Truncated L1 protein of human papillomavirus type 58

Abstract

Provided are an N-terminal truncated L1 protein of the Human Papillomavirus Type 58, a coding sequence and preparation method thereof, and a virus-like particle comprising the protein. Uses of the protein and the virus-like particle in the preparation of a pharmaceutical composition or a vaccine are further provided. The pharmaceutical composition or vaccine is used for prevention of HPV infection and a disease caused by HPV infection.

Claims

1. A truncated HPV58 L1 protein, wherein said truncated HPV58 L1 protein has the amino acid sequence as set forth in SEQ ID NO: 1.

2. An isolated nucleic acid, encoding the truncated HPV58 L1 protein according to claim 1.

3. A vector comprising the isolated nucleic acid according to claim 2.

4. A host cell, wherein said host cell comprises the isolated nucleic acid according to claim 2 or a vector comprising the isolated nucleic acid.

5. A HPV58 virus-like particle, comprising or consisting of the truncated HPV58 L1 protein according to claim 1.

6. A pharmaceutical composition or vaccine comprising the HPV58 virus-like particle according to claim 5, and optionally comprising pharmaceutically acceptable carriers and/or excipients.

7. The pharmaceutical composition or vaccine according to claim 6, wherein the HPV58 virus-like particle is present at an amount effective for preventing HPV infection or cervical cancer.

8. A method of preventing HPV infection or a disease caused by HPV infection comprising administering the HPV58 virus-like particle according to claim 5.

9. The method according to claim 8, wherein HPV infection is HPV58 infection.

10. The method according to claim 9, wherein the disease caused by HPV infection is cervical cancer.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the SDS-PAGE result of the HPV58N35C-L1 protein obtained during different steps of Example 2 of the invention. Lane M: protein molecular weight marker; Lane 1: supernatant of disrupted bacteria (i.e. the supernatant obtained by centrifuging the disrupted bacteria); Lane 2: precipitate product free of salts (i.e. the precipitate obtained by centrifugation after dialysis); Lane 3: re-dissolved supernatant (i.e. the supernatant obtained by centrifuging the solution resulted from re-dissolving the precipitate product free of salts); Lane 4: precipitant obtained after re-dissolution (i.e. the precipitate obtained by centrifuging the solution resulted from re-dissolving the precipitate product free of salts). The result showed that the purity of HPV58N35C-L1 protein was increased from about 10% (see Lane 1) to about 70% (see Lane 3) after the steps of precipitation and re-dissolution.

(2) FIG. 2 shows the SDS-PAGE result of HPV58N35C-L1 purified by cation exchange chromatography and CHT-II (hydroxyapatite chromatography) in Example 3. Lane M: protein molecular weight marker; Lane 1: sample before purification with CHT-II column; Lane 2: flow-through fraction during purification with CHT-II column; Lane 3: elution fraction eluted with 500 mmol/L NaCl; Lane 4: elution fraction eluted with 1000 mmol/L NaCl (the loading volume was 10 μL); Lane 5: elution fraction eluted with 1000 mmol/L NaCl (the loading volume was 20 μL). The result showed that after purification with CHT-II, HPV58N35C-L1 protein eluted with 1000 mmol/L NaCl reached a purity of about 98%.

(3) FIG. 3 shows the transmission electron microscopy (TEM) photograph of HPV58N35C-L1 VLPs obtained in Example 4 (taken at 50,000× magnification, Bar=0.2 μm). A large number of VLPs with a radius of about 25 nm were observed in visual field, wherein the particle size was consistent with the theoretic size and the particles were homogenous.

(4) FIG. 4 shows the dynamic light-scattering measurement result of HPV58N35C-L1 VLPs obtained in Example 4. The result showed that HPV58N35C-L1 VLPs had a hydrodynamic radius of 25.64 nm and a particle assembly rate of 100%.

(5) FIG. 5 shows neutralization titers of antibodies in serum at different stages after vaccination of rabbits with HPV58N35C-L1 VLPs as determined in Example 5. The neutralization titers of antibodies increased significantly 2 months after the first vaccination, and reached a peak level of 10.sup.5 after a booster. The longitudinal axis represents the neutralization titers of antibodies; the horizontal axis represents the time of blood sampling.

(6) FIG. 6 shows the SDS-PAGE results of the HPV58 L1 proteins having 5, 15, 27, 40, 60 or 70 amino acids truncated at the N-terminal, respectively, i.e. HPV58N5C-L1, HPV58N15C-L1, HPV58N27C-L1, HPV58N40C-L1, HPV58N60C-L1, HPV58N70C-L1 (their amino acid sequences were set forth in SEQ ID NOS:2, 3, 4, 5, 6 and 7, respectively), as obtained in Example 6. Lane M: protein molecular weight marker; Lane 1: the truncated HPV58 L1 protein HPV58N5C-L1 (the loading volume was 10 μL); Lane 2: the truncated HPV58 L1 protein HPV58N15C-L1 (the loading volume was 10 μL); Lane 3: the truncated HPV58 L1 protein HPV58N27C-L1 (the loading volume was 10 μL); Lane 4: the truncated HPV58 L1 protein HPV58N40C-L1 (the loading volume was 10 μL); Lane 5: the truncated HPV58 L1 protein HPV58N60C-L1 (the loading volume was 10 μL); Lane 6: the truncated HPV58 L1 protein HPV58N70C-L1 (the loading volume was 10 μL). The results show that the HPV58 L1 proteins having 5, 15, 27, 40, 60 or 70 amino acids truncated at the N-terminal, respectively, i.e. HPV58N5C-L1, HPV58N15C-L1, HPV58N27C-L1, HPV58N40C-L1, HPV58N60C-L1, HPV58N70C-L1, as obtained in Example 6, reached a purity above 98%.

(7) FIGS. 7A-F show the transmission electron microscopy (TEM) photographs of HPV58N5C-L1, HPV58N15C-L1, HPV58N27C-L1, HPV58N40C-L1, HPV58N60C-L1, and HPV58N70C-L1 VLPs obtained in Example 6 (taken at 50,000× magnification, Bar=0.2 μm). FIG. 7A, HPV58N5C-L1 VLPs; FIG. 7B, HPV58N15C-L1 VLPs; FIG. 7C, HPV58N27C-L1 VLPs; FIG. 7D, HPV58N40C-L1 VLPs; FIG. 7E, HPV58N60C-L1 VLPs; FIG. 7F, HPV58N70C-L1 VLPs. The results showed that a large number of VLPs with a radius of about 25 nm were observed in visual field in FIGS. 7A-7F, wherein the particle size was consistent with the theoretic size and the particles were homogenous.

(8) FIGS. 8A-F show the dynamic light-scattering measurement results of HPV58N5C-L1, HPV58N15C-L1, HPV58N27C-L1, HPV58N40C-L1, HPV58N60C-L1, and HPV58N70C-L1 VLPs obtained in Example 6. FIG. 8A, HPV58N5C-L1 VLPs; FIG. 8B, HPV58N15C-L1 VLPs; FIG. 8C, HPV58N27C-L1 VLPs; FIG. 8D, HPV58N40C-L1 VLPs; FIG. 8E, HPV58N60C-L1 VLPs; FIG. 8F, HPV58N70C-L1 VLPs. The results showed that HPV58N5C-L1, HPV58N15C-L1, HPV58N27C-L1, HPV58N40C-L1, HPV58N60C-L1, and HPV58N70C-L1 VLPs had a hydrodynamic radius of about 25 nm and a particle assembly rate of 100%.

SEQUENCE INFORMATION

(9) The information on the sequences involved in the invention is provided in the following Table 1.

