WO2022142524A1 - 一种人乳头瘤病毒31型嵌合蛋白及其用途 - Google Patents

一种人乳头瘤病毒31型嵌合蛋白及其用途 Download PDF

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WO2022142524A1
WO2022142524A1 PCT/CN2021/120603 CN2021120603W WO2022142524A1 WO 2022142524 A1 WO2022142524 A1 WO 2022142524A1 CN 2021120603 W CN2021120603 W CN 2021120603W WO 2022142524 A1 WO2022142524 A1 WO 2022142524A1
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protein
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amino acid
hpv31
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许雪梅
郝亚茹
张婷
马铭饶
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中国医学科学院基础医学研究所
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Priority to EP21913269.3A priority patent/EP4273174A1/en
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Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to a human papillomavirus chimeric protein, and a pentamer or virus-like particle formed therefrom, and the human papillomavirus chimeric protein, its pentamer or virus-like particle in Use in the preparation of a vaccine for preventing papillomavirus infection and infection-induced disease in a subject.
  • HPV Human papillomavirus
  • HPV16 and HPV18 have the highest detection rates in cervical cancer worldwide, at 55.4% and 14.6%, respectively.
  • HPV31 is a relatively common HR-HPV worldwide, with a detection rate of 3.5% in cervical cancer tissue, ranking sixth; in cervical high-grade precancerous lesions, the detection rate is 10.4%, ranking first. The third place, second only to HPV16 (45.1%) and HPV52 (11%); the detection rate in cervical tissue with normal cytology was 1.3%, ranking third, second only to HPV16 (2.9%) and HPV58 (1.5%).
  • the detection rate of HPV31 in high-grade cervical precancerous lesions is as high as 12.4%, second only to HPV16 (46.8%); in addition, the detection rate of HPV31 in normal cytological cervical tissue in Latin America Second only to HPV16 (3.3%) at 1.2%.
  • the C-terminus of HPV L1 protein contains a nuclear localization signal.
  • L1 protein is expressed in eukaryotic cells, it is mediated by the nuclear localization signal into the nucleus and assembled into virus-like particles in the nucleus.
  • the study found that by removing the nuclear localization signal by deletion method, the L1 protein could be distributed in the cytoplasm after translation. Therefore, on the basis of not affecting the C-terminal helix 5 domain of the L1 protein involved in VLP assembly, the nuclear localization signal was removed, and the L1 C-terminal truncated gene was obtained.
  • L1 VLP After expression in a eukaryotic expression system (such as insect cells), L1 VLP mainly Distributed in the cytoplasm, which is beneficial to cell disruption and downstream purification.
  • bovine papilloma virus type 1 (BPV1) L1 mutant with 24 amino acid truncated C-terminal, HPV16 L1 mutant with 23 amino acid truncated C-terminal, HPV58 L1 C-terminal truncated mutant The 25 amino acid mutants did not affect the activity of L1 protein assembly into VLP, and the assembly efficiency of BPV1 L1 truncated protein mutant was increased by 3 times, and the expression of HPV58 L1 truncated protein was increased by 2 times.
  • the effects of other types of truncation mutants on expression level, assembly activity and yield have not been reported.
  • the C-terminus of the L1 gene that uses the Escherichia coli expression system to express the L1 VLP in the existing reports is the natural complete sequence.
  • the expression level was increased by 32 times and 16 times, and the yield was increased by 39 times and 42 times, respectively), and the expression level of another variant (Alg1) was increased by 8 times compared with the prototype, and the yield was increased by 24 times.
  • the remaining 2 strains The expression levels and yields were comparable to those of the prototype.
  • the VLP expression levels of the two 16L1 mutant strains (B27 and T3) were analyzed by yeast expression system, and it was found that the VLP production of the two mutant strains was significantly higher than that of the prototype control group.
  • amino acid analysis found that the amino acid primary sequences of the above seven 16L1 mutants were all different, two mutants with higher expression levels in yeast species and three mutants with higher expression levels in insect cells, although their common feature was 202
  • the amino acids at position 266 and position 266 are the same (Asp and Ala, respectively), but the expression level and VLP production of Fra25, which has the same characteristics, are very low (comparable to the control group), indicating that the characteristic constituent amino acids in different variants may affect L1 expression levels, which are unpredictable.
  • the above data show that through the analysis of the expression levels of different mutants, the sequences of mutants with increased VLP expression can be found, which can be used in the production of L1 VLP vaccines, which is expected to reduce the cost of vaccine production. So far, there is no report on the effect of other types of variant strain sequences on the expression level.
  • coli expression system was used to compare the expression levels of nine types of L1 N-terminal truncation mutants and their corresponding types of N-terminal full-length L1, including two 16L1 N-terminal truncation mutants ( ⁇ N5, ⁇ N10), the expression levels of which were significantly higher than those of the control group; two 18L1 N-terminal truncation mutants ( ⁇ N5, ⁇ N10), in which the expression level of ⁇ N5 was significantly increased, and the expression level of ⁇ N10 was significantly decreased; 31L1 N-terminal truncation mutants ( ⁇ N5, ⁇ N10), the expression levels of which were comparable to the control group; 3 33L1 N-terminal truncation mutants ( ⁇ N5, ⁇ N10, ⁇ N15), in which the expression level of ⁇ N10 was significantly increased , the expression levels of the other two were decreased; the expression levels of two 45L1 N-terminal truncation mutants ( ⁇ N4, ⁇ N9) were reduced; the
  • N-terminal truncation mutant of L1 can affect the expression level of the protein in E. coli, and the effect of the length of the N-terminal truncation on the expression level is irregular and unpredictable. So far, there is no research on the expression level of N-terminal truncation mutants in insect cells. In addition, there is no N-terminal truncation using the C-terminal truncated L1 mutant, and the effect of the N-terminal truncation strategy on the expression level of the truncated protein in the eukaryotic expression system has been studied.
  • L1 VLPs are regular icosahedrons with a diameter of 55 nm assembled from 72 L1 pentamers (360 L1 monomers).
  • the L1-dependent neutralizing antibody epitopes are regularly and densely arranged on its surface at certain intervals, and each epitope is repeated 360 times. Therefore, L1VLP is beneficial to the cross-linking of BCR and the production of high-titer neutralizing antibodies.
  • Animal experiments and clinical research data show that HPV L1VLP can induce high titers of persistent L1-specific neutralizing antibodies. After the three marketed VLP vaccines have been applied in more than 100 countries and regions around the world, no intratype variant has been found. The escape phenomenon suggests that VLP induction is directed against multiple L1 epitopes.
  • VLP can also be used as a carrier for epitope peptide vaccines.
  • HPV16 L1 as a carrier, the constructed HPV16 cVLP vaccines displaying 16RG-1, 58RG-1, 33RG-1 and 31RG-1 on the surface are relatively successful and can induce different types of HPV16 at the same time. Allo-RG-1-dependent cross-neutralizing antibodies and HPV16L1-dependent type-specific neutralizing antibodies, the neutralizing antibody titers of HPV16 were comparable to those of 16L1 VLP, and the RG-1-dependent cross-neutralizing antibody titers of each type were relatively The high and neutralization range covers many types (more than 10), and other literatures have also reported some research data on HPV16 cVLP vaccines.
  • the chimeric epitopes include other epitope regions of 16RG-1 and 16L2, but due to insertion
  • the 16cVLP-induced cross-neutralization activity obtained by different sites, insertion strategies, and lengths of selected epitopes was relatively poor.
  • Replacing 16VLP in Cervarix (HPV16/18 L1VLP) with 16cVLP to prepare a 16cVLP/18VLP mixed vaccine preliminary studies show that the vaccine can not only induce high titers of HPV16/18 neutralizing antibodies at the same time, but also induce crossover against HPV58 protective activity. It is suggested that the use of HPV cVLP vaccine for the development of a new generation of preventive vaccines is expected to expand the scope of vaccine protection and reduce the cost of vaccines.
  • the research of cVLP vaccine faces many challenges.
  • the first is to select a highly immunogenic chimeric L2 conserved epitope, including the RG-1 epitope and other conserved epitopes in the L2 protein.
  • the selection of RG-1 epitope is empirical, and the RG-1 epitope of the dominant popular strain type is often selected instead of the RG-1 epitope with strong immunogenicity. The main reason is the lack of different types of RG-1.
  • the sequence flanking the epitope core sequence has an impact on the correct display of the epitope on the surface of the chimeric protein.
  • the different insertion sites and insertion methods of epitope peptides have a great impact on the assembly and activity of chimeric proteins. Insertion site; the different insertion methods include direct insertion, substitution insertion, and whether there is modification of the backbone amino acids in the insertion site region (including whether to add a linker).
  • the tolerance of HPV31 L1 VLP vectors to chimeric epitopes and their effects on immune activity In addition to the three cases listed above, there are many other challenges, mainly due to the structural characteristics of HPV31 L1 VLPs and the The main neutralizing antibody epitope regions are not clear.
  • HPV16 and 18 cVLPs are DE loops.
  • the appropriate sites for HPV31 L1 VLPs to display foreign epitopes are unclear, if the insertion site is not selected properly , will affect the immunogenicity of the HPV31 L1VLP backbone.
  • the HPV31 neutralizing antibody titer induced by 31cVLP is significantly lower than that of HPV31 L1 VLP.
  • the advantages of immune protection against HPV31 were also lost in the preparation of other cVLP mixed vaccines.
  • the type-specific L1-dependent neutralizing antibody titer is preferably comparable to that induced by the corresponding type of L1 VLP, so that in the study of mixed vaccines of different types of cVLP, the protection advantage against the backbone type can be maintained ;
  • the cross-neutralizing activity covers many types and the titer is higher, and its dominant cross-neutralizing type has characteristics compared with other types of cVLP reported so far.
  • the purpose of the present invention is to provide a papillomavirus chimeric protein for preparing a vaccine for preventing papillomavirus infection and infection-induced diseases in subjects.
  • the inventors have unexpectedly found that appropriate truncation, point mutation and/or C-terminal amino acid modification of the HPV31 L1 protein backbone can improve its expression level to varying degrees without affecting its activity to assemble into VLPs; Or the surface region of truncated HPV31 L1 protein or its mutant is inserted into the HPV73 L2 protein polypeptide, which can improve the immunogenicity of the HPV73 L2 protein polypeptide, and the obtained chimeric protein can be expressed in Escherichia coli or insect cells. Horizontally expressed, this chimeric protein can be assembled into VLPs and can induce broad-spectrum protective immune responses against multiple types of HPV from different genera/subgenus.
  • the present invention provides a human papillomavirus chimeric protein comprising an HPV31 type L1 protein or a mutant of the HPV31 type L1 protein, and a HPV31 type L1 protein or HPV31 type L1 protein inserted into the HPV31 type L1 protein
  • the HPV31 type L1 protein is from but not limited to P17388.1, AEI61021.1, AEI60949.1, AAA92894.1, AIG59245 in the NCBI database .1, AIG59235.1 and other L1 proteins from HPV31 prototype or variant strains.
  • the amino acid sequence of the HPV31 type L1 protein is shown in SEQ ID No.1.
  • the HPV31 type L1 protein mutant of the present invention compared with the HPV31 type L1 protein shown in SEQ ID No. 1, comprises:
  • Truncating mutations that truncate the N-terminus by 2, 4, 5, 8 or 10 amino acids; and/or
  • the number in the middle represents the amino acid position compared to a control sequence (e.g., the amino acid sequence shown in SEQ ID No. 1)
  • the letter before the number represents the amino acid residue before mutation
  • the letters after the numbers represent the mutated amino acid residues.
  • mutant of the HPV31 type L1 protein is selected from:
  • T The 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 is replaced by asparagine (N), and its sequence is shown in SEQ ID No.3;
  • T The 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 is replaced with asparagine (N), and the N-terminal of the amino acid sequence is truncated by 4 amino acids, and its sequence is as SEQ ID No.4 shown;
  • SEQ ID No.1 The C-terminal of the amino acid sequence shown in SEQ ID No.1 is truncated by 29 amino acids, and its sequence is shown in SEQ ID No.5;
  • the 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 is replaced by asparagine (N), and the C-terminal of the amino acid sequence is truncated by 29 amino acids.
  • the mutant, its sequence is as SEQ ID No.6 shown;
  • T The 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 was replaced by asparagine (N), the C-terminal of the amino acid sequence was truncated by 29 amino acids, and the N of the amino acid sequence was truncated by 29 amino acids.
  • N asparagine
  • T The 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 was replaced by asparagine (N), the C-terminal of the amino acid sequence was truncated by 29 amino acids, and the N of the amino acid sequence was truncated by 29 amino acids.
  • N asparagine
  • T The 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 was replaced by asparagine (N), the C-terminal of the amino acid sequence was truncated by 29 amino acids, and the N of the amino acid sequence was truncated by 29 amino acids.
  • N asparagine
  • T The 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 was replaced by asparagine (N), the C-terminal of the amino acid sequence was truncated by 29 amino acids, and the N of the amino acid sequence was truncated by 29 amino acids.
  • N asparagine
  • the 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 is replaced by asparagine (N), the N-terminal of the amino acid sequence is truncated by 4 amino acids, and the 475th position, Arginine (R) at positions 483 and 496 was replaced by glycine (G), lysine (K) at positions 477, 497 and 501 was replaced by serine (S), and positions 479 and 501 were replaced by serine (S). 482 and 498 lysine (K) is replaced by alanine (A), the 495th and 500th lysine (K) is replaced by a mutant of glycine (G), its sequence is as SEQ ID No. .12 shown;
  • the 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 was replaced with asparagine (N), the N-terminal of the amino acid sequence was truncated by 4 amino acids, and the 473rd position, Arginine (R) at positions 475, 483 and 496 was replaced by glycine (G), lysine (K) at positions 477, 497 and 501 was replaced by serine (S), Mutants in which lysine (K) at positions 479, 482 and 498 are replaced by alanine (A), and lysine (K) at positions 495 and 500 are replaced by glycine (G), the sequence of which is As shown in SEQ ID No.13;
  • the 274th threonine (T) of the amino acid sequence shown in SEQ ID No.1 is replaced by asparagine (N), the N-terminal of the amino acid sequence is truncated by 4 amino acids, and the 475th position, Arginine (R) at positions 483 and 496 was replaced by glycine (G), lysine (K) at positions 477, 497 and 501 was replaced by serine (S), and positions 482 and 501 were replaced by serine (S).
  • the 498-position lysine (K) is replaced by alanine (A), and the 495-position and 500-position lysine (K) is replaced by a mutant of glycine (G), the sequence of which is shown in SEQ ID No.14 .
