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

Abstract

The present invention relates to a truncated human papillomavirus type 16 L1 protein and its application. Specifically, the present invention relates to a truncated HPV16 L1 protein, a nucleotide encoding the same, a vector comprising the nucleotide, a recombinant Bacmid, a recombinant baculovirus, an insect cell comprising the recombinant baculovirus, A virus-like particle composed of the HPV16L1 protein, a vaccine containing the virus-like particle and a vaccine adjuvant, and use thereof in preventing HPV infection or HPV infection-related diseases.

Description

Truncated human papillomavirus type 16 L1 protein and its application

technical field

The present invention relates to a novel truncated human papillomavirus type 16 L1 protein, a virus-like particle composed thereof, a vaccine containing the virus-like particle and a vaccine adjuvant, and their use in preventing HPV infection or HPV infection-related pathology the use of.

Background technique

At present, more than 200 types of human papillomavirus (HPV) have been isolated and identified, which are divided into mucosa-tropic group and dermatophilic group. HPV in the mucosa group mainly infects the mucosa and surrounding skin of the urogenital tract, perianal and oropharynx, and induces various benign and malignant lesions. According to the nature of the induced lesions, it can be divided into high-risk types (including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, etc.), suspected high-risk types ( HPV26, 30, 53, 66, 67, 69, 70, 73, 82, 85, etc.), undetermined types (HPV34, 42, 43, 54, 71, 81, 83, 97, 102, 114, etc.), and induced verrucous hyperplasia, etc. Low-risk types of benign lesions (HPV6, 7, 11, 13, 32, 40, 42, 44, 61, 62, 72, 74, 81, 83, 84, 86, 87, 89, 90, 91, 106, etc.). The dermatophilic group mainly infects skin tissues other than the above-mentioned parts, induces skin verrucous hyperplasia, and is closely related to the occurrence of some skin cancers.

The malignant tumors associated with high-risk HPV infection have been identified as follows: cervical cancer, vaginal cancer, labia cancer, penile cancer, perianal cancer, oropharyngeal cancer, tonsil cancer, oral cancer, among which cervical cancer is the most harmful. Cervical cancer is the third most common malignant tumor in women worldwide, with an annual incidence of about 527,000, of which 285,000 are in Asia; the annual incidence in China is 75,000.

The study found that the main coat protein L1 of the virus can be assembled into L1 virus-like particles (VLPs) after in vitro expression. The structure and shape of L1VLPs are similar to the natural structure of virus particles and have strong immunogenicity. The three HPV preventive vaccines currently listed abroad are all HPV L1 VLP vaccines, including GlaxoSmithKline's HPV16/18 bivalent vaccine, Merck's HPV16/18/6/11 quadrivalent vaccine and HPV16/18/58/52 /31/33/45/6/11 9-valent vaccine, after the population is immunized, the immune activity is good, and the protection efficiency against infection and infection-related diseases is high. Given that HPV16 is the predominant viral strain worldwide, about 53.5% of cervical cancers are caused by HPV16 infection, and the remaining 46.5% of cervical cancers are caused by infection with about 22 other high-risk and suspected high-risk HPV types. Therefore, it is meaningful to study the HPV16 L1VLP vaccine in depth.

At present, a variety of different expression systems, including insect cell expression system, yeast expression system, E. coli expression system and plant expression system, can be used for the expression and production of HPV16 L1VLP. The study found that HPV16 L1 full-length protein (White W, Wilson S, et al. J. Virol. 1999; 73 (6): 4882-4889.), N-terminal truncated mutant protein (Chen XS, Casini G, et al. al.J.Mol.Biol.2011; 307:173-182.), C-terminal truncated mutants (patents CN1976718A, CN102586287, CN104418942A), and combined N-terminal truncated 10 amino acids and C-terminal truncated 22 amino acids The mutant proteins of HPV16L1 (Varsani A, Williamson AL, et al.Vir.Res.2006; 122:154-163.) can be assembled into HPV16L1 VLPs using specific techniques (the full-length HPV16 L1 protein sequence is the NCBI database AAC09292. 1 sequence, the N-terminal truncation starts from the first amino acid M at the N-terminal). The study also found a variety of methods to increase the expression level of HPV16 L1 protein. For example, truncation of 31 amino acids at the C-terminal can increase the expression level by 1.58 times, and eliminating the inhibitory DNA sequence within the first 129 nucleotides of the N-terminal can also improve the expression. The combined use of codon optimization, elimination of potential transcription termination sites, elimination of potential splicing sites, and simplification of mRNA secondary structure can also improve expression levels.

