Truncated L1 protein of human papillomavirus type 6

Abstract

The invention relates to a truncated L1 protein of the Human Papillomavirus Type 6, a virus-like particle consisting of the truncated L1 protein, a vaccine comprising said virus-like particle, and the use of the vaccine in the prevention of condyloma acuminatum or HPV infections.

Claims

1. A method for producing a human papillomavirus (HPV) L1 protein, consisting of: a) expressing a HPV L1 gene encoding the HPV L1 protein in an E. coli expression system; b) disrupting the E. coli, which has expressed the HPV L1 protein, in a salt solution having a salt concentration of from 200 mM to 500 mM, centrifuging the solution comprising the disrupted E. coli, and isolating the supernatant without using chromatography; c) precipitating the HPV L1 protein from the supernatant of b) by decreasing the salt concentration of the supernatant of b) to from 100 mM to 0, inclusive, by using water or a low salt solution, and collecting the resultant precipitate; d) redissolving the precipitate of c) in a salt solution having a salt concentration of from 150 mM to 25006 mM, adding a reductant to it, and then centrifuging the resultant solution, and isolating the supernatant without using chromatography, wherein the supernatant contains the HPV L1 protein with a purity of at least 50%.

2. The method of claim 1, wherein the HPV L1 protein is HPV6 L1 protein.

3. The method of claim 2, wherein the HPV L1 protein is HPV6 L1 protein with 2, 3, 4, or 5 amino acids truncated at its N-terminal.

4. The method o claim 1, wherein the HPV L1 protein is HPV6 L1 protein having a sequence set forth in SEQ ID NOs:1, 2, 3, or 4.

5. The method of claim 1, wherein the salt concentration in step d) is 200 mM-2000 mM.

6. The method of claim 1, wherein the purity is at least 70%.

7. The method of claim 1, wherein the salt in steps b), c) or d) is one or more of neutral salts.

8. The method of claim 1, wherein the salt in steps b), c) or d) is selected from alkali metal salt, ammonium salts, hydrochlorides, sulfates, bicarbonates, phosphate salts or hydrogenphosphates.

9. The method of claim 1, wherein the salt in steps b), c) or d) is selected from NaCl, KCl, NH.sub.4Cl, and (NH.sub.4).sub.2SO.sub.4.

10. The method of claim 2, wherein the salt concentration in step d) is 200 mM-2000 mM.

11. The method of claim 2, wherein the purity is at least 70%.

12. The method of claim 2, wherein the salt in steps b), c) or d) is one or more of neutral salts.

13. The method of claim 2, wherein the salt in steps b), c) or d) is selected from alkali metal salt, ammonium salts, hydrochlorides, sulfates, bicarbonates, phosphate salts or hydrogenphosphates.

14. The method of claim 2, wherein the salt in steps b), c) or d) is selected from NaCl, KCl, NH.sub.4Cl, and (NH.sub.4) SO.sub.4.

15. A method for obtaining a virus-like particles (VLP) of a human papillomavirus (HPV) 6 L1 protein, the method consisting of: a) expressing a HPV L1 gene encoding the HPV6 L1 protein in an E. coli expression system; b) disrupting the E. coli, which has expressed the HPV6 L1 protein, in a salt solution having a salt concentration of from 200 mM to 600 mM, centrifuging the solution comprising the disrupted E. coli, and isolating the supernatant without using chromatography; c) precipitating the HPV L1 protein from the supernatant of b) by decreasing the salt concentration of the supernatant of b) to from 100 mM to 0, inclusive, by using water or a low salt solution, and collecting the resultant precipitate; d) redissolving the precipitate of c) in a salt solution having a salt concentration of from 150 mM to 2500 mM, adding a reductant to it, and then centrifuging the resultant solution, and isolating the supernatant without using chromatography, wherein the supernatant contains the HPV6 L1protein with a purity of at least 50%; e) further purifying the HPV 6 L1 protein with a purity of at least 50% obtained in step d) by chromatography; and f) removing the reductant from the HPV 6L1 protein obtained in step e).

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the SDS-PAGE result of HPV6N3C-L1 protein during steps a)-d) of the method according to the invention. Lane 1: Lysate supernatant; Lane 2: HPV6N3C-L1 protein precipitated by tangential flow; Lane 3: Redissolved HPV6N3C-L1 in a re-suspension solution. The result shows that the purity of HPV6N3C-L1 reached about 70% following the steps of precipitation and re-dissolution.

(2) FIG. 2 shows the SDS-PAGE result of HPV6N3C-L1 that was obtained in step d) and was further purified according to step e). Lane 1: HPV6N3C-L1 purified according to step e), 10 μL; Lane 2: HPV6N3C-L1 purified according to step e), 20 μL. The result shows that HPV6N3C-L1 purified according to step e) reached a purity of about 98%.

(3) FIG. 3 shows the transmission electron microscopy (TEM) photograph of HPV6N3C-L1 VLPs obtained in step f), taken at 50,000× magnification. A great deal of VLPs in a radius of about 25 nm were observed in visual field, wherein the particle size was consistant with the theoretic size and the particles were homogenous.

(4) FIG. 4 shows the dynamic light-scattering measurement result of HPV6N3C-L1 VLPs obtained in step f). The result shows that HPV6N3C-L1 VLP had a hydrodynamic radius of 24.70 nm and a particle assembly rate of 100%.

(5) FIG. 5 shows titers of neutralizing antibodies in serum at different stages after vaccination of goat with HPV6N3C-L1 VLPs. Vaccination times are indicated with arrows. The titer of neutralizing antibodies increased rapidly a week after the first vaccination, and reached a peak level of 10.sup.7-10.sup.8 after a booster.

(6) FIG. 6 shows titers of neutralizing antibodies in serum at different stages a week after vaccination of rabbit with HPV6N3C-L1 VLPs. Vaccination times are indicated with arrows. The titer of neutralizing antibodies increased rapidly a week after the first vaccination, and reached a peak level of 10.sup.6 after a booster.

(7) FIG. 7 shows the titers of neutralizing antibodies against HPV 6 and HPV11 in serum at different times after vaccination of mice with HPV6/11 bivalent vaccine obtained in Example 5. Vaccine was administered at 0 and 2 weeks. The titers of neutralizing antibodies against HPV6 and HPV 11 increased rapidly after the first vaccination, reaching 10.sup.4-10.sup.5.

(8) FIG. 8 shows the titers of neutralizing antibodies against HPV 6, HPV 11, HPV 16 and HPV 18 in serum at different times after vaccination of mice with HPV6/11/16/18 quadrivalent vaccine obtained in Example 5. Vaccine was administered at 0 and 2 weeks. The titers of neutralizing antibodies against HPV 6, HPV 11, HPV 16 and HPV 18 increased rapidly after the first vaccination, reaching 10.sup.5-10.sup.6.

