WO2012000454A1 - 截短的人乳头瘤病毒52型l1蛋白 - Google Patents

截短的人乳头瘤病毒52型l1蛋白 Download PDF

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WO2012000454A1
WO2012000454A1 PCT/CN2011/076763 CN2011076763W WO2012000454A1 WO 2012000454 A1 WO2012000454 A1 WO 2012000454A1 CN 2011076763 W CN2011076763 W CN 2011076763W WO 2012000454 A1 WO2012000454 A1 WO 2012000454A1
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protein
hpv52
virus
truncated
seq
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PCT/CN2011/076763
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English (en)
French (fr)
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李少伟
莫小兵
魏旻希
潘晖榕
张军
夏宁邵
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厦门大学
厦门万泰沧海生物技术有限公司
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Application filed by 厦门大学, 厦门万泰沧海生物技术有限公司 filed Critical 厦门大学
Priority to EP11800199.9A priority Critical patent/EP2589604B1/en
Priority to US13/807,858 priority patent/US9499591B2/en
Priority to DK11800199.9T priority patent/DK2589604T3/en
Priority to IN536CHN2013 priority patent/IN2013CN00536A/en
Priority to BR112013000031A priority patent/BR112013000031A2/pt
Publication of WO2012000454A1 publication Critical patent/WO2012000454A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/892—Reproductive system [uterus, ovaries, cervix, testes]
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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
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    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20023—Virus like particles [VLP]
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    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20051—Methods of production or purification of viral material

Definitions

  • the invention relates to the field of molecular virology and immunology.
  • the present invention relates to a truncated human papillomavirus type 52 L1 protein, its coding sequence and preparation method, and virus-like particles comprising the same, which can be used for the prevention of HPV (especially HPV5) 2 ) infections and diseases caused by HPV (especially HPV5 2 infection) such as cervical cancer.
  • the invention further relates to the use of the above proteins and virus-like particles for the preparation of a pharmaceutical composition or vaccine for the prevention of HPV (especially HPV5 2 ) infection and infection by HPV (particularly HPV 5 2 ) Caused by diseases such as cervical cancer.
  • Human papillomavirus (Human Papi l lomavi rus, HPV) belongs to papillomavirus (Papi l lomavi r idae) and is a non-enveloped DNA virus.
  • the viral genome is a double-strand closed-loop DNA with a size of about 7. 2 ⁇ 8 kb and 8 open reading frames.
  • the viral genome can be divided into three regions according to their functions: 1 early region (E), about 4. 5 kb, encoding 6 non-structural proteins related to viral replication, transcription and transformation, including El, E2, E4 ⁇ E7; 2 late region (L), about 2.
  • HPV virus particles are 45 to 55 nm in diameter, and the nucleocapsid is icosahedral. There are 72 shell particles consisting of L1 and L2.
  • HPV has more than 100 subtypes, which cause skin and mucous membrane lesions in the human population. According to their relationship with tumorigenesis, HPV can be divided into 3 groups: 1 low or no carcinogenic risk group, including HPV6, 11, 39, 41, 42, 43; 2 moderate The carcinogenic risk group, including HPV 31, 33, 35, 51; 3 highly carcinogenic risk groups, including HPV 16, 18, 58, 45, 52.
  • HPV molecular epidemiological survey confirmed that high-risk HPV infection is an important trigger for cervical cancer.
  • the detection rate of HPV DM is over 80%.
  • Cervical cancer is a common female malignant tumor, and its incidence is second only to breast cancer, which is a serious threat to women's health. According to statistics, there are about 490,000 new cases worldwide each year, and about 270,000 people die from the disease (Boley, P., and J. Fer lay. Ann Oncol 2005, 16: 481-8) . Of all cervical cancer cases, approximately 83% occur in developing countries, where cervical cancer can even account for 15% of female malignancies. In developed countries, this figure only accounts for 1.5%.
  • HPV16, 18 subtype is the most common type of cervical cancer
  • HPV52 subtype is the sixth most common high-risk HPV.
  • HPV52 is a high-risk carcinogenic HPV type after HPV16, 33, and 18.
  • HPV vaccines are Merck's Gardas i l® and GSK's Cervar ix®, which contain HPV6/11/16/18 and HPV16/18 VLPs, respectively, but do not include HPV52 VLPs.
  • HPV L1 protein is the major capsid protein with a molecular weight of 55-60 kDa and is the main target protein of HPV vaccine.
  • HPV L1 protein expressed in various expression systems can form a virus-like morphologically similar to natural virus particles without the aid of L2 protein.
  • Virus-Like Particle VLP
  • the virus-like particle is an icosahedral stereo-symmetric structure composed of 72 pentamers of L1 protein. It retains the natural epitope of the virion, has strong immunogenicity, and induces neutralizing antibodies against the same type of HPV virus (Kirnbauer, R., F. Booy, et al. 1992 Proc Natl Acad Sci USA 89 (24 ): 12180-4).
  • the virus-like particles do not carry viral nucleic acid, have no potential carcinogenic risk, and have good safety. Therefore, VLP vaccine has become the main direction of HPV vaccine development.
  • VLP vaccines The key to the development of HPV VLP vaccines is the ability to efficiently and efficiently prepare VLP samples.
  • the more commonly used VLP expression systems can be divided into eukaryotic expression systems and prokaryotic expression systems.
  • Common eukaryotic expression systems include a poxvirus expression system, an insect baculovirus expression system, and a yeast expression system.
  • the HPVL1 protein expressed in the eukaryotic expression system has little natural conformational destruction and can spontaneously form VLPs. It is often necessary to perform a simple density gradient centrifugation to obtain a purified VLP, which provides great convenience for purification work.
  • Due to the low expression level of eukaryotic expression systems and high cultivation costs it has brought great difficulties to large-scale industrial production.
  • the currently marketed HPV vaccine Gardasil® uses a Saccharomyces cerevisiae expression system with low expression and high production costs, so the price of this product is high, which affects its wide application.
  • HPVL 1 protein by the E. coli expression system in prokaryotic expression systems has been reported.
  • expression of HPV16 L1 protein by E. coli has been reported (Banks, L., G. Matlashewski, et al. (1987). J Gen Virol 68 (Pt 12): 3081-9).
  • most of the HPV LI proteins expressed by E. coli lose their natural conformation and cannot produce protective antibodies against HPV.
  • HPV VLPs can also be obtained (Kelsall, SR and JK Kulski (1995).
  • HPVL1 protein can also be expressed in E. coli in a proper conformation and soluble in the lysed supernatant of the cell, but its expression is low, and the amount of the heteroprotein in the supernatant is large and large, it is purified from the purpose. The protein is quite difficult. Although it has been reported in the literature that the expression of L1 protein in the supernatant can be increased by GST fusion expression, and the purification of the target protein is facilitated (Li, M., TP Cripe, et al. (1997). J Virol 71 (4) : 2988-95 ), but the cutting of fusion proteins often requires expensive enzymes and still cannot be applied to large-scale production.
  • the present invention is based, at least in part, on the surprising findings of the inventors:
  • the E. coli expression system is capable of expressing in large amounts a truncated HPV52 L1 protein that can induce neutralizing antibodies against HPV52, which has high yields and is purified.
  • the purity of the post-protein can be at least 50% or higher (for example, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%), and the purified protein is Further treatment provides virus-like particles that can induce protective antibodies against HPV52.
  • the invention relates to the N-terminal truncation of 27-42 amino acids, such as 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41 or 42 amino acids of the HPV52 L1 protein or variant thereof.
  • the invention relates to a truncated HPV52 L1 protein or variant thereof, which has a N-terminal truncation of 27-42 amino acids compared to the wild-type HPV52 L1 protein, eg, 27, 28, 29 , 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41 or 42 amino acids.
  • the N-terminus of the truncated HPV52 L1 protein is truncated by 27-42 amino acids (eg, 35-42 amino acids), such as 27, 35, compared to the wild-type HPV52 L1 protein. , 38, 40 or 42 amino acids.
  • the N-terminus of the truncated HPV52 L1 protein is truncated by 40 amino acids compared to the wild-type HPV52 L1 protein.
  • the truncated HPV52 L1 protein (hereinafter also simply referred to as a truncated protein) has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO:
  • the amino acid sequence shown by 13 has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 13.
  • the truncated protein has the amino acid sequence set forth in SEQ ID NO: 12.
  • the invention features a polynucleotide encoding a truncated protein of the invention, or a variant thereof, and a vector comprising the polynucleotide.
  • 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, a cosmid, and the like.
  • the invention also relates to a host cell comprising the above polynucleotide or vector.
  • 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 (e.g., 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 invention relates to a HPV52 virus-like particle, wherein the virus-like particle comprises or consists of a truncated protein of the invention or a variant thereof.
  • the HPV52 virus-like particle of the invention comprises a N-terminal truncation of 27-42 amino acids compared to the wild-type HPV52 L1 protein, such as 27, 35, 38, 40 or 42 An amino acid truncated HPV52 L1 protein, either composed or formed of the protein.
  • the HPV52 virus-like particle of the invention comprises or consists of or comprises a truncated HPV52 L1 protein having the sequence set forth in SEQ ID NO: 1, 7, 10, 12 or 13. .
  • the invention also relates to a composition
  • a composition comprising the above-described truncated protein or variant thereof, or the above polynucleotide or vector or host cell or HPV52 virus-like particle.
  • the composition comprises a truncated protein of the invention or a variant thereof.
  • the composition comprises HPV52 virus-like particles of the invention.
  • the present invention is also a pharmaceutical composition or vaccine comprising the HPV52 virus-like particle of the present invention, optionally further comprising a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition or vaccine of the present invention can be used for the prevention of HPV (especially HPV52) infection or diseases caused by HPV (especially HPV52) infection such as cervical cancer.
  • the HPV52 virus-like particle is present in an amount effective to prevent HPV infection or cervical cancer.
  • the pharmaceutical composition or vaccine of the present invention further comprises at least one virus-like particle selected from the group consisting of: HPV6 L1 protein virus-like particle, HPV11 L1 protein virus-like particle, HPV16 L1 protein virus-like particle , HPV18 L1 protein virus-like particles, HPV31 L1 protein virus-like particles, HPV33 L1 protein virus-like particles, HPV45 L1 protein virus-like particles, And HPV58 L1 protein virus-like particles; preferably, these virus-like particles are each independently present in an amount effective to prevent cervical cancer or a corresponding HPV subtype infection.
  • 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 HPV52 virus-like particles contained per unit dose is 5 g - 8 (g, preferably 2 ( ⁇ g - 4 ( ⁇ g.
  • the invention relates to obtaining A method of truncating a protein of the present invention, which comprises expressing a truncated protein of the present invention using an E. coli expression system, and then purifying the cleavage supernatant containing the truncated protein.
  • the method of obtaining a truncated protein of the invention comprises a) expressing said truncated protein in E. coli,
  • the Escherichia coli expressing the truncated protein is disrupted in a solution having a salt concentration of 100 mM to 600 mM, and the supernatant is separated.
  • c) reduce the salt concentration of the supernatant obtained in b) to 100 mM or less with water or a low salt solution, as low as 0, and collect the precipitate.
  • the salt concentration in step b) above is from 200 mM to 500 mM.
  • the present invention also relates to a method for obtaining the HPV52 virus-like particle of the present invention, which comprises the steps of obtaining the truncated protein of the present invention, comprising the steps of:
  • the invention also relates to a method of preparing a vaccine comprising mixing a HPV52 virus-like particle of the invention with a pharmaceutically acceptable carrier and/or excipient, optionally further mixing one or more selected from the group consisting of HPV6, 1 HPV-type virus-like particles of 1, 16, 18, 31, 33, 45 and 58.
  • the vaccine obtained can be used to prevent HPV (especially HPV52) infection or diseases caused by HPV (especially HPV52) infection such as cervical cancer and the like.
  • 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 a HPV52 virus-like particle or pharmaceutical composition or vaccine according to the present invention to a subject .
  • the HPV infection is an HPV52 infection.
  • the disease caused by HPV infection includes, but is not limited to, cervical cancer.
  • the subject is a mammal, such as a human.
  • the invention relates to the use of a truncated protein or variant thereof or HPV52 virus-like particle according to the invention for the preparation of a pharmaceutical composition or vaccine for the prevention of HPV infection or infection by HPV
  • HPV infection is an HPV52 infection.
  • the disease caused by HPV infection includes, but is not limited to, cervical cancer.
  • HPV infection is an HPV 52 infection.
  • disease caused by HPV infection comprises But not limited to, cervical cancer. Description and explanation of related terms in the present invention
  • the expression "the protein whose N-terminally truncated X amino acids” means that the methionine residue encoded by the initiation codon (for initiation of protein translation) replaces the 1-X of the N-terminus of the protein.
  • the HPV52 L1 protein whose N-terminal is truncated by 27 amino acids refers to a protein obtained by replacing the amino acid residues 1 to 27 of the N-terminus of the wild-type HPV52 L1 protein with a methionine residue encoded by the initiation codon. .
  • the term "variant” refers to a protein having an amino acid sequence identical to the amino acid sequence of the truncated HPV52 L1 protein of the invention (such as the protein of SEQ ID NO: 1, 7, 10, 12 or 13).
  • the sequence has one or more (eg, 1-10 or 1-5 or 1-3) amino acid differences (eg, conservative amino acid substitutions) or has at least 60%, 80%, 85%, 90%, 95%, 96 %, 97%, 98%, or 99% identity, and it retains the necessary properties of the truncated protein.