(10) TABLE-US-00001 TABLE 1  Depiction of sequences SEQ ID NO: depiction  1 a HPV58 L1 protein having 35 amino acids truncated at its N-terminal, HPV58N35C-L1  2 a HPV58 L1 protein having 5 amino acids truncated at its N-terminal, HPV58N5C-L1  3 a HPV58 L1 protein having 15 amino acids truncated at its N-terminal, HPV58N15C-L1  4 a HPV58 L1 protein having 27 amino acids truncated at its N-terminal, HPV58N27C-L1  5 a HPV58 L1 protein having 40 amino acids truncated at its N-terminal, HPV58N40C-L1  6 a HPV58 L1 protein having 60 amino acids truncated at its N-terminal, HPV58N60C-L1  7 a HPV58 L1 protein having 70 amino acids truncated at its N-terminal, HPV58N70C-L1  8 HPV58 Ll gene sequence (1575 bp)  9 a DNA sequence encoding SEQ ID NO: 1 10 a DNA sequence encoding SEQ ID NO: 2 11 a DNA sequence encoding SEQ ID NO: 3 12 a DNA sequence encoding SEQ ID NO: 4 13 a DNA sequence encoding SEQ ID NO: 5 14 a DNA sequence encoding SEQ ID NO: 6 15 a DNA sequence encoding SEQ ID NO: 7 16 primer 17 primer Sequence 1 (SEQ ID NO: 1): MTVYLPPVPVSKVVSTDEYVSRTSIYYYAGSSRLLAVGNPYFSIKSPNNNKKVLVPKVSGLQYRVFRVRLP DPNKFGFPDTSFYNPDTQRLVWACVGLEIGRGQPLGVGVSGHPYLNKFDDTETSNRYPAQPGSDNRECLSM DYKQTQLCLIGCKPPTGEHWGKGVACNNNAAATDCPPLELFNSIIEDGDMVDTGFGCMDFGTLQANKSDVP IDICNSTCKYPDYLKMASEPYGDSLFFFLRREQMFVRHFFNRAGKLGEAVPDDLYIKGSGNTAVIQSSAFF PTPSGSIVTSESQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTLCTEVTKEGTYKNDNFKEYV RHVEEYDLQFVFQLCKITLTAEIMTYIHTMDSNILEDWQFGLTPPPSASLQDTYRFVTSQAITCQKTAPPK EKEDPLNKYTFWEVNLKEKFSADLDQFPLGRKFLLQSGLKAKPRLKRSAPTTRAPSTKRKKVKK Sequence 2 (SEQ ID NO: 2): MCCTLAILFCVADVNVFHIFLQMSVWRPSEATVYLPPVPVSKVVSTDEYVSRTSIYYYAGSSRLLAVGNPY FSIKSPNNNKKVLVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPDTQRLVWACVGLEIGRGQPLGVGVSG HPYLNKFDDTETSNRYPAQPGSDNRECLSMDYKQTQLCLIGCKPPTGEHWGKGVACNNNAAATDCPPLELF NSIIEDGDMVDTGFGCMDFGTLQANKSDVPIDICNSTCKYPDYLKMASEPYGDSLFFFLRREQMFVRHFFN RAGKLGEAVPDDLYIKGSGNTAVIQSSAFFPTPSGSIVTSESQLFNKPYWLQRAQGHNNGICWGNQLFVTV VDTTRSTNMTLCTEVTKEGTYKNDNFKEYVRHVEEYDLQFVFQLCKITLTAEIMTYIHTMDSNILEDWQFG LTPPPSASLQDTYRFVTSQAITCQKTAPPKEKEDPLNKYTFWEVNLKEKFSADLDQFPLGRKFLLQSGLKA KPRLKRSAPTTRAPSTKRKKVKK Sequence 3 (SEQ ID NO: 3): MADVNVFHIFLQMSVWRPSEATVYLPPVPVSKVVSTDEYVSRTSIYYYAGSSRLLAVGNPYFSIKSPNNNK KVLVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPDTQRLVWACVGLEIGRGQPLGVGVSGHPYLNKFDDT ETSNRYPAQPGSDNRECLSMDYKQTQLCLIGCKPPTGEHWGKGVACNNNAAATDCPPLELFNSIIEDGDMV DTGFGCMDFGTLQANKSDVPIDICNSTCKYPDYLKMASEPYGDSLFFFLRREQMFVRHFFNRAGKLGEAVP DDLYIKGSGNTAVIQSSAFFPTPSGSIVTSESQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMT LCTEVTKEGTYKNDNFKEYVRHVEEYDLQFVFQLCKITLTAEIMTYIHTMDSNILEDWQFGLTPPPSASLQ DTYRFVTSQAITCQKTAPPKEKEDPLNKYTFWEVNLKEKFSADLDQFPLGRKFLLQSGLKAKPRLKRSAPT TRAPSTKRKKVKK Sequence 4 (SEQ ID NO: 4): MSVWRPSEATVYLPPVPVSKVVSTDEYVSRTSIYYYAGSSRLLAVGNPYFSIKSPNNNKKVLVPKVSGLQY RVFRVRLPDPNKFGFPDTSFYNPDTQRLVWACVGLEIGRGQPLGVGVSGHPYLNKFDDTETSNRYPAQPGS DNRECLSMDYKQTQLCLIGCKPPTGEHWGKGVACNNNAAATDCPPLELFNSIIEDGDMVDTGFGCMDFGTL QANKSDVPIDICNSTCKYPDYLKMASEPYGDSLFFFLRREQMFVRHFFNRAGKLGEAVPDDLYIKGSGNTA VIQSSAFFPTPSGSIVTSESQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTLCTEVTKEGTYK NDNFKEYVRHVEEYDLQFVFQLCKITLTAEIMTYIHTMDSNILEDWQFGLTPPPSASLQDTYRFVTSQAIT CQKTAPPKEKEDPLNKYTFWEVNLKEKFSADLDQFPLGRKFLLQSGLKAKPRLKRSAPTTRAPSTKRKKVK K Sequence 5 (SEQ ID NO: 5): MPVPVSKVVSTDEYVSRTSIYYYAGSSRLLAVGNPYFSIKSPNNNKKVLVPKVSGLQYRVFRVRLPDPNKF GFPDTSFYNPDTQRLVWACVGLEIGRGQPLGVGVSGHPYLNKFDDTETSNRYPAQPGSDNRECLSMDYKQT QLCLIGCKPPTGEHWGKGVACNNNAAATDCPPLELFNSIIEDGDMVDTGFGCMDFGTLQANKSDVPIDICN STCKYPDYLKMASEPYGDSLFFFLRREQMFVRHFFNRAGKLGEAVPDDLYIKGSGNTAVIQSSAFFPTPSG SIVTSESQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTLCTEVTKEGTYKNDNFKEYVRHVEE YDLQFVFQLCKITLTAEIMTYIHTMDSNILEDWQFGLTPPPSASLQDTYRFVTSQAITCQKTAPPKEKEDP LNKYTFWEVNLKEKFSADLDQFPLGRKFLLQSGLKAKPRLKRSAPTTRAPSTKRKKVKK Sequence 6 (SEQ ID NO: 6): MYYAGSSRLLAVGNPYFSIKSPNNNKKVLVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPDTQRLVWACV GLEIGRGQPLGVGVSGHPYLNKFDDTETSNRYPAQPGSDNRECLSMDYKQTQLCLIGCKPPTGEHWGKGVA CNNNAAATDCPPLELFNSIIEDGDMVDTGFGCMDFGTLQANKSDVPIDICNSTCKYPDYLKMASEPYGDSL FFFLRREQMFVRHFFNRAGKLGEAVPDDLYIKGSGNTAVIQSSAFFPTPSGSIVTSESQLFNKPYWLQRAQ GHNNGICWGNQLFVTVVDTTRSTNMTLCTEVTKEGTYKNDNFKEYVRHVEEYDLQFVFQLCKITLTAEIMT YIHTMDSNILEDWQFGLTPPPSASLQDTYRFVTSQAITCQKTAPPKEKEDPLNKYTFWEVNLKEKFSADLD QFPLGRKFLLQSGLKAKPRLKRSAPTTRAPSTKRKKVKK Sequence 7 (SEQ ID NO: 