Truncated L1 protein of human papillomavirus type 11

Abstract

The invention relates to a truncated L1 protein of the Human Papillomavirus Type 11, a virus-like particle consisting of the 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 N-terminally truncated HPV11 L1 protein consisting of SEQ ID NO.: 2.

2. A polynucleotide encoding the N-terminally truncated protein according to claim 1.

3. A vector comprising the polynucleotide according to claim 2.

4. A cell comprising the vector according to claim 3.

5. A composition comprising the N-terminally truncated HPV11 L1 protein according to claim 1.

6. A HPV 11 virus-like particle (VLP) comprising a N-terminally truncated HPV11 L1 protein consisting of SEQ ID NO.: 2.

7. A method for producing a N-terminally truncated HPV11 L1 protein consisting of SEQ ID NO.: 2, comprising: a) expressing a HPV L1 gene encoding the N-terminally truncated HPV L1 protein in an E. coli expression system; b) disrupting the E. coli, which has expressed the N-terminally truncated HPV L1 protein, in a solution at a salt concentration of from 100 mM to 600 mM, and isolating the supernatant; c) 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 a precipitate; and d) redissolving the precipitation of c) in a solution at a salt concentration of from 150 mM to 2500 mM, adding a reductant to it, and then isolating the resultant solution, wherein the solution contains the N-terminally truncated HPV L1 protein with a purity of at least 50%.

8. A vaccine for prevention of condyloma acuminatum or HPV infections, comprising HPV11 VLP according to claim 6 and carriers or excipients useful for vaccines.

9. The vaccine for prevention of condyloma acuminatum or HPV infections according to claim 8 further comprising at least one HPV VLP selected from the group consisting of VLPs of HPV type 6, 16, 18, 31, 33, 45, 52, and 58.

10. The vaccine for prevention of condyloma acuminatum or HPV infections according to claim 8, further comprising a HPV 16 VLP comprising a protein having an amino acid sequence set forth in SEQ ID No: 8, and a HPV 18 VLP comprising a protein having an amino acid sequence set forth in SEQ ID No: 9.

11. A method of claim 7, further comprising: e) further purifying the N-terminally truncated HPV 11 L1 protein with a purity of at least 50% by a chromatography; and f) removing the reductant from the HPV 11 L1 protein obtained in e).

12. A method for producing a vaccine for prevention of condyloma acuminatum or HPV infections, comprising blending the VLP according to claim 6, with carriers or excipients useful for vaccines.

13. A method of claim 12 comprising blending the VLP according to claim 6, and one or more VLPs selected from the group consisting of VLPs of HPV types 6, 16, 18, 31, 33, 45, 52, and 58, with carriers or excipients useful for vaccines.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the SDS-PAGE result of HPV11N4C-L1 protein during steps a)-d) of the method according to the invention. Lane 1: Lysate supernatant; Lane 2: HPV11N4C-L1 protein precipitated by tangential flow; Lane 3: Redissolved HPV11N4C-L1 in a re-suspension solution. The result shows that the purity of HPV11N4C-L1 reached about 70% following the steps of precipitation and re-dissolution.

(2) FIG. 2 shows the SDS-PAGE result of HPV11N4C-L1 that was obtained in step d) and was further purified according to step e). Lane 1: HPV11N4C-L1 purified according to step e), 10 L; Lane 2: HPV11N4C-L1 purified according to step e), 20 L. The result shows that HPV11N4C-L1 purified according to step e) reached a purity of about 98%.

(3) FIG. 3 shows the transmission electron microscopy (TEM) photograph of HPV11N4C-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 HPV11N4C-L1 VLPs obtained in step f). The result shows that HPV11N4C-L1 VLP had a hydrodynamic radius of 27.19 nm and a particle assembly rate of 96.7%.

(5) FIG. 5 shows titers of neutralizing antibodies in serum at different stages after vaccination of rabbit with HPV11N4C-L1 VLPs. Vaccination times are indicated with arrows. The titer of neutralizing antibodies reached a peak level of 10.sup.5, 1-2 months after a booster.

(6) FIG. 6 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.

(7) FIG. 7 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.

(8) FIG. 8 shows the SDS-PAGE results of HPV11N3C-L1, HPV11N5C-L1 and HPV11N6C-L1 protein separately having 3, 5 and 6 amino acids truncated at the N-terminal of HPV 11 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: HPV11N3C-L1 purified according to step a)-e), 10 L; Lane 3: HPV11N5C-L1 purified according to step a)-e), 10 L; Lane 4: HPV11N6C-L1 purified according to step a)-e), 10 L. The result shows that the purity of HPV11N3C-L1, HPV11N5C-L1 and HPV11N6C-L1 protein having 3, 5 and 6 amino acids truncated at the N-terminal of HPV 11 L1 protein respectively, reached about 95% following the steps a)-e).

