CN107188932B

CN107188932B - Truncated human papilloma virus 16 type L1 protein and application thereof

Info

Publication number: CN107188932B
Application number: CN201710153290.7A
Authority: CN
Inventors: 许雪梅; 张婷; 陈雪; 刘洪洋; 周艳; 望朔
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of CAMS
Priority date: 2016-03-15
Filing date: 2017-03-15
Publication date: 2020-02-11
Anticipated expiration: 2037-03-15
Also published as: CN107188932A; CN107184973A

Abstract

The invention relates to a truncated human papilloma virus 16 type L1 protein and application thereof. Specifically, the invention relates to a truncated HPV16L1 protein, nucleotide for coding the protein, a vector containing the nucleotide, recombinant Bacmid, recombinant baculovirus, insect cells containing the recombinant baculovirus, virus-like particles consisting of the HPV16L1 protein, a vaccine containing the virus-like particles and a vaccine adjuvant, and application of the protein and the vector in preventing HPV infection or HPV infection-related diseases.

Description

Truncated human papilloma virus 16 type L1 protein and application thereof

Technical Field

The invention relates to a novel truncated human papilloma virus 16 type L1 protein, a virus-like particle consisting of the protein, a vaccine containing the virus-like particle and a vaccine adjuvant, and application of the protein and the vaccine adjuvant in preventing HPV infection or HPV infection-related lesions.

Background

More than 200 types of Human Papilloma Viruses (HPV) have been isolated and identified, and are classified into a mucophilic group and a dermophilic group. HPV in the mucosal group mainly infects mucous membranes and surrounding skin in the urogenital tract, around anus and oropharynx, and induces various benign and malignant lesions. According to the nature of the induced lesion, the high-risk type inducing malignant tumor (including HPV16,18,31,33,35,39,45,51,52,56,58,59,68, etc.), suspicious high-risk type (HPV26,30,53,66,67,69,70,73,82,85, etc.), unidentified type (HPV34,42,43,54,71,81,83,97,102,114, etc.), and low-risk type inducing benign lesion such as verrucous hyperplasia (HPV6,7,11,13,32,40,42,44,61,62,72,74,81,83,84,86,87,89,90,91,106, etc.) can be classified. The skin-addicted group mainly infects skin tissues except the above-mentioned parts, induces warty hyperplasia of the skin, and is closely related to the occurrence of certain skin cancers.

Malignant tumors associated with high-risk HPV infection are currently identified as: cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal and perianal cancer, oropharyngeal cancer, tonsil cancer, and oral cancer, wherein the harm of cervical cancer is greatest. Cervical cancer is a third highest worldwide malignancy in women with an annual incidence of about 52.7 ten thousand, with 28.5 thousand in asian regions; the annual incidence of China is 7.5 ten thousand.

The research finds that the main coat protein L1 of the virus can be assembled into L1 virus-like particles (VLP) after being expressed in vitro, and the structure and the shape of the L1VLP are similar to the natural structure of the virus particles, so that the L1VLP has strong immunogenicity. At present, three HPV preventive vaccines sold in the market abroad are HPV L1VLP vaccines, including HPV16/18 bivalent vaccine of Kuraring smith, HPV16/18/6/11 tetravalent vaccine of Mushadong and HPV16/18/58/52/31/33/45/6/11 ninth vaccine, and after the people are immunized, the immunity activity is good, and the protection efficiency for preventing infection and related diseases is high. Given that HPV16 is the predominant strain prevalent worldwide, about 53.5% of cervical cancers are caused by HPV16 infection, and the remaining 46.5% of cervical cancers are caused by HPV infection of about the other 22 high-risk and suspected high-risk types. Therefore, the intensive research on HPV16L 1VLP vaccine is significant.

At present, various expression systems, including insect cell expression systems, yeast expression systems, escherichia coli expression systems and plant expression systems, can be used for expression production of HPV16L1 VLPs. It was found that HPV16L1 full-length protein (White W, Wilson S, et al.J.Virol.1999; 73(6):4882-4889.), N-terminally truncated mutant protein (Chen XS, Casini G, et al.J.mol.biol.2011; 307:173-182.), C-terminally truncated mutant (patent CN1976718A, CN102586287, CN104418942A), and mutant protein combining 10 amino acids N-terminally truncated and 22 amino acids C-terminally truncated (Varsani A, Williamson AL, et al.Vir.Res.2006; 122:154-163.) could be assembled into HPV16L 1VLP using specific techniques (full-length HPV16L1 protein sequence is NCBI database AAC09292.1 sequence, N-terminally from the first amino acid M). Researches also find various methods for improving the expression level of HPV16L1 protein, for example, the C-terminal is truncated by 31 amino acids, so that the expression level can be improved by 1.58 times, inhibitory DNA sequences in the first 129 nucleotides at the N terminal are eliminated, the expression level can also be improved, and the expression level can also be improved by combining methods such as codon optimization, elimination of potential transcription termination sites, elimination of potential shearing sites, simplification of mRNA secondary structures and the like.

At present, reports on whether the HPV16L1 mutant with 2, 3,5 or 7 amino acids truncated at the N terminal can form VLPs and whether the HPV16L1 mutant with 1-9 amino acids truncated at the N terminal and 1-34 amino acids truncated at the C terminal can form VLPs have not been found. Therefore, the analysis of the expression level of the truncated mutant obtained by the above method is not clear. The research shows that the expression level of the HPV16L1 protein can be obviously influenced even if 1 or a few amino acids of the HPV protein are different, the expression level of the L1 mutant with different lengths and C-terminal truncation is obviously different, and the expression level of the L1 mutant with 34 amino acids of C-terminal truncation is obviously improved compared with that of the wild type.

The number of C-terminal truncated amino acids adopted by the invention is different from that of the research, and the expression level of the truncated protein cannot be predicted; in addition, the combination of N-terminal truncation of amino acids with different lengths, the composition of the obtained HPV16L1 mutant amino acid sequence is more different from the method reported at present, and how and whether the expression level can be assembled into VLP cannot be predicted, and the determination can be carried out only by depending on specific experimental research. Therefore, the research value of the HPV16L1 mutant in the HPV16L 1VLP vaccine designed by the invention can be determined after experimental exploration.

Disclosure of Invention

The invention aims to provide a novel truncated HPV16L1 protein, a virus-like particle consisting of the protein and a vaccine containing the virus-like particle, and researches the application of the vaccine in preventing HPV infection and infection-related diseases.

