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WO2019233415A1 - 一种人乳头瘤病毒39型l1蛋白的突变体 - Google Patents

一种人乳头瘤病毒39型l1蛋白的突变体 Download PDF

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WO2019233415A1
WO2019233415A1 PCT/CN2019/089988 CN2019089988W WO2019233415A1 WO 2019233415 A1 WO2019233415 A1 WO 2019233415A1 CN 2019089988 W CN2019089988 W CN 2019089988W WO 2019233415 A1 WO2019233415 A1 WO 2019233415A1
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protein
hpv39
hpv
type
wild
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PCT/CN2019/089988
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English (en)
French (fr)
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李少伟
王大宁
王致萍
柳欣林
张军
夏宁邵
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厦门大学
厦门万泰沧海生物技术有限公司
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Application filed by 厦门大学, 厦门万泰沧海生物技术有限公司 filed Critical 厦门大学
Priority to BR112020024694-8A priority Critical patent/BR112020024694A2/pt
Priority to JP2021517892A priority patent/JP2021526851A/ja
Priority to US15/734,715 priority patent/US11427618B2/en
Priority to EP19815471.8A priority patent/EP3805253A4/en
Priority to KR1020207038070A priority patent/KR20210018351A/ko
Publication of WO2019233415A1 publication Critical patent/WO2019233415A1/zh
Priority to JP2023146255A priority patent/JP2023182596A/ja

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    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N2710/20071—Demonstrated in vivo effect

Definitions

  • the invention relates to the fields of molecular virology and immunology.
  • the present invention relates to a mutant HPV39 L1 protein (or a variant thereof), a coding sequence and a preparation method thereof, and a virus-like particle including the same, which protein (or a variant thereof) and the virus-like particle are capable of inducing Neutralizing antibodies against at least two types of HPV (eg, HPV39 and HPV68, or HPV39, HPV68, and HPV70), which can be used to prevent the at least two types of HPV infection and diseases caused by the infection Examples include cervical cancer and genital warts.
  • the present invention also relates to the use of the above-mentioned protein and virus-like particles for the preparation of a pharmaceutical composition or vaccine, which can be used to prevent the at least two types of HPV infections and diseases caused by the infections Examples include cervical cancer and genital warts.
  • HPV Human Papillomavirus
  • HPV Human Papillomavirus
  • the high-risk HPV infection has been proven to be the main cause of genital cancers including cervical cancer in women.
  • the low-risk type mainly causes genital warts.
  • the most effective way to prevent and control HPV infection is to administer an HPV vaccine, especially a vaccine against high-risk HPV that can cause cervical cancer.
  • HPV VLP Virus-Like Particles
  • HPV VLP is a 20-hedral stereosymmetric structure consisting of pentamers of 72 major capsid proteins L1 (Doorbar, J. and P.H. Gallimore. 1987. J. Virol, 61 (9): 2793-9).
  • the structure of HPVVLP is highly similar to that of natural HPV. It retains most of the neutralizing epitopes of natural viruses and can induce high titer neutralizing antibodies (Kirnbauer, R., F.Booy, et al. 1992 Proc Natl Acad Sci U.S.A. 89 (24): 12180-4).
  • HPV VLP mainly induces neutralizing antibodies against homotype HPV, and produces protective immunity against homotype HPV, while there is only a low cross-protection effect between some types with high homology (Sara L. Bissett, Giada Mattiuzzo, et al. 2014 Vaccine. 32: 6548-6555). Therefore, the scope of protection of existing HPV vaccines is very limited. In general, one type of HPV VLP can only be used to prevent HPV infection of that type. In this case, if the scope of protection of the HPV vaccine is to be expanded, more types of HPV VLPs can only be added to the vaccine.
  • HPV vaccines currently on the market, including Merck's (It is a tetravalent vaccine against HPV 16, 18, 6, and 11) (Which is a bivalent vaccine against HPV 16, 18) and Merck's (It is a nine-valent vaccine against HPV 6, 11, 16, 18, 31, 33, 45, 52, 58), all of which are made by mixing multiple types of HPV VLPs.
  • Merck's It is a tetravalent vaccine against HPV 16, 18, 6, and 11
  • Which is a bivalent vaccine against HPV 16, 18
  • Merck's It is a nine-valent vaccine against HPV 6, 11, 16, 18, 31, 33, 45, 52, 58
  • HPV virus-like particles capable of inducing protective neutralizing antibodies against multiple types of HPV in order to more economically and effectively prevent multiple types of HPV infections and diseases such as cervical cancer And genital warts.
  • the present invention is based at least in part on the inventor's unexpected discovery that a specific segment in the human papilloma virus (HPV) type 39 L1 protein is replaced with a corresponding segment of the second type HPV (eg, HPV68) L1 protein Later, the obtained mutant HPV39 L1 protein can induce the body to produce high titer neutralizing antibodies against HPV39 and type II HPV (such as HPV68), and its protective effect is similar to that of mixed HPV39 and VLP and type II HPV.
  • VLP is equivalent
  • its protective effect against HPV68 is equivalent to that of HPV68 alone
  • its protective effect against the second type of HPV (eg, HPV68) is equivalent to that of HPV alone.
  • HPV39 L1 protein can be further replaced with the corresponding segment of the third type HPV (for example, HPV70) L1 protein.
  • HPV70 third type HPV
  • the mutant HPV39 L1 protein can induce the body to produce high titer neutralizing antibodies against HPV39, type II HPV (such as HPV68) and type III HPV (such as HPV70). Its protective effect is mixed with that of HPV39 VLP.
  • the second type of HPV and VLP are equivalent to the third type of HPV and VLP; and the protection effect against HPV39 is equivalent to that of HPV39 alone, and the protection effect against second type HPV (such as HPV68) is similar to that of separate HPV
  • the second type of HPV and VLP are equivalent, and the protection effect against the third type of HPV (for example, HPV70) is equivalent to that of the third type of HPV and VLP alone.
  • the present invention provides a mutant HPV39 L1 protein or a variant thereof, wherein the mutant HPV39 L1 protein has the following mutations compared to the wild-type HPV39 L1 protein:
  • N terminus is truncated by 1-25 amino acids, such as 1-5, 1-10, 1-15, 1-20, 5-15, 10-15, 10-20, 15-20 amino acids;
  • the variant differs from the mutant HPV39 L1 protein by only one or a few (e.g., one, two, three, four, five, six, seven, eight, or nine Amino acid substitutions (preferably conservative substitutions), additions or deletions, and the function of the mutated HPV39 L1 protein is retained, that is, capable of inducing HPV (for example, HPV39 and HPV68, or HPV39, HPV68 and HPV70).
  • the mutated HPV39 L1 protein optionally further has the following mutations:
  • the mutant HPV39 L1 protein is truncated by three, five, eight, ten, twelve, fifteen, and eighteen N-terminally compared to the wild-type HPV39 tL1 protein. , 20 or 22 amino acids. In certain preferred embodiments, the mutant HPV39 L1 protein is truncated by 15 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein.
  • the second type of wild-type HPV is HPV68.
  • the amino acid residues at the corresponding positions described in (2) are amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein.
  • the third type of wild-type HPV is HPV70.
  • the amino acid residues at the corresponding positions described in (3) (a) are amino acid residues at positions 117-141 of the wild-type HPV70 L1 protein.
  • the amino acid residues at the corresponding positions described in (3) (b) are the amino acid residues at positions 170-182 of the wild-type HPV70 L1 protein.
  • the amino acid residues at the corresponding positions described in (3) (c) are amino acid residues at positions 348-359 of the wild-type HPV70 L1 protein.
  • the wild-type HPV39 L1 protein has an amino acid sequence as shown in SEQ ID NO: 1.
  • the wild-type HPV68 L1 protein has an amino acid sequence as shown in SEQ ID NO: 2.
  • the wild-type HPV70 L1 protein has an amino acid sequence as shown in SEQ ID NO: 3.
  • sequence of amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein is shown in SEQ ID NO: 25.
  • sequence of amino acid residues at positions 117-141 of the wild-type HPV70 L1 protein is shown in SEQ ID NO: 26.
  • sequence of amino acid residues 170-182 of the wild-type HPV70 L1 protein is shown in SEQ ID NO: 27.
  • sequence of amino acid residues 348-359 of the wild-type HPV70 L1 protein is shown in SEQ ID NO: 28.
  • the mutated HPV39 L1 protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 10, 11 and 12.
  • the invention provides an isolated nucleic acid encoding a mutant HPV39 L1 protein or a variant thereof as described above.
  • the invention provides a vector comprising the isolated nucleic acid.
  • the isolated nucleic acid of the invention has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 19, 22, 23, and 24.
  • Vectors useful for inserting a polynucleotide of interest include, but are not limited to, cloning vectors and expression vectors.
  • the vector is, for example, a plasmid, cosmid, phage, and the like.
  • the invention also relates to a host cell comprising an isolated nucleic acid or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the host cells of the invention may also be cell lines, such as 293T and 293TT cells.
  • the present invention relates to an HPV virus-like particle, wherein the virus-like particle contains the mutant HPV39 L1 protein or a variant thereof of the invention, or is composed or formed of the mutant HPV39 L1 protein or a variant thereof of the invention .
  • the HPV virus-like particles of the present invention comprise a mutated HPV39 L1 protein, which is truncated by 1-25 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein, such as 1-5, 1-10, 1-15, 1-20, 5-15, 10-15, 10-20, 15-20 amino acids, such as 3, 5, 8, 11, 11 , 15, 18, 20, or 22 amino acids, and the amino acid residues at positions 269-288 of the wild-type HPV39 L1 protein were replaced with amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein.
  • a mutated HPV39 L1 protein which is truncated by 1-25 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein, such as 1-5, 1-10, 1-15, 1-20, 5-15, 10-15, 10-20, 15-20 amino acids, such as 3, 5, 8, 11, 11 , 15, 18, 20, or 22 amino acids, and the amino acid residues at positions 269-288 of the wild-type HPV
  • the HPV virus-like particles of the invention comprise a mutated HPV39 L1 protein, which is truncated by 1-25 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein, such as 1-05, 1-10, 1-15, 1-20, 5-15, 10-15, 10-20, 15-20 amino acids, such as 3, 5, 8, 11, 13 , 15, 18, 20, or 22 amino acids, and the amino acid residues at positions 269-288 of the wild-type HPV39 L1 protein were replaced with amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein, and are located at The amino acid residues at positions 117-140 of the wild-type HPV39 L1 protein were replaced with amino acid residues at positions 117-141 of the wild-type HPV70 L1 protein.
  • a mutated HPV39 L1 protein which is truncated by 1-25 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein, such as 1-05, 1-10, 1-15, 1
  • the HPV virus-like particles of the present invention comprise a mutated HPV39 L1 protein, which is truncated by 1-25 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein, such as 1-5, 1-10, 1-15, 1-20, 5-15, 10-15, 10-20, 15-20 amino acids, such as 3, 5, 8, 11, 11 , 15, 18, 20, or 22 amino acids, and the amino acid residues at positions 269-288 of the wild-type HPV39 L1 protein were replaced with amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein, and The amino acid residues at positions 169-181 of the wild-type HPV39 L1 protein were replaced with amino acid residues at positions 170-182 of the wild-type HPV70 L1 protein.
  • a mutated HPV39 L1 protein which is truncated by 1-25 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein, such as 1-5, 1-10, 1-15, 1-20,
  • the HPV virus-like particles of the present invention comprise a mutated HPV39 L1 protein, which is truncated by 1-25 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein, such as 1-5, 1-10, 1-15, 1-20, 5-15, 10-15, 10-20, 15-20 amino acids, such as 3, 5, 8, 11, 11 , 15, 18, 20, or 22 amino acids, and the amino acid residues at positions 269-288 of the wild-type HPV39 L1 protein were replaced with amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein, and The amino acid residues at positions 347-358 of the wild-type HPV39 L1 protein were replaced with amino acid residues at positions 348-359 of the wild-type HPV70 L1 protein.
  • mutated HPV39 L1 protein which is truncated by 1-25 amino acids at the N-terminus compared to the wild-type HPV39 L1 protein, such as 1-5, 1-10, 1-15, 1-20, 5-15
  • the HPV virus-like particle of the present invention comprises a mutated HPV39 L1 protein having the sequences shown in SEQ ID NOs: 7, 10, 11 and 12.
  • the present invention also relates to a composition
  • a composition comprising the mutant HPV39 L1 protein or a variant thereof, or the isolated nucleic acid or vector or host cell or HPV virus-like particle described above.
  • the composition comprises a mutant HPV39 L1 protein of the invention or a variant thereof.
  • the composition comprises an HPV virus-like particle of the invention.
  • the invention also relates to a pharmaceutical composition or vaccine comprising the HPV virus-like particles of the invention, optionally further comprising a pharmaceutically acceptable carrier and / or excipient.
  • the pharmaceutical composition or vaccine of the present invention can be used to prevent HPV infection or diseases caused by HPV infection such as cervical cancer and genital warts.
  • the HPV virus-like particles are present in an effective amount to prevent HPV infection or a disease caused by HPV infection.
  • the HPV infection is one or more types of HPV infection (eg, HPV39 infection, HPV68 infection, and / or HPV70 infection).
  • the disease caused by HPV infection is selected from cervical cancer and genital warts.
  • composition or vaccine of the present invention can be administered by methods known in the art, such as, but not limited to, oral administration or injection.
  • a particularly preferred mode of administration is injection.
  • the pharmaceutical composition or vaccine of the invention is administered in a unit dosage form.
  • the amount of HPV virus-like particles contained in each unit dose is 5 ⁇ g to 80 ⁇ g, preferably 20 ⁇ g to 40 ⁇ g.
  • the present invention relates to a method for preparing a mutant HPV39 L1 protein or a variant thereof as described above, which comprises expressing the mutant HPV39 L1 protein or a variant thereof in a host cell, and The mutant HPV39 L1 protein or a variant thereof is recovered from the host cell culture.
  • the host cell is E. coli.
  • the method comprises the steps of: expressing the mutant HPV39 L1 protein or a variant thereof in E. coli, and then purifying the lysed supernatant of the E. coli to obtain the mutant HPV39 L1 protein or a variant thereof.
  • the mutant HPV39L1 is recovered from the lysed supernatant of the E. coli by chromatography (e.g., cation exchange chromatography, hydroxyapatite chromatography, and / or hydrophobic interaction chromatography). Protein or a variant thereof.
  • the invention in another aspect, relates to a method of preparing a vaccine comprising mixing an HPV virus-like particle of the invention with a pharmaceutically acceptable carrier and / or excipient.
  • the present invention relates to a method for preventing HPV infection or a disease caused by HPV infection, comprising administering to a subject a prophylactically effective amount of an HPV virus-like particle or a pharmaceutical composition or vaccine according to the present invention.
  • the HPV infection is one or more types of HPV infections (eg, HPV39 infection, HPV68 infection, and / or HPV70 infection).
  • the diseases caused by HPV infection include, but are not limited to, cervical cancer and genital warts.
  • the subject is a mammal, such as a human.
  • the present invention also relates to the use of the mutated HPV39 L1 protein or a variant thereof or HPV virus-like particles of the present invention in the preparation of a pharmaceutical composition or vaccine for preventing HPV infection or Diseases caused by HPV infection.
  • the HPV infection is one or more types of HPV infections (eg, HPV39 infection, HPV68 infection, and / or HPV70 infection).
  • the diseases caused by HPV infection include, but are not limited to, cervical cancer and genital warts.
  • the present invention also relates to the mutated HPV39 L1 protein of the present invention or a variant or HPV virus-like particle thereof for use in preventing HPV infection or a disease caused by HPV infection.
  • the HPV infection is one or more types of HPV infections (eg, HPV39 infection, HPV68 infection, and / or HPV70 infection).
  • the diseases caused by HPV infection include, but are not limited to, cervical cancer and genital warts.
  • wild type HPV of the second type refers to another type of wild type HPV different from HPV39.
  • the wild type HPV of the second type is preferably wild type HPV68.
  • wild-type HPV of the third type refers to another type of wild-type HPV different from HPV39 and different from the wild-type HPV of the second type.
  • the third type of wild-type HPV is preferably wild-type HPV70.
  • the expression "corresponding position” refers to the equivalent position in the compared sequences when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity.
  • wild-type HPV39 L1 protein refers to the major capsid protein L1 naturally occurring in human papillomavirus type 39 (HPV39).