  • the polypeptide from the HPV73 type L2 protein is any continuous 8 in the region of amino acids aa.1-50 shown in SEQ ID No.2 -33 amino acid fragments; preferably, the polypeptide is the HPV73 type L2 protein RG-1 epitope peptide or its mutant epitope peptide shown in SEQ ID No. 2; more preferably, the polypeptide is The polypeptide of amino acids 17 to 39 shown in SEQ ID No. 2, or the N-terminal extension or truncation of the polypeptide of amino acids 17 to 39 shown in SEQ ID No. 2 by 1 to 6 amino acids and/or C-terminal extension or truncation Mutants that are 1 to 6 amino acids shorter.
  • polypeptide from HPV73 type L2 protein is shown in SEQ ID No.15, SEQ ID No.16 or SEQ ID No.17.
  • polypeptide from HPV73 type L2 protein can also be more than 60%, preferably more than 70%, with the amino acid sequence shown in SEQ ID No. 15, SEQ ID No. 16 or SEQ ID No. 17, Polypeptides with greater than 80%, greater than 90%, even more preferably greater than 95% sequence identity.
  • the polypeptide from the HPV73 type L2 protein is inserted into the surface region of the HPV31 type L1 protein or the HPV31 type L1 protein mutant, preferably into the HPV31 type L1 protein or the HPV31 type L1 protein
  • the DE loop or h4 region of the L1 protein mutant is more preferably inserted between amino acid 132 and amino acid 133, or amino acid 134 of the HPV31 type L1 protein or the HPV31 type L1 protein mutant by direct insertion and 135, or between amino acids 136 and 137, or between amino acids 137 and 138, or between amino acids 432 and 433, or between amino acids 434 and 435, or between amino acids 435 and 436; or by non-isometric
  • the HPV31 type L1 protein or the HPV31 type L1 protein mutant is inserted into the amino acid 132 to 136 region, or the amino acid 135 to 139 region, or the amino acid 428-431 region, or the amino acid 431-434 region by substitution.
  • direct insertion refers to the insertion of a selected peptide fragment between two adjacent amino acids.
  • a direct insertion between amino acid 132 and amino acid 133 of SEQ ID NO. 1 refers to inserting the selected peptide fragment directly between amino acid 132 and amino acid 133 of SEQ ID NO. 1.
  • non-isometric substitution refers to the insertion of a selected peptide fragment into a specified amino acid interval after deletion of the sequence in the specified amino acid interval.
  • a non-isometric substitution in the region of amino acids 132 to 136 of SEQ ID NO. 1 means that after deletion of amino acids 133-135 of SEQ ID NO. 1, the selected peptide fragment is inserted into the Amino acids between amino acids 132 to 136.
  • the polypeptide from the HPV73 type L2 protein comprises a linker of 1 to 3 amino acid residues at its N-terminus and/or C-terminus.
  • the linker is composed of any combination of amino acids selected from glycine (G), serine (S), alanine (A) and proline (P).
  • G glycine
  • S serine
  • A alanine
  • P proline
  • the N-terminal linker consists of G(glycine)P(proline)
  • the C-terminal linker consists of P(proline).
  • the amino acid sequence of the polypeptide from the HPV73 type L2 protein is SEQ ID No. 15, SEQ ID No. 16 or SEQ ID No. 17, and the insertion site is the The N-terminal intact HPV31 type L1 protein and said mutants are between amino acids 137 and 138 or between amino acids 432 and 433.
  • the amino acid sequence of the polypeptide from the HPV73 type L2 protein is SEQ ID No. 15, SEQ ID No. 16 or SEQ ID No. 17, and the insertion site is the HPV31 type L1 protein with N-terminal truncated 4 amino acids and said mutants between amino acids 134 and 135 or between amino acids 429 and 430.
  • the amino acid sequence of the polypeptide from the HPV73 type L2 protein is SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID NO. No. 16 or the sequence shown in SEQ ID No. 17, the insertion site is between amino acids 137 and 138 or between amino acids 432 and 433 of the N-terminal complete HPV31 type L1 protein and the mutant.
  • the amino acid sequence of the polypeptide from the HPV73 type L2 protein is SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID No. 15, SEQ ID NO. No. 16 or the sequence shown in SEQ ID No. 17, the insertion site is the HPV31 type L1 protein truncated by 4 amino acids at the N-terminal and between amino acids 134 and 135 or between amino acids 429 and 430 of the mutant.
  • the N-terminal complete HPV31 type L1 protein and the amino acid 136-138 region are deleted.
  • the polypeptide from the HPV73 type L2 protein has a glycine-proline linker added to the N-terminus, and the polypeptide derived from the HPV73 type L2 protein
  • the amino acid sequence of is shown in SEQ ID No.15, SEQ ID No.16 or SEQ ID No.17.
  • polypeptide derived from the HPV73 type L2 protein was inserted, and the N-terminal of the polypeptide derived from the HPV73 type L2 protein added a glycine-proline linker, so The amino acid sequence of the polypeptide from the HPV73 type L2 protein is shown in SEQ ID No.15, SEQ ID No.16 or SEQ ID No.17.
  • the N-terminal complete HPV31 type L1 protein and all amino acids 432-433 are deleted.
  • amino acids 431 and 434 of the mutant is inserted a polypeptide from the HPV73 type L2 protein, the amino acid sequence of the polypeptide from the HPV73 type L2 protein is as shown in SEQ ID No.15, SEQ ID No.16 or SEQ ID No.17 Show.
  • the polypeptide of type L2 protein after deleting the amino acid 133-135 region of the HPV31 type L1 protein mutant, between amino acids 132 and 136 of the HPV31 type L1 protein mutant is inserted from HPV73
  • the polypeptide of type L2 protein, the polypeptide from HPV73 type L2 protein has a glycine-proline linker added to its N-terminus, and the amino acid sequence of the polypeptide from HPV73 type L2 protein is as SEQ ID No. 15 or SEQ ID No.
  • the obtained chimeric protein amino acid sequence is such as SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.23, ID No. 24 or SEQ ID No. 25.
  • the amino acid sequence of the polypeptide from HPV73 type L2 protein is shown in SEQ ID No.16 or SEQ ID No.17
  • the amino acid sequence of the obtained chimeric protein is shown in SEQ ID No.26, SEQ ID No. .27, SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ ID No.32, or SEQ ID No.33.
  • Another aspect of the present invention relates to a polynucleotide encoding the aforementioned human papillomavirus chimeric protein.
  • the present invention also provides a vector comprising the above-mentioned polynucleotide, and a cell comprising the vector.
  • polynucleotide sequences encoding the above-mentioned human papillomavirus chimeric proteins involved in the present invention are suitable for different expression systems.
  • these nucleotide sequences are fully gene-optimized using E. coli codons, which can be expressed at a high level in an E. coli expression system; or whole-gene optimization using insect cell codons, which can be expressed at a high level in an insect cell expression system.
  • the present invention also provides a polymer, preferably, the polymer is a human papillomavirus chimeric pentamer or a chimeric virus-like particle, wherein the multimer comprises the human papilloma of the present invention Viral chimeric protein, or formed by the human papillomavirus chimeric protein of the present invention.
  • the present invention also provides the above-mentioned papillomavirus chimeric protein, papillomavirus chimeric pentamer or the above-mentioned papillomavirus chimeric virus-like particle in preparation for preventing papillomavirus infection and/or induction of papillomavirus infection
  • the use in the vaccine of the diseases, preferably, the diseases induced by the papillomavirus infection include but are not limited to cervical cancer, vaginal cancer, labia cancer, penile cancer, perianal cancer, oropharyngeal cancer, tonsil cancer and oral cancer ;
  • the papillomavirus infection is one or more infections selected from the following human papillomavirus types: HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56 , HPV58, HPV59, HPV66, HPV68, HPV70, HPV73; HPV6, HPV11, HPV2, HPV5, HPV27 and HPV57.
  • human papillomavirus types selected from the following human papillomavirus types: HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56 , HPV58, HPV59, HPV66, HPV68, HPV70, HPV73; HPV6, HPV11, HPV2, HPV5, HPV27 and HPV57.
  • the present invention also provides a vaccine for preventing papillomavirus infection and infection-induced diseases, comprising the above papillomavirus chimeric pentamer or chimeric virus-like particle, an adjuvant, and an excipient for the vaccine Or the vector, preferably, further comprising a virus-like particle or a chimeric virus-like particle of at least one mucotropic and/or dermatophilic HPV.
  • the content of these virus-like particles is respectively an effective amount capable of inducing a protective immune response.
  • the adjuvant is a human adjuvant.
  • insect cell expression system includes insect cells, recombinant baculovirus, recombinant Bacmid and expression vectors.
  • the insect cells are derived from commercially available cells, such as but not limited to: Sf9, Sf21, High Five.
  • prokaryotic expression system includes, but is not limited to, E. coli expression systems.
  • the expression host bacteria are derived from commercially available strains, such as but not limited to: BL21(DE3), BL21(DE3) plysS, C43(DE3), and Rosetta-gami B(DE3).
  • full-length HPV31 type L1 protein examples include, but are not limited to, the proteins numbered P17388.1, AEI61021.1, AEI60949.1, AAA92894.1, AIG59245.1, AIG59235.1 in the NCBI database, etc. Long full-length L1 protein.
  • a gene fragment of "truncated HPV31 type 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 HPV31 type L1 protein gene , wherein the full-length sequence of "wild-type HPV31 type L1 protein” is such as but not limited to the following sequences in the NCBI database: P17388.1, AEI61021.1, AEI60949.1, AAA92894.1, AIG59245.1, AIG59235.1, etc.
  • the term "vaccine excipient or carrier” refers to one or more selected from the group consisting of, but not limited to, pH adjusters, surfactants, and ionic strength enhancers.
  • 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.
  • adjuvant for human use 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 adjuvants, MF59 and various forms of adjuvant compositions.
  • 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.
  • the vaccine of the present invention is preferably used in unit dosage form, wherein the dose of protein virus-like particles in the unit dosage form is 5 ⁇ g-100 ⁇ g, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ⁇ g, and the range between any two of the above values; preferably 30 ⁇ g-60 ⁇ g.
  • 1A-1B Expression identification of the 31-type L1 protein mutant and the chimeric protein comprising the same in insect cells in Example 7 of the present invention. The results showed that 11 31-type L1 proteins, mutants and 16 chimeric proteins could be expressed in insect cells.
  • Figure 1A Expression identification of type 31 L1 protein and its mutant proteins in insect cells: 1 is 31L1; 2 is T 274 N; 3 is 31L1M ⁇ C; 4 is T 274 N ⁇ C; 5 is T 267 A ⁇ C; 6 is T 267 AT 274 N ⁇ C; 7 is T 274 N ⁇ N2C; 8 is T 274 N ⁇ N4C; 9 is T 274 N ⁇ N5C; 10 is T 274 N ⁇ N8C; 11 is T 274 N ⁇ N10C;
  • Figure 1B Expression identification in insect cells of chimeric proteins comprising mutants of type 31 L1 protein: 1 is 31L1DE 132-136 /dE; 2 is 31L1DE 132-136 /dES; 3 is 31L1h4 428-431 /dE; 4 31L1h4 428-431 /dES; 5 is 31L1DE 132-136 /dE-CS1; 6 is 31L1DE 132-136 /dES-CS1; 7 is 31L1h4 428-431 /dE-CS1; 8 is 31L1h4 428-431 /dES- CS1, 9 is 31L1DE 132-136 /dE-CS2; 10 is 31L1DE 132-136 /dES-CS2; 11 is 31L1h4 428-431 /dE-CS2; 12 is 31L1h4 428-431 /dES-CS2; 13 is 31L1DE 132 -136 /dE-CS3; 14 is 31L1DE 132-1
  • Example 8 Dynamic light scattering analysis results of VLPs and cVLPs obtained after purification in Example 8 of the present invention.
  • the results showed that the hydration kinetic diameters of virus-like particles formed by 31L1M ⁇ C, T 274 N ⁇ C, T 274 N ⁇ N4C, 31L1DE 132-136 /dE, 31L1h4 428-431 /dE and 31L1h4 428-431 /dE-CS1 recombinant proteins were 103.3 nm, respectively. , 99.78nm, 106.8nm, 104.59nm, 47.8nm and 42.4nm, the percentage of particle assembly is 100%.
  • Figure 2A 31L1M ⁇ C
  • Figure 2B T 274 N ⁇ C
  • Figure 2C T 274 N ⁇ N4C
  • Figure 2D 31L1DE 132-136 /dE
  • Figure 2E 31L1h4 428-431 /dE
  • Figure 2F 31L1h4 428-431 /dE-CS1 .
  • Figure 3A 31L1M ⁇ C
  • Figure 3B T274N ⁇ N4C
  • Figure 3C 31L1DE 132-136 /dE
  • Figure 3D 31L1h4 428-431 /dE
  • Figure 3E 31L1h4 428-431 /dE-CS1.
  • Figure 4 The results of the neutralizing antibody titer detection of the mouse immune serum described in Example 11 of the present invention using HPV31 pseudovirus. ns: No statistical difference (P>0.05).
  • amino acids 267 and 274 53% were mutated from threonine (T) to alanine (A) at position 267, and mutated from threonine (T) to asparagine at position 274 (N) accounted for 89%, so A and T were the dominant amino acids at positions 267 and 274, respectively.
  • the RG-1 epitope peptides of HPV35, -39, -51, -53, -56, -68, -73, -82 were synthesized by chemical synthesis.
  • the epitope peptide sequences are shown in Table 1.
  • each synthetic peptide was synthesized by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, CAS No.25952-53- 8) Coupling with keyhole limpet hemocyanin (KLH) after activation of the carboxyl group.
  • New Zealand white rabbits weighing 2.0-2.5 kg were taken and randomly divided into 2-4 groups, and 15 mg of inactivated DH5a (containing an equal volume of complete Freund's adjuvant) was subcutaneously injected at multiple points on the back 4 days before immunization. 0.5% v/v formaldehyde in PBS, treated at 37°C for 24-48h) for immunostimulation.
  • 1 mg of KLH-polypeptide mixed with an equal volume of Freund's complete adjuvant was subcutaneously injected at multiple points on the back and inner thigh.
  • the booster immunization was performed 4 times with an interval of 2 weeks.
  • the antigen for booster immunization was 0.5 mg of KLH-polypeptide thoroughly mixed with an equal volume of incomplete Freund's adjuvant. Blood was collected 2 weeks after the last immunization, and serum was separated.
  • the neutralizing antibody titers of immune sera were detected using 17 HPV pseudoviruses, and the results are shown in Table 3.
  • the 73RG-1 epitope peptide has the best immune activity, and its antiserum can neutralize all 17 types of detection, among which the neutralizing antibody titers of HPV45, -18, -16 are all above 10 3 , HPV68, -57 ,-59,-39,-5 neutralizing antibody titers between 500-1000.