(9) FIG. 9 shows the SDS-PAGE results of HPV6N2C-L1, HPV6N4C-L1 and HPV6N5C-L1 protein separately having 2, 4 and 5 amino acids truncated at the N-terminal of HPV 6 L1 protein (the amino acid sequences thereof set forth in SEQ ID Nos: 2, 3 and 4, respectively) during steps a)-e) of the method according to the invention. Lane 1: Molecular Weight Marker; Lane 2: HPV6N2C-L1 purified according to step a)-e), 10 μL; Lane 3: HPV6N4C-L1 purified according to step a)-e), 10 μL; Lane 4: HPV6N5C-L1 purified according to step a)-e), 10 μL. The result shows that the purity of HPV6N2C-L1, HPV6N4C-L1 and HPV6N5C-L1 protein separately having 2, 4 and 5 amino acids truncated at the N-terminal of HPV 6 L1 protein, reached about 98% following the steps a)-e).

(10) FIG. 10 the transmission electron microscopy (TEM) photographs of the VLPs of HPV6N2C-L1, HPV6N4C-L1 and HPV6N5C-L1 protein separately having 2, 4 and 5 amino acids truncated at the N-terminal of HPV 6 L1 protein obtained after steps a)-f), taken at 50,000× magnification. 1. The transmission electron microscopy (TEM) photographs of HPV6N2C-L1 VLPs obtained after steps a)-f), taken at 50,000× magnification. 2 The transmission electron microscopy (TEM) photographs of HPV6N4C-L1 VLPs obtained after steps a)-f), taken at 50,000× magnification. 3. The transmission electron microscopy (TEM) photographs of HPV6N5C-L1 VLPs obtained after steps a)-f), taken at 50,000× magnification. The results show that a great deal of VLPs in a radius of about 25 nm were observed in visual field, wherein the particle size was consistant with the theoretic size and the particles were homogenous.

(11) FIG. 11 shows the dynamic light-scattering measurement result of the VLPs of HPV6N2C-L1, HPV6N4C-L1 and HPV6N5C-L1 protein separately having 2, 4 and 5 amino acids truncated at the N-terminal of HPV 6 L1 protein obtained after steps a)-f). 1. The dynamic light-scattering measurement result of HPV6N2C-L1 VLPs obtained after steps a)-f). 2. The dynamic light-scattering measurement result of HPV6N4C-L1 VLPs obtained after steps a)-f). 3. The dynamic light-scattering measurement result of HPV6N5C-L1 VLPs obtained after steps a)-f). The result shows that HPV6N2C-L1 VLPs, HPV6N4C-L1 VLPs and HPV6N5C-L1 VLPs had a hydrodynamic radius of about 25 nm and a particle assembly rate of 100%.