(9) FIG. 9 the transmission electron microscopy (TEM) photographs of the VLPs of HPV11N3C-L1, HPV11N5C-L1 and HPV11N6C-L1 protein separately having 3, 5 and 6 amino acids truncated at the N-terminal of HPV 11 L1 protein obtained following steps a)-f), taken at 50,000 magnification. 1. The transmission electron microscopy (TEM) photographs of HPV11N3C-L1 VLPs obtained following steps a)-f). 2. The transmission electron microscopy (TEM) photographs of HPV11N5C-L1 VLPs obtained following steps a)-f). 3. The transmission electron microscopy (TEM) photographs of HPV11N6C-L1 VLPs obtained following steps a)-f). 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.

(10) FIG. 10 shows the dynamic light-scattering measurement result of the VLPs of HPV11N3C-L1, HPV11N5C-L1 and HPV11N6C-L1 protein separately having 3, 5 and 6 amino acids truncated at the N-terminal of HPV 11 L1 protein obtained following steps a)-f). 1. The dynamic light-scattering measurement result of HPV11N3C-L1 VLPs obtained following steps a)-f). 2. The dynamic light-scattering measurement result of HPV11N5C-L1 VLPs obtained following steps a)-f). 3. The dynamic light-scattering measurement result of HPV11N6C-L1 VLPs obtained following steps a)-f). The result shows that HPV11N3C-L1 VLPs, HPV11N5C-L1 VLPs and HPV11N6C-L1 VLPs had a hydrodynamic radius of about 25 nm and a particle assembly rate of above 80%.