  • Type synthetic peptide sequence SEQ ID NO. HPV35 TQLYRTCKAAGTCPPDVIPKVEG 53 HPV39 STLYRTCKQSGTCPPDVVDKVEG 54 HPV51 TQLYSTCKAAGTCPPDVVNKVEG 55 HPV53 TQLYQTCKQSGTCPEDVINKIEH 56 HPV56 TQLYKTCKLSGTCPEDVVNKIEQ 57 HPV68 STLYKTCKQSGTCPPDVINKVEG 58 HPV73 TQLYKTCKQAGTCPPDVIPKVEG 59 HPV82 TQLYSTCKAAGTCPPDVIPKVKG 60
  • HPV31L1 proteins and mutants are:
  • 31L1 prototype its amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence encoding the 31L1 prototype protein is designed by codon optimization in insect cells and constructed by means of total gene synthesis;
  • T 274 N mutant the 274th threonine of SEQ ID No. 1 is mutated to asparagine, and its amino acid sequence is shown in SEQ ID No. 3, the nucleotides encoding the T 274 N mutant The sequence is designed by codon optimization of insect cells and constructed by whole gene synthesis;
  • 31L1M ⁇ C mutant the C-terminal of HPV31L1 is truncated by 29 amino acids, and its amino acid sequence is shown in SEQ ID No. 5.
  • the nucleotide sequence encoding 31L1M ⁇ C is designed by codon optimization of insect cells, and the whole gene synthesis method is adopted. Construction, its nucleotide sequence is shown in SEQ ID No.34;
  • T274N ⁇ C mutant the 274th threonine of the sequence of SEQ ID No. 5 is mutated to asparagine, and its amino acid sequence is shown in SEQ ID No. 6, and the nucleotide sequence encoding T274N ⁇ C is purified by insect cells Codon-optimized design, constructed by whole gene synthesis, and its nucleotide sequence is shown in SEQ ID No.35;
  • T267A ⁇ C mutant The 267th threonine of SEQ ID No.5 sequence was mutated to alanine, and the nucleotide sequence encoding T267A ⁇ C was designed by codon optimization in insect cells and constructed by whole gene synthesis ;
  • T 267 AT 274 N ⁇ C mutant the 267th threonine of SEQ ID No. 6 was mutated to alanine, and the nucleotide sequence encoding T267AT 274 N ⁇ C was designed by codon optimization in insect cells, using the whole gene synthetically constructed;
  • T 274 N ⁇ N2C mutant the N-terminal of the sequence shown in SEQ ID No. 6 is truncated by 2 amino acids, and its sequence is shown in SEQ ID No. 7, and the nucleotide sequence encoding T 274 N ⁇ N2C is encoded by insect cell code. Suboptimal design, constructed by whole gene synthesis;
  • T 274 N ⁇ N4C mutant the N-terminal of the sequence shown in SEQ ID No. 6 is truncated by 4 amino acids, and its sequence is shown in SEQ ID No. 8, and the nucleotide sequence encoding T 274 N ⁇ N4C is encoded by insect cell code. Suboptimal design, constructed by whole gene synthesis, and its nucleotide sequence is shown in SEQ ID No.36;
  • T 274 N ⁇ N5C mutant the N-terminal of the sequence shown in SEQ ID No. 6 is truncated by 5 amino acids, and its sequence is shown in SEQ ID No. 9, and the nucleotide sequence encoding T 274 N ⁇ N5C is encoded by insect cell code. Suboptimal design, constructed by whole gene synthesis;
  • T 274 N ⁇ N8C mutant the N-terminal of the sequence shown in SEQ ID No. 6 is truncated by 8 amino acids, and its sequence is shown in SEQ ID No. 10, and the nucleotide sequence encoding T 274 N ⁇ N8C is encoded by insect cell code. Suboptimal design, constructed by whole gene synthesis;
  • T 274 N ⁇ N10C mutant the N-terminal of the sequence shown in SEQ ID No. 6 is truncated by 10 amino acids, and its sequence is shown in SEQ ID No. 11, and the nucleotide sequence encoding T 274 N ⁇ N10C is encoded by insect cell code.
  • the suboptimal design was constructed by whole gene synthesis.
  • the codon-optimized HPV31L1 protein and mutant gene in insect cells were digested with BamHI/XbaI, and then inserted into the commercial expression vector pFastBac1 (produced by Invitrogen).
  • the expression vectors containing the chimeric protein gene were obtained, respectively: pFastBac1-31L1, pFastBac1-T 274 N, pFastBac1-31L1M ⁇ C, pFastBac1-T 274 N ⁇ C, pFastBac1-T 267 A ⁇ C, pFastBac1-T 267 AT 274 N ⁇ C, pFastBac1-T 274 N ⁇ N2C, pFastBac1-T 274 N ⁇ N4C, pFastBac1-T 274 N ⁇ N5C, pFastBac1-T
  • Chimeric L1 protein 31L1DE 132-136 /dE the backbone is T 274 N ⁇ N4C (that is, on the basis of mutating threonine at position 274 to asparagine, the N-terminal is truncated by 4 amino acids, and the C-terminal is truncated by 29 amino acids. , whose sequence is shown in SEQ ID No.
  • Chimeric L1 protein 31L1DE 132-136 /dES the backbone is T 274 N ⁇ N4C (its sequence is shown in SEQ ID No. 8), its aa.133-135 region is deleted, and it is fused between aa.132/136
  • the polynucleotide sequence encoding 31L1DE 132-136 /dES was designed by codon optimization in insect cells and constructed by whole gene synthesis, and its sequence is shown in SEQ ID No.38;
  • Chimeric L1 protein 31L1DE 132-136 /dE-CS1 the backbone is T 274 N ⁇ N4C-CS1 (that is, the N-terminal 4 amino acids are truncated on the basis of the mutation of threonine at position 274 to asparagine, and the C Replace the basic amino acids within 29 amino acids of the terminal, its sequence is shown in SEQ ID No.
  • Chimeric L1 protein 31L1DE 132-136 /dES-CS1 the backbone is T 274 N ⁇ N4C-CS1 (its sequence is shown in SEQ ID No. 12), its aa.133-135 region is deleted, and aa.132/ The aa.19-35 polypeptide of HPV73 type L2 protein containing a GP linker at the N-terminus between 136 (in the aa.132-136 region of SEQ ID No. 12, a non-isometric substitution insertion), the amino acid sequence of the inserted fragment is SEQ ID No. 12. Glycine-proline was added to the N-terminus of the sequence shown in ID No.
  • 31L1DE 132-136 /dES-CS1 chimeric protein was shown in SEQ ID No. 21.
  • the polynucleotide sequence encoding 31L1DE 132-136 /dES-CS1 was designed by codon optimization in insect cells and constructed by whole gene synthesis, and its sequence is shown in SEQ ID No.40;
  • Chimeric L1 protein 31L1DE 132-136 /dE-CS2 the backbone is T 274 N ⁇ N4C-CS2 (that is, on the basis of mutating threonine at position 274 to asparagine, the N-terminal is truncated by 4 amino acids, and the C Replace the basic amino acids within 29 amino acids of the terminal, its sequence is shown in SEQ ID No.
  • Chimeric L1 protein 31L1DE 132-136 /dES-CS2 the backbone is T 274 N ⁇ N4C-CS2 (its sequence is shown in SEQ ID No. 13), its aa.133-135 region is deleted, and aa.132/ The aa.19-35 polypeptide of the HPV73 type L2 protein containing the GP linker at the N-terminal fused between 136 (in the aa.132-136 region of SEQ ID No. 13, the aa.132-136 region is inserted by non-isometric substitution), and the amino acid sequence of the inserted fragment is SEQ ID No. 13. Glycine-proline was added to the N-terminus of the sequence shown in ID No.
  • 31L1DE 132-136 /dES-CS2 chimeric protein was shown in SEQ ID No. 23.
  • the polynucleotide sequence encoding 31L1DE 132-136 /dES-CS2 was designed by codon optimization in insect cells and constructed by whole gene synthesis, and its sequence is shown in SEQ ID No.42;
  • Chimeric L1 protein 31L1DE 132-136 /dE-CS3 the backbone is T 274 N ⁇ N4C-CS3 (that is, on the basis of mutating threonine at position 274 to asparagine, the N-terminal is truncated by 4 amino acids, and the C Replace the basic amino acid within 29 amino acids of the terminal, its sequence is shown in SEQ ID No.
  • Chimeric L1 protein 31L1DE 132-136 /dES-CS3 the backbone is T 274 N ⁇ N4C-CS3 (its sequence is shown in SEQ ID No. 14), its aa.133-135 region is deleted, and aa.132/ The aa.19-35 polypeptide of the HPV73 type L2 protein containing the GP linker at the N-terminal fused between 136 (in the aa.132-136 region of SEQ ID No.14, the aa.132-136 region is substituted by non-isometric insertion), and the amino acid sequence of the inserted fragment is SEQ ID No. 14. Glycine-proline was added to the N-terminus of the sequence shown in ID No.
  • 31L1DE 132-136 /dES-CS3 chimeric protein was shown in SEQ ID No. 25.
  • the polynucleotide sequence encoding 31L1DE 132-136 /dES-CS3 was designed by codon optimization in insect cells and constructed by whole gene synthesis, and its sequence is shown in SEQ ID No.44;
  • Chimeric L1 protein 31L1h4 428-431 /dE the backbone is T 274 N ⁇ N4C (that is, on the basis of mutating threonine at position 274 to asparagine, the N-terminal is truncated by 4 amino acids, and the C-terminal is truncated by 29 amino acids.
  • Chimeric L1 protein 31L1h4 428-431 /dES the backbone is T 274 N ⁇ N4C (its sequence is shown in SEQ ID No. 8), its aa.429-430 region is deleted, and it is fused between aa.428/431
  • the aa.19-35 polypeptide of HPV73 type L2 protein in the aa.428-431 region of SEQ ID No.8 is inserted by non-isometric substitution), the amino acid sequence of the inserted fragment is the sequence shown in SEQ ID No.17, 31L1h4 428-
  • the amino acid sequence of the 431 /dES chimeric protein is shown in SEQ ID No.27.
  • the polynucleotide sequence encoding 31L1h4 428-431 /dES was designed by codon optimization in insect cells and constructed by whole gene synthesis, and its sequence is shown in SEQ ID No.46;
  • Chimeric L1 protein 31L1h4 428-431 /dE-CS1 the backbone is T 274 N ⁇ N4C-CS1 (that is, the N-terminal 4 amino acids are truncated on the basis of the mutation of threonine at position 274 to asparagine, and the C Replace the basic amino acids within the terminal 29 amino acids, the sequence of which is shown in SEQ ID No.
  • Chimeric L1 protein 31L1h4 428-431 /dES-CS1 the backbone is T 274 N ⁇ N4C-CS1 (its sequence is shown in SEQ ID No. 12), its aa.429-430 region is deleted, and aa.428/ The aa.19-35 polypeptides of HPV73 type L2 protein are fused between 431 (non-isometric substitutions are inserted in the aa.428-431 region of SEQ ID No.12), and the amino acid sequence of the inserted fragment is the sequence shown in SEQ ID No.17 , the amino acid sequence of 31L1h4 428-431 /dES-CS1 chimeric protein is shown in SEQ ID No.29.
  • the polynucleotide sequence encoding 31L1h4 428-431 /dES-CS1 was designed by codon optimization in insect cells and constructed by whole gene synthesis, and its sequence is shown in SEQ ID No.48;
  • Chimeric L1 protein 31L1h4 428-431 /dE-CS2 the backbone is T 274 N ⁇ N4C-CS2 (that is, on the basis of mutating threonine at position 274 to asparagine, the N-terminal is truncated by 4 amino acids, and the C Replace the basic amino acids within the terminal 29 amino acids, the sequence of which is shown in SEQ ID No.
  • Chimeric L1 protein 31L1h4 428-431 /dES-CS2 the backbone is T 274 N ⁇ N4C-CS2 (its sequence is shown in SEQ ID No. 13), its aa.429-430 region is deleted, and aa.428/ The aa.19-35 polypeptides of HPV73 type L2 protein were fused between 431 (non-isometric substitution insertion in the aa.428-431 region of SEQ ID No.13), and the amino acid sequence of the inserted fragment was the sequence shown in SEQ ID No.17 , the amino acid sequence of 31L1h4 428-431 /dES-CS2 chimeric protein is shown in SEQ ID No.31.
  • the polynucleotide sequence encoding 31L1h4 428-431 /dES-CS2 was designed by codon optimization in insect cells and constructed by whole gene synthesis, and its sequence is shown in SEQ ID No.50;
  • Chimeric L1 protein 31L1h4 428-431 /dE-CS3 the backbone is T 274 N ⁇ N4C-CS3 (that is, on the basis of mutating threonine at position 274 to asparagine, the N-terminal is truncated by 4 amino acids, and the C Replace the basic amino acids within the terminal 29 amino acids, the sequence of which is shown in SEQ ID No. 14), delete the aa.429-430 region, and fuse the aa of the HPV73 L2 protein between aa.428/431 .19-39 polypeptide (in the aa.428-431 region of SEQ ID No.
  • Chimeric L1 protein 31L1h4 428-431 /dES-CS3 the backbone is T 274 N ⁇ N4C-CS3 (its sequence is shown in SEQ ID No. 14), its aa.429-430 region is deleted, and aa.428/ The aa.19-35 polypeptides of HPV73 type L2 protein are fused between 431 (non-isometric substitutions are inserted in the aa.428-431 region of SEQ ID No.14), and the amino acid sequence of the inserted fragment is the sequence shown in SEQ ID No.17 , the amino acid sequence of 31L1h4 428-431 /dES-CS3 chimeric protein is shown in SEQ ID No.33.
  • the polynucleotide sequence encoding 31L1h4 428-431 /dES-CS3 was designed by codon optimization in insect cells and constructed by whole gene synthesis, and its sequence is shown in SEQ ID No.52.
  • the codon-optimized HPV31L1 protein and mutant gene in insect cells were digested with BamHI/XbaI, and then inserted into the commercial expression vector pFastBac1 (produced by Invitrogen).
  • the expression vectors containing the chimeric protein gene were obtained, respectively: pFastBac1-31L1DE 132-136 /dE, pFastBac1-31L1DE 132-136 /dES, pFastBac1-31L1DE 132-136 /dE-CS1, pFastBac1-31L1DE 132-136 /dES -CS1, pFastBac1-31L1DE 132-136 /dE-CS2, pFastBac1-31L1DE 132-136 /dES-CS2, pFastBac1-31L1DE 132-136 /dES-CS2, pFastBac1-3
  • amino acid sequence involved in the present invention is as follows:
  • Example 5 Construction of L1 protein, recombinant Bacmid of chimeric L1 protein gene and recombinant baculovirus
  • Example 6 Expression of L1 protein and chimeric L1 protein gene in Sf9 cells
  • Sf9 cells were inoculated with 11 recombinant baculoviruses containing 31L1 protein or mutant gene, or 16 recombinant baculoviruses containing chimeric L1 gene, respectively, for protein expression, cultured at 27°C for about 88 hours, and then the fermentation broth was harvested and centrifuged at 3000 rpm. After 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, for example patent CN 101148661B.