  • HPV39 human papillomavirus type 39
  • the sequence of the wild-type HPV39 L1 protein is well known in the art and can be found in various public databases (for example, NCBI database accession number P24838.1, ARQ82617.1, AGU90549.1, and AEP23084.1).
  • amino acid sequence of the wild-type HPV39 L1 protein when referring to the amino acid sequence of the wild-type HPV39 L1 protein, the description is made with reference to the sequence shown in SEQ ID NO: 1.
  • amino acid residues 53-61 of the wild-type HPV39 L1 protein refers to amino acid residues 53-61 of the polypeptide represented by SEQ ID NO: 1.
  • wild-type HPV39 may include a variety of isolates, and there may be differences between the amino acid sequences of the L1 proteins of the various isolates.
  • wild-type HPV39 L1 protein includes not only the protein shown in SEQ ID NO: 1, but also the L1 protein of various HPV39 isolates (for example, P24838.1, ARQ82617.1, AGU90549. 1 or HPV39 L1 protein as shown in AEP23084.1).
  • sequence fragments of the wild-type HPV39 L1 protein when describing the sequence fragments of the wild-type HPV39 L1 protein, it includes not only the sequence fragments of SEQ ID NO: 1 but also the corresponding sequence fragments in the L1 protein of various HPV39 isolates.
  • amino acid residues 53-61 of the wild-type HPV39 L1 protein includes amino acid residues 53-61 of SEQ ID NO: 1 and corresponding fragments in the L1 protein of various HPV39 isolates.
  • wild-type HPV68 L1 protein refers to the major capsid protein L1 naturally occurring in human papillomavirus type 68 (HPV68).
  • HPV68 human papillomavirus type 68
  • the sequence of the wild-type HPV68L1 protein is well known in the art and can be found in various public databases (for example, NCBI database accession numbers AAZ39498.1, AGU90717.1, P4669.1, and AGU90703.1).
  • amino acid sequence of the wild-type HPV68 L1 protein when referring to the amino acid sequence of the wild-type HPV68 L1 protein, the description is made with reference to the sequence shown in SEQ ID NO: 2.
  • amino acid residues 53 to 61 of the wild-type HPV68 L1 protein refers to amino acid residues 53 to 61 of the polypeptide represented by SEQ ID NO: 2.
  • wild-type HPV68 may include a variety of isolates, and there may be differences between the amino acid sequences of the L1 proteins of the various isolates.
  • wild-type HPV68 L1 protein includes not only the protein shown in SEQ ID NO: 2 but also the L1 protein of various HPV68 isolates (for example, AAZ39498.1, AGU90717.1, P4669. 1 or HPV68 L1 protein shown in AGU90703.1).
  • sequence fragments of the wild-type HPV68 L1 protein when describing the sequence fragments of the wild-type HPV68 L1 protein, it includes not only the sequence fragments of SEQ ID NO: 2 but also the corresponding sequence fragments in the L1 protein of various HPV68 isolates.
  • amino acid residues 53-61 of the wild-type HPV68 L1 protein includes amino acid residues 53-61 of SEQ ID NO: 2 and corresponding fragments in the L1 protein of various HPV68 isolates.
  • wild-type HPV70 L1 protein refers to the major capsid protein L1 naturally present in human papillomavirus type 70 (HPV70).
  • HPV70 human papillomavirus type 70
  • sequence of the wild-type HPV70 L1 protein is well known in the art and can be found in various public databases (for example, NCBI database accession numbers AGU90846.1, AGU90854.1, AAC54879.1, and P50793.1).
  • amino acid sequence of the wild-type HPV70 L1 protein when referring to the amino acid sequence of the wild-type HPV70 L1 protein, the description is made with reference to the sequence shown in SEQ ID NO: 3.
  • amino acid residues 117-141 of the wild-type HPV70 L1 protein means amino acid residues 117-141 of the polypeptide represented by SEQ ID NO: 3.
  • wild-type HPV70 may include a variety of isolates, and there may be differences between the amino acid sequences of the L1 proteins of the various isolates.
  • wild-type HPV70 L1 protein includes not only the protein shown in SEQ ID NO: 3, but also the L1 protein of various HPV70 isolates (for example, AGU90846.1, AGU90854.1, AAC54879. 1 or HPV70 L1 protein shown in P50793.1).
  • sequence fragments of the wild-type HPV70 L1 protein when describing the sequence fragments of the wild-type HPV70 L1 protein, it includes not only the sequence fragments of SEQ ID NO: 3, but also the corresponding sequence fragments in the L1 protein of various HPV70 isolates.
  • amino acid residues 117-141 of the wild-type HPV70 L1 protein includes amino acid residues 117-141 of SEQ ID NO: 3 and corresponding fragments in the L1 protein of various HPV70 isolates.
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percent identity, the compared sequences are located at equivalent positions Fragment.
  • the expression "the N-terminus is truncated by X amino acids” means that the methionine residue encoded by the start codon (for initiating protein translation) is used to replace the amino acids 1-X at the N-terminus of the protein. Residues.
  • HPV39 L1 protein truncated by 15 amino acids at the N-terminus is obtained by replacing the amino acid residues 1 to 15 at the N-terminus of the wild-type HPV39 L1 protein with a methionine residue encoded by the start codon. protein.
  • the term "variant” refers to a protein whose amino acid sequence is compared with the amino acid sequence of the mutated HPV39 L1 protein (such as the proteins shown by SEQ ID NO: 7, 10, 11, and 12) of the present invention. , Substitutions (preferably conservative substitutions), additions or deletions with one or several (eg, 1, 2, 3, 4, 5, 5, 7, 7, 8 or 9) amino acids, or Has at least 90%, 95%, 96%, 97%, 98%, or 99% identity, and it retains the function of the mutated HPV39 L1 protein.
  • the term "function of mutated HPV39 L1 protein” refers to the ability to induce the body to produce neutralizing antibodies against at least two types of HPV (e.g., HPV39 and HPV68, or HPV39, HPV68, and HPV70).
  • HPV39 and HPV68, or HPV39, HPV68, and HPV70 are a measure of the similarity of a nucleotide sequence or an amino acid sequence. Sequences are usually arranged for maximum matching. "Identity” itself has a well-known meaning in the art and can be calculated using published algorithms such as BLAST.
  • the term "identity" is used to refer to a sequence match between two polypeptides or between two nucleic acids.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or two Each position of the polypeptide is occupied by lysine)
  • the molecules are identical at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of compared positions x 100. For example, if 6 of the 10 positions of two sequences match, the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 positions out of a total of 6 positions match).
  • comparisons are made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc).
  • Align program DNAstar, Inc.
  • the algorithm of E.Meyers and W.Miller Comput.Appl.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithm integrated into the GAP program of the GCG software package can be used, using the Blossom 62 matrix or PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the necessary properties of a protein / polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residue (e.g., have similar size, shape, charge, chemical properties, including The ability to form covalent or hydrogen bonds, etc.).
  • a family of amino acid residues with similar side chains has been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and uncharged polar side chains (e.g., glycine). , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • a conservative substitution generally refers to replacing the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12 (10): 879-884 (1999) ; And Burks et al. Proc. Natl. Acad. Set USA 94: 412-417 (1997), which is incorporated herein by reference).
  • E. coli expression system refers to an expression system composed of E. coli (strain) and a vector, wherein E. coli (strain) is derived from a commercially available strain, such as, but not limited to: ER2566, BL21 ( DE3), B834 (DE3), BLR (DE3).
  • the term "vector” refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements carried by the vector can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phages; cosmids and the like.
  • the term "pharmaceutically acceptable carrier and / or excipient” refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient and is in the art It is well known (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th Ed., Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80; adjuvants include, but are not limited to, aluminum adjuvants (such as hydroxide Aluminum), Freund's adjuvant (eg, complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.
  • surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80
  • adjuvants include, but are not limited to, aluminum adjuvants (such as hydroxide Aluminum), Freund's adjuvant (eg, complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.
  • an effective amount refers to an amount effective to achieve the intended purpose.
  • an effective amount for preventing a disease e.g., HPV infection
  • an effective amount effective to prevent, prevent, or delay the occurrence of a disease e.g., HPV infection. Determining such an effective amount is well within the capabilities of those skilled in the art.
  • chromatographic chromatography includes, but is not limited to: ion exchange chromatography (such as cation exchange chromatography), hydrophobic interaction chromatography, adsorption chromatography (such as hydroxyapatite chromatography), gel filtration (gel chromatography Resistance) chromatography, affinity chromatography.
  • the term "lysed supernatant” refers to a solution produced by the steps of: disrupting a host cell (eg, E. coli) in a lysate, and then insoluble matter in the lysate containing the disrupted host cell Remove.
  • a host cell eg, E. coli
  • Various lysates are well known to those skilled in the art, including but not limited to Tris buffer, phosphate buffer, HEPES buffer, MOPS buffer and the like.
  • host cell disruption can be achieved by various methods well known to those skilled in the art, including, but not limited to, homogenizer disruption, homogenizer disruption, ultrasonic treatment, grinding, high-pressure extrusion, lysozyme treatment, and the like. Methods for removing insolubles in the lysate are also well known to those skilled in the art, including but not limited to filtration and centrifugation.
  • the present invention provides a mutated HPV39 L1 protein and HPV virus-like particles formed therefrom.
  • the HPV virus-like particles of the present invention can provide significant cross-protection capabilities between HPV39 and other types of HPV (such as HPV68 and HPV70).
  • the HPV virus-like particles of the present invention can induce the body to produce high titer neutralizing antibodies against at least two types of HPV (eg, HPV39 and HPV68, or HPV39, HPV68 and HPV70), And the effect is equivalent to a mixture of multiple types of HPV and VLP (for example, a mixture of HPV39 and HPV68VLP, or a mixture of HPV39 and VLP, HPV68 and HPV70).
  • the HPV virus-like particles of the present invention can be used to prevent infection of at least two types of HPV (for example, HPV39 and HPV68, or HPV39, HPV68, and HPV70) and related diseases at the same time, and have significant advantageous technical effects. .
  • HPV39 and HPV68, or HPV39, HPV68, and HPV70 are types of HPV
  • HPV70 HPV39, HPV68, and HPV70
  • FIG. 1 shows the results of SDS polyacrylamide gel electrophoresis of the purified mutein in Example 1.
  • FIG. Lane 1 Protein molecular weight marker
  • Lane 2 HPV39N15 (HPV39 L1 protein with N-terminal truncated 15 amino acids);
  • Lane 3 HPV68N0 (HPV68 L1 protein with N-terminal truncated 0 amino acids, that is, the full-length wild type HPV68 L1 protein);
  • lane 4 H39N15-68T1; lane 5: H39N15-68T2;
  • lane 6 H39N15-68T3;
  • lane 7 H39N15-68T4;
  • lane 8 H39N15-68T5;
  • lane 9 protein molecular weight marker;
  • lane 10 H39N15-68T4;
  • Lane 11 HPV70N10 (HPV70L1 protein with N-terminal truncated by 10 amino acids);
  • Lane 12 H39N15-68T
  • Figure 2 shows the detection of the mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, and broad-spectrum antibody 4B3. Results of Western blot detection of H39N15-68T4-70S3 and H39N15-68T4-70S5.
  • Lane 1 HPV39N15; Lane 2: HPV68N0; Lane 3: H39N15-68T1; Lane 4: H39N15-68T2; Lane 5: H39N15-68T3; Lane 6: H39N15-68T4; Lane 7: H39N15-68T5; Lane 8: H39N15-68 68T4; lane 9: HPV70N10; lane 10: H39N15-68T4-70S1; lane 11: H39N15-68T4-70S2; lane 12: H39N15-68T4-70S3; lane 13: H39N15-68T4-70S5.
  • mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, H39N15-68T4-70S5 are all able to Specific recognition by broad-spectrum antibody 4B3.
  • Figure 3 shows the results of molecular sieve chromatography analysis of samples containing proteins HPV39N15, H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5.
  • the results showed that the first protein peaks in the samples containing proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, and H39N15-68T5 were all about 13-14min, which was equivalent to HPV39N15. This indicates that these proteins can be assembled into VLPs.
  • Figure 4 shows the results of molecular sieve analysis of samples containing the proteins HPV39N15, HPV68L1N0, HPV70N10, H39N15-68T4, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, H39N15-68T4-70S5.
  • Figures 5A-5L show HPV39N15, VLP, HPV68L1N0, VLP, and HPV70N10, VLP and H39N15-68T1, VLP, H39N15-68T2, VLP, H39N15-68T3, VLP, H39N15-68T4, VLP, H39N15-68T5N1, V5 68T4-70S2 VLP, H39N15-68T4-70S3 VLP, H39N15-68T4-70S5 VLP settlement result analysis.
  • Figure 5A HPV39N15, VLP; Figure 5B, HPV68L1N0, VLP; Figure 5C, H39N15-68T1, VLP; Figure 5D, H39N15-68T2, VLP; Figure 5E, H39N15-68T3, VLP; Figure 5F, H39N15-68T4, VLP; Figure 5G -68T5 VLP; Figure 5H, HPV70N10 VLP; Figure 5I, H39N15-68T4-70S1 VLP; Figure 5J, H39N15-68T4-70S2 VLP; Figure 5K, H39N15-68T4-70S3 VLP; Figure 5L, H39N15-68T4-70S5 VLP.
  • mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S5 and H39N15-68T4-70S5 can each Assembled into virus-like particles of similar size and shape to wild-type VLPs (HPV39N15, VLP, 115S; HPV68N0, VLP, 153S; HPV70N10, VLP, 144S).
  • Figure 6A VLP assembled by HPV39N15
  • Figure 6B VLP assembled by HPV68L1N0
  • Figure 6C VLP assembled by HPV70N10
  • Figure 6D VLP assembled by H39N15-68T1
  • Figure 6E VLP assembled by H39N15-68T2
  • Figure 6F VLP assembled by H39N15-68T3
  • Figure 6G VLP assembled by H39N15-68T4
  • Figure 6H VLP assembled by H39N15-68T5
  • Figure 6I VLP assembled by H39N15-68T4-70S1
  • Figure 6J VLP assembled from H39N15-68T4-70S2
  • Figure 6K VLP assembled from H39N15-68T4-70S3
  • Figure 6L VLP assembled from H39N15-68T4-70S5.
  • FIGS. 7A-7C show the evaluation results of neutralizing antibody titers in mouse serum after immunizing mice with H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, and H39N15-68T5 VLP.
  • Figure 7A Aluminum adjuvant group 1 (immunized dose of 5 ⁇ g, using aluminum adjuvant);
  • Figure 7B Aluminum adjuvant group 2 (immunized dose of 1 ⁇ g, using aluminum adjuvant);
  • Figure 7C Aluminum adjuvant group 3 (immunization The dose was 0.2 ⁇ g, using an aluminum adjuvant).
  • H39N15-68T4 VLP can induce high titer neutralizing antibodies against HPV39 in mice, although its protective effect is slightly weaker than the same dose of HPV39N15 VLP alone, but it is significantly better than the same dose of HPV68N0 VLP alone; And it can induce mice to produce high titer neutralizing antibodies against HPV68. Although its protective effect is slightly weaker than the same dose of HPV68N0 VLP alone, it is significantly better than the same dose of HPV39N15 VLP alone. This indicates that H39N15-68T4 VLP has good cross-immunogenicity and cross-protection against HPV39 and HPV68.
  • Figures 8A-8C show the neutralizing antibody titers in mouse serum after immunizing mice with H39N15-68T4-70S1 VLP, H39N15-68T4-70S2, VLP, H39N15-68T4-70S3, VLP, and H39N15-68T4-70S5. Evaluation results.
  • Figure 8A Aluminum adjuvant group 1 (immunization dose is 5 ⁇ g, using aluminum adjuvant);
  • Figure 8B Aluminum adjuvant group 2 (immunization dose is 1 ⁇ g, using aluminum adjuvant);
  • Figure 8C Aluminum adjuvant group 3 (immunization The dose was 0.2 ⁇ g, using an aluminum adjuvant).