At present, whether HPV16 L1 mutants with N-terminal truncated 2, 3, 5 or 7 amino acids can form VLPs, and HPV16 L1 mutations with N-terminal truncated 1-9 amino acids combined with C-terminal truncated 1-34 amino acids Whether the body can form VLP has not been reported yet. Therefore, the analysis of the expression level of the truncated mutant obtained by the above-mentioned method is also unclear. The study found that even the difference of one or several amino acids in HPV16L1 protein can significantly affect its expression level. The expression levels of L1 mutants with different lengths of C-terminal truncation are significantly different. The L1 mutant had significantly higher expression levels than the wild type.

The number of amino acids truncated at the C-terminal used in the present invention is different from this study, and the expression level of the truncated protein is unpredictable; in addition, the amino acid sequence composition of the obtained HPV16 L1 mutant obtained by combining amino acids with different lengths of truncated N-terminal is the same as that of the currently known HPV16 L1 mutant. The reported methods are more different, and the expression levels and whether they can be assembled into VLPs are also unpredictable, and need to rely on specific experimental studies to determine. Therefore, the research value of the various HPV16L1 mutants designed in the present invention in the HPV16 L1 VLP vaccine can only be determined after experimental exploration.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new truncated HPV16 L1 protein, a virus-like particle composed thereof and a vaccine containing the virus-like particle, and to study the application of the vaccine in preventing HPV infection and infection-related diseases.

The inventors have unexpectedly discovered through research that appropriate truncation of the N-terminal and/or C-terminal of HPV16 L1 protein can increase the expression of HPV16 L1 protein in an insect cell expression system, and the truncated protein can be assembled into VLPs , and can induce a protective immune response against HPV16. The present invention is based on the above findings, now completed, and the data are provided in the Examples herein.

Therefore, the first aspect of the present invention relates to a 2, 3, 5 or 7 amino acid truncated N-terminus compared to the wild-type HPV16 L1 protein (eg, the starting ATG is the same as the sequence of NCBI database AAC09292.1), and the C-terminus remains intact The amino acids encoded by the reading frame or the HPV16L1 protein truncated by 29, 31, and 33 amino acids.优选地该截短蛋白选自HPV16 L1ΔN2、HPV16L1ΔN3、HPV16 L1ΔN5、HPV16 L1ΔN7、HPV16 L1ΔN2C29、HPV16 L1ΔN3C29、HPV16 L1ΔN5C29、HPV16L1ΔN7C29、HPV16 L1ΔN2C31、HPV16 L1ΔN3C31、HPV16 L1ΔN5C31、HPV16 L1ΔN7C31、HPV16 L1ΔN2C33、HPV16 L1ΔN3C33、HPV16 L1ΔN5C33 and HPV16 L1ΔN7C33.

Specifically, the present invention provides a truncated HPV16 L1 protein, wherein the truncated HPV16 L1 protein is truncated by 2, 3, 5 or 7 amino acids at the N-terminus compared with the wild-type HPV16 L1 protein; Compared with the wild-type HPV16 L1 protein, the truncated HPV16 L1 protein retains the amino acids encoded by the complete reading frame at the C-terminus or is truncated by 29, 31 or 33 amino acids.

Preferably, the truncated HPV16 L1 protein of the present invention is truncated based on the sequence of NCBI database AAC09292.1; particularly preferably, the truncated HPV16 L1 protein is selected from HPV16 L1ΔN5C31 (SEQ ID No. 1) and HPV16 L1ΔN7C33 (SEQ ID No. 2).

Wild-type HPV16 L1 protein can also be derived from, but not limited to, the L1 protein of HPV16Phi1, Tha7, Alg1, Sen32, Fra25, Fra63, 114K, 114B, Z-1194 and other mutant strains (Touze A, Mehdaoui SE, et al. J. Clin. Micr 1998;36(7):2046-2051), the truncated L1 protein of the corresponding variant is truncated at the equivalent position of the above-mentioned truncated HPV16 L1 protein, as assessed by sequence comparison.

A second aspect of the present invention relates to a polynucleotide encoding the truncated HPV16 L1 protein of the present invention.