The inventor has surprisingly found that suitably truncating the N-terminus and/or C-terminus of the HPV16L1 protein increases the expression of the HPV16L1 protein in insect cell expression systems, and the truncated protein can assemble into VLPs and induce a protective immune response against HPV 16. The present invention is based on the above findings, which have now been completed, and data is provided in the examples herein.

Thus, in a first aspect the invention relates to an HPV16L1 protein which is truncated by 2, 3,5 or 7 amino acids at the N-terminus and by the amino acids encoded by the complete reading frame or by 29, 31 or 33 amino acids at the C-terminus compared to the wild-type HPV16L1 protein (e.g. the starting ATG versus NCBI database AAC09292.1 sequence). Preferably the truncated protein is selected from HPV16L1 Δ N2, HPV16L1 Δ N3, HPV16L1 Δ N5, HPV16L1 Δ N7, HPV16L1 Δ N2C29, HPV16L1 Δ N3C29, HPV16L1 Δ N5C29, HPV16L1 Δ N7C29, HPV16L1 Δ N2C31, HPV16L1 Δ N3C31, HPV16L1 Δ N5C31, HPV16L1 Δ N7C31, HPV16L1 Δ N2C33, HPV16L1 Δ N3C33, HPV16L1 Δ N5C33 and HPV16L1 Δ N7C 33.

Specifically, the invention provides a truncated HPV16L1 protein, wherein the N end of the truncated HPV16L1 protein is truncated by 2, 3,5 or 7 amino acids compared with the wild type HPV16L1 protein; wherein the truncated HPV16L1 protein is C-terminally conserved with the amino acids encoded by the complete reading frame or truncated by 29, 31 or 33 amino acids compared to the wild-type HPV16L1 protein.

Preferably, the truncated HPV16L1 protein is truncated on the basis of the NCBI database AAC09292.1 sequence; particularly preferably, the truncated HPV16L1 protein is selected from HPV16L1 Δ N5C31(SEQ ID No.1) and HPV16L1 Δ N7C33(SEQ ID No. 2).

Wild-type HPV16L1 protein may also be derived from, but not limited to, the L1 proteins of variants of HPV16Phi1, Tha7, Alg1, Sen32, Fra25, Fra63, 114K, 114B, Z-1194 (Touze A, Mehdaoui SE, et al. J. Clin. Micr. 1998; 36(7):2046 and 2051), and the truncated L1 protein of the corresponding variant is truncated at the equivalent position of the truncated HPV16L1 protein, as assessed by sequence comparison.

The second aspect of the present invention relates to a polynucleotide encoding the truncated HPV16L1 protein of the present invention.

The third aspect of the present invention relates to a vector comprising the polynucleotide of the second aspect described above, preferably said vector is selected from the group consisting of a plasmid, recombinant Bacmid and a recombinant baculovirus.

In a fourth aspect, the present invention relates to an insect cell comprising the vector described above.

The fifth aspect of the present invention relates to an HPV16L1 virus-like particle, which comprises the HPV16L1 protein of the first aspect or consists of the HPV16L1 protein of the first aspect.

The sixth aspect of the invention relates to a vaccine for preventing HPV infection or a lesion related to HPV infection, the vaccine comprising the HPV16L1 virus-like particle of the fifth aspect, wherein the HPV16L1 virus-like particle is present in an amount effective to induce a protective immune response. Preferably, the vaccine may further comprise at least one virus-like particle of HPV selected from the other mucophilic and/or dermophilic groups, each in an amount effective to elicit a protective immune response. Such vaccines typically further comprise a vaccine excipient or carrier.

Preferably, the vaccine comprises HPV16L1 virus-like particles of the fifth aspect and at least 1L 1 virus-like particle selected from HPV2, 5, 6,7,11, 18, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,39, 40, 43, 44, 45,51,52, 53, 56, 57, 58,59, 61, 66,67, 68, 69,70,73, 74, 77, 81, 82, 83, 85, 91 in amounts effective to induce a protective immune response.

Further preferably, the vaccine comprises HPV16L1 virus-like particles of the fifth aspect and L1 virus-like particles of HPV18, 26, 31,33,35,39,45,51,52,56,58,59,68 and 73, in amounts effective to induce a protective immune response.

Further preferably, the vaccine comprises the HPV16L1 virus-like particle of the fifth aspect and the L1 virus-like particles of HPV18, 31,33,35,39,45, 52 and 58, respectively, in amounts effective to induce a protective immune response.

Further preferably, the vaccine comprises the HPV16L1 virus-like particle of the fifth aspect and the L1 virus-like particles of HPV18, 52 and 58 in amounts respectively effective to induce a protective immune response.

Further preferably, the vaccine comprises the HPV16L1 virus-like particle of the fifth aspect and L1 virus-like particles of HPV18 and 58 in amounts effective to induce a protective immune response.

Particularly preferably, the vaccine comprises the HPV16L1 virus-like particles and the HPV 18L 1 virus-like particles in the fifth aspect, and the contents of the virus-like particles are respectively effective amounts for inducing protective immune response.

The seventh aspect of the present invention relates to a novel vaccine comprising the vaccine of the sixth aspect and an adjuvant, which can further enhance the immune response. Preferably, the adjuvant used is an adjuvant composition comprising an aluminium adjuvant, an oil-in-water or water-in-oil emulsion and a TLR stimulant.

Further preferably, the adjuvant used is a composition comprising an aluminum hydroxide adjuvant or an aluminum phosphate adjuvant with a polyinosinic-polycytidylic acid adjuvant and a stabilizer.

Further preferably, the adjuvant used is a composition comprising MF59 adjuvant, polyinosinic-polycytidylic acid adjuvant and a stabilizer.

In particular, the composite adjuvant is a composite formed by combining polyinosinic-polycytidylic acid and a stabilizing agent (PIKA) with an aluminum hydroxide adjuvant or a composite formed by combining polyinosinic-polycytidylic acid and a stabilizing agent (PIKA) with an MF59 adjuvant, and the two composite adjuvants can be used together with the HPV VLP vaccine to effectively improve the immunological activity of the vaccine.

An eighth aspect of the invention relates to the use of the vaccine of the sixth and seventh aspects for the prevention of HPV infection or a disease associated with HPV infection.

Description and explanation of related terms

According to the present invention, the term "insect cell expression system" includes insect cells, recombinant Bacmid and expression vectors. Wherein the insect cell is derived from a commercially available cell, exemplified herein but not limited to: sf9, Sf21, High Five.

