various modifications and alterations of the details of the invention can be made in light of the teachings of all disclosures, and all these modifications and alterations fall within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.