(12) TABLE-US-00001 SEQUENCES SEQ ID NO: 1: 1 MPSDSTVYVP PPNPVSKVVA TDAYVTRTNI FYHASSSRLL AVGHPYFSIK RANKTVVPKV 61 SGYQYRVFKV VLPDPNKFAL PDSSLFDPTT QRLVWACTGL EVGRGQPLGV GVSGHPFLNK 121 YDDVENSGSG GNPGQDNRVN VGMDYKQTQL CMVGCAPPLG EHWGKGKQCT NTPVQAGDCP 181 PLELITSVIQ DGDMVDTGFG AMNFADLQTN KSDVPIDICG TTCKYPDYLQ MAADPYGDRL 241 FFFLRKEQMF ARHFFNRAGE VGEPVPDTLI IKGSGNRTSV GSSIYVNTPS GSLVSSEAQL 301 FNKPYWLQKA QGHNNGICWG NQLFVTVVDT TRSTNMTLCA SVTTSSTYTN SDYKEYMRHV 361 EEYDLQFIFQ LCSITLSAEV VAYIHTMNPS VLEDWNFGLS PPPNGTLEDT YRYVQSQAIT 421 CQKPTPEKQK PDPYKNLSFW EVNLKEKFSS ELDQYPLGRK FLLQSGYRGR SSIRTGVKRP 481 AVSKASAAPK RKRAKTKR SEQ ID NO: 2 1 MRPSDSTVYV PPPNPVSKVV ATDAYVTRTN IFYHASSSRL LAVGHPYFSI KRANKTVVPK 61 VSGYQYRVFK VVLPDPNKFA LPDSSLFDPT TQRLVWACTG LEVGRGQPLG VGVSGHPFLN 121 KYDDVENSGS GGNPGQDNRV NVGMDYKQTQ LCMVGCAPPL GEHWGKGKQC TNTPVQAGDC 181 PPLELITSVI QDGDMVDTGF GAMNFADLQT NKSDVPIDIC GTTCKYPDYL QMAADPYGDR 241 LFFFLRKEQM FARHFFNRAG EVGEPVPDTL IIKGSGNRTS VGSSIYVNTP SGSLVSSEAQ 301 LFNKPYWLQK AQGHNNGICW GNQLFVTVVD TTRSTNMTLC ASVTTSSTYT NSDYKEYMRH 361 VEEYDLQFIF QLCSITLSAE VVAYIHTMNP SVLEDWNFGL SPPPNGTLED TYRYVQSQAI 421 TCQKPTPEKQ KPDPYKNLSF WEVNLKEKFS SELDQYPLGR KFLLQSGYRG RSSIRTGVKR 481 PAVSKASAAP KRKRAKTKR SEQ ID NO: 3 1 MSDSTVYVPP PNPVSKVVAT DAYVTRTNIF YHASSSRLLA VGHPYFSIKR ANKTVVPKVS 61 GYQYRVFKVV LPDPNKFALP DSSLFDPTTQ RLVWACTGLE VGRGQPLGVG VSGHPFLNKY 121 DDVENSGSGG NPGQDNRVNV GMDYKQTQLC MVGCAPPLGE HWGKGKQCTN TPVQAGDCPP 181 LELITSVIQD GDMVDTGFGA MNFADLQTNK SDVPIDICGT TCKYPDYLQM AADPYGDRLF 241 FFLRKEQMFA RHFFNRAGEV GEPVPDTLII KGSGNRTSVG SSIYVNTPSG SLVSSEAQLF 301 NKPYWLQKAQ GHNNGICWGN QLFVTVVDTT RSTNMTLCAS VTTSSTYTNS DYKEYMRHVE 361 EYDLQFIFQL CSITLSAEVV AYIHTMNPSV LEDWNFGLSP PPNGTLEDTY RYVQSQAITC 421 QKPTPEKQKP DPYKNLSFWE VNLKEKFSSE LDQYPLGRKF LLQSGYRGRS SIRTGVKRPA 481 VSKASAAPKR KRAKTKR SEQ ID NO: 4 1 MDSTVYVPPP NPVSKVVATD AYVTRTNIFY HASSSRLLAV GHPYFSIKRA NKTVVPKVSG 61 YQYRVFKVVL PDPNKFALPD SSLFDPTTQR LVWACTGLEV GRGQPLGVGV SGHPFLNKYD 121 DVENSGSGGN PGQDNRVNVG MDYKQTQLCM VGCAPPLGEH WGKGKQCTNT PVQAGDCPPL 181 ELITSVIQDG DMVDTGFGAM NFADLQTNKS DVPIDICGTT CKYPDYLQMA ADPYGDRLFF 241 FLRKEQMFAR HFFNRAGEVG EPVPDTLIIK GSGNRTSVGS SIYVNTPSGS LVSSEAQLFN 301 KPYWLQKAQG HNNGICWGNQ LFVTVVDTTR STNMTLCASV TTSSTYTNSD YKEYMRHVEE 361 YDLQFIFQLC SITLSAEVVA YIHTMNPSVL EDWNFGLSPP PNGTLEDTYR YVQSQAITCQ 421 KPTPEKQKPD PYKNLSFWEV NLKEKFSSEL DQYPLGRKFL LQSGYRGRSS IRTGVKRPAV 481 SKASAAPKRK RAKTKR SEQ ID NO: 5 1 ATGTGGCGGC CTAGCGACAG CACAGTATAT GTGCCTCCTC CTAACCCTGT ATCCAAAGTT 61 GTTGCCACGG ATGCTTATGT TACTCGCACC AACATATTTT ATCATGCCAG CAGTTCTAGA 121 CTTCTTGCAG TGGGTCATCC TTATTTTTCC ATAAAACGGG CTAACAAAAC TGTTGTGCCA 181 AAGGTGTCAG GATATCAATA CAGGGTATTT AAGGTGGTGT TACCAGATCC TAACAAATTT 241 GCATTGCCTG ACTCGTCTCT TTTTGATCCC ACAACACAAC GTTTGGTATG GGCATGCACA 301 GGCCTAGAGG TGGGCAGGGG ACAGCCATTA GGTGTGGGTG TAAGTGGACA TCCTTTCCTA 361 AATAAATATG ATGATGTTGA AAATTCAGGG AGTGGTGGTA ACCCTGGACA GGATAACAGG 421 GTTAATGTTG GTATGGATTA TAAACAAACA CAATTATGCA TGGTTGGATG TGCCCCCCCT 481 TTGGGCGAGC ATTGGGGTAA AGGTAAACAG TGTACTAATA CACCTGTACA GGCTGGTGAC 541 TGCCCGCCCT TAGAACTTAT TACCAGTGTT ATACAGGATG GCGATATGGT TGACACAGGC 601 TTTGGTGCTA TGAATTTTGC TGATTTGCAG ACCAATAAAT CAGATGTTCC TATTGACATA 661 TGTGGCACTA CATGTAAATA TCCAGATTAT TTACAAATGG CTGCAGACCC ATATGGTGAT 721 AGATTATTTT TTTTTCTACG GAAGGAACAA ATGTTTGCCA GACATTTTTT TAACAGGGCT 781 GGCGAGGTGG GGGAACCTGT GCCTGATACT CTTATAATTA AGGGTAGTGG AAATCGAACG 841 TCTGTAGGGA GTAGTATATA TGTTAACACC CCAAGCGGCT CTTTGGTGTC CTCTGAGGCA 901 CAATTGTTTA ATAAGCCATA TTGGCTACAA AAAGCCCAGG GACATAACAA TGGTATTTGT 961 TGGGGTAATC AACTGTTTGT TACTGTGGTA GATACCACAC GCAGTACCAA CATGACATTA 1021 TGTGCATCCG TAACTACATC TTCCACATAC ACCAATTCTG ATTATAAAGA GTACATGCGT 1081 CATGTGGAAG AGTATGATTT ACAATTTATT TTTCAATTAT GTAGCATTAC ATTGTCTGCT 1141 GAAGTAATGG CCTATATTCA CACAATGAAT CCCTCTGTTT TGGAAGACTG GAACTTTGGG 1201 TTATCGCCTC CCCCAAATGG TACATTAGAA GATACCTATA GGTATGTGCA GTCACAGGCC 1261 ATTACCTGTC AAAAGCCCAC TCCTGAAAAG CAAAAGCCAG ATCCCTATAA GAACCTTAGT 1321 TTTTGGGAGG TTAATTTAAA AGAAAAGTTT TCTAGTGAAT TGGATCAGTA TCCTTTGGGA 1381 CGCAAGTTTT TGTTACAAAG TGGATATAGG GGACGGTCCT CTATTCGTAC CGGTGTTAAG 1441 CGCCCTGCTG TTTCCAAAGC CTCTGCTGCC CCTAAACGTA AGCGCGCCAA AACTAAAAGG 1501 TAA SEQ ID NO: 6 1 ATGCCTAGCG ACAGCACAGT ATATGTGCCT CCTCCTAACC CTGTATCCAA 51 AGTTGTTGCC ACGGATGCTT ATGTTACTCG CACCAACATA TTTTATCATG 101 CCAGCAGTTC TAGACTTCTT GCAGTGGGTC ATCCTTATTT TTCCATAAAA 151 CGGGCTAACA AAACTGTTGT GCCAAAGGTG TCAGGATATC AATACAGGGT 201 ATTTAAGGTG GTGTTACCAG ATCCTAACAA ATTTGCATTG CCTGACTCGT 251 CTCTTTTTGA TCCCACAACA CAACGTTTGG TATGGGCATG CACAGGCCTA 301 GAGGTGGGCA GGGGACAGCC ATTAGGTGTG GGTGTAAGTG GACATCCTTT 351 CCTAAATAAA TATGATGATG TTGAAAATTC AGGGAGTGGT GGTAACCCTG 401 GACAGGATAA CAGGGTTAAT GTTGGTATGG ATTATAAACA AACACAATTA 451 TGCATGGTTG GATGTGCCCC CCCTTTGGGC GAGCATTGGG GTAAAGGTAA 501 ACAGTGTACT AATACACCTG TACAGGCTGG TGACTGCCCG CCCTTAGAAC 551 TTATTACCAG TGTTATACAG GATGGCGATA TGGTTGACAC AGGCTTTGGT 601 GCTATGAATT TTGCTGATTT GCAGACCAAT AAATCAGATG TTCCTATTGA 651 TATATGTGGC ACTACATGTA AATATCCAGA TTATTTACAA ATGGCTGCAG 701 ACCCTTATGG TGATAGATTA TTTTTTTTTC TACGGAAGGA ACAAATGTTT 751 GCCAGACATT TTTTTAACAG GGCTGGCGAG GTGGGGGAAC CTGTGCCTGA 801 TACTCTTATA ATTAAGGGTA GTGGAAATCG AACGTCTGTA GGGAGTAGTA 851 TATATGTTAA CACCCCAAGC GGCTCTTTGG TGTCCTCTGA GGCACAATTG 901 TTTAATAAGC CATATTGGCT ACAAAAAGCC CAGGGACATA ACAATGGTAT 951 TTGTTGGGGT AATCAACTGT TTGTTACTGT GGTAGATACC ACACGCAGTA 1001 CCAACATGAC ATTATGTGCA TCCGTAACTA CATCTTCCAC ATACACCAAT 1051 TCTGATTATA AAGAGTACAT GCGTCATGTG GAAGAGTATG ATTTACAATT 1101 TATTTTTCAA TTATGTAGCA TTACATTGTC TGCTGAAGTA GTGGCCTATA 1151 TTCACACAAT GAATCCCTCT GTTTTGGAAG ACTGGAACTT TGGGTTATCG 1201 CCTCCCCCAA ATGGTACATT AGAAGATACC TATAGGTATG TGCAGTCACA 1251 GGCCATTACC TGTCAAAAGC CCACTCCTGA AAAGCAAAAG CCAGATCCCT 1301 ATAAGAACCT TAGTTTTTGG GAGGTTAATT TAAAAGAAAA GTTTTCTAGT 1351 GAATTGGATC AGTATCCTTT GGGACGCAAG TTTTTGTTAC AAAGTGGATA 1401 TAGGGGACGG TCCTCTATTC GTACCGGTGT TAAGCGCCCT GCTGTTTCCA 1451 AAGCCTCTGC TGCCCCTAAA CGTAAGCGCG CCAAAACTAA AAGGTAA

