WO2019011331A1 - 一种人乳头瘤病毒16型l1蛋白的突变体 - Google Patents

一种人乳头瘤病毒16型l1蛋白的突变体 Download PDF

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WO2019011331A1
WO2019011331A1 PCT/CN2018/095632 CN2018095632W WO2019011331A1 WO 2019011331 A1 WO2019011331 A1 WO 2019011331A1 CN 2018095632 W CN2018095632 W CN 2018095632W WO 2019011331 A1 WO2019011331 A1 WO 2019011331A1
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protein
hpv
hpv16
vlp
type
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PCT/CN2018/095632
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French (fr)
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顾颖
李少伟
宋硕
何茂洲
李智海
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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Priority to EP18832785.2A priority Critical patent/EP3653638A4/en
Priority to JP2020501525A priority patent/JP7224332B2/ja
Priority to BR112020000833-8A priority patent/BR112020000833A2/pt
Priority to US16/630,673 priority patent/US11213580B2/en
Priority to KR1020207003760A priority patent/KR102567627B1/ko
Publication of WO2019011331A1 publication Critical patent/WO2019011331A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/01—DNA viruses
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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
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of molecular virology and immunology.
  • the present invention relates to a mutant HPV16 L1 protein (or variant thereof), a coding sequence thereof and a method of preparation, and a virus-like particle comprising the same, the protein (or variant thereof) and virus-like particles capable of inducing Neutralizing antibodies against at least two types of HPV (eg, HPV 16 and HPV 35, or HPV 16, HPV 35, and HPV 31), thereby being useful for preventing at least two types of HPV infections and diseases caused by the infections For example, cervical cancer and genital warts.
  • the invention further relates to the use of the above proteins and virus-like particles for the preparation of a pharmaceutical composition or vaccine, which pharmaceutical composition or vaccine is useful for preventing at least two types of HPV infections and diseases caused by said infections For example, cervical cancer and genital warts.
  • HPV Human papillomavirus
  • HPV Human papillomavirus
  • HPV Human papillomavirus
  • HPV can be divided into high-risk type and low-risk type, among which high-risk HPV infection is confirmed to be the main cause of genital cancer including cervical cancer in women; low-risk type mainly causes condyloma acuminata .
  • the most effective way to prevent and control HPV infection is to administer HPV vaccines, especially to high-risk HPV-producing vaccines that cause cervical cancer.
  • the major capsid protein L1 of HPV has the property of self-assembly into a virus-like particle (VLP).
  • the HPV VLP is a icosahedral stereo-symmetric structure composed of a pentamer of 72 major capsid proteins L1 (Doorbar, J. and P. H. Gallimore. 1987. J Virol, 61(9): 2793-9).
  • the structure of HPV VLPs is highly similar to that of native HPV, retaining most of the neutralizing epitopes of natural viruses and inducing high titers of neutralizing antibodies (Kirnbauer, R., F.Booy, et al. 1992 Proc Natl Acad Sci U S A 89(24): 12180-4).
  • HPV VLPs primarily induce neutralizing antibodies against homologous HPV, producing protective immunity against homologous HPV, while low cross-protection exists only between some highly homologous types (Sara L. Bissett, Giada Mattiuzzo, et al. 2014 Vaccine. 32: 6548-6555). Therefore, the scope of protection of existing HPV vaccines is very limited. Usually, a type of HPV VLP can only be used to prevent this type of HPV infection. In this case, if you want to expand the scope of HPV vaccine protection, you can only add more types of HPV VLPs to the vaccine.
  • HPV vaccines including Merck (It is a four-valent vaccine for HPV 16, 18, 6 and 11), GSK's (It is a bivalent vaccine for HPV 16, 18) and Merck (It is a nine-valent vaccine), which is made by mixing multiple types of HPV VLPs.
  • Merck It is a four-valent vaccine for HPV 16, 18, 6 and 11
  • GSK's It is a bivalent vaccine for HPV 16, 18
  • Merck It is a nine-valent vaccine
  • HPV virus-like particles capable of inducing protective neutralizing antibodies against multiple types of HPV to more effectively and effectively prevent multiple types of HPV infections and the resulting diseases such as cervical cancer. And genital warts.
  • the present invention is based, at least in part, on the inventors' surprising finding that a particular segment of the human papillomavirus (HPV) type 16 L1 protein is replaced with a corresponding segment of a second type of HPV (eg, HPV35) L1 protein. Thereafter, the obtained mutated HPV16 L1 protein is capable of inducing the body to produce high titer neutralizing antibodies against HPV16 and a second type of HPV (eg, HPV35) with a protective effect of mixed HPV16 VLPs and a second type of HPV.
  • the VLP is comparable and its protection against HPV 16 is comparable to that of a single HPV 16 VLP, and the protection against a second type of HPV (eg HPV 35) is comparable to a separate second type of HPV VLP.
  • another specific segment of the HPV16 L1 protein may be further substituted with a corresponding segment of the third type HPV (eg, HPV31) L1 protein, thereby obtaining a double substitution.
  • the mutated HPV16 L1 protein is capable of inducing the body to produce high titer neutralizing antibodies against HPV16, a second type of HPV (eg HPV35) and a third type of HPV (eg HPV31) with a protective effect and mixed HPV16 VLP
  • the second type of HPV VLP is equivalent to the third type of HPV VLP; and its protection against HPV16 is comparable to that of the HPV16 VLP alone, and the protection against the second type of HPV (eg HPV35) is separate.
  • the second type of HPV VLP is comparable, and the protection against the third type of HPV (such as HPV31) is comparable to the third type of HPV VLP alone.
  • the invention provides a mutant HPV16 L1 protein or variant thereof, wherein the mutated HPV16 L1 protein has the following mutations compared to the wild-type HPV16 L1 protein:
  • N-terminally truncated 4-50 amino acids such as 4, 6, 8, 10, 20, 30 or 40 amino acids
  • the mutated HPV16 L1 protein also has the following mutations:
  • the variant differs from the mutated HPV16 L1 protein by only one or several (eg, 1, 2, 3, 4, 5, 6, 7, 8, or 9) a substitution (preferably conservative substitution), addition or deletion of an amino acid, and retaining the function of the mutated HPV16 L1 protein, ie, capable of inducing HPV against at least two types (eg, HPV16 and HPV35, or HPV16, Neutralizing antibodies to HPV35 and HPV31).
  • a substitution preferably conservative substitution
  • HPV16 L1 protein capable of inducing HPV against at least two types (eg, HPV16 and HPV35, or HPV16, Neutralizing antibodies to HPV35 and HPV31).
  • the mutated HPV16 L1 protein has a N-terminal truncation of 30 or 40 amino acids compared to the wild-type HPV16 L1 protein.
  • the mutated HPV16 L1 protein is N-terminally truncated by 30 amino acids compared to the wild-type HPV16 L1 protein.
  • the second type of wild-type HPV is HPV35.
  • the amino acid residue at the corresponding position described in (2) is the amino acid residue at positions 266-288 of the wild-type HPV35 L1 protein.
  • the third type of wild-type HPV is HPV31.
  • the amino acid residue at the corresponding position described in (3) is the amino acid residue at positions 50-62 of the wild-type HPV31 L1 protein.
  • the amino acid residue at the corresponding position described in (4) is the amino acid residue at positions 127-142 of the wild-type HPV31 L1 protein.
  • the amino acid residue at the corresponding position described in (5) is the amino acid residue at positions 177-182 of the wild-type HPV31 L1 protein.
  • the wild type HPV 16 L1 protein has the amino acid sequence set forth in SEQ ID NO: 1.
  • the wild type HPV35 L1 protein has the amino acid sequence set forth in SEQ ID NO:2.
  • the wild type HPV31 L1 protein has the amino acid sequence set forth in SEQ ID NO:3.
  • sequence of the amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein is set forth in SEQ ID NO:25.
  • sequence of the amino acid residues at positions 50-62 of the wild-type HPV31 L1 protein is set forth in SEQ ID NO:26.
  • sequence of amino acid residues at positions 127-142 of the wild-type HPV31 L1 protein is set forth in SEQ ID NO:27.
  • sequence of the amino acid residues at positions 177-182 of the wild-type HPV31 L1 protein is set forth in SEQ ID NO:28.
  • the mutated HPV16 L1 protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, 10, 11.
  • the invention provides an isolated nucleic acid encoding a mutated HPV 16 L1 protein or variant thereof as described above.
  • the invention provides a vector comprising the isolated nucleic acid.
  • the isolated nucleic acids of the invention have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 19, 21, 22, 23.
  • Vectors useful for insertion of a polynucleotide of interest include, but are not limited to, cloning vectors and expression vectors.
  • the vector is, for example, a plasmid, a cosmid, a phage, and the like.
  • the invention also relates to a host cell comprising the isolated nucleic acid or vector 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 (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the invention may also be a cell line, such as a 293T cell.
  • the present invention relates to an HPV virus-like particle, wherein the virus-like particle comprises the mutated HPV16 L1 protein of the present invention or a variant thereof, or consists or is formed by the mutated HPV16 L1 protein of the present invention or a variant thereof .
  • the HPV virus-like particle of the invention comprises a mutated HPV16 L1 protein that is N-terminally truncated by 4-50 amino acids, such as 4, 6, 8 compared to the wild-type HPV16 L1 protein. , 10, 20, 30 or 40 amino acids, and the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein.
  • the HPV virus-like particle of the invention comprises a mutated HPV16 L1 protein that is N-terminally truncated by 4-50 amino acids, such as 4, 6, 8 compared to the wild-type HPV16 L1 protein. , 10, 20, 30 or 40 amino acids, and the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and are located in wild-type HPV16 The amino acid residues at positions 76-87 of the L1 protein were replaced with amino acid residues at positions 50-62 of the wild-type HPV31 L1 protein.
  • the HPV virus-like particle of the invention comprises a mutated HPV16 L1 protein that is N-terminally truncated by 4-50 amino acids, such as 4, 6, 8 compared to the wild-type HPV16 L1 protein. , 10, 20, 30 or 40 amino acids, and the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and are located in wild-type HPV16 The amino acid residues at positions 152-167 of the L1 protein were replaced with amino acid residues at positions 127-142 of the wild type HPV31 L1 protein.
  • the HPV virus-like particle of the invention comprises a mutated HPV16 L1 protein that is N-terminally truncated by 4-50 amino acids, such as 4, 6, 8 compared to the wild-type HPV16 L1 protein. , 10, 20, 30 or 40 amino acids, and the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and are located in wild-type HPV16 The amino acid residues at positions 202-207 of the L1 protein were replaced with amino acid residues at positions 177-182 of the wild type HPV31 L1 protein.
  • the HPV virus-like particle of the invention comprises a mutated HPV16 L1 protein having the sequence set forth in SEQ ID NOs: 7, 9, 10 or 11.
  • the invention also relates to a composition
  • a composition comprising the above-described mutated HPV16 L1 protein or variant thereof, or the above isolated nucleic acid or vector or host cell or HPV virus-like particle.
  • the composition comprises a mutated HPV16 L1 protein of the invention or a variant thereof.
  • the composition comprises HPV virus-like particles of the invention.
  • the invention also relates to a pharmaceutical composition or vaccine comprising an HPV virus-like particle 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 for preventing HPV infection or diseases caused by HPV infection such as cervical cancer and condyloma acuminatum.
  • the HPV virus-like particle is present in an amount effective to prevent HPV infection or a disease caused by HPV infection.
  • the HPV infection is one or more types of HPV infection (eg, HPV 16 infection, HPV 35 infection, and/or HPV 31 infection).
  • the disease caused by HPV infection is selected from the group consisting of cervical cancer and condyloma acuminatum.
  • compositions or vaccines of the invention may be administered by methods well known in the art such as, but not limited to, administration by oral or injection.
  • a particularly preferred mode of administration is injection.
  • the pharmaceutical compositions or vaccines of the invention are administered in unit dosage form.
  • the amount of HPV virus-like particles contained per unit dose is from 5 ⁇ g to 80 ⁇ g, preferably from 20 ⁇ g to 40 ⁇ g.
  • the present invention relates to a method of producing a mutant HPV16 L1 protein or variant thereof as described above, which comprises expressing the mutant HPV16 L1 protein or a variant thereof in a host cell, and then The mutated HPV16 L1 protein or variant thereof is recovered from the culture of the host cell.
  • the host cell is E. coli.
  • the method comprises the steps of: expressing the mutated HPV16 L1 protein or variant thereof in E. coli, and then purifying the mutated HPV 16 from the lysed supernatant of the E. coli L1 protein or a variant thereof.
  • the mutated HPV16 L1 is recovered from the lysed supernatant of the E. coli by chromatography (eg, cation exchange chromatography, hydroxyapatite chromatography, and/or hydrophobic interaction chromatography). Protein or 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 of preventing HPV infection or a disease caused by HPV infection comprising administering a prophylactically effective amount of an HPV virus-like particle or pharmaceutical composition or vaccine according to the present invention to a subject .
  • the HPV infection is one or more types of HPV infection (eg, HPV 16 infection, HPV 35 infection, and/or HPV 31 infection).
  • the disease caused by HPV infection includes, but is not limited to, cervical cancer and condyloma acuminata.