  • H39N15-68T4-70S2, H39N15-68T4-70S3, H39N15-68T4-70S5, VLP can induce mice to produce high titers of neutralizing antibodies against HPV39, although its protective effect is slightly weaker than the same dose of HPV39N15 alone VLP and mixed HPV39 / HPV68 / HPV70 VLP, but still significantly better than the same dose of HPV68N0 VLP or HPV70N10 VLP alone; and it can induce mice to produce high titers of neutralizing antibodies against HPV68, its protective effect It is equivalent to the same dose of HPV68N0 VLP alone and mixed HPV39 / HPV68 / HPV70 VLP, and is significantly better than the same dose of HPV39N15 VLP alone or HPV70N10 VLP alone; and it can induce mice to produce high titers against HPV70
  • the protective effect of the neutralizing antibody is equivalent to that of HPV70N10 and VLP alone and mixed HPV39
  • H39N15-68T4-70S2, VLP, H39N15-68T4-70S3, VLP, H39N15-68T4-70S5, VLP have good cross-immunogenicity and cross-protection against HPV39, HPV68, and HPV70.
  • Figure 9 shows HPV39N15 VLP, HPV68N0 VLP, HPV70N10 VLP and H39N15-68T4 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S5 thermal stability test results of VLP.
  • A is the thermal stability test result of HPV39N15 VLP
  • B is the thermal stability test result of HPV68L1N0 VLP
  • C is the thermal stability test result of HPV70N10 VLP
  • D is the thermal stability test result of H39N15-68T4 VLP
  • E is H39N15-68T4-70S2 VLP thermal stability test results
  • F is H39N15-68T4-70S5 VLP thermal stability test results.
  • the results show that the VLPs formed by each protein have extremely high thermal stability.
  • Figure 10 shows the results of cryo-electron microscopy of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP and their reconstructed three-dimensional structures.
  • A is a frozen electron microscope image of H39N15-68T4-70S2 VLP;
  • B is a reconstructed three-dimensional structure of VLP of H39N15-68T4-70S2;
  • C is a frozen electron microscope image of H39N15-68T4-70S5 VLP;
  • D is a H39N15-68T4-70S5 VLP Reconstructed three-dimensional structure.
  • all constituent subunits in the VLP structure of H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 are pentamers without hexamers, and
  • the outermost diameter of the two types of VLPs is about 55 nm.
  • Sequence 7 (SEQ ID NO: 7):
  • Sequence 8 (SEQ ID NO: 8):
  • Sequence 9 (SEQ ID NO: 9):
  • the molecular biology experimental methods and immunoassays used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compilation of a Guide to Molecular Biology Experiments, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes was in accordance with conditions recommended by the product manufacturer.
  • restriction enzymes was in accordance with conditions recommended by the product manufacturer.
  • a multi-point mutation PCR reaction was used to construct an expression vector encoding the HPV39 L1 protein containing a mutation derived from the HPV68 L1 protein segment.
  • the initial template used was the pTO-T7-HPV39N15C plasmid (which encodes a truncated N-terminus 15 amino acid HPV39 L1 protein; abbreviated 39L1N15 in Table 2).
  • the templates and primers used for each PCR reaction are shown in Table 2, and the amplification conditions for the PCR reaction are set to: 94 ° C denaturation for 10 minutes; 25 cycles (94 ° C denaturation for 50 seconds, specified temperature annealing for a certain time, 72 ° C extension) 7 minutes and 30 seconds); lastly extended at 72 ° C for 10 minutes.
  • the annealing temperatures and times are listed in Table 2.
  • the specific sequences of the PCR primers used are listed in Table 3.
  • coli was coated on a solid LB medium containing kanamycin (final concentration 25 ⁇ g / mL, hereinafter the same) (LB medium components: 10 g / L peptone, 5 g / L yeast powder, 10 g / L chlorine Sodium sulphate, the same below), and allowed to stand at 37 ° C for 10-12 hours until a single colony is clearly distinguishable.
  • a single colony was picked into a test tube containing 4 mL of liquid LB medium (containing kanamycin), and cultured with shaking at 220 rpm at 37 ° C for 10 hours. Subsequently, 1 mL of the bacterial solution was stored at -70 ° C.
  • the plasmid was extracted from E.
  • the nucleotide sequence of the target fragment inserted in the plasmid was sequenced using T7 primers.
  • the sequencing results showed that the nucleotide sequence of the target fragment inserted in the constructed plasmid (expression vector) was SEQ ID NO: 16, and the encoded amino acid sequence was SEQ ID NO: 4 (the corresponding protein was named H39N15-68T1 ).
  • the difference between the mutant proteins H39N15-68T1 and HPV39N15 is that the amino acid residues at positions 53-61 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 53-61 of the wild-type HPV68 L1 protein.
  • the initial template used includes pTO-T7-HPV39N15 plasmid (which encodes HPV39L1 protein with N-terminal truncation of 15 amino acids; abbreviated as 39L1N15 in Table 2), pTO-T7-HPV68L1 plasmid (which encodes HPV68 L1 protein; Abbreviated as 68L1N0 in Table 2), pTO-T7-H39N15-68T4 plasmid (which encodes the mutant protein H39N15-68T4; abbreviated as H39N15-68T4 in Table 2) and pTO-T7-HPV70N10 plasmid (which encodes a truncated N-terminus) 10 amino acid HPV70 L1 protein; abbreviated 70L1N10 in Table 2).
  • the templates and primers used for each PCR reaction are shown in Table 2, and the amplification conditions for PCR reactions used to amplify short fragments are set as follows: denaturation at 94 ° C for 10 minutes; 25 cycles (denaturation at 94 ° C for 50 seconds, annealing at the specified temperature) For a certain period of time, 72 ° C is extended for 1 minute); finally, 72 ° C is extended for 10 minutes.
  • the amplification conditions for PCR reactions used to amplify long fragments were set as follows: denaturation at 94 ° C for 10 minutes; 25 cycles (denaturation at 94 ° C for 50 seconds, annealing at a specified temperature for a certain time, and extension at 72 ° C for 7 minutes and 30 seconds); finally 72 ° C Extend for 10 minutes.
  • the specific sequences of the PCR primers used are listed in Table 3.
  • the amplified product was subjected to electrophoresis, and then a target fragment was recovered using a DNA recovery kit (BEYOTIME, article number: D0033) and its concentration was measured.
  • the amplified short and long fragments were mixed at a molar ratio of 2: 1 (total volume 3 ⁇ L), and then 3 ⁇ L 2X Gibson Assembly Premix Reagent (2X Gibson Assembly Master Mix, purchased from NEB, including T5 exonuclease, Phusion DNA polymerase, DNA polymerase, and reaction at 50 ° C for 1 hour.
  • 2X Gibson Assembly Master Mix purchased from NEB, including T5 exonuclease, Phusion DNA polymerase, DNA polymerase, and reaction at 50 ° C for 1 hour.
  • the assembled product (6 ⁇ L) was used to transform 40 ⁇ L of competent E. coli ER2566 (purchased from New England Biolabs) prepared by the calcium chloride method.
  • the transformed E. coli was spread on a kanamycin-containing solid LB medium, and cultured at 37 ° C for 10-12 hours until a single colony was clearly distinguishable.
  • a single colony was picked into a test tube containing 4 mL of liquid LB medium (containing kanamycin), and cultured with shaking at 220 rpm at 37 ° C for 10 hours. Subsequently, 1 mL of the bacterial solution was stored at -70 ° C.
  • the plasmid was extracted from E.
  • nucleotide sequence of the target fragment inserted in the plasmid was sequenced using T7 primers.
  • the sequencing results showed that the nucleotide sequences of the target fragments inserted in each of the constructed plasmids (expression vectors) were SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, and the encoded amino acid sequences SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12 (the corresponding proteins are named H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, H39N15-68T4-70S5).
  • mutant proteins H39N15-68T2 and HPV39N15 are that the amino acid residues at positions 117-150 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 117-151 of the wild-type HPV68 L1 protein.
  • mutant protein H39N15-68T3 and HPV39N15 is that the amino acid residues at positions 169-181 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 170-182 of the wild-type HPV68 L1 protein.
  • mutant proteins H39N15-68T4 and HPV39N15 are that the amino acid residues at positions 269-288 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein.
  • mutant proteins H39N15-68T5 and HPV39N15 are that the amino acid residues at positions 347-358 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 348-359 of the wild-type HPV68 L1 protein.
  • mutant protein H39N15-68T4-70S1 and HPV39N15 is that the amino acid residues at positions 269-288 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein and are located in the wild type
  • amino acid residues 53-61 of the HPV39 L1 protein were replaced with amino acid residues 53-61 of the wild-type HPV70 L1 protein.
  • mutant protein H39N15-68T4-70S2 and HPV39N15 is that the amino acid residues at positions 269-288 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein and are located in the wild type
  • amino acid residues at positions 117-140 of the HPV39 L1 protein were replaced with amino acid residues at positions 117-141 of the wild-type HPV70 L1 protein.
  • mutant protein H39N15-68T4-70S3 and HPV39N15 The difference between the mutant protein H39N15-68T4-70S3 and HPV39N15 is that the amino acid residues at positions 269-288 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein and are located in the wild type.
  • the amino acid residues at positions 169-181 of the HPV39 L1 protein were replaced with amino acid residues at positions 170-182 of the wild-type HPV70 L1 protein.
  • mutant protein H39N15-68T4-70S5 and HPV39N15 The difference between the mutant protein H39N15-68T4-70S5 and HPV39N15 is that the amino acid residues at positions 269-288 of the wild-type HPV39 L1 protein are replaced with amino acid residues at positions 270-289 of the wild-type HPV68 L1 protein and are located in the wild-type The amino acid residues 347-358 of HPV39 L1 protein were replaced with amino acid residues 348-359 of wild-type HPV70 L1 protein.
  • the cells obtained above were resuspended in a proportion of 1 g of cells corresponding to 10 mL of lysate (20 mM Tris buffer, pH 7.2, 300 mM NaCl).
  • the bacterial cells were disrupted with an ultrasonic instrument for 30 min.
  • the lysate containing the disrupted bacterial cells was centrifuged at 13,500 rpm (30000 g) for 15 min, and the supernatant was taken (that is, the bacterial cell broken supernatant).
  • Instrument system AKTA explorer 100 preparative liquid chromatography system produced by GE Healthcare (formerly Amershan Pharmacia).
  • Chromatographic media SP Sepharose 4 Fast Flow (GE Healthcare), CHT-II (purchased from Bio-RAD) and Butyl Sepharose 4 Fast Flow (GE Healthcare).
  • Buffer Buffer A (20 mM phosphate buffer, pH 8.0, 20 mM DTT); and Buffer B (20 mM phosphate buffer, pH 8.0, 20 mM DTT, 2M NaCl).
  • Buffer A (20 mM phosphate buffer, pH 8.0, 20 mM DTT)
  • Buffer B (20 mM phosphate buffer, pH 8.0, 20 mM DTT, 2M NaCl).
  • the buffers containing different concentrations of NaCl used in the following elution procedure are prepared by mixing buffers A and B in proportion.
  • step (3) Take 150 ⁇ L of the eluted fraction obtained in step (3), add 30 ⁇ L of 6X Loading Buffer (1M contains 1M TB, 6.8300ml, 100% glycerol 600ml, SDS 120g, bromophenol blue 6g, ⁇ -mercaptoethanol 50ml) and mix well And incubate for 10 min in a water bath at 80 ° C. Then 10 ⁇ l of the sample was electrophoresed in a 10% SDS-polyacrylamide gel at 120 V for 120 min; then electrophoretic bands were displayed by Coomassie blue staining. The electrophoresis results are shown in FIG. 1.
  • 6X Loading Buffer 1M contains 1M TB, 6.8300ml, 100% glycerol 600ml, SDS 120g, bromophenol blue 6g, ⁇ -mercaptoethanol 50ml
  • HPV39N15 protein was prepared and purified using E. coli and pTO-T7-HPV39N15 plasmid;
  • HPV68N0 protein was prepared and purified using E. coli and pTO-T7-HPV68L1N0 plasmid;
  • E. coli and pTO-T7-HPV70N10 plasmid were used And purified HPV70N10 protein.
  • H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, H39N15-68T4- The proteins were electrophoresed. After the end of the electrophoresis, Western Blot detection was performed using a broad-spectrum antibody 4B3 against HPV L1 protein, and the results are shown in FIG. 2.
  • H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, H39N15-68T4-70S5 are all capable of Spectrum antibody 4B3 specifically recognizes.
  • Example 2 Assembly and particle morphology detection of HPV virus-like particles
  • HPV39N15, HPV68N0 and HPV70N10 proteins were assembled into HPV39N15, VLP, HPV68N0, VLP and HPV70N10, respectively.
  • the instrument used for sedimentation rate analysis is a Beckman XL-A analytical ultracentrifuge, which is equipped with an optical detection system and An-50Ti and An-60Ti rotors.
  • mutant proteins H39N15-68T1, H39N15-68T2, H39N15-68T3, H39N15-68T4, H39N15-68T5, H39N15-68T4-70S1, H39N15-68T4-70S2, H39N15-68T4-70S3, and H39N15-68T4- 70S5 can each be assembled into virus-like particles of similar size and shape to wild-type VLPs (HPV39N15, VLP, 115S; HPV68N0, VLP, 153S; HPV70N10, VLP, 144S).
  • a 100 ⁇ L sample containing VLP was observed by transmission electron microscopy.
  • the instrument used is a 100kV transmission electron microscope produced by Japan Electronics Corporation, with a magnification of 100,000 times. Briefly, a 13.5 ⁇ L sample was taken, negatively stained with 2% phosphotungstic acid, pH 7.0, and fixed on a carbon sprayed copper mesh, followed by transmission electron microscope observation. The observation results are shown in Figs. 6A to 6L.
  • Virus-like particles are assembled, and the virus-like particles assembled by these mutant proteins are uniform in size, and the radius of the particles is about 25-30nm.
  • the virus-like particles assembled from wild-type HPV39N15, HPV68N0, and HPV70N10 also have a radius of about 25-30 nm and are uniform in size. This indicates that these mutant proteins are similar to the L1 proteins of wild-type HPV39, HPV68, and HPV70, and can form VLPs of uniform size.
  • Example 3 Evaluation of neutralizing antibody titers in mouse serum after immunization with virus-like particles 1
  • virus-like particles used were: H39N15-68T1 VLP, H39N15-68T2 VLP, H39N15-68T3 VLP, H39N15-68T4 VLP, and H39N15-68T5 VLP.
  • mice (6-week-old BalB / c female mice) were divided into 3 groups: aluminum adjuvant group 1 (immunized dose of 5 ⁇ g, using aluminum adjuvant), aluminum adjuvant group 2 (immunized dose of 1 ⁇ g, using Aluminum adjuvant), and aluminum adjuvant group 3 (immunized dose of 0.2 ⁇ g, using aluminum adjuvant).
  • Each group was further subdivided into 8 subgroups.
  • Control subgroups 1 and 2 were immunized with HPV39N15 and HPV68N0 VLP, respectively.
  • Control subgroup 3 was mixed with HPV39 / HPV68 and VLP (that is, HPV39N15 and HPV68N0).
  • mice / subgroup were immunized by intraperitoneal injection, the immunization doses were 5 ⁇ g, 1 ⁇ g and 0.2 ⁇ g, respectively, and the injection volume was 1 ml. All mice were initially immunized at week 0 and then boosted once at weeks 2 and 4 each. Orbital blood was collected from the mice at week 8 and the serum titers of anti-HPV39 and HPV68 antibodies were analyzed. The analysis results are shown in Figures 7A-7C.
  • H39N15-68T4 VLP can induce high titer neutralizing antibodies against HPV39 in mice, and its protective effect is slightly weaker than the same dose of HPV39N15 VLP alone, which is significantly better than the same dose of HPV68N0 VLP alone; and Can induce mice to produce high titers of neutralizing antibodies against HPV68, its protection effect is slightly weaker than the same dose of HPV68N0 VLP alone, significantly better than the same dose of HPV39N15 VLP alone. This indicates that H39N15-68T4 VLP has good cross-immunogenicity and cross-protection against HPV39 and HPV68.
  • virus-like particles used were H39N15-68T4 VLP.
  • mice were immunized in each group, and in the fifth week after immunization, ocular venous blood was drawn to detect HPV antibodies in the serum, and the Reed-Muench method (Reed LJ MH.A simple method of estimating fifty percent endpoints. Am J Hyg.1938; 27: 493-7) calculates ED induce seroconversion of each sample (i.e., an antibody induced in mice) of 50. The results are shown in Table 5-8.