According to the present invention, examples of the term "wild-type HPV16L1 protein" include, but are not limited to, the long full-length L1 protein of the protein numbered AAC09292.1 in the NCBI database.

A gene fragment of "truncated HPV16L1 protein" refers to a gene which is deleted at its 5 'end and/or 3' end of nucleotides encoding 1 or more amino acids compared to the wild-type HPV16L1 protein gene, wherein the full-length sequence of the "wild-type HPV16L1 protein" is, for example and without limitation, the following sequence in the NCBI database: AAC09292.1, AIQ82817.1, AAC61736.1, etc.

According to the invention, the expression "Δ NX" denotes a "protein truncated by X amino acids from the N-terminus", which refers to a protein counted with the starting methionine as the 1 st amino acid, and after X amino acids have been truncated from the N-terminus, the remaining sequence is supplemented with a further methionine as the starting site at the N-terminus. For example, "HPV 16L 1. DELTA.N 5" represents an HPV16L1 protein which is truncated at the N-terminus by 5 amino acids, i.e., a protein obtained by deleting 5 amino acids from the initial methionine and then supplementing the remaining sequence with a single methionine at the N-terminus.

According to the invention, the expression "Δ CY" denotes a "protein C-terminally truncated by Y amino acids", and refers to a protein C-terminally truncated by Y amino acids, counting from amino acid 505 of HPV16L 1. For example, "HPV 16L1 Δ C1" represents a protein obtained by C-terminal truncation of 1 amino acid at position 505.

According to the invention, the expression "Δ NXCY" denotes a protein truncated by X amino acids at the N-terminus and Y amino acids at the C-terminus, and refers to a protein truncated by Y amino acids, counting with the initial methionine as the 1 st amino acid, after X amino acids have been truncated from the N-terminus, supplemented with one more methionine at the N-terminus of the remaining sequence as the initial site, and counting with the HPV16L1 at the 505 th amino acid. For example, "HPV 16L1 Δ N5C 31" represents an HPV16L1 protein truncated by 5 amino acids at the N-terminus and 31 amino acids at the C-terminus, i.e., a protein supplemented with one more methionine at the N-terminus of the remaining sequence after deletion of 5 amino acids starting from the initial methionine and deleted at positions 475-.

According to the present invention, the term "excipient or carrier" means a substance selected from one or more of, including but not limited to: pH regulator, surfactant and ionic strength enhancer. For example, pH adjusting agents are exemplified by, but not limited to, phosphate buffers, surfactants include cationic, anionic, or nonionic surfactants, exemplified by, but not limited to, polysorbate 80(Tween-80), ionic strength enhancers are exemplified by, but not limited to, sodium chloride.

According to the present invention, the term "adjuvant" refers to an adjuvant that is clinically applicable to a human body, and includes various adjuvants that have been currently approved and may be approved in the future, such as, but not limited to, aluminum adjuvant, MF59, and various forms of adjuvant compositions.

According to the present invention, the term "emulsion" refers to a heterogeneous liquid dispersion system formed by mixing an aqueous phase component, an oil phase component and an emulsifier in a suitable ratio and emulsifying the mixture. Wherein the aqueous phase components include but are not limited to buffer systems such as phosphate buffer, HEPES buffer, etc.; the oil phase component is a metabolizable lipid including, but not limited to, vegetable oils, fish oils, animal oils, synthetic oils, and other lipid components (e.g., but not limited to squalene, tocopherol); emulsifiers are suitable surfactants such as, but not limited to, sorbitan trioleate (Span-85), polysorbate 80 (Tween-80).

According to the present invention, the term "stabilizer" refers to a component that can bind to polyinosinic-polycytidylic acid in an adjuvant and act as a stabilizer, including but not limited to antibiotics (such as but not limited to kanamycin, neomycin, gentamicin), inorganic salts (such as but not limited to calcium chloride, magnesium chloride, calcium phosphate), organic complexes of cations (such as but not limited to calcium stearate, calcium gluconate).

According to the present invention, the vaccine of the present invention may take a patient-acceptable form, including but not limited to oral administration or injection, preferably injection.

According to the invention, the vaccine of the invention is preferably used in a unit dosage form, wherein the dosage of the virus-like particles of the truncated HPV16L1 protein in the unit dosage form is 5 mug-80 mug, preferably 20 mug-40 mug.

Drawings

FIG. 1 shows the expression identification of truncated HPV16L1 in insect cells in example 4 of the present invention. The results show that all 16 truncated HPV16L1 can be expressed at high levels in insect cells.

1 to 16 represent HPV16L1 Δ N2, HPV16L1 Δ N3, HPV16L1 Δ N5, HPV16L1 Δ N7, HPV16L1 Δ N2C29, HPV16L1 Δ N3C29, HPV16L1 Δ N5C29, HPV16L1 Δ N7C29, HPV16L1 Δ N2C31, HPV16L1 Δ N3C31, HPV16L1 Δ N5C31, HPV16L1 Δ N7C31, HPV16L1 Δ N2C33, HPV16L1 Δ N3C33, HPV16L1 Δ N5C33 and HPV16L1 Δ N7C33, respectively.

Fig. 2A to 2B show the results of dynamic light scattering analysis of HPV16L1 Δ N5 and HPV16L1 Δ N5C31 mutant proteins obtained after purification in example 6 of the present invention. The result shows that the hydration kinetic diameters of the virus-like particles formed by the HPV16L1 delta N5 and the HPV16L1 delta N5C31 recombinant proteins are 129.6nm and 128nm respectively, and the percentage of particle assembly is 100 percent.

FIG. 2A represents HPV16L1 Δ N5; FIG. 2B shows HPV16L 1. delta.N 5C 31.

FIGS. 3A to 3P show the transmission electron microscopy observations of 16 truncated HPV16L1 VLPs obtained after purification in example 7 of the invention. A large number of virus-like particles with the diameter of about 55nm can be seen in a visual field, the size of the particles is consistent with a theoretical value, and the uniformity is good. Bar is 200 nm.

FIGS. 3A to 3P show HPV16L1 Δ N2, HPV16L1 Δ N3, HPV16L1 Δ N5, HPV16L1 Δ N7, HPV16L1 Δ N2C29, HPV16L1 Δ N3C29, HPV16L1 Δ N5C29, HPV16L1 Δ N7C29, HPV16L1 Δ N2C31, HPV16L1 Δ N3C31, HPV16L1 Δ N5C31, HPV16L1 Δ N7C31, HPV16L1 Δ N2C33, HPV16L1 Δ N3C33, HPV16L1 Δ N5C33 and HPV16L1 Δ N7C33, respectively.