  • the subject is a mammal, such as a human.
  • the invention relates to the use of a mutant HPV16 L1 protein or variant thereof or HPV virus-like particle according to the invention for the preparation of a pharmaceutical composition or vaccine for the prevention of HPV infection or by A disease caused by HPV infection.
  • the HPV infection is one or more types of HPV infection (eg, HPV 16 infection, HPV 35 infection, and/or HPV 31 infection).
  • the disease caused by HPV infection includes, but is not limited to, cervical cancer and condyloma acuminata.
  • the term "type 2 wild-type HPV” means another type of wild-type HPV different from HPV16.
  • the second type wild type HPV is preferably wild type HPV35.
  • the term “type 3 wild-type HPV” refers to another type of wild-type HPV that differs from HPV 16 and is different from the second type of wild-type HPV.
  • the third type of wild-type HPV is preferably wild type HPV31.
  • corresponding position means an equivalent position in a sequence to be compared when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage identity.
  • wild-type HPV16 L1 protein refers to the major capsid protein L1 naturally present in human papillomavirus type 16 (HPV16).
  • HPV16 human papillomavirus type 16
  • sequence of the wild-type HPV16 L1 protein is well known in the art and can be found in various public databases (e.g., NCBI database accession numbers ANA05496.1, ANA05539.1, AGC65525.1, AAV91659.1 and AAD33259.1).
  • amino acid residues 292 to 316 of the wild type HPV16 L1 protein means the amino acid residues 292 to 316 of the polypeptide represented by SEQ ID NO: 1.
  • wild-type HPV 16 may include a plurality of isolates, and there may be a difference between the amino acid sequences of the L1 proteins of the various isolates.
  • wild-type HPV16 L1 protein includes not only the protein represented by SEQ ID NO: 1, but also the L1 protein of various HPV16 isolates (for example, ANA05496.1, ANA05539.1, AGC65525. 1, HPV16 L1 protein shown in AAV91659.1 and AAD33259.1).
  • sequence fragment of the wild-type HPV16 L1 protein includes not only the sequence fragment of SEQ ID NO: 1, but also the corresponding sequence fragment in the L1 protein of various HPV16 isolates.
  • amino acid residues 292 to 316 of the wild-type HPV16 L1 protein includes the amino acid residues 292 to 316 of SEQ ID NO: 1, and the corresponding fragments of the L1 proteins of various HPV16 isolates.
  • wild-type HPV35 L1 protein refers to the major capsid protein L1 naturally present in human papillomavirus type 35 (HPV35).
  • HPV35 human papillomavirus type 35
  • the sequence of the wild-type HPV35 L1 protein is well known in the art and can be found in various public databases (e.g., NCBI database accession numbers P27232.2, ACV84022.1, AEI61365.1, AEI61429.1, and ACV84029.1).
  • amino acid residue at positions 266 to 288 of the wild-type HPV35 L1 protein means the amino acid residues 266 to 288 of the polypeptide represented by SEQ ID NO: 2.
  • wild-type HPV35 can include a variety of isolates, and there may be differences between the amino acid sequences of the L1 proteins of various isolates.
  • wild-type HPV35 L1 protein includes not only the protein represented by SEQ ID NO: 2 but also the L1 protein of various HPV35 isolates (for example, P27232.2, ACV84022.1, AEI61365. 1. HPV35 L1 protein shown in AEI61429.1 and ACV84029.1).
  • sequence fragment of the wild type HPV35 L1 protein includes not only the sequence fragment of SEQ ID NO: 2 but also the corresponding sequence fragment of the L1 protein of various HPV35 isolates.
  • amino acid residues 266-288 of the wild-type HPV35 L1 protein includes amino acid residues 266-288 of SEQ ID NO: 2, and corresponding fragments of the L1 proteins of various HPV35 isolates.
  • wild-type HPV31 L1 protein refers to the major capsid protein L1 naturally present in human papillomavirus type 31 (HPV31).
  • HPV31 human papillomavirus type 31
  • sequence of the wild-type HPV31 L1 protein is well known in the art and can be found in various public databases (e.g., NCBI database accession numbers P17388.1, AEI60965.1, ANB49655.1, and AEI61021.1).
  • amino acid residue at positions 177 to 182 of the wild type HPV31 L1 protein means the amino acid residues 177 to 182 of the polypeptide represented by SEQ ID NO: 3.
  • wild-type HPV31 may include a plurality of isolates, and there may be a difference between the amino acid sequences of the L1 proteins of the various isolates.
  • wild type HPV31 L1 protein includes not only the protein represented by SEQ ID NO: 3 but also the L1 protein of various HPV31 isolates (for example, P17388.1, AEI60965.1, ANB49655. 1 and HPV31 L1 protein shown in AEI61021.1).
  • sequence fragment of the wild type HPV31 L1 protein includes not only the sequence fragment of SEQ ID NO: 3 but also the corresponding sequence fragment of the L1 protein of various HPV31 isolates.
  • amino acid residues 177 to 182 of the wild type HPV31 L1 protein includes amino acid residues 177 to 182 of SEQ ID NO: 3, and corresponding fragments of the L1 proteins of various HPV31 isolates.
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percentage identity, the sequences to be compared are in the equivalent position. Fragment of.
  • the expression "the N-terminal truncation of X amino acids” means that the methionine residue encoded by the initiation codon (for initiation of protein translation) replaces the amino acid at position N of the N-terminus of the protein. Residues.
  • an HPV16 L1 protein having a N-terminally truncated 30 amino acid is obtained by replacing the amino acid residues 1-30 of the N-terminus of the wild-type HPV16 L1 protein with a methionine residue encoded by a start codon. protein.
  • the term "variant” refers to a protein having an amino acid sequence which is compared to the amino acid sequence of a mutated HPV16 L1 protein of the invention (such as the protein of SEQ ID NO: 7, 9, 10 or 11). a substitution (preferably conservative substitution), addition or deletion of one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acids, or It has at least 90%, 95%, 96%, 97%, 98%, or 99% identity and it retains the function of the mutated HPV16 L1 protein.
  • the term "function of a mutated HPV16 L1 protein” means that a neutralizing antibody capable of inducing production of at least two types of HPV (for example, HPV16 and HPV35, or HPV16, HPV35 and HPV31) can be induced.
  • the term “identity” is a measure of the similarity to a nucleotide sequence or amino acid sequence. The sequences are usually arranged to get the maximum match. "Identity" itself has a meaning that is well known in the art and can be calculated using published algorithms such as BLAST.
  • the term "identity” is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the 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 is conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight 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 essential 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 similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including Substitution of residues by formation of a covalent bond or a hydrogen bond, etc.).
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • conservative substitutions generally refer to the replacement of 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 consisting of E. coli (strain) and a vector, wherein E. coli (strain) is derived from commercially available strains such as, but not limited to, ER2566, BL21 ( DE3), B834 (DE3), BLR (DE3).
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of a protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby 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; phage; cosmid and the like.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient which is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which 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 adjusting agents, 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 nonionic surfactants such as Tween-80; adjuvants include, but are not limited to, aluminum adjuvants (eg, hydroxides) 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 nonionic surfactants such as Tween-80
  • adjuvants include, but are not limited to, aluminum adjuvants (eg, hydroxides) Aluminum), Freund's adjuvant (eg complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.
  • an effective amount means an amount effective to achieve the intended purpose.
  • an effective amount to prevent disease e.g., HPV infection
  • an effective amount that is effective to prevent, prevent, or delay the onset of a disease e.g., HPV infection. Determination of such effective amounts is well within the abilities of those skilled in the art.
  • chromatography includes, but is not limited to, ion exchange chromatography (eg cation exchange chromatography), hydrophobic interaction chromatography, adsorption chromatography (eg hydroxyapatite chromatography), gel filtration (gel discharge) Resistance) chromatography, affinity chromatography.
  • the term "lytic supernatant” refers to a solution produced by disrupting a host cell, such as E. coli, in a lysate, and then insolubles in the lysate containing the disrupted host cell. Remove.
  • a host cell such as E. coli
  • phosphate buffer such as HEPES buffer
  • MOPS buffer such as Tris buffer
  • disruption of host cells can be accomplished by a variety of methods well known to those skilled in the art including, but not limited to, homogenizer disruption, homogenizer disruption, sonication, milling, high pressure extrusion, lysozyme treatment, and the like.
  • Methods of removing insolubles from the lysate are also well known to those skilled in the art and include, but are not limited to, filtration and centrifugation.
  • the present invention provides a mutant HPV16 L1 protein and HPV virus-like particles formed therefrom.
  • the HPV virus-like particles of the present invention are capable of providing significant cross-protection between HPV 16 and other types of HPV (e.g., HPV 35 and HPV 31).
  • the HPV virus-like particles of the invention are capable of inducing the body to produce high titer neutralizing antibodies against at least two types of HPV (eg, HPV 16 and HPV 35, or HPV 16, HPV 35 and HPV 31), And its effect is comparable to a mixture of multiple types of HPV VLPs (eg, a mixture of HPV16 VLPs and HPV35 VLPs, or a mixture of HPV16 VLPs, HPV35 VLPs, and HPV31 VLPs).
  • HPV 16 and HPV 35 e.g, HPV 16 and HPV 35, or HPV 16, HPV 35 and HPV 31
  • HPV VLPs eg, a mixture of HPV16 VLPs and HPV35 VLPs, or a mixture of HPV16 VLPs, HPV35 VLPs, and HPV31 VLPs.
  • the HPV virus-like particles of the present invention can be used to simultaneously prevent infection of at least two types of HPV (for example, HPV16 and HPV35, or HPV16, HPV35, and HPV31) and diseases associated therewith, and have significant advantageous technical effects. .
  • HPV16 and HPV35 for example, HPV16 and HPV35, or HPV16, HPV35, and HPV31
  • diseases associated therewith for example, HPV16 and HPV35, or HPV16, HPV35, and HPV31
  • This has a particularly significant advantage in expanding the scope of protection of HPV vaccines and reducing the cost of production of HPV vaccines.
  • Figure 1 shows the results of SDS polyacrylamide gel electrophoresis of the purified mutant protein of Example 1.
  • Lane M protein molecular weight marker
  • Lane 1 HPV16N30 (HPV16 L1 protein with N-terminally truncated 30 amino acids)
  • Lane 2 H16N30-35T1
  • Lane 3 H16N30-35T2
  • Lane 4 H16N30-35T3
  • Lane 5 H16N30-35T4
  • Lane 6 H16N30-35T5
  • Lane 7 HPV16N30
  • Lane 8 H16N30-35T4-31S1
  • Lane 9 H16N30-35T4-31S2
  • Lane 10 H16N30-35T4-31S3
  • Lane 11 H16N30-35T4- 31S5.
  • Figure 2 shows the detection of H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30- prepared in Example 1 with broad-spectrum antibody 4B3.
  • Lane M Protein molecular weight marker; Lane 1: HPV16N30; Lane 2: H16N30-35T1; Lane 3: H16N30-35T2; Lane 4: H16N30-35T3; Lane 5: H16N30-35T4; Lane 6: H16N30-35T5; Lane 7: HPV16N30; Lane 8: H16N30-35T4-31S1; Lane 9: H16N30-35T4-31S2; Lane 10: H16N30-35T4-31S3; Lane 11: H16N30-35T4-31S5.
  • mutant proteins H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 can be Broad-spectrum antibody 4B3 is specifically recognized.
  • Figures 3A-3L show the inclusion proteins HPV16N30, HPV35 L1, HPV31 L1, H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30- The results of molecular sieve chromatography analysis of samples of 35T4-31S3 and H16N30-35T4-31S5.
  • Figure 3A HPV16N30; Figure 3B: HPV35 L1; Figure 3C: HPV31 L1; Figure 3D: H16N30-35T1; Figure 3E: H16N30-35T2; Figure 3F: H16N30-35T3; Figure 3G: H16N30-35T4; Figure 3H: H16N30- 35T5; Fig. 3I: H16N30-35T4-31S1; Fig. 3J: H16N30-35T4-31S2; Fig. 3K: H16N30-35T4-31S3; Fig. 3L: H16N30-35T4-31S5.
  • VLP HPV16N30 VLP
  • HPV35 VLP HPV35 L1 protein
  • HPV31 L1 protein HPV31
  • FIG 4A HPV16N30 VLP; Figure 4B, HPV35 VLP; Figure 4C, HPV31 VLP; Figure 4D, H16N30-35T1 VLP; Figure 4E, H16N30-35T2 VLP; Figure 4F, H16N30-35T3 VLP; Figure 4G, H16N30-35T4 VLP; Figure 4H, H16N30-35T5 VLP; Figure 4I, H16N30-35T4-31S1 VLP; Figure 4J, H16N30-35T4-31S2 VLP; Figure 4K, H16N30-35T4-31S3 VLP; Figure 4L, H16N30-35T4-31S5 VLP.
  • H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 are assembled into a size, Virus-like particles similar in morphology to wild-type VLPs.