The third aspect of the present invention relates to a vector containing the polynucleotide of the second aspect above, preferably, the vector is selected from plasmids, recombinant Bacmid and recombinant baculovirus.

A fourth aspect of the present invention relates to insect cells comprising the above-mentioned vector.

The fifth aspect of the present invention relates to a HPV16 L1 virus-like particle, the virus-like particle containing the HPV16L1 protein described in the first aspect above, or consisting of the HPV16 L1 protein described in the first aspect above.

The sixth aspect of the present invention relates to a vaccine for preventing HPV infection or HPV infection-related lesions, the vaccine contains the HPV16 L1 virus-like particles described in the fifth aspect, wherein the content of the HPV16 L1 virus-like particles is such that a protective immune response can be induced effective amount. Preferably, the vaccine may further comprise at least one virus-like particle of HPV selected from other mucophagic group and/or dermatophilic group, and the content of these virus-like particles is respectively an effective amount for inducing a protective immune response. The vaccines described above generally also contain a vaccine excipient or carrier.

Preferably, the vaccine contains the HPV16 L1 virus-like particle of the fifth aspect and at least one selected from the group consisting of 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, and 91 of L1 virus-like particles, and the content of these virus-like particles is respectively an effective amount that can induce a protective immune response.

Further preferably, the vaccine contains the HPV16 L1 virus-like particles described in the fifth aspect and the L1 viruses of HPV18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and 73 The content of these virus-like particles is an effective amount to induce a protective immune response, respectively.

Further preferably, the vaccine contains the HPV16L1 virus-like particles described in the fifth aspect and the L1 virus-like particles of HPV18, 31, 33, 35, 39, 45, 52 and 58, and the content of these virus-like particles is respectively enough to induce An effective amount for a protective immune response.

Further preferably, the vaccine contains the HPV16L1 virus-like particles described in the fifth aspect and the L1 virus-like particles of HPV18, 52 and 58, and the contents of these virus-like particles are respectively an effective amount capable of inducing a protective immune response.

Further preferably, the vaccine contains the HPV16 L1 virus-like particles described in the fifth aspect and the L1 virus-like particles of HPV18 and 58, and the content of these virus-like particles is respectively an effective amount capable of inducing a protective immune response.

Particularly preferably, the vaccine contains the HPV16 L1 virus-like particles described in the fifth aspect and the L1 virus-like particles of HPV18, and the content of these virus-like particles is respectively an effective amount capable of inducing a 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 improve the immune response. Preferably, the adjuvant used is an adjuvant composition comprising an aluminium adjuvant, an oil-in-water emulsion or a water-in-oil emulsion and a TLR stimulator.

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

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

In particular, the complex adjuvant involved in the present invention is the complex composed of polyinosinic acid-polycytidylic acid and stabilizer (PIKA) combined with aluminum hydroxide adjuvant described in the embodiment, or polyinosinic acid-polycytidylic acid adjuvant A complex composed of cytidine and stabilizer (PIKA) combined with MF59 adjuvant, the combined use of two complex adjuvants and HPV VLP vaccine can effectively improve the immune activity of the vaccine.

The eighth aspect of the present invention relates to the use of the vaccine according to the sixth and seventh aspects in preventing HPV infection or HPV infection-related diseases.

Explanation and explanation of related terms

According to the present invention, the term "insect cell expression system" includes insect cells, recombinant baculovirus, recombinant Bacmid and expression vectors. Insect cells are derived from commercially available cells, such as but not limited to: Sf9, Sf21, High Five.

According to the present invention, examples of the term "wild-type HPV16 L1 protein" include, but are not limited to, the full-length L1 protein equivalent to the protein numbered AAC09292.1 in the NCBI database.

The gene fragment of "truncated HPV16 L1 protein" refers to a nucleotide encoding 1 or more amino acids deleted at its 5' end and/or 3' end compared with the wild-type HPV16 L1 protein gene, wherein The full-length sequence of "wild-type HPV16 L1 protein" such as, but not limited to, the following sequences in the NCBI database: AAC09292.1, AIQ82817.1, AAC61736.1, etc.

According to the present invention, the expression "ΔNX" stands for "a protein with N-terminal truncated by X amino acids", which means that the starting methionine is counted as the first amino acid, and after X amino acids are truncated from the N-terminal, the remaining The N-terminus of the sequence is supplemented with a methionine as the start site of the protein. For example, "HPV16 L1ΔN5" represents the HPV16 L1 protein with the N-terminal truncated by 5 amino acids, that is, after deleting the 5 amino acids from the initial methionine, an additional methionine is added to the N-terminal of the remaining sequence. obtained protein.