  • essential property herein may be one or more of the following characteristics: ability to induce a neutralizing antibody against HPV52; capable of being solublely expressed in Escherichia coli; high yield can be obtained by the expression purification method of the present invention Purified protein.
  • the term "identity” is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in two sequences being compared is occupied by the same base or amino acid monomer subunit (eg, two DNA molecules)
  • Each position in each of them is occupied by adenine, or a position in each of the two 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 ⁇ ⁇ ⁇ . For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • 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, for example, the method of Needleman et al. (1970) /. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Al ign program (DNAs tar, Inc.). .
  • the algorithm of E. Meyers and W. Mi ller Comput.
  • Appl Biosci., 4: 11-17 (1988) which has been integrated into the ALIGN program (version 2.0), can also be used, using the PAM120 weight residue table ( Weight res idue table ), a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences.
  • the Needleman and Wunsch (J Mol Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package (available on www.gcg.com) 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 biological activity 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 in place of amino acid residues with amino acid residues having similar side chains, 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.). already at A family of amino acid residues having similar side chains is 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), P-branched 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, gly
  • 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: GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3), etc.
  • the term "vector” means a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector When the vector enables expression of a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector.
  • 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 "truncated HPV52 L1 protein” refers to a protein obtained by removing one or more amino acids at the N-terminus and/or C-terminus of the wild-type HPV52 L1 protein, wherein examples of the wild-type HPV52 L1 protein include but not Limited to: NCBI number According to ACX32362. 1, Q05138. 2 or ABU55790. 1 in the library, the full-length L1 protein.
  • the amino acid sequence of the wild-type HPV52 L1 protein can be as set forth in SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29.
  • truncated HPV52 L1 protein gene fragment refers to a gene fragment which lacks one or more amino acids at the 5' or 3' end as compared to the wild type HPV52 L1 protein gene.
  • Nucleotides, wherein the full-length sequence of the wild-type HPV52 L1 protein gene is, for example but not limited to, the following sequence in the NCBI database: EU077195. 1 , EU077194. 1 , FJ615303. 1 et al.
  • 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 Well-known (see, for example, Remington's Pharmaceutica ica l Sc iences. Edi ted by Gennaro AR, 19th ed. Pennsylvania: Mack Publ i shing Company, 1995), and includes but is not limited to: pH adjusters, surfactants, adjuvants , ionic strength enhancer.
  • 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 such as hydrogen Alumina), 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 a disease e.g., HPV infection
  • an 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 truncated HPV52 L1 protein of the present invention preferably passes the following steps Obtaining: Escherichia coli expressing the truncated HPV52 L1 protein is disrupted in a buffer solution having a salt concentration of 100 - 600 mM, preferably 200 - 500 mM, and the solution is centrifuged to obtain a supernatant; water or a low salt concentration (usually The solution below the salt concentration for the crushing reduces the salt concentration of the resulting supernatant to a salt concentration of 100 mM - OmM, whereby the truncated HPV52 L1 protein precipitates in the supernatant; the precipitate is contained in the reducing agent and the salt concentration is The solution containing 150-200 mM, preferably 200 mM or more, is re-dissolved to isolate a solution containing the truncated HPV52 L1 protein, wherein the protein has a purity of at least 50%, preferably at least 70%, more preferably at
  • Buffers useful in the methods of the invention are well known in the art and include, but are not limited to, Tr i s buffer, phosphate buffer, HEPES buffer, MOPS buffer, and the like.
  • 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. .
  • Salts useful in the process of the present invention include, but are not limited to, acid salts, basic salts, neutral salts such as alkali metal salts, alkaline earth metal salts, ammonium salts, hydrochloride salts, sulfate salts, hydrogencarbonates, phosphate salts. Or a hydrogen phosphate, in particular one or more of NaC l, KC1, NH 4 C 1 , (NH 4 ) 2 S0 4 . A particularly preferred salt is NaCl.
  • Reducing agents useful in the methods of the invention include, but are not limited to, DTT, 2-mercaptoethanol, in amounts including, but not limited to, 1 OmM-100 mM.
  • the HPV52 virus-like particle according to the present invention can be obtained by the following steps: further separating the truncated HPV52 L1 protein having a purity of at least 50% as described above by, for example, color transfer chromatography to obtain a purified truncated protein solution; removing the solution
  • the reducing agent gives the HPV52 virus-like particles.
  • the manner in which the reducing agent is removed is known in the art and includes, but is not limited to, dialysis, ultrafiltration or chromatography.
  • HPV virus-like particles can be divided into eukaryotic expression systems and prokaryotic expression systems.
  • HPV L1 protein expressed in the eukaryotic expression system has little natural conformational destruction and can spontaneously form VLPs, and it is often necessary to carry out a simple purification process to obtain a VLP having a correct conformation.
  • current eukaryotic expression systems such as baculovirus expression systems and yeast expression systems have shortcomings such as low expression levels and high cultivation costs, which have brought great difficulties to large-scale industrial production.
  • the E. coli expression system has the advantages of low culture cost and large expression amount.
  • the HPV L1 protein expressed in E. coli tends to lose its correct native conformation and is expressed in the form of inclusion bodies in the pellet.
  • the renaturation of proteins expressed in inclusion bodies is still a worldwide problem. Reconciliation difficulties and inefficiencies make it difficult to obtain VLPs with correct conformation from inclusion bodies in large-scale production and can only be limited to small-scale laboratory studies.
  • HPV L1 can also be solublely expressed in E. coli in the correct conformation, its expression level is low.
  • purification of HPV L1 protein from E. coli lysed supernatant containing a wide variety of soluble proteins is also quite difficult, often by means of fusion expression and affinity chromatography, which often require expensive enzymes. Industrial production is still not possible.
  • the N-terminally truncated HPV52 L1 protein provided by the present invention and a preparation method thereof effectively solve the above problems.
  • the present invention uses an E. coli expression system to express the N-terminally truncated HPV52 L1 protein, ensuring its high expression level.
  • the present invention selectively precipitates the truncated protein in the E. coli lysate supernatant by mild means, and then redissolves the truncated protein with a salt-containing buffer to thereby ensure the purity of the protein while maintaining the correct conformation of the truncated protein.
  • the obtained truncated protein solution can be directly passed through color chromatography such as ion exchange chromatography and hydrophobic exchange chromatography. Purification in one step yields a high purity protein of interest (eg, 80% purity). Further, the obtained high-purity truncated protein can be assembled into a virus-like particle, and the virus-like particle can induce a high titer neutralizing antibody against HPV52 in vivo, has good immunogenicity, and is a good vaccine. Form, can be used to prevent HPV52 infection of the human body.
  • the truncated protein of the present invention can realize a large amount of expression in an E. coli expression system while retaining the antigenicity, immunogenicity and particle assembly ability of the full-length HPV52 L1 protein;
  • the preparation method used does not require the use of expensive enzymes, and the cost is low;
  • the truncated protein has no undergoing a strong denaturing and refolding process during the purification process, and the loss is small and the yield is high;
  • the virus-like particles formed by the truncated protein can induce high A titer of protective antibodies against HPV that can be used to produce vaccines.
  • the truncated protein of the present invention and the preparation method thereof can be applied to large-scale industrial production, and it is possible to produce a large-scale industrial production of cervical cancer vaccine.
  • Figure 1 shows the results of SDS polyacrylamide gel electrophoresis of the HPV52N40C-L1 protein obtained in the different steps of Example 3 of the present invention.
  • Lane M Protein molecular weight marker
  • Lane 1 Bacterial supernatant (ie, supernatant obtained after centrifugation of the cells)
  • Lane 2 Salt-free precipitated product (ie, precipitate obtained by centrifugation after dialysis);
  • Lane 3 Heavy After dissolving the supernatant (ie, re-dissolving the salt-free precipitated product and then centrifuging The supernatant 4)
  • Lane 4 Precipitate after reconstitution (i.e., the precipitate obtained by centrifugation after re-dissolving the salt-free precipitated product).
  • the results showed that the purity of the HPV52N40C-L1 protein increased from about 10% (see lane 1) to about 70% after the step of precipitation and reconstitution (see lane 3).
  • Fig. 2 shows the results of SDS polyacrylamide gel electrophoresis of HPV52N40C-L1 obtained by cation exchange chromatography purification and CHT-I I purification in Example 4.
  • Lane M Protein molecular weight marker; Lane 1, HPV52N40C-L1 purified by the method of Example 4 (loading volume of 10 ⁇ M); Lane 2, HPV52N40C-L1 purified by the method of Example 4 (loading The volume is 20 ⁇ 1).
  • the results showed that the purity of HPV52N40C-L1 protein reached about 98% after the cation exchange color transfer purification and CHT-I I purification of Example 4.
  • Fig. 4 shows the results of cryo-electron microscopic observation of the HPV52N40C-L1 virus-like particles obtained in Example 5 described in Example 6, and the reconstructed three-dimensional structure thereof.
  • Figure 4A HPV52N40C-L1 virus-like particles
  • Figure 4B reconstructed three-dimensional structure of HPV52N40C-L1 virus-like particles.
  • HPV52N40C-L1 VLP structure Unlike the general icosahedral viral capsids that conform to the quasi-equivalent principle, all the constituent subunits in the HPV52N40C-L1 VLP structure are pentamers, but no hexamers are seen, and the outermost diameter of the VLP is about 60 nm.
  • This is a three-dimensional structure of HPV VLPs derived from previously reported natural HPV virions and eukaryotic expression systems (eg, poxvirus expression systems) (Baker TS, Newcomb WW, Olson NH. et al. Biophys J. (1991) , 60 (6) : 1445-1456; Hagensee ME, Ol son NH, Baker TS, et al. J Vi rol. (1994), 68 (7): 4503-4505; Buck CB, Cheng N, Thompson CD. et a l. J Vi rol. (2008) , 82 (11) : 5190- 7) Similar
  • Fig. 5 shows the results of dynamic light scattering observation of the HPV52N40C-L1 virus-like particles obtained in Example 5 described in Example 6. The results showed that the HPV52N40C-L1 virus-like particle had a hydration molecular dynamic radius of 24.39 nm and a particle assembly percentage of 100%.
  • Figure 6 shows the neutralizing antibody titer of the serum at different stages after inoculation of rabbits with HPV52N40C-L1 virus-like particles as determined in Example 7.
  • the arrows in the figure show the immunization time. In one month after the initial immunization, total antibody titers increased rapidly; after a first vaccination, titers of neutralizing antibodies that can achieve a high level of 105.
  • Figure 7 shows the amino acid sequence of HPV52 L1 protein ⁇ HPV52N27C-L1, HPV52N35C-LK HPV52N38C-LU HPV52N42C-LK with N-terminally truncated 27, 35, 38 or 42 amino acids obtained in Example 8.
  • ID NO: 1 Results of SDS polyacrylamide gel electrophoresis of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13).
  • Lane M protein molecular weight marker
  • Lane 1 HPV52N27C-L1 protein, loading volume ⁇ ⁇
  • Lane 2 HPV52N35C-L1 protein, loading volume ⁇ ⁇
  • Lane 4 HPV52N42C-L1 protein, loading volume is 10 ⁇ 1.
  • the results showed that the protein purity of the truncated proteins HPV52N27C-Ll and HPV52N35C-LK HPV52N38C-LU HPV52N42C-L1 obtained in Example 8 was about 98%.
  • Fig. 8 shows the results of transmission electron microscopic observation (50, 000 times, 100 nm) of HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1, and HPV52N42C-L1 virus-like particles obtained in Example 8.
  • Figure 8A HPV52N27C-L1 virus-like particles
  • Figure 8B HPV52N35C-L1 virus-like particles
  • Figure 8C HPV52N38C-L1 virus-like particles
  • Figure 8D HPV52N42C-L1 virus-like particles.
  • the results show that in these 4 figures A large number of virus-like particles with a radius of about 25 nm can be seen in the field of view, and the particle size is consistent with the theoretical size, and both are consistent.
  • Fig. 9 shows the results of dynamic light scattering observation of HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-LK HPV52N42C-L1 virus-like particles obtained in Example 8.
  • Figure 9A HPV52N27C-L1 virus-like particles
  • Figure 9B HPV52N35C-L1 virus-like particles
  • Figure 9C HPV52N38C-L1 virus-like particles
  • Figure 9D HPV52N42C-L1 virus-like particles.
  • DNA sequence encoding SEQ ID NO: 1 DNA sequence encoding SEQ ID NO: 2
  • DNA sequence encoding SEQ ID NO: 3 DNA sequence encoding SEQ ID NO: 4
  • the DNA sequence encoding SEQ ID NO: 5 encodes SEQ ID NO: The DNA sequence encoding 6
  • the DNA sequence encoding SEQ ID NO: 7 The DNA sequence encoding SEQ ID NO: 8
  • the DNA sequence encoding SEQ ID NO: 10 encodes the DNA sequence encoding SEQ ID NO: 11.
  • the DNA sequence of SEQ ID NO: 12 encodes the DNA sequence of SEQ ID NO:
  • NSGNPGDCPP LQLINSVIQD GDMVDTGFGC MDFNTLQASK SDVPIDICSS VCKYPDYLQM
  • AdHSVWOlAO dA3 ⁇ 43ASS3ia idAas3 ⁇ 4svm BTOdi aAwaoaOMS ⁇ 8 ⁇ dNOSNNNDdl 3 ⁇ 433 ⁇ 3 ⁇ 0 ⁇ 0 flat SO3 ai3d3 ⁇ 43VAM ⁇
  • the molecular biology experimental methods and immunoassays used in the present invention are basically referred to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, And according to the method described in FM Ausubel et al., Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995 or in accordance with product specifications.
  • the reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.