  • Figure 5A VLP assembled by HPV16N30;
  • Figure 5B VLP assembled by HPV35 L1;
  • Figure 5C VLP assembled by HPV31 L1;
  • Figure 5D VLP assembled by H16N30-35T1;
  • Figure 5E VLP assembled by H16N30-35T2
  • Figure 5F VLP assembled by H16N30-35T3;
  • Figure 5G VLP assembled by H16N30-35T4;
  • Figure 5H VLP assembled by H16N30-35T5;
  • Figure 5I VLP assembled by H16N30-35T4-31S1;
  • Figure 5J VLP assembled from H16N30-35T4-31S2;
  • Figure 5K VLP assembled from H16N30-35T4-31S3;
  • Figure 5L VLP assembled from H16N30-35T4-31S
  • HPV35 L1 is similar to the HPV31 L1 protein and can be assembled into VLPs with a radius of about 30 nm.
  • Figures 6A-6L show HPV16N30 VLP, HPV35 VLP, HPV31 VLP, H16N30-35T1 VLP, H16N30-35T2 VLP, H16N30-35T3 VLP, H16N30-35T4 VLP, H16N30-35T5 VLP, H16N30-35T4-31S1 VLP, H16N30-35T4 -31S2 VLP, H16N30-35T4-31S3 VLP, H16N30-35T4-31S5 VLP thermal stability evaluation results.
  • FIG. 6A HPV16N30 VLP; Figure 6B, HPV35 VLP; Figure 6C, HPV31 VLP; Figure 6D, H16N30-35T1 VLP; Figure 6E, H16N30-35T2 VLP; Figure 6F, H16N30-35T3 VLP; Figure 6G, H16N30-35T4 VLP; Figure 6H, H16N30-35T5 VLP; Figure 6I, H16N30-35T4-31S1 VLP; Figure 6J, H16N30-35T4-31S2 VLP; Figure 6K, H16N30-35T4-31S3 VLP; Figure 6L, H16N30-35T4-31S5 VLP.
  • the results show that the VLP formed by each protein has extremely high thermal stability.
  • Figure 7A shows experimental groups H16N30-35T1 VLP, H16N30-35T2 VLP, H16N30-35T3 VLP, H16N30-35T4 VLP, H16N30-35T5 VLP and control HPV16N30 VLP, HPV35 VLP, mixed HPV16/HPV35 VLP in mice The results of immunoprotective evaluation.
  • H16N30-35T4 VLP induced high titers of neutralizing antibodies against HPV16 and HPV35 in mice; and its protective effect against HPV16 was comparable to that of HPV16N30 VLP alone, mixed HPV16/HPV35 VLP, and was significantly higher The HPV35 VLP alone; and its protective effect against HPV35 is comparable to the HPV35 VLP, hybrid HPV16/HPV35 VLP alone, and significantly higher than the HPV16N30 VLP alone.
  • the H16N30-35T4 VLP can be used as an effective vaccine against HPV16 infection and HPV35 infection, and can be used to replace the mixed vaccine containing HPV16 VLP and HPV35 VLP.
  • Figure 7B shows experimental groups H16N30-35T4-31S1 VLP, H16N30-35T4-31S2 VLP, H16N30-35T4-31S3 VLP and H16N30-35T4-31S5 VLP, and control HPV16N30 VLP, HPV31 VLP, HPV35 VLP and mixed HPV16/ The results of immunoprotective evaluation of HPV35/HPV31 VLP in mice.
  • H16N30-35T4-31S1 VLP, H16N30-35T4-31S2 VLP and H16N30-35T4-31S3 VLPs can induce high titers of neutralizing antibodies against HPV16, HPV35 and HPV31 in mice; and their protection against HPV16
  • the effect is comparable to HPV16N30 VLP alone, mixed HPV16/HPV35/HPV31 VLP, and significantly higher than HPV35 VLP and HPV31 VLP alone; and its protective effect against HPV35 is independent of HPV35 VLP, mixed HPV16/HPV35/
  • the HPV31 VLP is comparable and significantly higher than the HPV16N30 VLP alone and the HPV31 VLP alone; and its protection against HPV31 is comparable to that of the HPV31 VLP alone, the hybrid HPV16/HPV35/HPV31 VLP, and significantly higher than the HPV16N30 VLP alone.
  • H16N30-35T4-31S1 VLP, H16N30-35T4-31S2 VLP and H16N30-35T4-31S3 VLP can be used as an effective vaccine against HPV16 infection, HPV35 infection and HPV31 infection, and can be used to replace HPV16 VLP, HPV35 VLP and HPV31.
  • Mixed vaccine for VLP can be used as an effective vaccine against HPV16 infection, HPV35 infection and HPV31 infection.
  • Figures 8A-8C show the results of evaluation of neutralizing antibody titers in mouse serum after immunization of mice with H16N30-35T4 VLP.
  • Figure 8A 10 ⁇ g dose group (immunization dose 10 ⁇ g, using aluminum adjuvant);
  • Figure 8B 1 ⁇ g dose group (immunization dose 1 ⁇ g, using aluminum adjuvant);
  • Figure 8C 0.1 ⁇ g dose group (immunization dose 0.1 ⁇ g, Use aluminum adjuvant).
  • H16N30-35T4 VLP induced high titers of neutralizing antibodies against HPV16 in mice, and its protective effect was comparable to that of the same dose of HPV16N30 VLP, mixed HPV16/HPV35 VLP, and significantly better than the same dose.
  • HPV35 VLP alone and it can induce high titers of neutralizing antibodies against HPV35 in mice, with the same protective effect as the same dose of HPV35 VLP alone, mixed HPV16/HPV35 VLP, and significantly better than the same dose Separate HPV16N30 VLP.
  • H16N30-35T4 VLP has good cross-immunogenicity and cross-protection against HPV16 and HPV35.
  • Figures 8D-8F show the results of evaluation of neutralizing antibody titers in mouse sera after immunization of mice with H16N30-35T4-31S3 VLP.
  • Figure 8D 10 ⁇ g dose group (immunization dose 10 ⁇ g, using aluminum adjuvant);
  • Figure 8E 1 ⁇ g dose group (immunization dose 1 ⁇ g, using aluminum adjuvant);
  • Figure 8F 0.1 ⁇ g dose group (immunization dose 0.1 ⁇ g, Use aluminum adjuvant).
  • H16N30-35T4-31S3 VLP could induce high titers of neutralizing antibodies against HPV16 in mice, and its protective effect was comparable to that of the same dose of HPV16N30 VLP and mixed HPV16/HPV35/HPV31 VLP, and was significantly better.
  • HPV35 VLP alone or HPV31 VLP alone; and it can induce high titers of neutralizing antibodies against HPV35 in mice, with the same protective effect as the same dose of HPV35 VLP alone and mixed HPV16/HPV35/
  • HPV31 VLP is comparable and significantly superior to the same dose of HPV16N30 VLP alone or HPV31 VLP alone; and it induces high titers of neutralizing antibodies against HPV31 in mice with a protective effect compared to HPV31 VLP alone.
  • HPV16/HPV35/HPV31 VLPs are comparable and significantly superior to the same dose of HPV16N30 VLP alone or HPV35 VLP alone; this indicates that H16N30-35T4-S3 VLPs have good cross-immunogenicity and cross-protection for HPV16, HPV35 and HPV31 Sex.
  • Sequence 7 (SEQ ID NO: 7):
  • Sequence 8 (SEQ ID NO: 8):
  • Sequence 9 (SEQ ID NO: 9):
  • a multi-point mutation PCR reaction was used to construct an expression vector encoding a HPV16 L1 protein containing a mutation derived from the segment 3 and segment 5 of the HPV35 L1 protein, wherein the initial template used was the pTO-T7-HPV16L1N30C plasmid (the coding thereof)
  • HPV16N30 The N-terminal truncated 30 amino acid HPV16 L1 protein (this protein was named HPV16N30); in Table 2 abbreviated as 16L1N30).
  • the template and primers used for each PCR reaction are shown in Table 2, and the amplification conditions of the PCR reaction were set to: 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, extension at 72 ° C) 7 minutes and 30 seconds); last 7 minutes at 72 ° C.
  • the specific sequences of the PCR primers used are listed in Table 3.
  • the sequencing results showed that the nucleotide sequences of the target fragments inserted in each of the constructed plasmids (expression vectors) were SEQ ID NOs: 18 and 20, respectively, and the encoded amino acid sequences were SEQ ID NOs: 6, 8 (corresponding to The proteins were named H16N30-35T3 and H16N30-35T5, respectively.
  • the mutein H16N30-35T3 differs from HPV16N30 in that amino acid residues at positions 199-210 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 173-184 of the wild-type HPV35 L1 protein.
  • the mutein H16N30-35T5 differs from HPV16N30 in that amino acid residues at positions 374-384 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 346-356 of the wild-type HPV35 L1 protein.
  • the initial template used was the pTO-T7-HPV16L1N30C plasmid and the pTO-T7-HPV35L1 plasmid (which encodes the HPV35 L1 protein; abbreviated as 35L1 in Table 2).
  • the templates and primers used for the respective PCR reactions are shown in Table 2, and the amplification conditions for the PCR reaction for amplifying the short fragments were set to: denaturation at 94 ° C for 10 minutes; 25 cycles (denaturation at 94 ° C for 50 seconds, specified temperature) Annealing for a certain period of time, extending at 72 ° C for 1 minute); finally extending at 72 ° C for 10 minutes.
  • the amplification conditions for the PCR reaction for amplifying the long fragment were set to: 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, extension at 72 ° C for 7 minutes and 30 seconds); °C extended 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 the target fragment was recovered using a DNA recovery kit and its concentration was determined.
  • the amplified short and long fragments were mixed at a molar ratio of 2:1 (total volume 3 ⁇ L), followed by the addition of 3 ⁇ L of 2X Gibson Assembly Premix (2X Gibson Assembly Master Mix, purchased from NEB, containing T5exonuclease, Phusion DNA polymerase , Taq DNA ligase), and reacted at 50 ° C for 1 hour.
  • 2X Gibson Assembly Master Mix purchased from NEB, containing T5exonuclease, Phusion DNA polymerase , Taq DNA ligase
  • nucleotide sequence of the target fragment inserted into the plasmid was sequenced using a T7 primer.
  • the sequencing results showed that the nucleotide sequences of the target fragments inserted in each of the constructed plasmids (expression vectors) were SEQ ID NOs: 16, 17, and 19, respectively, and the amino acid sequences encoded therein were SEQ ID NOs: 4, 5, and 7.
  • the corresponding proteins are named H16N30-35T1, H16N30-35T2 and H16N30-35T4, respectively).
  • the mutein H16N30-35T1 differs from HPV16N30 in that amino acid residues at positions 76-87 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 50-61 of the wild-type HPV35 L1 protein.
  • the mutein H16N30-35T2 differs from HPV16N30 in that amino acid residues at positions 158-167 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 132-141 of the wild-type HPV35 L1 protein.
  • the mutein H16N30-35T4 differs from HPV16N30 in that amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein.
  • An Gibson assembly was used to construct an expression vector encoding a double-substituted mutant HPV16 L1 protein containing a segment derived from HPV35 L1 and a segment derived from HPV31 L1. Briefly, a PCR reaction was first used to obtain a short fragment containing a mutation and a long fragment containing no mutation, and then the two fragments were ligated into a loop using a Gibson assembly system.
  • the initial template used included the pTO-T7-H16N30-35T4 plasmid (which encodes the mutant protein H16N30-35T4; abbreviated as H16N30-35T4 in Table 2), and the pTO-T7-HPV31L1 plasmid (which encodes the HPV31 L1 protein; 2 is abbreviated as 31L1).
  • the templates and primers used for the respective PCR reactions are shown in Table 2, and the amplification conditions for the PCR reaction for amplifying the short fragments were set to: denaturation at 94 ° C for 10 minutes; 25 cycles (denaturation at 94 ° C for 50 seconds, specified temperature) Annealing for a certain period of time, extending at 72 ° C for 1 minute); finally extending at 72 ° C for 10 minutes.
  • the amplification conditions for the PCR reaction for amplifying the long fragment were set to: 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, extension at 72 ° C for 7 minutes and 30 seconds); °C extended 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 the target fragment was recovered using a DNA recovery kit and its concentration was determined.
  • the amplified short and long fragments were mixed at a molar ratio of 2:1 (total volume 3 ⁇ L), followed by the addition of 3 ⁇ L of 2X Gibson Assembly Premix (2X Gibson Assembly Master Mix, purchased from NEB, containing T5exonuclease, Phusion DNA polymerase , Taq DNA ligase), and reacted at 50 ° C for 1 hour.
  • 2X Gibson Assembly Master Mix purchased from NEB, containing T5exonuclease, Phusion DNA polymerase , Taq DNA ligase
  • nucleotide sequence of the target fragment inserted into the plasmid was sequenced using a T7 primer.
  • the sequencing results showed that the nucleotide sequences of the desired fragment inserted in each plasmid (expression vector) constructed were SEQ ID NO: 21, 22, 23, and 24, respectively, and the encoded amino acid sequence was SEQ ID NO: 9, 10. 11, 12 (the corresponding proteins are named H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, and H16N30-35T4-31S5).
  • the mutein H16N30-35T4-31S1 differs from HPV16N30 in that amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and are located in the wild type.
  • the amino acid residues at positions 76-87 of the HPV16 L1 protein were replaced with amino acid residues at positions 50-62 of the wild-type HPV31 L1 protein.