(11) TABLE-US-00001 SEQUENCES SEQIDNO:1: 1 MSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVS 61 GYQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKY 121 DDVENSGGYGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCP 181 PLELITSVIQDGDMVDTGFGAMNFADLOTNKSDVPLDICGTVCKYPDYLQMAADPYGDRL 241 FFYLRKEQMFARHFFNRAGTVGEPVPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQL 301 FNKPYWLQKAQGHNNGICWGNHLFVTVVDTTRSTNMTLCASVSKSATYTNSDYKEYMRHV 361 EEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQAIT 421 CQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGYRGRTSARTGIKRP 481 AVSKPSTAPKRKRIKTKK SEQIDNO:2 1 MPSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKV 61 SGYQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNK 121 YDDVENSGGYGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDC 181 PPLELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDR 241 LFFYLRKEQMFARHFFNRAGTVGEPVPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQ 301 LFNKPYWLQKAQGHNNGICWGNHLFVTVVDTTRSTNMTLCASVSKSATYTNSDYKEYMRH 361 VEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQAI 421 TCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGYRGRTSARTGIKR 481 PAVSKPSTAPKRKRTKTKK SEQIDNO:3 1 MDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSG 61 YQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWAQTGLEVGRGQPLGVGVSGHPLLNKYD 121 DVENSGGYGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPP 181 LELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLF 241 FYLRKEQMFARHFFNRAGTVGEPVPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLF 301 NKPYWLQKAQGHNNGICWGNHLFVIVVDTTRSTNMILCASVSKSATYTNSDYKEYMRHVE 361 EFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQAITC 421 QKPIPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGYRGRTSARTGIKRPA 481 VSKPSTAPKRKRIKIKK SEQIDNO:4 1 MSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSGY 61 QYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKYDD 121 VENSGGYGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPPL 181 ELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFF 241 YLRKEQMFARHFFNRAGTVGEPVPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLFN 301 KPYWLQKAQGHNNGICWGNHLFVIVVDTIRSTNMTLCASVSKSATYTNSDYKEYMRHVEE 361 FDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGILEDTYRYVQSQAITCQ 421 KPTPEKEKQDPYKDMSFWEVNLKEKESSELDQFPLGRKFLLQSGYRGRTSARTGIKRPAV 481 SKPSTAPKRKRTKIKK SEQIDNO:5 1 ATGTGGCGGCCTAGCGACAGCACAGTATATGTGCCTCCTCCCAACCCTGTATCCAAGGTT 61 GTTGCCACGGATGCGTATGTTAAACGCACCAACATATTTTATCACGCCAGCAGTTCTAGA 121 CTCCTTGCTGTGGGACATCCATATTACTCTATCAAAAAAGTTAACAAAACAGTTGTACCA 181 AAGGTGTCTGGATATCAATATAGAGTGTTTAAGGTAGTGTTGCCAGATCCTAACAAGTTT 241 GCATTACCTGATTCATCTCTGTTTGACCCCACTACACAGCGTTTAGTATGGGCGTGCACA 301 GGGTTGGAGGTAGGCAGGGGTCAACCTTTAGGCGTTGGTGTTAGTGGGCATCCATTGCTA 361 AACAAATATGATGATGTAGAAAATAGTGGTGGGTATGGTGGTAATCCTGGTCAGGATAAT 421 AGGGTTAATGTAGGTATGGATTATAAACAAACCCAGCTATGTATGGTGGGCTGTGCTCCA 481 CCGTTAGGTGAACATTGGGGTAAGGGTACACAATGTTCAAATACCTTTGTACAAAATGGT 541 GACTGCCCCCCGTTGGAACTTATTACCAGTGTTATACAGGATGGGGACATGGTTGATACA 601 GGCTTTGGTGCTATGAATTTTGCAGACTTACAAACCAATAAATCGGATGTTCCCCTTGAT 661 ATTTGTGGAACTGTCTGCAAATATCCTGATTATTTGCAAATGGCAGCAGACCCTTATGGT 721 GATAGGTTGTTTTTTTATTTGCGAAAGGAACAAATGTTTGCTAGACACTTTTTTAATAGG 781 GCCGGTACTGTGGGGGAACCTGTGCCTGATGACCTGTTGGTAAAAGGGGGTAATAATAGA 841 TCATCTGTAGCTAGTAGTATTTATGTACATACACCTAGTGGCTCATTGGTGTCTTCAGAG 901 GCTCAATTATTTAATAAACCATATTGGCTTCAAAAGGCTCAGGGACATAACAATGGTATT 961 TGCTGGGGAAACCACTTGTTTGTTACTGTGGTAGATACCACACGCAGTACAAATATGACA 1021 CTATGTGCATCTGTGTCTAAATCTGCTACATACACTAATTCAGATTATAAGGAATACATG 1081 CGCCATGTGGAAGAGTTTGATTTACAGTTTATTTTTCAATTGTGTAGCATTACATTATCT 1141 GCAGAAGTCATGGCCTATATACACACAATGAATCCTTCTGTTTTGGAGGACTGGAACTTT 1201 GGTTTATCGCCTCCACCAAATGGTACACTGGAGGATACTTATAGATATGTACAGTCACAG 1261 GCCATTACCTGTCAGAAACCCACACCCGAAAAAGAAAAACAGGACCCCTATAAGGATATG 1321 AGTTTTTGGGAGGTTAACTTAAAAGAAAAGTTTTCTTATGAATTAGATCAGTTTCCCCTT 1381 GGACGTAAGTTTTTATTGCAAAGTGGATATCGAGGACGGACGTCTGCTCGTACAGGTATA 1441 AAGCGCCCAGCTGTGTCTAAGCCCTCTACAGCCCCCAAACGAAAACGTACCAAAACCAGA 1501 AAGTAA SEQIDNO:6 1 ATGAGCGACAGCACAGTATATGTGCCTCCTCCCAACCCTGTATCCAAGGTTGTTGCCACG 61 GATGCGTATGTTAAACGCACCAACATATTTTATCACGCCAGCAGTTCTAGACTCCTTGCT 121 GTOGGACATCCATATTACTCTATCAAAAAAGTTAACAAAACAGTTGTACCAAAGGTGTCT 181 GGATATCAATATAGAGTGTTTAAGGTAGTGTTGCCAGATCCTAACAAGTTTGCATTACCT 241 GATTCATCTCTGTTTGACCCCACTACACAGCTTTTAGTATGGGCGTGCACAGGGTTGGAG 301 GTAGGCAGGGGTCAACCTTTAGGCGTTGGTGTTAGTGGGCATCCATTGCTAAACAAATAT 361 GATGATGTAGAAAATAGTGGTGGGTATGGTGGTAATCCTGGTCAGGATAATAGGGTTAAT 421 GTAGGTATGGATTATAAACAAACCCAGCTATGTATGGTGGGCTGTGGTCCACCGTTAGGT 481 GAACATTGGGGTAAGGGTACACAATGTTCAAATACCTCTGTACAAAATGGTGACTGCCCC 541 CCGTTGGAACTTATTACCAGTGTTATACAGGATGGGGACATGGTTGATACAGGCTTTGGT 601 GCTATGAATTTTGCAGACTTACAAACCAATAAATCGGATGTTCCCCTTGATATTTGTGGA 661 ACTGTCTGCAAATATCCTGATTATTTGCAAATGGCAGGAGACCCTTATGGTGATAGGTTG 721 TTTTTTTATTTGCGAAAGGAACAAATGTTTGCTAGACACTTTTTTAATAGGGCCGGTACT 781 GTGGGGGAACCTGTGCCTGATGACCTGTTGGTAAAAGGGGGTAATAATAGATCATCTGTA 841 GCTAGTAGTATTTATGTACATACACCTAGTGGCTCATTGGTGTCTTCAGAGGCTCAATTA 901 TTTAATAAACCATATTGGCTTCAAAAGGCTCAGGGACATAACAATGGTATTTGOTGGGGA 961 AACCACTTGTTTGTTACTGTGGTAGATACCACACGCAGTACAAATATGACACTATGTGCA 1021 TCTGTGTCTAAATCTGCTACATACACTAATTCAGATTATAAGGAATACATGCGCCATGTG 1081 GAAGAGTTTGATTTACAGTTTATTTTTCAATTGTGTAGCATTACATTATCTGCAGAAGTC 1141 ATGGCCTATATACACACAATGAATCCTTCTGTTTTGGAGGACTGGAACTTTGGTTTATCG 1201 CCTCCACCAAATGGTACACTGGAGGATACTTATAGATATGTACAGTCACAGGCCATTACC 1261 TGTCAGAAACCCACACCCGAAAAAGAAAAACAGGACCCCTATAAGGATATGAGTTTTTGG 1321 GAGGTTAACTTAAAAGAAAAGTTTTCTTATGAATTAGATCAGTTTCCCCTTGGACGTAAG 1381 TTTTTATTGCAAAGTGGATATCGAGGACGGACGTCTGCTCGTACAGGTATAAAGCGCCCA 1441 GCTGTGTCTAAGCCCTCTACAGCCCCCAAACGAAAACGTACCAAAACCAGAAAGTAA