(13) The description is further illustrated in combination with the Examples, wherein it is not limited to the Examples.

EXAMPLE 1

Expression of the Truncated HPV6 L1 Protein (SEQ ID NO. 1)

(14) Preparation of HPV6 L1 Gene Fragments as PCR Template

(15) The full-length gene of HPV-6 L1 was synthesized by Shanghai Boya Bio Co. The synthesized gene fragment has a full length of 1503 by and has a sequence of SEQ ID NO:5. Based on the synthetic full-length gene fragment of HPV-6 L1, the truncated HPV 6 L1 protein according to the invention was prepared as a template.

(16) Construction of Non-Fusion Expression Vector of Truncated HPV6 L1 Gene

(17) The full-length gene fragment of HPV-6 L1 synthesized in the previous step were used as the template for the next PCR reaction. The forward primer was 6N3F: 5′-CAT ATG CCT AGC GAC AGC ACA GTA TA-3′ (SEQ ID NO:10), at the 5′ terminal of which the restriction endonuclease NdeI site was introduced. The sequence of NdeI site was CAT ATG, wherein ATG was the initiation codon in E. coli system. The reverse primer was 6CR: 5′-GTC GAC TTA CCT TTT AGT TTT GGC GC-3′ (SEQ ID NO:11), at the 5′ terminal of which the restriction endonuclease Sa1I site was introduced. Amplification was performed in a Biometra T3 PCR thermocycler using the following parameters:

(18) TABLE-US-00002 94° C. denaturation 5 min  1 cycle 94° C. denaturation 50 sec 25 cycles 57° C. annealing 50 sec 72° C. elongation 2 min 72° C. elongation 10 min  1 cycle

(19) The DNA fragments, about 1.5 kb in length, were obtained after amplification. The PCR products were linked to the pMD 18-T vector (Takara Biosciences). After digestion with NdeI/Sa1I, it was identified that positive colonies, wherein the truncated HPV6 L1 gene was inserted, were obtained, designated as pMD 18-T-HPV6N3C-L1.

(20) The nucleotide sequence of interest, which was inserted into the plasmid pMD 18-T-HPV6N3C-L1, was determined as SEQ ID NO: 6 by Shanghai Boya Bio Co. through using M13 +/− primers. SEQ ID NO:6 encodes the amino acid sequence set forth in SEQ ID NO:1 which corresponds to a HPV 6 L1 protein having 3 amino acids truncated at its N-terminal and no amino acid truncated at its C-terminal and was designated as HPV6N3C-L1.

(21) The truncated HPV6N3C-L1 gene fragments were obtained by NdeI/Sa1I digestion of plasmid pMD 18-T-HPV6N3C-L1 The fragments were linked to the prokaryotic expression vector pTrxFus digested with NdeI/Sa1I (Invitrogen). Since the fusion protein was cleaved, the protein of interest was expressed immediately after initiating the expression of the amino acid Met, without other fusion proteins included. Colonies were screened with NdeI/Sa1I digestion. Positive colonies containing the insert of the L1 gene fragment were labeled pTRX-HPV6N3C-L1. 1 μL plasmid pTRX-HPV6N3C-L1 (0.15 mg/ml) was used to transform 40 μL competent E. coli GI698 (Invitrogen) prepared by Calcium chloride method, and then were coated on solid CAA media (dissolving 6 g Na.sub.2HPO.sub.4, 3 g KH.sub.2PO.sub.4, 0.5 g NaCl, 1 g NH.sub.4Cl, 20 g casein hydrolysate, 0.095 MgCl.sub.2, 1.5 g agar powder, and 20 ml 50% glycerinin 900 ml deionized water, and was added) containing benzyl chloride (at a final concentration of 100 mg/ml, the same as below). Plates were incubated at 30° 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 IMC medium containing benzyl chloride. Cultures were incubated in a shaking incubator at 220 rpm for 10 h at 25° C., and then 1 ml bacterial solution was freeze-dried and stored at −70° C.

(22) Expression of HPV6N3C-L1 in Large Scale

(23) E. coli transformed with pTRX-HPV6N3C-L1 was taken from freeze-dried stain at −70° C., and diluted with a little sterile water, and then incubated in 50 mL IMC medium containing benzyl amine at 200 rpm and 30° C. for 8 h. Then, the cultures were transferred to ten flasks (5 ml cultures per flask), each of which contains 500 mL LB medium, and were incubated in a shaking incubator overnight at 200 rpm and 30° C.

(24) A 50 L fermenter made by Shanghai Baoxing Biological Ltd. was used in large-scale incubation. pH electrode was calibrated. 30 L LB medium was prepared and transferred into the fermenter, sterilized at 121° C. for 30 minutes. Dissolved oxygen electrode was calibrated, wherein the value was determined as 0 before introduction of air after sterilization and as 100% prior to vaccination after introduction of air while stirring at 100 rpm at the beginning.