  • the invention is described by way of example, and is not intended to limit the scope of the invention.
  • Example 1 Construction of a non-fusion expression vector expressing a truncated HPV52 L1 protein
  • the full-length HPV52 L1 gene (SEQ ID NO: 30) used as a template was synthesized by Shanghai Boya Company.
  • the synthesized gene fragment has a full length of 1590 bp.
  • a polynucleotide encoding the truncated HPV52 L1 protein of the present invention was prepared.
  • the synthetic full-length HPV52 L1 gene was used as a template for PCR reaction, and 52N40F: 5'-CAT ATg CCC GTG CCC GTG AGC AAG-3' (SEQ ID NO: 31) was used as a forward primer (the 5' end introduced a restriction Endonuclease CAT3 ⁇ 43 ⁇ 4 ⁇ site CAT ATG, ATG is the initiation codon in the E.
  • a size-specific DNA fragment of about 1.5 kb was amplified.
  • the PCR product was ligated with a commercially available pMD 18-T vector (manufactured by TAKARA Co., Ltd.), and transformed into Escherichia coli; a positive colony was screened, and a plasmid was extracted and identified by ⁇ / ⁇ digestion to obtain a truncated HPV52 L1 gene. Positive clone pMD 18-T- HPV52N40C-L1.
  • the nucleotide sequence of the inserted fragment of the pMD 18-T-HPV52N40C-L1 plasmid was determined as shown in SEQ ID NO: 25, The encoded amino acid sequence is set forth in SEQ ID NO: 12. The protein corresponding to this sequence is truncated by 40 amino acids at the N-terminus and the C-terminus is not truncated.
  • HPV52 LI protein named HPV52N40C-L1.
  • the above pMD18-T-HPV52N40C-L1 plasmid was digested with Ndel/Sail to obtain a HPV52N40C-L1 gene fragment.
  • the fragment was ligated with the prokaryotic expression vector ⁇ 0- ⁇ 7 (purchased from Invi trogen) digested with Mell Sa and transferred into ER2566 bacteria; the positive colonies were screened, and the plasmid was extracted and identified by ⁇ / ⁇ digestion.
  • the positive expression clone of the fragment was pTO-T7-HPV-52N40C-Ll.
  • the ⁇ - ⁇ 7-HPV-52N40C-L1 plasmid (0.15 mg/ml) of 1 was transformed into 40 ⁇ L of competent Escherichia coli ER2566 (purchased from Invi trogen) prepared by calcium chloride method, and coated with Kanamycin (final concentration 100 mg/mL, the same below) solid LB medium (LB medium composition: 10 g/L peptone, 5 g/L yeast powder, 10 g/L sodium chloride, the same below), and 37
  • the culture is allowed to stand for 10-12 hours until the single colony is clearly distinguishable.
  • Feed preparation Prepare 30% casein hydrolysate (30g dissolved to 100ml), 50% Glucose (50 g dissolved to 100 ml), sterilized at 121X for 20 min.
  • Feeding 50% glucose and 30% casein hydrolysate were mixed at a solute mass ratio of 2:1.
  • the flow acceleration is as follows (100% at 25mL/min):
  • the cells were resuspended in a ratio of 10 mL of lysate (20 mM Tris buffer, pH 7.2, 300 mM NaCl) to lg cells.
  • the cells were disrupted 5 times with a pressure of 600 bar using an APV homogenizer (An Invensys Group product).
  • APV homogenizer An Invensys Group product.
  • the bacterial cell disruption was centrifuged at 13500 rpm (30000 g) for 15 min, and the supernatant (ie, the supernatant of the bacteria) was taken.
  • the supernatant was detected by 10% SDS-polyacrylamide gel electrophoresis, and the purity of the HPV52N40C-L1 protein in the supernatant was about 10% (see Figure 1, lane 1).
  • the supernatant was dialyzed using a CENTRASETTE 5 tangential flow device (PALL product) with a molecular weight cut-off of 30 kDa, a dialysate of 10 mM phosphate buffer pH 6.0, a dialysis volume of three times the supernatant volume, and a running pressure of 0.5psi, flow rate is 500 mL/min, tangential flow rate was 200 ml for 7 min.
  • PALL product CENTRASETTE 5 tangential flow device
  • the pellet was harvested by centrifugation at 9,500 rpm (12,000 g) for 20 min using a JA-10 rotor (Beckman J25 high speed centrifuge) (i.e., salt-free precipitated product).
  • the pellet was resuspended in 1/10 supernatant volume of 10 mM phosphate buffer pH 7.0, 10 mM DTT, 300 mM NaCl, stirred for 30 min, and then centrifuged at 13500 rpm (30000 g) using a JA-14 rotor (Beckman J25 high speed centrifuge). After 20 min, the supernatant and pellet were harvested (i.e., precipitated after reconstitution).
  • the supernatant was filtered using a 0.22 ⁇ pore size filter and the obtained sample (i.e., the supernatant after re-dissolution) was used for cation exchange chromatography purification (as described in Example 4).
  • Take 150 ⁇ of the filtered sample add 30 ⁇ 6 ⁇ Loading Buffer ( 12% (w/v) SDS , 0.6% (w/v) bromophenol blue, 0 ⁇ 3M Tris-HCl ⁇ 6.8, 60% ( ⁇ / ⁇ ) glycerol , 5% ( ⁇ / ⁇ ) ⁇ -mercaptoethanol), mix and heat in an 80* ⁇ water bath for 10 min; then take ⁇ in a 10% SDS-polyacrylamide gel and electrophoresis at 120 V for 120 min; Staining shows the electrophoresis band.
  • Loading Buffer 12% (w/v) SDS , 0.6% (w/v) bromophenol blue, 0 ⁇ 3M Tris-HCl ⁇ 6.8,
  • AKTA explorer 100 preparative liquid phase color transfer system manufactured by GE Healthcare (formerly Amershan Pharmacia).
  • Buffer 20 mM phosphate buffer pH 8.0, lOmM DTT
  • the sample was a solution of HPV52N40C-L1 protein (3 L) having a purity of about 70%, which was filtered through a 0.22 ⁇ m pore size filter obtained in Example 3.
  • the elution procedure was as follows: 500 mM NaCl was used to elute the heteroprotein, and the target protein was eluted with 100 mM NaCl, and the eluted product of 100 mM NaCl was collected to obtain a total of 900 mL of the purified sample of HPV52N40C-L1.
  • AKTA explorer 100 preparative liquid phase color transfer system manufactured by GE Healthcare (formerly Amershan Pharmacia).
  • Chromatography media CHT-11 (purchased from Bio-RAD)
  • Buffer 20 mM phosphate buffer pH 8.0, 10 mM DTT
  • the sample was: the lOOOOmM NaCl eluted product obtained in the previous step, and the NaCl concentration was diluted to 500 mM with 20 mM phosphate buffer ⁇ 8.0, 10 mM DTT.
  • the elution procedure was as follows: 500 mM NaCl was used to elute the heteroprotein, and 100 mM NaCl was used to elute the target protein, and 100 mM NaCl was eluted to obtain a purified HPV52N40C-L1 protein sample of 800 mL.
  • the instrument system was a CENTRASETTE5 tangential flow system produced by PALL; the membrane molecular weight cutoff was 30 kDa; and the sample was HPV 52N40C-L1, 800 ml with a purity greater than 98% obtained in Example 4.
  • the tangential flow rate of the tangential flow system was adjusted to 50 mL/min, and the sample was concentrated to a total volume of 600 mL.
  • the sample buffer was fully exchanged with 10 L of refolding buffer (20 mMPB (sodium phosphate buffer) pH 6.0, 2 mM CaCl 2 , 2 mM MgCl 2 , 0.5 M NaCl, 0.003% Tween-80).
  • the tangential flow device was operated at a pressure of 0.5 psi and a tangential flow rate of 10 mL/min.
  • exchange was carried out with storage buffer (20 mM PB (sodium phosphate buffer) pH 6.5, 0.5 M NaCl), and the exchange volume was 20 L.
  • the tangential flow device was operated at a pressure of 0.5 psi and a tangential flow rate of 25 mL/min. After the exchange was completed, the sample was sterile-filtered using a PALL 0.22 ⁇ n filter to obtain HPV52N40C-L1 virus-like particles, which were placed in 4 for storage.
  • Example 6 Morphological detection of HPV52N40C-L1 VLP
  • the instrument used was a 100 kV transmission electron microscope manufactured by JEOL, with a magnification of 100,000 times.
  • the HPV52N40C-L1 virus-like particles obtained in Example 5 were negatively stained with 2% phosphotungstic acid pH 7.0, and fixed on a carbon-coated copper mesh for observation.
  • the results of the electron microscopy are shown in Fig. 3. A large number of virus-like particles with a radius of about 25 nm are visible, and the size is uniform, showing a hollow shape.
  • all the constituent subunits in the HPV52N40C-LI VLP structure are pentamers, and no hexamers are seen, and the outermost diameter of the VLP is about 60 nm. .
  • This is similar to the three-dimensional structure of previously reported HPV virions and HPV VLPs derived from eukaryotic expression systems (eg, poxvirus expression systems).
  • the instrument used was a DynaPro MS/X dynamic light scattering instrument (including temperature controller) manufactured by Protein Solut ions of the United States, and the algorithm used was the Regu lat ion algorithm.
  • the sample was the HPV52N40C-L1 virus-like particle obtained in Example 5.
  • the sample was filtered through a 0.22 ⁇ m filter and measured. The measurement results are shown in Figure 5. The results show that the hydration molecular dynamics radius of the HPV52N40C-L1 VLP is 24.39 nm.
  • Example 7 Immunogenicity determination of HPV52N40C-L1 VLP
  • HPV is difficult to culture in vitro, and the HPV host is highly specific, it is difficult to propagate on a host other than human, and there is no suitable animal model. Therefore, in order to quickly evaluate the immunoprotective effect of HPV vaccine, it is necessary to establish an effective in vitro body. Neutralize the experimental model.
  • In vitro infection model of pseudovir ions using HPV VLPs to non-specifically package nucleic acid properties, by expressing HPV L1 and L2 proteins in cells, by wrapping intracellular free viral DNA or exogenously introduced reporter plasmids, Composition of HPV pseudovirus (Yeager, M. D, As te-Amezaga, M. et al (2000) Virology (278) 570-7). Specific methods include recombinant viral expression system methods and multi-plasmid co-transfection methods. This example exemplifies the use of a multi-plasmid co-transfection method.
  • HPV system uses conventional methods: Optimizing the calcium phosphate transfection method for 293FT cells to achieve transfection efficiency of up to 90%, thereby facilitating large-scale production;
  • the expression plasmid of HPV structural protein is codon optimized to efficiently express HPV L1 and L2 genes in mammalian cells, thereby facilitating efficient assembly of pseudoviruses.
  • HPV pseudovirus is built as follows:
  • Plasmid p52Llh (pAAV vector carrying the nucleotide sequence encoding HPV52 L1 protein (NCBI database, accession number Q05138)), plasmid p52L2h (carrier encoding HPV52 L2 protein (NCBI database, accession number P36763) was purified by CsCl density gradient centrifugation.
  • the pAAV vector of the nucleotide sequence) and the plasmid pN31-EGFP with the green fluorescent protein gene (pN31-EGFP and the above pAAV vector were all given by Professor John T. Schiler of NIH). Methods for purifying plasmids using CsCl density gradient centrifugation are well known in the art, see Molecular Cloning: Third Edition.
  • 293FT cells (Invi trogen) grown in 10 cm cell culture dishes were co-transfected with purified p52Llh, p52L2h, pN31-EGFP 4 ( ⁇ g) using calcium phosphate transfection. Phosphotransfection is well known in the art. , see Molecular Cloning: Third Edition.
  • 293FT cells (Invitrogen) were plated in 96-well cell culture plates (1.5 x 107 wells). After 5 hr, the neutralization experiment was carried out as follows: The serum sample to be tested (including the antibody to be tested) was serially diluted with 10% DMEM, and then 5 ( ⁇ L each diluted sample was diluted with 50 ⁇ in 10% DMEM, respectively.
  • Infection inhibition rate (1 - infection rate of wells added to serum / infection rate of wells not added to serum) ⁇ %.
  • the positive region is defined as: The GFP signal measured by flow cytometry is at least 10 times higher than the control cell.
  • Antibody neutralization titers are defined as: Maximum dilution factor that achieves greater than 50% infection inhibition. Antibodies that still achieve 50% inhibition of infection after 50-fold dilution are considered to have neutralizing ability.
  • the immunized animals were 6-8 week old female rabbits (purchased from the Guangxi Provincial Center for Disease Control and Prevention).
  • the HPV52N40C-L1 virus-like particles prepared in Example 5 were combined with an equal volume of complete Freund's adjuvant (for primary immunization) or an equal volume of incomplete Freund's adjuvant (for Strengthen immunity) Mix evenly.
  • the immunization schedule was: initial immunization at 0 weeks; each boost at 4 and 10 weeks.
  • the immunization method was intramuscular injection, the initial immunization dose was 100 ⁇ 8 /piece, and the booster dose was 50 ⁇ 8 /piece.
  • peripheral venous blood is taken weekly, serum is separated, and stored for examination.
  • the antibody neutralizing titer against HPV52 pseudovirus in rabbit serum was then determined according to the method described above.
  • Figure 6 shows serum neutralizing antibody titers at various stages after inoculation of HPV52N40C-L1 virus-like particles into rabbits, and the arrows indicate the immunization time.
  • the total serum antibody titers increased rapidly; after a first vaccination, titers of neutralizing antibodies that can achieve a high level of 105.