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

Example 1: Expression of the Truncated HPV11 L1 Protein (SEQ ID NO. 1)

(13) Preparation of HPV11 L1 Gene Fragments as PCR Template

(14) The full-length gene of HPV-11 L1 was synthesized by Shanghai Boya Bio Co. The synthesized gene fragment has a full length of 1506 bp and has a sequence of SEQ ID NO:5. Based on the synthetic full-length gene fragment of HPV-11 L1, the truncated HPV 11 L1 protein according to the invention was prepared as a template.

(15) Construction of Non-Fusion Expression Vector of Truncated HPV11 L1 Gene

(16) The full-length gene fragment of HPV-11 L1 synthesized in the previous step were used as the template for the next PCR reaction. The forward primer was 11N4F: 5-CAT ATG AGC GAC AGC ACA GTA TAT GTG-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 CTT TCT GGT TTT GGT ACG TTT-3 (SEQ ID NO: 11), at the 5 terminal of which the restriction endonuclease SalI site was introduced. Amplification was performed in a Biometra T3 PCR thermocycler using the following parameters:

(17) 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

(18) 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/SalI, it was identified that positive colonies, wherein the truncated HPV11 L1 gene was inserted, were obtained, designated as pMD 18-T-HPV11N4C-L1.

(19) The nucleotide sequence of interest, which was inserted into the plasmid pMD 18-T-HPV11N4C-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 11 L1 protein having 4 amino acids truncated at its N-terminal and no amino acid truncated at its C-terminal and was designated as HPV11N4C-L1.

(20) The truncated HPV11N4C-L1 gene fragments were obtained by NdeI/SalI digestion of plasmid pMD 18-T-HPV11N4C-L1. The fragments were linked to Non-Fusion Expression Vector pTO-T7 (Luo Wenxin et al., Chinese Journal of Biotechnology, 2000, 16:53-57). Colonies were screened with NdeI/SalI digestion. Positive colonies containing the insert of the L1 gene fragment were labeled pTO-T7-HPV11N4C-L1. 1 L plasmid pTO-T7-HPV11N4C-L1 (0.15 mg/ml) was used to transform 40 L competent E. coli ER2566 (New England Labs) prepared by Calcium chloride method, and then was coated on solid LB medium containing kanamycin (at a final concentration of 25 mg/mL, the same as below). The plates were incubated at 37 C. for about 10-12 h until single colonies could be observed clearly. Single colonies were transferred to a tube containing 4 ml liquid LB medium 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 freeze-dried and stored at 70 C.

(21) Expression of HPV11N4C-L1 in Large Scale

(22) E. coli transformed with pTO-T7-HPV11N4C-L1 was taken from the freeze-dried strain at 70 C. and diluted with a little sterile water, and then incubated in 50 mL LB medium containing Kanamycin at 200 rpm and 37 C. for 8 h. Then, the cultures were transferred to ten flasks (5 ml culture per flask), each of which contains 500 mL LB medium, and were incubated in a shaking incubator overnight at 200 rpm and 30 C. The cultures were the starter cultures.

(23) TABLE-US-00003 LB medium: Peptone: 10 g Yeast extract: 5 g NaCl: 10 g

(24) The above components were dissolved in IL deionized water; the resultant solution was adjusted to pH 7.2 by addition of NaOH, sterilized at 121 C. for 30 minutes and cooled to 50 C.