  • the mutein H16N30-35T4-31S2 differs from HPV16N30 in that the amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and are located in the wild type.
  • the amino acid residues at positions 152-167 of the HPV16 L1 protein were replaced with amino acid residues at positions 127-142 of the wild type HPV31 L1 protein.
  • the mutein H16N30-35T4-31S3 differs from HPV16N30 in that amino acid residues at positions 292-316 of the wild-type HPV16L1 protein are replaced with amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and are located in wild-type HPV16.
  • the amino acid residues at positions 202-207 of the L1 protein were replaced with amino acid residues at positions 177-182 of the wild type HPV31 L1 protein.
  • the mutein H16N30-35T4-31S5 differs from HPV16N30 in that amino acid residues at positions 292-316 of the wild-type HPV16 L1 protein are replaced with amino acid residues at positions 266-288 of the wild-type HPV35 L1 protein, and are located in the wild type.
  • the amino acid residues at positions 375-384 of the HPV16 L1 protein were replaced with amino acid residues at positions 350-359 of the wild-type HPV31 L1 protein.
  • the recombinant plasmids pTO-T7-H16N30-35T1, pTO-T7-H16N30-35T2, pTO-T7-H16N30-35T3, pTO-T7-H16N30-35T4, pTO-T7-H16N30-35T5, were taken out from the -70 °C refrigerator.
  • the culture temperature was lowered to 25 ° C, and 500 ⁇ L of IPTG was added to each flask, and the culture was continued for 8 hours. After the completion of the culture, the cells were collected by centrifugation.
  • the cells expressing H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 protein were obtained. .
  • the above-obtained cells were resuspended in a ratio of 1 g of the bacterium to 10 mL of a lysate (20 mM Tris buffer, pH 7.2, 300 mM NaCl).
  • the cells were disrupted by a sonicator for 30 min.
  • the lysate containing the disrupted cells was centrifuged at 13500 rpm (30000 g) for 15 min to remove the supernatant (i.e., the bacterial cell disrupted supernatant).
  • AKTA explorer 100 preparative liquid chromatography system manufactured by GE Healthcare (formerly Amershan Pharmacia).
  • Chromatographic media SP Sepharose 4 Fast Flow (GE Healthcare), CHT-II (available from Bio-RAD), and Butyl Sepharose 4 Fast Flow (GE Healthcare).
  • Buffer 20 mM phosphate buffer, pH 8.0, 20 mM DTT; and, 20 mM phosphate buffer, pH 8.0, 20 mM DTT, 2 M NaCl.
  • the eluted fraction obtained in the step (3) was 150 ⁇ L, added to 30 ⁇ L of 6X Loading Buffer, mixed, and incubated in a water bath at 80 ° C for 10 min. 10 ⁇ l of the sample was then electrophoresed in a 10% SDS-polyacrylamide gel at 120 V for 120 min; then the electrophoresis band was visualized by Coomassie blue staining. The results of electrophoresis are shown in Figure 1.
  • HPV16N30 protein was prepared and purified by E. coli and pTO-T7-HPV16L1N30C plasmid by similar methods; HPV35 L1 protein (SEQ ID NO: 2) was prepared and purified using E. coli and pTO-T7-HPV35L1 plasmid; Escherichia coli was used.
  • the HPV31L1 protein (SEQ ID NO: 3) was prepared and purified from the pTO-T7-HPV31L1 plasmid.
  • H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 Perform electrophoresis. After the end of the electrophoresis, Western Blot detection was carried out using a broad-spectrum antibody 4B3 against HPV L1 protein, and the results are shown in Fig. 2.
  • H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 can be broad-spectrum antibodies 4B3 specific recognition.
  • Example 2 Assembly and particle morphology detection of HPV virus-like particles
  • HPV16N30, HPV35 L1 and HPV31 L1 proteins were assembled into HPV16N30 VLP, HPV35 VLP and HPV31 VLP, respectively.
  • dialyzed samples were analyzed by molecular sieve chromatography using an Agilent 1120 Compact LC system, wherein the analytical column used was TSK Gel PW5000xl 7.8 x 300 mm. The results of the analysis are shown in Figures 3A-3L.
  • the instrument used for sedimentation rate analysis was a Beckman XL-A analytical ultracentrifuge equipped with an optical inspection system and An-50Ti and An-60Ti rotors.
  • H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 are assembled into a size, Virus-like particles similar in morphology to wild-type VLPs.
  • H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 can be assembled into Virus-like particles.
  • the results also showed that the particles formed by these mutant proteins have a radius of about 30 nm and a uniform size. This indicates that these mutant proteins are similar to the L1 proteins of HPV16, HPV35 and HPV31, and are capable of forming VLPs of uniform size.
  • H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30- were evaluated using a differential temperature calorimeter VP Capillary DSC purchased from GE Corporation (formerly MicroCal Corporation).
  • Each protein was scanned at 90 °C.
  • the test results are shown in Figures 6A-6L. The results show that the VLP formed by each protein has extremely high thermal stability.
  • mice to evaluate the formation of H16N30-35T1, H16N30-35T2, H16N30-35T3, H16N30-35T4, H16N30-35T5, H16N30-35T4-31S1, H16N30-35T4-31S2, H16N30-35T4-31S3, H16N30-35T4-31S5 The immunoprotective properties of VLPs.
  • the animals used for immunization were 5-6 week old BalB/c normal mice (purchased from Shanghai Sleek Experimental Animal Co., Ltd.).
  • Mice were divided into 8 groups according to different immunogens, and each group contained 5 mice.
  • the immunization procedure was: primary immunization at 0 weeks; booster immunizations at 2 and 4 weeks.
  • the immunological method was intraperitoneal injection, and the immunogen and dose used were as shown in Table 4.
  • H16N30-35T4 VLP induced high titers of neutralizing antibodies against HPV16 and HPV35 in mice; and its protective effect against HPV16 was comparable to that of HPV16N30 VLP alone, mixed HPV16/HPV35 VLP, and was significantly higher The HPV35 VLP alone; and its protective effect against HPV35 is comparable to the HPV35 VLP, hybrid HPV16/HPV35 VLP alone, and significantly higher than the HPV16N30 VLP alone.
  • H16N30-35T4 VLP can be used as an effective vaccine against HPV16 infection and HPV35 infection, and can be used to replace the mixed vaccine containing HPV16 VLP and HPV35 VLP.
  • H16N30-35T4-31S1 VLP, H16N30-35T4-31S2 VLP, H16N30-35T4-31S3 VLP, H16N30-35T4-31S5 VLP, HPV16N30 VLP, HPV31 VLP, HPV35 VLP and mixed HPV16/HPV35/HPV31 prepared as above were prepared.
  • VLPs i.e., mixed HPV 16N30 VLPs, HPV35 VLPs, and HPV31 VLPs
  • Mice were divided into 8 groups according to different immunogens, and each group contained 5 mice.
  • the immunization procedure was: primary immunization at 0 weeks; booster immunizations at 2 and 4 weeks.
  • the mode of immunization was intraperitoneal injection, and the immunogen and dose used were as shown in Table 5.
  • the ocular venous blood was taken, and the serum was separated, and then the titer of the neutralizing antibody in the serum was measured. The test results are shown in Fig. 7B.
  • H16N30-35T4-31S1 VLP, H16N30-35T4-31S2 VLP and H16N30-35T4-31S3 VLPs can induce high titers of neutralizing antibodies against HPV16, HPV35 and HPV31 in mice; and their protection against HPV16
  • the effect is comparable to HPV16N30 VLP alone, mixed HPV16/HPV35/HPV31 VLP, and significantly higher than HPV35 VLP and HPV31 VLP alone; and its protective effect against HPV35 is independent of HPV35 VLP, mixed HPV16/HPV35/
  • the HPV31 VLP is comparable and significantly higher than the HPV16N30 VLP alone and the HPV31 VLP alone; and its protection against HPV31 is comparable to that of the HPV31 VLP alone, the hybrid HPV16/HPV35/HPV31 VLP, and significantly higher than the HPV16N30 VLP alone.
  • H16N30-35T4-31S1 VLP, H16N30-35T4-31S2 VLP and H16N30-35T4-31S3 VLP can be used as an effective vaccine against HPV16 infection, HPV35 infection and HPV31 infection, and can be used to replace HPV16 VLP, HPV35 VLP and HPV31.
  • Mixed vaccine for VLP can be used as an effective vaccine against HPV16 infection, HPV35 infection and HPV31 infection.
  • mice 6-week-old BalB/c female mice (8 rats) were immunized with aluminum adjuvant and single intraperitoneal injection.
  • the experimental group used H16N30-35T4 VLP (immunization doses were 0.300 ⁇ g, 0.100 ⁇ g, 0.033 ⁇ g, 0.011).
  • the control group used HPV16N30 VLP alone and HPV35 VLP alone (immunization dose 0.300 ⁇ g, 0.100 ⁇ g, 0.033 ⁇ g, 0.011 ⁇ g, 0.004 ⁇ g), or mixed HPV16/HPV35 VLP (for each VLP)
  • the immunization doses were 0.300 ⁇ g, 0.100 ⁇ g, 0.033 ⁇ g, 0.011 ⁇ g, 0.004 ⁇ g each; the immunological volume was 1 mL.
  • H16N30-35T4 VLP induced in mice ED 50 of the anti-HPV16 alone HPV16N30 VLP, mixed HPV16 / HPV35 VLP considerable, and significantly superior to separate HPV35 VLP; and which induced in mice ED 50 of the anti-HPV35 HPV35 VLPs alone, a mixed HPV16 / HPV35 VLP considerable, and significantly superior to separate HPV16N30 VLP.
  • H16N30-35T4 VLP has good cross-immunogenicity and cross-protection against HPV16 and HPV35.
  • mice 6-week-old BalB/c female mice (8 rats) were immunized with aluminum adjuvant and single intraperitoneal injection.
  • the experimental group used H16N30-35T4-31S3 VL (immunization dose was 0.300 ⁇ g, 0.100 ⁇ g, 0.033 ⁇ g).
  • the control group used HPV16N30 VLP alone, HPV35 VLP alone, HPV31 VLP alone (immunization dose 0.300 ⁇ g, 0.100 ⁇ g, 0.033 ⁇ g, 0.011 ⁇ g, 0.004 ⁇ g), and mixed HPV16/ HPV35/HPV31 VLP (immunization dose of each VLP was 0.300 ⁇ g, 0.100 ⁇ g, 0.033 ⁇ g, 0.011 ⁇ g, 0.004 ⁇ g); the immunological volume was 1 mL.
  • mice after 5 weeks of immunization of mice, the ED 50 of H16N30-35T4-31S3 VLP-induced anti-HPV16 antibody in mice was comparable to HPV16N30 VLP alone and mixed HPV16/HPV35/HPV31 VLP, and was significantly better than HPV35 alone.
  • H16N30-35T4-31S3 VLP has good cross-immunogenicity and cross-protection against HPV16, HPV35 and HPV31.
  • mice (6-week old BalB/c female mice) were divided into 3 groups: 10 ⁇ g dose group (immunization dose 10 ⁇ g, using aluminum adjuvant), 1 ⁇ g dose group (immunization dose 1 ⁇ g, using aluminum adjuvant) , and a 0.1 ⁇ g dose group (immunization dose 0.1 ⁇ g, using an aluminum adjuvant).
  • Each group was subdivided into 4 subgroups, control subgroups 1 and 2 were immunized with HPV16N30 VLP and HPV35 VLP alone, and control subgroup 3 was immunized with mixed HPV16/HPV35 VLP.
  • the experimental subgroup was H16N30. -35T4 VLP for immunization.
  • mice/subgroups were immunized by intraperitoneal injection, and the immunization doses were 10 ⁇ g, 1 ⁇ g, and 0.1 ⁇ g, respectively, and the injection volume was 1 ml. All mice were immunized initially at week 0 and then boosted once each at weeks 2 and 4. The mice were subjected to eyelid blood sampling at week 8, and the titers of anti-HPV16 and HPV35 antibodies in the serum were analyzed. The results of the analysis are shown in Figures 8A-8C.
  • H16N30-35T4 VLP induced high titers of neutralizing antibodies against HPV16 in mice, and its protective effect was comparable to that of the same dose of HPV16N30 VLP, mixed HPV16/HPV35 VLP, and significantly better than the same dose.
  • HPV35 VLP alone and it can induce high titers of neutralizing antibodies against HPV35 in mice, with the same protective effect as the same dose of HPV35 VLP alone, mixed HPV16/HPV35 VLP, and significantly better than the same dose Separate HPV16N30 VLP.
  • H16N30-35T4 VLP has good cross-immunogenicity and cross-protection against HPV16 and HPV35.
  • mice (6-week old BalB/c female mice) were divided into 3 groups: 10 ⁇ g dose group (immunization dose 10 ⁇ g, using aluminum adjuvant), 1 ⁇ g dose group (immunization dose 1 ⁇ g, using aluminum adjuvant) , and a 0.1 ⁇ g dose group (immunization dose 0.1 ⁇ g, using an aluminum adjuvant).
  • Each group was subdivided into 6 subgroups, and control subgroups 1, 2, and 3 were immunized with HPV16N30 VLP alone, HPV35 VLP alone, and HPV31 VLP alone, and control subgroup 4 with mixed HPV16/HPV35/HPV31.