7): MVGNPYFSIKSPNNNKKVLVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPDTQRLVWACVGLEIGRGQPL GVGVSGHPYLNKFDDTETSNRYPAQPGSDNRECLSMDYKQTQLCLIGCKPPTGEHWGKGVACNNNAAATDC PPLELFNSIIEDGDMVDTGFGCMDFGTLQANKSDVPIDICNSTCKYPDYLKMASEPYGDSLFFFLRREQMF VRHFFNRAGKLGEAVPDDLYIKGSGNTAVIQSSAFFPTPSGSIVTSESQLFNKPYWLQRAQGHNNGICWGN QLFVTVVDTTRSTNMTLCTEVTKEGTYKNDNFKEYVRHVEEYDLQFVFQLCKITLTAEIMTYIHTMDSNIL EDWQFGLTPPPSASLQDTYRFVTSQAITCQKTAPPKEKEDPLNKYTFWEVNLKEKFSADLDQFPLGRKFLL QSGLKAKPRLKRSAPTTRAPSTKRKKVKK Sequence 8 (SEQ ID NO: 8): ATGGTGCTGATCCTGTGCTGCACCCTGGCCATCCTGTTCTGCGTGGCCGACGTGAACGTGTTCCACATCTT CCTGCAGATGAGCGTGTGGAGGCCCAGCGAGGCCACCGTGTACCTGCCCCCCGTGCCCGTGAGCAAGGTGG TGAGCACCGACGAGTACGTGAGCAGGACCAGCATCTACTACTACGCCGGCAGCAGCAGGCTGCTGGCCGTG GGCAACCCCTACTTCAGCATCAAGAGCCCCAACAACAACAAGAAGGTGCTGGTGCCCAAGGTGAGCGGCCT GCAGTACAGGGTGTTCAGGGTGAGGCTGCCCGACCCCAACAAGTTCGGCTTCCCCGACACCAGCTTCTACA ACCCCGACACCCAGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGATCGGCAGGGGCCAGCCCCTGGGCGTG GGCGTGAGCGGCCACCCCTACCTGAACAAGTTCGACGACACCGAGACCAGCAACAGGTACCCCGCCCAGCC CGGCAGCGACAACAGGGAGTGCCTGAGCATGGACTACAAGCAGACCCAGCTGTGCCTGATCGGCTGCAAGC CCCCCACCGGCGAGCACTGGGGCAAGGGCGTGGCCTGCAACAACAACGCCGCCGCCACCGACTGCCCCCCC CTGGAGCTGTTCAACAGCATCATCGAGGACGGCGACATGGTGGACACCGGCTTCGGCTGCATGGACTTCGG CACCCTGCAGGCCAACAAGAGCGACGTGCCCATCGACATCTGCAACAGCACCTGCAAGTACCCCGACTACC TGAAGATGGCCAGCGAGCCCTACGGCGACAGCCTGTTCTTCTTCCTGAGGAGGGAGCAGATGTTCGTGAGG CACTTCTTCAACAGGGCCGGCAAGCTGGGCGAGGCCGTGCCCGACGACCTGTACATCAAGGGCAGCGGCAA CACCGCCGTGATCCAGAGCAGCGCCTTCTTCCCCACCCCCAGCGGCAGCATCGTGACCAGCGAGAGCCAGC TGTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTG TTCGTGACCGTGGTGGACACCACCAGGAGCACCAACATGACCCTGTGCACCGAGGTGACCAAGGAGGGCAC CTACAAGAACGACAACTTCAAGGAGTACGTGAGGCACGTGGAGGAGTACGACCTGCAGTTCGTGTTCCAGC TGTGCAAGATCACCCTGACCGCCGAGATCATGACCTACATCCACACCATGGACAGCAACATCCTGGAGGAC TGGCAGTTCGGCCTGACCCCCCCCCCCAGCGCCAGCCTGCAGGACACCTACAGGTTCGTGACCAGCCAGGC CATCACCTGCCAGAAGACCGCCCCCCCCAAGGAGAAGGAGGACCCCCTGAACAAGTACACCTTCTGGGAGG TGAACCTGAAGGAGAAGTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGGAAGTTCCTGCTGCAGAGC GGCCTGAAGGCCAAGCCCAGGCTGAAGAGGAGCGCCCCCACCACCAGGGCCCCCAGCACCAAGAGGAAGAA GGTGAAGAAGTGA Sequence 9 (SEQ ID NO: 9): ATGACCGTGTACCTGCCCCCCGTGCCCGTGAGCAAGGTGGTGAGCACCGACGAGTACGTGAGCAGGACCAG CATCTACTACTACGCCGGCAGCAGCAGGCTGCTGGCCGTGGGCAACCCCTACTTCAGCATCAAGAGCCCCA ACAACAACAAGAAGGTGCTGGTGCCCAAGGTGAGCGGCCTGCAGTACAGGGTGTTCAGGGTGAGGCTGCCC GACCCCAACAAGTTCGGCTTCCCCGACACCAGCTTCTACAACCCCGACACCCAGAGGCTGGTGTGGGCCTG CGTGGGCCTGGAGATCGGCAGGGGCCAGCCCCTGGGCGTGGGCGTGAGCGGCCACCCCTACCTGAACAAGT TCGACGACACCGAGACCAGCAACAGGTACCCCGCCCAGCCCGGCAGCGACAACAGGGAGTGCCTGAGCATG GACTACAAGCAGACCCAGCTGTGCCTGATCGGCTGCAAGCCCCCCACCGGCGAGCACTGGGGCAAGGGCGT GGCCTGCAACAACAACGCCGCCGCCACCGACTGCCCCCCCCTGGAGCTGTTCAACAGCATCATCGAGGACG GCGACATGGTGGACACCGGCTTCGGCTGCATGGACTTCGGCACCCTGCAGGCCAACAAGAGCGACGTGCCC ATCGACATCTGCAACAGCACCTGCAAGTACCCCGACTACCTGAAGATGGCCAGCGAGCCCTACGGCGACAG CCTGTTCTTCTTCCTGAGGAGGGAGCAGATGTTCGTGAGGCACTTCTTCAACAGGGCCGGCAAGCTGGGCG AGGCCGTGCCCGACGACCTGTACATCAAGGGCAGCGGCAACACCGCCGTGATCCAGAGCAGCGCCTTCTTC CCCACCCCCAGCGGCAGCATCGTGACCAGCGAGAGCCAGCTGTTCAACAAGCCCTACTGGCTGCAGAGGGC CCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTGGTGGACACCACCAGGAGCA CCAACATGACCCTGTGCACCGAGGTGACCAAGGAGGGCACCTACAAGAACGACAACTTCAAGGAGTACGTG AGGCACGTGGAGGAGTACGACCTGCAGTTCGTGTTCCAGCTGTGCAAGATCACCCTGACCGCCGAGATCAT GACCTACATCCACACCATGGACAGCAACATCCTGGAGGACTGGCAGTTCGGCCTGACCCCCCCCCCCAGCG CCAGCCTGCAGGACACCTACAGGTTCGTGACCAGCCAGGCCATCACCTGCCAGAAGACCGCCCCCCCCAAG GAGAAGGAGGACCCCCTGAACAAGTACACCTTCTGGGAGGTGAACCTGAAGGAGAAGTTCAGCGCCGACCT GGACCAGTTCCCCCTGGGCAGGAAGTTCCTGCTGCAGAGCGGCCTGAAGGCCAAGCCCAGGCTGAAGAGGA GCGCCCCCACCACCAGGGCCCCCAGCACCAAGAGGAAGAAGGTGAAGAAGTAA Sequence 10 (SEQ ID NO: 10): ATGTGCTGCACCCTGGCCATCCTGTTCTGCGTGGCCGACGTGAACGTGTTCCACATCTTCCTGCAGATGAG CGTGTGGAGGCCCAGCGAGGCCACCGTGTACCTGCCCCCCGTGCCCGTGAGCAAGGTGGTGAGCACCGACG AGTACGTGAGCAGGACCAGCATCTACTACTACGCCGGCAGCAGCAGGCTGCTGGCCGTGGGCAACCCCTAC TTCAGCATCAAGAGCCCCAACAACAACAAGAAGGTGCTGGTGCCCAAGGTGAGCGGCCTGCAGTACAGGGT GTTCAGGGTGAGGCTGCCCGACCCCAACAAGTTCGGCTTCCCCGACACCAGCTTCTACAACCCCGACACCC AGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGATCGGCAGGGGCCAGCCCCTGGGCGTGGGCGTGAGCGGC CACCCCTACCTGAACAAGTTCGACGACACCGAGACCAGCAACAGGTACCCCGCCCAGCCCGGCAGCGACAA CAGGGAGTGCCTGAGCATGGACTACAAGCAGACCCAGCTGTGCCTGATCGGCTGCAAGCCCCCCACCGGCG AGCACTGGGGCAAGGGCGTGGCCTGCAACAACAACGCCGCCGCCACCGACTGCCCCCCCCTGGAGCTGTTC