According to the present invention, the expression "ΔCY" stands for "protein truncated by Y amino acids at the C-terminal", and refers to a protein that is truncated by Y amino acids, counting from the 505th amino acid of HPV16 L1. For example, "HPV16 L1ΔC1" represents a protein obtained by truncating the 505th amino acid at the C-terminal.

According to the present invention, the expression "ΔNXCY" stands for "a protein with X amino acids truncated at the N-terminal and Y amino acids at the C-terminal", which means that the starting methionine is counted as the first amino acid, and the truncated amino acid from the N-terminal is counted. After removing X amino acids, a methionine was added to the N-terminus of the remaining sequence as a starting site, and the protein of Y amino acids was truncated by counting from the 505th amino acid of HPV16 L1. For example, "HPV16L1ΔN5C31" represents the HPV16 L1 protein with 5 amino acids truncated at the N-terminus and 31 amino acids at the C-terminus, that is, after deleting 5 amino acids starting from the initial methionine, it is added at the N-terminus of the remaining sequence. A methionine was added, and the protein with a total of 31 amino acids at positions 475-505 of the C-terminal was deleted at the same time.

According to the present invention, the term "excipient or carrier" refers to one or more selected from the group consisting of, but not limited to: pH adjusters, surfactants, ionic strength enhancers. For example, pH adjusters are exemplified but not limited to phosphate buffers, surfactants include cationic, anionic or nonionic surfactants, such as but not limited to polysorbate 80 (Tween-80), ionic strength enhancers are exemplified but not limited to Limited to sodium chloride.

According to the present invention, the term "adjuvant" refers to an adjuvant that is clinically applicable to humans, including various adjuvants currently approved and those that may be approved in the future, such as but not limited to aluminum adjuvant, MF59 and various adjuvants. A form of adjuvant composition.

According to the present invention, the term "emulsion" refers to a heterogeneous liquid dispersion system formed by mixing a water phase component, an oil phase component and an emulsifier in an appropriate ratio after emulsification. The water phase components include but are not limited to buffer systems such as phosphate buffer and HEPES buffer; the oil phase components are metabolizable lipids, including but not limited to vegetable oil, fish oil, animal oil, synthetic oil and other lipid components (such as but not limited to 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 acid-polycytidylic acid in an adjuvant and play a stabilizing role, 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), cationic organic complexes (such as but not limited to calcium stearate, calcium gluconate).

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

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

Description of drawings

Figure 1 shows the expression identification of truncated HPV16L1 in insect cells in Example 4 of the present invention. The results showed that all 16 truncated HPV16 L1s could be expressed at high levels in insect cells.

1至16分别表示HPV16 L1ΔN2、HPV16 L1ΔN3、HPV16 L1ΔN5、HPV16 L1ΔN7、HPV16 L1ΔN2C29、HPV16 L1ΔN3C29、HPV16 L1ΔN5C29、HPV16 L1ΔN7C29、HPV16 L1ΔN2C31、HPV16 L1ΔN3C31、HPV16 L1ΔN5C31、HPV16 L1ΔN7C31、HPV16 L1ΔN2C33、HPV16L1ΔN3C33、HPV16 L1ΔN5C33和HPV16 L1ΔN7C33.

2A to 2B show the dynamic light scattering analysis results of the HPV16 L1ΔN5 and HPV16 L1ΔN5C31 mutant proteins obtained after purification in Example 6 of the present invention. The results showed that the hydration kinetic diameters of virus-like particles formed by HPV16 L1ΔN5 and HPV16 L1ΔN5C31 recombinant proteins were 129.6 nm and 128 nm, respectively, and the percentage of particle assembly was 100%.

Figure 2A represents HPV16 L1ΔN5; Figure 2B represents HPV16 L1ΔN5C31.

3A to 3P show the results of transmission electron microscopy of 16 truncated HPV16 L1 VLPs obtained after purification in Example 7 of the present invention. A large number of virus-like particles with a diameter of about 55 nm can be seen in the field of vision. The size of the particles is consistent with the theoretical value, and the uniformity is good. Bar=200nm.