  • the HPV52N40C-L1 VLP obtained according to the methods described in Examples 1-5 has strong immunogenicity and is capable of inducing a high titer neutralizing antibody against HPV52 in animals, and is useful as an effective vaccine for preventing HPV52 infection. .
  • the vaccine may also use other adjuvants known in the art, such as aluminum hydroxide or aluminum phosphate adjuvants.
  • Example 8 HPV52N27C—Ll, HPV52N35C—Ll, HPV52N38C-LK Preparation and morphological observation of HPV52N42C-L1 protein and virus-like particles
  • HPV52N27C-L1, HPV52N35C-LU HPV52N38C-L1 and HPV52N42C-L1 proteins were assembled into virus-like particles according to the method described in Example 5, respectively, referred to as HPV52N27C-LI, HPV52N35C-L1, HPV52N38C-L1, respectively. And HPV52N42C-L1 virus-like particles.
  • HPV52N27C-L1, HPV52N35C-LU HPV52N38C-L1 and HPV52N42C-L1 virus-like particles were observed by transmission electron microscopy and dynamic light scattering, respectively, according to the method described in Example 6. The results are shown in Figures 8 and 9.
  • Figure 8 shows that these truncated proteins can form a large number of virus-like particles with a radius of about 25 nm, and the particle size is consistent with the theoretical size and uniform.
  • Figure 9 shows that the HPM52N27C-Ll, HPV52N35C-LU HPV52N38C-L1 and HPV52N42C-L1 virus-like particles have a hydration molecular dynamic radius of about 25 nm and a particle assembly percentage of 100%.
  • HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1 and HPV52N42C-L1 virus-like particles obtained by the present invention also have good immunogenicity and can be induced high in animals.
  • the neutralizing antibody of titer can be used as a vaccine for preventing HPV infection. While the invention has been described in detail, it will be understood by those skilled in the art . The full scope of the invention is indicated by the appended claims and any equivalents thereof.

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Abstract

提供了一种截短的人乳头瘤病毒（HPV）52型L1蛋白，其与野生型HPV52L1蛋白相比，N端截短了27-42个氨基酸。还提供了截短的HPV52L1蛋白的编码序列、包含该蛋白的病毒样颗粒（VLP）以及利用大肠杆菌表达系统制备该蛋白和VLP的方法。截短的HPV52L1蛋白和组装形成的VLP可用于预防HPV52感染及由HPV52感染所导致的疾病，如宫颈癌等。

Description

截短的人乳头瘤病毒 52型 L1 蛋白 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 一种截短的人乳头瘤病毒 52型 L1蛋白，其编码序列和制备方法， 以及包含其的病毒样颗粒， 所述蛋白和病毒样颗粒可用于预防 HPV (特别是 HPV52 )感染及由 HPV (特别是 HPV52 )感染所导致 的疾病例如宫颈癌等。 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用于预防 HPV (特别是 HPV52 )感染及由 HPV (特别是 HPV52 )感染所导致 的疾病例如宫颈癌等。 背景技术
人乳头瘤病毒（Human Papi l lomavi rus , HPV)属于乳头瘤病毒 ^ (Papi l lomavi r idae) , 为无包膜 DNA病毒。 该病毒基因组为双 链闭环 DNA, 大小约为 7. 2 ~ 8kb, 具有 8个开放阅读框。 该病毒 基因组按功能的不同可以分为三个区域：①早期区（E) ,约 4. 5kb, 编码 El、 E2、 E4 ~ E7共 6个与病毒复制， 转录及转化有关的非结 构蛋白； ②晚期区（L) , 约 2. 5kb, 编码主要衣壳蛋白 L1和次要 衣壳蛋白 L2; ③长调控区（LCR) , 位于 L区末端与 E区起始端之 间， 长约 800 ~ 900bp， 不编码任何蛋白， 含 DNA复制和表达调控 元件。 HPV病毒颗粒直径为 45 ~ 55nm, 核衣壳呈 20面体对称， 有 72个壳微粒， 由 L1及 L2组成。
目前已知的 HPV约有 100多种亚型，在人群中主要引起皮肤， 粘膜的疣状病变。 根据其与肿瘤发生的关系， HPV可分为 3组： ①低或无致癌风险组， 包括 HPV6、 11、 39、 41、 42、 43; ②中度 致癌风险组， 包括 HPV31、 33、 35、 51 ; ③高度致癌风险组， 包 括 HPV16、 18、 58、 45、 52。
HPV分子流行病学调查证实， 高危型 HPV感染是宫颈癌发生 的重要启动因子。在所有宫颈癌标本中， HPV DM检出率高达 80 % 以上。 宫颈癌是一种常见的女性恶性肿瘤， 其发病率仅次于乳腺 癌， 是严重威胁女性健康的杀手。 据统计， 每年世界范围内约有 490， 000例的新发病例，约有 270， 000人死于该疾病（ Boyle, P. , and J. Fer lay. Ann Oncol 2005 , 16: 481-8 ) 。 在所有宫颈癌 病例中， 发生于发展中国家的占了约 83 %， 在这些国家中， 宫颈 癌甚至能够占到女性恶性肿瘤的 15 %。 而在发达国家， 这个数字 仅占到 1. 5 %。 在撒哈拉以南地区， 中南亚， 拉丁美洲， 东亚均 为宫颈癌的高发区。 我国也属于宫颈癌高发区。 在陕西略阳县， 已婚妇女中宫颈癌的发病率高达 1026/100000。
HPV 的型别分布表现一定的地理分布及人群特点。 在世界范 围内， HPV16、 18亚型是宫颈癌中最常见的型别， 并且 HPV52亚 型是第 6种最常见的高危型 HPV。 在我国的某些地区， 诸如广东 省等省份， HPV52是仅次于 HPV16、 33、 18的高危致癌性 HPV型 别。
目前已上市的 HPV 疫苗为 Merck 的 Gardas i l®及 GSK 的 Cervar ix®, 上述两种疫苗分别含有 HPV6/11/16/18及 HPV16/18 VLP, 但均不包含 HPV52型别 VLP。
因此， 开发覆盖范围更广泛、 适合中国人群的高危型疫苗必 须将针对 HPV52型别的疫苗包含在内。
HPV L1 蛋白为主要衣壳蛋白， 分子量为 55-60kDa , 是 HPV 疫苗主要靶蛋白。 在多种表达系统中表达的 HPV L1蛋白无需 L2 蛋白辅助即可形成在形态结构上与天然病毒颗粒相似的病毒样颗 粒 ( Virus-Like Particle, VLP) 。 该病毒样颗粒为二十面体立 体对称结构， 由 72个 L1蛋白的五聚体组成。 其保留了病毒颗粒 的天然表位， 具有较强的免疫原性， 可诱导针对同型 HPV病毒的 中和抗体（Kirnbauer, R. , F. Booy, et al. 1992 Proc Natl Acad Sci U S A 89 (24): 12180-4)。 并且， 病毒样颗粒不带有病毒核 酸， 无潜在致癌危险， 具有良好的安全性。 因此， VLP 疫苗已成 为 HPV疫苗发展的主要方向。
HPV VLP疫苗研制的关键是能够大量高效制备 VLP样品。 目 前较为常用的 VLP表达系统可以分为真核表达系统及原核表达系 统。
常用的真核表达系统有痘病毒表达系统、 昆虫杆状病毒表达 系统、 酵母表达系统。 在真核表达系统中所表达的 HPVL1蛋白天 然构象破坏少， 能自发形成 VLP, 往往只需进行简单的密度梯度 离心即可得到纯化的 VLP, 为纯化工作提供极大的便利。 但是由 于真核表达系统的表达量低， 培养成本高， 给大规模工业化生产 带来了极大困难。 目前已上市的 HPV疫苗 Gardasil®采用了酿酒 酵母表达系统， 其表达量低， 生产成本高， 因此该产品价位偏高， 影响其广泛应用。
在原核表达系统中利用大肠杆菌表达系统表达 HP V L 1蛋白已 有报道。 例如已报道利用大肠杆菌表达 HPV16 L1蛋白 （Banks, L. , G. Matlashewski, et al. (1987). J Gen Virol 68 (Pt 12): 3081-9 )。但是大肠杆菌表达的 HPV LI蛋白大多失去其天然构象， 不能产生针对 HPV的保护抗体。 或者尽管上述蛋白通过包含体纯 化， 复性等步骤也可得到 HPV VLP (Kelsall, S. R. and J. K. Kulski (1995). J Virol Methods 53 (1): 75-90 ) ， 但是在复 性过程中蛋白损失量大， 得率低， 因此， 难以在大规模生产上应 用。 虽然 HPVLl蛋白也可以在大肠杆菌中以正确构象可溶性地表 达， 溶解于菌体的裂解上清中， 但是其表达量较低， 而且上清中 杂蛋白种类多且量大， 要从中純化出目的蛋白难度相当大。 虽然 也有文献报道通过 GST融合表达的方式可以增加上清中 L1蛋白的 表达量， 而且有助目的蛋白的纯化（Li, M. , T. P. Cripe, et al. (1997). J Virol 71 (4): 2988-95 ) , 但融合蛋白的切割往往需 要价格昂贵的酶， 依然无法应用于大规模生产。
因此， 本领域仍然需要能够低成本获得且能够诱导针对 HPV 的保护性抗体的 HPVL1蛋白及由其组成的病毒样颗粒，从而使大 规模工业化生产宫颈癌疫苗成为可能。 发明内容
本发明至少部分基于发明人的出人意料的发现： 利用大肠杆 菌表达系统能大量表达可以诱导针对 HPV52的中和抗体的截短的 HPV52 L1蛋白， 该截短的 HPV52 L1蛋白具有高产率， 且经纯化 后蛋白的纯度可达到至少 50%或更高 （例如 60%, 70%, 80%, 90%, 95%, 96%, 97%， 98%, 99% ) , 并且纯化后的所述蛋 白经进一步处理可得到可诱导针对 HPV52的保护性抗体的病毒样 颗粒。
因此，在一个方面，本发明涉及 N端截短了 27-42个氨基酸， 例如 27个、 28个、 29个、 30个、 31个、 32个、 33个、 34个、 35个、 36个、 37个、 38个、 39个、 40个、 41个或 42个氨基酸 的 HPV52 L1蛋白或其变体。
在一个方面，本发明涉及一种截短的 HPV52 L1蛋白或其变体， 其与野生型 HPV52 L1蛋白相比， N端截短了 27-42个氨基酸， 例 如 27个、 28个、 29个、 30个、 31个、 32个、 33个、 34个、 35 个、 36个、 37个、 38个、 39个、 40个、 41个或 42个氨基酸。 在一个优选的实施方案中， 与野生型 HPV52 L1蛋白相比， 该 截短的 HPV52 L1蛋白的 N端截短了 27-42个氨基酸（例如， 35-42 个氨基酸） , 例如 27个、 35个、 38个、 40个或 42个氨基酸。 在另一个优选的实施方案中， 与野生型 HPV52 L1蛋白相比， 该截 短的 HPV52 L1蛋白的 N端截短了 40个氨基酸。
在另一个优选的实施方案中， 该截短的 HPV52 L1蛋白 （以下 也简称为截短蛋白）具有 SEQ ID NO: 1、 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 5、 SEQ ID NO: 6、 SEQ ID NO: 7、 SEQ ID NO: 8、 SEQ ID NO: 9、 SEQ ID NO: 10、 SEQ ID NO: 11、 SEQ ID NO: 12或 SEQ ID NO: 13所示的氨基酸序列， 例如 具有 SEQ ID NO: 1、 SEQ ID NO: 7、 SEQ ID NO: 10、 SEQ ID NO: 12或者 SEQ ID NO: 13所示的氨基酸序列。 在另一个优选的实施 方案中， 该截短蛋白具有如 SEQ ID NO: 12所示的氨基酸序列。 在另一个方面， 本发明涉及编码本发明的截短蛋白或其变体 的多核苷酸以及含有该多核苷酸的载体。
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， 柯斯质粒等等。
在另一个方面， 本发明还涉及包含上述多核苷酸或载体的宿 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 HPV52病毒样颗粒， 其中该 病毒样颗粒含有本发明的截短蛋白或其变体， 或者由本发明的截 短蛋白或其变体组成或形成。