AACAGCATCATCGAGGACGGCGACATGGTGGACACCGGCTTCGGCTGCATGGACTTCGGCACCCTGCAGGC CAACAAGAGCGACGTGCCCATCGACATCTGCAACAGCACCTGCAAGTACCCCGACTACCTGAAGATGGCCA GCGAGCCCTACGGCGACAGCCTGTTCTTCTTCCTGAGGAGGGAGCAGATGTTCGTGAGGCACTTCTTCAAC AGGGCCGGCAAGCTGGGCGAGGCCGTGCCCGACGACCTGTACATCAAGGGCAGCGGCAACACCGCCGTGAT CCAGAGCAGCGCCTTCTTCCCCACCCCCAGCGGCAGCATCGTGACCAGCGAGAGCCAGCTGTTCAACAAGC CCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTG GTGGACACCACCAGGAGCACCAACATGACCCTGTGCACCGAGGTGACCAAGGAGGGCACCTACAAGAACGA CAACTTCAAGGAGTACGTGAGGCACGTGGAGGAGTACGACCTGCAGTTCGTGTTCCAGCTGTGCAAGATCA CCCTGACCGCCGAGATCATGACCTACATCCACACCATGGACAGCAACATCCTGGAGGACTGGCAGTTCGGC CTGACCCCCCCCCCCAGCGCCAGCCTGCAGGACACCTACAGGTTCGTGACCAGCCAGGCCATCACCTGCCA GAAGACCGCCCCCCCCAAGGAGAAGGAGGACCCCCTGAACAAGTACACCTTCTGGGAGGTGAACCTGAAGG AGAAGTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGGAAGTTCCTGCTGCAGAGCGGCCTGAAGGCC AAGCCCAGGCTGAAGAGGAGCGCCCCCACCACCAGGGCCCCCAGCACCAAGAGGAAGAAGGTGAAGAAGTG A Sequence 11 (SEQ ID NO: 11): ATGGCCGACGTGAACGTGTTCCACATCTTCCTGCAGATGAGCGTGTGGAGGCCCAGCGAGGCCACCGTGTA CCTGCCCCCCGTGCCCGTGAGCAAGGTGGTGAGCACCGACGAGTACGTGAGCAGGACCAGCATCTACTACT ACGCCGGCAGCAGCAGGCTGCTGGCCGTGGGCAACCCCTACTTCAGCATCAAGAGCCCCAACAACAACAAG AAGGTGCTGGTGCCCAAGGTGAGCGGCCTGCAGTACAGGGTGTTCAGGGTGAGGCTGCCCGACCCCAACAA GTTCGGCTTCCCCGACACCAGCTTCTACAACCCCGACACCCAGAGGCTGGTGTGGGCCTGCGTGGGCCTGG AGATCGGCAGGGGCCAGCCCCTGGGCGTGGGCGTGAGCGGCCACCCCTACCTGAACAAGTTCGACGACACC GAGACCAGCAACAGGTACCCCGCCCAGCCCGGCAGCGACAACAGGGAGTGCCTGAGCATGGACTACAAGCA GACCCAGCTGTGCCTGATCGGCTGCAAGCCCCCCACCGGCGAGCACTGGGGCAAGGGCGTGGCCTGCAACA ACAACGCCGCCGCCACCGACTGCCCCCCCCTGGAGCTGTTCAACAGCATCATCGAGGACGGCGACATGGTG GACACCGGCTTCGGCTGCATGGACTTCGGCACCCTGCAGGCCAACAAGAGCGACGTGCCCATCGACATCTG CAACAGCACCTGCAAGTACCCCGACTACCTGAAGATGGCCAGCGAGCCCTACGGCGACAGCCTGTTCTTCT TCCTGAGGAGGGAGCAGATGTTCGTGAGGCACTTCTTCAACAGGGCCGGCAAGCTGGGCGAGGCCGTGCCC GACGACCTGTACATCAAGGGCAGCGGCAACACCGCCGTGATCCAGAGCAGCGCCTTCTTCCCCACCCCCAG CGGCAGCATCGTGACCAGCGAGAGCCAGCTGTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACA ACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTGGTGGACACCACCAGGAGCACCAACATGACC CTGTGCACCGAGGTGACCAAGGAGGGCACCTACAAGAACGACAACTTCAAGGAGTACGTGAGGCACGTGGA GGAGTACGACCTGCAGTTCGTGTTCCAGCTGTGCAAGATCACCCTGACCGCCGAGATCATGACCTACATCC ACACCATGGACAGCAACATCCTGGAGGACTGGCAGTTCGGCCTGACCCCCCCCCCCAGCGCCAGCCTGCAG GACACCTACAGGTTCGTGACCAGCCAGGCCATCACCTGCCAGAAGACCGCCCCCCCCAAGGAGAAGGAGGA CCCCCTGAACAAGTACACCTTCTGGGAGGTGAACCTGAAGGAGAAGTTCAGCGCCGACCTGGACCAGTTCC CCCTGGGCAGGAAGTTCCTGCTGCAGAGCGGCCTGAAGGCCAAGCCCAGGCTGAAGAGGAGCGCCCCCACC ACCAGGGCCCCCAGCACCAAGAGGAAGAAGGTGAAGAAGTGA Sequence 12 (SEQ ID NO: 12): ATGAGCGTGTGGAGGCCCAGCGAGGCCACCGTGTACCTGCCCCCCGTGCCCGTGAGCAAGGTGGTGAGCAC CGACGAGTACGTGAGCAGGACCAGCATCTACTACTACGCCGGCAGCAGCAGGCTGCTGGCCGTGGGCAACC CCTACTTCAGCATCAAGAGCCCCAACAACAACAAGAAGGTGCTGGTGCCCAAGGTGAGCGGCCTGCAGTAC AGGGTGTTCAGGGTGAGGCTGCCCGACCCCAACAAGTTCGGCTTCCCCGACACCAGCTTCTACAACCCCGA CACCCAGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGATCGGCAGGGGCCAGCCCCTGGGCGTGGGCGTGA GCGGCCACCCCTACCTGAACAAGTTCGACGACACCGAGACCAGCAACAGGTACCCCGCCCAGCCCGGCAGC GACAACAGGGAGTGCCTGAGCATGGACTACAAGCAGACCCAGCTGTGCCTGATCGGCTGCAAGCCCCCCAC CGGCGAGCACTGGGGCAAGGGCGTGGCCTGCAACAACAACGCCGCCGCCACCGACTGCCCCCCCCTGGAGC TGTTCAACAGCATCATCGAGGACGGCGACATGGTGGACACCGGCTTCGGCTGCATGGACTTCGGCACCCTG CAGGCCAACAAGAGCGACGTGCCCATCGACATCTGCAACAGCACCTGCAAGTACCCCGACTACCTGAAGAT GGCCAGCGAGCCCTACGGCGACAGCCTGTTCTTCTTCCTGAGGAGGGAGCAGATGTTCGTGAGGCACTTCT TCAACAGGGCCGGCAAGCTGGGCGAGGCCGTGCCCGACGACCTGTACATCAAGGGCAGCGGCAACACCGCC GTGATCCAGAGCAGCGCCTTCTTCCCCACCCCCAGCGGCAGCATCGTGACCAGCGAGAGCCAGCTGTTCAA CAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGA CCGTGGTGGACACCACCAGGAGCACCAACATGACCCTGTGCACCGAGGTGACCAAGGAGGGCACCTACAAG AACGACAACTTCAAGGAGTACGTGAGGCACGTGGAGGAGTACGACCTGCAGTTCGTGTTCCAGCTGTGCAA GATCACCCTGACCGCCGAGATCATGACCTACATCCACACCATGGACAGCAACATCCTGGAGGACTGGCAGT TCGGCCTGACCCCCCCCCCCAGCGCCAGCCTGCAGGACACCTACAGGTTCGTGACCAGCCAGGCCATCACC TGCCAGAAGACCGCCCCCCCCAAGGAGAAGGAGGACCCCCTGAACAAGTACACCTTCTGGGAGGTGAACCT GAAGGAGAAGTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGGAAGTTCCTGCTGCAGAGCGGCCTGA AGGCCAAGCCCAGGCTGAAGAGGAGCGCCCCCACCACCAGGGCCCCCAGCACCAAGAGGAAGAAGGTGAAG AAGTGA Sequence 13 (SEQ ID NO: 13): ATGCCCGTGCCCGTGAGCAAGGTGGTGAGCACCGACGAGTACGTGAGCAGGACCAGCATCTACTACTACGC CGGCAGCAGCAGGCTGCTGGCCGTGGGCAACCCCTACTTCAGCATCAAGAGCCCCAACAACAACAAGAAGG TGCTGGTGCCCAAGGTGAGCGGCCTGCAGTACAGGGTGTTCAGGGTGAGGCTGCCCGACCCCAACAAGTTC