(25) 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 in situ 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.

(26) Preparation of the feed: 20 g peptone and 10 g yeast extract were dissolved in 100 ml deionized water to prepare a mixture of peptone and yeast extract (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.

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

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

(29) Flow rates were as follows:

(30) 25 mL/min was defined as 100%.

(31) 1 h: 5%

(32) 2 h: 10%

(33) 3 h: 20%

(34) 4 h: 40%

(35) 5 h to the end: 60%

(36) When OD.sub.600nm reached about 10, the culture temperature was lowered to 25 C. and 4 g IPTG was added to begin induction culture of 4 h. Fermentation was halted when OD.sub.600nm reached about 60. The culture was then centrifuged to obtain target strains expressing the HPV11N4C-L1 protein (about 2.7 kg).

Example 2: Preparation HPV11N4C-L1 with a Purity of about 70%

(37) 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 HPV11N4C-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 8.0 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 m filter membrane. The sample was further subjected to cation exchange chromatography. 30 L of 6 loading buffer was added to 150 L of the filtered supernatant, and the result solution was mixed. After heating 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. 1. According to the analysis of SDS-PAGE, HPV11N4C-L1 protein was purified and enriched after the steps of precipitation and re-dissolution, with the purity increased to about 70%.

Example 3: Chromatography Purification of HPV11N4C-L1

(38) Cation Exchange Chromatography of HPV11N4C-L1

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

(40) Chromatographic media: SP Sepharose 4 Fast Flow

(41) Column Volume: 5.5 cm20 cm

(42) Buffer: 20 mM phosphate buffer pH 8.0, 10 mM DTT

(43) 20 mM phosphate buffer pH 8.0, 10 mM DTT, 2M NaCl

(44) Flow Rate: 25 mL/min

(45) Detector Wavelength: 280 nm

(46) Sample: 3 L, about 70% HPV11N4C-L1 solution

(47) Elution protocol: eluting undesired proteins with 200 mM NaCl, eluting the protein of interest with 800 mM NaCl, collecting 500 mM NaCl elutate, and finally getting about 700 mL purified HPV11N4C-L1 sample.

(48) Purification of HPV11N4C-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 cm20 cm

(52) Buffer: 10 mM phosphate buffer pH7.0, 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 HPV11N4C-L1, was collected and about 300 mL purified HPV11N4C-L1 was obtained. 30 L 6 loading buffer was added to 150 L HPV11N4C-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 0.3 mg/ml, and the purity was greater than 98% according to SDS-PAGE.

Example 4: Assembly of HPV11N4C-L1 VLPs

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

(59) Sample: 300 mL HPV11N4C-L1 obtained in Example 3

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

(61) 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 m). The HPV11N4C-L1 VLPs were obtained and were stored at 4 for further use.

Example 5: Determination of the Morphology of HPV11N4C-L1 VLPs

(62) Transmission Electron Microscopy (TEM) of HPV11N4C-L1 VLPs

(63) The equipment was a JEOL 100 kV Transmission Electron Microscope (100,000 magnification). HPV11N4C-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 HPV11N4C-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 m filter membrane prior to the measurement. Results were shown in FIG. 4. The result shows that HPV11N4C-L1 VLPs had a Hydrodynamic radius of 27.19 nm and a particle assembly rate of 96.7%.

(66) Establishment of Pseudovirion Neutralization Assay for HPV11

(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 293FT 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 structural protein could express HPV L1 and L2 gene efficiently in mammalian cell lines, facilitating efficient assembly of pseudovirion.

(70) Construction of HPV Pseudovirion:

(71) P11L1h, p11L2h and pN31-EGFP (donated by Professor John T. Schiller of NIH) contain genes for HPV11L1, HPV11L2, 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:

(72) Plasmids were used to transform E. coli DH5;

(73) Single colonies were transferred into 500 mL LB culture medium and incubated in a shaking flask at 37 C. for 16 h;

(74) Culture medium was centrifuged at 9,000 g for 5 min and the stains were collected;

(75) The following substances were successively added to bacteria in each 1000 mL LB: 40 mL solution I (50 mM glucose, 25 mM Tris-CI pH 8.0, 10 mM EDTA pH 8.0) and 2 ml 1 g/L RNase A), 40 mL solution 11 (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);

(76) After placing on ice for 10 min. the mixture was centrifuged at 15,000 g for 20 min at 4 C.;

(77) The supernatant was mixed with 0.6 volume of isopropyl alcohol, then was centrifuged again at 15,000 g for 30 min at 4 C.;

(78) The supernatant was decanted into waste and the precipitation was washed with 70% ethanol;