(25) Preparation of the feed: 30 g casein hydrolysates was dissolved in 100 mL deionized water to prepare a solution (30%), and 50 g glucose was dissolved in 100 ml deionized water to prepared a glucose solution (50%). The two mixtures were sterilized at 121° C. for 20 min.

(26) On the second day, the starter cultures in the ten flasks (for a total of 5 L) were transferred to the fermenter. At 30° C. and pH 7.0, the dissolved O.sub.2 was maintained at >40% by regulating agitation rate or air supply manually.

(27) Flow Feed: 50% glucose and 30% casein hydrolysates were mixed at a 2:1 mass ratio.

(28) Flow rates were as follows:

(29) 25 ml/min was defined as 100%.

(30) 1 h: 5%

(31) 2 h: 10%

(32) 3 h: 20%

(33) 4 h: 40%

(34) 5 h to the end: 60%

(35) When OD.sub.600 reached about 10.0, the culture temperature was lowered to 25° C. and 4 g tryptophan was added to begin an induction culture of 4 h. Fermentation was halted when OD.sub.600 reached about 40. The culture was then centrifuged to obtain strains (about 2.5 kg).

(36) IMC medium (1 liter):

(37) TABLE-US-00003 Na.sub.2HPO.sub.4 6 g KH.sub.2PO.sub.4 3 g NaCl 0.5 g   NH.sub.4Cl 1 g Casein Hydrolysates 20 g  MgCl.sub.2 0.095 g   

EXAMPLE 2

Preparation of HPV6N3C-L1 with a Purity of about 70%

(38) 1 g strains were re-suspended in 10 ml lysis buffer (20 mM tris buffer pH 7.2, 300 mM NaCl). Strains were disrupted by passing through a APV homogenizer (Invensys Group) for five times at a pressure of 600 bar. The homogenate was centrifuged at 30,000 g (13,500 rpm in JA-14 rotor) for 15 min. The supernatant was subjected to SDS-PAGE on a 10% gel. At this stage, the HPV6N3C-L1 had a purity of about 10%. 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 retention molecular weight was 30 kDa, the dialysate was 10 mM phosphate buffer pH 6.0, and the dialysis volume was three times as large as the volume of supernatant. After thorough dialysis, the mixture was centrifuged at 12,000 g (9500 rpm in JA-10 rotor (Beckman J25 high speed centrifuge)) for 20 min, and the precipitation was collected. The precipitation was re-suspended in 10 mM phosphate buffer pH 7.0 containing 10 mM DTT and 300 mM NaCl, wherein the volume of the buffer was 1/10 times as large as the volume of the supernatant. The mixture was stirred for 30 min and centrifuged at 30,000 g (13,500 rpm in JA-14 rotor (Beckman J25 high speed centrifuge)) for 20 min. The supernatant passes through a 0.22 μm filter membrane. The sample was used for cation exchange chromatography of the next step. 30 μL of 6× loading buffer was added to 150 μL of the filtered supernatant, and the result solution was mixed. After heating in a water bath at 80° C. for 10 min, 10 ul of the sample was subjected to SDS-PAGE on a 10% gel at 120V for 120 min. The electrophoretic bands were stained by Coomassie brilliant blue. The result was shown in FIG. 1. According to the analysis of SDS-PAGE, HPV6N3C-L1 protein was purified and enriched after the steps of precipitation and re-dissolution, with the purity of about 70%.

EXAMPLE 3

Chromatography Purification of HPV6N3C-L1

(39) Purification of HPV6N3C-L1 by Cation exchange Chromatography

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

(41) Chromatographic media: SP Sepharose 4 Fast Flow

(42) Column Volume: 5.5 cm×20 cm

(43) Buffer: 20 mM phosphate buffer pH 7.0, 10 mM DTT 20 mM phosphate buffer pH 7.0, 10 mM DTT, 2M NaCl

(44) Flow Rate: 25 mL/min

(45) Detector Wavelength: 280 nm

(46) Sample: 3 L 70% pure HPV6N3C-L1 solution

(47) Elution protocol: eluting undesired proteins with 200 mM NaCl, eluting the protein of interest with 500 mM NaCl, collecting 500 mM NaCl elutate, and finally getting about 900 mL purified HPV6N3C-L1 sample.

(48) Purification of HPV6N3C-L1 by CHT-II Chromatography

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

(50) Chromatographic media: CHT-II (Bio-Rad)

(51) Column Volume: 5.5 cm×20 cm

(52) Buffer: 10 mM phosphate buffer pH 7.0, 10 mM DTT, 0.5M NaCl

(53) Flow Rate: 20 mL/min

(54) Detector Wavelength: 280 nm

(55) Sample: 500 mM NaCl elutate from SP Sepharose 4 Fast Flow

(56) Elution protocol: directly collecting the pass-through containing the protein of interest.

(57) The pass-through, which contains HPV6N3C-L1, was collected and about 1000 mL purified HPV6N3C-L1 was obtained. 30 μL 6× loading buffer was added to 150 μL HPV6N3C-L1 sample purified according to the method of the Example, and then the result solution was mixed thoroughly. After heating the solution in a water bath at 80° C. for 10 min, a 10 uL sample was subjected to SDS-PAGE on a 10% gel at 120V for 120 min. The electrophoretic bands were stained by Coomassie brilliant blue. The result was shown in FIG. 2. The concentration of the protein of interest was about 0.7 mg/ml, and the purity was greater than 98% according to SDS-PAGE.

EXAMPLE 4

Assembly of HPV6N3C-L1 VLPs

(58) Equipment: Centrasette 5 Tangential Flow Filter (Pall Co.), retention MW 30 kDa.

(59) Sample: 1000 mL HPV6N3C-L1 obtained in Example 3

(60) Sample Concentration Sample was concentrated to 800 mL with the system tangential flow rate was adjusted to 50 mL/min

(61) Sample renaturation: Sample Renaturation: Sample buffer was exchanged with 10 L renaturation buffer (20 mM PB pH 6.0, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2, 0.5M NaCl, 0.003% Tween-80) thoroughly. When running the Tangential Flow Filter, the pressure was 0.5 psi and the tangential flow rate was 10 mL/min. When exchange was finished, the sample buffer was replaced with storage buffer (20 L PBS: 20 mM PB pH 6.5, 0.5M NaCl). The exchange volume was 20 L. The running pressure was 0.5 psi and the tangential flow rate was 25 mL/min. When the liquid exchange was finished, the sample was aseptically filtrated with a Pall filter (0.20 μm), and thereby obtaining HPV6N3C-L1 VLPs. The HPV6N3C-L1 VLPs were stored at 4° C. for further use.