  • Example 7 Expression and identification of L1 protein and chimeric L1 protein
  • Example 8 Comparison of the expression levels of L1 protein and chimeric L1 protein in insect cells
  • the HPV31L1 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 HPV31L1 VLP standard was also serially diluted, with a concentration ranging from 2 ⁇ g/ml to 0.0625 ⁇ g/ml, added to the ELISA plate, 100 ⁇ l per well, and incubated at 37°C for 1 h.
  • the plate was washed three times with PBST, and 1:3000 diluted HPV31L1 rabbit polyclonal antibody was added, 100 ⁇ l 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 ⁇ l 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 ⁇ l of 2M sulfuric acid, and the absorbance was measured at 490 nm.
  • the concentration of 31L1 protein, 31L1 protein mutant or chimeric L1 protein in the lysis supernatant was calculated according to the standard curve.
  • the results are shown in Table 4.
  • the expression level of the 31L1 mutant protein (31L1M ⁇ C) with 29 amino acids truncated at the C-terminal of the present invention is significantly higher than that of the HPV31L1 full-length protein; the expression level of the mutant protein obtained by point mutation of the 31L1 protein.
  • the expression level of the T 274 N mutant was significantly higher than that of the HPV31L1 prototype protein, and the expression level of the T 274 N ⁇ C mutant protein was also further increased than that of the 31L1M ⁇ C protein, indicating that the 274th threonine was mutated to asparagine.
  • the expression levels of the chimeric proteins constructed on the basis of T 274 N ⁇ N4C (31L1DE 132-136 /dE, 31L1DE 132-136 /dES, 31L1h4 428-431 /dE, 31L1h4 428-431 /dES) were all the same as those of their backbone T 274 N ⁇ N4C.
  • the expression levels of 12 chimeric proteins with C-terminal replacement 31L1 mutant as backbone were higher than the corresponding C-terminal truncated chimeric proteins.
  • Example 9 Purification and dynamic light scattering particle size analysis of L1 protein and chimeric L1 protein
  • VLPs were depolymerized by adding 4% ⁇ -mercaptoethanol (w/w) to the lysate, then the samples were filtered using a 0.22 ⁇ m filter, followed by DMAE anion exchange chromatography or CM cation exchange chromatography (20 mM Tris, 180 mM NaCl, 4% ⁇ -ME, pH 7.9 elution), TMAE anion exchange chromatography or Q cation exchange chromatography (20 mM Tris, 180 mM NaCl, 4% ⁇ -ME, pH 7.9 elution) and hydroxyapatite chromatography (eluted with 100 mM NaH2PO4 , 30 mM NaCl, 4 % ⁇ -ME, pH 6.0).
  • the purified product was concentrated using a Planova ultrafiltration system and buffer exchange (20 mM NaH 2 PO 4 , 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 HPV31L1 protein, 31L1 mutant protein and chimeric L1 protein could be assembled efficiently.
  • the assembled protein solution was taken for DLS particle size analysis ( Zetasizer Nano ZS 90 dynamic light scattering instrument, Malvern Company), and the results are shown in Table 5.
  • DLS analysis plots of 31L1h4 428-431 /dE, are shown in Figures 2A to 2F.
  • Example 10 Transmission electron microscope observation of VLP and chimeric VLP
  • the recombinant proteins were purified respectively, and the copper mesh was prepared by using post-assembly chimera, which was stained with 1% uranyl acetate, fully dried and observed using a JEM-1400 electron microscope (Olympus). .
  • HPV31L1, T 274 N, 31L1M ⁇ C, T 274 N ⁇ C, T 267 A ⁇ C and T 267 AT 274 N ⁇ C proteins expressed in insect cells could be assembled into VLPs with a diameter of about 50-60 nm; N-terminal truncation combined with C-terminal truncation Short 31L1 protein mutants can be assembled into VLPs with a diameter of 17-35 nm; chimeric proteins with 73L2 polypeptides inserted in the DE loop surface region can be assembled into 30-50 nm cVLPs; chimeric proteins with 73L2 polypeptide inserted in the h4 region can be assembled into cVLPs approximately 17-30 nm in diameter.
  • Example 11 Mouse immunization of HPV31L1 or mutant VLPs and determination of neutralizing antibody titers
  • mice aged 4-6 weeks were randomly divided into 5 groups, and the mice were immunized with 0.1 ⁇ g VLP. Subcutaneous injection, immunization on the 0th and 2nd week, a total of 2 times. Two weeks after the second immunization, blood was collected from the tail vein, and the serum was separated.
  • Example 12 Mouse immunization of chimeric VLPs and determination of neutralizing antibody titers
  • mice aged 4-6 weeks were randomly divided into 5 groups, and the mice were immunized with 10 ⁇ g cVLP combined with 50 ⁇ g Al(OH) 3 and 5 ⁇ g MPL adjuvant. Subcutaneous injection, immunization at 0, 4, 7, 10 weeks, a total of 4 times. Two weeks after the fourth immunization, blood was collected from the tail vein, and the serum was separated.
  • the neutralizing antibody titers of 17 kinds of HPV pseudoviruses were used to detect the antiserum.
  • the results showed that after immunizing mice with various cVLPs, the levels and neutralization ranges of cross-neutralizing antibodies induced were different.
  • the neutralizing antibody titer of backbone type HPV31 type induced by the cVLP of the chimeric epitope in the h4 surface region is comparable to that of HPV31L1 VLP, and the cross-neutralizing antibody titer of its antiserum is high, which can neutralize 17 types of detection.
  • the HPV31-type neutralizing antibody titer induced by the chimeric epitope in the DE loop surface region was one order of magnitude lower than that of the 31L1VLP, and the cross-neutralizing spectrum of the immune serum was also relatively narrow.
  • the cross-neutralizing activities of some cVLP immune sera are shown in Table 5, among which 31L1h4 428-431 /dE, 31L1h4 428-431 /dES and 31L1h4 428-431 /dE-CS1 antisera can neutralize at least 17 types of pseudosera.
  • Viruses, 31L1DE 132-136 /dE and 31L1DE 132-136 /dES antisera neutralized only 10 and 8 types of pseudoviruses, respectively.
  • the cVLP constructed by the C-terminal substituted 31L1 mutant in the present invention after immunizing mice with the above strategy, the induced cross-neutralizing antibody level and neutralization range are consistent with the corresponding C-terminal truncated cVLP.
  • pseudovirus preparation and pseudovirus neutralization experiments are disclosed, for example, patent CN104418942A.

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Abstract

本发明涉及一种人乳头瘤病毒31型嵌合蛋白及其用途。具体地，本发明涉及一种人乳头瘤病毒嵌合蛋白，其包含HPV31型L1蛋白或HPV31型L1蛋白的突变体、以及插入所述HPV31型L1蛋白或HPV31型L1蛋白的突变体中的来自HPV73型L2蛋白的多肽，或由其组成，其中所述HPV31型L1蛋白如SEQ ID No.1所示，所述HPV73型L2蛋白如SEQ ID No.2所示。

Description

一种人乳头瘤病毒31型嵌合蛋白及其用途 技术领域
本发明涉及生物技术领域。具体地，本发明涉及一种人乳头瘤病毒嵌合蛋白，及由其形成的五聚体或病毒样颗粒，以及所述人乳头瘤病毒嵌合蛋白、其五聚体或病毒病毒样颗粒在制备预防受试者中乳头瘤病毒感染及感染诱发的疾病的疫苗中的用途。
背景技术
人乳头瘤病毒(human papillomavirus，HPV)是一类感染上皮组织的无包膜小DNA病毒，目前已鉴定200余型，其中40余型主要感染肛周、泌尿生殖器及口咽部的粘膜及附近皮肤；根据感染诱发病变性质不同，分为诱发恶性肿瘤的致癌型(HPV16/-18/-31/-33/-45/-52/-58等)和诱发疣状增生的低危型(HPV 6/-11等)。分子流行病学研究发现，致癌型HPV持续感染可诱发约100％的宫颈癌、88％的肛门癌、70％的阴道癌、50％的阴茎癌、43％的阴户癌及72％的头颈癌。目前已鉴定20余型，在宫颈癌组织中常见的12种型别，如HPV16/-18/-31/-33/-35/-39/-45/-51/-52/-56/-58/-59，又称为高危型(high-risk HPV，HR-HPV)。HR-HPV累计诱发95.2％～96.5％的宫颈癌；其余致癌性HPV相对少见，除HPV68以外，单型检出率小于0.5％。HPV16和HPV18在世界范围内宫颈癌中的检出率最高，分别为55.4％及14.6％。HPV31是世界范围内相对常见的HR-HPV，在宫颈癌组织中的检出率为3.5％，位列第六位；在宫颈高度癌前病变组织中的检出率为10.4％，位列第三位，仅次于HPV16(45.1％)及HPV52(11％)；在细胞学正常的宫颈组织中的检出率为1.3％，位列第三位，仅次于HPV16(2.9％)及HPV58(1.5％)。值得注意的是，在某些发达地区宫颈高度癌前病变中，HPV31的检出率高达12.4％，仅次于HPV16(46.8％)；另外在拉丁美洲细胞学正常宫颈组织中HPV31的检出率仅次于HPV16(3.3％)，为1.2％。