(79) The precipitation was dissolved in TE and the content of DNA was determined;

(80) CsCl was dissolved in the solution of DNA (1 g DNA per 1.01 g CsCl), and then 100 L 10 mg/mL EB solution was also dissolved in it;

(81) The mixture was centrifuged using a Beckman NVT65 centrifuge at 62,000 rpm for 10 hr at 20 C.;

(82) Closed circle DNA section was collected using an injector pinhead:

(83) EB was extracted with equivalent volume of Isoamyl alcohol repeatedly for four times;

(84) 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.;

(85) The DNA precipitation was collected and washed with 75% ethanol, and then dissolved in 1 mL TE;

(86) The concentration of the DNA solution was determined, then the solution was stored in small packages at 20 C.

(87) The purified p11L1h, p11L2h and pN31-EGFP co-transfected 293FT cells (Invitrogen) cultured on a 10 cm cell culture plate by calcium phosphate method. The calcium phosphate method was described as follows. 40 g p11L1h, 40 g p11L2h, and 40 g pN31-EGFP were separately added to the mixture of 1 mL HEPES solution (125 L 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 2HeBS 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 293FT 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.

(88) 293FT cells (Invitrogen) were spread on a 96-well cell culture plate (1.510.sup.4 cells/well). Neutralization assay was performed five hours later. Serum samples were serially diluted with 10% DMEM half-by-half. 50 L diluted samples were separately mixed with 50 L 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 293FT 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=(1infection percentage of sample cell/infection percentage of negative cell)100%

(89) 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.

(90) Measurement of Immune Protection of Animals Vaccinated with HPV11 VLPs:

(91) Female rabbits (general level), 6-8 weeks old, were purchased from the Disease Prevention and Control Center of Guangxi province, where they were raised. HPV11N4C-L1 VLPs prepared in Example 4, were mixed with equal amount of complete Freund's Adjuvant for the first immunization. For the booster, HPV11N4C-L1 VLPs were mixed with incomplete Freund's Adjuvant. Rabbits were immunized via muscle injection, with 100 g per rabbit for the first immunization, and with 50 g 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.

(92) Neutralization titers of the anti-serums were evaluated using a pseudovirion-based neutralization cell model assay. As shown in FIG. 5, the vaccine produced by mixing HPV11N4C-L1 VLPs prepared in Example 4, could induce neutralizing antibodies with a high titer in animals, and could be used as an effective vaccine for the prevention of HPV infection.

(93) Measurement of Immune Protection of Rhesus Monkeys Vaccinated with HPV6/11 Bivalent Vaccine

(94) Four SPF BALB/c mice, 4-5 weeks old, were used. HPV6N5C-L1 VLPs and HPV11N4C-L1 VLPs, which were 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 g/mL and 80 g/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.

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

(96) After immunization, external vein blood was collected every week and serum was separated. The titers of neutralizing antibodies against HPV6 and HPV11 in immunized mice were separately determined according to the method of Example 5.

(97) Results were shown in FIG. 6, indicating that HPV6/11 bivalent vaccine, prepared by blending HPV6N5C-L1 and HPV11N4C-L1 VLPs prepared according to the method as described 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 VLPs of HPV16N5C-L1 and HPV11N4C-L1 with aluminum hydroxide or aluminum phosphate adjuvants available commercially or self-prepared).

(98) The Amino Acid Sequence of L1 of HPV6N5C-L1 is showed in SEQ ID NO: 7 as follows.