EXAMPLE 5

Determination of the Morphology and Immunogenicity of HPV6N3C-L1 VLPs

(62) Transmission Electron Microscopy (TEM) of HPV6N3C-L1 VLPs

(63) The equipment was a JEOL 100 kV Transmission Electron Microscope (100,000× magnification). HPV6N3C-L1 VLPs were negatively stained with 2% phosphotungstic acid at pH 7.0, and fixed on a copper grid. Results were shown in FIG. 3. It could be seen that the VLPs obtained in Example 4 had a radius of approximately 25 nm, and were homogenous and in a hollow form.

(64) Dynamic Light-Scattering Measurement of HPV6N3C-L1 VLPs

(65) 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 one obtained in Example 4. The sample was passed through a 0.22 μm filter membrane prior to the measurement. Results were shown in FIG. 4. The result shows that HPV6N3C-L1 VLPs had a Hydrodynamic radius of 25.46 nm.

(66) Establishment a Model of Pseudovirion Neutralization Assay for HPV6:

(67) HPV can hardly be cultured in vitro, and the HPV host had a strong specificity. 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 productivity of HPV vaccine quickly, there was a need to establish a efficient model for in vitro neutralization assays.

(68) In Vitro Infection Model of Pseudovirion: According to the characteristic that HPV VLP can package nucleic acids non-specifically, HPV pseudivirion was formed by expressing HPV L1 and L2 protein in cells, and by packaging viral DNA of episome or introducing reporter plasmids heterologously. Methods include expression systems based on recombinant viruses and cotransfection of multi-plasmids (see Yeager, M. D, Aste-Amezaga, M. et al (2000) Virology (278) 570-7).

(69) The invention utilizes cotransfection of a multi-plasmid system. Some improvements were made as follows. An optimized calcium phosphate transfection method was established for the 293 FT cell line, with a transfection efficiency of above 90%, which facilitate the production on a large scale. The resultant codon-optimized expression plasmid of HPV protein could express HPV L1 and L2 gene efficiently in mammalian cell lines, facilitating efficient assembly of pseudovirion.

(70) 1. Construction of HPV Pseudovirion:

(71) P6L1h, p6L2h and pN31-EGFP (donated by Professor John T. Schiller of NIH) contain genes for HPV6L1, HPV6L2, and GFP, respectively. These plasmids were purified using CsCl density gradient centrifugation as described in The Molecular Cloning Experiment Guide, (3rd edition). The purification procedure was as follows: Plasmids were used to transform E. coli DH5α; Single colonies were transferred into 500 mL LB culture medium and incubated in a shaking flask at 37° C. for 16 h; Culture medium was centrifuged at 9,000 g for 5 min and the stains were collected; The following substances were successively added to bacteria in each 1000 mL LB: 40 mL solution I (50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA pH 8.0) and 2 ml 1 μg/μL RNase A), 40 mL solution II (0.2M NaOH, 1% SDS), and 48 mL solution III (60.0 mL 5M potassium acetate, 11.5 mL acetic acid, and 28.5 mL deionized water); After placing on ice for 10 min, the mixture was centrifuged at 15,000 g for 20 min at 4° C.; The supernatant was mixed with 0.6 volume of isopropyl alcohol, then was centrifuged again at 15,000 g for 30 min; The supernatant was decanted into waste and the precipitation was washed with 70% ethanol; The precipitation was dissolved in TE and the content of DNA was determined; CsCl was dissolved in the solution of DNA (1 g DNA per 1.01 g CsCl), and then 100 μL 10 mg/mL EB solution was also dissolved in it; The mixture was centrifuged using a Beckman NVT65 centrifuge at 62,000 rpm for 10 hr at 20° C.; Closed circle DNA section was collected using an injector pinhead; EB was extracted with equivalent volume of Isoamyl alcohol repeatedly for four times; Three volumes of deionized water and eight volumes of dry ethanol were added to one volume of DNA solution, and then the mixture was centrifuged at 20000 g for 30 min at 4° C.; The precipitation was collected and washed with 75% ethanol, and then dissolved in 1 mL TE; The concentration of the DNA solution was determined, then the solution was stored in small packages at −20° C.

(72) The purified p6L1h, p6L2h and pN31-EGFP co-transfected 293 FT cells (Invitrogen) cultured on a 10 cm cell culture plate by calcium phosphate method. The calcium phosphate method was described as follows. 40 μg p6L1h, 40 μg p6L2h and 40 μg pN31-EGFP were separately added to the mixture of 1 mL HEPES solution (125 μL 1M HEPES/50 mL deionized water, at pH7.3 and 4° C.) and 1 mL 0.5M CaCl.sub.2 solution. After mixing, 2 mL 2× HeBS solution (0.28M NaCl (16.36 g), 0.05M HEPES (11.9 g), 1.5 mM Na.sub.2HPO.sub.4 (0.213 g), dissolved in 1000 mL deionized water, at pH 6.96 and −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 293 FT cells were cultured. The original culture medium was replaced with 10 ml complete medium (Invitrogen Co.) 6 hours later. 48 hours after transfection, 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 suspended in 1 mL cytolytic solution (0.25% Brij58, 9.5 mM MgCl.sub.2). After lysing, cell lysate was centrifugated at 5,000 g for 10 min and the supernatant was collected. The Pseudovirion solution was obtained after adding 5M NaCl to the supernatant to a final concentration of 850 mM, then was stored in small packages at −20° C.

(73) 293 FT cells (Invitrogen) were spread on a 96-well cell culture plate (1.5×10.sup.4 cells/well), Neutralization assay was performed five hours later. Serum samples were serially diluted with 10% DMEM half-by-half. 50 μL diluted samples were separately mixed with 50 μL Pseudovirion solutions diluted with 10% DMEM (moi=0.1). After incubating at 4° C. for 1 h, the mixture was added to the 96-well cell culture plate spread with 293 FT cells. The mixture was then incubated for 72 h at 37° C. Neutralization titers of samples were estimated by observing fluorescence. Infection percentage of cells in each well was checked by flow cytometry (EPICS XL, American Beckman Coulter Co.). The exact titers of monoclonal antibodies or polyclonal antibodies were calculated. Infection percentage was the percentage of cells in the positive region minus the uninfected cells in the positive region.
Infection control percentage=(1−infection percentage of sample cell/infection percentage of negative cell)×100%

(74) Neutralization titer was defined as the highest dilution multiple by which the infection control percentage was just above 50%. Monoclonal and polyclonal antibodies were considered as having neutralizing capacity if their infection control percentage was above 50% after 50 times dilutions.