  • H39N15-68T4 VLP mice induce anti-HPV39 antibodies ED 50 is quite HPV39N15 VLP alone, and significantly better than single HPV68N0 VLP; and that anti-HPV68 induced in mice ED 50 antibody alone, although weaker in HPV68N0 VLP, but significantly better than separate HPV39N15 VLP. This indicates that the H39N15-68T4 VLP has good cross-immunogenicity and cross-protection against HPV68 and HPV39.
  • Example 5 Evaluation of neutralizing antibody titers in mouse serum after immunization with virus-like particles 2
  • virus particles used were H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, H39N15-68T4-70S5 VLP.
  • mice (6-week-old BalB / c female mice) were divided into 3 groups: aluminum adjuvant group 1 (immunized dose of 5 ⁇ g, using aluminum adjuvant), aluminum adjuvant group 2 (immunized dose of 1 ⁇ g, using Aluminum adjuvant), and aluminum adjuvant group 3 (immunized dose of 0.2 ⁇ g, using aluminum adjuvant).
  • Each group was further subdivided into 8 subgroups.
  • the control subgroups 1, 2 and 3 were immunized with HPV39N15, HPV68N0, VLP, and HPV70N10, respectively.
  • the control subgroups were mixed with 4 mixed HPV39 / HPV68 / HPV70.
  • VLPs ie, a mixture of HPV39N15, VLP, HPV68N0, VLP, and HPV70N10, where each VLP is administered at a specified immunization dose
  • the experimental subgroups 1, 2, 3, and 4 were immunized with H39N15-68T4-70S1 VLP, H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP respectively.
  • mice / subgroup were immunized by intraperitoneal injection, the immunization doses were 5 ⁇ g, 1 ⁇ g, 0.2 ⁇ g, and the injection volume was 1 ml. All mice were initially immunized at week 0 and then boosted once at weeks 2 and 4 each. Orbital blood was collected from mice at week 8 and the titers of anti-HPV39, HPV68, and HPV70 antibodies in serum were analyzed. The analysis results are shown in Figures 8A-8C.
  • H39N15-68T4-70S2, VLP, H39N15-68T4-70S3, VLP, H39N15-68T4-70S5, VLP can induce mice to produce high titers of neutralizing antibodies against HPV39, although the protective effect is slightly weaker than the same dose HPV39N15 VLP alone and mixed HPV39 / HPV68 / HPV70 VLP, but still significantly better than the same dose of HPV68N0 VLP alone or HPV70N10 VLP alone; and it can induce mice to produce high titers of neutralizing antibodies against HPV68, Its protective effect is equivalent to the same dose of HPV68N0 VLP alone and mixed HPV39 / HPV68 / HPV70 VLP, and it is significantly better than the same dose of HPV39N15 VLP alone or HPV70N10 VLP alone; and it can induce high titer in mice
  • the neutralizing antibody against HPV70 has the same protective effect as the single dose of HPV70N10 VLP and
  • H39N15-68T4-70S2, VLP, H39N15-68T4-70S3, VLP, H39N15-68T4-70S5, VLP have good cross-immunogenicity and cross-protection against HPV39, HPV68, and HPV70.
  • Example 6 Evaluation of virus-like particle-induced seroconverted ED 50 2
  • virus-like particles used were H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP, and H39N15-68T4-70S5 VLP.
  • mice (8) 6-week-old BalB / c female mice (8) were immunized with aluminum adjuvant and single intraperitoneal injection.
  • the experimental group used H39N15-68T4-70S2 VLP, H39N15-68T4-70S3 VLP or H39N15-68T4- 70S5 VLP (immunized doses are 0.900 ⁇ g, 0.300 ⁇ g, 0.100 ⁇ g, 0.033 ⁇ g, or 0.011 ⁇ g); control group uses HPV39N15 VLP alone, HPV68N0 VLP or HPV70N10 VLP alone (immunized doses are 0.900 ⁇ g, 0.300 ⁇ g, 0.100 ⁇ g, 0.033 ⁇ g, or 0.011 ⁇ g), or mixed HPV39 / HPV68 / HPV70 VLP (that is, a mixture of HPV39N15 VLP, HPV68N0 VLP, and HPV70N10 VLP, the immune dose of each VLP is 0.900
  • mice were immunized in each group, and in the fifth week after immunization, ocular venous blood was drawn to detect HPV antibodies in the serum, and the Reed-Muench method (Reed LJ MH.A simple method of estimating fifty percent endpoints. Am J Hyg.1938; 27: 493-7) calculates ED induce seroconversion of each sample (i.e., an antibody induced in mice) of 50. The results are shown in Table 10-16.
  • H39N15-68T4-70S3 VLP induces mice to produce anti-HPV39, anti-HPV68 and anti-HPV70 antibodies (seroconverted) ED 50
  • the differential temperature calorimeter VP Capillary DSC purchased from GE of the United States (formerly MicroCal) was used to evaluate HPV39N15 protein, HPV68N0 protein, HPV70N10 protein, H39N15-68T4 protein, H39N15-68T4-70S2 protein, H39N15-68T4-70S5 protein laboratory
  • the thermostability of the formed VLP was performed using a storage buffer of the protein as a control, and each protein was scanned at a temperature increase rate of 1.5 ° C / min in the range of 10 ° C to 90 ° C.
  • the test results are shown in Figure 9. The results show that the VLPs formed by H39N15-68T4 protein, H39N15-68T4-70S2 protein, and H39N15-68T4-70S5 protein have extremely high thermal stability.
  • Example 8 H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 three-dimensional structure reconstruction of VLP
  • H39N15-68T4-70S2 VLP Reconstruction of H39N15-68T4-70S2 VLP by three-dimensional structure reconstruction experiments using cryo-electron microscopy (Wolf M, Garcea RL, Grigorieff N. et al. Proc Natl Acad Sci US A. (2010), 107 (14): 6298-303) And the three-dimensional structure of H39N15-68T4-70S5 VLP.
  • 6410 particles of uniform size and diameter over 50nm were selected for computer overlap and structural reconstruction to obtain the H39N15-68T4-70S2 VLP.
  • Three-dimensional structure The obtained three-dimensional structure is shown as B in FIG.
  • H39N15-68T4-70S2 VLP and H39N15-68T4-70S5 VLP are pentamers, without hexamers
  • the outermost diameter of the two types of VLPs is about 60 nm. This is in contrast to the three-dimensional structure of previously reported natural HPV virus particles and HPV VLPs prepared by eukaryotic expression systems (e.g., poxvirus expression system) (Baker TS, Newcomb WW, Olson NH. Et al. Biophys J.

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Abstract

本发明涉及一种突变的HPV39 L1蛋白(或其变体)，其编码序列和制备方法，以及包含其的病毒样颗粒，所述蛋白(或其变体)和病毒样颗粒能够诱发抗至少两个型别的HPV(例如，HPV39和HPV68，或者HPV39、HPV68和HPV70)的中和抗体，从而可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。本发明还涉及上述蛋白和病毒样颗粒用于制备药物组合物或疫苗的用途，所述药物组合物或疫苗可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。

Description

一种人乳头瘤病毒39型L1蛋白的突变体
本申请是以CN申请号为201810563378.0，申请日为2018年6月4日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本发明涉及分子病毒学和免疫学领域。具体地，本发明涉及一种突变的HPV39 L1蛋白(或其变体)，其编码序列和制备方法，以及包含其的病毒样颗粒，所述蛋白(或其变体)和病毒样颗粒能够诱发抗至少两个型别的HPV(例如，HPV39和HPV68，或者HPV39、HPV68和HPV70)的中和抗体，从而可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。本发明还涉及上述蛋白和病毒样颗粒用于制备药物组合物或疫苗的用途，所述药物组合物或疫苗可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。
背景技术
人乳头瘤病毒(Human Papillomavirus，HPV)主要引起皮肤和粘膜的疣状病变。根据其与肿瘤发生的关系，HPV可分为高危型与低危型，其中高危型的HPV感染被证实是诱发包括女性宫颈癌在内的生殖器癌症的主要原因；低危型则主要引起尖锐湿疣。预防与控制HPV感染的最有效方式是施用HPV疫苗，特别是针对能引起宫颈癌的高危型HPV的疫苗。
HPV的主要衣壳蛋白L1具有自组装为空心病毒样颗粒(Virus-Like Particle,VLP)的特性。HPV VLP是由72个主要衣壳蛋白L1的五聚体构成的20面体立体对称结构(Doorbar,J.and P.H.Gallimore.1987.J Virol,61(9):2793-9)。HPV VLP的结构与天然HPV高度相似，保留了天然病毒的绝大多数中和表位，可诱导高滴度的中和抗体(Kirnbauer，R.，F.Booy，et al.1992 Proc Natl Acad Sci U S A 89(24):12180-4)。
然而，现有的研究显示，HPV VLP主要诱导针对同型HPV的中和抗体，产生针对同型HPV的保护性免疫，而仅在一些同源性高的型别之间 存在低的交叉保护作用(Sara L.Bissett,Giada Mattiuzzo,et al.2014 Vaccine.32:6548-6555)。因此，现有的HPV疫苗的保护范围非常有限。通常，一个型别的HPV VLP只能用于预防该型别的HPV感染。在这种情况下，如果要扩大HPV疫苗的保护范围，那就只能在疫苗中增加更多型别的HPV VLP。目前已上市的HPV疫苗，包括Merck公司的
Figure PCTCN2019089988-appb-000001
(其为针对HPV16，18，6和11的四价疫苗)，GSK公司的
Figure PCTCN2019089988-appb-000002
(其为针对HPV16，18的二价疫苗)和Merck公司的
Figure PCTCN2019089988-appb-000003
(其为针对HPV6，11，16，18，31，33，45，52，58的九价疫苗)，均是通过混合多个型别的HPV VLP而制成的。然而，这种方案将导致HPV疫苗的生产成本大大提高，并且可能因为免疫剂量的增加而导致潜在的安全性问题。
因此，本领域需要开发能够诱导针对多个型别的HPV的保护性中和抗体的HPV病毒样颗粒，以更经济、有效地预防多个型别的HPV感染和由此导致的疾病例如宫颈癌和尖锐湿疣。
发明内容
本发明至少部分基于发明人的下述出人意料的发现：将人乳头瘤病毒(HPV)39型L1蛋白中的一个特定区段置换为第二型别的HPV(例如HPV68)L1蛋白的相应区段后，所获得的突变的HPV39 L1蛋白能够诱导机体产生针对HPV39和第二型别的HPV(例如HPV68)的高滴度中和抗体，其保护效果与混合的HPV39 VLP和第二型别的HPV VLP相当，并且其针对HPV68的保护效果与单独的HPV68 VLP相当，且针对第二型别的HPV(例如HPV68)的保护效果与单独的第二型别的HPV VLP相当。
此外，在上述置换的基础上，还可以将HPV39 L1蛋白中的另一个特定区段进一步置换为第三型别的HPV(例如HPV70)L1蛋白的相应区段，由此所获得的含有双置换的突变的HPV39 L1蛋白能够诱导机体产生针对HPV39、第二型别的HPV(例如HPV68)和第三型别的HPV(例如HPV70)的高滴度中和抗体，其保护效果与混合的HPV39 VLP、第二型别的HPV VLP和第三型别的HPV VLP相当；并且，其针对HPV39的保 护效果与单独的HPV39 VLP相当，针对第二型别的HPV(例如HPV68)的保护效果与单独的第二型别的HPV VLP相当，且针对第三型别的HPV(例如HPV70)的保护效果与单独的第三型别的HPV VLP相当。
因此，在一个方面，本发明提供了一种突变的HPV39 L1蛋白或其变体，其中，所述突变的HPV39 L1蛋白与野生型HPV39 L1蛋白相比，具有下述突变：
(1)N端截短了1-25个氨基酸，例如1-5个、1-10个、1-15个、1-20个、5-15个、10-15个、10-20个、15-20个氨基酸；和