在一个优选的实施方案中， 本发明的 HPV52病毒样颗粒包含 与野生型 HPV52 L1蛋白相比， N端截短了 27- 42个氨基酸， 例如 27个、 35个、 38个、 40个或 42个氨基酸的截短的 HPV52 L1蛋 白， 或由所述蛋白组成或形成。 在一个特别优选的实施方案中， 本发明的 HPV52病毒样颗粒包含具有 SEQ ID NO: 1 , 7 , 10 , 12 或 13所示序列的截短的 HPV52 L1蛋白， 或由所述蛋白组成或形 成。
在另一个方面， 本发明还涉及包含上述截短蛋白或其变体， 或上述多核苷酸或载体或宿主细胞或 HPV52 病毒样颗粒的组合 物。 在一个优选的实施方案中， 所述组合物包含本发明的截短蛋 白或其变体。 在另一个优选的实施方案中， 所述组合物包含本发 明的 HPV52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 HPV52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 (特 别是 HPV52 )感染或由 HPV (特别是 HPV52 )感染所导致的疾病例 如宫颈癌等。
在一个优选的实施方案中，所述 HPV52病毒样颗粒以预防 HPV 感染或宫颈癌有效量存在。 在另一个优选的实施方案中， 本发明 的药物组合物或疫苗还包含至少一种选自下列的病毒样颗粒： HPV6 L1蛋白病毒样颗粒， HPV11 L1蛋白病毒样颗粒， HPV16 L1 蛋白病毒样颗粒， HPV18 L1蛋白病毒样颗粒， HPV31 L1蛋白病毒 样颗粒， HPV33 L1蛋白病毒样颗粒， HPV45 L1蛋白病毒样颗粒， 和 HPV58 L1蛋白病毒样颗粒； 优选地， 这些病毒样颗粒各自独立 地以预防宫颈癌或者相应 HPV亚型感染有效量存在。
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。
在一个优选的实施方案中， 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 HPV52病毒样颗粒的量为 5 g - 8 ( g , 优选 2(^g - 4 (^g。 在另一个方面， 本发明涉及一种获得本发明的截短蛋白的方 法， 其包括利用大肠杆菌表达系统表达本发明的截短蛋白， 然后 将含有该截短蛋白的裂解上清进行纯化处理。
在一个优选实施方案中， 获得本发明的截短蛋白的方法包括 a)在大肠杆菌中表达所述截短蛋白，
b)将表达所述截短蛋白的大肠杆菌在盐浓度为 100mM-600mM 的溶液中破碎， 分离得到上清液，
c)用水或低盐溶液将 b )获得的上清液的盐浓度降至 l OOmM 或以下， 最低至 0， 并收集沉淀，
d)将 c )获得的沉淀在 150mM - 250mM盐溶液中重新溶解， 同 时加入还原剂， 分离得到溶液， 该溶液中含纯度至少 50 %的截短 的 HPV52 L1蛋白。
在本发明的一个实施方案中， 上述步骤 b ) 中的所述盐浓度 为 200mM- 500mM。
本发明还涉及一种获得本发明的 HPV52病毒样颗粒的方法， 其在获得本发明的截短蛋白的基础上， 包括步骤：
e)将纯度至少 50 %的本发明的截短的 HPV52 L1蛋白进一步 通过色谱层析进行纯化，
f)将步骤 e)中得到的截短蛋白去除还原剂。
本发明还涉及一种制备疫苗的方法， 其包括将本发明的 HPV52病毒样颗粒与药学可接受的载体和 /或赋形剂混合，任选地 还混合一种或多种选自 HPV6 , 1 1 , 16 , 18 , 31 , 33 , 45和 58的 HPV 型别的病毒样颗粒。 如上所论述的， 所获得的疫苗可以用于 预防 HPV (特别是 HPV52 )感染或由 HPV (特别是 HPV52 )感染所 导致的疾病例如宫颈癌等。 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 HPV52 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 HPV52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。 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截短蛋白或其变体或 HPV52 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 HPV52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。
在另一个方面， 还涉及根据本发明的截短蛋白或其变体或 HPV52病毒样颗粒， 其用于预防 HPV感染或由 HPV感染所导致的 疾病。 在一个优选的实施方案中， 所述 HPV感染是 HPV52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。 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， 表述 "N端截短了 X个氨基酸的蛋白质" 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位氨基酸残基所获得的蛋白质。例如 N端截短 了 27个氨基酸的 HPV52 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 HPV52 L1蛋白 N末端的第 1-27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 与本发明的截短的 HPV52 L1蛋白 （如 SEQ ID NO: 1 , 7 , 10, 12 或 13所示的蛋白） 的氨基酸序列具有一个或多个（例如 1-10个 或 1-5个或 1-3个）氨基酸不同 （例如保守氨基酸置换）或者具 有至少 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 或 99%的同 一性， 并且其保留了所述截短蛋白的必要特性。此处术语"必要特 性，，可以是如下特性中的一个或者多个：能够诱导针对 HPV52的中 和抗体； 能够在大肠杆菌中可溶性地表达； 利用本发明所涉及的 表达纯化方法能够获得高产率的纯化蛋白。
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 χ ΐ οο 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 Al ign程序（ DNAs tar, Inc. )方便地进行的 Needleman 等人（ 1970 ) /. 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. Mi l ler (Comput. Appl Biosci. , 4: 11-17 (1988) )的算法, 使用 PAM120 权重残基表（weight res idue 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 Mol 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 /多肽的生物学活性的氨基酸置换。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、 纈氨酸、 亮氨酸、 异亮氨酸、 脯氨酸、 苯丙氨酸、 甲硫氨酸） 、 P分支侧链（例如， 苏氨酸、 纈氨酸、 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， 优选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， Brummel 1等人， Biochem. 32: 1180-1187 (1993) ; Kobayashi 等 人 Protein Eng. 12 (10) : 879-884 (1999) ; 和 Burks等人 Pro Nat l 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： GI698、 ER2566、 BL21 (DE3)、 B834 (DE3)、 BLR (DE3)等等。
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 "截短的 HPV52 L1 蛋白" 是指在野生型 HPV52 L1蛋白的 N端和 /或 C端去掉一个或者多个氨基酸后的蛋 白质， 其中野生型 HPV52 L1蛋白的例子包括但不限于： NCBI数 据库中的 ACX32362. 1、 Q05138. 2或 ABU55790. 1等全长 L1蛋白。 例如， 野生型 HPV52 L1蛋白的氨基酸序列可如 SEQ ID NO: 27 , SEQ ID NO: 28或 SEQ ID NO: 29所示。
在本发明中， 术语 "截短的 HPV52 L1蛋白基因片段" 是指这 样的基因片段， 其与野生型 HPV52 L1蛋白基因相比， 在 5'端或 3'端缺失编码一个或多个氨基酸的核苷酸， 其中野生型 HPV52 L1 蛋白基因的全长序列例如但不限于 NCBI 数据库中的如下序列： EU077195. 1 , EU077194. 1 , FJ615303. 1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 Pharmaceut ica l Sc iences. Edi ted by Gennaro AR, 19th ed. Pennsylvania: Mack Publ i shing 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 限于磷酸盐緩冲液； 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，本发明的截短的 HPV52 L1蛋白优选通过如下步 骤获得： 将表达截短的 HPV52 L1蛋白的大肠杆菌在盐浓度为 100 - 600mM,优选 200 - 500mM的緩冲液中进行破碎， 离心破碎溶液， 得到上清液； 用水或低盐浓度（通常低于破碎用的盐浓度） 的溶 液降低所得上清液的盐浓度至盐浓度 l OOmM - OmM, 从而截短的 HPV52 L1蛋白在上清液中沉淀；将沉淀在含还原剂及盐浓度为 150 - 200mM,优选 200mM以上的溶液中重新溶解，从而分离得到含截 短的 HPV52 L1蛋白的溶液， 其中所述蛋白的纯度为至少 50 % , 优选至少 70 % , 更优选至少 80 %。
可用于本发明的方法中的緩冲液是本领域公知的， 包括但不 限于， Tr i s緩冲液， 磷酸盐緩冲液， HEPES緩冲液， MOPS緩冲液 等等。
根据本发明， 宿主细胞的破碎可通过本领域技术人员熟知的 各种方法来实现， 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。
可用于本发明的方法中的盐包括但不限于酸式盐， 碱式盐， 中性盐， 例如碱金属盐、 碱土金属盐、 铵盐、 盐酸盐、 硫酸盐、 碳酸氢盐、磷酸盐或磷酸氢盐，特别是 NaC l、 KC1、 NH4C 1、 (NH4) 2S04 中的一种或几种。 特别优选的盐是 NaC l。 可用于本发明的方法中 的还原剂包括但不限于 DTT, 2 -巯基乙醇， 其用量包括但不限于 l OmM- 100mM。
根据本发明的 HPV52病毒样颗粒可通过如下步骤获得： 将上 述纯度至少 50 %的截短的 HPV52 L1蛋白通过例如色傳层析进行 进一步分离， 得到经纯化的截短蛋白溶液； 去除该溶液中的还原 剂， 得到所述 HPV52病毒样颗粒。 去除还原剂的方式是本领域已 知的， 包括但不限于， 透析， 超滤或者层析等。 发明的有益效果
目前用于制备 HPV病毒样颗粒的表达系统可以分为真核表达 系统和原核表达系统。
在真核表达系统中表达的 HPV L1蛋白天然构象破坏少， 能自 发形成 VLP , 往往只需进行简单的纯化过程即可获得具有正确构 象的 VLP。 但目前真核表达系统例如杆状病毒表达系统和酵母表 达系统存在表达量低， 培养成本高等缺陷， 给大规模工业化生产 带来了极大困难。
在原核表达系统中， 大肠杆菌表达系统具有培养成本低， 表 达量大的优点。 然而， 在大肠杆菌中表达的 HPV L1蛋白往往失去 正确的天然构象， 以包含体形式表达于沉淀中。 目前对表达于包 含体中的蛋白进行复性依然是一个世界性难题。 复性困难和效率 低下使得从包含体中获得有正确构象的 VLP难以在大规模生产中 实施， 只能局限于小规模的实验室研究中。 虽然 HPV L1也可以以 正确构象可溶性地表达于大肠杆菌中，但是其表达量低下。并且， 从包含种类繁多的可溶性蛋白的大肠杆菌裂解上清中纯化出 HPV L1蛋白也相当困难， 往往需要借助融合表达及亲和层析等手段， 而这些手段又往往需要昂贵的酶， 因此， 仍然无法实现工业化生 产。
本发明提供的 N端截短的 HPV52 L1蛋白和其制备方法有效地 解决了上述问题。 首先， 本发明使用大肠杆菌表达系统来表达 N 端截短的 HPV52 L1蛋白， 确保了其高表达量。 其次， 本发明采用 温和的手段选择性沉淀大肠杆菌裂解上清中的截短蛋白， 然后采 用含盐緩冲液重新溶解截短蛋白， 从而在保持截短蛋白的正确构 象的前提下使蛋白纯度有了显著提高， 并且获得的截短蛋白溶液 可以直接通过色讲层析例如离子交换层析及疏水交换层析进行进 一步纯化， 获得高纯度的目的蛋白 （例如纯度达到 80% ) 。 进一 步， 所获得的高纯度截短蛋白可以组装为病毒样颗粒， 并且该病 毒样颗粒可以在体内诱导高滴度的针对 HPV52的中和抗体， 具有 良好的免疫原性， 是一种良好的疫苗形式， 可用于预防 HPV52对 人体的感染。
由此可见， 本发明具有以下优点： 本发明的截短蛋白在保留 全长 HPV52 L1蛋白的抗原性、 免疫原性及颗粒组装能力的同时， 可在大肠杆菌表达系统中实现大量表达； 本发明所采用的制备方 法无需使用昂贵的酶， 成本低廉； 截短蛋白在纯化过程中构象没 有经过剧烈的变性复性过程， 损失小， 产率高； 截短蛋白所形成 的病毒样颗粒能够诱导高滴度的针对 HPV的保护性抗体， 可用于 生产疫苗。 因此， 本发明的截短蛋白和其制备方法可应用于大规 模工业化生产， 并且使大规模工业化生产宫颈癌疫苗成为可能。 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 显示了在本发明实施例 3 的不同步骤中获得的 HPV52N40C-L1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 : 破菌上清（即， 破碎菌体后离心获得 的上清）； 泳道 2: 无盐沉淀产物（即，透析后离心获得的沉淀）； 泳道 3: 重溶后上清 （即， 将无盐沉淀产物重新溶解后离心获得 的上清） ； 泳道 4: 重溶后沉淀（即， 将无盐沉淀产物重新溶解 后离心获得的沉淀）。结果显示， HPV52N40C-L1蛋白在通过沉淀， 重溶的步骤之后， 纯度从之前的约 10 % (参见泳道 1 )提高到了 约 70 % (参见泳道 3 ) 。