(99) TABLE-US-00004 MetAspSerThrValTyrValProProProAsnProValSerLysVal 151015 ValAlaThrAspAlaTyrValThrArgThrAsnIlePheTyrHisAla 202530 SerSerSerArgLeuLeuAlaValGlyHisProTyrPheSerIleLys 354045 ArgAlaAsnLysThrValValProLysValSerGlyTyrGlnTyrArg 505560 ValPheLysValValLeuProAspProAsnLysPheAlaLeuProAsp 65707580 SerSerLeuPheAspProThrThrGlnArgLeuValTrpAlaCysThr 859095 GlyLeuGluValGlyArgGlyGlnProLeuGlyValGlyValSerGly 100105110 HisProPheLeuAsnLysTyrAspAspValGlnAsnSerGlySerGly 115120125 GlyAsnProGlyGlnAspAsnArgValAsnValGlyMetAspTyrLys 130135140 GlnThrGlnLeuCysMetValGlyCysAlaProProLeuGlyGlnHis 145150155160 TrpGlyLysGlyLysGlnCysThrAsnThrProValGlnAlaGlyAsp 165170175 CysProProLeuGluLeuIleThrSerValIleGlnAspGlyAspMet 180185190 ValAspThrGlyPheGlyAlaMetAsnPheAlaAspLeuGlnThrAsn 195200205 LysSerAspValProIleAspIleCysGlyThrThrCysLysTyrPro 210215220 AspTyrLeuGlnMetAlaAlaAspProTyrGlyAspArgLeuPhePhe 225230235240 PheLeuArgLysGluGlnMetPheAlaArgHisPhePheAsnArgAla 245250255 GlyGluValGlyGluProValProAspThrLeuIleIleLysGlySer 260265270 GlyAsnArgThrSerValGlySerSerIleTyrValAsnThrProSer 275280285 GlySerLeuValSerSerGluAlaGlnLeuPheAsnLysProTyrTrp 290295300 LeuGlnLysAlaGlnGlyHisAsnAsnGlyIleCysTrpGlyAsnGln 305310315320 LeuPheValThrValValAspThrThrArgSerThrAsnMetThrLeu 325330335 CysAlaSerValThrThrSerSerThrTyrThrAsnSerAspTyrLys 340345350 GluTyrMetArgHisValGlnGluTyrAspLeuGlnPheIlePheGln 355360365 LeuCysSerIleThrLeuSerAlaGluValValAlaTyrIleHisThr 370375380 MetAsnProSerValLeuGluAspTrpAsnPheGlyLeuSerProPro 385390395400 ProAsnGlyThrLeuGluAspThrTyrArgTyrValGlnSerGlnAla 405410415 IleThrCysGlnLysProThrProGluLysGlnLysProAspProTyr 420425430 LysAsnLeuSerPheTrpGluValAsnLeuLysGluLysPheSerSer 435440445 GluLeuAspGlnTyrProLeuGlyArgLysPheLeuLeuGlnSerGly 450455460 TyrArgGlyArgSerSerIleArgThrGlyValLysArgProAlaVal 465470475480 SerLysAlaSerAlaAlaProLysArgLysArgAlaLysThrLysArg 485490495

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

(101) 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 g/mL, 80 g/mL, 80 g/mL and 40 g/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.

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

(103) After immunization, external vein blood was collected every week and serum was separated. The titers of neutralizing antibodies against HPV6, HPV11, HPV16 and HPV18 in immunized mice were separately determined according to the method of Example 5.

(104) Results were shown in FIG. 7, indicating that HPV6/11/16/18 quadrivalent vaccine, prepared by blending HPV6N5C-L1. HPV11N4C-L1, HPV16N30C-L1 and HPV18N65C-L1 VLPs prepared according to the method as described 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 (besides the Freund's adjuvants used in the experiments, the vaccine may be prepared by blending the four VLPs of HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1 and HPV18N65C-L1 VLPs with aluminum hydroxide or aluminum phosphate adjuvants available commercially or self-prepared).

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

(106) TABLE-US-00005 MetLeuProSerGluAlaThrValTyrLeuProProValProValSer 151015 LysValValSerThrAspGluTyrValAlaArgThrAsnIleTyrTyr 202530 HisAlaGlyThrSerArgLeuLeuAlaValGlyHisProTyrPhePro 354045 IleLysLysProAsnAsnAsnLysIleLeuValProLysValSerGly 505560 LeuGlnTyrArgValPheArgIleHisLeuProAspProAsnLysPhe 65707580 GlyPheProAspThrSerPheTyrAsnProAspThrGlnArgLeuVal 859095 TrpAlaCysValGlyValGluValGlyArgGlyGlnProLeuGlyVal 100105110 GlyIleSerGlyHisProLeuLeuAsnLysLeuAspAspThrGluAsn 115120125 AlaSerAlaTyrAlaAlaAsnAlaGlyValAspAsnArgGluCysIle 130135140 SerMetAspTyrLysGlnThrGlnLeuCysLeuIleGlyCysLysPro 145150155160 ProIleGlyGluHisTrpGlyLysGlySerProCysThrAsnValAla 165170175 ValAsnProGlyAspCysProProLeuGluLeuIleAsnThrValIle 180185190 GlnAspGlyAspMetValAspThrGlyPheGlyAlaMetAspPheThr 195200205 ThrLeuGlnAlaAsnLysSerGluValProLeuAspIleCysThrSer 210215220 IleCysLysTyrProAspTyrIleLysMetValSerGluProTyrGly 225230235240 AspSerLeuPhePheTyrLeuArgArgGluGlnMetPheValArgHis 245250255 LeuPheAsnArgAlaGlyAlaValGlyAspAsnValProAspAspLeu 260265270 TyrIleLysGlySerGlySerThrAlaAsnLeuAlaSerSerAsnTyr 275280285 PheProThrProSerGlySerMetValThrSerAspAlaGlnIlePhe 290295300 AsnLysProTyrTrpLeuGlnArgAlaGlnGlyHisAsnAsnGlyIle 305310315320 CysTrpGlyAsnGlnLeuPheValThrValValAspThrThrArgSer 325330335 ThrAsnMetSerLeuCysAlaAlaIleSerThrSerGluThrThrTyr 340345350 LysAsnThrAsnPheLysGluTyrLeuArgHisGlyGluGluTyrAsp 355360365 LeuGlnPheIlePheGlnLeuCysLysIleThrLeuThrAlaAspIle 370375380 MetThrTyrIleHisSerMetAsnSerThrIleLeuGluAspTrpAsn 385390395400 PheGlyLeuGlnProProProGlyGlyThrLeuGluAspThrTyrArg 405410415 PheValThrSerGlnAlaIleAlaCysGlnLysHisThrProProAla 420425430 ProLysGluAspProLeuLysLysTyrThrPheTrpGluValAsnLeu 435440445 LysGluLysPheSerAlaAspLeuAspGlnPheProLeuGlyArgLys 450455460 PheLeuLeuGlnAlaGlyLeuGluAlaLysProLysPheThrLeuGly 465470475480 LysArgLysAlaThrProThrThrSerSerThrSerThrThrAlaLys 485490495 ArgLysLysArgLysLeu 500