FIG. 4 shows the analysis of the neutralizing antibody titer of HPV16 in the immune serum of mice vaccinated with HPV16L 1. delta.N 5 and HPV16L 1. delta.N 5C31 VLPs in example 8 of the present invention.

Fig. 5 shows the analysis of the neutralizing antibody titers of the immune sera of mice vaccinated with HPV16L1 Δ N5C31VLP protein in example 9 of the invention in combination with PIKA-containing co-adjuvant: p <0.05 compared to the non-adjuvanted group; **: p <0.01 compared to the non-adjuvanted group; ***: p <0.001 compared to the non-adjuvanted group; #: compared with the pure PIKA group, P is less than 0.05; # ##: compared with the pure PIKA group, the P is less than 0.001; and &: compared with the pure Alum group, P is less than 0.05; and & & &: compared with the pure Alum group, the product has the P < 0.001.

Detailed Description

The invention will now be further illustrated by the following non-limiting examples, and it will be apparent to those skilled in the art that many modifications can be made without departing from the spirit of the invention, such modifications also falling within the scope of the invention. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, as the embodiments are necessarily diverse. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be defined in the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. The following experimental procedures are all the ones described in the conventional methods or product specifications unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and/or materials in the publications.

Example 1: amplification of truncated HPV16L1 gene and construction of expression vector

The full-length HPV16L1 gene used as the template was synthesized from the whole gene of Shanghai Biotechnology engineering services, Inc. (SEQ ID NO: 3), and its corresponding amino acid sequence was the sequence numbered AAC09292.1 in NCBI database.

The primers for constructing the truncated HPV16L1 gene were synthesized by Shanghai Biotechnology engineering services, Inc., as follows:

16N2F：5’-GAATTCGCCGCCACCATGCTCTGGTTGCCATCCGAAGC-3’；

16N3F：5’-GAATTCGCCGCCACCATGTGGTTGCCATCCGAAGCTAC-3’；

16N5F：5’-GAATTCGCCGCCACCATGCCATCCGAAGCTACAGTCTATC-3’；16N7F：5’-GAATTCGCCGCCACCATGGAAGCTACAGTCTATCTCCC-3’；

16CR：5’-TCTAGATTACAGCTTACGTTTTTTGCG-3’；

16C29R：5’-TCTAGAATTAGGCCTTGAGACCAGCTTGCA AC-3’

16C31R：5’-TCTAGAATTAGAGACCAGCTTGC-3’；

16C33R：5’-TCTAGAATTAAGCTTGCAAC AGGAATTTC-3’,

the primer sequences are shown in the sequence table SEQ ID NO: 6-13.

As set forth in SEQ ID NO: 3 as a template, and 16N2F/16CR as a primer, and amplifying the HPV16L1 delta N2 gene by PCR; PCR amplification of HPV16L 1. delta.N 3 gene using 16N3F/16CR as primers; PCR amplification of HPV16L 1. delta.N 5 gene using 16N5F/16CR as primers; PCR amplification of HPV16L 1. delta.N 7 gene using 16N7F/16CR as primers; PCR amplification of HPV16L1 Δ N2C29 gene using 16N2F/16C29R as primers; PCR amplification of HPV16L1 Δ N3C29 gene using 16N3F/16C29R as primers; PCR amplification of HPV16L1 Δ N5C29 gene using 16N5F/16C29R as primers; PCR amplification of HPV16L1 Δ N7C29 gene using 16N7F/16C29R as primers; PCR amplification of HPV16L1 Δ N2C31 gene using 16N2F/16C31R as primers; PCR amplification of HPV16L1 Δ N3C31 gene using 16N3F/16C31R as primers; PCR amplification of the HPV16L1 Δ N5C31 gene (SEQ ID NO: 4) using 16N5F/16C31R as primers; PCR amplification of HPV16L1 Δ N7C31 gene using 16N7F/16C31R as primers; PCR amplification of HPV16L1 Δ N2C33 gene using 16N2F/16C33R as primers; PCR amplification of HPV16L1 Δ N3C33 gene using 16N3F/16C33R as primers; PCR amplification of HPV16L1 Δ N5C33 gene using 16N5F/16C33R as primers; HPV16L 1. delta.N 7C33 gene (SEQ ID NO: 5) was PCR amplified using 16N7F/16C33R as primers. Methods for PCR amplification are well known, for example, from patent CN 101293918B.

The genes obtained by PCR amplification are respectively digested by EcoRI/Xba I restriction sites, and are respectively inserted into a commercial expression vector pFastBac1 (manufactured by Invitrogen company), so as to obtain a recombinant expression vector containing the truncated HPV16L1 gene: pFastBac1-16L1 delta N2, pFastBac1-16L1 delta N1, pFastBac1-16L1 delta N2C 1, pFastBac1-16L1 delta N3C 1, pFastBac1-16L1 delta N5C 1, pFastBac1-16L1 delta N7C 1, pFastBac1-16L1 delta N2C 1, pFastBac1-16L1 delta N3C 1, pFastBac1-16L1 delta N5C 1, pFastBac tBac1-16L1 delta N7C 1, pFastBac1-16L1 delta N1, pFastBac1 delta N1-16L 1 delta N1, pFastBac1 delta C1-16L1, pFastBac1 delta N1-3C. The above methods of cleavage, ligation and cloning are well known, for example, from patent CN 101293918B.

Example 2: recombinant Bacmid of truncated HPV16L1 gene and recombinant baculovirus construct

Respectively using recombinant expression vectors containing truncated HPV16L gene, pFastBac-16L delta N2C, pFastBac-16L delta N3C, pFastBac-16L delta N5C, pFastBac-16L delta N7C, pFastBacac-16L delta N2C, pFastBacac-16L delta N3C, pFastBac-16L delta N5C and pFastBac-16L delta N7C to transform bacillus BacDH 10, then screening to obtain recombinant mids transfected by SastBus in Sasf recombinant virus cells, and amplifying SastBus. Screening of recombinant Bacmid and methods for amplifying recombinant baculoviruses are well known, for example, from patent CN 101148661B.

Example 3: expression of truncated HPV16L1 gene in Sf9 cells

Sf9 cells are respectively inoculated with 16 recombinant baculoviruses of truncated HPV16L1 genes to express the truncated HPV16L1 protein, cultured at 27 ℃ for about 88h, then fermented, centrifuged at 3000rpm for 15min, the supernatant is discarded, and the cells are washed by PBS for expression identification and purification. Methods for infection expression are disclosed, for example, in patent CN 101148661B.