图 2显示了实施例 4 中经过阳离子交换色谱纯化和 CHT-I I 纯化后获得的 HPV52N40C- L1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， 经实施例 4的方法纯化获得的 HPV52N40C-L1 (上样体积为 10 μ ΐ) ; 泳道 2， 经实施例 4的方法 纯化获得的 HPV52N40C- L1 (上样体积为 20 μ 1)。 结果显示， 经过 实施例 4的阳离子交换色傳纯化和 CHT- I I纯化后，HPV52N40C-L1 蛋白纯度达到了 98 %左右。
图 3显示了实施例 6中所述的实施例 5所得的 HPV52N40C-L1 病毒样颗粒的透射电镜观察（放大 50， 000倍， Bar=100nm ) 的结 果。视野中可见大量半径为 25nm左右的病毒样颗粒，颗粒大小与 理论大小相符， 且均勾一致。
图 4显示了实施例 6中所述的实施例 5所得的 HPV52N40C-L1 病毒样颗粒的冷冻电镜观察的结果及其重建的三维结构。 图 4A, HPV52N40C-L1病毒样颗粒； 图 4B, HPV52N40C-L1病毒样颗粒的 重建的三维结构。 重建的三维结构显示， HPV52N40C- LI VLP是由 72个壳粒（形态亚单位，五聚体）形成的 T=7的二十面体结构（ h=l， k=2 ) 。 与一般的符合准等价原理的二十面体病毒衣壳不同， HPV52N40C-L1 VLP结构中所有的组成亚单位均为五聚体， 而未见 六聚体，且 VLP的最外围直径为约 60nmo这与之前报道的天然 HPV 病毒颗粒及真核表达系统（例如，痘病毒表达系统）来源的 HPV VLP 的三维结构 ( Baker TS， Newcomb WW, Ol son NH. et al. Biophys J. (1991) , 60 (6) : 1445-1456; Hagensee ME, Ol son NH, Baker TS， et al. J Vi rol. (1994)， 68 (7) : 4503-4505; Buck CB， Cheng N， Thompson CD. et a l. J Vi rol. (2008) , 82 (11) : 5190-7 ) 类似。
图 5显示了实施例 6中所述的实施例 5所得的 HPV52N40C-L1 病毒样颗粒的动态光散射观测结果。 结果显示， HPV52N40C-L1病 毒样颗粒的水化分子动力学半径为 24. 39nm, 颗粒组装百分比为 100%。
图 6显示了实施例 7中测定的用 HPV52N40C-L1病毒样颗粒接 种兔后不同阶段血清的中和抗体滴度。图中箭头所示为免疫时间。 在初次免疫一个月后， 总抗体滴度即有明显上升； 在经过一次加 强免疫后， 中和抗体的滴度即能达到 105的高水平。
图 7显示了实施例 8得到的 N端截短了 27个、 35个、 38个 或 42个氨基酸的 HPV52 L1蛋白 ~HPV52N27C-L1、 HPV52N35C-LK HPV52N38C-LU HPV52N42C-LK其氨基酸序列分别是 SEQ ID NO: 1、 SEQ ID NO: 7、 SEQ ID NO: 10、 SEQ ID NO: 13 ) 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， HPV52N27C-L1 蛋白， 上样体积为 Ι ΟμΙ ; 泳道 2 , HPV52N35C-L1 蛋白， 上样体积为 Ι ΟμΙ ; 泳道 3, HPV52N38C-L1蛋白， 上样体积 为 Ι ΟμΙ ; 泳道 4 , HPV52N42C-L1蛋白， 上样体积为 10μ1。 结果 显示， 实施例 8所得的截短蛋白 HPV52N27C- Ll、 HPV52N35C-LK HPV52N38C-LU HPV52N42C-L1的蛋白纯度均达到了 98 %左右。
图 8显示了实施例 8中得到的 HPV52N27C- L1、HPV52N35C-L1、 HPV52N38C-L1 , HPV52N42C-L1 病毒样颗粒的透射电镜观察 ( 50， 000倍, l OOnm )的结果。 图 8A, HPV52N27C-L1病毒样颗粒; 图 8B， HPV52N35C-L1病毒样颗粒； 图 8C, HPV52N38C-L1病毒样 颗粒； 图 8D, HPV52N42C-L1病毒样颗粒。 结果显示， 在这 4个图 的视野中均可见大量半径为 25nm左右的病毒样颗粒，颗粒大小与 理论大小相符， 且均勾一致。
图 9显示了实施例 8中得到的 HPV52N27C- L1、HPV52N35C-L1、 HPV52N38C-LK HPV52N42C-L1 病毒样颗粒的动态光散射观测结 果。 图 9A， HPV52N27C-L1病毒样颗粒； 图 9B， HPV52N35C-L1病 毒样颗粒； 图 9C , HPV52N38C-L1 病毒样颗粒； 图 9D， HPV52N42C-L1 病毒样颗粒。 结果显示， HPV52N27C- L1、 HPV52N35C-LU HPV52N38C-L1和 HPV52N42C- L1病毒样颗粒的水 化分子动力学半径均为 25nm左右， 颗粒组装百分比为 100%。 序列信息
本发明涉及的序列的信息提供于下面的表 1中。 表 1: 序列的描述
Figure imgf000019_0001
编码 SEQ ID NO: 1的 DNA序列 编码 SEQ ID NO: 2的 DNA序列 编码 SEQ ID NO: 3的 DNA序列 编码 SEQ ID NO: 4的 DNA序列 编码 SEQ ID NO: 5的 DNA序列 编码 SEQ ID NO: 6的 DNA序列 编码 SEQ ID NO: 7的 DNA序列 编码 SEQ ID NO: 8的 DNA序列 编码 SEQ ID NO: 9的 DNA序列 编码 SEQ ID NO: 10的 DNA序列 编码 SEQ ID NO: 11的 DNA序列 编码 SEQ ID NO: 12的 DNA序列 编码 SEQ ID NO: 13的 DNA序列
ACX32362. 1的氨基酸序列
Q05138. 2的氨基酸序列
ABU55790. 1的氨基酸序列
HPV-52 L1基因序列
引物
引物 EQ ID NO: 1)：
MSVWRPSEAT VYLPPVPVSK VVSTDEYVSR TSIYYYAGSS RLLTVGHPYF SIKNTSSGNG
KKVLVPKVSG LQYRVFRIKL PDPNKFGFPD TSFYNPETQR LVWACTGLEI GRGQPLGVGI
SGHPLLNKFD DTETSNKYAG KPGIDNRECL SMDYKQTQLC ILGCKPPIGE HWGKGTPCNN
NSGNPGDCPP LQLINSVIQD GDMVDTGFGC MDFNTLQASK SDVPIDICSS VCKYPDYLQM
ASEPYGDSLF FFLRREQMFV RHFFNRAGTL GDPVPGDLYI QGSNSGNTAT VQSSAFFPTP
SGSMVTSESQ LFNKPYWLQR AQGHNNGICW GNQLFVTVVD TTRSTNMTLC AEVKKESTYK
NENFKEYLRH GEEFDLQFIF QLCKITLTAD VMTYIHKMDA TILEDWQFGL TPPPSASLED
TYRFVTSTAI TCQKNTPPKG KEDPLKDYMF WEVDLKEKFS ADLDQFPLGR KFLLQAGLQA
RPKLKRPASS APRTSTKKKK VKR -03- M SIHdVSSVdH
)n¾davOiov oaiavsd¾3¾ ΊαΛ3ΜΜ(Η M03IIVISI IZf cramva腿 H ΐΑΐ ανπι I¾3l0dId0T
¾dN3N¾AIS3 MA3VCm觀 ISHIiaAAIA NHOOVHOlMA dMdlOSHSI Toe
AWSOSdlddd VSS0AIVIN3 SNSOOlAiaO IfZ
AdHSVWOlAO dA¾3ASS3ia IdAaS¾SVOl aAwaoaOMS ΐ8ΐ dNOSNMDdl ¾33ΊΙ3Ί0Ι0 扁 SO3 ai3d¾3VAM ιζι
NAdSiaddOd AHA0l3SA¾d 19
IMISdAdHO AmassovA AAISIHSAA3 QISAA¾SAdA ddlAAIVHSW I
: (t^ :ON ai OHS) ^ iadvssvda¾ aiavsd¾3)n αΛ3 Μ(Ι)Π ¾03IIVISIA IZf dddllOdOitta 3ΊΙ環腿 I Αΐ ανπιι d33簡 Ί人 3 dN3N¾AIS3¾ SHIiaAAIAd HOOVHOlMAd MdlOSHSIA Toe
WSOSdldddV SS0AIVIN3S IfZ dHSVWOlAOd 人) DASSMdl dAas¾svmi AwaoaOiAS ΐ8ΐ
NOSNNNDdIO 33ΊΙ3Ί0Ι0¾ 扁 SO3丽 I3d¾3VAMS ιζι dHOSIOAOld 題 cTraiMA HA0l3SA¾dA 19
MISdAdHOA ITOSSOVAA AISIHSAAHa ISAA¾SAdAd dlAAIVHSdW I
: (ε :ON ai OHS) £
HdVSSVdMT iavsd¾3)na Λ3Μ匪) ΙΉ ODIIVISIAd IZf mva腿 I人 i avniDi 人 3 d 誦 AHVDII IS 0N3M3I3NNH NdlOSHSIAW Toe
SOSdldddVS S0AIVIN3SN SOOlAiaOdA IfZ
HSVWOlAOdA ¾3ASS3iaid AaS¾SVOlIN waoaOMSNi ΐ8ΐ
3SNNN3dI3¾ cms™丽 I 3d¾3VAMSI ιζι
HOSIOAOldO A0l3SA¾dAT 19
ISIHSAAHQI SAA¾SAdAdd ΊΜ Sd測 I
: (i :ON ai
£9l9lO/llOZ 3/13d OOO Z OAV -\ z-
VcM)n νδ 層 Ή(Ί3 IZf
ISVSdddllO cramva HIAI aV
lA3¾dN3N¾A IS3¾¾A3V3T I3NNH30VH0 IMAdMdlOS Toe
HSIAWSOSdl dddVSSOAIV IN3SNS30IA 3V腿 dHMd dddl IfZ
SaOAdHSVWO Ί編人) DASS 3iaidAas¾s IASNHOldd ΐ8ΐ
DaOd OS Idd¾331I31 δΐΰ 扁 SO 3Mai3d¾3V AMSIHiaad ιζι
MlldHOSIO IHdNAdSiad 19
OSSIMISdA dHOAmass OVAAAISIHS AA3QISAA¾S AdAddlAAIW I
: :0N ai 0HS) /,
¾ISIHdVSSV da)n¾davOT 3)naA3MdM (Dracra d dIMODIIVI IZf
SVSdddllOd δ壓 m爾 皿人愿 (TO lll¾3l0dld
A3¾dN3N¾AI S3¾¾A3V31I ONNHOOVHOT MAdMdlOSH Toe
SIAWSOSdld ddVSSOAIVI N3SNS30IA1 V丽 IfZ 編人) DASS iaidAas¾sv ASNHOlddO ΐ8ΐ aOdNOSNNND J 扁 SO3 丽 ΙΜ ιζι
NlldHOSIOA HdNAdSiadd 19
SSIMISdAd VAAAISIHSA A3QISAA¾SA dAddlAAIVW I
: (9 :0N QI 0HS) 9
ISIHdVSSVd
ΊαΛ3ω匪 IM03IIVIS IZf
VSdddllOdO 腿 mv醒 ΗΐΑΐ ανπ II¾3l0dId0
3¾dN3 ¾AIS 3¾¾A3V31IW NISHIiaAAI NNHOOVHOlM AdMdlOSHS Toe
!AWSOSdldd dVSSOAIVIN OSNSOOlAia IfZ
OAdHSVWOlA M人) DASS I aidAas¾svo S nOiddoa ΐ8ΐ
OdNOSMNDd Nai3d¾3VA¾ ιζι dNAdSIOddO 19
SIMISdAdH AAAISIHSAA 3QISAA¾SAd AddlAAIVHW I
: (S :0N QI 0HS) S
£9L9LO/llOZ l3/13d OOO Z OAV -zz-
IIVISIAdHA xaaisvsddd 環腿 I人 ΙΛ Λανπιι¾3Ί T9e
MAIS3MA3 VJU丽 lOAAIAdlON Toe lOSHSIAWSO SdldddVSSO AIVIN3SNS3 OiAiaodAda d IfZ 環 編人) D ASS3皿編 S¾SVOlINda oaoiASNno ΐ8ΐ
Ι Ίδΐ 扁 ¾3VAMSI3I ιζι
OIHIOIDVMA IHOlHdNAdS 19
¾ )SSJ I SdAdHOAm HSS3VAAAIS IHSAAHQISA A¾SAdAddlW I
: (01 :0N GI 03S) 0 T
MMISIHdV ssvda)n¾da V0l3V0nd¾ Sd¾3)naAai 愚 a)raa3 3¾ddIN¾03I IZf
IVISIAdHAI aaisvsdddi va腿 HI人愿 avnii¾3io
人 3 d誦 ¾AIS3¾ 3V OAAIAdlONO M3I3NNH30V Toe
0S3SIAWS3S dldddVSSOA IVIN3SNS30 Ad«33 dd IfZ
«3Ί編人) DA ssoiaidAas 願 3 aoiAS nm ΐ8ΐ ddDaOdNOS 33Idd¾331I Ίδΐ 扁 S Ί33丽 I3cH OVAMSIHia ιζι
HOlHdNAdSI 19
3N3SSIMIS SS3VAAAISI HSAAHQISAA ¾SAdAddlAW I
: (6 :0N GI 03S) 6
¾MISIHdVS svda)n¾dav a)nda3¾o ¾ddI ¾03II IZf
VISIAdHAia HlSVSdddlT 画 HI人愿 a Vnil¾3l0d
AIS3¾¾A3V3 AAIAdlONOM OlMAdMdlO Toe
SHSIAWSOSd IdddVSSOAI VIN3SNS30I IfZ δΊ編人) DAS S3iaidAas¾ OlASNHOld ΐ8ΐ dDaOd OS 3Idd¾331I3 33丽 Ι ¾) VAMSiaiaa ιζι
OlHdNAdSia I ΜδΈ) 19
NOSSIMISd AdHOAmas S3VAAAISIH SAA3QISAA¾ SAdAddlAAW I
: (8 :0N GI 03S) 8
MISIHdVSS
C9 .9 .0/llOZN3/X3d OOO Z OAV (M33簡 Ί人 3 MN3MAIS3 重 扁 NHOOVHOlMA Toe dMdlOSHSI AWSOSdlddd VSS0AIVIN3 SNSOOlAiaO IfZ
AdHSVWOlAO dA¾3ASS3ia idAas¾svm (BTOdi aAwaoaOMS ΐ8ΐ dNOSNNNDdl ¾33ΊΙ3Ί0Ι0 扁 SO3 ai3d¾3VAM ιζι
19
AHA0l3SA¾d IMISdAdHO AITOSSOVA AAISIHSAA3 QISAA¾SAdW I