(2)位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为第二型别的野生型HPV的L1蛋白的相应位置的氨基酸残基；
并且，所述变体与所述突变的HPV39 L1蛋白相异仅在于一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失，且保留了所述突变的HPV39 L1蛋白的功能，即，能够诱导针对至少两个型别的HPV(例如，HPV39和HPV68，或者HPV39、HPV68和HPV70)的中和抗体。
在某些优选的实施方案中，所述突变的HPV39 L1蛋白任选地还具有下述突变：
(3)(a)位于野生型HPV39 L1蛋白第117-140位的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；或
(b)位于野生型HPV39 L1蛋白第169-181位的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基。
(c)位于野生型HPV39 L1蛋白第347-358位的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基。
在某些优选的实施方案中，所述突变的HPV39 L1蛋白与野生型HPV39 L1蛋白相比，N端截短了3个、5个、8个、10个、12个、15个、18个、20个或22个氨基酸。在某些优选的实施方案中，所述突变的HPV39 L1蛋白与野生型HPV39 L1蛋白相比，N端截短了15个氨基酸。
在某些优选的实施方案中，所述第二型别的野生型HPV为HPV68。在某些优选的实施方案中，(2)中所述的相应位置的氨基酸残基为野生型HPV68 L1蛋白第270-289位的氨基酸残基。在某些优选的实施方案中， 所述第三型别的野生型HPV为HPV70。在某些优选的实施方案中，(3)(a)中所述的相应位置的氨基酸残基为野生型HPV70 L1蛋白第117-141位的氨基酸残基。在某些优选的实施方案中，(3)(b)中所述的相应位置的氨基酸残基为野生型HPV70 L1蛋白第170-182位的氨基酸残基。在某些优选的实施方案中，(3)(c)中所述的相应位置的氨基酸残基为野生型HPV70 L1蛋白第348-359位的氨基酸残基。
在某些优选的实施方案中，所述野生型HPV39 L1蛋白具有如SEQ ID NO:1所示的氨基酸序列。
在某些优选的实施方案中，所述野生型HPV68 L1蛋白具有如SEQ ID NO:2所示的氨基酸序列。
在某些优选的实施方案中，所述野生型HPV70 L1蛋白具有如SEQ ID NO:3所示的氨基酸序列。
在某些优选的实施方案中，所述野生型HPV68 L1蛋白第270-289位的氨基酸残基的序列如SEQ ID NO:25所示。
在某些优选的实施方案中，所述野生型HPV70 L1蛋白第117-141位的氨基酸残基的序列如SEQ ID NO:26所示。
在某些优选的实施方案中，所述野生型HPV70 L1蛋白第170-182位的氨基酸残基的序列如SEQ ID NO:27所示。
在某些优选的实施方案中，所述野生型HPV70 L1蛋白第348-359位的氨基酸残基的序列如SEQ ID NO:28所示。
在某些优选的实施方案中，所述突变的HPV39 L1蛋白具有选自下列的氨基酸序列：SEQ ID NO:7、10、11和12。
在另一个方面，本发明提供了一种分离的核酸，其编码如上所述的突变的HPV39 L1蛋白或其变体。在另一个方面，本发明提供了一种载体，其包含所述分离的核酸。在某些优选的实施方案中，本发明的分离的核酸具有选自下列的核苷酸序列：SEQ ID NO:19、22、23和24。
可用于插入目的多核苷酸的载体是本领域公知的，包括但不限于克隆载体和表达载体。在一个实施方案中，载体是例如质粒，粘粒，噬菌体等等。
在另一个方面，本发明还涉及包含上述分离的核酸或载体的宿主细胞。 此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的宿主细胞还可以是细胞系，例如293T和293TT细胞。
在另一个方面，本发明涉及一种HPV病毒样颗粒，其中该病毒样颗粒含有本发明的突变的HPV39 L1蛋白或其变体，或者由本发明的突变的HPV39 L1蛋白或其变体组成或形成。
在某些优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV39 L1蛋白，其与野生型HPV39 L1蛋白相比，N端截短了1-25个氨基酸，例如1-5个、1-10个、1-15个、1-20个、5-15个、10-15个、10-20个、15-20个氨基酸，例如3个、5个、8个、11个、13个、15个、18个、20个或22个氨基酸，并且位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为野生型HPV68 L1蛋白第270-289位的氨基酸残基。
在某些优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV39 L1蛋白，其与野生型HPV39 L1蛋白相比，N端截短了1-25个氨基酸，例如1-05个、1-10个、1-15个、1-20个、5-15个、10-15个、10-20个、15-20氨基酸，例如3个、5个、8个、11个、13个、15个、18个、20个或22个氨基酸，并且位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为野生型HPV68 L1蛋白第270-289位的氨基酸残基，并且位于野生型HPV39 L1蛋白第117-140位的氨基酸残基被替换为野生型HPV70 L1蛋白第117-141位的氨基酸残基。
在某些优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV39 L1蛋白，其与野生型HPV39 L1蛋白相比，N端截短了1-25个氨基酸，例如1-5个、1-10个、1-15个、1-20个、5-15个、10-15个、10-20个、15-20个氨基酸，例如3个、5个、8个、11个、13个、15个、18个、20个或22个氨基酸，并且位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为野生型HPV68 L1蛋白第270-289位的氨基酸残基，并且位于野生型HPV39 L1蛋白第169-181位的氨基酸残基被替换为野生型HPV70 L1蛋白第170-182位的氨基酸残基。
在某些优选的实施方案中，本发明的HPV病毒样颗粒包含突变的 HPV39 L1蛋白，其与野生型HPV39 L1蛋白相比，N端截短了1-25个氨基酸，例如1-5个、1-10个、1-15个、1-20个、5-15个、10-15个、10-20个、15-20个氨基酸，例如3个、5个、8个、11个、13个、15个、18个、20个或22个氨基酸，并且位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为野生型HPV68 L1蛋白第270-289位的氨基酸残基，并且位于野生型HPV39 L1蛋白第347-358位的氨基酸残基被替换为野生型HPV70 L1蛋白第348-359位的氨基酸残基。
在一个特别优选的实施方案中，本发明的HPV病毒样颗粒包含突变的HPV39 L1蛋白，其具有SEQ ID NO:7、10、11和12所示的序列。
在另一个方面，本发明还涉及包含上述突变的HPV39 L1蛋白或其变体，或上述分离的核酸或载体或宿主细胞或HPV病毒样颗粒的组合物。在某些优选的实施方案中，所述组合物包含本发明的突变的HPV39 L1蛋白或其变体。在某些优选的实施方案中，所述组合物包含本发明的HPV病毒样颗粒。
在另一个方面，本发明还涉及一种药物组合物或疫苗，其包含本发明的HPV病毒样颗粒，任选地还包含药学可接受的载体和/或赋形剂。本发明的药物组合物或疫苗可以用于预防HPV感染或由HPV感染所导致的疾病例如宫颈癌和尖锐湿疣。
在某些优选的实施方案中，所述HPV病毒样颗粒以预防HPV感染或由HPV感染导致的疾病的有效量存在。在某些优选的实施方案中，所述HPV感染是一个或多个型别的HPV感染(例如，HPV39感染、HPV68感染和/或HPV70感染)。在某些优选的实施方案中，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
本发明的药物组合物或疫苗可通过本领域公知的方法进行施用，例如但不限于通过口服或者注射进行施用。在本发明中，特别优选的施用方式是注射。
在某些优选的实施方案中，本发明的药物组合物或疫苗以单位剂量形式进行施用。例如但不意欲限定本发明，每单位剂量中包含的HPV病毒样颗粒的量为5μg－80μg，优选20μg－40μg。
在另一个方面，本发明涉及一种制备如上所述的突变的HPV39 L1蛋 白或其变体的方法，其包括，在宿主细胞中表达所述突变的HPV39 L1蛋白或其变体，然后从所述宿主细胞的培养物中回收所述突变的HPV39 L1蛋白或其变体。
在某些优选的实施方案中，所述宿主细胞为大肠杆菌。
在某些优选的实施方案中，所述方法包括步骤：在大肠杆菌中表达所述突变的HPV39 L1蛋白或其变体，然后从所述大肠杆菌的裂解上清中纯化得到所述突变的HPV39 L1蛋白或其变体。在某些优选的实施方案中，通过色谱法(例如，阳离子交换色谱，羟基磷灰石色谱和/或疏水相互作用色谱)，从所述大肠杆菌的裂解上清中回收所述突变的HPV39 L1蛋白或其变体。
在另一个方面，本发明涉及一种制备疫苗的方法，其包括将本发明的HPV病毒样颗粒与药学可接受的载体和/或赋形剂混合。
在另一个方面，本发明涉及一种预防HPV感染或由HPV感染所导致的疾病的方法，其包括将预防有效量的根据本发明的HPV病毒样颗粒或药物组合物或疫苗施用给受试者。在一个优选的实施方案中，所述HPV感染是一个或多个型别的HPV感染(例如，HPV39感染、HPV68感染和/或HPV70感染)。在另一个优选的实施方案中，所述由HPV感染所导致的疾病包括但不限于，宫颈癌和尖锐湿疣。在另一个优选的实施方案中，所述受试者是哺乳动物，例如人。
在另一个方面，本发明还涉及本发明的突变的HPV39 L1蛋白或其变体或HPV病毒样颗粒在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于预防HPV感染或由HPV感染所导致的疾病。在一个优选的实施方案中，所述HPV感染是一个或多个型别的HPV感染(例如，HPV39感染、HPV68感染和/或HPV70感染)。在另一个优选的实施方案中，所述由HPV感染所导致的疾病包括但不限于，宫颈癌和尖锐湿疣。
在另一个方面，本发明还涉及本发明的突变的HPV39 L1蛋白或其变体或HPV病毒样颗粒，其用于预防HPV感染或由HPV感染所导致的疾病。在一个优选的实施方案中，所述HPV感染是一个或多个型别的HPV感染(例如，HPV39感染、HPV68感染和/或HPV70感染)。在另一个优选的实施方案中，所述由HPV感染所导致的疾病包括但不限于，宫颈 癌和尖锐湿疣。
本发明中相关术语的说明及解释
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
根据本发明，术语“第二型别的野生型HPV”是指，不同于HPV39的另一型别的野生型HPV。在本发明中，第二型别的野生型HPV优选为野生型HPV68。
根据本发明，术语“第三型别的野生型HPV”是指，不同于HPV39，且不同于第二型别的野生型HPV的另一型别的野生型HPV。在本发明中，第三型别的野生型HPV优选为野生型HPV70。
根据本发明，表述“相应位置”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中的等同位置。
根据本发明，术语“野生型HPV39 L1蛋白”是指，天然存在于人乳头瘤病毒39型(HPV39)中的主要衣壳蛋白L1。野生型HPV39 L1蛋白的序列是本领域公知的，并且可参见各种公共数据库(例如NCBI数据库登录号P24838.1、ARQ82617.1、AGU90549.1和AEP23084.1)。
在本发明中，当提及野生型HPV39 L1蛋白的氨基酸序列时，参照SEQ ID NO：1所示的序列来进行描述。例如，表述“野生型HPV39 L1蛋白的第53-61位氨基酸残基”是指，SEQ ID NO：1所示的多肽的第53-61位氨基酸残基。然而，本领域技术人员理解，野生型HPV39可包括多种分离株，并且各种分离株的L1蛋白的氨基酸序列之间可能存在着差异。进一步，本领域技术人员理解，尽管可能存在着序列差异，但是HPV39的不同分离株的L1蛋白在氨基酸序列上具有极高的同一性(通常高于95％，例如高于96％，高于97％，高于98％，或高于99％)，并且具有实质上相同的生物学功能。因此，在本发明中，术语“野生型HPV39 L1蛋白”不仅包括SEQ ID NO:1所示的蛋白，而且应包括各种HPV39分离株的L1蛋白(例如P24838.1、ARQ82617.1、AGU90549.1或AEP23084.1所示的HPV39 L1蛋白)。并且，当描述野生型HPV39 L1蛋白的序列片 段时，其不仅包括SEQ ID NO：1的序列片段，还包括各种HPV39分离株的L1蛋白中的相应序列片段。例如，表述“野生型HPV39 L1蛋白的第53-61位氨基酸残基”包括，SEQ ID NO：1的第53-61位氨基酸残基，以及各种HPV39分离株的L1蛋白中的相应片段。
根据本发明，术语“野生型HPV68 L1蛋白”是指，天然存在于人乳头瘤病毒68型(HPV68)中的主要衣壳蛋白L1。野生型HPV68L1蛋白的序列是本领域公知的，并且可参见各种公共数据库(例如NCBI数据库登录号AAZ39498.1，AGU90717.1，P4669.1和AGU90703.1)。
在本发明中，当提及野生型HPV68 L1蛋白的氨基酸序列时，参照SEQ ID NO：2所示的序列来进行描述。例如，表述“野生型HPV68 L1蛋白的第53-61位氨基酸残基”是指，SEQ ID NO：2所示的多肽的第53-61位氨基酸残基。然而，本领域技术人员理解，野生型HPV68可包括多种分离株，并且各种分离株的L1蛋白的氨基酸序列之间可能存在着差异。进一步，本领域技术人员理解，尽管可能存在着序列差异，但是HPV68的不同分离株的L1蛋白在氨基酸序列上具有极高的同一性(通常高于95％，例如高于96％，高于97％，高于98％，或高于99％)，并且具有实质上相同的生物学功能。因此，在本发明中，术语“野生型HPV68 L1蛋白”不仅包括SEQ ID NO:2所示的蛋白，而且应包括各种HPV68分离株的L1蛋白(例如AAZ39498.1、AGU90717.1、P4669.1或AGU90703.1所示的HPV68 L1蛋白)。并且，当描述野生型HPV68 L1蛋白的序列片段时，其不仅包括SEQ ID NO：2的序列片段，还包括各种HPV68分离株的L1蛋白中的相应序列片段。例如，表述“野生型HPV68 L1蛋白的第53-61位氨基酸残基”包括，SEQ ID NO：2的第53-61位氨基酸残基，以及各种HPV68分离株的L1蛋白中的相应片段。
根据本发明，术语“野生型HPV70 L1蛋白”是指，天然存在于人乳头瘤病毒70型(HPV70)中的主要衣壳蛋白L1。野生型HPV70 L1蛋白的序列是本领域公知的，并且可参见各种公共数据库(例如NCBI数据库登录号AGU90846.1、AGU90854.1、AAC54879.1和P50793.1)。
在本发明中，当提及野生型HPV70 L1蛋白的氨基酸序列时，参照SEQ ID NO：3所示的序列来进行描述。例如，表述“野生型HPV70 L1蛋 白的第117-141位氨基酸残基”是指，SEQ ID NO：3所示的多肽的第117-141位氨基酸残基。然而，本领域技术人员理解，野生型HPV70可包括多种分离株，并且各种分离株的L1蛋白的氨基酸序列之间可能存在着差异。进一步，本领域技术人员理解，尽管可能存在着序列差异，但是HPV70的不同分离株的L1蛋白在氨基酸序列上具有极高的同一性(通常高于95％，例如高于96％，高于97％，高于98％，或高于99％)，并且具有实质上相同的生物学功能。因此，在本发明中，术语“野生型HPV70 L1蛋白”不仅包括SEQ ID NO:3所示的蛋白，而且应包括各种HPV70分离株的L1蛋白(例如AGU90846.1、AGU90854.1、AAC54879.1或P50793.1所示的HPV70 L1蛋白)。并且，当描述野生型HPV70 L1蛋白的序列片段时，其不仅包括SEQ ID NO：3的序列片段，还包括各种HPV70分离株的L1蛋白中的相应序列片段。例如，表述“野生型HPV70 L1蛋白的第117-141位氨基酸残基”包括，SEQ ID NO：3的第117-141位氨基酸残基，以及各种HPV70分离株的L1蛋白中的相应片段。
根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
根据本发明，表述“N端截短了X个氨基酸”是指，用起始密码子(用于起始蛋白质翻译)编码的甲硫氨酸残基置换蛋白质N末端的第1－X位氨基酸残基。例如，N端截短了15个氨基酸的HPV39 L1蛋白是指，用起始密码子编码的甲硫氨酸残基置换野生型HPV39 L1蛋白N末端的第1－15位氨基酸残基所获得的蛋白质。
根据本发明，术语“变体”是指这样的蛋白，其氨基酸序列与本发明的突变的HPV39 L1蛋白(如SEQ ID NO：7、10、11和12所示的蛋白)的氨基酸序列相比，具有一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失，或者具有至少90％，95％，96％，97％，98％，或99％的同一性，并且其保留了所述突变的HPV39 L1蛋白的功能。在本发明中，术语“突变的HPV39 L1蛋白的功能”是指：能够诱导机体产生针对至少两个型别的HPV(例如，HPV39和HPV68，或者HPV39、HPV68和HPV70)的中 和抗体。术语“同一性”是对核苷酸序列或氨基酸序列的相似性的量度。通常将序列排列起来，以获得最大限度的匹配。“同一性”本身具有本领域公知的意义并且可用公开的算法(例如BLAST)来计算。
根据本发明，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具 有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，保守置换通常是指，用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
根据本发明，术语“大肠杆菌表达系统”是指由大肠杆菌(菌株)与载体组成的表达系统，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：ER2566，BL21(DE3)，B834(DE3)，BLR(DE3)。
根据本发明，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸所编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌体；柯斯质粒等等。
根据本发明，术语“药学可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)；离子强度增强剂包括但不限于氯化钠。
根据本发明，术语“有效量”是指能够有效实现预期目的的量。例如，预防疾病(例如HPV感染)有效量是指，能够有效预防，阻止，或延迟 疾病(例如HPV感染)的发生的量。测定这样的有效量在本领域技术人员的能力范围之内。