图3A至3P分别表示HPV16 L1ΔN2、HPV16 L1ΔN3、HPV16L1ΔN5、HPV16 L1ΔN7、HPV16 L1ΔN2C29、HPV16 L1ΔN3C29、HPV16 L1ΔN5C29、HPV16 L1ΔN7C29、HPV16 L1ΔN2C31、HPV16 L1ΔN3C31、HPV16 L1ΔN5C31、HPV16 L1ΔN7C31、HPV16 L1ΔN2C33、HPV16L1ΔN3C33、HPV16 L1ΔN5C33和HPV16 L1ΔN7C33.

Figure 4 shows the analysis of HPV16 neutralizing antibody titers in immune serum after inoculating mice with HPV16 L1ΔN5 and HPV16 L1ΔN5C31 VLPs in Example 8 of the present invention.

Figure 5 shows the analysis of the neutralizing antibody titer of immune serum after inoculating mice with HPV16 L1ΔN5C31 VLP protein combined with PIKA-containing compound adjuvant in Example 9 of the present invention, *: compared with the no-adjuvant group, P<0.05; **: Compared with the unadjuvanted group, with P<0.01; ***: compared with the unadjuvanted group, with P<0.001; #: compared with the pure PIKA group, with P<0.05; ### : Compared with the pure PIKA group, compared with P<0.001; &: compared with the pure Alum group, compared with P<0.05; &&&: compared with the pure Alum group, compared with P<0.001.

Detailed ways

The present invention will be further illustrated by non-limiting examples below. It is well known to those skilled in the art that many modifications can be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention. The following examples are only intended to illustrate the present invention and should not be construed to limit the scope of the present invention, since the embodiments are necessarily varied. The phraseology used in this specification is for the purpose of describing particular embodiments only, not limiting, and the scope of the invention is defined in the appended claims.

Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this application belongs. Preferred methods and materials of the present invention are described below, but any methods and materials similar or equivalent to those described in this specification can be used in the practice or testing of the present invention. Unless otherwise specified, the following experimental methods are conventional methods or methods described in the product specification, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified. All publications mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in the publications.

Example 1: Amplification of truncated HPV16 L1 gene and construction of expression vector

The full-length HPV16L1 gene used as a template was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. (SEQ ID NO: 3), and its corresponding amino acid sequence was the sequence numbered AAC09292.1 in the NCBI database.

The primers used to construct the truncated HPV16L1 gene were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., as follows:

16N2F: 5'-GAATTCGCCGCCACCATGCTCTGGTTTGCCATCCGAAGC-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’-TCTAGAATTAAGCTTGCAACAGGAATTTC-3’,

The above primer sequences are shown in SEQ ID NOs: 6-13 of the Sequence Listing.

Using SEQ ID NO: 3 as a template, using 16N2F/16CR as primers, PCR amplification of HPV16 L1ΔN2 gene; using 16N3F/16CR as primers, PCR amplification of HPV16 L1ΔN3 gene; using 16N5F/16CR as primers, PCR amplification of HPV16 L1ΔN5 gene ; Using 16N7F/16CR as primers, PCR amplification of HPV16 L1ΔN7 gene; using 16N2F/16C29R as primers, PCR amplification of HPV16 L1ΔN2C29 gene; using 16N3F/16C29R as primers, PCR amplification of HPV16 L1ΔN3C29 gene; using 16N5F/16C29R as primers, PCR-amplified HPV16 L1ΔN5C29 gene; using 16N7F/16C29R as primers, PCR-amplified HPV16 L1ΔN7C29 gene; using 16N2F/16C31R as primers, PCR-amplified HPV16 L1ΔN2C31 gene; using 16N3F/16C31R as primers, PCR-amplified HPV16 L1ΔN3C31 gene; Using 16N5F/16C31R as primers, PCR amplification of HPV16 L1ΔN5C31 gene (SEQ ID NO: 4); using 16N7F/16C31R as primers, PCR amplification of HPV16 L1ΔN7C31 gene; using 16N2F/16C33R as primers, PCR amplification of HPV16 L1ΔN2C33 gene; using 16N3F Using /16C33R as primers, PCR amplification of HPV16 L1ΔN3C33 gene; using 16N5F/16C33R as primers, PCR amplification of HPV16 L1ΔN5C33 gene; using 16N7F/16C33R as primers, PCR amplification of HPV16 L1ΔN7C33 gene (SEQ ID NO: 5). The methods of PCR amplification are well known, such as patent CN 101293918B.