HPV L1蛋白的C端含有核定位信号，L1蛋白在真核细胞表达后，经核定位信号的介导入核，在细胞核内组装成病毒样颗粒。研究发现，通过删除法去除核定位信号，可使L1蛋白翻译后分布在胞浆。因此，在不影响参与VLP组装的L1蛋白C端helix 5结构域的基础上，去除核定位信号，获得的L1C端截短基因，在真核表达体系(如昆虫细胞)表达后，L1 VLP主要分布在胞浆，利于细胞的破碎及下游纯化。利用昆虫细胞表达体系研究发现，牛乳头瘤病毒1型(BPV1)L1截短C端24个氨基酸的突变体、HPV16 L1的C端截短23个氨基酸的突变体、HPV58 L1的C端截短25个氨基酸的突变体，均不影响L1蛋白组装成VLP的活性，且BPV1 L1截短蛋白突变体的组装效率提高了3倍，HPV58 L1截短蛋白的表达量提高了2倍。其他型别的截短突变体对表达水平、组装活性及产量的影响尚 未见有报道。现有报道的采用大肠杆菌表达体系进行L1 VLP表达的L1基因的C端均为天然完整序列。
根据L1氨基酸序列的差异，每种HPV型别的L1存在许多不同的型内变异株。现有报道的数据显示，HPV16L1的变异株在昆虫细胞及酵母细胞中的表达水平存在差异。有报道分析了5株16L1变异株在昆虫细胞内L1 VLP的表达水平，发现其中2株变异株(Phil1和Fra63)的L1表达水平及VLP的产量显著高于原型L1对照组(Phil1和Fra63的表达水平较原型分别提高了32倍和16倍，产量分别提高了39倍和42倍)，另一变异株(Alg1)的表达水平较原型提高了8倍，产量提高了24倍，其余2株的表达水平和产量与原型的相当。采用酵母表达体系对2株16L1变异株(B27和T3)的VLP表达水平进行分析，发现2株变异株的VLP产量均显著高于原型对照组。氨基酸分析发现，上述7株16L1变异株的氨基酸一级序列均不相同，酵母种表达水平较高的2个变异株及昆虫细胞中表达水平较高的3个变异株，虽然其共同特征是202位和266位的氨基酸相同(分别是Asp和Ala)，但与其有相同特征的Fra25的表达水平及VLP产量很低(与对照组相当)，表明不同变异株中的特征性构成氨基酸可能影响L1的表达水平，这是无法预测的。上述数据表明，通过对不同变异株的表达水平分析，可以发现VLP表达量提高的变异株序列，用于L1 VLP疫苗的生产，可望降低疫苗的生产成本。目前尚未见有其他型别变异株序列对表达水平的影响的报道。
采用大肠杆菌表达体系对C端完整的HPV 16L1的表达研究显示，N端分别截短4、6、8、9、10个氨基酸，不影响L1 VLP的组装(X.Chen et al,Journal of molecular biology,2001)。采用大肠杆菌表达体系对合计9种型别L1的N端截短突变体的表达水平与其相应型别N端全长L1表达水平进行比较分析，包括2种16L1的N端截短突变体(△N5、△N10)，其表达水平均较对照组的显著提高；2种18L1N端截短突变体(△N5、△N10)，其中△N5的表达水平显著提高，△N10的显著降低；2种31L1N端截短突变体(△N5、△N10)，其表达水平均与对照组相当；3种33L1N端截短突变体(△N5、△N10、△N15)，其中△N10的表达水平显著提高，其余2种的表达水平降低；2种45L1N端截短突变体(△N4、△N9)，其表达水平均降低；4种58L1N端截短突变体(△N5、△N10、△N15、△N19)，其中△N15的表达水平显著提高，其余3种与对照组相当；3种52L1N端截短突变体(△N5、△N10、△N15)，其表达水平均显著提高；3种6L1N端截短突变体(△N3、△N6、△N9)，其中△N6的表达水平显著提高，△N9表达水平不变，△N3表达水平降低；2种11 1N端截短突变体(△N5、△N9)，其中△N5的表达水平显著提高，△N9的表达水平不变(M.Wei et al,Emerging Microbes&Infections,2018)。上述数据显示，L1的N端截短突变体可影响蛋白在大肠杆菌中的表达水平，N端截短的长度对表达水平的影响是没有规律的、不 可预测的。目前尚未见有N端截短突变体在昆虫细胞中表达水平的研究。另外，目前也未见有利用C端截短L1突变体进行N端截短，研究N端截短策略对截短蛋白在真核表达体系中表达水平的影响。
L1 VLP是由72个L1五聚体(360个L1单体)组装成的直径为55nm的正二十面体。L1依赖的中和抗体表位按一定间隔在其表面规律性密集排列，每种表位重复360次，因此L1VLP利于BCR的交联及高滴度中和抗体的产生。动物实验及临床研究数据显示，HPV L1VLP可诱发产生高滴度的持久性的L1特异的中和抗体，三种上市的VLP疫苗在全球100多个国家和地区应用后，未见型内变异株逃逸的现象，表明VLP诱发的是针对多个L1表位的。
另外，VLP还可以作为表位肽疫苗的载体，以HPV16 L1为载体，构建的表面展示16RG-1、58RG-1、33RG-1及31RG-1的HPV16 cVLP疫苗相对成功，可同时诱发不同型别RG-1依赖的交叉中和抗体及HPV16L1依赖的型别特异性中和抗体，其中HPV16的中和抗体滴度与16L1VLP的相当，各型RG-1依赖的交叉中和抗体滴度相对较高，中和范围涵盖的型别较多(大于10种)，其他文献也报道了一些HPV16 cVLP疫苗的研究数据，嵌合的表位包括16RG-1及16L2其他的表位区，但由于插入位点、插入策略及选择表位的长度不同，其获得的16cVLP诱发的交叉中和活性相对较差。用16cVLP替换Cervarix(HPV16/18 L1VLP)中的16VLP，配制16cVLP/18VLP混合疫苗，初步研究显示，疫苗不仅可同时诱发高滴度的HPV16/18的中和抗体，同时还可诱发针对HPV58的交叉保护活性。提示，采用HPV cVLP疫苗进行新一代的预防性疫苗的研发，可望扩大疫苗保护范围的同时降低疫苗的成本。GSK公司在其二价苗Cervarix成功的基础上，又进行了HPV16 cVLP及HPV18 cVLP疫苗的研究，但数据显示其嵌合33RG-1的16cVLP的表达量较低，交叉中和范围也不理想；其嵌合33RG-1的18cVLP疫苗交叉中和范围虽然仅涵盖7种型别(4种高危型、2种低危型、1种皮肤型)，交叉中和抗体滴度除HPV58的中和滴度较高之外，其余6种型别的中和滴度均相对较低，但其免疫活性显著优于目前Huber等报道的嵌合45RG-1的18cVLP(B.Huber et al,PLOS ONE,2017；M.Boxus et al,Journal of virology,2016)。上述数据提示，16cVLP已相对成功，18cVLP的实验研究处于初级阶段，尚需进一步优化插入表位、插入位点，以期进一步提高其免疫保护活性涵盖的范围；其他型别的cVLP疫苗尚未见有报道。
依据目前报道的HPV16/18 cVLP疫苗的研究数据可以看出，cVLP疫苗的研究面临许多挑战。首先是需要选择免疫原性强的嵌合L2保守表位，包括RG-1表位及L2蛋白中其他保守表位。目前RG-1表位的选择是经验性的，常选择优势流行株型别的RG-1表位，而不是免疫原性强的RG-1表位，主要原因是目前缺乏不同型别RG-1表位免疫原性比较的数据。其次，RG-1表位肽的长度，即表位核心 序列的旁侧序列对表位在嵌合蛋白表面的正确展示具有影响。第三，表位肽的插入位点和插入方式的不同对嵌合蛋白的组装及活性影响很大，插入位点的不同包括在L1的不同表面区的插入位点、同一表面区不同位置的插入位点；插入方式的不同包括直接插入、置换插入、插入位点区骨架氨基酸是否存在修饰(包括是否增加连接子)。第四，HPV31 L1 VLP载体对嵌合表位的耐受性及其免疫活性的影响除了存在上述所列的3种情况之外，还存在许多其他挑战，主要是由于HPV31 L1 VLP的结构特点及主要中和抗体表位区均不清楚，目前成功的HPV16和18 cVLP的插入位点均是DE loop，鉴于HPV31 L1 VLP合适的展示外源表位的位点不清楚，如果插入位点选择不当，将会影响HPV31 L1VLP骨架的免疫原性，其31cVLP诱发产生的HPV31中和抗体滴度较HPV31 L1 VLP的显著降低，即使获得了嵌合表位依赖的广谱中和抗体，其在不同型别cVLP混合疫苗的配制中也失去了对HPV31的免疫保护优势。
因而，目前需要开发新型别的HPV cVLP，首先要求其表达量高、具有研发优势，另外还需要同时诱发高滴度的针对载体型别的中和抗体，同时诱发的交叉中和抗体的活性较强，其中型别特异性的L1依赖的中和抗体滴度最好与其相应型别L1 VLP诱发的相当，这样在不同型别cVLP混合疫苗的研究中，才可保持针对骨架型别的保护优势；交叉中和活性涵盖的型别多、滴度较高，而且其优势交叉中和型别与目前报道的其他型别的cVLP相比具有特点。
发明内容
有鉴于此，本发明的目的在于提供一种乳头瘤病毒嵌合蛋白，用于制备预防受试者中乳头瘤病毒感染及感染诱发的疾病的疫苗。
本发明人出人意料地发现，对HPV31 L1蛋白骨架进行适当的截短、点突变和/或C端氨基酸改造，可不同程度的提高其表达水平，且不影响其组装成VLP的活性；在全长或截短型HPV31型L1蛋白或其突变体的表面区插入HPV73型L2蛋白多肽，可提高HPV73型L2蛋白多肽的免疫原性，获得的嵌合蛋白在大肠杆菌或昆虫细胞表达系统中可高水平表达，该嵌合蛋白可组装成VLP，并可诱发针对来自不同属/亚属的多种型别HPV的广谱保护性免疫反应。
因而，在第一方面，本发明提供了一种人乳头瘤病毒嵌合蛋白，其包含HPV31型L1蛋白或HPV31型L1蛋白的突变体、以及插入所述HPV31型L1蛋白或HPV31型L1蛋白的突变体中的来自HPV73型L2蛋白的多肽，或由其组成，其中所述HPV31型L1蛋白如SEQ ID No.1所示，所述HPV73型L2蛋白如SEQ ID No.2所示。
在本发明的人乳头瘤病毒嵌合蛋白的优选的实施方案中，所述的HPV31型L1蛋白来自但不限于NCBI数据库中的P17388.1、AEI61021.1、AEI60949.1、 AAA92894.1、AIG59245.1、AIG59235.1等来自HPV31原型或变异株的L1蛋白。优选地，所述的HPV31型L1蛋白的氨基酸序列如SEQ ID No.1所示。
在本发明的人乳头瘤病毒嵌合蛋白的进一步优选的实施方案中，本发明所述的HPV31型L1蛋白的突变体与SEQ ID No.1所示的HPV31型L1蛋白相比，包含：
选自以下的任何一种或多种置换突变：
T274N、R475G、R483G、R496G、K477S、K497S、K501S、K479A、K482A、K498A、K495G、K500G和R473G；和/或
N端截短2、4、5、8或10个氨基酸的截短突变；和/或
C端截短29个氨基酸的截短突变。
在本文所使用的置换突变的表示中，中间的数字代表与对照序列相比(例如，SEQ ID No.1所示的氨基酸序列)的氨基酸位置，数字前面的字母代表突变前的氨基酸残基，数字后的字母代表突变后的氨基酸残基。
在进一步优选的实施方案中，所述的HPV31型L1蛋白的突变体选自：
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，其序列如SEQ ID No.3所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，并将氨基酸序列的N端截短4个氨基酸，其序列如SEQ ID No.4所示；
将SEQ ID No.1所示的氨基酸序列的C端截短29个氨基酸的突变体，其序列如SEQ ID No.5所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，并将氨基酸序列的C端截短29个氨基酸的突变体，其序列如SEQ ID No.6所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，将氨基酸序列的C端截短29个氨基酸的突变体，并将氨基酸序列的N端截短2个氨基酸的突变体，其序列如SEQ ID No.7所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，将氨基酸序列的C端截短29个氨基酸的突变体，并将氨基酸序列的N端截短4个氨基酸的突变体，其序列如SEQ ID No.8所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，将氨基酸序列的C端截短29个氨基酸的突变体，并将氨基酸序列的N端截短5个氨基酸的突变体，其序列如SEQ ID No.9所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，并将氨基酸序列的C端截短29个氨基酸的突变体，并将氨基酸序列的N端截短8个氨基酸的突变体，其序列如SEQ ID No.10所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)， 将氨基酸序列的C端截短29个氨基酸的突变体，并将氨基酸序列的N端截短10个氨基酸的突变体，其序列如SEQ ID No.11所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，将氨基酸序列的N端截短4个氨基酸，并将氨基酸序列的第475位、第483位及496位的精氨酸(R)替换为甘氨酸(G)，第477位、第497位及第501位的赖氨酸(K)替换为丝氨酸(S)，第479位、第482位及第498位赖氨酸(K)替换为丙氨酸(A)，第495位及第500位赖氨酸(K)替换为甘氨酸(G)的突变体，其序列如SEQ ID No.12所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，将氨基酸序列的N端截短4个氨基酸，并将氨基酸序列的第473位、第475位、第483位及496位的精氨酸(R)替换为甘氨酸(G)，第477位、第497位及第501位的赖氨酸(K)替换为丝氨酸(S)，第479位、第482位及第498位赖氨酸(K)替换为丙氨酸(A)，第495位及第500位赖氨酸(K)替换为甘氨酸(G)的突变体，其序列如SEQ ID No.13所示；
将SEQ ID No.1所示的氨基酸序列第274位苏氨酸(T)替换为天冬酰胺(N)，将氨基酸序列的N端截短4个氨基酸，并将氨基酸序列的第475位、第483位及496位的精氨酸(R)替换为甘氨酸(G)，第477位、第497位及第501位的赖氨酸(K)替换为丝氨酸(S)，第482位及第498位赖氨酸(K)替换为丙氨酸(A)，第495位及第500位赖氨酸(K)替换为甘氨酸(G)的突变体，其序列如SEQ ID No.14所示。
在本发明的人乳头瘤病毒嵌合蛋白的进一步优选的实施方案中，所述的来自HPV73型L2蛋白的多肽为SEQ ID No.2所示氨基酸aa.1-50区域内的任意连续的8-33个氨基酸的片段；优选地，所述的多肽为SEQ ID No.2所示的HPV73型L2蛋白RG-1表位肽或其突变体表位肽；更优选地，所述的多肽为SEQ ID No.2所示的氨基酸17至39的多肽，或SEQ ID No.2所示的氨基酸17至39的多肽的N端延长或截短1至6个氨基酸和/或C端延长或截短1至6个氨基酸的突变体。
优选地，所述的来自HPV73型L2蛋白的多肽如SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示。
可选地，所述的来自HPV73型L2蛋白的多肽还可以是与SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示的氨基酸序列具有大于60％、优选大于70％、大于80％、大于90％，甚至更优选大于95％的序列同一性的多肽。