根据本发明，术语“色谱层析”包括但不限于：离子交换色谱(例如阳离子交换色谱)、疏水相互作用色谱、吸附层析法(例如羟基磷灰石色谱)、凝胶过滤(凝胶排阻)层析、亲和层析法。
根据本发明，术语“裂解上清”是指通过下述步骤所产生的溶液：将宿主细胞(例如大肠杆菌)在裂解液中破碎，然后将含有经破碎的宿主细胞的裂解液中的不溶物去除。各种裂解液是本领域技术人员公知的，包括但不限于Tris缓冲液，磷酸盐缓冲液，HEPES缓冲液，MOPS缓冲液等等。此外，可通过本领域技术人员熟知的各种方法来实现宿主细胞的破碎，包括但不限于匀浆器破碎、均质机破碎、超声波处理、研磨、高压挤压、溶菌酶处理等等。去除裂解液中的不溶物的方法也是本领域技术人员公知的，包括但不限于过滤和离心。
发明的有益效果
研究表明，虽然HPV39和其他型别的HPV(例如HPV68和HPV70)之间存在一定的交叉保护，但是这种交叉保护的能力很低，通常低于自身型别的VLP的保护水平的百分之一，甚至低于千分之一。因此，对于接种了HPV39疫苗的受试者来说，其感染其他型别的HPV(例如HPV68和HPV70)的风险依然很高。
本发明提供了一种突变的HPV39 L1蛋白以及由其形成的HPV病毒样颗粒。本发明的HPV病毒样颗粒能够在HPV39和其他型别的HPV(例如HPV68和HPV70)之间提供显著的交叉保护能力。特别地，在同等免疫剂量下，本发明的HPV病毒样颗粒能够诱发机体产生针对至少两个型别的HPV(例如，HPV39和HPV68，或者HPV39、HPV68和HPV70)的高滴度中和抗体，并且其效果与多个型别的HPV VLP的混合物(例如，HPV39 VLP和HPV68VLP的混合物，或者HPV39 VLP、HPV68 VLP和HPV70 VLP的混合物)相当。因此，本发明的HPV病毒样颗粒能够用于同时预防至少两个型别的HPV(例如，HPV39和HPV68，或者HPV39、HPV68和HPV70)的感染以及与此相关的疾病，具有显著的有利技术效果。这在扩大HPV疫苗的保护范围和降低HPV疫苗的生产成本等方面具 有特别显著的优势。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了实施例1中经纯化的突变蛋白的SDS聚丙烯酰胺凝胶电泳的结果。泳道1：蛋白分子量标记；泳道2:HPV39N15(N端截短了15个氨基酸的HPV39 L1蛋白)；泳道3:HPV68N0(N端截短了0个氨基酸的HPV68 L1蛋白，即全长的野生型HPV68 L1蛋白)；泳道4:H39N15-68T1；泳道5:H39N15-68T2；泳道6:H39N15-68T3；泳道7:H39N15-68T4；泳道8:H39N15-68T5；泳道9：蛋白分子量标记；泳道10:H39N15-68T4；泳道11:HPV70N10(N端截短了10个氨基酸的HPV70L1蛋白)；泳道12:H39N15-68T4-70S1；泳道13:H39N15-68T4-70S2；泳道14:H39N15-68T4-70S3；泳道15:H39N15-68T4-70S5。结果显示，经过色谱纯化后，H39N15-68T1、H39N15-68T2、H39N15-68T3、H39N15-68T4、H39N15-68T5、H39N15-68T4-70S1、H39N15-68T4-70S2、H39N15-68T4-70S3、H39N15-68T4-70S5蛋白的纯度均达到90％以上。
图2显示了用广谱抗体4B3检测实施例1中制备的突变蛋白H39N15-68T1、H39N15-68T2、H39N15-68T3、H39N15-68T4、H39N15-68T5、H39N15-68T4-70S1、H39N15-68T4-70S2、H39N15-68T4-70S3、H39N15-68T4-70S5的蛋白质免疫印迹检测的结果。泳道1:HPV39N15；泳道2:HPV68N0；泳道3:H39N15-68T1；泳道4:H39N15-68T2；泳道5:H39N15-68T3；泳道6:H39N15-68T4；泳道7:H39N15-68T5；泳道8:H39N15-68T4；泳道9:HPV70N10；泳道10:H39N15-68T4-70S1；泳道11:H39N15-68T4-70S2；泳道12:H39N15-68T4-70S3；泳道13:H39N15-68T4-70S5。结果显示，突变蛋白H39N15-68T1、H39N15-68T2、H39N15-68T3、H39N15-68T4、H39N15-68T5、H39N15-68T4-70S1、H39N15-68T4-70S2、H39N15-68T4-70S3、H39N15-68T4-70S5均能够被广谱抗体4B3特异性识别。
图3显示了包含蛋白HPV39N15、H39N15-68T1、H39N15-68T2、H39N15-68T3、H39N15-68T4、H39N15-68T5的样品的分子筛层析分析的结果。结果显示，包含蛋白H39N15-68T1、H39N15-68T2、H39N15-68T3、H39N15-68T4、H39N15-68T5的样品最先出现的蛋白峰均在13-14min左右，与HPV39N15相当。这表明，这些蛋白均可组装成VLP。
图4显示了包含蛋白HPV39N15、HPV68L1N0、HPV70N10、H39N15-68T4、H39N15-68T4-70S1、H39N15-68T4-70S2、H39N15-68T4-70S3、H39N15-68T4-70S5的样品的分子筛层析分析的结果。结果显示，包含蛋白H39N15-68T4-70S1、H39N15-68T4-70S2、H39N15-68T4-70S3、H39N15-68T4-70S5的样品最先出现的蛋白峰均在13-14min左右，与HPV39N15、HPV68L1N0、HPV70N10及H39N15-68T4 VLP相当。这表明，这些蛋白均可组装成VLP。
图5A-图5L显示了HPV39N15 VLP、HPV68L1N0 VLP和HPV70N10 VLP与H39N15-68T1 VLP、H39N15-68T2 VLP、H39N15-68T3 VLP、H39N15-68T4 VLP、H39N15-68T5 VLP、H39N15-68T4-70S1 VLP、H39N15-68T4-70S2 VLP、H39N15-68T4-70S3 VLP、H39N15-68T4-70S5 VLP的沉降速率分析的结果。其中：图5A，HPV39N15 VLP；图5B，HPV68L1N0 VLP；图5C，H39N15-68T1 VLP；图5D，H39N15-68T2 VLP；图5E，H39N15-68T3 VLP；图5F，H39N15-68T4 VLP；图5G，H39N15-68T5 VLP；图5H，HPV70N10 VLP；图5I，H39N15-68T4-70S1 VLP；图5J，H39N15-68T4-70S2 VLP；图5K，H39N15-68T4-70S3 VLP；图5L，H39N15-68T4-70S5 VLP。结果显示，H39N15-68T1 VLP，H39N15-68T2 VLP，H39N15-68T3 VLP，H39N15-68T4 VLP，H39N15-68T5 VLP，H39N15-68T4-70S1 VLP，H39N15-68T4-70S2 VLP，H39N15-68T4-70S3 VLP和H39N15-68T4-70S5 VLP的沉降系数分别为136S、151S、138S、145S、135S、124S、108S、99S和127S。这表明，突变蛋白H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3和H39N15-68T4-70S5各自能够组装成大小、形态与野生型VLP(HPV39N15 VLP，115S；HPV68N0  VLP，153S；HPV70N10 VLP，144S)相似的病毒样颗粒。
图6A-图6L显示了各种VLP样品的透射电镜观察结果(放大倍数为100,000倍，Bar＝0.1μm)。其中：图6A，由HPV39N15组装的VLP；图6B，由HPV68L1N0组装的VLP；图6C，由HPV70N10组装的VLP；图6D，由H39N15-68T1组装的VLP；图6E，由H39N15-68T2组装的VLP；图6F，由H39N15-68T3组装的VLP；图6G，由H39N15-68T4组装的VLP；图6H，由H39N15-68T5组装的VLP；图6I，由H39N15-68T4-70S1组装的VLP；图6J，由H39N15-68T4-70S2组装的VLP；图6K，由H39N15-68T4-70S3组装的VLP；图6L，由H39N15-68T4-70S5组装的VLP。结果显示，H39N15-68T1、H39N15-68T2、H39N15-68T3、H39N15-68T4、H39N15-68T5、H39N15-68T4-70S1、H39N15-68T4-70S2、H39N15-68T4-70S3、H39N15-68T4-70S5与HPV39N15、HPV68L1N0和HPV70N10类似，都能组装成半径为25-30nm左右的VLP。
图7A-图7C显示了用H39N15-68T1 VLP、H39N15-68T2 VLP、H39N15-68T3 VLP、H39N15-68T4 VLP、H39N15-68T5 VLP免疫小鼠后小鼠血清中的中和抗体滴度的评价结果。图7A：铝佐剂组1(免疫剂量为5μg，使用铝佐剂)；图7B：铝佐剂组2(免疫剂量为1μg，使用铝佐剂)；图7C：铝佐剂组3(免疫剂量为0.2μg，使用铝佐剂)。结果显示，H39N15-68T4 VLP能诱导小鼠产生高滴度的针对HPV39的中和抗体，其保护效果虽稍微弱于同剂量的单独的HPV39N15 VLP，但显著优于同剂量的单独的HPV68N0 VLP；并且其能诱导小鼠产生高滴度的针对HPV68的中和抗体，其保护效果虽稍微弱于同剂量的单独的HPV68N0 VLP，但显著优于同剂量的单独的HPV39N15 VLP。这表明，H39N15-68T4 VLP对HPV39和HPV68具有良好的交叉免疫原性和交叉保护性。
图8A-图8C显示了用H39N15-68T4-70S1 VLP、H39N15-68T4-70S2 VLP、H39N15-68T4-70S3 VLP、H39N15-68T4-70S5 VLP免疫小鼠后小鼠血清中的中和抗体滴度的评价结果。图8A：铝佐剂组1(免疫剂量为5μg，使用铝佐剂)；图8B：铝佐剂组2(免疫剂量为1μg，使用铝佐剂)；图8C：铝佐剂组3(免疫剂量为0.2μg，使用铝佐剂)。结果显示，H39N15-68T4-70S2、H39N15-68T4-70S3、H39N15-68T4-70S5 VLP能诱 导小鼠产生高滴度的针对HPV39的中和抗体，其保护效果虽然略微弱于同剂量的单独的HPV39N15 VLP以及混合的HPV39/HPV68/HPV70 VLP，但仍显著优于同剂量的单独的HPV68N0 VLP或单独的HPV70N10 VLP；并且其能诱导小鼠产生高滴度的针对HPV68的中和抗体，其保护效果与同剂量的单独的HPV68N0 VLP以及混合的HPV39/HPV68/HPV70 VLP相当，且显著优于同剂量的单独的HPV39N15 VLP或单独的HPV70N10 VLP；并且其能诱导小鼠产生高滴度的针对HPV70的中和抗体，其保护效果与同剂量的单独的HPV70N10 VLP以及混合的HPV39/HPV68/HPV70 VLP相当，且显著优于同剂量的单独的HPV39N15 VLP或单独的HPV68N0 VLP。这表明，H39N15-68T4-70S2 VLP、H39N15-68T4-70S3 VLP、H39N15-68T4-70S5 VLP对HPV39、HPV68和HPV70具有良好的交叉免疫原性和交叉保护性。
图9显示了HPV39N15 VLP、HPV68N0 VLP和HPV70N10 VLP与H39N15-68T4 VLP、H39N15-68T4-70S2 VLP、H39N15-68T4-70S5 VLP热稳定性的检测结果。其中A为HPV39N15 VLP的热稳定性检测结果；B为HPV68L1N0 VLP的热稳定性检测结果；C为HPV70N10 VLP的热稳定性检测结果；D为H39N15-68T4 VLP VLP的热稳定性检测结果；E为H39N15-68T4-70S2 VLP的热稳定性检测结果；F为H39N15-68T4-70S5 VLP的热稳定性检测结果。结果显示，各个蛋白所形成的VLP均具有极高的热稳定性。
图10显示了H39N15-68T4-70S2 VLP、H39N15-68T4-70S5 VLP的冷冻电镜观察结果及其重建的三维结构。其中A是H39N15-68T4-70S2 VLP的冷冻电镜图像；B是H39N15-68T4-70S2 VLP的重建的三维结构；C是H39N15-68T4-70S5 VLP的冷冻电镜图像；D是H39N15-68T4-70S5 VLP的重建的三维结构。重建的三维结构显示，H39N15-68T4-70S2 VLP和H39N15-68T4-70S5 VLP均是由72个壳粒(形态亚单位，五聚体)形成的T＝7的二十面体结构(h＝1,k＝2)。与一般的符合准等价原理的二十面体病毒衣壳不同，H39N15-68T4-70S2 VLP和H39N15-68T4-70S5 VLP结构中所有组成亚单位均为五聚体，而不存在六聚体，并且，上述两种VLP的最外围直径均为约55nm。这与之前报道的天然HPV病毒颗粒及真核表达 系统(例如，痘病毒表达系统)制备的HPV VLP的三维结构(Baker TS,Newcomb WW,Olson NH.et al.Biophys J.(1991),60(6):1445-1456.Hagensee ME,Olson NH,Baker TS,et al.J Virol.(1994),68(7):4503-4505.Buck CB,Cheng N,Thompson CD.et al.J Virol.(2008),82(11):5190-7)类似。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2019089988-appb-000004
Figure PCTCN2019089988-appb-000005
序列1(SEQ ID NO:1)：
Figure PCTCN2019089988-appb-000006
序列2(SEQ ID NO:2)：
Figure PCTCN2019089988-appb-000007
序列3(SEQ ID NO:3)：
Figure PCTCN2019089988-appb-000008
序列4(SEQ ID NO:4)：
Figure PCTCN2019089988-appb-000009
序列5(SEQ ID NO:5)：
Figure PCTCN2019089988-appb-000010
序列6(SEQ ID NO:6)：
Figure PCTCN2019089988-appb-000011
序列7(SEQ ID NO:7)：
Figure PCTCN2019089988-appb-000012
序列8(SEQ ID NO:8)：
Figure PCTCN2019089988-appb-000013
序列9(SEQ ID NO:9)：
Figure PCTCN2019089988-appb-000014
序列10(SEQ ID NO:10)：
Figure PCTCN2019089988-appb-000015
Figure PCTCN2019089988-appb-000016
序列11(SEQ ID NO:11)：
Figure PCTCN2019089988-appb-000017
序列12(SEQ ID NO:12)：
Figure PCTCN2019089988-appb-000018
序列13(SEQ ID NO:13)：
Figure PCTCN2019089988-appb-000019
序列14(SEQ ID NO:14)：
Figure PCTCN2019089988-appb-000020
Figure PCTCN2019089988-appb-000021
序列15(SEQ ID NO:15)：
Figure PCTCN2019089988-appb-000022
序列16(SEQ ID NO:16)：
Figure PCTCN2019089988-appb-000023
Figure PCTCN2019089988-appb-000024
序列17(SEQ ID NO:17)：
Figure PCTCN2019089988-appb-000025
序列18(SEQ ID NO:18)：
Figure PCTCN2019089988-appb-000026
序列19(SEQ ID NO:19)：
Figure PCTCN2019089988-appb-000027
Figure PCTCN2019089988-appb-000028
序列20(SEQ ID NO:20)：
Figure PCTCN2019089988-appb-000029
序列21(SEQ ID NO:21)：
Figure PCTCN2019089988-appb-000030
Figure PCTCN2019089988-appb-000031
序列22(SEQ ID NO:22)：
Figure PCTCN2019089988-appb-000032
序列23(SEQ ID NO:23)：
Figure PCTCN2019089988-appb-000033
Figure PCTCN2019089988-appb-000034
序列24(SEQ ID NO:24)：
Figure PCTCN2019089988-appb-000035
序列25(SEQ ID NO:25)：
Figure PCTCN2019089988-appb-000036
序列26(SEQ ID NO:26)：
Figure PCTCN2019089988-appb-000037
序列27(SEQ ID NO:27)：
Figure PCTCN2019089988-appb-000038
序列28(SEQ ID NO:28)：
Figure PCTCN2019089988-appb-000039
序列29(SEQ ID NO:29)：
Figure PCTCN2019089988-appb-000040
序列30(SEQ ID NO:30)：
Figure PCTCN2019089988-appb-000041
Figure PCTCN2019089988-appb-000042
序列31(SEQ ID NO:31)：
Figure PCTCN2019089988-appb-000043
序列32(SEQ ID NO:32)：
Figure PCTCN2019089988-appb-000044
序列33(SEQ ID NO:33)：
Figure PCTCN2019089988-appb-000045
序列34(SEQ ID NO:34)：
Figure PCTCN2019089988-appb-000046
序列35(SEQ ID NO:35)：
Figure PCTCN2019089988-appb-000047
序列36(SEQ ID NO:36)：
Figure PCTCN2019089988-appb-000048
序列37(SEQ ID NO:37)：
Figure PCTCN2019089988-appb-000049
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1.突变的HPV39 L1蛋白的表达与纯化