(75) Measurement of Immune Protection of Animals Vaccinated with HPV6 VLPs

(76) Female rabbits (general level), 6-8 weeks old, were purchased from the Disease Prevention and Control Center of Guangxi province, where they were raised. HPV6N3C-L1 VLPs prepared in Example 4, were mixed with equal amount of complete Freund's Adjuvant for the first immunization. For the booster, HPV6N3C-L1 VLPs were mixed with incomplete Freund's Adjuvant. Rabbits were immunized via muscle injection, with 100 μg per rabbit for the first immunization, and with 50 μg per rabbit for the booster separately at week 4, 10. After immunization, external vein blood was collected every week, and serum was separated and stored for detection.

(77) Female goats (general level), 6-8 weeks old, were purchased from the Disease Prevention and Control Center of Guangxi province, where they were raised. HPV6N3C-L1 VLPs as prepared in Example 4, were mixed with equal amount of complete Freund's adjuvant for the first immunization. For the booster, HPV6N3C-L1 VLPs were mixed with incomplete Freund's adjuvant. Goats were immunized via muscle injection, with 1 mg per goat for the first immunization, and with 0.5 mg per goat for the booster separately at weeks 4, and 18. After immunization, external vein blood was collected, and serum was separated and stored for detection.

(78) Neutralization titers of the anti-serums were evaluated using a pseudovirion-based neutralization cell model assay. As shown in FIGS. 5 and 6, the vaccine produced by mixing HPV6N3C-L1 VLPs prepared in Example 4 with Freund's adjuvants (aluminum hydroxide or aluminum phosphate adjuvants available commercially or self-prepared may be used besides Freund's adjuvants) had good immunogenicity, could induce neutralizing antibodies with a high titer in animals, and could be used as an effective vaccine for the prevention of HPV infection.

(79) Measurement of Immune Protection of Mice Vaccinated with HPV6/11 Bivalent Vaccine.

(80) Four SPF BALB/c mice, 4-5 weeks old, were used. HPV6N5C-L1 and HPV11N4C-L1 VLPs, prepared according to the method similar to that of Examples 1-4, were mixed at a ratio of 1:2 (by weight), wherein the final concentrations of them were 40 μg/mL and 80 μg/mL, respectively. The vaccine was mixed with an equal amount of complete Freund's adjuvant for the first immunization, and was mixed with an equal amount of incomplete Freund's adjuvant for the booster.

(81) Mice were immunized by muscle injection. The amount for the first immunization was 10 μg HPV6N5C-L1 and 20 μg HPV11N4C-L1 per mouse. The booster was administered every two weeks. The amount for the booster was 20 μg HPV6N5C-L1 and 40 μg HPV11N4C-L1 per mouse.

(82) After immunization, external vein blood was collected every week and serum was separated. The titers of neutralizing antibodies against HPV6 and HPV 11 in immunized mice were separately determined according to the method of Example 5.

(83) Results were shown in FIG. 7, indicating that HPV6/11 bivalent vaccine, prepared by blending HPV6N5C-L1 and HPV11N4C-L1 VLPs as prepared in Examples 1-4, had good immunogenicity, could induce neutralizing antibodies with a high titer against HPV 6 and HPV 11 in animals, and could be used as an effective vaccine for the prevention of HPV6/HPV11 infection (besides the Freund's adjuvants used in the experiments, the vaccine may be prepared by blending the two HPV6N5C-L1 and HPV11N4C-L1, with aluminum hydroxide or aluminum phosphate adjuvants available commercially or self-prepared).

(84) The Amino Acid Sequence of HPV11N4C-L1 is showed in SEQ ID NO: 7 as follows.

(85) TABLE-US-00004 Met Ser Asp Ser Thr Val Tyr Val Pro Pro Pro Asn Pro Val Ser Lys 1                 5                      10                    15 Val Val Ala Thr Asp Ala Tyr Val Lys Arg Thr Asn Ile Phe Tyr His                20                    25                     30 Ala Ser Ser Ser Arg Len Leu Ala Val Gly His Pro Tyr Tyr Ser Ile            35                   40                     45 Lys Lys Val Asn Lys Thr Val Val Pro Lys Val Ser Gly Tyr Gln Tyr     50                      55                     60 Arg Val Phe Lys Val Val Leu Pro Asp Pro Asn Lys Phe Ala Leu Pro 65                     70                    75                 80 Asp Ser Ser Leu Phe Asp Pro Thr Thr Gln Arg Leu Val Trp Ala Cys                  85                    90                    95 Thr Gly Leu Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Val Ser              100                   105                   110 Gly His Pro Leu Leu Asn Lys Tyr Asp Asp Val Glu Asn Ser Gly Gly          115                   120                   125 Tyr Gly Gly Asn Pro Gly Gln Asp Asn Arg Val Asn Val Gly Met Asp 130                       135                   140 Tyr Lys Gln Thr Gln Leu Cys Met Val Gly Cys Ala Pro Pro Leu Gly 145                  150                    155                 160 Glu His Trp Gly Lys Gly Thr Gln Cys Ser Asn Thr Ser Val Gln Asn                  165                    170                    175 Gly Asp Cys Pro Pro Leu Glu Leu Ile Thr Ser Val Ile Gln Asp Gly              180                    185                     190 Asp Met Val Asp Thr Gly Phe Gly Ala Met Asn Phe Ala Asp Leu Gln         195                   200                   205 Thr Asn Lys Ser Asp Val Pro Leu Asp Ile Cys Gly Thr Val Cys Lys     210                    215                   220 Tyr Pro Asp Tyr Leu Gln Met Ala Ala Asp Pro Tyr Gly Asp Arg Leu 225                   230                   235                240 Phe Phe Tyr Leu Arg Lys Glu Gln Met Phe Ala Arg His Phe Phe Asn                  245                   250                   255 Arg Ala Gly Thr Val Gly Glu Pro Val Pro Asp Asp Leu Leu Val Lys              260                    265                  270 Gly Gly Asn Asn Arg Ser Ser Val Ala Ser Ser Ile Tyr Val His Thr          275                    280                     285 Pro Ser Gly Ser Leu Val Ser Ser Glu Ala Gln Leu Phe Asn Lys Pro     290                     295                   300 Tyr Trp Leu Gln Lys Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly 305                  310                    315                320 Asn His Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met                  325                    330                  335 Thr Leu Cys Ala Ser Val Ser Lys Ser Ala Thr Tyr Thr Asn Ser Asp             340                     345                    350 Tyr Lys Glu Tyr Met Arg His Val Glu Glu Phe Asp Leu Gln Phe Ile          355                   360                   365 Phe Gln Len Cys Ser Ile Thr Leu Ser Ala Glu Val Met Ala Tyr Ile     370                    375                   380 His Thr Met Asn Pro Ser Val Leu Glu Asp Trp Asn Phe Gly Leu Ser 385                   390                   395                400 Pro Pro Pro Asn Gly Thr Leu Glu Asp Thr Tyr Arg Tyr Val Gln Ser                   405                    410                  415 Gln Ala Ile Thr Cys Gln Lys Pro Thr Pro Glu Lys Glu Lys Gln Asp               420                   425                   430 Pro Tyr Lys Asp Met Ser Phe Trp Glu Val Asn Leu Lys Glu Lys Phe          435                   440                   445 Ser Ser Glu Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln      450                 455                    460 Ser Gly Tyr Arg Gly Arg Thr Ser Ala Arg Thr Gly Ile Lys Arg Pro 465                   470                   475                 480 Ala Val Ser Lys Pro Ser Thr Ala Pro Lys Arg Lys Arg Thr Lys Thr                   485                    490                   495 Lys Lys