  • VLPs were immunized and the experimental subgroups were immunized with H16N30-35T4-31S3 VLP alone.
  • mice/subgroups were immunized by intraperitoneal injection, and the immunization doses were 10 ⁇ g, 1 ⁇ g, and 0.1 ⁇ g, respectively, and the injection volume was 1 ml. All mice were immunized initially at week 0 and then boosted once each at weeks 2 and 4. The mice were subjected to eyelid blood sampling at week 8, and the titers of anti-HPV16, HPV35 and HPV31 antibodies in the serum were analyzed. The results of the analysis are shown in Figures 8D-8F.
  • H16N30-35T4-31S3 VLP could induce high titers of neutralizing antibodies against HPV16 in mice, and its protective effect was comparable to that of the same dose of HPV16N30 VLP and mixed HPV16/HPV35/HPV31 VLP, and was significantly better.
  • HPV35 VLP alone or HPV31 VLP alone; and it can induce high titers of neutralizing antibodies against HPV35 in mice, with the same protective effect as the same dose of HPV35 VLP alone and mixed HPV16/HPV35/
  • HPV31 VLP is comparable and significantly superior to the same dose of HPV16N30 VLP alone or HPV31 VLP alone; and it induces high titers of neutralizing antibodies against HPV31 in mice with protection from the same dose of HPV31 VLP alone
  • the hybrid HPV16/HPV35/HPV31 VLP is comparable and significantly superior to the same dose of HPV16N30 VLP alone or HPV35 VLP alone. This indicates that the H16N30-35T4-31S3 VLP has good cross-immunogenicity and cross-protection against HPV16, HPV35 and HPV31.

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Abstract

提供一种突变的HPV16 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，HPV16和HPV35，或者HPV16、HPV35和HPV31)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。还提供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16型L1蛋白的突变体 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HPV16 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，HPV16和HPV35，或者HPV16、HPV35和HPV31)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.1992Proc 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.2014Vaccine.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 PCTCN2018095632-appb-000001
(其为针对HPV16，18，6和11的四价疫苗)，GSK公司的
Figure PCTCN2018095632-appb-000002
(其为针对HPV16，18的二价疫苗)和Merck公司的
Figure PCTCN2018095632-appb-000003
(其为九价疫苗)，均是通过混合多个型别的HPV VLP而制成。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)16型L1蛋白中的一个特定区段置换为第二型别的HPV(例如HPV35)L1蛋白的相应区段后，所获得的突变的HPV16 L1蛋白能够诱导机体产生针对HPV16和第二型别的HPV(例如HPV35)的高滴度中和抗体，其保护效果与混合的HPV16 VLP和第二型别的HPV VLP相当，并且其针对HPV16的保护效果与单独的HPV16 VLP相当，且针对第二型别的HPV(例如HPV35)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HPV16 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HPV31)L1蛋白的相应区段，由此所获得的含有双置换的突变的HPV16 L1蛋白能够诱导机体产生针对HPV16、第二型别的HPV(例如HPV35)和第三型别的HPV(例如HPV31)的高滴度中和抗体，其保护效果与混合的HPV16 VLP、第二型别的HPV VLP和第三型别的HPV VLP相当；并且，其针对HPV16的保护效果与单独的HPV16 VLP相当，针对第二型别的HPV(例如HPV35)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HPV31)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HPV16 L1蛋白或其变体，其中，所述突变的HPV16 L1蛋白与野生型HPV16 L1蛋白相比，具有下述突变：
(1)N端截短了4-50个氨基酸，例如4、6、8、10、20、30或40个氨基酸；和
(2)位于野生型HPV16 L1蛋白第292-316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；
任选地，所述突变的HPV16 L1蛋白还具有下述突变：
(3)位于野生型HPV16 L1蛋白第76-87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；
(4)位于野生型HPV16 L1蛋白第152-167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
(5)位于野生型HPV16 L1蛋白第202-207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；
并且，所述变体与所述突变的HPV16 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HPV16 L1蛋白的功能，即，能够诱导针对至少两个型别的HPV(例如，HPV16和HPV35，或者HPV16、HPV35和HPV31)的中和抗体。
在某些优选的实施方案中，所述突变的HPV16 L1蛋白与野生型HPV16 L1蛋白相比，N端截短了30个或40个氨基酸。
在某些优选的实施方案中，所述突变的HPV16 L1蛋白与野生型HPV16 L1蛋白相比，N端截短了30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HPV35。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HPV35 L1蛋白第266-288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HPV31。在某些优选的实施方案中，(3)中所述的相应位置的氨基酸残基为野生型HPV31 L1蛋白第50-62位的氨基酸残基。在某些优选的实施方案中，(4)中所述的相应位置的氨基酸残基为野生型HPV31 L1蛋白第127-142位的氨基酸残基。在某些优选的实施方案中，(5)中所述的相应位置的氨基酸残基为野生型HPV31 L1蛋白第177-182位的氨基酸残基。
在某些优选的实施方案中，所述野生型HPV16 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HPV35 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HPV31 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HPV35 L1蛋白第266-288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HPV31 L1蛋白第50-62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HPV31 L1蛋白第127-142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HPV31 L1蛋白第177-182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HPV16 L1蛋白具有选自下列的氨基酸序列：SEQ ID NO:7、9、10、11。
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HPV16L1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、21、22、23。
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HPV16 L1蛋白或其变体，或者由本发明的突变的HPV16 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HPV16 L1蛋白，其与野生型HPV16 L1蛋白相比，N端截短了4-50个氨基酸，例如4、6、8、10、20、30或40个氨基酸，并且位于野生型HPV16 L1蛋白第292-316位的氨基酸残基被替换为野生型HPV35 L1蛋白第266-288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HPV16 L1蛋白，其与野生型HPV16 L1蛋白相比，N端截短了4-50个氨基酸，例如4、6、8、 10、20、30或40个氨基酸，并且位于野生型HPV16 L1蛋白第292-316位的氨基酸残基被替换为野生型HPV35 L1蛋白第266-288位的氨基酸残基，并且位于野生型HPV16 L1蛋白第76-87位的氨基酸残基被替换为野生型HPV31 L1蛋白第50-62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HPV16 L1蛋白，其与野生型HPV16 L1蛋白相比，N端截短了4-50个氨基酸，例如4、6、8、10、20、30或40个氨基酸，并且位于野生型HPV16 L1蛋白第292-316位的氨基酸残基被替换为野生型HPV35 L1蛋白第266-288位的氨基酸残基，并且位于野生型HPV16 L1蛋白第152-167位的氨基酸残基被替换为野生型HPV31 L1蛋白第127-142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HPV16 L1蛋白，其与野生型HPV16 L1蛋白相比，N端截短了4-50个氨基酸，例如4、6、8、10、20、30或40个氨基酸，并且位于野生型HPV16 L1蛋白第292-316位的氨基酸残基被替换为野生型HPV35 L1蛋白第266-288位的氨基酸残基，并且位于野生型HPV16 L1蛋白第202-207位的氨基酸残基被替换为野生型HPV31 L1蛋白第177-182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HPV16 L1蛋白，其具有SEQ ID NOs:7、9、10或11所示的序列。
在另一个方面，本发明还涉及包含上述突变的HPV16 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HPV16 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，HPV16感染、HPV35感染和/或HPV31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HPV16 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HPV16 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HPV16 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HPV16 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HPV16 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HPV16 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，HPV16感染、HPV35感染和/或HPV31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，还涉及根据本发明的突变的HPV16 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，HPV16感染、HPV35感染和/或HPV31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HPV16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HPV35。根据本发明，术语“第三型别的野生型HPV”是指，不同于HPV16，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HPV31。
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HPV16 L1蛋白”是指，天然存在于人乳头瘤病毒16型(HPV16)中的主要衣壳蛋白L1。野生型HPV16 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ANA05496.1，ANA05539.1，AGC65525.1，AAV91659.1和AAD33259.1)。
在本发明中，当提及野生型HPV16 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HPV16 L1蛋白的第292-316位氨基酸残基”是指，SEQ ID NO：1所示的多肽的第292-316位氨基酸残基。然而，本领域技术人员理解，野生型HPV16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HPV16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HPV16 L1蛋白”不仅包括SEQ ID NO:1所示的蛋白，而且应包括各种HPV16分离株的L1蛋白(例如ANA05496.1，ANA05539.1，AGC65525.1，AAV91659.1和AAD33259.1所示的HPV16 L1蛋白)。并且，当描述野生型HPV16 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HPV16分离株的L1蛋白中的相应序列片段。例如，表述“野生型HPV16 L1蛋白的第292-316位氨基酸残基”包括，SEQ ID NO：1的第292-316位氨 基酸残基，以及各种HPV16分离株的L1蛋白中的相应片段。
根据本发明，术语“野生型HPV35 L1蛋白”是指，天然存在于人乳头瘤病毒35型(HPV35)中的主要衣壳蛋白L1。野生型HPV35 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P27232.2、ACV84022.1、AEI61365.1、AEI61429.1和ACV84029.1)。
在本发明中，当提及野生型HPV35 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HPV35 L1蛋白的第266-288位氨基酸残基”是指，SEQ ID NO：2所示的多肽的第266-288位氨基酸残基。然而，本领域技术人员理解，野生型HPV35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HPV35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HPV35 L1蛋白”不仅包括SEQ ID NO:2所示的蛋白，而且应包括各种HPV35分离株的L1蛋白(例如P27232.2、ACV84022.1、AEI61365.1、AEI61429.1和ACV84029.1所示的HPV35 L1蛋白)。并且，当描述野生型HPV35 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HPV35分离株的L1蛋白中的相应序列片段。例如，表述“野生型HPV35 L1蛋白的第266-288位氨基酸残基”包括，SEQ ID NO：2的第266-288位氨基酸残基，以及各种HPV35分离株的L1蛋白中的相应片段。
根据本发明，术语“野生型HPV31 L1蛋白”是指，天然存在于人乳头瘤病毒31型(HPV31)中的主要衣壳蛋白L1。野生型HPV31 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P17388.1、AEI60965.1、ANB49655.1和AEI61021.1)。