GGCTTCCCCGACACCAGCTTCTACAACCCCGACACCCAGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGAT CGGCAGGGGCCAGCCCCTGGGCGTGGGCGTGAGCGGCCACCCCTACCTGAACAAGTTCGACGACACCGAGA CCAGCAACAGGTACCCCGCCCAGCCCGGCAGCGACAACAGGGAGTGCCTGAGCATGGACTACAAGCAGACC CAGCTGTGCCTGATCGGCTGCAAGCCCCCCACCGGCGAGCACTGGGGCAAGGGCGTGGCCTGCAACAACAA CGCCGCCGCCACCGACTGCCCCCCCCTGGAGCTGTTCAACAGCATCATCGAGGACGGCGACATGGTGGACA CCGGCTTCGGCTGCATGGACTTCGGCACCCTGCAGGCCAACAAGAGCGACGTGCCCATCGACATCTGCAAC AGCACCTGCAAGTACCCCGACTACCTGAAGATGGCCAGCGAGCCCTACGGCGACAGCCTGTTCTTCTTCCT GAGGAGGGAGCAGATGTTCGTGAGGCACTTCTTCAACAGGGCCGGCAAGCTGGGCGAGGCCGTGCCCGACG ACCTGTACATCAAGGGCAGCGGCAACACCGCCGTGATCCAGAGCAGCGCCTTCTTCCCCACCCCCAGCGGC AGCATCGTGACCAGCGAGAGCCAGCTGTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAA CGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTGGTGGACACCACCAGGAGCACCAACATGACCCTGT GCACCGAGGTGACCAAGGAGGGCACCTACAAGAACGACAACTTCAAGGAGTACGTGAGGCACGTGGAGGAG TACGACCTGCAGTTCGTGTTCCAGCTGTGCAAGATCACCCTGACCGCCGAGATCATGACCTACATCCACAC CATGGACAGCAACATCCTGGAGGACTGGCAGTTCGGCCTGACCCCCCCCCCCAGCGCCAGCCTGCAGGACA CCTACAGGTTCGTGACCAGCCAGGCCATCACCTGCCAGAAGACCGCCCCCCCCAAGGAGAAGGAGGACCCC CTGAACAAGTACACCTTCTGGGAGGTGAACCTGAAGGAGAAGTTCAGCGCCGACCTGGACCAGTTCCCCCT GGGCAGGAAGTTCCTGCTGCAGAGCGGCCTGAAGGCCAAGCCCAGGCTGAAGAGGAGCGCCCCCACCACCA GGGCCCCCAGCACCAAGAGGAAGAAGGTGAAGAAGTAA Sequence 14 (SEQ ID NO: 14): ATGTACTACGCCGGCAGCAGCAGGCTGCTGGCCGTGGGCAACCCCTACTTCAGCATCAAGAGCCCCAACAA CAACAAGAAGGTGCTGGTGCCCAAGGTGAGCGGCCTGCAGTACAGGGTGTTCAGGGTGAGGCTGCCCGACC CCAACAAGTTCGGCTTCCCCGACACCAGCTTCTACAACCCCGACACCCAGAGGCTGGTGTGGGCCTGCGTG GGCCTGGAGATCGGCAGGGGCCAGCCCCTGGGCGTGGGCGTGAGCGGCCACCCCTACCTGAACAAGTTCGA CGACACCGAGACCAGCAACAGGTACCCCGCCCAGCCCGGCAGCGACAACAGGGAGTGCCTGAGCATGGACT ACAAGCAGACCCAGCTGTGCCTGATCGGCTGCAAGCCCCCCACCGGCGAGCACTGGGGCAAGGGCGTGGCC TGCAACAACAACGCCGCCGCCACCGACTGCCCCCCCCTGGAGCTGTTCAACAGCATCATCGAGGACGGCGA CATGGTGGACACCGGCTTCGGCTGCATGGACTTCGGCACCCTGCAGGCCAACAAGAGCGACGTGCCCATCG ACATCTGCAACAGCACCTGCAAGTACCCCGACTACCTGAAGATGGCCAGCGAGCCCTACGGCGACAGCCTG TTCTTCTTCCTGAGGAGGGAGCAGATGTTCGTGAGGCACTTCTTCAACAGGGCCGGCAAGCTGGGCGAGGC CGTGCCCGACGACCTGTACATCAAGGGCAGCGGCAACACCGCCGTGATCCAGAGCAGCGCCTTCTTCCCCA CCCCCAGCGGCAGCATCGTGACCAGCGAGAGCCAGCTGTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAG GGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTGGTGGACACCACCAGGAGCACCAA CATGACCCTGTGCACCGAGGTGACCAAGGAGGGCACCTACAAGAACGACAACTTCAAGGAGTACGTGAGGC ACGTGGAGGAGTACGACCTGCAGTTCGTGTTCCAGCTGTGCAAGATCACCCTGACCGCCGAGATCATGACC TACATCCACACCATGGACAGCAACATCCTGGAGGACTGGCAGTTCGGCCTGACCCCCCCCCCCAGCGCCAG CCTGCAGGACACCTACAGGTTCGTGACCAGCCAGGCCATCACCTGCCAGAAGACCGCCCCCCCCAAGGAGA AGGAGGACCCCCTGAACAAGTACACCTTCTGGGAGGTGAACCTGAAGGAGAAGTTCAGCGCCGACCTGGAC CAGTTCCCCCTGGGCAGGAAGTTCCTGCTGCAGAGCGGCCTGAAGGCCAAGCCCAGGCTGAAGAGGAGCGC CCCCACCACCAGGGCCCCCAGCACCAAGAGGAAGAAGGTGAAGAAGTGA Sequence 15 (SEQ ID NO: 15): ATGGTGGGCAACCCCTACTTCAGCATCAAGAGCCCCAACAACAACAAGAAGGTGCTGGTGCCCAAGGTGAG CGGCCTGCAGTACAGGGTGTTCAGGGTGAGGCTGCCCGACCCCAACAAGTTCGGCTTCCCCGACACCAGCT TCTACAACCCCGACACCCAGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGATCGGCAGGGGCCAGCCCCTG GGCGTGGGCGTGAGCGGCCACCCCTACCTGAACAAGTTCGACGACACCGAGACCAGCAACAGGTACCCCGC CCAGCCCGGCAGCGACAACAGGGAGTGCCTGAGCATGGACTACAAGCAGACCCAGCTGTGCCTGATCGGCT GCAAGCCCCCCACCGGCGAGCACTGGGGCAAGGGCGTGGCCTGCAACAACAACGCCGCCGCCACCGACTGC CCCCCCCTGGAGCTGTTCAACAGCATCATCGAGGACGGCGACATGGTGGACACCGGCTTCGGCTGCATGGA CTTCGGCACCCTGCAGGCCAACAAGAGCGACGTGCCCATCGACATCTGCAACAGCACCTGCAAGTACCCCG ACTACCTGAAGATGGCCAGCGAGCCCTACGGCGACAGCCTGTTCTTCTTCCTGAGGAGGGAGCAGATGTTC GTGAGGCACTTCTTCAACAGGGCCGGCAAGCTGGGCGAGGCCGTGCCCGACGACCTGTACATCAAGGGCAG CGGCAACACCGCCGTGATCCAGAGCAGCGCCTTCTTCCCCACCCCCAGCGGCAGCATCGTGACCAGCGAGA GCCAGCTGTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAAC CAGCTGTTCGTGACCGTGGTGGACACCACCAGGAGCACCAACATGACCCTGTGCACCGAGGTGACCAAGGA GGGCACCTACAAGAACGACAACTTCAAGGAGTACGTGAGGCACGTGGAGGAGTACGACCTGCAGTTCGTGT TCCAGCTGTGCAAGATCACCCTGACCGCCGAGATCATGACCTACATCCACACCATGGACAGCAACATCCTG GAGGACTGGCAGTTCGGCCTGACCCCCCCCCCCAGCGCCAGCCTGCAGGACACCTACAGGTTCGTGACCAG CCAGGCCATCACCTGCCAGAAGACCGCCCCCCCCAAGGAGAAGGAGGACCCCCTGAACAAGTACACCTTCT GGGAGGTGAACCTGAAGGAGAAGTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGGAAGTTCCTGCTG CAGAGCGGCCTGAAGGCCAAGCCCAGGCTGAAGAGGAGCGCCCCCACCACCAGGGCCCCCAGCACCAAGAG GAAGAAGGTGAAGAAGTGA Sequence 16 (SEQ ID NO: 16): CATATgAccgTgTAccTgccc Sequence 17 (SEQ ID NO: 17): gTCgACTTACTTCTTCACCTTCTTCC