可选地，所述的来自HPV73型L2蛋白的多肽插入所述的HPV31型L1蛋白或所述HPV31型L1蛋白的突变体的表面区，优选插入所述的HPV31型L1蛋白或所述HPV31型L1蛋白的突变体的DE环或h4区域，更优选为通过直接插入的方式插入所述的HPV31型L1蛋白或所述HPV31型L1蛋白的突变体的氨基酸132 和氨基酸133之间、或氨基酸134和135之间、或氨基酸136和137之间、或氨基酸137和138之间、或氨基酸432和433之间、或氨基酸434和435之间、或氨基酸435和436之间；或者通过非等长置换的方式插入所述的HPV31型L1蛋白或所述HPV31型L1蛋白的突变体的氨基酸132至136区域、或氨基酸135至139区域、或氨基酸428-431区域、或氨基酸431-434区域。
如本文所用，术语“直接插入”是指在相邻两个氨基酸之间插入所选择的肽片段。例如，在SEQ ID NO.1的氨基酸132和氨基酸133之间的直接插入指的是将所选择的肽片段直接插入到SEQ ID NO.1的氨基酸132和氨基酸133之间。
如本文所用，术语“非等长置换”指的是在删除指定氨基酸区间的序列后，将所选的肽片段插入到指定的氨基酸区间。例如，在SEQ ID NO.1的氨基酸132至136区域的非等长置换指的是，删除SEQ ID NO.1的氨基酸133-135之后，将所选择的肽片段插入到SEQ ID NO.1的氨基酸氨基酸132至136之间。
可选地，在所述直接插入或非等长置换的方式中，所述来自HPV73型L2蛋白的多肽在其N端和/或C端包含1至3个氨基酸残基长的连接子。
可选地，所述的连接子由选自甘氨酸(G)、丝氨酸(S)、丙氨酸(A)及脯氨酸(P)的氨基酸任意组合构成。优选地，N端的连接子由G(甘氨酸)P(脯氨酸)组成，C端的连接子由P(脯氨酸)组成。
可选地，在所述直接插入的方式中，所述来自HPV73型L2蛋白的多肽的氨基酸序列是SEQ ID No.15、SEQ ID No.16或SEQ ID No.17，插入位点为所述N端完整的HPV31型L1蛋白及所述突变体的氨基酸137和氨基酸138之间或氨基酸432和433之间。
可选地，在所述直接插入的方式中，所述来自HPV73型L2蛋白的多肽的氨基酸序列是SEQ ID No.15、SEQ ID No.16或SEQ ID No.17，插入位点为所述N端截短4个氨基酸的HPV31型L1蛋白及所述突变体的氨基酸134和氨基酸135之间或氨基酸429和430之间。
可选地，在所述直接插入的方式中，所述来自HPV73型L2蛋白的多肽的氨基酸序列是N端含有GP连接子和/或C端含有P连接子的SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示序列，插入位点为所述N端完整的HPV31型L1蛋白及所述突变体的氨基酸137和氨基酸138之间或氨基酸432和433之间。
可选地，在所述直接插入的方式中，所述来自HPV73型L2蛋白的多肽的氨基酸序列是N端含有GP连接子和/或C端含有P连接子的SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示序列，插入位点为N端截短4个氨基酸的HPV31型L1蛋白及所述突变体的氨基酸134和氨基酸135之间或氨基酸429和430之间。
可选地，在所述非等长置换的方式中，删除N端完整的HPV31型L1蛋白及所述突变体的氨基酸136-138区域后，在所述N端完整的HPV31型L1蛋白及所 述突变体的氨基酸135及139之间插入来自HPV73型L2蛋白的多肽，所述来自HPV73型L2蛋白的多肽其N端增加了甘氨酸-脯氨酸连接子，所述来自HPV73型L2蛋白的多肽的氨基酸序列如SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示。
可选地，在所述非等长置换的方式中，删除N端截短4个氨基酸的HPV31型L1蛋白及所述突变体的氨基酸133-135区域后，在所述N端截短4个氨基酸的HPV31型L1蛋白及所述突变体的氨基酸132及136之间插入来自HPV73型L2蛋白的多肽，所述来自HPV73型L2蛋白的多肽其N端增加了甘氨酸-脯氨酸连接子，所述来自HPV73型L2蛋白的多肽的氨基酸序列如SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示。
可选地，在所述非等长置换的方式中，删除N端完整的HPV31型L1蛋白及所述突变体的氨基酸432-433区域后，在所述N端完整的HPV31型L1蛋白及所述突变体的氨基酸431及434之间插入来自HPV73型L2蛋白的多肽，所述来自HPV73型L2蛋白的多肽的氨基酸序列如SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示。
可选地，在所述非等长置换的方式中，删除N端截短4个氨基酸的HPV31型L1蛋白及所述突变体的氨基酸429-430区域后，在所述N端截短4个氨基酸的HPV31型L1蛋白及所述突变体的氨基酸428及431之间插入来自HPV73型L2蛋白的多肽，所述来自HPV73型L2蛋白的多肽的氨基酸序列如SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示。
优选地，在所述非等长置换的方式中，删除所述HPV31型L1蛋白突变体的氨基酸133-135区域后，在所述HPV31型L1蛋白突变体的氨基酸132及136之间插入来自HPV73型L2蛋白的多肽，所述来自HPV73型L2蛋白的多肽其N端增加了甘氨酸-脯氨酸连接子，所述来自HPV73型L2蛋白的多肽的氨基酸序列如SEQ ID No.15或SEQ ID No.17所示，获得的嵌合蛋白氨基酸序列如SEQ ID No.18、SEQ ID No.19、SEQ ID No.20、SEQ ID No.21、SEQ ID No.22、SEQ ID No.23、SEQ ID No.24或SEQ ID No.25所示。
优选地，在所述非等长置换的方式中，删除所述HPV31型L1蛋白突变体的氨基酸429-430区域后，在所述HPV31型L1蛋白突变体的氨基酸428及431之间插入来自HPV73型L2蛋白的多肽，所述来自HPV73型L2蛋白的多肽的氨基酸序列如SEQ ID No.16或SEQ ID No.17所示，获得的嵌合蛋白氨基酸序列如SEQ ID No.26、SEQ ID No.27、SEQ ID No.28、SEQ ID No.29、SEQ ID No.30、SEQ ID No.31、SEQ ID No.32或SEQ ID No.33所示。
本发明的另一方面涉及编码上述的人乳头瘤病毒嵌合蛋白的多核苷酸。
本发明还提供了包含上述的多核苷酸的载体，以及包含所述的载体的细胞。
本发明涉及的编码上述的人乳头瘤病毒嵌合蛋白的多核苷酸序列适用于不同的表达系统。可选地，这些核苷酸序列采用大肠杆菌密码子进行全基因优化，可在大肠杆菌表达系统中高水平表达；或采用昆虫细胞密码子进行全基因优化，可在昆虫细胞表达系统中高水平表达。
本发明还提供了一种多聚物，优选地，所述多聚物为人乳头瘤病毒嵌合五聚体或嵌合病毒样颗粒，其中所述多聚物包含本发明所述的人乳头瘤病毒嵌合蛋白，或者由本发明所述的人乳头瘤病毒嵌合蛋白所形成。
本发明还提供了上述的乳头瘤病毒嵌合蛋白、乳头瘤病毒嵌合五聚体或上述的乳头瘤病毒嵌合病毒样颗粒在制备预防乳头瘤病毒感染和/或所述乳头瘤病毒感染诱发的疾病的疫苗中的用途，优选地，所述乳头瘤病毒感染诱发的疾病包括但不限于宫颈癌、阴道癌、阴唇癌、阴茎癌、肛门肛周癌、口咽癌、扁桃体癌及口腔癌；
优选地，所述乳头瘤病毒感染为一种或多种选自以下人乳头瘤病毒型别的感染：HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV68、HPV70、HPV73；HPV6、HPV11、HPV2、HPV5、HPV27和HPV57。
本发明还提供了一种用于预防乳头瘤病毒感染及感染诱发的疾病的疫苗，其包含上述的乳头瘤病毒嵌合五聚体或嵌合病毒样颗粒、佐剂、以及疫苗用赋形剂或载体，优选地，还包含至少一种嗜黏膜组和/或嗜皮肤组的HPV的病毒样颗粒或嵌合病毒样颗粒。其中，这些病毒样颗粒的含量分别为能诱发保护性免疫反应的有效量。
可选地，所述佐剂为人用佐剂。
发明中相关术语的说明及解释
根据本发明，术语“昆虫细胞表达系统”包括昆虫细胞、重组杆状病毒、重组Bacmid及表达载体。其中昆虫细胞来源于市场上可得到的细胞，在此举例但不限于：Sf9，Sf21，High Five。
根据本发明，术语“原核表达系统”包括但不限于大肠杆菌表达系统。其中表达宿主菌来源于市场上可得到的菌株，在此举例但不限于：BL21(DE3)，BL21(DE3)plysS，C43(DE3)，Rosetta-gami B(DE3)。
根据本发明，术语“全长HPV31型L1蛋白”的例子包括但不限于NCBI数据库中编号为P17388.1、AEI61021.1、AEI60949.1、AAA92894.1、AIG59245.1、AIG59235.1的蛋白等长的全长L1蛋白。
“截短型HPV31型L1蛋白”的基因片段指的是其与野生型HPV31型L1蛋白基因相比，在其5’端和/或3’端缺失编码1个或多个氨基酸的核苷酸，其中“野生型HPV31型L1蛋白”的全长序列例如但不限于NCBI数据库中的如下序列： P17388.1、AEI61021.1、AEI60949.1、AAA92894.1、AIG59245.1、AIG59235.1等。
根据本发明，术语“疫苗用赋形剂或载体”是指选自一种或多种，包括但不限于：pH调节剂，表面活性剂，离子强度增强剂。例如，pH调节剂举例但不限于磷酸盐缓冲液，表面活性剂包括阳离子、阴离子或非离子型表面活性剂，举例但不限于聚山梨酯80(Tween-80)，离子强度增强剂举例但不限于氯化钠。
根据本发明，术语“人用佐剂”是指在临床上可应用于人体的佐剂，包括当前已获得批准的和将来可能获得批准的各种佐剂，例如但不限于铝佐剂、MF59及各种形式的佐剂组合物。
根据本发明，本发明的疫苗可采用患者可接受的形式，包括但不限于口服或者注射，优选注射。
根据本发明，本发明疫苗优选单位剂型使用，其中单位剂型中蛋白病毒样颗粒的剂量为5μg-100μg，例如5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100μg、以及上述任意两个数值之间的范围；优选30μg-60μg。
附图说明
图1A-图1B：本发明实施例7中31型L1蛋白突变体及包含其的嵌合蛋白在昆虫细胞中的表达鉴定。结果显示，11种31型L1蛋白及突变体、16种嵌合蛋白均可在昆虫细胞中表达。
图1A：31型L1蛋白及其突变体蛋白在昆虫细胞中的表达鉴定：1为31L1；2为T 274N；3为31L1MΔC；4为T 274NΔC；5为T 267AΔC；6为T 267AT 274NΔC；7为T 274NΔN2C；8为T 274NΔN4C；9为T 274NΔN5C；10为T 274NΔN8C；11为T 274NΔN10C；
图1B：包含31型L1蛋白突变体的嵌合蛋白在昆虫细胞中的表达鉴定：1为31L1DE 132-136/dE；2为31L1DE 132-136/dES；3为31L1h4 428-431/dE；4为31L1h4 428-431/dES；5为31L1DE 132-136/dE-CS1；6为31L1DE 132-136/dES-CS1；7为31L1h4 428-431/dE-CS1；8为31L1h4 428-431/dES-CS1，9为31L1DE 132-136/dE-CS2；10为31L1DE 132-136/dES-CS2；11为31L1h4 428-431/dE-CS2；12为31L1h4 428-431/dES-CS2；13为31L1DE 132-136/dE-CS3；14为31L1DE 132-136/dES-CS3；15为31L1h4 428-431/dE-CS3；16为31L1h4 428-431/dES-CS3。
图2A-图2F：本发明实施例8中纯化后获得的VLP及cVLP的动态光散射分析结果。结果显示31L1MΔC、T 274NΔC、T 274NΔN4C、31L1DE 132-136/dE、31L1h4 428-431/dE及31L1h4 428-431/dE-CS1重组蛋白形成的病毒样颗粒水化动力学直径分别为103.3nm、99.78nm、106.8nm、104.59nm、47.8nm及42.4nm，颗粒组 装的百分比均为100％。
图2A：31L1MΔC；图2B：T 274NΔC；图2C：T 274NΔN4C；图2D：31L1DE 132-136/dE；图2E：31L1h4 428-431/dE；图2F：31L1h4 428-431/dE-CS1。
图3A-图3E：本发明实施例8中纯化后获得的VLP及cVLP的透射电镜观察结果。视野中可见大量的病毒样颗粒。Bar＝50nm。
图3A：31L1MΔC；图3B：T 274NΔN4C；图3C：31L1DE 132-136/dE；图3D：31L1h4 428-431/dE；图3E：31L1h4 428-431/dE-CS1。
图4：本发明实施例11所述的小鼠免疫血清采用HPV31假病毒进行中和抗体滴度检测结果。ns：无统计学差异(P>0.05)。
具体实施方式
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。下面的实施例仅用于说明本发明，而不应视为限定本发明的范围，因为实施方案必然是多样的。本说明书中使用的用语仅是为了阐述特定的实施方案，而非作为限制，本发明的范围已界定在所附的权利要求中。
除非特别说明，本说明书中所使用的所有技术和科学用语均和本案所属技术领域的技术人员所普遍明了的意义相同。下面就本发明的优选方法和材料加以叙述，但是与本说明书中所述方法和材料类似或等效的任何方法和材料均可用以实施或测试本发明。下述实验方法如无特别说明，均为常规方法或产品说明书所描述的方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。本说明书中所提到的所有公开文献均被并入于此作为参考，以揭示并说明所述公开文献中的方法和/或材料。
实施例1：HPV31变异株的L1序列分析
在NCBI Genbank中输入“major capsid protein L1[Human papillomavirus type 31]”或“late protein L1[Human papillomavirus type 31]”关键词，获得19例自然界中存在的HPV31 L1变异株，利用DNAMAN软件进行氨基酸序列比对(表1)，发现L1的第15、179、181、194、267、274、432、439位氨基酸发生了变异，其中第267位(突变频率53％)和第274位(突变频率89％)为高频变异位点，其他位点氨基酸突变频率在5％～15％之间。对于第267位和第274位氨基酸而言，第267位由苏氨酸(T)突变为丙氨酸(A)的占53％，第274位由苏氨酸(T)突变为天冬酰胺(N)的占89％，因此A和T分别是第267位和第274位的优势氨基酸。
表1.不同HPV31 L1变异株的氨基酸序列比对
Figure PCTCN2021120603-appb-000001
*以连字号(-)表示的氨基酸与HPV31L1原型对应位置的氨基酸相同。
实施例2：不同型别RG-1表位肽的免疫活性检测
采用化学合成法合成HPV35、-39、-51、-53、-56、-68、-73、-82的RG-1表位肽，表位肽序列如表1所示，多肽由上海吉尔生化有限公司合成，为了提高合成肽的免疫原性，各合成肽通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC，CAS No.25952-53-8)活化羧基后与钥孔血蓝蛋白(KLH)偶联。
取2.0-2.5kg体重的新西兰大白兔，随机分组，每组2-4只，于免疫前4天背部多点皮下注射15mg灭活的与等体积弗氏完全佐剂充分混匀的DH5a(含0.5％v/v甲醛的PBS，37℃处理24-48h)进行免疫刺激，首次免疫采用背部、大腿内侧多点皮下注射1mg与等体积弗氏完全佐剂充分混匀的KLH-多肽。加强免疫4次，每次间隔2周，加强免疫的抗原为0.5mg与等体积弗氏不完全佐剂充分混匀的KLH-多肽。最后一次免疫后2周采血，分离血清。
使用17种HPV假病毒对免疫血清的中和抗体滴度进行检测，结果如表3所示。73RG-1表位肽的免疫活性最好，其抗血清可中和所有17种检测型别，其中HPV45,-18,-16的中和抗体的滴度均在10 3以上，HPV68,-57,-59,-39,-5的中和抗体滴度在500-1000之间。
多肽合成、假病毒制备及假病毒中和实验的方法均是公开的，例如专利CN104418942A及108676057A。
表2.合成的不同型别RG-1表位肽的序列
型别 合成肽序列 SEQ ID NO.