在本发明中，当提及野生型HPV31 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HPV31 L1蛋白的第177-182位氨基酸残基”是指，SEQ ID NO：3所示的多肽的第177-182位氨基酸残基。然而，本领域技术人员理解，野生型HPV31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HPV31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HPV31 L1蛋白”不仅包括SEQ ID NO:3所示的蛋白，而且应包括各种HPV31分离株的L1蛋白(例如P17388.1、AEI60965.1、ANB49655.1和AEI61021.1所示的HPV31 L1蛋白)。并且，当描述野生型HPV31 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HPV31分离株的L1蛋白中的相应序列片段。例如，表述“野生型HPV31 L1蛋白的第177-182位氨基酸残基”包括，SEQ ID NO：3的第177-182位氨基酸残基，以及各种HPV31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30个氨基酸的HPV16 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HPV16 L1蛋白N末端的第1-30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HPV16 L1蛋白(如SEQ ID NO：7、9、10或11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HPV16 L1蛋白的功能。在本发明中，术语“突变的HPV16 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，HPV16和HPV35，或者HPV16、HPV35和HPV31)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HPV16和其他型别的HPV(例如HPV35和HPV31)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HPV16疫苗的受试者来说，其感染其他型别的HPV(例如HPV35和HPV31)的风险依然很高。
本发明提供了一种突变的HPV16 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HPV16和其他型别的HPV(例如HPV35和HPV31)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，HPV16和HPV35，或者HPV16、HPV35和HPV31)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，HPV16 VLP和HPV35 VLP的混合物，或者HPV16 VLP、HPV35 VLP和HPV31 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，HPV16和HPV35，或者HPV16、HPV35和HPV31)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M：蛋白分子量标记；泳道1:HPV16N30(N端截短了30个氨基酸的HPV16 L1蛋白)；泳道2:H16N30-35T1；泳道3:H16N30-35T2；泳道4:H16N30-35T3；泳道5:H16N30-35T4；泳道6:H16N30-35T5；泳道7:HPV16N30；泳道8:H16N30-35T4-31S1；泳道9:H16N30-35T4-31S2；泳道10:H16N30-35T4-31S3；泳道11:H16N30-35T4-31S5。结果显示，经过色谱纯化后，蛋白H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5的纯度达到95％以上。
图2显示了用广谱抗体4B3检测实施例1中制备的H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5的蛋白质免疫印迹检测 的结果。泳道M：蛋白分子量标记；泳道1：HPV16N30；泳道2：H16N30-35T1；泳道3：H16N30-35T2；泳道4：H16N30-35T3；泳道5：H16N30-35T4；泳道6：H16N30-35T5；泳道7：HPV16N30；泳道8：H16N30-35T4-31S1；泳道9：H16N30-35T4-31S2；泳道10：H16N30-35T4-31S3；泳道11：H16N30-35T4-31S5。结果显示，突变蛋白H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5能够被广谱抗体4B3特异性识别。
图3A-3L显示了包含蛋白HPV16N30、HPV35 L1、HPV31 L1、H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5的样品的分子筛层析分析的结果。图3A：HPV16N30；图3B：HPV35 L1；图3C：HPV31 L1；图3D：H16N30-35T1；图3E：H16N30-35T2；图3F：H16N30-35T3；图3G：H16N30-35T4；图3H：H16N30-35T5；图3I：H16N30-35T4-31S1；图3J：H16N30-35T4-31S2；图3K：H16N30-35T4-31S3；图3L：H16N30-35T4-31S5。结果显示，各个样品的最先出现的蛋白峰均在12min左右，与由HPV16N30蛋白组装的VLP(HPV16N30 VLP)、由HPV35 L1蛋白组装的VLP(HPV35 VLP)和由HPV31 L1蛋白组装的VLP(HPV31 VLP)相当。这表明，上述突变蛋白组装均可组装成VLP。
图4A-4L显示了HPV16N30 VLP、HPV35 VLP、HPV31 VLP、H16N30-35T1 VLP、H16N30-35T2 VLP、H16N30-35T3 VLP、H16N30-35T4 VLP、H16N30-35T5 VLP、H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP、H16N30-35T4-31S3 VLP、H16N30-35T4-31S5 VLP的沉降速率分析的结果。图4A，HPV16N30 VLP；图4B，HPV35 VLP；图4C，HPV31 VLP；图4D，H16N30-35T1 VLP；图4E，H16N30-35T2 VLP；图4F，H16N30-35T3 VLP；图4G，H16N30-35T4 VLP；图4H，H16N30-35T5 VLP；图4I，H16N30-35T4-31S1 VLP；图4J，H16N30-35T4-31S2 VLP；图4K，H16N30-35T4-31S3 VLP；图4L，H16N30-35T4-31S5 VLP。结果显示，H16N30-35T1 VLP、H16N30-35T2 VLP、H16N30-35T3 VLP、H16N30-35T4 VLP、H16N30-35T5 VLP、H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP、H16N30-35T4-31S3 VLP、H16N30-35T4-31S5 VLP的沉降系数与HPV16N30 VLP、HPV35 VLP、HPV31 VLP的沉降系数相近。这表明，H16N30-35T1、 H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5组装成大小、形态与野生型VLP相似的病毒样颗粒。
图5A-5L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)。图5A，由HPV16N30组装的VLP；图5B，由HPV35 L1组装的VLP；图5C，由HPV31 L1组装的VLP；图5D，由H16N30-35T1组装的VLP；图5E，由H16N30-35T2组装的VLP；图5F，由H16N30-35T3组装的VLP；图5G，由H16N30-35T4组装的VLP；图5H，由H16N30-35T5组装的VLP；图5I，由H16N30-35T4-31S1组装的VLP；图5J，由H16N30-35T4-31S2组装的VLP；图5K，由H16N30-35T4-31S3组装的VLP；图5L，由H16N30-35T4-31S5组装的VLP。结果显示，H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3和H16N30-35T4-31S5与HPV16N30、HPV35 L1和HPV31 L1蛋白类似，都能够组装成半径为30nm左右的VLP。
图6A-6L显示了HPV16N30 VLP、HPV35 VLP、HPV31 VLP、H16N30-35T1 VLP、H16N30-35T2 VLP、H16N30-35T3 VLP、H16N30-35T4 VLP、H16N30-35T5 VLP、H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP、H16N30-35T4-31S3 VLP、H16N30-35T4-31S5 VLP的热稳定性评价的结果。图6A，HPV16N30 VLP；图6B，HPV35 VLP；图6C，HPV31 VLP；图6D，H16N30-35T1 VLP；图6E，H16N30-35T2 VLP；图6F，H16N30-35T3 VLP；图6G，H16N30-35T4 VLP；图6H，H16N30-35T5 VLP；图6I，H16N30-35T4-31S1 VLP；图6J，H16N30-35T4-31S2 VLP；图6K，H16N30-35T4-31S3 VLP；图6L，H16N30-35T4-31S5 VLP。结果显示，各个蛋白所形成的VLP均具有极高的热稳定性。
图7A显示了实验组H16N30-35T1 VLP、H16N30-35T2 VLP、H16N30-35T3 VLP、H16N30-35T4 VLP、H16N30-35T5 VLP和对照组HPV16N30 VLP、HPV35 VLP、混合的HPV16/HPV35 VLP在小鼠体内的免疫保护性的评价结果。结果显示，H16N30-35T4 VLP可在小鼠体内诱导高滴度的针对HPV16和HPV35的中和抗体；并且其针对HPV16的保护效果与单独的HPV16N30 VLP、混合的HPV16/HPV35 VLP相当，且显著高于单独的HPV35 VLP；并且其针对HPV35的保护效果与单独的HPV35 VLP、混合的HPV16/HPV35 VLP相当，且显著高于单独的HPV16N30  VLP。这表明，H16N30-35T4 VLP可用作预防HPV16感染和HPV35感染的有效疫苗，可用于代替含有HPV16 VLP和HPV35 VLP的混合疫苗。
图7B显示了实验组H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP、H16N30-35T4-31S3 VLP和H16N30-35T4-31S5 VLP，以及对照组HPV16N30 VLP、HPV31 VLP、HPV35 VLP和混合的HPV16/HPV35/HPV31 VLP在小鼠体内的免疫保护性的评价结果。结果显示，H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP和H16N30-35T4-31S3 VLP可在小鼠体内诱导高滴度的针对HPV16、HPV35和HPV31的中和抗体；并且其针对HPV16的保护效果与单独的HPV16N30 VLP、混合的HPV16/HPV35/HPV31 VLP相当，且显著高于单独的HPV35 VLP和单独的HPV31 VLP；并且其针对HPV35的保护效果与单独的HPV35 VLP、混合的HPV16/HPV35/HPV31 VLP相当，且显著高于单独的HPV16N30 VLP和单独的HPV31 VLP；并且其针对HPV31的保护效果与单独的HPV31 VLP、混合的HPV16/HPV35/HPV31 VLP相当，且显著高于单独的HPV16N30 VLP和单独的HPV35 VLP。这表明，H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP和H16N30-35T4-31S3 VLP可用作预防HPV16感染、HPV35感染和HPV31感染的有效疫苗，可用于代替含有HPV16 VLP、HPV35 VLP和HPV31 VLP的混合疫苗。
图8A-8C显示了用H16N30-35T4 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：10μg剂量组(免疫剂量为10μg，使用铝佐剂)；图8B：1μg剂量组(免疫剂量为1μg，使用铝佐剂)；图8C：0.1μg剂量组(免疫剂量为0.1μg，使用铝佐剂)。结果显示，H16N30-35T4 VLP能诱导小鼠产生高滴度的针对HPV16的中和抗体，其保护效果与同剂量的单独的HPV16N30 VLP、混合的HPV16/HPV35 VLP相当，且显著优于同剂量的单独的HPV35 VLP；并其能诱导小鼠产生高滴度的针对HPV35的中和抗体，其保护效果与同剂量的单独的HPV35 VLP、混合的HPV16/HPV35 VLP相当，且显著优于同剂量的单独的HPV16N30 VLP。这表明，H16N30-35T4 VLP对HPV16和HPV35具有良好的交叉免疫原性和交叉保护性。
图8D-8F显示了用H16N30-35T4-31S3 VLP免疫小鼠后小鼠血清中的中和抗体滴度的评价结果。图8D：10μg剂量组(免疫剂量为10μg，使用铝佐剂)；图8E：1μg剂量组(免疫剂量为1μg，使用铝佐剂)；图8F：0.1μg剂量组(免疫剂量为0.1μg，使用铝佐剂)。结果显示，H16N30-35T4-31S3 VLP能诱导小鼠产生高滴度的针对HPV16的中和抗体，其保护效果与同剂量的单独的HPV16N30 VLP以及混合的 HPV16/HPV35/HPV31 VLP相当，且显著优于同剂量的单独的HPV35 VLP或单独的HPV31 VLP；并且其能诱导小鼠产生高滴度的针对HPV35的中和抗体，其保护效果与同剂量的单独的HPV35 VLP以及混合的HPV16/HPV35/HPV31 VLP相当，且显著优于同剂量的单独的HPV16N30 VLP或单独的HPV31 VLP；并且其能诱导小鼠产生高滴度的针对HPV31的中和抗体，其保护效果与单独的HPV31 VLP以及混合的HPV16/HPV35/HPV31 VLP相当，且显著优于同剂量的单独的HPV16N30 VLP或单独的HPV35 VLP；这表明，H16N30-35T4-S3 VLP对HPV16、HPV35和HPV31具有良好的交叉免疫原性和交叉保护性。
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 PCTCN2018095632-appb-000004
Figure PCTCN2018095632-appb-000005
序列1(SEQ ID NO:1)：
Figure PCTCN2018095632-appb-000006
序列2(SEQ ID NO:2)：
Figure PCTCN2018095632-appb-000007
序列3(SEQ ID NO:3)：
Figure PCTCN2018095632-appb-000008
序列4(SEQ ID NO:4)：
Figure PCTCN2018095632-appb-000009
序列5(SEQ ID NO:5)：
Figure PCTCN2018095632-appb-000010
序列6(SEQ ID NO:6)：
Figure PCTCN2018095632-appb-000011
Figure PCTCN2018095632-appb-000012
序列7(SEQ ID NO:7)：
Figure PCTCN2018095632-appb-000013
序列8(SEQ ID NO:8)：
Figure PCTCN2018095632-appb-000014
序列9(SEQ ID NO:9)：
Figure PCTCN2018095632-appb-000015
序列10(SEQ ID NO:10)：
Figure PCTCN2018095632-appb-000016
Figure PCTCN2018095632-appb-000017
序列11(SEQ ID NO:11)：
Figure PCTCN2018095632-appb-000018
序列12(SEQ ID NO:12)：
Figure PCTCN2018095632-appb-000019
序列13(SEQ ID NO:13)：
Figure PCTCN2018095632-appb-000020
Figure PCTCN2018095632-appb-000021
序列14(SEQ ID NO:14)：
Figure PCTCN2018095632-appb-000022
Figure PCTCN2018095632-appb-000023
序列15(SEQ ID NO:15)：
Figure PCTCN2018095632-appb-000024
序列16(SEQ ID NO:16)：
Figure PCTCN2018095632-appb-000025
Figure PCTCN2018095632-appb-000026
序列17(SEQ ID NO:17)：
Figure PCTCN2018095632-appb-000027
Figure PCTCN2018095632-appb-000028
序列18(SEQ ID NO:18)：
Figure PCTCN2018095632-appb-000029
Figure PCTCN2018095632-appb-000030
序列19(SEQ ID NO:19)：
Figure PCTCN2018095632-appb-000031
序列20(SEQ ID NO:20)：
Figure PCTCN2018095632-appb-000032
Figure PCTCN2018095632-appb-000033
序列21(SEQ ID NO:21)：
Figure PCTCN2018095632-appb-000034
Figure PCTCN2018095632-appb-000035
序列22(SEQ ID NO:22)：
Figure PCTCN2018095632-appb-000036
Figure PCTCN2018095632-appb-000037
序列23(SEQ ID NO:23)：
Figure PCTCN2018095632-appb-000038
序列24(SEQ ID NO:24)：
Figure PCTCN2018095632-appb-000039
Figure PCTCN2018095632-appb-000040
序列25(SEQ ID NO:25)：
Figure PCTCN2018095632-appb-000041
序列26(SEQ ID NO:26)：
Figure PCTCN2018095632-appb-000042
序列27(SEQ ID NO:27)：
Figure PCTCN2018095632-appb-000043
序列28(SEQ ID NO:28)：
Figure PCTCN2018095632-appb-000044