Example 4: expression identification of truncated HPV16L1

Cells expressing different truncated HPVs 16L1 as described in example 3 were each taken at 1X 10 ⁶And the cells were resuspended in 200. mu.l of PBS solution, and 6 XLoading buffer (50. mu.l) was added, denatured at 75 ℃ for 8 minutes, and 10. mu.l of each was subjected to SDS-PAGE and Western blot analysis. As shown in figure 1, 16 truncated HPV16L1 proteins can be expressed in insect cells at high levels, wherein the sizes of HPV16L1 delta N2, HPV16L1 delta N3, HPV16L1 delta N5, HPV16L1 delta N7 are about 55kDa, and the sizes of the other 12 proteins are about 50 kDa. Methods for SDS-PAGE electrophoresis and Western blot identification are disclosed, for example, in patent CN 101148661B.

Example 5: comparison of expression levels of truncated HPV16L1 protein and wild HPV16L1 protein

Cells expressing the truncated HPV16L1 protein and wild-type HPV16L1 described in example 3 were each 1X 10 ⁶The cells were resuspended in 200. mu.l of PBS solution, disrupted by ultrasonication (Ningbo Xinzhi ultrasonication apparatus, 2# probe, 100W, ultrasonication for 5s, interval of 7s, total time 3min), and centrifuged at 12000rpm for 10 minutes at high speed. The lysate supernatant is collected and assayed for L1 content using a sandwich ELISA method, which is well known, for example, from CN 104513826A.

The HPV16L1 monoclonal antibody prepared by the inventor is used for coating an enzyme label plate, 80 ng/hole and is incubated overnight at 4 ℃; blocking was performed using 5% BSA-PBST for 2h at room temperature, and the plate was washed 3 times with PBST. The lysed supernatant was serially diluted 2-fold in PBS and the HPV16L 1VLP standard was also diluted in a gradient from 2. mu.g/ml to 0.0625. mu.g/ml, added to each microplate well at 100. mu.l per well and incubated at 37 ℃ for 1 h. Wash plates 3 times with PBST, add 1: HPV16L1 rabbit polyclonal antibody at 3000 dilution, 100. mu.l per well, was incubated at 37 ℃ for 1 h. Wash plates 3 times with PBST, add 1: 3000 dilutions of HRP-labeled goat anti-mouse IgG (1: 3000 dilution, Mitsuga bridge) were incubated at 37 ℃ for 45 min. The plate was washed 5 times with PBST, 100. mu.l of OPD substrate (Sigma) was added to each well, color was developed at 37 ℃ for 5 minutes, the reaction was stopped with 50. mu.l of 2M sulfuric acid, and the absorbance was measured at 490 nm. The concentrations of truncated HPV16L1 protein and wild-type HPV16L1 protein in the lysis supernatant were calculated according to a standard curve.

The results are shown in table 1, the expression level of the truncated HPV16L1 protein is higher than that of the wild-type HPV16L1 protein, and higher than that of C-terminal truncated mutant HPV16L 1-498(HPV 16L1 Δ C7) and HPV16L1-483(HPV 16L1 Δ C22) in patent CN 104418942A.

TABLE 1HPV16L1 protein expression analysis

Example 6: purification and dynamic light scattering particle size analysis of truncated HPV16L1 protein

Taking 50ml of cell fermentation broth of truncated HPV16L1, resuspending the cells with 10ml PBS, adding PMSF to a final concentration of 1mg/ml, ultrasonication (Ningbo New glossy ganoderma ultrasonicator, 6# Probe, 100W, ultrasonication 5s, 7s apart, total time 5min), taking the supernatant of the disruption, purification at room temperature, disaggregation of VLPs by adding 4% β -mercaptoethanol (W/W) to the lysate, filtration of the sample with 0.22 μm filter, DMAE anion exchange chromatography (20mM Tris,180mM NaCl, 4% β -ME, pH7.9 elution), TMAE anion exchange chromatography (20mM Tris,180mM NaCl, 4% β -ME, pH7.9 elution) and hydroxyapatite chromatography (100mM NaH H, 100mM Tris, 4% β -ME) in this order ₂PO ₄30mM NaCl, 4% β -ME, pH 6.0 elute purified product was concentrated using a Planova ultrafiltration system and buffer (20mM NaH) was changed ₂PO ₄500mM NaCl, pH 6.0) facilitates VLP assembly. The above purification methods are disclosed in, for example, patents CN101293918B, CN1976718A, etc.

The purified truncated HPV16L1 protein solution was subjected to DLS particle size analysis (Zetasizer Nano ZS 90 dynamic light scattering instrument, Malvern corporation), the results are shown in Table 2, wherein the DLS analysis chart of HPV16L1 Δ N5 and HPV16L1 Δ N5C31 is shown in FIG. 2.

TABLE 2 truncated HPV16L1 protein DLS analysis

Name of protein	Hydraulic diameter (nm)	PDI
			HPV16 L1ΔN2	128.4	0.148
HPV16 L1ΔN3	127.2	0.153
			HPV16 L1ΔN5	129.6	0.167
HPV16 L1ΔN7	129.1	0.151
			HPV16 L1ΔN2C29	128.5	0.146
HPV16 L1ΔN3C29	129.3	0.133
			HPV16 L1ΔN5C29	127.4	0.145
HPV16 L1ΔN7C29	126.9	0.138
			HPV16 L1ΔN2C31	128.3	0.141
HPV16 L1ΔN3C31	127.8	0.138
			HPV16 L1ΔN5C31	128	0.146
HPV16 L1ΔN7C31	128.4	0.129
			HPV16 L1ΔN2C33	127.5	0.137
HPV16 L1ΔN3C33	127	0.147
			HPV16 L1ΔN5C33	128.8	0.150
HPV16 L1ΔN7C33	128.5	0.138

Example 7: transmission electron microscopy of truncated HPV16L 1VLP

The truncated HPV16L1 VLPs were purified separately as described in example 6, and the dialyzed VLPs were used to prepare copper meshes, stained with 1% uranium acetate, dried thoroughly and visualized using JEM-1400 electron microscopy (Olympus). As shown in FIG. 3, the truncated HPV16L 1VLP had a diameter of about 55nm, uniform size and regular shape. The preparation of copper mesh and the observation by electron microscope are disclosed, for example, in patent CN 101148661B.