: (ε ι :0N GI 0HS) £ T
ISf
HdVSSVdMT νδΉνδΊ iavsd¾3)na A3Md a)nd IZf
ODIIVISIAd lIIVa HIA i avnu¾
人 3 d N3N¾AIS3¾¾ 0N3M3I3NNH Toe
NdlOSHSIAW SOSdldddVS S0AIVIN3SN SOOlAiaOdA IfZ
HSVWOlAOdA ¾3ASS3iaid AaS¾SVOlIN waoaOMSNi ΐ8ΐ
3SNNN3dI3¾ 3ittH33Idd¾3 3ΊΙ3Ί0Ι0¾Α cms™丽 I 3d¾3VAMSI ιζι
HOSIOAOldO 19
A0l3SA¾dAT A¾¾3N3SSIN ¾ISdAdH3AI ISIHSAAHQI SAA¾SAdAdW I
: (Z l :0N GI 0HS) 11
dvssvda)n¾ avsd¾3)naA 3MdMa)nda IZf
DIIVISIAdH Aiaaisvsdd I環腿 IM avnii¾3
33 人 3 IiaAAIAdlO OVHOTIAdM Toe dlOSHSIAWS OSdldddVSS 0AIVIN3SNS IfZ
SVWOlAadA¾ DASSDiaidA as¾svoiiM aoaOiASNn ΐ8ΐ
SNNN3dI3¾3 ΊΙ ΊδΙΰ爾 d¾0VAMSI3 ιζι
19
0l3SA¾dAlA M3N3SSIM ISdAdHOAIT 1HSS3VAAAI SIHSAAHQIS AA¾SAdAddW I
: (Π :ON GI 0HS) π
VSd¾3)naA3 IZf
£9L9LO/llOZ l3/13d OOO Z OAV
Figure imgf000025_0001
Figure imgf000025_0002
Figure imgf000025_0003
Figure imgf000025_0004
序列 15 (SEQ ID NO: 15)：
1 ATGAGGCCCA GCGAGGCCAC CGTGTACCTG CCCCCCGTGC CCGTGAGCAA GGTGGTGAGC
61 ACCGACGAGT ACGTGAGCAG GACCAGCATC TACTACTACG CCGGCAGCAG CAGGCTGCTG
121 ACCGTGGGCC ACCCCTACTT CAGCATCAAG AACACCAGCA GCGGCAACGG CAAGAAGGTG
181 CTGGTGCCCA AGGTGAGCGG CCTGCAGTAC AGGGTGTTCA GGATCAAGCT GCCCGACCCC
241 AACAAGTTCG GCTTCCCCGA CACCAGCTTC TACAACCCCG AGACCCAGAG GCTGGTGTGG
301 GCCTGCACCG GCCTGGAGAT CGGCAGGGGC CAGCCCCTGG GCGTGGGCAT CAGCGGCCAC
361 CCCCTGCTGA ACAAGTTCGA CGACACCGAG ACCAGCAACA AGTACGCCGG CAAGCCCGGC
421 ATCGACAACA GGGAGTGCCT GAGCATGGAC TACAAGCAGA CCCAGCTGTG CATCCTGGGC
481 TGCAAGCCCC CCATCGGCGA GCACTGGGGC AAGGGCACCC CCTGCAACAA CAACAGCGGC
541 AACCCCGGCG ACTGCCCCCC CCTGCAGCTG ATCAACAGCG TGATCCAGGA CGGCGACATG
601 GTGGACACCG GCTTCGGCTG CATGGACTTC AACACCCTGC AGGCCAGCAA GAGCGACGTG
661 CCCATCGACA TCTGCAGCAG CGTGTGCAAG TACCCCGACT ACCTGCAGAT GGCCAGCGAG
721 CCCTACGGCG ACAGCCTGTT CTTCTTCCTG AGGAGGGAGC AGATGTTCGT GAGGCACTTC
781 TTCAACAGGG CCGGCACCCT GGGCGACCCC GTGCCCGGCG ACCTGTACAT CCAGGGCAGC
841 AACAGCGGCA ACACCGCCAC CGTGCAGAGC AGCGCCTTCT TCCCCACCCC CAGCGGCAGC
901 ATGGTGACCA GCGAGAGCCA GCTGTTCAAC AAGCCCTACT GGCTGCAGAG GGCCCAGGGC
961 CACAACAACG GCATCTGCTG GGGCAACCAG CTGTTCGTGA CCGTGGTGGA CACCACCAGG
1021 AGCACCAACA TGACCCTGTG CGCCGAGGTG AAGAAGGAGA GCACCTACAA GAACGAGAAC
1081 TTCAAGGAGT ACCTGAGGCA CGGCGAGGAG TTCGACCTGC AGTTCATCTT CCAGCTGTGC
1141 AAGATCACCC TGACCGCCGA CGTGATGACC TACATCCACA AGATGGACGC CACCATCCTG
1201 GAGGACTGGC AGTTCGGCCT GACCCCCCCC CCCAGCGCCA GCCTGGAGGA CACCTACAGG
1261 TTCGTGACCA GCACCGCCAT CACCTGCCAG AAGAACACCC CCCCCAAGGG CAAGGAGGAC
1321 CCCCTGAAGG ACTACATGTT CTGGGAGGTG GACCTGAAGG AGAAGTTCAG CGCCGACCTG
1381 GACCAGTTCC CCCTGGGCAG GAAGTTCCTG CTGCAGGCCG GCCTGCAGGC CAGGCCCAAG
1441 CTGAAGAGGC CCGCCAGCAG CGCCCCCAGG ACCAGCACCA AGAAGAAGAA GGTGAAGAGG
1501 TGA 序列 16 (SEQ ID NO: 16)：
1 ATGCCCAGCG AGGCCACCGT GTACCTGCCC CCCGTGCCCG TGAGCAAGGT GGTGAGCACC
61 GACGAGTACG TGAGCAGGAC CAGCATCTAC TACTACGCCG GCAGCAGCAG GCTGCTGACC o o ^ o
σ>
Q (SO:E ID N
841 ACCGCCACCG TGCAGAGCAG CGCCTTCTTC CCCACCCCCA GCGGCAGCAT GGTGACCAGC
901 GAGAGCCAGC TGTTCAACAA GCCCTACTGG CTGCAGAGGG CCCAGGGCCA CAACAACGGC
961 ATCTGCTGGG GCAACCAGCT GTTCGTGACC GTGGTGGACA CCACCAGGAG CACCAACATG
1021 ACCCTGTGCG CCGAGGTGAA GAAGGAGAGC ACCTACAAGA ACGAGAACTT CAAGGAGTAC
1081 CTGAGGCACG GCGAGGAGTT CGACCTGCAG TTCATCTTCC AGCTGTGCAA GATCACCCTG
1141 ACCGCCGACG TGATGACCTA CATCCACAAG ATGGACGCCA CCATCCTGGA GGACTGGCAG
1201 TTCGGCCTGA CCCCCCCCCC CAGCGCCAGC CTGGAGGACA CCTACAGGTT CGTGACCAGC
1261 ACCGCCATCA CCTGCCAGAA GAACACCCCC CCCAAGGGCA AGGAGGACCC CCTGAAGGAC
1321 TACATGTTCT GGGAGGTGGA CCTGAAGGAG AAGTTCAGCG CCGACCTGGA CCAGTTCCCC
1381 CTGGGCAGGA AGTTCCTGCT GCAGGCCGGC CTGCAGGCCA GGCCCAAGCT GAAGAGGCCC
1441 GCCAGCAGCG CCCCCAGGAC CAGCACCAAG AAGAAGAAGG TGAAGAGGTG A 序列 20 (SEQ ID NO: 20)：
1 ATGACCGTGT ACCTGCCCCC CGTGCCCGTG AGCAAGGTGG TGAGCACCGA CGAGTACGTG
61 AGCAGGACCA GCATCTACTA CTACGCCGGC AGCAGCAGGC TGCTGACCGT GGGCCACCCC
121 TACTTCAGCA TCAAGAACAC CAGCAGCGGC AACGGCAAGA AGGTGCTGGT GCCCAAGGTG
181 AGCGGCCTGC AGTACAGGGT GTTCAGGATC AAGCTGCCCG ACCCCAACAA GTTCGGCTTC
241 CCCGACACCA GCTTCTACAA CCCCGAGACC CAGAGGCTGG TGTGGGCCTG CACCGGCCTG
301 GAGATCGGCA GGGGCCAGCC CCTGGGCGTG GGCATCAGCG GCCACCCCCT GCTGAACAAG
361 TTCGACGACA CCGAGACCAG CAACAAGTAC GCCGGCAAGC CCGGCATCGA CAACAGGGAG
421 TGCCTGAGCA TGGACTACAA GCAGACCCAG CTGTGCATCC TGGGCTGCAA GCCCCCCATC
481 GGCGAGCACT GGGGCAAGGG CACCCCCTGC AACAACAACA GCGGCAACCC CGGCGACTGC
541 CCCCCCCTGC AGCTGATCAA CAGCGTGATC CAGGACGGCG ACATGGTGGA CACCGGCTTC
601 GGCTGCATGG ACTTCAACAC CCTGCAGGCC AGCAAGAGCG ACGTGCCCAT CGACATCTGC
661 AGCAGCGTGT GCAAGTACCC CGACTACCTG CAGATGGCCA GCGAGCCCTA CGGCGACAGC
721 CTGTTCTTCT TCCTGAGGAG GGAGCAGATG TTCGTGAGGC ACTTCTTCAA CAGGGCCGGC
781 ACCCTGGGCG ACCCCGTGCC CGGCGACCTG TACATCCAGG GCAGCAACAG CGGCAACACC
841 GCCACCGTGC AGAGCAGCGC CTTCTTCCCC ACCCCCAGCG GCAGCATGGT GACCAGCGAG
901 AGCCAGCTGT TCAACAAGCC CTACTGGCTG CAGAGGGCCC AGGGCCACAA CAACGGCATC
961 TGCTGGGGCA ACCAGCTGTT CGTGACCGTG GTGGACACCA CCAGGAGCAC CAACATGACC
1021 CTGTGCGCCG AGGTGAAGAA GGAGAGCACC TACAAGAACG AGAACTTCAA GGAGTACCTG 1081 AGGCACGGCG AGGAGTTCGA CCTGCAGTTC ATCTTCCAGC TGTGCAAGAT CACCCTGACC
1141 GCCGACGTGA TGACCTACAT CCACAAGATG GACGCCACCA TCCTGGAGGA CTGGCAGTTC
1201 GGCCTGACCC CCCCCCCCAG CGCCAGCCTG GAGGACACCT ACAGGTTCGT GACCAGCACC
1261 GCCATCACCT GCCAGAAGAA CACCCCCCCC AAGGGCAAGG AGGACCCCCT GAAGGACTAC
1321 ATGTTCTGGG AGGTGGACCT GAAGGAGAAG TTCAGCGCCG ACCTGGACCA GTTCCCCCTG
1381 GGCAGGAAGT TCCTGCTGCA GGCCGGCCTG CAGGCCAGGC CCAAGCTGAA GAGGCCCGCC
1441 AGCAGCGCCC CCAGGACCAG CACCAAGAAG AAGAAGGTGA AGAGGTGA 序列 21 (SEQ ID NO: 21)：
1 ATGGTGTACC TGCCCCCCGT GCCCGTGAGC AAGGTGGTGA GCACCGACGA GTACGTGAGC
61 AGGACCAGCA TCTACTACTA CGCCGGCAGC AGCAGGCTGC TGACCGTGGG CCACCCCTAC
121 TTCAGCATCA AGAACACCAG CAGCGGCAAC GGCAAGAAGG TGCTGGTGCC CAAGGTGAGC
181 GGCCTGCAGT ACAGGGTGTT CAGGATCAAG CTGCCCGACC CCAACAAGTT CGGCTTCCCC
241 GACACCAGCT TCTACAACCC CGAGACCCAG AGGCTGGTGT GGGCCTGCAC CGGCCTGGAG
301 ATCGGCAGGG GCCAGCCCCT GGGCGTGGGC ATCAGCGGCC ACCCCCTGCT GAACAAGTTC
361 GACGACACCG AGACCAGCAA CAAGTACGCC GGCAAGCCCG GCATCGACAA CAGGGAGTGC
421 CTGAGCATGG ACTACAAGCA GACCCAGCTG TGCATCCTGG GCTGCAAGCC CCCCATCGGC
481 GAGCACTGGG GCAAGGGCAC CCCCTGCAAC AACAACAGCG GCAACCCCGG CGACTGCCCC
541 CCCCTGCAGC TGATCAACAG CGTGATCCAG GACGGCGACA TGGTGGACAC CGGCTTCGGC
601 TGCATGGACT TCAACACCCT GCAGGCCAGC AAGAGCGACG TGCCCATCGA CATCTGCAGC
661 AGCGTGTGCA AGTACCCCGA CTACCTGCAG ATGGCCAGCG AGCCCTACGG CGACAGCCTG
721 TTCTTCTTCC TGAGGAGGGA GCAGATGTTC GTGAGGCACT TCTTCAACAG GGCCGGCACC
781 CTGGGCGACC CCGTGCCCGG CGACCTGTAC ATCCAGGGCA GCAACAGCGG CAACACCGCC
841 ACCGTGCAGA GCAGCGCCTT CTTCCCCACC CCCAGCGGCA GCATGGTGAC CAGCGAGAGC
901 CAGCTGTTCA ACAAGCCCTA CTGGCTGCAG AGGGCCCAGG GCCACAACAA CGGCATCTGC
961 TGGGGCAACC AGCTGTTCGT GACCGTGGTG GACACCACCA GGAGCACCAA CATGACCCTG
1021 TGCGCCGAGG TGAAGAAGGA GAGCACCTAC AAGAACGAGA ACTTCAAGGA GTACCTGAGG