SPECIFIC MODES FOR CARRYING OUT THE INVENTION

(11) The present invention is further illustrated in detail by reference to the examples as follows. It is understood by those skilled in the art that the examples are used only for the purpose of illustrating the present invention, rather than limiting the protection scope of the present invention.

(12) 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, 3.sup.rd Edition, John Wiley & Sons, Inc., 1995, or in accordance with the product instructions. The reagents and instruments used in the present invention without marking out their manufacturers are all conventional products commercially available from markets. 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 of the Truncated HPV58 L1 Proteins with a Sequence as Set Forth in SEQ ID NO:1

(13) Preparation of Full-Length HPV58 L1 Gene as a Template

(14) The full-length HPV58 L1 gene as a template was synthesized by Shanghai Boya Bio Co. The synthesized gene fragment has a full length of 1575 bp, and its sequence is as set forth in SEQ ID NO:8. On the basis of the synthetic full-length HPV58 L1 gene fragment, the polynucleotides encoding the truncated HPV58 L1 proteins according to the invention were prepared.

(15) Construction of Non-Fusion Expression Vectors for Expressing the Truncated HPV58 L1 Proteins

(16) The full-length HPV58 L1 gene as synthesized in the previous step was used as the template for the PCR reaction. The forward primer was 58N35F: 5′-cAT ATg Acc gTg TAc cTg ccc-3′ (SEQ ID NO: 16), at the 5′ terminal of which the restriction endonuclease NdeI site CAT ATG was introduced, wherein ATG was the initiation codon in E. coli system. The reverse primer was 58CR: 5′-gTC gAC TTA CTT CTT CAC CTT CTT CC-3′ (SEQ ID NO: 17), at the 5′ terminal of which the restriction endonuclease SalI site was introduced. The PCR reaction was performed in a PCR thermocycler (Biometra T3) under the following conditions:

(17) TABLE-US-00002 94° C. denaturation for 10 min 1 cycle 94° C. denaturation for 50 sec 15 cycles 56° C. annealing for 50 sec 72° C. elongation for 1.5 min 72° C. elongation for 10 min 1 cycle

(18) The DNA fragments, about 1.5 kb in length, were obtained after amplification. The PCR products were linked into the commercially available pMD 18-T vector (Takara Biosciences), and were then transformed into E. coli DH5α. Positive bacterial colonies were screened, and plasmids were extracted. After digestion with NdeI/SalI, it was identified that positive clones, designated as pMD 18-T-HPV58N35C-L1, were obtained, wherein the truncated HPV58 L1 gene was inserted.

(19) The nucleotide sequence of the fragment of interest, which was inserted into the plasmid pMD 18-T-HPV58N35C-L1, was determined as SEQ ID NO:9 by Shanghai Boya Bio Co. using M13 (+)/(−) primers, and the amino acid sequence encoded thereby was set forth in SEQ ID NO:1. The sequence corresponded to a HPV58 L1 protein having 35 amino acids truncated at its N-terminal and no amino acid truncated at its C-terminal, designated as HPV58N35C-L1.

(20) The HPV58N35C-L1 gene fragment was obtained by NdeI/SalI digestion of the plasmid pMD 18-T-HPV58N35C-L1. The fragment was linked to the non-fusion expression vector pT0-T7 (Luo wenxin et al. Chinese Journal of Biotechnology, 2000, 16: 53-57) digested with NdeI/SalI, and was then transformed into E. coli ER2566. Positive bacterial colonies were screened, and plasmids were extracted. After digestion with NdeI/SalI, it was identified that positive clones, designated as pT0-T7-HPV58N35C-L1, were obtained, wherein the fragment of interest was inserted.

(21) 1 μl of the plasmid pT0-T7-HPV58N35C-L1 (0.15 mg/ml) was taken to transform 40 μL competent E. coli ER2566 (purchased from New England Biolabs) prepared by Calcium chloride method, and then the bacteria were plated on solid LB medium (the components of the LB medium: 10 g/L peptone, 5 g/L yeast powder, and 10 g/L NaCl, the same as below) containing kanamycin (at a final concentration of 25 mg/ml, the same as below). Plates were statically incubated at 37° C. for about 10-12 h until single colonies could be observed clearly. Single colonies from the plates were transferred to a tube containing 4 ml liquid LB media containing kanamycin. Cultures were incubated in a shaking incubator at 220 rpm for 10 h at 37° C., and then 1 ml bacterial solution was taken and stored at −70° C.

(22) Expression of HPV58N35C-L1 Protein on Large Scale

(23) The E. coli solution carrying the recombinant plasmid pTO-T7-HPV58N35C-L1 at −70° C. as prepared in the previous step was seeded in 50 mL LB liquid medium containing kanamycin and incubated at 200 rpm and 37° C. for about 8 h. Then, the cultures were transferred to ten flasks (5 ml cultures per flask), each of which contained 500 mL LB medium containing kanamycin, and then were incubated in a shaking incubator overnight at 200 rpm and 37° C., as a starter culture.

(24) A 50 L fermenter made by Shanghai Baoxing Biological Ltd was used in large-scale culture. PH electrode of the fermenter was calibrated. 30 L LB medium was loaded into the fermenter, in situ sterilized at 121° C. for 30 minutes. Oxygen-dissolved electrode was calibrated, wherein the value was determined as 0 prior to introduction of air after sterilization and as 100% prior to vaccination after introduction of air while stirring at an initial rate of 100 rpm.

(25) Preparation of the feed: The mixture of peptone and yeast extract at a concentration of 30% is prepared (20 g peptone and 10 g yeast extract were dissolved in 100 mL); a glucose solution of 50% is prepared (50 g glucose was dissolved in 100 ml). The two solutions were sterilized at 121° C. for 20 min.

(26) On the next day, the starter cultures in the ten flasks (for a total of 5 L) were transferred to the fermenter. A temperature of 37° C. and a pH value of 7.0 were set, the dissolved O.sub.2 was maintained at >40% by regulating agitation rate and air supply manually.

(27) Flow Feed: the glucose solution (50%) and the mixture of peptone and yeast extract (30%) were mixed at a solute mass ratio of 2:1.

(28) Flow rates were as followed (25 ml/min was defined as 100%):

(29) 1.sup.st h: 5%

(30) 2.sup.nd h: 10%

(31) 3.sup.rd h: 20%

(32) 4.sup.th h: 40%

(33) 5.sup.th h to the end: 60%

(34) When the bacterial concentration reached an OD.sub.600 of about 10.0, the culturing temperature was lowered to 25° C. and 4 g IPTG was added to initiate an induction culture of 4 h. Fermentation was halted when the final concentration reached an OD.sub.600 of about 60. The bacteria were collected by centrifugation. The bacteria expressing HPV58N35C-L1 protein were obtained, weighted about 2.5 kg.

Example 2: Preparation of HPV58N35C-L1 Protein with a Purity of about 70%

(35) Bacteria were re-suspended at a proportion of 1 g bacteria corresponding to 10 ml lysis buffer (20 mM Tris buffer pH 7.2, 300 mM NaCl). Bacteria were disrupted by an APV homogenizer (Invensys Group) for five times at a pressure of 600 bar. The homogenate was centrifuged at 13,500 rpm (30,000 g) using JA-14 rotor for 15 min, and the supernatant (i.e. the supernatant of disrupted bacteria) was obtained. The supernatant was subjected to 10% SDS-PAGE. At this stage, the HPV58N35C-L1 protein in the supernatant had a purity of about 10% (see FIG. 1, Lane 1).