Example 8: mouse immunization of truncated HPV16L1 VLPs and determination of neutralizing antibody titers

BALB/c mice of 4-6 weeks of age were taken and randomized into groups of 4 mice each, immunized with PBS, truncated HPV16L 1VLP and HPV16L 1wtVLP, respectively. Intramuscular injection of L1VLP at an immunization dose of 0.1. mu.g, 2 weeks after immunization, for 2 times. Blood was collected from the tail vein 2 weeks after the 2 nd immunization, and serum was isolated.

The HPV16 pseudovirus was used to test the titer of neutralizing antibodies against HPV16 in immune serum, and the results are shown in FIG. 4, and the neutralizing antibodies were induced efficiently by immunizing mice with HPV16L1 Δ N5VLP (16L1 Δ N5), HPV16L1 Δ N5C31VLP (16L1 Δ N5C31) and HPV16L 1wtVLP (16L1wt), and the titer of neutralizing antibodies was not statistically different. After mice were immunized with the other 14 truncated HPV16L1 mutant VLPs included in the invention by adopting the above strategy, the induced HPV16 neutralizing antibody level was between 400-1600, and there was no difference compared with the HPV16L1 wtVLP. Methods for preparing pseudoviruses and neutralizing the pseudoviruses are disclosed, for example, in patent CN 104418942A.

Example 9: HPV16L1 delta N5C31VLP combined adjuvant immune mice and neutralizing antibody titer determination

BALB/C mice of 4-6 weeks old were selected, randomly divided into 11 groups, and immunized with HPV16L 1. DELTA.N 5C31VLP in combination with polyinosinic acid-polycytidylic acid injection (PIKA, south China pharmaceutical industry) and aluminum hydroxide adjuvant or MF59 adjuvant (4.3% squalene, 0.5% Tween-80, 0.5% Span-85), respectively, the specific groups and immunization doses are shown in Table 3. Subcutaneous injection, immunization at

week

0, 2 for 2 total times. Blood was collected from the tail vein 2 weeks after the 2 nd immunization, and serum was isolated.

Table 3 adjuvant experimental mice groups and doses

Group name	Vaccine compositions and dosages
		Adjuvant-free group	HPV16 L1ΔN5C31 VLP 0.1μg
Alum	HPV16 L1ΔN5C31 VLP 0.1μg，Al(OH) ₃ 1μg
		PIKA	HPV16 L1ΔN5C31 VLP 0.1μg，PIKA 25μg
Alum+PIKA 1μg	HPV16 L1ΔN5C31 VLP 0.1μg，Al(OH) ₃ 1μg，PIKA 1μg
		Alum+PIKA 10μg	HPV16 L1ΔN5C31 VLP 0.1μg，Al(OH) ₃ 1μg，PIKA 10μg
Alum+PIKA 25μg	HPV16 L1ΔN5C31 VLP 0.1μg，Al(OH) ₃ 1μg，PIKA 25μg
		Alum+PIKA 50μg	HPV16 L1ΔN5C31 VLP 0.1μg，Al(OH) ₃ 1μg，PIKA 50μg
MF59+PIKA 1μg	HPV16 L1ΔN5C31 VLP 0.1μg，MF59 100μl，PIKA 1μg
		MF59+PIKA 10μg	HPV16 L1ΔN5C31 VLP 0.1μg，MF59 100μl，PIKA 10μg
MF59+PIKA 25μg	HPV16 L1ΔN5C31 VLP 0.1μg，MF59 100μl，PIKA 25μg
		MF59+PIKA 50μg	HPV16 L1ΔN5C31 VLP 0.1μg，MF59 100μl，PIKA 50μg

The HPV16 pseudovirus is used for detecting the HPV16 neutralizing antibody titer of immune serum, and the result is shown in figure 5, the aluminum hydroxide adjuvant or polyinosinic acid-polycytidylic acid adjuvant (PIKA adjuvant) has no obvious enhancement effect on the neutralizing antibody level induced by HPV16L 1VLP, but the aluminum hydroxide or MF59 combined with the PIKA adjuvant can obviously improve the neutralizing antibody level induced by the VLP, and compared with the aluminum hydroxide alone or the PIKA adjuvant alone, the neutralizing antibody level is also obviously improved. The Alum/PIKA composite adjuvant or MF59/PIKA composite adjuvant can obviously improve the immunocompetence of the vaccine, and has application prospect (SPSS software, One-way ANOVA analysis is used).