表达载体的构建
采用多点突变PCR反应来构建编码含有来源于HPV68 L1蛋白的区段的突变的HPV39 L1蛋白的表达载体，其中，所使用的初始模板为pTO-T7-HPV39N15C质粒(其编码N端截短了15个氨基酸的HPV39 L1蛋白；在表2中简写为39L1N15)。用于各个PCR反应的模板和引物见表2，并且，PCR反应的扩增条件设为：94℃变性10分钟；25个循环的(94℃变性50秒，指定温度退火一定时间，72℃延伸7分30秒)；最后72℃延伸10分钟。退火温度和时间列于表2。所使用的PCR引物的具体序列列于表3。
向扩增产物(50μL)中加入2μL DpnI限制性内切酶(Fermentas(MBI)，货号：FD1704，2500U/管)，并在37℃温育60min。取10μL酶切产物，用于转化40μL以氯化钙法制备的感受态大肠杆菌ER2566(购自新英格兰生物实验室公司)。将经转化的大肠杆菌涂布于含卡那霉素(终浓度25μg/mL，下同)的固体LB培养基(LB培养基成分：10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，下同)，并在37℃静置培养10-12小时，直至单菌落清晰可辨。挑取单菌落至含有4mL液体LB培养基(含卡那霉素)的试管中，并在37℃220转/分钟下振荡培养10小时。随后，取1mL菌液 于-70℃保存。从大肠杆菌中提取质粒，并利用T7引物对质粒中插入的目的片段的核苷酸序列进行测序。测序结果显示，所构建的质粒(表达载体)中插入的目的片段的核苷酸序列为SEQ ID NO：16，其编码的氨基酸序列为SEQ ID NO：4(所对应的蛋白命名为H39N15-68T1)。突变蛋白H39N15-68T1与HPV39N15的区别在于：位于野生型HPV39 L1蛋白第53-61位的氨基酸残基被替换为野生型HPV68 L1蛋白第53-61位的氨基酸残基。
采用Gibson装配(Gibson DG,Young L,Chuang RY,Venter JC,Hutchison CA,Smith HO.Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods.2009；6:343-5.doi:10.1038/nmeth.1318)来构建编码其他的突变HPV39 L1蛋白的表达载体，所述突变的HPV39 L1蛋白含有来源于HPV68 L1的特定区段和/或来源于HPV70L1的特定区段。简言之，首先采用PCR反应来获得一个包含突变的短片段和一个不包含突变的长片段，然后再采用Gibson装配体系将这两个片段连接成环。所使用的初始模板包括pTO-T7-HPV39N15质粒(其编码N端截短了15个氨基酸的HPV39 L1蛋白；在表2中简写为39L1N15)、pTO-T7-HPV68L1质粒(其编码HPV68 L1蛋白；在表2中简写为68L1N0)、pTO-T7-H39N15-68T4质粒(其编码突变蛋白H39N15-68T4；在表2中简写为H39N15-68T4)和pTO-T7-HPV70N10质粒(其编码N端截短了10个氨基酸的HPV70 L1蛋白；在表2中简写为70L1N10)。用于各个PCR反应的模板和引物见表2，并且，用于扩增短片段的PCR反应的扩增条件设为：94℃变性10分钟；25个循环(94℃变性50秒，指定温度退火一定时间，72℃延伸1分钟)；最后72℃延伸10分钟。用于扩增长片段的PCR反应的扩增条件设为：94℃变性10分钟；25个循环(94℃变性50秒，指定温度退火一定时间，72℃延伸7分30秒)；最后72℃延伸10分钟。所使用的PCR引物的具体序列列于表3。将扩增产物进行电泳，随后使用DNA回收试剂盒(BEYOTIME(碧云天)，货号：D0033)回收目的片段并测定其浓度。按2:1的摩尔比将扩增得到的短片段和长片段混合(总体积3μL)，随后添加3μL 2X Gibson装配预混试剂(2 X Gibson Assembly Master Mix，购自NEB，包含T5  exonuclease，Phusion DNA polymerase，Taq DNA ligase)，并在50℃反应1小时。
用装配后的产物(6μL)转化40μL以氯化钙法制备的感受态大肠杆菌ER2566(购自新英格兰生物实验室公司)。将经转化的大肠杆菌涂布于含卡那霉素的固体LB培养基，并在37℃静置培养10-12小时，直至单菌落清晰可辨。挑取单菌落至含有4mL液体LB培养基(含卡那霉素)的试管中，并在37℃220转/分钟下振荡培养10小时。随后，取1mL菌液于-70℃保存。从大肠杆菌中提取质粒，并利用T7引物对质粒中插入的目的片段的核苷酸序列进行测序。测序结果显示，所构建的各个质粒(表达载体)中插入的目的片段的核苷酸序列分别为SEQ ID NO：17、18、19、20、21、22、23、24，其编码的氨基酸序列为SEQ ID NO：5、6、7、8、9、10、11、12(所对应的蛋白分别命名为H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3，H39N15-68T4-70S5)。
突变蛋白H39N15-68T2与HPV39N15的区别在于：位于野生型HPV39 L1蛋白第117-150位的氨基酸残基被替换为野生型HPV68 L1蛋白第117-151位的氨基酸残基。突变蛋白H39N15-68T3与HPV39N15的区别在于：位于野生型HPV39 L1蛋白第169-181位的氨基酸残基被替换为野生型HPV68 L1蛋白第170-182位的氨基酸残基。突变蛋白H39N15-68T4与HPV39N15的区别在于：位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为野生型HPV68 L1蛋白第270-289位的氨基酸残基。突变蛋白H39N15-68T5与HPV39N15的区别在于：位于野生型HPV39 L1蛋白第347-358位的氨基酸残基被替换为野生型HPV68 L1蛋白第348-359位的氨基酸残基。
突变蛋白H39N15-68T4-70S1与HPV39N15的区别在于：位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为野生型HPV68 L1蛋白第270-289位的氨基酸残基，并且位于野生型HPV39 L1蛋白第53-61位的氨基酸残基被替换为野生型HPV70 L1蛋白的第53-61位的氨基酸残基。突变蛋白H39N15-68T4-70S2与HPV39N15的区别在于：位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为野生型HPV68 L1蛋 白第270-289位的氨基酸残基，并且位于野生型HPV39 L1蛋白第117-140位的氨基酸残基被替换为野生型HPV70 L1蛋白的第117-141位的氨基酸残基。突变蛋白H39N15-68T4-70S3与HPV39N15的区别在于：位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为野生型HPV68 L1蛋白第270-289位的氨基酸残基，并且位于野生型HPV39 L1蛋白第169-181位的氨基酸残基被替换为野生型HPV70 L1蛋白的第170-182位的氨基酸残基。突变蛋白H39N15-68T4-70S5与HPV39N15的区别在于：位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为野生型HPV68 L1蛋白第270-289位的氨基酸残基，并且位于野生型HPV39 L1蛋白第347-358位的氨基酸残基被替换为野生型HPV70 L1蛋白的第348-359位的氨基酸残基。
表2.用于构建表达载体的PCR反应的模板和引物
Figure PCTCN2019089988-appb-000050
表3：所使用的引物的具体序列(SEQ ID NO:38-71)
Figure PCTCN2019089988-appb-000051
Figure PCTCN2019089988-appb-000052
突变蛋白的大量表达
从-70℃冰箱中取出携带重组质粒pTO-T7-H39N15-68T1、pTO-T7-H39N15-68T2、pTO-T7-H39N15-68T3、pTO-T7-H39N15-68T4、pTO-T7-H39N15-68T5、pTO-T7-H39N15-68T4-70S1、pTO-T7-H39N15-68T4-70S2、pTO-T7-H39N15-68T4-70S3、pTO-T7-H39N15-68T4-70S5的大肠杆菌菌液，分别接种入100ml含卡那霉素的LB液体培养基中，在200rpm，37℃下培养大约8小时；然后分别转接入500ml含卡那霉素的LB培养基中(接入1ml菌液)，并继续进行培养。当细菌浓度达到OD 600为0.6左右时，将培养温度降至25℃，并向各培养瓶中加入500μL IPTG，继续培养8小时。培养结束后，离心收集菌体。获得表达了H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3，H39N15-68T4-70S5蛋白的菌体。
表达突变蛋白的菌体破碎
按1g菌体对应10mL裂解液(20mM Tris缓冲液，pH7.2，300mM NaCl)的比例重悬上述得到的菌体。用超声波仪破碎菌体30min。以13500rpm(30000g)离心含有经破碎的菌体的裂解液15min，留取上清(即，菌体破碎上清)。
突变蛋白的色谱纯化
仪器系统：GE Healthcare公司(原Amershan Pharmacia公司)生产的AKTA explorer 100型制备型液相色谱系统。
层析介质：SP Sepharose 4 Fast Flow(GE Healthcare公司)、CHT-Ⅱ(购自Bio－RAD)和Butyl Sepharose 4 Fast Flow(GE Healthcare公司)。
缓冲液：缓冲液A(20mM磷酸盐缓冲液，pH8.0，20mM DTT)；以及缓冲液B(20mM磷酸盐缓冲液，pH8.0，20mM DTT，2M NaCl)。 下面的洗脱程序中用到的含有不同浓度NaCl的缓冲液是由缓冲液A和B按比例混合配置而成。
样品：如上获得的含有H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3，H39N15-68T4-70S5的菌体破碎上清。
洗脱程序为：
(1)用SP Sepharose 4 Fast Flow对菌体破碎上清进行阳离子交换纯化：将样品上柱，然后用含有400mM NaCl的缓冲液(80％缓冲液A+20％缓冲液B)洗脱杂蛋白，然后用含有800mM NaCl的缓冲液(60％缓冲液A+40％缓冲液B)洗脱目的蛋白，并收集由含有800mM NaCl的缓冲液洗脱的级分；
(2)用CHT Ⅱ(羟基磷灰石色谱)对前一步获得的洗脱级分进行色谱纯化：对前一步骤获得的洗脱级分进行稀释，以使得NaCl的浓度降至0.5M；将样品上柱，然后用含有500mM NaCl的缓冲液(75％缓冲液A+25％缓冲液B)洗脱杂蛋白，然后用含有1000mM NaCl(50％缓冲液A+50％缓冲液B)的缓冲液洗脱目的蛋白，并收集由含有1000mM NaCl的缓冲液洗脱的级分；
(3)用HIC(疏水相互作用色谱)对前一步骤获得的洗脱级分进行色谱纯化：将样品上柱，然后用含有1000mM NaCl的缓冲液洗脱杂蛋白，然后用含有200mM NaCl的缓冲液(90％缓冲液A+10％缓冲液B)洗脱目的蛋白，并收集由含有200mM NaCl的缓冲液洗脱的级分。
取步骤(3)获得的洗脱级分150μL，加入30μL 6X Loading Buffer(1L中含有1M TB 6.8 300ml、100％甘油600ml、SDS 120g、溴酚蓝6g、β-巯基乙醇50ml)中，混匀，并于80℃水浴中温育10min。然后取10μl样品于10％SDS-聚丙烯酰胺凝胶中以120V电压电泳120min；然后以考马斯亮兰染色显示电泳条带。电泳结果示于图1中。结果显示，经过上述纯化步骤后，H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3，H39N15-68T4-70S5蛋白的纯度均大于90％。
通过类似的方法，使用大肠杆菌和pTO-T7-HPV39N15质粒制备和纯 化了HPV39N15蛋白；使用大肠杆菌和pTO-T7-HPV68L1N0质粒制备和纯化了HPV68N0蛋白；使用大肠杆菌和pTO-T7-HPV70N10质粒制备和纯化了HPV70N10蛋白。
突变蛋白的免疫印迹实验
按上述方法对经纯化的H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3，H39N15-68T4-70S5蛋白进行电泳。电泳结束后，使用抗HPV L1蛋白的广谱抗体4B3进行Western Blot检测，结果示于图2中。结果显示，H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3，H39N15-68T4-70S5均能够被广谱抗体4B3特异性识别。
实施例2：HPV病毒样颗粒的组装与颗粒形态学检测
HPV病毒样颗粒的组装
取一定体积(约2ml)的蛋白H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3和H39N15-68T4-70S5，分别依次透析至(1)2L储存缓冲液(20mM磷酸钠缓冲液pH 6.5，0.5M NaCl)；(2)2L复性缓冲液(50mM磷酸钠缓冲液pH 6.0，2mM CaCl 2，2mM MgCl 2，0.5M NaCl)；和(3)20mM磷酸钠缓冲液pH 7.0，0.5M NaCl中。在三种缓冲液中各自进行透析12h。
通过类似的方法，将HPV39N15、HPV68N0和HPV70N10蛋白分别组装为HPV39N15 VLP、HPV68N0 VLP和HPV70N10 VLP。
分子筛层析分析
用美国安捷伦公司的1120 Compact LC高效液相色谱系统对经透析的样品进行分子筛层析分析，其中，所使用的分析柱为TSK Gel PW5000xl 7.8x300 mm。分析结果如图3和图4所示。结果显示，包含蛋白H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3，H39N15-68T4-70S5的样品最先出现的蛋白峰均在13-14min左右，与HPV39N15 VLP、HPV68N0 VLP以及HPV70N10 VLP相当。这表明，这些蛋白均可组装成VLP。
沉降速率分析
沉降速率分析所使用的仪器为Beckman XL-A分析型超速离心机，其配有光学检测系统及An-50Ti和An-60Ti转头。采用沉降速率法分析HPV39N15 VLP、HPV68N0 VLP、HPV70N10 VLP以及H39N15-68T1 VLP，H39N15-68T2 VLP，H39N15-68T3 VLP，H39N15-68T4 VLP，H39N15-68T5 VLP，H39N15-68T4-70S1 VLP，H39N15-68T4-70S2 VLP，H39N15-68T4-70S3 VLP，H39N15-68T4-70S5 VLP的沉降系数。结果如图5A-图5L所示。结果显示，H39N15-68T1 VLP，H39N15-68T2 VLP，H39N15-68T3 VLP，H39N15-68T4 VLP，H39N15-68T5 VLP，H39N15-68T4-70S1 VLP，H39N15-68T4-70S2 VLP，H39N15-68T4-70S3 VLP和H39N15-68T4-70S5 VLP的沉降系数分别为136S、151S、138S、145S、135S、124S、108S、99S和127S。这表明，以上制备的突变蛋白H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3和H39N15-68T4-70S5均各自能够组装成大小、形态与野生型VLP(HPV39N15 VLP，115S；HPV68N0 VLP，153S；HPV70N10 VLP，144S)相似的病毒样颗粒。
病毒样颗粒的形态学检测
取100μL含有VLP的样品进行透射电镜观察。所使用的仪器为日本电子公司生产的100kV透射电镜，放大倍数为100,000倍。简言之，取13.5μL样品，用2％磷钨酸pH7.0进行负染，并固定于喷碳的铜网上，然后进行透射电镜观察。观察结果如图6A-图6L所示。结果显示，突变蛋白H39N15-68T1，H39N15-68T2，H39N15-68T3，H39N15-68T4，H39N15-68T5，H39N15-68T4-70S1，H39N15-68T4-70S2，H39N15-68T4-70S3和H39N15-68T4-70S5均可以组装成病毒样颗粒，并且这些突变蛋白所组装成的病毒样颗粒大小均一，颗粒的半径均在25-30nm左右。野生型的HPV39N15，HPV68N0，HPV70N10所组装形成的病毒样颗粒的半径也在 25-30nm左右，大小均一。这表明，这些突变蛋白与野生型HPV39、HPV68和HPV70的L1蛋白类似，能够形成大小均一的VLP。
实施例3：用病毒样颗粒免疫后小鼠血清中的中和抗体滴度的评价1
本实验中，所用病毒样颗粒为：H39N15-68T1 VLP，H39N15-68T2 VLP，H39N15-68T3 VLP，H39N15-68T4 VLP和H39N15-68T5 VLP。
在本实验中，免疫方案如表4所示。将所有小鼠(6周龄BalB/c雌性小鼠)分为3个组:铝佐剂组1(免疫剂量为5μg，使用铝佐剂)，铝佐剂组2(免疫剂量为1μg，使用铝佐剂)，和铝佐剂组3(免疫剂量为0.2μg，使用铝佐剂)。各个组又细分为8个亚组，对照亚组1和2分别用单独的HPV39N15 VLP和单独的HPV68N0 VLP进行免疫，对照亚组3用混合的HPV39/HPV68 VLP(即，HPV39N15 VLP和HPV68N0 VLP的混合物，其中每种VLP均以指定的免疫剂量施用)进行免疫。实验亚组1、2、3、4、5分别用H39N15-68T1 VLP、H39N15-68T2 VLP、H39N15-68T3 VLP、H39N15-68T4 VLP、H39N15-68T5 VLP进行免疫。
对于铝佐剂组1-3，采用腹腔注射方式免疫5只小鼠/亚组，免疫剂量分别为5μg、1μg和0.2μg，注射体积为1ml。所有小鼠均在第0周进行初次免疫，然后在第2和4周各自进行加强免疫一次。在第8周对小鼠进行眼眶采血，并分析血清中的抗HPV39和HPV68抗体的滴度。分析结果如图7A-7C所示。结果显示，H39N15-68T4 VLP能诱导小鼠产生高滴度的针对HPV39的中和抗体，其保护效果稍微弱于同剂量的单独的HPV39N15 VLP，显著优于同剂量的单独的HPV68N0 VLP；并且其能诱导小鼠产生高滴度的针对HPV68的中和抗体，其保护效果稍微弱于同剂量的单独的HPV68N0 VLP，显著优于同剂量的单独的HPV39N15 VLP。这表明，H39N15-68T4 VLP对HPV39和HPV68具有良好的交叉免疫原性和交叉保护性。
表4免疫方案
Figure PCTCN2019089988-appb-000053
Figure PCTCN2019089988-appb-000054
实施例4：病毒样颗粒诱导血清转换的ED 50的评价1
本实验中，所用病毒样颗粒为H39N15-68T4 VLP。