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HPV16 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HPV35 L1蛋白的区段3和区段5的突变的HPV16 L1蛋白的表达载体，其中，所使用的初始模板为pTO-T7-HPV16L1N30C质粒(其编码N端截短了30个氨基酸的HPV16 L1蛋白(该蛋白命名为HPV16N30)；在表2中简写为16L1N30)。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。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，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25mg/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：18、20，其编码的氨基酸序列为SEQ ID NO：6、8(所对应的蛋白分别命名为H16N30-35T3和H16N30-35T5)。
突变蛋白H16N30-35T3与HPV16N30的区别在于：位于野生型HPV16 L1蛋白第199-210位的氨基酸残基被替换为野生型HPV35 L1蛋白第173-184位的氨基酸残基。突变蛋白H16N30-35T5与HPV16N30的区别在于：位于野生型HPV16 L1蛋白第374-384位的氨基酸残基被替换为野生型HPV35 L1蛋白第346-356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)来构建含有来源于HPV35 L1 蛋白的区段1、区段2和区段4的突变的HPV16 L1蛋白的表达载体。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。所使用的初始模板为pTO-T7-HPV16L1N30C质粒和pTO-T7-HPV35L1质粒(其编码HPV35 L1蛋白；在表2中简写为35L1)。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个循环的(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。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(2X Gibson Assembly Master Mix，购自NEB，包含T5exonuclease，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：16、17、19，其编码的氨基酸序列为SEQ ID NO：4、5、7(所对应的蛋白分别命名为H16N30-35T1，H16N30-35T2和H16N30-35T4)。
突变蛋白H16N30-35T1与HPV16N30的区别在于：位于野生型HPV16 L1蛋白第76-87位的氨基酸残基被替换为野生型HPV35 L1蛋白第50-61位的氨基酸残基。突变蛋白H16N30-35T2与HPV16N30的区别在于：位于野生型HPV16 L1蛋白第158-167位的氨基酸残基被替换为野生型HPV35 L1蛋白第132-141位的氨基酸残基。突变蛋白H16N30-35T4与HPV16N30的区别在于：位于野生型HPV16 L1蛋白第292-316位的氨基酸残基被替换为野生型HPV35 L1蛋白第266-288位的氨基酸残基。
采用Gibson装配来构建编码双置换的突变的HPV16 L1蛋白的表达载体，所述突变的HPV16 L1蛋白含有来源于HPV35 L1的区段和来源于HPV31 L1的区段。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-H16N30-35T4质粒(其编码突变蛋白H16N30-35T4；在表2中简写为H16N30-35T4)，和pTO-T7-HPV31L1质粒(其编码HPV31 L1蛋白；在表2中简写为31L1)。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个循环的(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。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(2X Gibson Assembly Master Mix，购自NEB，包含T5exonuclease，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：21、22、23、24，其编码的氨基酸序列为SEQ ID NO：9、10、11、12(所对应的蛋白分别命名为H16N30-35T4-31S1，H16N30-35T4-31S2，H16N30-35T4-31S3，和H16N30-35T4-31S5)。
突变蛋白H16N30-35T4-31S1与HPV16N30的区别在于：位于野生型HPV16 L1蛋白第292-316位的氨基酸残基被替换为野生型HPV35 L1蛋白第266-288位的氨基酸残基，并且位于野生型HPV16 L1蛋白第76-87位的氨基酸残基被替换为野生型HPV31 L1蛋白的第50-62位的氨基酸残基。突变蛋白H16N30-35T4-31S2与HPV16N30的区别在于：位于野生型HPV16 L1蛋白第292-316位的氨基酸残基被替 换为野生型HPV35 L1蛋白第266-288位的氨基酸残基，并且位于野生型HPV16 L1蛋白第152-167位的氨基酸残基被替换为野生型HPV31 L1蛋白的第127-142位的氨基酸残基。突变蛋白H16N30-35T4-31S3与HPV16N30的区别在于：位于野生型HPV16L1蛋白第292-316位的氨基酸残基被替换为野生型HPV35 L1蛋白第266-288位的氨基酸残基，并且位于野生型HPV16 L1蛋白第202-207位的氨基酸残基被替换为野生型HPV31 L1蛋白的第177-182位的氨基酸残基。突变蛋白H16N30-35T4-31S5与HPV16N30的区别在于：位于野生型HPV16 L1蛋白第292-316位的氨基酸残基被替换为野生型HPV35 L1蛋白第266-288位的氨基酸残基，并且位于野生型HPV16 L1蛋白第375-384位的氨基酸残基被替换为野生型HPV31 L1蛋白的第350-359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 PCTCN2018095632-appb-000045
Figure PCTCN2018095632-appb-000046
表3：所使用的引物的具体序列(SEQ ID NO:29-60)
Figure PCTCN2018095632-appb-000047
Figure PCTCN2018095632-appb-000048
突变蛋白的大量表达
从-70℃冰箱中取出携带重组质粒pTO-T7-H16N30-35T1、pTO-T7-H16N30-35T2、pTO-T7-H16N30-35T3、pTO-T7-H16N30-35T4、pTO-T7-H16N30-35T5、pTO-T7-H16N30-35T4-31S1、pTO-T7-H16N30-35T4-31S2、pTO-T7-H16N30-35T4-31S3、pTO-T7-H16N30-35T4-31S5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H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5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 4Fast Flow(GE Healthcare公司)、CHT-Ⅱ(购自Bio-RAD)和Butyl Sepharose 4Fast Flow(GE Healthcare公司)。
缓冲液：20mM磷酸盐缓冲液，pH8.0，20mM DTT；以及，20mM磷酸盐缓冲液，pH8.0，20mM DTT，2M NaCl。
样品：如上获得的含有H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5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的缓冲液洗脱杂蛋白，然后用含有800mM NaCl的缓冲液洗脱目的蛋白，并收集由含有800mM NaCl的缓冲液洗脱的级分；
(2)用CHTⅡ(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的缓冲液洗脱杂蛋白，然后用含有1000mM NaCl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，H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5蛋白的纯度大于95％。
通过类似的方法，使用大肠杆菌和pTO-T7-HPV16L1N30C质粒制备和纯化了HPV16N30蛋白；使用大肠杆菌和pTO-T7-HPV35L1质粒制备和纯化了HPV35 L1蛋白(SEQ ID NO:2)；使用大肠杆菌和pTO-T7-HPV31L1质粒制备和纯化了HPV31L1蛋白(SEQ ID NO:3)。
突变蛋白的免疫印迹实验
按上述方法对经纯化的H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5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H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5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H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5，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pH7.0，0.5M NaCl中。在三种缓冲液中各自进行透析12h。
通过类似的方法，将HPV16N30、HPV35 L1和HPV31 L1蛋白分别组装为HPV16N30 VLP、HPV35 VLP和HPV31 VLP。
分子筛层析分析
用美国安捷伦公司的1120Compact 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 7.8x300mm。分析结果如图3A-3L所示。结果显示，包含蛋白H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5的样品最先出现的蛋白峰均在12min左右，与HPV16N30 VLP、HPV35 VLP和HPV31 VLP相当。这表明，如上制备的蛋白均可组装成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HPV16N30 VLP、HPV35 VLP、HPV31 VLP、H16N30-35T1 VLP、H16N30-35T2 VLP、H16N30-35T3 VLP、H16N30-35T4 VLP、H16N30-35T5 VLP、H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP、H16N30-35T4-31S3 VLP、H16N30-35T4-31S5 VLP的沉降系数。结果如图4A-4L所示。结果显示，H16N30-35T1 VLP、H16N30-35T2 VLP、H16N30-35T3 VLP、H16N30-35T4 VLP、H16N30-35T5 VLP、H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP、H16N30-35T4-31S3 VLP、H16N30-35T4-31S5 VLP的沉降系数与HPV16N30 VLP、HPV35 VLP、HPV31 VLP的沉降系数相近。这表明，H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5组装成大小、形态与野生型VLP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，并固定于喷炭的铜网上，然后进行透射电镜观察。观察结果如图5A-5L所示。结果显示，H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5均可组装成病毒样颗粒。此外，结果还显示，这些突变蛋白所组装形成的颗粒的半径均在30nm左右，大小均一。这表明，这些突变蛋白与HPV16、HPV35和HPV31的L1蛋白类似，能够形成大小均一的VLP。
实施例3：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H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5所形成的VLP的热稳定性，其中，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6A-6L所示。结果显示，各个蛋白所形成的VLP均具有极高的热稳定性。
实施例4：病毒样颗粒在动物体内的免疫保护性的评价1
使用小鼠来评价由H16N30-35T1、H16N30-35T2、H16N30-35T3、H16N30-35T4、H16N30-35T5、H16N30-35T4-31S1、H16N30-35T4-31S2、H16N30-35T4-31S3、H16N30-35T4-31S5形成的VLP的免疫保护性。用于免疫接种的动物为，5-6周龄BalB/c普通级小鼠(购自上海斯莱康实验动物有限公司)。
将如上制备的H16N30-35T1 VLP、H16N30-35T2 VLP、H16N30-35T3 VLP、H16N30-35T4 VLP、H16N30-35T5 VLP、HPV16N30 VLP、HPV35 VLP以及混合的HPV16/HPV35 VLP(即，混合的HPV16N30 VLP和HPV35 VLP)分别吸附于铝佐剂上。按不同的免疫原将小鼠分为8组，每组包含5只小鼠。免疫程序为：在0周时进行初次免疫；在第2和4周时各进行加强免疫一次。免疫方式为腹腔注射，所使用的免疫原与剂量如表4所示。在初次免疫后的第8周，抽取眼球静脉血，并分离血清，随后检测血清中的中和抗体的滴度。检测结果如图7A所示。结果显示，H16N30-35T4 VLP可在小鼠体内诱导高滴度的针对HPV16和HPV35的中和抗体；并且其针对HPV16的保护效果与单独的HPV16N30 VLP、混合的HPV16/HPV35 VLP相当，且显著高于单独的HPV35 VLP；并且其针对HPV35的保护效果与单独的HPV35  VLP、混合的HPV16/HPV35 VLP相当，且显著高于单独的HPV16N30 VLP。这表明，H16N30-35T4 VLP可用作预防HPV16感染和HPV35感染的有效疫苗，可用于代替含有HPV16 VLP和HPV35 VLP的混合疫苗。
表4.免疫方案
Figure PCTCN2018095632-appb-000049
另外，将如上制备的H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP、H16N30-35T4-31S3 VLP、H16N30-35T4-31S5 VLP、HPV16N30 VLP、HPV31 VLP、HPV35 VLP和混合的HPV16/HPV35/HPV31 VLP(即，混合的HPV16N30 VLP、HPV35 VLP和HPV31 VLP)分别吸附于铝佐剂上。按不同的免疫原将小鼠分为8组，每组包含5只小鼠。免疫程序为：在0周时进行初次免疫；在第2和4周时各进行加强免疫一次。免疫方式为腹腔注射，所使用的免疫原与剂量如表5所示。在初次免疫后的第8周，抽取眼球静脉血，并分离血清，随后检测血清中的中和抗体的滴度。检测结果如图7B所示。结果显示，H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP和H16N30-35T4-31S3 VLP可在小鼠体内诱导高滴度的针对HPV16、HPV35和HPV31的中和抗体；并且其针对HPV16的保护效果与单独的HPV16N30 VLP、混合的HPV16/HPV35/HPV31 VLP相当，且显著高于单独的HPV35 VLP和单独的HPV31 VLP；并且其针对HPV35的保护效果与单独的HPV35 VLP、混合的HPV16/HPV35/HPV31 VLP相当，且显著高于单独的HPV16N30 VLP和单独的HPV31 VLP；并且其针对HPV31的保护效果与单独的HPV31 VLP、混合的 HPV16/HPV35/HPV31 VLP相当，且显著高于单独的HPV16N30 VLP和单独的HPV35 VLP。这表明，H16N30-35T4-31S1 VLP、H16N30-35T4-31S2 VLP和H16N30-35T4-31S3 VLP可用作预防HPV16感染、HPV35感染和HPV31感染的有效疫苗，可用于代替含有HPV16 VLP、HPV35 VLP和HPV31 VLP的混合疫苗。
表5.免疫方案
Figure PCTCN2018095632-appb-000050
实施例5：病毒样颗粒在动物体内的免疫保护性的评价2
H16N30-35T4 VLP的ED50
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H16N30-35T4 VLP(免疫剂量为0.300μg、0.100μg、0.033μg、0.011μg、0.004μg)，对照组使用单独的HPV16N30 VLP和单独的HPV35 VLP(免疫剂量为0.300μg、0.100μg、0.033μg、0.011μg、0.004μg)，或混合的HPV16/HPV35 VLP(每种VLP的免疫剂量各为0.300μg、0.100μg、0.033μg、0.011μg、0.004μg)；免疫体积为1mL。在免疫后第五周，抽取眼球静脉血，对血液中的HPV抗体进行检测，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6-9所示。
表6.HPV16N30 VLP诱导小鼠产生抗HPV16、抗HPV35抗体的ED 50
Figure PCTCN2018095632-appb-000051
表7.HPV35 VLP诱导小鼠产生抗HPV16、抗HPV35抗体的ED 50
Figure PCTCN2018095632-appb-000052
表8.混合的HPV16/HPV35 VLP诱导小鼠产生抗HPV16、抗HPV35抗体的ED 50
Figure PCTCN2018095632-appb-000053
Figure PCTCN2018095632-appb-000054
表9.H16N30-35T4 VLP诱导小鼠产生抗HPV16、抗HPV35抗体的ED 50
Figure PCTCN2018095632-appb-000055
结果显示，在免疫小鼠5周后，H16N30-35T4 VLP诱导小鼠产生抗HPV16的ED 50与单独的HPV16N30 VLP、混合的HPV16/HPV35 VLP相当，且显著优于单独的HPV35 VLP；并且，其诱导小鼠产生抗HPV35的ED 50与单独的HPV35 VLP、混合的HPV16/HPV35 VLP相当，且显著优于单独的HPV16N30 VLP。这表明，H16N30-35T4 VLP对HPV16和HPV35具有良好的交叉免疫原性和交叉保护性。
H16N30-35T4-31S3 VLP的ED50
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H16N30-35T4-31S3 VL(免疫剂量为0.300μg、0.100μg、0.033μg、0.011μg、0.004μg)；对照组使用单独的HPV16N30 VLP、单独的HPV35 VLP、单独的HPV31 VLP(免疫剂量为0.300μg、0.100μg、0.033μg、0.011μg、0.004μg)，以及混合的HPV16/HPV35/HPV31 VLP(每种VLP的免疫剂量各为0.300μg、0.100μg、0.033μg、0.011μg、0.004μg)；免疫体积为1mL。在免疫后第五周，抽取眼球静脉血，对血液中的HPV抗体进行检测，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10-14所 示。