HPV35 TQLYRTCKAAGTCPPDVIPKVEG 53
HPV39 STLYRTCKQSGTCPPDVVDKVEG 54
HPV51 TQLYSTCKAAGTCPPDVVNKVEG 55
HPV53 TQLYQTCKQSGTCPEDVINKIEH 56
HPV56 TQLYKTCKLSGTCPEDVVNKIEQ 57
HPV68 STLYKTCKQSGTCPPDVINKVEG 58
HPV73 TQLYKTCKQAGTCPPDVIPKVEG 59
HPV82 TQLYSTCKAAGTCPPDVIPKVKG 60
表3.不同的RG1-KLH偶联肽在兔子中诱发的血清中和抗体滴度
Figure PCTCN2021120603-appb-000002
实施例3：HPV31L1蛋白及其突变体的基因的合成及表达载体构建
合计11种HPV31L1蛋白及突变体，分别为：
1)31L1原型：其氨基酸序列如SEQ ID No.1所示，编码31L1原型蛋白的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
2)T 274N突变体：将SEQ ID No.1序列的第274位苏氨酸突变为天冬酰胺，其氨基酸序列如SEQ ID No.3所示，编码T 274N突变体的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
3)31L1MΔC突变体：将HPV31L1的C端截短29个氨基酸，其氨基酸序列如SEQ ID No.5所示，编码31L1MΔC的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其核苷酸序列如SEQ ID No.34所示；
4)T 274NΔC突变体：将SEQ ID No.5序列的第274位苏氨酸突变为天冬酰胺，其氨基酸序列如SEQ ID No.6所示，编码T274NΔC的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其核苷酸序列如SEQ ID No.35所示；
5)T 267AΔC突变体：将SEQ ID No.5序列的第267位苏氨酸突变为丙氨酸，编码T267AΔC的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
6)T 267AT 274NΔC突变体：将SEQ ID No.6序列的第267位苏氨酸突变为丙氨酸，编码T267AT 274NΔC的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
7)T 274NΔN2C突变体：将SEQ ID No.6所示序列的N端截短2个氨基酸，其序列如SEQ ID No.7所示，编码T 274NΔN2C的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
8)T 274NΔN4C突变体：将SEQ ID No.6所示序列的N端截短4个氨基酸，其序列如SEQ ID No.8所示，编码T 274NΔN4C的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其核苷酸序列如SEQ ID No.36所示；
9)T 274NΔN5C突变体：将SEQ ID No.6所示序列的N端截短5个氨基酸，其序列如SEQ ID No.9所示，编码T 274NΔN5C的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
10)T 274NΔN8C突变体：将SEQ ID No.6所示序列的N端截短8个氨基酸，其序列如SEQ ID No.10所示，编码T 274NΔN8C的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建；
11)T 274NΔN10C突变体：将SEQ ID No.6所示序列的N端截短10个氨基酸，其序列如SEQ ID No.11所示，编码T 274NΔN10C的核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建。
昆虫细胞密码子优化的HPV31L1蛋白及突变体基因经BamHI/XbaI酶切后，分别插入商业化表达载体pFastBac1(Invitrogen公司生产)中。得到包含嵌合蛋白 基因的表达载体，分别为：pFastBac1-31L1，pFastBac1-T 274N，pFastBac1-31L1MΔC，pFastBac1-T 274NΔC，pFastBac1-T 267AΔC，pFastBac1-T 267AT 274NΔC，pFastBac1-T 274NΔN2C，pFastBac1-T 274NΔN4C，pFastBac1-T 274NΔN5C，pFastBac1-T 274NΔN8C，pFastBac1-T 274NΔN10C。上述酶切、连接及克隆构建的方法都是公知的，例如专利CN101293918B。
实施例4：HPV31L1嵌合蛋白及其突变体的基因的合成及表达载体构建
合计16种嵌合蛋白及突变体，分别为：
1)嵌合L1蛋白31L1DE 132-136/dE：骨架为T 274NΔN4C(即在第274位苏氨酸突变为天冬酰胺的基础上N端截短4个氨基酸，C端截短29个氨基酸，其序列如SEQ ID No.8所示)，删除其aa.133-135区域，并在aa.132/136之间融合N端包含GP连接子的HPV73型L2蛋白的aa.18-38多肽(在SEQ ID No.8的aa.132-136区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.15所示序列的N端添加甘氨酸-脯氨酸，31L1DE 132-136/dE嵌合蛋白的氨基酸序列如SEQ ID No.18所示。编码31L1DE 132-136/dE的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.37所示；
2)嵌合L1蛋白31L1DE 132-136/dES：骨架为T 274NΔN4C(其序列如SEQ ID No.8所示)，删除其aa.133-135区域，并在aa.132/136之间融合N端包含GP连接子的HPV73型L2蛋白的aa.19-35多肽(在SEQ ID No.8的aa.132-136区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.17所示序列的N端添加甘氨酸-脯氨酸，31L1DE 132-136/dES嵌合蛋白的氨基酸序列如SEQ ID No.19所示。编码31L1DE 132-136/dES的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.38所示；
3)嵌合L1蛋白31L1DE 132-136/dE-CS1：骨架为T 274NΔN4C-CS1(即在第274位苏氨酸突变为天冬酰胺的基础上N端截短4个氨基酸，并对C端29个氨基酸之内的碱性氨基酸进行置换，其序列如SEQ ID No.12所示)，删除其aa.133-135区域，并在aa.132/136之间融合N端包含GP连接子的HPV73型L2蛋白的aa.18-38多肽(在SEQ ID No.12的aa.132-136区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.15所示序列的N端添加甘氨酸-脯氨酸，31L1DE 132-136/dE-CS1嵌合蛋白的氨基酸序列如SEQ ID No.20所示。编码31L1DE 132-136/dE-CS1的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.39所示；
4)嵌合L1蛋白31L1DE 132-136/dES-CS1：骨架为T 274NΔN4C-CS1(其序列如SEQ ID No.12所示)，删除其aa.133-135区域，并在aa.132/136之间融合N端包含GP连接子的HPV73型L2蛋白的aa.19-35多肽(在SEQ ID No.12的aa.132-136 区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.17所示序列的N端添加甘氨酸-脯氨酸，31L1DE 132-136/dES-CS1嵌合蛋白的氨基酸序列如SEQ ID No.21所示。编码31L1DE 132-136/dES-CS1的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.40所示；
5)嵌合L1蛋白31L1DE 132-136/dE-CS2：骨架为T 274NΔN4C-CS2(即在第274位苏氨酸突变为天冬酰胺的基础上N端截短4个氨基酸，并对C端29个氨基酸之内的碱性氨基酸进行置换，其序列如SEQ ID No.13所示)，删除其aa.133-135区域，并在aa.132/136之间融合N端包含GP连接子的HPV73型L2蛋白的aa.18-38多肽(在SEQ ID No.13的aa.132-136区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.15所示序列的N端添加甘氨酸-脯氨酸，31L1DE 132-136/dE-CS2嵌合蛋白的氨基酸序列如SEQ ID No.22所示。编码31L1DE 132-136/dE-CS2的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.41所示；
6)嵌合L1蛋白31L1DE 132-136/dES-CS2：骨架为T 274NΔN4C-CS2(其序列如SEQ ID No.13所示)，删除其aa.133-135区域，并在aa.132/136之间融合N端包含GP连接子的HPV73型L2蛋白的aa.19-35多肽(在SEQ ID No.13的aa.132-136区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.17所示序列的N端添加甘氨酸-脯氨酸，31L1DE 132-136/dES-CS2嵌合蛋白的氨基酸序列如SEQ ID No.23所示。编码31L1DE 132-136/dES-CS2的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.42所示；
7)嵌合L1蛋白31L1DE 132-136/dE-CS3：骨架为T 274NΔN4C-CS3(即在第274位苏氨酸突变为天冬酰胺的基础上N端截短4个氨基酸，并对C端29个氨基酸之内的碱性氨基酸进行置换，其序列如SEQ ID No.14所示)，删除其aa.133-135区域，并在aa.132/136之间融合N端包含GP连接子的HPV73型L2蛋白的aa.18-38多肽(在SEQ ID No.14的aa.132-136区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.15所示序列的N端添加甘氨酸-脯氨酸，31L1DE 132-136/dE-CS3嵌合蛋白的氨基酸序列如SEQ ID No.24所示。编码31L1DE 132-136/dE-CS3的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.43所示；
8)嵌合L1蛋白31L1DE 132-136/dES-CS3：骨架为T 274NΔN4C-CS3(其序列如SEQ ID No.14所示)，删除其aa.133-135区域，并在aa.132/136之间融合N端包含GP连接子的HPV73型L2蛋白的aa.19-35多肽(在SEQ ID No.14的aa.132-136区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.17所示序列的N端添加甘氨酸-脯氨酸，31L1DE 132-136/dES-CS3嵌合蛋白的氨基酸序列如SEQ ID No.25所示。编码31L1DE 132-136/dES-CS3的多核苷酸序列经昆虫细胞密码子优化 设计，采用全基因合成的方式构建，其序列如SEQ ID No.44所示；
9)嵌合L1蛋白31L1h4 428-431/dE：骨架为T 274NΔN4C(即在第274位苏氨酸突变为天冬酰胺的基础上N端截短4个氨基酸，C端截短29个氨基酸，其序列如SEQ ID No.8所示)，删除其aa.429-430区域，并在aa.428/431之间融合HPV73型L2蛋白的aa.19-39多肽(在SEQ ID No.8的aa.428-431区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.16所示，31L1h4 428-431/dE嵌合蛋白的氨基酸序列如SEQ ID No.26所示。编码31L1h4 428-431/dE的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.45所示；
10)嵌合L1蛋白31L1h4 428-431/dES：骨架为T 274NΔN4C(其序列如SEQ ID No.8所示)，删除其aa.429-430区域，并在aa.428/431之间融合HPV73型L2蛋白的aa.19-35多肽(在SEQ ID No.8的aa.428-431区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.17所示序列，31L1h4 428-431/dES嵌合蛋白的氨基酸序列如SEQ ID No.27所示。编码31L1h4 428-431/dES的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.46所示；
11)嵌合L1蛋白31L1h4 428-431/dE-CS1：骨架为T 274NΔN4C-CS1(即在第274位苏氨酸突变为天冬酰胺的基础上N端截短4个氨基酸，并对C端29个氨基酸之内的碱性氨基酸进行置换，其序列如SEQ ID No.12所示)，删除其aa.429-430区域，并在aa.428/431之间融合HPV73型L2蛋白的aa.19-39多肽(在SEQ ID No.12的aa.428-431区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.16所示，31L1h4 428-431/dE-CS1嵌合蛋白的氨基酸序列如SEQ ID No.28所示。编码31L1h4 428-431/dE-CS1的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.47所示；
12)嵌合L1蛋白31L1h4 428-431/dES-CS1：骨架为T 274NΔN4C-CS1(其序列如SEQ ID No.12所示)，删除其aa.429-430区域，并在aa.428/431之间融合HPV73型L2蛋白的aa.19-35多肽(在SEQ ID No.12的aa.428-431区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.17所示序列，31L1h4 428-431/dES-CS1嵌合蛋白的氨基酸序列如SEQ ID No.29所示。编码31L1h4 428-431/dES-CS1的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.48所示；
13)嵌合L1蛋白31L1h4 428-431/dE-CS2：骨架为T 274NΔN4C-CS2(即在第274位苏氨酸突变为天冬酰胺的基础上N端截短4个氨基酸，并对C端29个氨基酸之内的碱性氨基酸进行置换，其序列如SEQ ID No.13所示)，删除其aa.429-430区域，并在aa.428/431之间融合HPV73型L2蛋白的aa.19-39多肽(在SEQ ID No.13的aa.428-431区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.16所示，31L1h4 428-431/dE-CS2嵌合蛋白的氨基酸序列如SEQ ID No.30所示。编码 31L1h4 428-431/dE-CS2的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.49所示；
14)嵌合L1蛋白31L1h4 428-431/dES-CS2：骨架为T 274NΔN4C-CS2(其序列如SEQ ID No.13所示)，删除其aa.429-430区域，并在aa.428/431之间融合HPV73型L2蛋白的aa.19-35多肽(在SEQ ID No.13的aa.428-431区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.17所示序列，31L1h4 428-431/dES-CS2嵌合蛋白的氨基酸序列如SEQ ID No.31所示。编码31L1h4 428-431/dES-CS2的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.50所示；
15)嵌合L1蛋白31L1h4 428-431/dE-CS3：骨架为T 274NΔN4C-CS3(即在第274位苏氨酸突变为天冬酰胺的基础上N端截短4个氨基酸，并对C端29个氨基酸之内的碱性氨基酸进行置换，其序列如SEQ ID No.14所示)，删除其aa.429-430区域，并在aa.428/431之间融合HPV73型L2蛋白的aa.19-39多肽(在SEQ ID No.13的aa.428-431区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.16所示，31L1h4 428-431/dE-CS3嵌合蛋白的氨基酸序列如SEQ ID No.32所示。编码31L1h4 428-431/dE-CS3的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.51所示；
16)嵌合L1蛋白31L1h4 428-431/dES-CS3：骨架为T 274NΔN4C-CS3(其序列如SEQ ID No.14所示)，删除其aa.429-430区域，并在aa.428/431之间融合HPV73型L2蛋白的aa.19-35多肽(在SEQ ID No.14的aa.428-431区域非等长置换插入)，插入片段的氨基酸序列为SEQ ID No.17所示序列，31L1h4 428-431/dES-CS3嵌合蛋白的氨基酸序列如SEQ ID No.33所示。编码31L1h4 428-431/dES-CS3的多核苷酸序列经昆虫细胞密码子优化设计，采用全基因合成的方式构建，其序列如SEQ ID No.52所示。