(36) The supernatant was dialyzed by a CENTRASETTE 5 Tangential Flow Filter (Pall Co.) running at a pressure of 0.5 psi, a flow rate of 500 ml/min, and a tangential flow rate of 200 mL/min, wherein the membrane retention molecular weight was 30 kDa, the dialysis solution was 10 mM phosphate buffer pH 6.0, and the dialysis volume was three times of the volume of the supernatant.

(37) After thorough dialysis, the mixture was centrifuged at 9500 rpm (12,000 g) using JA-10 rotor (Beckman J25 high speed centrifuge)) for 20 min, and the precipitate (i.e. the precipitate product free of salts) was collected. The precipitate was re-suspended in 20 mM phosphate buffer (pH 8.0) containing 20 mM DTT and 600 mM NaCl, wherein the volume of the buffer was 1/10 of the volume of the supernatant. The mixture was stirred for 30 min and centrifuged at 13,500 rpm (30,000 g) using JA-14 rotor (Beckman J25 high speed centrifuge) for 20 min. The supernatant and precipitate (i.e. the precipitate obtained after re-dissolution) were collected. The supernatant was diluted with 20 mM phosphate buffer (pH 8.0) containing 20 mM DTT of an equal volume, resulting in the final NaCl concentration of 0.3 M. Then, the diluted supernatant was filtered using a filter membrane with an aperture of 0.22 μm. The sample obtained (i.e. re-dissolved supernatant) was purified by cation exchange chromatography (as described in Example 3). 30 μL of 6× loading buffer (12% (w/v) SDS, 0.6% (w/v) bromophenol blue, 0.3M Tris-HCl pH 6.8, 60% (v/v) glycerin, 5% (v/v) β-mercaptoethanol) was added to 150 μL filtered supernatant, and the result solution was mixed homogeneously and was placed in a water bath at 80° C. for 10 min. Then, 10 μl sample was subjected to 10% SDS-PAGE at 120V for 120 min. The electrophoretic bands were stained by Coomassie brilliant blue. The electrophoretic result was shown in FIG. 1. The result showed that HPV58N35C-L1 protein was purified and enriched after the steps of precipitation and re-dissolution, with its purity increased from about 10% to about 70% (see FIG. 1, Lane 1 and Lane 3).

Example 3: Chromatographic Purification of HPV58N35C-L1 Protein

(38) 1) Purification of HPV58N35C-L1 Protein by Cation Exchange Chromatography

(39) Equipment: AKTA Explorer 100 preparative liquid chromatography system produced by GE Healthcare (i.e. the original Amershan Pharmacia Co.)

(40) Chromatographic media: SP Sepharose 4 Fast Flow (GE Healthcare Co.)

(41) Column Volume: 5.5 cm×20 cm

(42) Buffer: 20 mM phosphate buffer pH 8.0, 20 mM DTT 20 mM phosphate buffer pH 8.0, 20 mM DTT, 2M NaCl

(43) Flow Rate: 25 mL/min

(44) Detector Wavelength: 280 nm

(45) Sample: about 70% pure HPV58N35C-L1 protein solution, as filtered through a filter membrane with an aperture of 0.22 μm in Example 2.

(46) Elution protocol: eluting undesired proteins with 500 mM NaCl, eluting the protein of interest with 1000 mM NaCl, collecting eluate eluted with 1000 mM NaCl, and finally getting about 900 mL purified HPV58N35C-L1 sample.

(47) 2) Purification of HPV58N35C-L1 by CHT-II Chromatography (Hydroxyapatite Chromatography)

(48) Equipment: AKTA Explorer 100 preparative liquid chromatography system produced by GE Healthcare (i.e. the original Amershan Pharmacia Co.)

(49) Chromatographic media: CHT-II (purchased from Bio-Rad)

(50) Column Volume: 5.5 cm×20 cm

(51) Buffer: 20 mM phosphate buffer pH8.0, 20 mM DTT, 20 mM phosphate buffer pH 8.0, 20 mM DTT, 2M NaCl

(52) Flow Rate: 20 mL/min

(53) Detector Wavelength: 280 nm

(54) Sample: 1000 mM NaCl elution product obtained in the previous step, diluted to a NaCl concentration of 0.3M with 20 mM phosphate buffer pH 8.0, 20 mM DTT.

(55) Elution protocol: eluting undesired proteins with 500 mM NaCl, eluting the protein of interest with 1000 mM NaCl, collecting eluate eluted with 1000 mM NaCl, and finally getting 800 mL purified HPV58N35C-L1 sample.

(56) 30 μL 6× loading buffer was added to 150 μL HPV58N35C-L1 sample as purified by the method in the present Example, and then the result solution was mixed homogeneously. After incubating the solution in a water bath at 80° C. for 10 min, a 10 μL sample was subjected to 10% SDS-PAGE at 120V for 120 min. The electrophoretic bands were stained by Coomassie brilliant blue. The electrophoretic result was shown in FIG. 2. The result showed that after said purification step, the concentration of HPV58N35C-L1 protein was about 1.0 mg/ml, with a purity of greater than 98%.

Example 4: Assembly of HPV58N35C-L1 VLPs

(57) Equipment: CENTRASETTE 5 Tangential Flow Filter (Pall Co.), wherein the membrane retention molecular weight was 30 kDa. Sample: HPV58N35C-L1 with a purity of greater than 98% as obtained in Example 3.

(58) Sample Renaturation: Sample buffer was exchanged with 10 L renaturation buffer (50 mM PB (sodium phosphate buffer) pH 6.0, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2, 0.5M NaCl, 0.003% Tween-80) thoroughly. The Tangential Flow Filter was run at a pressure of 0.5 psi and a tangential flow rate of 10 mL/min. When the exchange with renaturation buffer was finished, the renaturation buffer was exchanged with storage buffer (20 L PBS: 20 mM PB pH 6.5, 0.5M NaCl) with an exchange volume of 20 L. The Tangential Flow Filter was run at a pressure of 0.5 psi and a tangential flow rate of 25 mL/min. When the exchange was finished, the sample was aseptically filtrated with a Pall filter (0.20 μm), and thereby obtaining HPV58N35C-L1 VLPs. The HPV58N35C-L1 VLPs were stored at 4° C. for further use.

Example 5: Determination of the Morphology of HPV58N35C-L1 VLPs and Determination of Immunogenicity Thereof

(59) Transmission Electron Microscopy (TEM) of HPV58N35C-L1 VLPs

(60) The equipment was a JEOL 100 kV Transmission Electron Microscope (100,000× magnification). HPV58N35C-L1 VLPs obtained in Example 4 were negatively stained with 2% phosphotungstic acid at pH 7.0, and fixed on a copper grid for observation. Results were shown in FIG. 3. A large number of VLPs with a radius of approximately 25 nm, which were homogenous and in a hollow form, were observed.

(61) Dynamic Light-Scattering Measurement of HPV58N35C-L1 VLPs

(62) DynaPro MS/X dynamic light-scattering instrument (including a temperature controller) (US Protein Solutions Co.) was used for light-scattering measurements. The regulation algorithm was used in the measurements. The sample was the HPV58N35C-L1 VLPs obtained in Example 4. The sample was passed through a 0.22 μm filter membrane prior to the measurement. The result was shown in FIG. 4. The result showed that HPV58N35C-L1 VLPs had a Hydrodynamic radius of 25.64 nm.

(63) Establishment of a Cellular Model for HPV58 Pseudovirion Neutralization

(64) HPV can hardly be cultured in vitro, and the HPV host is strongly specific. Thus, HPV can hardly be propagated in hosts other than human. That is, there was not an appropriate animal model for HPV. Therefore, in order to evaluate the immune protection of HPV vaccines quickly, it is urgent to establish an effective model for in vitro neutralization assays.

(65) In Vitro Model of Pseudovirion Infection: by means of the characteristic that HPV VLP can package nucleic acids non-specifically, HPV pseudovirion was formed by expressing HPV L1 and L2 protein in cells, and by packaging episomal viral DNA or reporter plasmids introduced heterologously (Yeager, M. D, Aste-Amezaga, M. et al (2000) Virology (278) 570-7). The concrete methods include methods of recombinant viral expression systems and methods of co-transfection of multi-plasmids. Methods of co-transfection of multi-plasmids were used in the Example exemplarily.