The EcoRI/Xba I restriction site was used to digest the gene amplified by the above-mentioned PCR, respectively, and inserted into the commercial expression vector pFastBac1 (produced by Invitrogen) to obtain a recombinant expression vector comprising the truncated HPV16L1 gene: pFastBac1-16L1ΔN2、pFastBac1-16L1ΔN3、pFastBac1-16L1ΔN5、pFastBac1-16L1ΔN7、pFastBac1-16L1ΔN2C29、pFastBac1-16L1ΔN3C29、pFastBac1-16L1ΔN5C29、pFastBac1-16L1ΔN7C29、pFastBac1-16L1ΔN2C31、pFastBac1-16L1ΔN3C31、pFastBac1-16L1ΔN5C31、pFastBac1-16L1ΔN7C31、pFastBac1- 16L1ΔN2C33, pFastBac1-16L1ΔN3C33, pFastBac1-16L1ΔN5C33, pFastBac1-16L1ΔN7C33. The above-mentioned methods of enzyme digestion, ligation and cloning construction are well known, such as patent CN101293918B.

Example 2: Construction of recombinant Bacmid and recombinant baculovirus of truncated HPV16 L1 gene

分别使用包含截短型HPV16L1基因的重组表达载体pFastBac1-16L1ΔN2、pFastBac1-16L1ΔN3、pFastBac1-16L1ΔN5、pFastBac1-16L1ΔN7、pFastBac1-16L1ΔN2C29、pFastBac1-16L1ΔN3C29、pFastBac1-16L1ΔN5C29、pFastBac1-16L1ΔN7C29、pFastBac1-16L1ΔN2C31、pFastBac1- 16L1ΔN3C31、pFastBac1-16L1ΔN5C31、pFastBac1-16L1ΔN7C31、pFastBac1-16L1ΔN2C33、pFastBac1-16L1ΔN3C33、pFastBac1-16L1ΔN5C33、pFastBac1-16L1ΔN7C33转化大肠杆菌DH10Bac感受态，筛选获得重组Bacmid，然后用重组Bacmid转染昆虫细胞Sf9，在Sf9内Amplification of recombinant baculovirus. Screening of recombinant Bacmid and amplification methods of recombinant baculovirus are well known, such as patent CN 101148661B.

Example 3: Expression of truncated HPV16 L1 gene in Sf9 cells

Sf9 cells were inoculated with 16 recombinant baculoviruses of truncated HPV16 L1 gene, and the truncated HPV16L1 protein was expressed. After culturing at 27 °C for about 88 h, the fermentation broth was collected, centrifuged at 3000 rpm for 15 min, the supernatant was discarded, and the cells were washed with PBS. , for expression identification and purification. The method of infection expression is disclosed, eg patent CN 101148661B.

Example 4: Expression identification of truncated HPV16 L1

Take 1×10 ⁶ cells expressing different truncated HPV16 L1 described in Example 3, resuspend in 200 μl of PBS solution, add 50 μl of 6× Loading buffer, denature at 75°C for 8 minutes, and take 10 μl respectively for SDS- PAGE electrophoresis and Western blot identification. The results are shown in Figure 1. All 16 truncated HPV16 L1 proteins can be expressed at high levels in insect cells. Among them, HPV16 L1ΔN2, HPV16 L1ΔN3, HPV16 L1ΔN5, and HPV16 L1ΔN7 are about 55 kDa in size, and the remaining 12 proteins are about 50 kDa in size. The methods of SDS-PAGE electrophoresis and Western blot identification are disclosed, such as patent CN101148661B.

Example 5: Comparison of expression levels of truncated HPV16 L1 protein and wild-type HPV16 L1 protein

Take 1 × 10 ⁶ cells each expressing truncated HPV16L1 protein and wild-type HPV16L1 described in Example 3, resuspend in 200 μl PBS solution, and use ultrasonication method (Ningbo Xinzhi ultrasonic crusher, 2# probe, 100W, ultrasonic for 5s, interval 7s, total time 3min) to disrupt cells, and centrifuge at 12000rpm for 10 minutes. The lysis supernatant is collected, and the L1 content in the supernatant is detected by a sandwich ELISA method, which is well known, such as patent CN104513826A.