昆虫细胞密码子优化的HPV31L1蛋白及突变体基因经BamHI/XbaI酶切后，分别插入商业化表达载体pFastBac1(Invitrogen公司生产)中。得到包含嵌合蛋白基因的表达载体，分别为：pFastBac1-31L1DE 132-136/dE，pFastBac1-31L1DE 132-136/dES，pFastBac1-31L1DE 132-136/dE-CS1，pFastBac1-31L1DE 132-136/dES-CS1，pFastBac1-31L1DE 132-136/dE-CS2，pFastBac1-31L1DE 132-136/dES-CS2，pFastBac1-31L1DE 132-136/dE-CS3，pFastBac1-31L1DE 132-136/dES-CS3，pFastBac1-31L1h4 428-431/dE，pFastBac1-31L1h4 428-431/dES，pFastBac1-31L1h4 428-431/dE-CS1，pFastBac1-31L1h4 428-431/dES-CS1，pFastBac1-31L1h4 428-431/dE-CS2，pFastBac1-31L1h4 428-431/dES-CS2，pFastBac1-31L1h4 428-431/dE-CS3，pFastBac1-31L1h4 428-431/dES-CS3。上述酶切、连接及克隆构建的方法都是公知 的，例如专利CN101293918B。
本发明涉及的氨基酸序列如下所述：
Figure PCTCN2021120603-appb-000003
Figure PCTCN2021120603-appb-000004
Figure PCTCN2021120603-appb-000005
Figure PCTCN2021120603-appb-000006
Figure PCTCN2021120603-appb-000007
Figure PCTCN2021120603-appb-000008
Figure PCTCN2021120603-appb-000009
Figure PCTCN2021120603-appb-000010
Figure PCTCN2021120603-appb-000011
Figure PCTCN2021120603-appb-000012
Figure PCTCN2021120603-appb-000013
Figure PCTCN2021120603-appb-000014
Figure PCTCN2021120603-appb-000015
Figure PCTCN2021120603-appb-000016
Figure PCTCN2021120603-appb-000017
Figure PCTCN2021120603-appb-000018
Figure PCTCN2021120603-appb-000019
Figure PCTCN2021120603-appb-000020
Figure PCTCN2021120603-appb-000021
Figure PCTCN2021120603-appb-000022
实施例5：L1蛋白、嵌合L1蛋白基因的重组Bacmid及重组杆状病毒的构建
分别使用包含L1基因的重组表达载体pFastBac1-31L1，pFastBac1-T 274N，pFastBac1-31L1MΔC，pFastBac1-T 274NΔC，pFastBac1-T 267AΔC，pFastBac1-T 267AT 274NΔC，pFastBac1-T 274NΔN2C，pFastBac1-T 274NΔN4C，pFastBac1-T 274NΔN5C，pFastBac1-T 274NΔN8C，pFastBac1-T 274NΔN10C；或嵌合L1基因的重组表达载体pFastBac1-31L1DE 132-136/dE，pFastBac1-31L1DE 132-136/dES，pFastBac1-31L1DE 132-136/dE-CS1，pFastBac1-31L1DE 132-136/dES-CS1，pFastBac1-31L1DE 132-136/dE-CS2，pFastBac1-31L1DE 132-136/dES-CS2，pFastBac1-31L1DE 132-136/dE-CS3，pFastBac1-31L1DE 132-136/dES-CS3，pFastBac1-31L1h4 428-431/dE，pFastBac1-31L1h4 428-431/dES，pFastBac1-31L1h4 428-431/dE-CS1，pFastBac1-31L1h4 428-431/dES-CS1，pFastBac1-31L1h4 428-431/dE-CS2，pFastBac1-31L1h4 428-431/dES-CS2，pFastBac1-31L1h4 428-431/dE-CS3，pFastBac1-31L1h4 428-431/dES-CS3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内扩增重组杆状病毒。重组Bacmid的筛选及重组杆状病毒的扩增方法都是公知的，例如专利CN101148661B。
实施例6：L1蛋白、嵌合L1蛋白的基因在Sf9细胞中的表达
Sf9细胞分别接种11种含31L1蛋白或突变体基因的重组杆状病毒，或16种 含嵌合L1基因的重组杆状病毒，进行蛋白的表达，27℃培养约88h后收发酵液，3000rpm离心15min，弃上清，用PBS洗涤细胞后，用于表达鉴定及纯化。感染表达的方法是公开的，例如专利CN 101148661B。
实施例7：L1蛋白、嵌合L1蛋白的表达鉴定
取实施例6中所述表达不同L1蛋白或嵌合L1蛋白的细胞各1×10 6个，重悬于200μl PBS溶液中，加入6×Loading Buffer 50μl，75℃变性8分钟，分别取10μl进行SDS-PAGE电泳及Western blot鉴定。结果如图1A至图1B所示，11种31L1蛋白或突变体、16种嵌合L1蛋白均可在昆虫细胞中高水平表达，其中31L1、T 274N、31L1DE 132-136/dE、31L1DE 132-136/dES、31L1h4 428-431/dE、31L1h4 428-431/dES蛋白大小约55kDa，31L1MΔC、T 274NΔC、T 267AΔC、T 267AT 274NΔC、T 274NΔN2C、T 274NΔN4C、T 274NΔN5C、T 274NΔN8C、T 274NΔN10C蛋白大小约50kD，31L1DE 132-136/dE-CS1、31L1DE 132-136/dES-CS1、31L1DE 132-136/dE-CS2、31L1DE 132-136/dES-CS2、31L1DE 132-136/dES-CS3、31L1DE 132-136/dES-CS3、31L1h4 428-431/dE-CS1、31L1h4 428-431/dES-CS1、31L1h4 428-431/dE-CS2、31L1h4 428-431/dES-CS2、31L1h4 428-431/dE-CS3、31L1h4 428-431/dES-CS3a大小约59kDa。SDS-PAGE电泳及Western blot鉴定的方法是公开的，例如专利CN101148661B。
实施例8：L1蛋白、嵌合L1蛋白在昆虫细胞中的表达量比较
取实施例6所述的表达不同重组蛋白的细胞各1×10 6个，重悬于200μl PBS溶液中，采用超声破碎法(宁波新芝超声破碎仪，2#探头，100W，超声5s，间隔7s，总时间3min)破碎细胞，12000rpm高速离心10分钟。收取裂解上清，采用夹心ELISA法检测上清中的L1含量，该方法是公知的，例如专利CN104513826A。
使用本发明人制备的HPV31L1单克隆抗体包被酶标板，80ng/孔，4℃孵育过夜；使用5％BSA-PBST室温封闭2h，再用PBST洗板3次。用PBS将裂解上清进行连续2倍稀释，并且将HPV31L1 VLP标准品也进行梯度稀释，浓度从2μg/ml-0.0625μg/ml，分别加入酶标板，每孔100μl，37℃孵育1h。用PBST洗板3次，加入1：3000稀释的HPV31L1兔多抗，每孔100μl，37℃孵育1h。用PBST洗板3次，加入1：3000稀释的HRP标记的山羊抗小鼠IgG(1：3000稀释，中杉金桥公司)，37℃孵育45分钟。用PBST洗板5次，每孔加入100μl OPD底物(Sigma公司)，37℃显色5分钟，用50μl 2M硫酸终止反应，在490nm处测定吸光值。依据标准曲线计算裂解上清中31L1蛋白、31L1蛋白突变体或嵌合L1蛋白的浓度。
结果如表4所示，本发明的C端截短29个氨基酸的31L1突变体蛋白 (31L1MΔC)的表达量显著高于HPV31L1全长蛋白；对31L1蛋白进行点突变获得的突变体蛋白的表达量也各不相同，其中T 274N突变体的表达量显著高于HPV31L1原型蛋白，T 274NΔC突变体蛋白的表达量也较31L1MΔC蛋白的进一步提高，表明第274位苏氨酸突变为天冬酰胺可提高31L1蛋白的表达量；在T 274NΔC的基础上进行不同的N端截短，发现不同的截短对表达量的影响各不相同，其中N端截短4个氨基酸(T 274NΔN4C)或N端截短8个氨基酸(T 274NΔN8C)获得的截短突变的表达量分别是T 274NΔC的2倍和1.28倍。在T 274NΔN4C的基础上构建的嵌合蛋白(31L1DE 132-136/dE、31L1DE 132-136/dES、31L1h4 428-431/dE、31L1h4 428-431/dES)表达量均与其骨架T 274NΔN4C的相当；此外，12种以C端置换的31L1突变体为骨架的嵌合蛋白表达量均高于相应的C端截短的嵌合蛋白。
表4. 31L1蛋白、31L1蛋白突变体及嵌合L1蛋白表达量分析
Figure PCTCN2021120603-appb-000023
Figure PCTCN2021120603-appb-000024
实施例9：L1蛋白、嵌合L1蛋白的纯化及动态光散射粒径分析
取表达上述重组蛋白的细胞发酵液适量，使用10ml PBS重悬细胞，加PMSF至终浓度1mg/ml，超声破碎(宁波新芝超声破碎仪，6#探头，200W，超声5s，间隔7s，总时间10min)，取破碎上清进行纯化，纯化步骤在室温进行。在裂解液中加入4％β-巯基乙醇(w/w)对VLP进行解聚，然后使用0.22μm滤器过滤样品，依次使用DMAE阴离子交换层析或CM阳离子交换层析(20mM Tris,180mM NaCl,4％β-ME,pH7.9洗脱)、TMAE阴离子交换层析或Q阳离子交换层析(20mM Tris,180mM NaCl,4％β-ME,pH7.9洗脱)及羟基磷灰石层析(100mM NaH 2PO 4，30mM NaCl，4％β-ME，pH 6.0洗脱)纯化。纯化产物采用Planova超滤系统进行浓缩，并更换缓冲液(20mM NaH 2PO 4，500mM NaCl，pH6.0)促使VLP组装。以上纯化方法均是公开的，例如专利CN101293918B、CN1976718A等。
纯化后的HPV31L1蛋白、31L1突变体蛋白及嵌合L1蛋白均可有效组装。取组装后的蛋白溶液进行DLS粒径分析(Zetasizer Nano ZS 90动态光散射仪，Malvern公司)，结果如表5所示，其中31L1MΔC、T 274NΔC、T 274NΔN4C、31L1DE 132-136/dE、31L1h4 428-431/dE、的DLS分析图如图2A至图2F所示。
表5.L1蛋白及嵌合L1蛋白的DLS分析
Figure PCTCN2021120603-appb-000025
Figure PCTCN2021120603-appb-000026
实施例10：VLP、嵌合VLP的透射电镜观察
按实施例9所述的层析纯化方法，分别纯化重组蛋白，使用组装后嵌合制备铜网，并用1％醋酸铀进行染色，充分干燥后使用JEM-1400电镜(奥林巴斯)进行观察。结果显示，昆虫细胞表达的HPV31L1、T 274N、31L1MΔC、T 274NΔC、T 267AΔC及T 267AT 274NΔC蛋白均可组装成直径约为50-60nm的VLP；N端截短联合C端截短的31L1蛋白突变体可组装成直径17-35nm的VLP；在DE环表面区插入73L2多肽的嵌合蛋白可组装成30-50nm的cVLP；在h4区域插入73L2多肽的嵌合蛋白可组装成直径约为17-30nm的cVLP。其中31L1MΔC、T 274NΔN4C、31L1DE 132-136/dE、31L1h4 428-431/dE、31L1h4 428-431/dE-CS1的VLP或cVLP的电镜图片如图3A至图3E所示。铜网制备及电镜观察的方法均是公开的，例如专利CN101148661B。
实施例11：HPV31L1或突变体VLP的小鼠免疫及中和抗体滴度测定
取4-6周龄的BALB/c小鼠，随机分组，每组5只，用0.1μg VLP免疫小鼠。皮下注射，于第0，2周免疫，共2次。第2次免疫后2周尾静脉采血，分离血清。
使用HPV31假病毒对免疫血清的中和抗体滴度进行检测，结果各种31L1突变体的VLP免疫小鼠后，诱发HPV31特异性中和抗体水平均与原型相当。其中31L1MΔC、T 274NΔC、T 274NΔN4C的免疫结果如图4所示。
实施例12：嵌合VLP的小鼠免疫及中和抗体滴度测定
取4-6周龄的BALB/c小鼠，随机分组，每组5只，用10μg cVLP联合Al(OH) 3 50μg及MPL佐剂5μg免疫小鼠。皮下注射，于第0，4，7，10周免疫，共4次。第4次免疫后2周尾静脉采血，分离血清。
使用17种HPV假病毒对抗血清的中和抗体滴度进行检测，结果显示各种cVLP免疫小鼠后，诱发的交叉中和抗体水平及中和范围各有不同。其中，在h4表面区嵌合表位的cVLP诱发的骨架型别HPV31型中和抗体滴度与HPV31L1VLP的相当，且其抗血清的交叉中和抗体滴度高、可中和17种检测型别的假病毒；在DE loop表面区嵌合表位的cVLP诱发的HPV31型中和抗体滴度较31L1VLP的降 低了1个数量级，其免疫血清的交叉中和谱也相对较窄。部分cVLP免疫血清的交叉中和活性如表5所示，其中31L1h4 428-431/dE、31L1h4 428-431/dES及31L1h4 428-431/dE-CS1抗血清可中和至少17个型别的假病毒，31L1DE 132-136/dE及31L1DE 132-136/dES抗血清分别仅中和10个及8个型别的假病毒。值得一提的是，31L1h4 428-431/dE、31L1h4 428-431/dES及31L1h4 428-431/dE-CS1抗血清对HPV16，-18及-45型的中和滴度均大于10 3，31L1h4 428-431/dE-CS1抗血清对HPV73型的中和抗体滴度也大于10 3。
此外，本发明中采用C端置换的31L1突变体构建的cVLP，采用上述策略免疫小鼠后，诱发的交叉中和抗体水平及中和范围均与相应的C端截短的cVLP的一致。
假病毒制备及假病毒中和实验的方法均是公开的，例如专利CN104418942A。
表6.不同cVLP在小鼠中诱发的中和抗体滴度
Figure PCTCN2021120603-appb-000027

Claims (10)

  1. 一种人乳头瘤病毒嵌合蛋白，其包含HPV31型L1蛋白或HPV31型L1蛋白的突变体、以及插入所述HPV31型L1蛋白或HPV31型L1蛋白的突变体中的来自HPV73型L2蛋白的多肽，或由其组成，其中所述HPV31型L1蛋白如SEQ ID No.1所示，所述HPV73型L2蛋白如SEQ ID No.2所示；
    优选地，所述来自HPV73型L2蛋白的多肽如SEQ ID No.15、SEQ ID No.16或SEQ ID No.17所示；
    进一步优选地，所述来自HPV73型L2蛋白的多肽插入所述的HPV31型L1蛋白或所述HPV31型L1蛋白的突变体的表面区，优选插入所述的HPV31型L1蛋白或所述HPV31型L1蛋白的突变体的DE环或h4区域；
    更优选地，所述来自HPV73型L2蛋白的多肽通过直接插入的方式插入所述的HPV31型L1蛋白或所述HPV31型L1蛋白的突变体的氨基酸132和氨基酸133之间、或氨基酸134和135之间、或氨基酸136和137之间、或氨基酸137和138之间、或氨基酸432和433之间、或氨基酸434和435之间、或氨基酸435和436之间；或者通过非等长置换的方式插入所述的HPV31型L1蛋白或所述HPV31型L1蛋白的突变体的氨基酸132至136区域、或氨基酸135至139区域、或氨基酸428-431区域、或氨基酸431-434区域；
    更优选地，所述来自HPV73型L2蛋白的多肽在其N端和/或C端包含1至3个氨基酸残基长的连接子，更优选地，所述连接子由1-3个选自甘氨酸、丝氨酸、丙氨酸和脯氨酸的氨基酸组成；更优选地，N端的连接子由甘氨酸-脯氨酸组成，C端的连接子由脯氨酸组成；
    进一步优选地，所述HPV31型L1蛋白的突变体与SEQ ID No.1所示的HPV31型L1蛋白相比，包含选自i)至iii)中的任何一种或多种突变：
    i)选自以下的任何一种或多种的置换突变：
    T274N、R475G、R483G、R496 G、K477S、K497S、K501S、K479A、K482A、K498A、K495G、K500 G和R473G；
    ii)N端截短2、4、5、8或10个氨基酸的截短突变；和
    iii)C端截短29个氨基酸的截短突变；
    进一步优选地，所述HPV31型L1蛋白的突变体选自SEQ ID No.3、4、5、6、7、8、9、10、11、12、13和14所示的任一种变体。
  2. 根据权利要求1所述的人乳头瘤病毒嵌合蛋白，其中所述的人乳头瘤病毒嵌合蛋白的氨基酸序列如SEQ ID NO：18-33中的任一项所示。
  3. 一种多核苷酸，其编码权利要求1或2所述的人乳头瘤病毒嵌合蛋白，优 选地，所述多核苷酸的序列采用大肠杆菌密码子进行全基因优化或采用昆虫细胞密码子进行全基因优化，更优选地，所述多核苷酸的序列如SEQ ID No.37至SEQ ID No.52中任一项所示。
  4. 一种载体，其包含如权利要求3所述的多核苷酸。
  5. 一种细胞，其包含如权利要求4所述的载体。
  6. 一种多聚物，该多聚物为嵌合五聚体或嵌合病毒样颗粒，其含有权利要求1或2所述的人乳头瘤病毒嵌合蛋白，或者由权利要求1或2所述的人乳头瘤病毒嵌合蛋白所形成。
  7. 如权利要求1或2所述的人乳头瘤病毒嵌合蛋白或如权利要求6所述的多聚物在制备用于预防受试者中乳头瘤病毒感染和/或乳头瘤病毒感染诱发的疾病的疫苗中的用途，优选地，乳头瘤病毒感染诱发的疾病选自宫颈癌、阴道癌、阴唇癌、阴茎癌、肛门肛周癌、口咽癌、扁桃体癌和口腔癌，优选地，所述受试者为人受试者；
    优选地，所述乳头瘤病毒感染为一种或多种选自以下人乳头瘤病毒型别的感染：HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV68、HPV70、HPV73、HPV6、HPV11、HPV2、HPV5、HPV27和HPV57。
  8. 一种用于预防乳头瘤病毒感染和/或乳头瘤病毒感染诱发的疾病的疫苗，其包含权利要求1或2所述的人乳头瘤病毒嵌合蛋白或如权利要求6所述的多聚物、佐剂、以及疫苗用赋形剂或载体。
  9. 根据权利要求8所述的用于预防乳头瘤病毒感染和/或乳头瘤病毒感染诱发的疾病的疫苗，还包含至少一种嗜黏膜组和/或嗜皮肤组的HPV的病毒样颗粒或嵌合病毒样颗粒。
  10. 根据权利要求8或9所述的用于预防乳头瘤病毒感染和/或乳头瘤病毒感染诱发的疾病的疫苗，其中所述佐剂为人用佐剂。
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