(66) In addition, some improvement directed to HPV systems were made by conventional methods as followed. The calcium phosphate transfection method for 293FT cell line was optimized to obtain a transfection efficiency of up to more than 90%, thereby facilitating large-scale production. The expression plasmid for expressing HPV structural proteins was codon-optimized to express HPV L1 and L2 gene efficiently in mammalian cells, thereby facilitating high efficient assembly of pseudovirion.

(67) Construction of HPV Pseudovirion was as follows:

(68) Plasmid p58L1h (the pAAV vector carrying the nucleotide sequence encoding HPV58 L1 protein (NCBI database, Accession Number: P26535.1)), plasmid p58L2h (the pAAV vector carrying the nucleotide sequence encoding HPV58 L2 protein (NCBI database, Accession Number: P26538.1)), and plasmid pN31-EGFP carrying green fluorescent protein gene, were purified by CsCl density gradient centrifugation, wherein said pN31-EGFP and said pAAV vectors were donated by Professor John T. Schiller of NIH. Methods for purifying plasmids using CsCl density gradient centrifugation were well known in the art (see The Molecular Cloning Experiment Guide, 3rd edition).

(69) 293FT cells (Invitrogen) cultured on a 10 cm cell culture plate were co-transfected with the purified p58L1h, p58L2h and pN31-EGFP (40 μg for each) by calcium phosphate transfection method. Calcium phosphate transfection method was well known in the art (see The Molecular Cloning Experiment Guide, 3rd edition). In brief, p58L1h, p58L2h and pN31-EGFP (40 μg for each) were added to the mixture of 1 mL HEPES solution (125 μL 1M HEPES pH7.3 per 50 mL deionized water, stored at 4° C.) and 1 mL 0.5M CaCl.sub.2 solution. After mixing homogeneously, 2 mL 2× HeBS solution (0.28M NaCl (16.36 g), 0.05M HEPES (11.9 g), and 1.5 mM Na.sub.2HPO.sub.4 (0.213 g), dissolved in 1000 mL deionized water, pH 6.96, stored at −70° C.) was added dropwise. After standing at room temperature for 1 min, the mixture was added to the 10 cm cell culture plate where the 293FT cells were cultured. After culturing for 6 hr, the original culture medium was decanted and 10 ml fresh complete medium (Invitrogen Co.) was added. After transfection for 48 hours, the medium was decanted and the cells were washed twice with PBS. Then, the cells were collected and counted. Every 10.sup.8 cells were re-suspended in 1 mL lysis solution (0.25% Brij58, 9.5 mM MgCl.sub.2). After lysing, cell lysate was centrifuged at 5,000 g for 10 min and the supernatant was collected. The Pseudovirion solution was obtained after adding 5M NaCl to a final concentration of 850 mM, and then was stored in small packages at −20° C.

(70) Determination of the Neutralization Titers of Antibodies

(71) 293FT cells (Invitrogen) were plated on a 96-well cell culture plate (1.5×10.sup.4 cells/well). Neutralization assay was performed five hours later. Serum samples comprising antibodies to be tested were serially diluted with 10% DMEM half-by-half. The diluted samples (50 μL for each) were respectively mixed with 50 μL Pseudovirion solution diluted in 10% DMEM as prepared above (moi=0.1). After incubating at 4° C. for 1 h, the mixture was added to the 96-well cell culture plate with 293FT cells. The mixture was then incubated for 72 h at 37° C. Antibody titers of samples were estimated by observing fluorescence. Infection percentage of cells in each well was then checked by flow cytometry (EPICS XL, American Beckman Coulter Co.). The exact antibody titers of serums were calculated. Infection percentage was the percentage of cells in the positive region of the cell sample to be tested minus that in the positive region of the uninfected control cell sample.
Infection-inhibition percentage=(1−infection percentage of wells with serum/infection percentage of wells without serum)×100%

(72) The positive region was defined as the cell region having a GFP signal determined by flow cytometry at least 10 times higher than the signal of the control cells.

(73) Neutralization titer of antibodies was defined as the highest dilution fold under which the infection-inhibition percentage reached above 50%. Antibodies were considered as having neutralizing capacity if their infection-inhibition percentage was above 50% after 50 times dilutions.

(74) Evaluation of Immune Protection of Vaccination of Animals with HPV58 VLPs

(75) Rabbits were used to evaluate the immune protection of the HPV58 VLPs according to the invention. Animals for vaccination were 5 female rabbits (general grade), 6-8 weeks old, purchased from the Disease Prevention and Control Center of Guangxi province. HPV58N35C-L1 VLPs (at a concentration of 0.1 mg/ml) prepared in Example 4, were mixed with equal volume of complete Freund's Adjuvant for the first vaccination, or with equal volume of incomplete Freund's Adjuvant for the booster. The vaccination procedure was as followed: the first vaccination at Month 0, and the booster at Month 1, 2 and 3, respectively. Rabbits were vaccinated via muscle injection, with an amount of 200 μg HPV58N35C-L1 VLPs prepared in Example 4 per rabbit.

(76) After the first vaccination, peripheral venous blood was collected every week, and serum was separated and stored for test. The neutralization titers of antibodies against HPV58 pseudovirion in the rabbit serum were determined by the method above.

(77) The result was shown in FIG. 5. FIG. 5 showed that neutralization titers of antibodies in serum at different stages after vaccination of rabbits with HPV58N35C-L1 VLPs prepared in Example 4. It could be seen that the neutralization titers of antibodies increased significantly 2 months after the first vaccination, and reached a peak level of 10.sup.5 after a booster. It suggested that HPV58N35C-L1 VLPs as prepared in Example 4 had good immunogenicity, could induce the generalization of neutralization antibodies against HPV58 with a high titer in animals, and could be used as an effective vaccine for the prevention of HPV58 infection. In addition to Freund's Adjuvant, other adjuvants well known in the art might also be used in the vaccines, for example, aluminum hydroxide or aluminum phosphate adjuvants.

Example 6: Preparation and Morphologic Observation of Other Truncated Proteins and VLPs

(78) HPV58 L1 proteins having 5, 15, 27, 40, 60 or 70 amino acids truncated at the N-terminal, respectively, i.e. HPV58N5C-L1, HPV58N15C-L1, HPV58N27C-L1, HPV58N40C-L1, HPV58N60C-L1, HPV58N70C-L1 (their amino acid sequences were set forth in SEQ ID NOS:2, 3, 4, 5, 6 and 7, respectively; their DNA sequences were set forth in SEQ ID NOS:10, 11, 12, 13, 14 and 15, respectively), were prepared and purified basically by the methods as described in Examples 1-3. The proteins thus obtained had a purity of above 98% (see FIG. 6).

(79) The purified HPV58N5C-L1, HPV58N15C-L1, HPV58N27C-L1, HPV58N40C-L1, HPV58N60C-L1, and HPV58N70C-L1 proteins were assembled into VLPs basically by the method as described in Example 4, respectively, designated as HPV58N5C-L1 VLPs, HPV58N15C-L1 VLPs, HPV58N27C-L1 VLPs, HPV58N40C-L1 VLPs, HPV58N60C-L1 VLPs, and HPV58N70C-L1 VLPs, respectively.

(80) HPV58N5C-L1 VLPs, HPV58N15C-L1 VLPs, HPV58N27C-L1 VLPs, HPV58N40C-L1 VLPs, HPV58N60C-L1 VLPs, and HPV58N70C-L1 VLPs were subjected to transmission electron microscopy and dynamic light scattering observation basically by the method as described in Example 5, respectively. The results were shown in FIG. 7 and FIG. 8. FIG. 7 showed that the truncated proteins could form a large number of VLPs with a radius of about 25 nm, wherein the particle size was consistent with the theoretic size and the particles were homogenous. FIG. 8 showed that HPV58N5C-L1, HPV58N15C-L1, HPV58N27C-L1, HPV58N40C-L1, HPV58N60C-L1, and HPV58N70C-L1 VLPs had a hydrodynamic radius of about 25 nm and a particle assembly rate of 100%.

(81) In addition, it was demonstrated by the method as described in Example 5 that the HPV58N5C-L1, HPV58N15C-L1, HPV58N27C-L1, HPV58N40C-L1, HPV58N60C-L1, and HPV58N70C-L1 VLPs obtained in the invention also had good immunogenicity, could induce the generalization of neutralization antibodies with a high titer in animals, and therefore could be used as an effective vaccine for the prevention of HPV infection.

(82) 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 without departing from the sprit or scope of the present invention as generally described, 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.