表10.HPV16N30 VLP诱导小鼠产生抗HPV16、抗HPV35和抗HPV31抗体的ED 50
Figure PCTCN2018095632-appb-000056
表11.HPV35 VLP诱导小鼠产生抗HPV16、抗HPV35和抗HPV31抗体的ED 50
Figure PCTCN2018095632-appb-000057
Figure PCTCN2018095632-appb-000058
表12.HPV31 VLP诱导小鼠产生抗HPV16、抗HPV35和抗HPV31抗体的ED 50
Figure PCTCN2018095632-appb-000059
表13.混合的HPV16/HPV35/HPV31 VLP诱导小鼠产生抗HPV16、抗HPV35和抗HPV31抗体的ED 50
Figure PCTCN2018095632-appb-000060
Figure PCTCN2018095632-appb-000061
表14.H16N30-35T4-31S3 VLP诱导小鼠产生抗HPV16、抗HPV35和抗HPV31抗体的ED 50
Figure PCTCN2018095632-appb-000062
结果显示，在免疫小鼠5周后，H16N30-35T4-31S3 VLP诱导小鼠产生抗HPV16抗体的ED 50与单独的HPV16N30 VLP以及混合的HPV16/HPV35/HPV31 VLP相当，且显著优于单独的HPV35 VLP以及单独的HPV31 VLP；并且，其诱导小鼠产生抗HPV35抗体的ED 50与单独的HPV35 VLP以及混合的HPV16/HPV35/HPV31 VLP相当，且显著优于单独的HPV16N30 VLP以及单独的HPV31 VLP；并且，其诱导小鼠产生抗HPV31抗体的ED 50与单独的HPV31 VLP以及混合的HPV16/HPV35/HPV31 VLP相当，且显著优于单独的HPV16N30 VLP以及单独的HPV35 VLP。这表明，H16N30-35T4-31S3 VLP对HPV16、HPV35以及HPV31具有 良好的交叉免疫原性和交叉保护性。
用H16N30-35T4 VLP免疫小鼠后血清中的中和抗体滴度的评价
在本实验中，免疫方案如表15所示。将所有小鼠(6周龄BalB/c雌性小鼠)分为3个组：10μg剂量组(免疫剂量为10μg，使用铝佐剂)，1μg剂量组(免疫剂量为1μg，使用铝佐剂)，和0.1μg剂量组(免疫剂量为0.1μg，使用铝佐剂)。各个组又细分为4个亚组，对照亚组1和2分别用单独的HPV16N30 VLP和单独的HPV35 VLP进行免疫，对照亚组3用混合的HPV16/HPV35 VLP进行免疫，实验亚组用H16N30-35T4 VLP进行免疫。
采用腹腔注射方式免疫6只小鼠/亚组，免疫剂量分别为10μg、1μg、0.1μg，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HPV16和HPV35抗体的滴度。分析结果如图8A-8C所示。结果显示，H16N30-35T4 VLP能诱导小鼠产生高滴度的针对HPV16的中和抗体，其保护效果与同剂量的单独的HPV16N30 VLP、混合的HPV16/HPV35 VLP相当，且显著优于同剂量的单独的HPV35 VLP；并其能诱导小鼠产生高滴度的针对HPV35的中和抗体，其保护效果与同剂量的单独的HPV35 VLP、混合的HPV16/HPV35 VLP相当，且显著优于同剂量的单独的HPV16N30 VLP。这表明，H16N30-35T4 VLP对HPV16和HPV35具有良好的交叉免疫原性和交叉保护性。
表15.免疫方案
Figure PCTCN2018095632-appb-000063
Figure PCTCN2018095632-appb-000064
用H16N30-35T4-31S3 VLP免疫小鼠后血清中的中和抗体滴度的评价
在本实验中，免疫方案如表16所示。将所有小鼠(6周龄BalB/c雌性小鼠)分为3个组：10μg剂量组(免疫剂量为10μg，使用铝佐剂)，1μg剂量组(免疫剂量为1μg，使用铝佐剂)，和0.1μg剂量组(免疫剂量为0.1μg，使用铝佐剂)。各个组又细分为6个亚组，对照亚组1、2和3分别用单独的HPV16N30 VLP、单独的HPV35 VLP和单独的HPV31 VLP进行免疫，对照亚组4用混合的HPV16/HPV35/HPV31 VLP进行免疫，实验亚组用单独的H16N30-35T4-31S3 VLP进行免疫。
采用腹腔注射方式免疫6只小鼠/亚组，免疫剂量分别为10μg、1μg、0.1μg，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HPV16、HPV35和HPV31抗体的滴度。分析结果如图8D-8F所示。结果显示，H16N30-35T4-31S3 VLP能诱导小鼠产生高滴度的针对HPV16的中和抗体，其保护效果与同剂量的单独的HPV16N30 VLP以及混合的HPV16/HPV35/HPV31 VLP相当，且显著优于同剂量的单独的HPV35 VLP或单独的HPV31 VLP；并且其能诱导小鼠产生高滴度的针对HPV35的中和抗体，其保护效果与同剂量的单独的HPV35 VLP以及混合的HPV16/HPV35/HPV31 VLP相当，且显著优于同剂量的单独的HPV16N30 VLP或单独的HPV31 VLP；并且其能诱导小鼠产生高滴度的针对HPV31的中和抗体，其保护效果与同剂量的单独的HPV31 VLP以及混合的HPV16/HPV35/HPV31 VLP相当，且显著优于同剂量的单独的HPV16N30 VLP或单独的HPV35 VLP。这表明，H16N30-35T4-31S3 VLP对HPV16、HPV35和HPV31具有良好的交叉免疫原性和交叉保护性。
表16.免疫方案
Figure PCTCN2018095632-appb-000065
Figure PCTCN2018095632-appb-000066
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 (11)

  1. 一种突变的HPV16 L1蛋白或其变体，其中，所述突变的HPV16 L1蛋白与野生型HPV16 L1蛋白相比，具有下述突变：
    (1)N端截短了4-50个氨基酸，例如4、6、8、10、20、30或40个氨基酸；和
    (2)位于野生型HPV16 L1蛋白第292-316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；
    任选地，所述突变的HPV16 L1蛋白还具有下述突变：
    (3)位于野生型HPV16 L1蛋白第76-87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；
    (4)位于野生型HPV16 L1蛋白第152-167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
    (5)位于野生型HPV16 L1蛋白第202-207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；
    并且，所述变体与所述突变的HPV16 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HPV16 L1蛋白的功能，即，能够诱导针对至少两个型别的HPV(例如，HPV16和HPV35，或者HPV16、HPV35和HPV31)的中和抗体；
    优选地，所述突变的HPV16 L1蛋白与野生型HPV16 L1蛋白相比，N端截短了30个或40个氨基酸；
    优选地，所述突变的HPV16 L1蛋白与野生型HPV16 L1蛋白相比，N端截短了30个氨基酸；
    优选地，所述第二型别的野生型HPV为HPV35；优选地，(2)中所述的相应位置的氨基酸残基为野生型HPV35 L1蛋白第266-288位的氨基酸残基；
    优选地，所述第三型别的野生型HPV为HPV31；优选地，(3)中所述的相应位置的氨基酸残基为野生型HPV31 L1蛋白第50-62位的氨基酸残基；优选地，(4)中所述的相应位置的氨基酸残基为野生型HPV31 L1蛋白第127-142位的氨基酸残基；优选地，(5)中所述的相应位置的氨基酸残基为野生型HPV31 L1蛋白第177-182位的氨基酸残基；
    优选地，所述野生型HPV16 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HPV35 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HPV31 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HPV16 L1蛋白具有选自下列的氨基酸序列：SEQ ID NOs:7、9、10、11。
  2. 一种分离的核酸，其编码权利要求1所述的突变的HPV16 L1蛋白或其变体。
  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HPV16 L1蛋白或其变体，或者由权利要求1所述的突变的HPV16 L1蛋白或其变体组成。
  6. 一种组合物，其包含权利要求1所述的突变的HPV16 L1蛋白或其变体，或权利要求2的分离的核酸，或权利要求3的载体，或权利要求4的宿主细胞，或权利要求5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，HPV16感染、HPV35感染和/或HPV31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HPV16 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HPV16 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HPV16 L1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HPV16 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HPV16 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HPV16 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的HPV病毒样颗粒或权利要求7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，HPV16感染、HPV35感染和/或HPV31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HPV16 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，HPV16感染、HPV35感染和/或HPV31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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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6562351B2 (en) * 1998-02-20 2003-05-13 Medigene Ag Papilloma truncated L1 protein and fusion protein constructs
US6689366B1 (en) * 1998-12-17 2004-02-10 Merck & Co., Inc. Synthetic virus like particles with heterologous epitopes
CN101293918A (zh) * 2007-04-29 2008-10-29 北京万泰生物药业股份有限公司 截短的人乳头瘤病毒16型l1蛋白
CN101518647A (zh) * 2008-02-29 2009-09-02 江阴艾托金生物技术有限公司 人乳头瘤病毒预防性疫苗、构建方法及应用
EP2377879A1 (en) * 2010-04-14 2011-10-19 Deutsches Krebsforschungszentrum N-terminal HPV E7 fusion proteins
CN102747047A (zh) * 2012-02-28 2012-10-24 厦门大学 人乳头瘤病毒型别杂合病毒样颗粒及其制备方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020168372A1 (en) * 1993-07-16 2002-11-14 Matthias Durst Dna sequence encoding a papillomavirus l1 protein capable of efficiently forming virus-like particles
JP2001506485A (ja) * 1996-10-04 2001-05-22 メルク エンド カンパニー インコーポレーテッド 合成ｈｐｖ１６ウイルス様粒子
US7182947B2 (en) 1998-02-20 2007-02-27 Medigene Ag Papillomavirus truncated L1 protein and fusion protein constructs
CN101148661B (zh) 2006-09-18 2013-01-02 中国医学科学院基础医学研究所 人乳头瘤病毒16型外壳蛋白病毒样颗粒及其制备方法和用途
CA2703066A1 (en) 2007-10-22 2009-04-30 University Of Rochester Respiratory syncytial virus vaccine based on chimeric papillomavirus virus-like particles or capsomeres
WO2010118424A2 (en) * 2009-04-10 2010-10-14 The Johns Hopkins University Papillomavirus-like particles (vlp) as broad spectrum human papillomavirus (hpv) vaccines
CN102497880A (zh) * 2009-06-25 2012-06-13 葛兰素史密丝克莱恩生物有限公司 新的人乳头状瘤病毒(hpv)蛋白构建体及其在预防hpv疾病中的用途
CN103483447B (zh) * 2012-06-08 2015-11-25 厦门大学 抗hpv l1蛋白的广谱单克隆抗体或其抗原结合片段及它们的用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6562351B2 (en) * 1998-02-20 2003-05-13 Medigene Ag Papilloma truncated L1 protein and fusion protein constructs
US6689366B1 (en) * 1998-12-17 2004-02-10 Merck & Co., Inc. Synthetic virus like particles with heterologous epitopes
CN101293918A (zh) * 2007-04-29 2008-10-29 北京万泰生物药业股份有限公司 截短的人乳头瘤病毒16型l1蛋白
CN101518647A (zh) * 2008-02-29 2009-09-02 江阴艾托金生物技术有限公司 人乳头瘤病毒预防性疫苗、构建方法及应用
EP2377879A1 (en) * 2010-04-14 2011-10-19 Deutsches Krebsforschungszentrum N-terminal HPV E7 fusion proteins
CN102747047A (zh) * 2012-02-28 2012-10-24 厦门大学 人乳头瘤病毒型别杂合病毒样颗粒及其制备方法

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
"NCBI", Database accession no. AEI61021.1
BISHOP, B. ET AL.: "Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 282, no. 43, 26 October 2007 (2007-10-26), pages 31803 - 31811, XP055565345 *
BRUMMELL ET AL., BIOCHEM., vol. 32, 1993, pages 1180 - 1187
BURKS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 94, 1997, pages 412 - 417
DOORBAR, J.P. H. GALLIMORE, J VIROL, vol. 61, no. 9, 1987, pages 2793 - 9
E. MEYERSW. MILLER, COMPUT. APPL. BIOSCI., vol. 4, 1988, pages 11 - 17
F. M. AUSUBEL ET AL.: "Short Protocols in Molecular Biology", 1995, JOHN WILEY & SONS, INC.
GIBSON DGYOUNG LCHUANG RYVENTER JCHUTCHISON CASMITH HO: "Enzymatic assembly of DNA molecules up to several hundred kilobases", NAT METHODS, vol. 6, 2009, pages 343 - 5, XP055224105, DOI: 10.1038/nmeth.1318
J. SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
KIRNBAUER, R.F. BOOY ET AL., PROC NATL ACAD SCI USA, vol. 89, no. 24, 1992, pages 12180 - 4
KOBAYASHI ET AL., PROTEIN ENG., vol. 12, no. 10, 1999, pages 879 - 884
NEEDLEMAN ET AL., J. MOL. BIOL., vol. 48, 1970, pages 444 - 453
REED LJ MH: "A simple method of estimating fifty percent endpoints", AM J HYG., vol. 27, 1938, pages 493 - 7
SARA L. BISSETTGIADA MATTIUZZO ET AL., VACCINE, vol. 32, 2014, pages 6548 - 6555

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