采用铝佐剂、单次腹腔注射方式对6周龄的BalB/c雌鼠(8只)进行免疫，其中，实验组使用H39N15-68T4 VLP(免疫剂量为0.900μg、0.300μg、0.100μg、0.033μg或0.011μg)，对照组使用单独的HPV68N0 VLP(免疫剂量为0.900μg、0.300μg、0.100μg、0.033μg或0.011μg)或单独的HPV39N15 VLP(免疫剂量为0.900μg、0.300μg、0.100μg、0.033μg或0.011μg)或混合的HPV39/HPV68 VLP(即，HPV39N15 VLP与HPV68 N0 VLP的混合物，每种VLP的免疫剂量分别为0.900μg、0.300μg、0.100μg、0.033μg或0.011μg)；免疫体积为1mL。另外，还将用于稀释疫苗的稀释液用作空白对照。每组免疫8只小鼠，并且在免疫后第五周，抽取眼球静脉血，检测血清中的HPV抗体，并通过Reed-Muench法(Reed LJ MH.A simple method of estimating fifty percent  endpoints.Am J Hyg.1938；27:493-7)来计算各个样品诱导血清转换(即，诱导小鼠产生抗体)的ED 50。结果如表5-8所示。
表5.HPV39N15 VLP诱导小鼠产生抗HPV39、抗HPV68抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000055
表6.HPV68N0 VLP诱导小鼠产生抗HPV39、抗HPV68抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000056
表7.H39N15-68T4 VLP诱导小鼠产生抗HPV39、抗HPV68抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000057
表8.混合的HPV39/HPV68 VLP诱导小鼠产生抗HPV39、抗HPV68抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000058
结果显示，在免疫小鼠5周后，H39N15-68T4 VLP诱导小鼠产生抗HPV39抗体的ED 50与单独的HPV39N15 VLP相当，且显著优于单独的HPV68N0 VLP；并且，其诱导小鼠产生抗HPV68抗体的ED 50虽稍弱于单独的HPV68N0 VLP，但显著优于单独的HPV39N15 VLP。这表明，H39N15-68T4 VLP对HPV68和HPV39具有良好的交叉免疫原性和交叉保护性。
实施例5：用病毒样颗粒免疫后小鼠血清中的中和抗体滴度的评价2
本实验中，所用的病毒颗粒为H39N15-68T4-70S1 VLP、H39N15-68T4-70S2 VLP、H39N15-68T4-70S3 VLP、H39N15-68T4-70S5 VLP。
在本实验中，免疫方案如表9所示。将所有小鼠(6周龄BalB/c雌性小鼠)分为3个组:铝佐剂组1(免疫剂量为5μg，使用铝佐剂)，铝佐剂组2(免疫剂量为1μg，使用铝佐剂)，和铝佐剂组3(免疫剂量为0.2μg，使用铝佐剂)。各个组再细分为8个亚组，对照亚组1、2、3分别用单独的HPV39N15 VLP、单独的HPV68N0 VLP、单独的HPV70N10 VLP进行免疫，对照亚组用4混合的HPV39/HPV68/HPV70 VLP(即，HPV39N15 VLP、HPV68N0 VLP和HPV70N10 VLP的混合物，其中每种VLP均以指定的免疫剂量施用)进行免疫。实验亚组1、2、3、4分别用H39N15-68T4-70S1 VLP、H39N15-68T4-70S2 VLP、H39N15-68T4-70S3 VLP和H39N15-68T4-70S5 VLP进行免疫。
对于铝佐剂组1-3，采用腹腔注射方式免疫5只小鼠/亚组，免疫剂量分别为5μg、1μg、0.2μg，注射体积为1ml。所有小鼠均在第0周进行初次免疫，然后在第2和4周各自进行加强免疫一次。在第8周对小鼠进行眼眶采血，并分析血清中的抗HPV39、HPV68和HPV70抗体的滴度。分析结果如图8A-8C所示。结果显示，H39N15-68T4-70S2 VLP、H39N15-68T4-70S3 VLP、H39N15-68T4-70S5 VLP都能诱导小鼠产生高滴度的针对HPV39的中和抗体，其保护效果虽然略微弱于同剂量的单独的HPV39N15 VLP以及混合的HPV39/HPV68/HPV70 VLP，但仍显著优于同剂量的单独的HPV68N0 VLP或单独的HPV70N10 VLP；并且其能诱导小鼠产生高滴度的针对HPV68的中和抗体，其保护效果与同剂量的单独的HPV68N0 VLP以及混合的HPV39/HPV68/HPV70 VLP相当，且显著优于同剂量的单独的HPV39N15 VLP或单独的HPV70N10 VLP；并且其能诱导小鼠产生高滴度的针对HPV70的中和抗体，其保护效果与同剂量的单独的HPV70N10 VLP以及混合的HPV39/HPV68/HPV70 VLP相当，且显著优于同剂量的单独的HPV39N15 VLP或单独的HPV68N0 VLP。这表明，H39N15-68T4-70S2 VLP、H39N15-68T4-70S3 VLP、H39N15- 68T4-70S5 VLP对HPV39、HPV68和HPV70具有良好的交叉免疫原性和交叉保护性。
表9免疫方案
Figure PCTCN2019089988-appb-000059
实施例6：病毒样颗粒诱导血清转换的ED 50的评价2
本实验中，所用病毒样颗粒为H39N15-68T4-70S2 VLP、H39N15-68T4-70S3 VLP和H39N15-68T4-70S5 VLP。
采用铝佐剂、单次腹腔注射方式对6周龄的BalB/c雌鼠(8只)进行免疫，其中，实验组使用H39N15-68T4-70S2 VLP、H39N15-68T4-70S3 VLP或H39N15-68T4-70S5 VLP(免疫剂量为0.900μg、0.300μg、 0.100μg、0.033μg或0.011μg)；对照组使用单独的HPV39N15 VLP、单独的HPV68N0 VLP或单独的HPV70N10 VLP(免疫剂量为0.900μg、0.300μg、0.100μg、0.033μg或0.011μg)，或者混合的HPV39/HPV68/HPV70 VLP(即，HPV39N15 VLP、HPV68N0 VLP和HPV70N10 VLP的混合物，每种VLP的免疫剂量各为0.900μg、0.300μg、0.100μg、0.033μg、0.011μg)；免疫体积为1mL。另外，还将用于稀释疫苗的稀释液用作空白对照。每组免疫8只小鼠，并且在免疫后第五周，抽取眼球静脉血，检测血清中的HPV抗体，并通过Reed-Muench法(Reed LJ MH.A simple method of estimating fifty percent endpoints.Am J Hyg.1938；27:493-7)来计算各个样品诱导血清转换(即，诱导小鼠产生抗体)的ED 50。结果如表10-16所示。
表10.HPV39N15 VLP诱导小鼠产生抗HPV39、抗HPV68抗HPV70抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000060
表11.HPV68N0 VLP诱导小鼠产生抗HPV39、抗HPV68抗HPV70抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000061
Figure PCTCN2019089988-appb-000062
表12.HP70N10 VLP诱导小鼠产生抗HPV39、抗HPV68抗HPV70抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000063
表13.混合的HPV39/HPV68/HPV70 VLP诱导小鼠产生抗HPV39、抗HPV68和抗HPV70抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000064
Figure PCTCN2019089988-appb-000065
表14.H39N15-68T4-70S2 VLP诱导小鼠产生抗HPV39、抗HPV68和抗HPV70抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000066
表15.H39N15-68T4-70S3 VLP诱导小鼠产生抗HPV39、抗HPV68和抗HPV70抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000067
表16.H39N15-68T4-70S5 VLP诱导小鼠产生抗HPV39、抗HPV68和抗HPV70抗体(血清转换)的ED 50
Figure PCTCN2019089988-appb-000068
结果显示，在免疫小鼠5周后，H39N15-68T4-70S2 VLP和H39N15-68T4-70S5 VLP诱导小鼠产生抗HPV39的ED 50与单独的HPV39N15 VLP以及混合的HPV39/HPV68/HPV70 VLP相当，且显著优于单独的HPV68N0 VLP以及单独的HPV70N10 VLP；并且，其诱导小鼠产生抗HP68的ED 50与单独的HPV68N0 VLP以及混合的HPV39/HPV68/HPV70 VLP相当，且显著优于单独的HPV39N15 VLP以及单独的HPV70N10 VLP；并且，其诱导小鼠产生抗HPV70的ED 50与单独的HPV70N10 VLP以及混合的HPV39/HPV68/HPV70 VLP相当，且显著优于单独的HPV39N15 VLP以及单独的HPV68N0 VLP。这表明，H39N15-68T4-70S2 VLP和H39N15-68T4-70S5 VLP对HPV39、HPV68以及HPV70具有良好的交叉免疫原性和交叉保护性。
实施例7：病毒样颗粒的热稳定性的评价
使用购自美国GE公司(原MicroCal公司)的差温量热仪VP Capillary DSC来评价HPV39N15蛋白、HPV68N0蛋白、HPV70N10蛋白、H39N15-68T4蛋白、H39N15-68T4-70S2蛋白、H39N15-68T4-70S5蛋白所形成的VLP的热稳定性，使用所述蛋白的储存缓冲液作为对照，并 且以1.5℃/min的升温速率在10℃-90℃区间对各蛋白进行扫描。检测结果如图9所示。结果显示，H39N15-68T4蛋白、H39N15-68T4-70S2蛋白、H39N15-68T4-70S5蛋白所形成的VLP均具有极高的热稳定性。
实施例8：H39N15-68T4-70S2 VLP和H39N15-68T4-70S5 VLP的三维结构的重建
通过使用冷冻电镜的三维结构重建实验(Wolf M,Garcea RL,Grigorieff N.et al.Proc Natl Acad Sci U S A.(2010),107(14):6298-303)来重建H39N15-68T4-70S2 VLP和H39N15-68T4-70S5 VLP的三维结构。简而言之，在H39N15-68T4-70S2的冷冻电镜图像中(图10中的A)选取6410个大小均一、直径超过50nm的颗粒进行计算机重叠和结构重建，从而获取H39N15-68T4-70S2 VLP的三维结构。所获得的三维结构如图10中的B所示(分辨率为
Figure PCTCN2019089988-appb-000069
)。另外，在H39N15-68T4-70S5 VLP的冷冻电镜图像中(图10中的C)选取617个大小均一、直径超过50nm的颗粒进行计算机重叠和结构重建，从而获取H39N15-68T4-70S5 VLP的三维结构。所获得的三维结构如图10中的D所示(分辨率为
Figure PCTCN2019089988-appb-000070
)。结果显示，H39N15-68T4-70S2 VLP和H39N15-68T4-70S5 VLP均是由72个壳粒(形态亚单位，五聚体)形成的T＝7的二十面体结构(h＝1,k＝2)。与一般的符合准等价原理的二十面体病毒衣壳不同，H39N15-68T4-70S2 VLP和H39N15-68T4-70S5 VLP结构中的所有组成亚单位均为五聚体，而不存在六聚体，并且，上述两种VLP的最外围直径均为约60nm。这与之前报道的天然HPV病毒颗粒及通过真核表达系统(例如，痘病毒表达系统)制备的HPV VLP的三维结构(Baker TS,Newcomb WW,Olson NH.et al.Biophys J.(1991),60(6):1445-1456；Hagensee ME,Olson NH,Baker TS,et al.J Virol.(1994),68(7):4503-4505；Buck CB,Cheng N,Thompson CD.et al.J Virol.(2008),82(11):5190-7)类似。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (12)

  1. 一种突变的HPV39 L1蛋白或其变体，其中，所述突变的HPV39 L1蛋白与野生型HPV39 L1蛋白相比，具有下述突变：
    (1)N端截短了1-25个氨基酸，例如1-5个、1-10个、1-15个、1-20个、5-15个、10-15个、10-20个、15-20个氨基酸；和
    (2)位于野生型HPV39 L1蛋白第269-288位的氨基酸残基被替换为第二型别的野生型HPV的L1蛋白的相应位置的氨基酸残基；
    并且，所述变体与所述突变的HPV39 L1蛋白相异仅在于一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失，且保留了所述突变的HPV39 L1蛋白的功能，即，能够诱导针对至少两个型别的HPV(例如，HPV39和HPV68，或者HPV39、HPV68和HPV70)的中和抗体；
    优选地，所述突变的HPV39 L1蛋白任选地还具有下述突变：
    (3)(a)位于野生型HPV39 L1蛋白第117-140位的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；或
    (b)位于野生型HPV39 L1蛋白第169-181的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；
    (c)位于野生型HPV39 L1蛋白第347-358的氨基酸残基被替换为第三型别的野生型HPV L1蛋白的相应位置的氨基酸残基；
    优选地，所述突变的HPV39 L1蛋白与野生型HPV39 L1蛋白相比，N端截短了N端截短了3个、5个、8个、10个、12个、15个、18个、20个或22个氨基酸；
    优选地，所述突变的HPV39 L1蛋白与野生型HPV39 L1蛋白相比，N端截短了15个氨基酸；
    优选地，所述第二型别的野生型HPV为HPV68；优选地，(2)中所述的相应位置的氨基酸残基为野生型HPV68 L1蛋白第270-289位的氨基酸残基；
    优选地，所述第三型别的野生型HPV为HPV70；优选地，(3)(a)中所述的相应位置的氨基酸残基为野生型HPV70 L1蛋白第117-141位的氨基酸残基；优选地，(3)(b)中所述的相应位置的氨基酸残基为野生型 HPV70 L1蛋白第170-182位的氨基酸残基；优选地，(3)(c)中所述的相应位置的氨基酸残基为野生型HPV70 L1蛋白第348-359位的氨基酸残基；
    优选地，所述野生型HPV39 L1蛋白具有如SEQ ID NO:1所示的氨基酸序列；
    优选地，所述野生型HPV68 L1蛋白具有如SEQ ID NO:2所示的氨基酸序列；
    优选地，所述野生型HPV70 L1蛋白具有如SEQ ID NO:3所示的氨基酸序列；
    优选地，所述突变的HPV39 L1蛋白具有选自下列的氨基酸序列：SEQ ID NO:7、10、11和12。
  2. 一种分离的核酸，其编码权利要求1所述的突变的HPV39 L1蛋白或其变体，
    优选地，所述分离的核酸具有选自下列的核苷酸序列：SEQ ID NO:19、22、23和24。
  3. 包含权利要求2所述的分离的核酸的载体。
  4. 包含权利要求2所述的分离的核酸和/或权利要求3所述的载体的宿主细胞。
  5. 一种HPV病毒样颗粒，其含有权利要求1所述的突变的HPV39 L1蛋白或其变体，或者由权利要求1所述的突变的HPV39 L1蛋白或其变体组成或形成。
  6. 一种组合物，其包含权利要求1所述的突变的HPV39 L1蛋白或其变体，或权利要求2所述的分离的核酸，或权利要求3所述的载体，或权利要求4所述的宿主细胞，或权利要求5所述的HPV病毒样颗粒。
  7. 一种药物组合物或疫苗，其包含权利要求5所述的HPV病毒样颗 粒，任选地还包含药学可接受的载体和/或赋形剂，
    优选地，所述HPV病毒样颗粒以预防HPV感染或由HPV感染导致的疾病的有效量存在；
    优选地，所述HPV感染是一个或多个型别的HPV感染(例如，HPV39感染、HPV68感染和/或HPV70感染)；
    优选地，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
  8. 制备权利要求1所述的突变的HPV39L1蛋白或其变体的方法，其包括，在宿主细胞中表达所述突变的HPV39 L1蛋白或其变体，然后从所述宿主细胞的培养物中回收所述突变的HPV 39L1蛋白或其变体；
    优选地，所述宿主细胞为大肠杆菌；
    优选地，所述方法包括步骤：在大肠杆菌中表达所述突变的HPV39 L1蛋白或其变体，然后从所述大肠杆菌的裂解上清中纯化得到所述突变的HPV39 L1蛋白或其变体；优选地，通过色谱法(例如，阳离子交换色谱，羟基磷灰石色谱和/或疏水相互作用色谱)，从所述大肠杆菌的裂解上清中回收所述突变的HPV39 L1蛋白或其变体。
  9. 一种制备疫苗的方法，其包括将权利要求5所述的HPV病毒样颗粒与药学可接受的载体和/或赋形剂混合。
  10. 一种预防HPV感染或由HPV感染所导致的疾病的方法，其包括将预防有效量的权利要求5所述的HPV病毒样颗粒或权利要求7所述的药物组合物或疫苗施用给受试者，
    优选地，所述HPV感染是一个或多个型别的HPV感染(例如，HPV39感染、HPV68感染和/或HPV70感染)；
    优选地，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
  11. 权利要求1所述的突变的HPV39 L1蛋白或其变体或权利要求5的HPV病毒样颗粒在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于预防HPV感染或由HPV感染所导致的疾病，
    优选地，所述HPV感染是一个或多个型别的HPV感染(例如，HPV39感染、HPV68感染和/或HPV70感染)；
    优选地，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
  12. 权利要求1所述的突变的HPV39 L1蛋白或其变体或权利要求5的HPV病毒样颗粒在制备药物组合物或疫苗，其用于预防HPV感染或由HPV感染所导致的疾病，
    优选地，所述HPV感染是一个或多个型别的HPV感染(例如，HPV39感染、HPV68感染和/或HPV70感染)；
    优选地，所述由HPV感染所导致的疾病选自宫颈癌和尖锐湿疣。
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