The HPV16 L1 monoclonal antibody prepared by the inventors was used to coat the ELISA plate, 80ng/well, incubated at 4°C overnight; blocked with 5% BSA-PBST at room temperature for 2 hours, and then washed with PBST for 3 times. The lysis supernatant was serially 2-fold diluted with PBS, and the HPV16 L1 VLP standard was also serially diluted, with concentrations ranging from 2 μg/ml to 0.0625 μg/ml, added to the ELISA plate, 100 μl per well, and incubated at 37°C for 1 h. The plate was washed three times with PBST, 1:3000 diluted HPV16 L1 rabbit polyclonal antibody was added, 100 μl per well, and incubated at 37°C for 1 h. The plate was washed three times with PBST, and a 1:3000 dilution of HRP-labeled goat anti-mouse IgG (1:3000 dilution, Zhongshan Jinqiao Co., Ltd.) was added, and incubated at 37°C for 45 minutes. The plate was washed 5 times with PBST, 100 μl of OPD substrate (Sigma) was added to each well, the color was developed at 37° C. for 5 minutes, the reaction was terminated with 50 μl of 2M sulfuric acid, and the absorbance was measured at 490 nm. The concentration of truncated HPV16 L1 protein and wild-type HPV16 L1 protein in the lysis supernatant was calculated according to the standard curve.

The results are shown in Table 1. The expression level of the truncated HPV16 L1 protein of the present invention is higher than that of the wild-type HPV16 L1 protein, and higher than the C-terminal truncated mutant HPV16 L1-498 (HPV16 L1ΔC7) and HPV16L1-483 (HPV16 L1[Delta]C22).

Table 1 Analysis of HPV16L1 protein expression

Example 6: Purification of truncated HPV16 L1 protein and dynamic light scattering particle size analysis

Take 50ml of truncated HPV16 L1 cell fermentation broth, resuspend the cells in 10ml PBS, add PMSF to a final concentration of 1mg/ml, and ultrasonically disrupt (Ningbo Xinzhi ultrasonic disruptor, 6# probe, 100W, ultrasonic for 5s, interval 7s, The total time was 5 min), and the crushed supernatant was taken for purification, and the purification step was carried out at room temperature. The VLPs were depolymerized by adding 4% β-mercaptoethanol (w/w) to the lysate, then the samples were filtered using a 0.22 μm filter, followed by DMAE anion exchange chromatography (20 mM Tris, 180 mM NaCl, 4% β-ME, pH 7.9 elution), TMAE anion exchange chromatography (20 mM Tris, 180 mM NaCl, 4% β-ME, pH 7.9 elution) and hydroxyapatite chromatography (100 mM NaH ₂ PO ₄ , 30 mM NaCl, 4% β- ME, pH 6.0 elution) purification. The purified product was concentrated using a Planova ultrafiltration system and buffer exchange ( ₂₀ mM _NaH2PO4 , 500 mM NaCl, pH 6.0) facilitated VLP assembly. The above purification methods are all disclosed, such as patents CN101293918B, CN1976718A and the like.

The purified truncated HPV16 L1 protein solution was taken for DLS particle size analysis (Zetasizer Nano ZS 90 dynamic light scattering instrument, Malvern Company). shown.

Table 2 DLS analysis of truncated HPV16 L1 protein

protein name 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 microscope observation of truncated HPV16 L1VLP

According to the chromatographic purification method described in Example 6, the truncated HPV16 L1 VLPs were purified respectively, and the dialyzed VLPs were used to prepare copper meshes, which were stained with 1% uranyl acetate, and fully dried using a JEM-1400 electron microscope (Olin Bath) to observe. The results are shown in Fig. 3, the truncated HPV16 L1VLPs were about 55 nm in diameter, uniform in size and regular in shape. The methods of copper mesh preparation and electron microscope observation are disclosed, such as patent CN 101148661 B.

Example 8: Mouse immunization of truncated HPV16 L1 VLPs and determination of neutralizing antibody titers

BALB/c mice aged 4-6 weeks were selected and randomly divided into groups of 4. The mice were immunized with PBS, truncated HPV16 L1VLP and HPV16 L1wtVLP, respectively. Intramuscular injection, the immunization dose of L1VLP was 0.1 μg, and the immunization was performed twice on the 0th and 2nd week. Two weeks after the second immunization, tail vein blood was collected, and serum was separated.