Sequence listing

<110> institute of basic medicine of Chinese academy of medical sciences

<120> truncated human papilloma virus 16 type L1 protein and application thereof

<130>370078CG-360032

<160>13

<170>PatentIn version 3.3

<210>1

<211>470

<212>PRT

<213> truncated HPV16

<400>1

Met Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys

1 5 10 15

Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr His

20 25 30

Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile

35 40 45

Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys Val Ser Gly Leu

50 55 60

Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro Asn Lys Phe Gly

65 70 75 80

Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp

85 90 95

Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly

100 105 110

Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala

115 120 125

Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser

130 135 140

Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro

145 150 155 160

Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn Val Ala Val

165 170 175

Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr Val Ile Gln

180 185 190

Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Thr

195 200 205

Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile

210 215 220

Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp

225 230 235 240

Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His Leu

245 250 255

Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro Asp Asp Leu Tyr

260 265 270

Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr Phe

275 280 285

Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Ile Phe Asn

290 295 300

Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys

305 310 315 320

Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr

325 330 335

Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr Lys

340 345 350

Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp Leu

355 360 365

Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met

370 375 380

Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu Asp Trp Asn Phe

385390 395 400

Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg Phe

405 410 415

Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr Pro Pro Ala Pro

420 425 430

Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys

435 440 445

Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe

450 455 460

Leu Leu Gln Ala Gly Leu

465 470

<210>2

<211>466

<212>PRT

<213> truncated HPV16

<400>2

Met Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val

1 5 10 15

Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr His Ala Gly

20 25 30

Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile Lys Lys

35 40 45

Pro Asn Asn Asn Lys Ile Leu Val Pro Lys Val Ser Gly Leu Gln Tyr

50 55 60

Arg Val Phe Arg Ile His Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro

65 70 75 80

Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys

85 90 95

Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser

100 105 110

Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala Ser Ala

115 120 125

Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser Met Asp

130 135 140

Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Ile Gly

145 150 155 160

Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn Val Ala Val Asn Pro

165 170 175

Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr Val Ile Gln Asp Gly

180 185 190

Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Thr Leu Gln

195 200 205

Ala Asn Lys Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys

210215 220

Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser Leu

225 230 235 240

Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His Leu Phe Asn

245 250 255

Arg Ala Gly Ala Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile Lys

260 265 270

Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr Phe Pro Thr

275 280 285

Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Ile Phe Asn Lys Pro

290 295 300

Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly

305 310 315 320

Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met

325 330 335

Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr Lys Asn Thr

340 345 350

Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp Leu Gln Phe

355 360 365

Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met Thr Tyr

370375 380

Ile His Ser Met Asn Ser Thr Ile Leu Glu Asp Trp Asn Phe Gly Leu

385 390 395 400

Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg Phe Val Thr

405 410 415

Ser Gln Ala Ile Ala Cys Gln Lys His Thr Pro Pro Ala Pro Lys Glu

420 425 430

Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys

435 440 445

Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu

450 455 460

Gln Ala

465

<210>3

<211>1518

<212>DNA

<213> full-Length HPV16

<400>3

atgtccctct ggttgccatc cgaagctaca gtctatctcc caccagtccc agtcagtaaa 60

gttgttagta cagatgaata tgtcgctagg acaaacatct actaccatgc tggcacaagt 120

aggctcctcg ctgtcggcca cccatacttc cccatcaaga agcccaacaa caacaagatc 180

ctggtcccca aggtgagcgg cctgcagtat cgcgtgttcc gcatccacct gcccgatcca 240

aacaaattcg gcttccccga tacaagcttc tacaaccccg acacccaaag actcgtctgg 300

gcatgcgtgg gcgtcgaagt gggcaggggc caacccctgg gcgtcggtat ctccggtcac 360

cccctgctga acaagctcga cgataccgag aacgcctccg cctacgctgc caacgctggc 420

gtcgacaacc gcgagtgcat cagcatggac tataagcaga cccagctctg cctgatcggc 480

tgtaagcccc ccatcggtga gcattggggt aaaggcagtc cctgcacaaa cgtcgctgtg 540

aaccccggtg actgcccccc cctggaactg atcaataccg tcatccaaga cggtgacatg 600

gtcgacaccg gtttcggtgc catggatttc accaccctgc aagccaacaa gtccgaggtc 660

cccctcgaca tctgcaccag catctgtaag taccccgact acatcaagat ggtctccgag 720

ccctacggtg attcactgtt cttctacctg aggcgcgagc agatgttcgt ccgccacctg 780

ttcaaccgcg ctggcgctgt cggtgagaac gtgcccgatg acctgtatat caagggtagc 840

ggtagcaccg ccaacctggc atccagcaac tacttcccca ccccctccgg cagcatggtc 900

acaagcgacg cacagatctt taacaagccc tactggctgc agagggccca aggtcataac 960

aacggtatct gctggggcaa tcagttgttc gtcaccgtcg tcgacaccac caggtccacc 1020

aacatgagcc tgtgcgccgc aatcagcacc agcgagacca cctacaagaa caccaatttc 1080

aaagaatact tgaggcacgg tgaagagtac gacctgcaat tcatcttcca actctgtaag 1140

atcacactga ccgccgatgt catgacctac atccacagca tgaacagcac catcctcgaa 1200

gattggaact tcggcttgca gcccccaccc ggcggtacct tggaggacac ctaccgcttc 1260

gtgaccagtc aagccatcgc ctgccagaag cacacccccc ccgcccccaa ggaagaccca 1320

ttgaagaagt ataccttctg ggaagtgaac ctgaaagaga aattcagcgc tgatttggac 1380

caattcccac tgggaaggaa attcctgttg caagctggtc tcaaggccaa accaaaattt 1440

acattaggaa aacgaaaagc tacacccacc acctcatcta cctctacaac tgctaaacgc 1500

aaaaaacgta agctgtaa 1518

<210>4

<211>1414

<212>DNA

<213> truncated HPV16

<400>4

atgccatccg aagctacagt ctatctccca ccagtcccag tcagtaaagt tgttagtaca 60

gatgaatatg tcgctaggac aaacatctac taccatgctg gcacaagtag gctcctcgct 120