(86) Measurement of Immune Protection of Mice Vaccinated with HPV6/11/16/18Quadrivalent Vaccine

(87) Four SPF BALB/c mice, 4-5 weeks old, were used. HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1 and HPV18N65C-L1 VLPs, prepared according to the method similar to that of Examples 1-4, were mixed at a ratio of 1:2:2:1 (by weight), wherein the final concentrations of them were 40 μg/mL, 80 μg/mL, 80 μg/mL and 40 μg/mL, respectively. The vaccine was mixed with an equal amount of complete Freund's adjuvant for the first immunization, and was mixed with an equal amount of incomplete Freund's adjuvant for the booster.

(88) Mice were immunized by muscle injection. The amount for the first immunization was 10 μg HPV6N5C-L1, 10 μg HPV18N65C-L1, 20 μg HPV11N4C-L1, and 20 μg HPV16N30C-L1 per mouse. The booster was administered every two weeks. The amount for the booster was 20 μg HPV6N5C-L1, 20 μg HPV18N65C-L1, 40 μg HPV11N4C-L1, and 40 μg HPV16N30C-L1 per mouse.

(89) After immunization, external vein blood was collected every week and serum was separated. The titers of neutralizing antibodies against HPV6, HPV 11, HPV 16 and HPV 18 in immunized mice were separately determined according to the method of Example 5.

(90) Results were shown in FIG. 8, indicating that HPV6/11/16/18 quadrivalent vaccine, prepared by blending HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1 and HPV18N65C-L1 VLPs as prepared in Examples 1-4, had good immunogenicity, could induce neutralizing antibodies with a high titer against HPV 6, HPV 11, HPV 16, and HPV 18 in animals, and could be used as a effective vaccine for the prevention of HPV6/HPV11/HPV16/HPV18 infection (in addition to the Freund's adjuvants used in the experiments, the vaccine could be prepared by blending the four HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1 and HPV18N65C-L1 VLPs with aluminum hydroxide or aluminum phosphate adjuvants available commercially or self-prepared).

(91) The Amino Acid Sequence of L1 of HPV6N5C-L1 is showed in SEQ ID NO 4 as follows.

(92) The Amino Acid Sequence of L1 of HPV16N30C-L1 is showed in SEQ ID NO 8 as follows.

(93) TABLE-US-00005 Met Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser 1                5                      10                     15 Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr               20                     25                     30 His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe Pro          35                     40                     45 Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys Val Ser Gly      50                     55                     60 Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro Asn Lys Phe 65                    70                     75                80 Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val                  85                     90                   95 Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val              100                    105                    110 Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn          115                    120                   125 Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys Ile     130                   135                    140 Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro 145                  150                    155                 160 Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn Val Ala                   165                    170                   175 Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr Val Ile              180                   185                    190 Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Thr         195                   200                    205 Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile Cys Thr Ser     210                   215                    220 Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly 225                    230                    235               240 Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His                  245                   250                   255 Leu Phe Asn Arg Ala Gly Ala Val Gly Asp Asn Val Pro Asp Asp Leu              260                    265                   270 Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr          275                     280                   285 Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Ile Phe     290                   295                    300 Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile 305                  310                    315                 320 Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser                  325                   330                    335 Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr              340                    345                   350 Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp         355                   360                   365 Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp Ile     370                   375                     380 Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu Asp Trp Asn 385                    390                    395               400 Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg                  405                   410                   415 Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr Pro Pro Ala              420                    425                     430 Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu Val Asn Len          435                   440                    445 Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys     450                    455                   460 Phe Leu Leu Gln Ala Gly Leu Glu Ala Lys Pro Lys Phe Thr Leu Gly 465                  470                    475                 480 Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys                   485                   490                    495 Arg Lys Lys Arg Lys Leu              500

(94) The Amino Acid Sequence of L1 of HPV18N65C-L1 is showed in SEQ ID NO 9 as follows.

(95) TABLE-US-00006 Met Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro Ser Val Ala 1                 5                     10                     15 Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser Ile Phe Tyr              20                     25                     30 His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro Tyr Phe Arg          35                     40                    45 Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys Val Ser Ala      50                    55                     60 Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro Asn Lys Phe 65                    70                    75                  80 Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln Arg Leu Val                  85                      90                   95 Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro Leu Gly Val              100                     105                  110 Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu Ser          115                    120                  125 Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val      130                    135                    140 Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro 145                    150                   155                160 Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro                    165                   170                   175 Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu              180                   185                   190 Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser         195                   200                    205 Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser     210                  215                    220 Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly 225                   230                    235                240 Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His                  245                  250                   255 Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu              260                   265                   270 Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr          275                   280                    285 Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe      290                    295                     300 Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val 305                   310                   315                 320 Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser                  325                    330                    335 Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln             340                     345                    350 Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr          355                   360                    365 Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp     370                   375                    380 Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu Glu Asp Trp 385                     390                    395              400 Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr                   405                   410                   415 Arg Phe Val Gln Ser Val Ala Ile Ala Cys Gln Lys Asp Ala Ala Pro              420                     425                    430 Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp          435                   440                   445 Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg     450                    455                  460 Lys Phe Leu Val Gln Ala Gly Leu Arg Arg Lys Pro Thr Ile Gly Pro 465                   470                   475                 480 Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ala Ser Lys Pro Ala Lys                  485                     490                   495 Arg Val Arg Val Arg Ala Arg Lys              500

(96) The Amino Acid Sequence of HPV11N4C-L1 VLP is shown in SEQ ID NO:7, as described above.

EXAMPLE 6

(97) The truncated HPV6L1 proteins set forth in SEQ ID NOs: 2, 3 and 4 were prepared according to the techniques used in examples 1-5. All these truncated proteins could be purified to an extent of above 98% and could be assembled into VLPs with a radius of about 25 nm. The results are shown in FIGS. 9, 10 and 11.