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

(108) TABLE-US-00006 MetArgProSerAspAsnThrValTyrLeuProProProSerValAla 151015 ArgValValAsnThrAspAspTyrValThrArgThrSerIlePheTyr 202530 HisAlaGlySerSerArgLeuLeuThrValGlyAsnProTyrPheArg 354045 ValProAlaGlyGlyGlyAsnLysGlnAspIleProLysValSerAla 505560 TyrGlnTyrArgValPheArgValGlnLeuProAspProAsnLysPhe 65707580 GlyLeuProAspThrSerIleTyrAsnProGlnThrGlnArgLeuVal 859095 TrpAlaCysAlaGlyValGlnIleGlyArgGlyGlnProLeuGlyVal 100105110 GlyLeuSerGlyHisProPheTyrAsnLysLeuAspAspThrGluSer 115120125 SerHisAlaAlaThrSerAsnValSerGlnAspValArgAspAsnVal 130135140 SerValAspTyrLysGlnThrGlnLeuCysIleLeuGlyCysAlaPro 145150155160 AlaIleGlyGlnHisTrpAlaLysGlyThrAlaCysLysSerArgPro 165170175 LeuSerGlnGlyAspCysProProLeuGluLeuLysAsnThrValLeu 180185190 GluAspGlyAspMetValAspThrGlyTyrGlyAlaMetAspPheSer 195200205 ThrLeuGlnAspThrLysCysGluValProLeuAspIleCysGlnSer 210215220 IleCysLysTyrProAspTyrLeuGlnMetSerAlaAspProTyrGly 225230235240 AspSerMetPhePheCysLeuArgArgGluGlnLeuPheAlaArgHis 245250255 PheTrpAsnArgAlaGlyThrMetGlyAspThrValProGlnSerLeu 260265270 TyrIleLysGlyThrGlyMetArgAlaSerProGlySerCysValTyr 275280285 SerProSerProSerGlySerIleValThrSerAspSerGlnLouPhe 290295300 AsnLysProTyrTrpLeuHisLysAlaGlnGlyHisAsnAsnGlyVal 305310315320 CysTrpHisAsnGlnLeuPheValThrValValAspThrThrArgSer 325330335 ThrAsnLeuThrIleCysAlaSerThrGlnSerProValProGlyGln 340345350 TyrAspAlaThrLysPheLysGlnTyrSerArgHisValGluGluTyr 355360365 AspLeuGlnPheIlePheGlnLeuCysThrIleThrLeuThrAlaAsp 370375380 ValMetSerTyrIleHisSerMetAsnSerSerIleLeuGluAspTrp 385390395400 AsnPheGlyValProProProProThrThrSerLeuValAspThrTyr 405410415 ArgPheValGlnSerValAlaIleAlaCysGlnLysAspAlaAlaPro 420425430 AlaGluAsnLysAspProTyrAspLysLeuLysPheTrpAsnValAsp 435440445 LeuLysGluLysPheSerLeuAspLeuAspGlnTyrProLeuGlyArg 450455460 LysPheLeuValGlnAlaGlyLeuArgArgLysProThrIleGlyPro 465470475480 ArgLysArgSerAlaProSerAlaThrThrAlaSerLysProAlaLys 485490495 ArgValArgValArgAlaArgLys 500

(109) The Amino Acid Sequence of L1 of HPV6N5C-L1 VLP is shown in SEQ ID NO:7, as described above.

Example 6

(110) The truncated HPV11L1 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. 8, 9 and 10.