gtcggccacc catacttccc catcaagaag cccaacaaca acaagatcct ggtccccaag 180

gtgagcggcc tgcagtatcg cgtgttccgc atccacctgc ccgatccaaa caaattcggc 240

ttccccgata caagcttcta caaccccgac acccaaagac tcgtctgggc atgcgtgggc 300

gtcgaagtgg gcaggggcca acccctgggc gtcggtatct ccggtcaccc cctgctgaac 360

aagctcgacg ataccgagaa cgcctccgcc tacgctgcca acgctggcgt cgacaaccgc 420

gagtgcatca gcatggacta taagcagacc cagctctgcc tgatcggctg taagcccccc 480

atcggtgagc attggggtaa aggcagtccc tgcacaaacg tcgctgtgaa ccccggtgac 540

tgcccccccc tggaactgat caataccgtc atccaagacg gtgacatggt cgacaccggt 600

ttcggtgcca tggatttcac caccctgcaa gccaacaagt ccgaggtccc cctcgacatc 660

tgcaccagca tctgtaagta ccccgactac atcaagatgg tctccgagcc ctacggtgat 720

tcactgttct tctacctgag gcgcgagcag atgttcgtcc gccacctgtt caaccgcgct 780

ggcgctgtcg gtgagaacgt gcccgatgac ctgtatatca agggtagcgg tagcaccgcc 840

aacctggcat ccagcaacta cttccccacc ccctccggca gcatggtcac aagcgacgca 900

cagatcttta acaagcccta ctggctgcag agggcccaag gtcataacaa cggtatctgc 960

tggggcaatc agttgttcgt caccgtcgtc gacaccacca ggtccaccaa catgagcctg 1020

tgcgccgcaa tcagcaccag cgagaccacc tacaagaaca ccaatttcaa agaatacttg 1080

aggcacggtg aagagtacga cctgcaattc atcttccaac tctgtaagat cacactgacc 1140

gccgatgtca tgacctacat ccacagcatg aacagcacca tcctcgaaga ttggaacttc 1200

ggcttgcagc ccccacccgg cggtaccttg gaggacacct accgcttcgt gaccagtcaa 1260

gccatcgcct gccagaagca cacccccccc gcccccaagg aagacccatt gaagaagtat 1320

accttctggg aagtgaacct gaaagagaaa ttcagcgctg atttggacca attcccactg 1380

ggaaggaaat tcctgttgca agctggtctc taat 1414

<210>5

<211>1402

<212>DNA

<213> truncated HPV16

<400>5

atggaagcta cagtctatct cccaccagtc ccagtcagta aagttgttag tacagatgaa 60

tatgtcgcta ggacaaacat ctactaccat gctggcacaa gtaggctcct cgctgtcggc 120

cacccatact tccccatcaa gaagcccaac aacaacaaga tcctggtccc caaggtgagc 180

ggcctgcagt atcgcgtgtt ccgcatccac ctgcccgatc caaacaaatt cggcttcccc 240

gatacaagct tctacaaccc cgacacccaa agactcgtct gggcatgcgt gggcgtcgaa 300

gtgggcaggg gccaacccct gggcgtcggt atctccggtc accccctgct gaacaagctc 360

gacgataccg agaacgcctc cgcctacgct gccaacgctg gcgtcgacaa ccgcgagtgc 420

atcagcatgg actataagca gacccagctc tgcctgatcg gctgtaagcc ccccatcggt 480

gagcattggg gtaaaggcag tccctgcaca aacgtcgctg tgaaccccgg tgactgcccc 540

cccctggaac tgatcaatac cgtcatccaa gacggtgaca tggtcgacac cggtttcggt 600

gccatggatt tcaccaccct gcaagccaac aagtccgagg tccccctcga catctgcacc 660

agcatctgta agtaccccga ctacatcaag atggtctccg agccctacgg tgattcactg 720

ttcttctacc tgaggcgcga gcagatgttc gtccgccacc tgttcaaccg cgctggcgct 780

gtcggtgaga acgtgcccga tgacctgtat atcaagggta gcggtagcac cgccaacctg 840

gcatccagca actacttccc caccccctcc ggcagcatgg tcacaagcga cgcacagatc 900

tttaacaagc cctactggct gcagagggcc caaggtcata acaacggtat ctgctggggc 960

aatcagttgt tcgtcaccgt cgtcgacacc accaggtcca ccaacatgag cctgtgcgcc 1020

gcaatcagca ccagcgagac cacctacaag aacaccaatt tcaaagaata cttgaggcac 1080

ggtgaagagt acgacctgca attcatcttc caactctgta agatcacact gaccgccgat 1140

gtcatgacct acatccacag catgaacagc accatcctcg aagattggaa cttcggcttg 1200

cagcccccac ccggcggtac cttggaggac acctaccgct tcgtgaccag tcaagccatc 1260

gcctgccaga agcacacccc ccccgccccc aaggaagacc cattgaagaa gtataccttc 1320

tgggaagtga acctgaaaga gaaattcagc gctgatttgg accaattccc actgggaagg 1380

aaattcctgt tgcaagctta at 1402

<210>6

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>6

gaattcgccg ccaccatgct ctggttgcca tccgaagc 38

<210>7

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>7

gaattcgccg ccaccatgtg gttgccatcc gaagctac 38

<210>8

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>8

gaattcgccg ccaccatgcc atccgaagct acagtctatc 40

<210>9

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>9

gaattcgccg ccaccatgga agctacagtc tatctccc 38

<210>10

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>10

tctagattac agcttacgtt ttttgcg 27

<210>11

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>11

tctagaatta ggccttgaga ccagcttgca ac 32

<210>12

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>12

tctagaatta gagaccagct tgc 23

<210>13

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>13

tctagaatta agcttgcaac aggaatttc 29

Claims

1. A truncated HPV16L1 protein expressed by an insect cell expression system, wherein the truncated HPV16L1 protein is truncated by 2, 3,5 or 7 amino acids at the N-terminal and 29, 31 or 33 amino acids at the C-terminal compared with the wild type HPV16L1 protein.

2. The truncated HPV16L1 protein of claim 1, wherein the truncated HPV16L1 protein is truncated based on NCBI database AAC09292.1 sequence; the truncated HPV16L1 protein is selected from HPV16L1 delta N2C29, HPV16L1 delta N3C29, HPV16L1 delta N5C29, HPV16L1 delta N7C29, HPV16L1 delta N2C31, HPV16L1 delta N3C31, HPV16L1 delta N5C31, HPV16L1 delta N7C31, HPV16L1 delta N2C33, HPV16L1 delta N3C33, HPV16L1 delta N5C33 and HPV16L1 delta N7C 33.

3. A polynucleotide encoding the truncated HPV16L1 protein of claim 1 or 2.

4. A vector comprising the polynucleotide of claim 3.

5. The vector of claim 4, wherein the vector is a recombinant baculovirus.

6. An insect cell comprising the vector of claim 4 or 5.

7. An HPV16L1 virus-like particle, wherein the HPV16L1 virus-like particle comprises the truncated HPV16L1 protein of claim 1 or 2.

8. An HPV16L1 virus-like particle, wherein the HPV16L1 virus-like particle consists of the truncated HPV16L1 protein of claim 1 or 2.

9. A vaccine for preventing HPV infection or a disease associated with HPV infection, wherein the vaccine comprises the HPV16L1 virus-like particle of claim 7 or 8 and an excipient or adjuvant.

10. The vaccine of claim 9, wherein the vaccine further comprises at least one virus-like particle of HPV selected from the other mucophilic and dermophilic groups.

11. The vaccine of claim 10, wherein the vaccine further comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15L 1 virus-like particles selected from HPV2, 5, 6,7,11, 18, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,39, 40, 43, 44, 45,51,52, 53, 56, 57, 58,59, 61, 66,67, 68, 69,70,73, 74, 77, 81, 82, 83, 85, 91.

12. The vaccine of any one of claims 9-11, wherein the adjuvant is an aluminum adjuvant, an oil-in-water emulsion, or a water-in-oil emulsion and adjuvant composition of a TLR stimulant.

13. The vaccine of any one of claims 9-11, wherein the adjuvant is an aluminum hydroxide adjuvant or a combination of an aluminum phosphate adjuvant with a polyinosinic-polycytidylic acid adjuvant and a stabilizer.

14. The vaccine of any one of claims 9-11, wherein the adjuvant is a combination of MF59 adjuvant with polyinosinic-polycytidylic acid adjuvant and a stabilizer.

15. Use of the truncated HPV16L1 protein according to claim 1 or 2, the HPV16L1 virus-like particle according to claim 7 or 8, or the vaccine according to any one of claims 9-14 for the manufacture of a medicament for the prevention of HPV infection and diseases associated with HPV infection.