The HPV16 neutralizing antibody titer of the immune serum was detected by HPV16 pseudovirus. The results are shown in Figure 4. HPV16 L1ΔN5VLP (16 L1ΔN5), HPV16 L1ΔN5C31VLP (16 L1ΔN5C31) and HPV16 L1wtVLP (16L1wt) were all effective after immunizing mice Neutralizing antibodies were induced, and the titers of neutralizing antibodies were not statistically different. After immunizing mice with other 14 truncated HPV16 L1 mutant VLPs included in the present invention, the induced HPV16 neutralizing antibody levels were between 400-1600, and there was no difference compared with HPV16 L1wtVLP. The methods of pseudovirus preparation and pseudovirus neutralization experiments are disclosed, for example, patent CN 104418942A.

Example 9: HPV16 L1ΔN5C31 VLP combined with adjuvant immunized mice and neutralizing antibody titer determination

BALB/c mice aged 4-6 weeks were randomly divided into 11 groups and were treated with HPV16 L1ΔN5C31VLP combined with polyinosinic acid-polycytidylic acid injection (PIKA, Nanguo Pharmaceutical Co., Ltd.) and aluminum hydroxide adjuvant or MF59 adjuvant. (4.3% squalene, 0.5% Tween-80, 0.5% Span-85) immunized mice, and the specific groups and immunization doses are shown in Table 3. Subcutaneous injection, immunization on the 0th and 2nd week, a total of 2 times. Two weeks after the second immunization, tail vein blood was collected, and serum was separated.

Table 3 Grouping and dosage of adjuvant experimental mice

group name Vaccine ingredients and doses no adjuvant 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 neutralizing antibody titers in immune serum were detected using HPV16 pseudovirus. The results are shown in Figure 5. The use of aluminum hydroxide adjuvant or polyinosinic acid-polycytidylic acid adjuvant (PIKA adjuvant) alone was used to detect HPV16 The level of neutralizing antibody induced by L1VLP had no significant enhancement effect, but the use of aluminum hydroxide or MF59 combined with PIKA adjuvant could significantly increase the level of neutralizing antibody induced by VLP, and compared with the use of aluminum hydroxide or PIKA adjuvant alone, Neutralizing antibody levels also increased significantly. It shows that Alum/PIKA compound adjuvant or MF59/PIKA compound adjuvant can significantly improve the immune activity of vaccine, and has application prospect (using SPSS software, One-way ANOVA analysis).

sequence listing

<110> Institute of Basic Medicine, Chinese Academy of Medical Sciences

<120> Truncated human papillomavirus type 16 L1 protein and its application

<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

385 390 395 400

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

210 215 220

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

370 375 380

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 gccccccaccc 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 ccccgcccccc aaggaagacc cattgaagaa gtataccttc 1320

tgggaagtga acctgaaaga gaaattcagc gctgatttgg accaattccc actgggaagg 1380

aaattcctgt tgcaagctta at 1402

<210> 6

<211> 38

<212> DNA

<213> Artificial sequences

<220>

<223> primers

<400> 6

gaattcgccg ccaccatgct ctggttgcca tccgaagc 38

<210> 7

<211> 38

<212> DNA

<213> Artificial sequences

<220>

<223> primers

<400> 7

gaattcgccg ccaccatgtg gttgccatcc gaagctac 38

<210> 8

<211> 40

<212> DNA

<213> Artificial sequences

<220>

<223> primers

<400> 8

gaattcgccg ccaccatgcc atccgaagct acagtctatc 40

<210> 9

<211> 38

<212> DNA

<213> Artificial sequences

<220>

<223> primers

<400> 9

gaattcgccg ccaccatgga agctacagtc tatctccc 38

<210> 10

<211> 27

<212> DNA

<213> Artificial sequences

<220>

<223> primers

<400> 10

tctagattac agcttacgtt ttttgcg 27

<210> 11

<211> 32

<212> DNA

<213> Artificial sequences

<220>

<223> primers

<400> 11

tctagaatta ggccttgaga ccagcttgca ac 32

<210> 12

<211> 23

<212> DNA

<213> Artificial sequences

<220>

<223> primers

<400> 12

tctagaatta gagaccagct tgc 23

<210> 13

<211> 29

<212> DNA

<213> Artificial sequences

<220>

<223> primers

<400> 13

tctagaatta agcttgcaac aggaatttc 29

Claims (15)

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.

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