1081 CACGGCGAGG AGTTCGACCT GCAGTTCATC TTCCAGCTGT GCAAGATCAC CCTGACCGCC
1141 GACGTGATGA CCTACATCCA CAAGATGGAC GCCACCATCC TGGAGGACTG GCAGTTCGGC
1201 CTGACCCCCC CCCCCAGCGC CAGCCTGGAG GACACCTACA GGTTCGTGAC CAGCACCGCC
1261 ATCACCTGCC AGAAGAACAC CCCCCCCAAG GGCAAGGAGG ACCCCCTGAA GGACTACATG 1321 TTCTGGGAGG TGGACCTGAA GGAGAAGTTC AGCGCCGACC TGGACCAGTT CCCCCTGGGC
1381 AGGAAGTTCC TGCTGCAGGC CGGCCTGCAG GCCAGGCCCA AGCTGAAGAG GCCCGCCAGC
1441 AGCGCCCCCA GGACCAGCAC CAAGAAGAAG AAGGTGAAGA GGTGA 序列 22 (SEQ ID NO: 22)：
1 ATGTACCTGC CCCCCGTGCC CGTGAGCAAG GTGGTGAGCA CCGACGAGTA CGTGAGCAGG
61 ACCAGCATCT ACTACTACGC CGGCAGCAGC AGGCTGCTGA CCGTGGGCCA CCCCTACTTC
121 AGCATCAAGA ACACCAGCAG CGGCAACGGC AAGAAGGTGC TGGTGCCCAA GGTGAGCGGC
181 CTGCAGTACA GGGTGTTCAG GATCAAGCTG CCCGACCCCA ACAAGTTCGG CTTCCCCGAC
241 ACCAGCTTCT ACAACCCCGA GACCCAGAGG CTGGTGTGGG CCTGCACCGG CCTGGAGATC
301 GGCAGGGGCC AGCCCCTGGG CGTGGGCATC AGCGGCCACC CCCTGCTGAA CAAGTTCGAC
361 GACACCGAGA CCAGCAACAA GTACGCCGGC AAGCCCGGCA TCGACAACAG GGAGTGCCTG
421 AGCATGGACT ACAAGCAGAC CCAGCTGTGC ATCCTGGGCT GCAAGCCCCC CATCGGCGAG
481 CACTGGGGCA AGGGCACCCC CTGCAACAAC AACAGCGGCA ACCCCGGCGA CTGCCCCCCC
541 CTGCAGCTGA TCAACAGCGT GATCCAGGAC GGCGACATGG TGGACACCGG CTTCGGCTGC
601 ATGGACTTCA ACACCCTGCA GGCCAGCAAG AGCGACGTGC CCATCGACAT CTGCAGCAGC
661 GTGTGCAAGT ACCCCGACTA CCTGCAGATG GCCAGCGAGC CCTACGGCGA CAGCCTGTTC
721 TTCTTCCTGA GGAGGGAGCA GATGTTCGTG AGGCACTTCT TCAACAGGGC CGGCACCCTG
781 GGCGACCCCG TGCCCGGCGA CCTGTACATC CAGGGCAGCA ACAGCGGCAA CACCGCCACC
841 GTGCAGAGCA GCGCCTTCTT CCCCACCCCC AGCGGCAGCA TGGTGACCAG CGAGAGCCAG
901 CTGTTCAACA AGCCCTACTG GCTGCAGAGG GCCCAGGGCC ACAACAACGG CATCTGCTGG
961 GGCAACCAGC TGTTCGTGAC CGTGGTGGAC ACCACCAGGA GCACCAACAT GACCCTGTGC
1021 GCCGAGGTGA AGAAGGAGAG CACCTACAAG AACGAGAACT TCAAGGAGTA CCTGAGGCAC
1081 GGCGAGGAGT TCGACCTGCA GTTCATCTTC CAGCTGTGCA AGATCACCCT GACCGCCGAC
1141 GTGATGACCT ACATCCACAA GATGGACGCC ACCATCCTGG AGGACTGGCA GTTCGGCCTG
1201 ACCCCCCCCC CCAGCGCCAG CCTGGAGGAC ACCTACAGGT TCGTGACCAG CACCGCCATC
1261 ACCTGCCAGA AGAACACCCC CCCCAAGGGC AAGGAGGACC CCCTGAAGGA CTACATGTTC
1321 TGGGAGGTGG ACCTGAAGGA GAAGTTCAGC GCCGACCTGG ACCAGTTCCC CCTGGGCAGG
1381 AAGTTCCTGC TGCAGGCCGG CCTGCAGGCC AGGCCCAAGC TGAAGAGGCC CGCCAGCAGC
1441 GCCCCCAGGA CCAGCACCAA GAAGAAGAAG GTGAAGAGGT GA 序列 23 (SEQ ID NO: 23)： >
NO:
Figure imgf000035_0001
Ί0Ν3Μ3Ι3ΝΝ HOOVHOTIAd MdlOSHSIA WSOSdldddV SS0AIVIN3S TOG dHSVWOlAOd 人) DASS 皿 dAas¾svoii
AwaoaOiAS NOSNNNDdIO 33ΊΙ3Ί0Ι0¾ ΐ8ΐ 扁 s™丽 I3d¾3VAMS dHOSIOAOld ιζι 腿 cn radA HA0l3SA¾dA lAMONOSSI MISdAdHOA massovAA 19
AISIHSAAHa ISAA¾SAdAd dlAAIVHSdH ANAOVAAAdl I
: (U :ON GI 03S) %Z
MdVSSV aiavsd¾3)n αΛ3Μ匪) Π ¾03IIVISIA dddllOdOitta amv画 HI IZf
Αΐ ανπιι dN3N¾AIS3¾ M3VC 皿
Ί0Ν3Μ3Ι3ΝΝ HOOVHOTIAd MdlOSHSIA WSOSdldddV SS0AIVIN3S Toe dHSVWOlAOd 人) DASS 皿 dAas¾svosi IfZ
AwaoaOiAS NOSNNNDdIO 33ΊΙ3Ί0Ι0¾ ΐ8ΐ 扁 s™丽 I3d¾3VAMS dHOSIOAOld ιζι 腿 cn radA HA0l3SA¾dA lAMONOSSI MISdAdHOA massovAA 19
AISIHSAAHa ISAA¾SAdAd d!AAIVHSdH ANAOVAAAdl I
: (L I :ON GI 03S) LI
VV3I33VV3V V3VV3VV33V IfU
33V33V30V3 3VV3I33VV3 im
II3VV33V33 I3VV3V33VV I ZZl
V3VI3V33VV V33333333V
3VV3VV3V33 3I33V3IV33 333V30V33V VI33V3V33V
IV33V3333V 33IV3VV3V3 ΐΗ ΐ
3IV3VI33V3 IV3I33V333 333V0I333V 3IV3VV33I3 I33V33II3I V3II3V33I3 ΐ80ΐ
V3I33VI3V3 3VV3II3VV3 V33W3VV3V I33V33V3V3 ΐ20ΐ
3VV3VV3I33 333V3IV3VV 33V33V30V3 3V33V3V33I Ϊ96
3II3I33V33 I3IV3333VV 3VV3V33333 Ϊ06
3333VV3VV3 V3V333V33V V3333II3II ΐ 8
V33I033V33 333V3VV333 33V3VV33V3 Ϊ8Ζ
3333103333 3VV3II3II3
C9 .9 .0/llOZN3/X3d OOO Ζ OAV -9e-
3II3II3V33 IfS
LLLLOJJLLOLLOOO 33VI3V0333 Ϊ8Ζ
3VI3VV33I3 I3IV3V33IV IZL
3VV3II3V33 V3I333V3VV Ϊ99
3IV3I33V33 1333333330 3VV33333V3 VV3W3VV33 I33333V330 Ϊ09
V3333333VV IV33I3I33V 333V3V33VV ΐ½
3VI3V33IV3 VV3333333V I3VV3VV33V ΐ8
33V3V033V3 V33V33II3V V3VV3I33I3
V3V33333VV
3VI3II33V3 3V3V03333I I3333II3VV 3VV3333V33 333I33VV3I V33V3II3I3 Toe
33V3VI3V33 VV3333VV33 333V30V33V IfZ
3VV3VV3IV3 3V3II3VI33 33333VI3VI ΐ8ΐ
3VI3IV33V3 3333010333
IV3V33I33I I3IV3V33II ΐ9
I3VI3II3IV VI3II3I33I V3V33I33IV ΐ
Figure imgf000037_0001
MdVSSV aiavsd¾3)n ΐ8 αΛ3Μ匪) Π ¾03IIVISIA amv画 HI IZf
Αΐ αννιιι dN3N¾AIS3¾ M3VC 皿
Ί0Ν3Μ3Ι3ΝΝ HOOVHOTIAd MdlOSHSIA WSOSdldddV SS0AIVIN3S Toe
dHSVWOlAOd 人) DASS 皿 dAas¾svoii IfZ
AwaoaOiAS NOSNNNDdIO 33ΊΙ3Ί0Ι0¾ ΐ8ΐ 扁 s™丽 I3d¾3VAMS dHOSIOAOld
腿 cn radA HA0l3SA¾dA lAMONOSSI MISdAdHOA massovAA Ϊ9
AISIHSAAHa ISAA¾SAdAd d!AAIVHSdH ANAOVAAAdl ΐ
: (6 1 :O GI 0HS) 6 Z
MdVSSV nd OiddO aiavsd¾3)n
(IA3iM匪) Π ¾03IIVISIA dddnodOMQ 3mv画 HI IZf
AlMQVnil dN3N¾AIS3¾ M3v:m丽 1 SHIIQAAIAd T9e
£9L9LO/llOZ l3/13d OOO Z OAV 901 CCCGGCGACC TGTACATCCA GGGCAGCAAC AGCGGCAACA CCGCCACCGT GCAGAGCAGC
961 GCCTTCTTCC CCACCCCCAG CGGCAGCATG GTGACCAGCG AGAGCCAGCT GTTCAACAAG
1021 CCCTACTGGC TGCAGAGGGC CCAGGGCCAC AACAACGGCA TCTGCTGGGG CAACCAGCTG
1081 TTCGTGACCG TGGTGGACAC CACCAGGAGC ACCAACATGA CCCTGTGCGC CGAGGTGAAG
1141 AAGGAGAGCA CCTACAAGAA CGAGAACTTC AAGGAGTACC TGAGGCACGG CGAGGAGTTC
1201 GACCTGCAGT TCATCTTCCA GCTGTGCAAG ATCACCCTGA CCGCCGACGT GATGACCTAC
1261 ATCCACAAGA TGGACGCCAC CATCCTGGAG GACTGGCAGT TCGGCCTGAC CCCCCCCCCC
1321 AGCGCCAGCC TGGAGGACAC CTACAGGTTC GTGACCAGCA CCGCCATCAC CTGCCAGAAG
1381 AACACCCCCC CCAAGGGCAA GGAGGACCCC CTGAAGGACT ACATGTTCTG GGAGGTGGAC
1441 CTGAAGGAGA AGTTCAGCGC CGACCTGGAC CAGTTCCCCC TGGGCAGGAA GTTCCTGCTG
1501 CAGGCCGGCC TGCAGGCCAG GCCCAAGCTG AAGAGGCCCG CCAGCAGCGC CCCCAGGACC
1561 AGCACCAAGA AGAAGAAGGT GAAGAGGTGA 具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。 本领 域技术人员将会理解， 下面的实施例仅用于说明本发明， 而不应 视为限定本发明的范围。
除非特别指明， 否则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 Wi ley & Sons , Inc. , 1995中所述的方法进行或者按照产品说明书进行。 所用试 剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。 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: 表达截短的 HPV52 L1蛋白的非融合表达载体的构 建 用做模板的全长 HPV52 L1基因 （SEQ ID NO: 30 ) 由上海博 亚公司合成。 所合成的基因片段全长为 1590bp。 在此人工合成的 全长 HPV52 L1 基因片段的基础上， 制备编码本发明的截短的 HPV52 L1蛋白的多核苷酸。
将合成的全长 HPV52 L1基因用做 PCR反应的模板，以 52N40F: 5' - CAT ATg CCC GTG CCC GTG AGC AAG- 3' ( SEQ ID NO: 31 ) 为正向引物 （其 5'端引入限制性内切酶 Λ¾¾Ι位点 CAT ATG , ATG 为大肠杆菌系统中的起始密码子）， 以 52CR: 5' -GTC GAC TCA CCT CTT CAC CTT CTT C -V ( SEQ ID NO: 32 ) 为反向引物 （其 5' 端引入限制性内切酶 S& 位点），在 PCR热循环仪（ Biometra T3 ) 中按照如下条件进行 PCR反应。
Figure imgf000039_0001
扩增得到 1. 5kb左右的大小特异的 DNA片段。 将该 PCR产物 与商售的 pMD 18- T载体（ TAKARA公司生产）连接， 转入大肠杆 菌； 筛选阳性菌落， 提取质粒， 经 Λ Ι/ Ι酶切鉴定， 得到插 入截短的 HPV52 L1基因的阳性克隆 pMD 18-T- HPV52N40C-L1。
在上海博亚生物工程公司， 利用 M13 (+) I (-)引物， 测得 pMD 18-T-HPV52N40C-L1 质粒中插入的目的片段的核苷酸序列如 SEQ ID NO: 25所示， 其编码的氨基酸序列如 SEQ ID NO: 12所示。 该序列对应的蛋白质为 N端被截短 40个氨基酸、 C端未被截短的 HPV52 LI蛋白， 将其命名为 HPV52N40C-L1。
将上述的 pMD 18-T-HPV52N40C-L1质粒进行 Ndel/ Sail酶切， 获得 HPV52N40C-L1基因片段。再将该片段与经 Mell Sa 酶切的 原核表达载体 ρΤ0-Τ7(购自 Invi trogen公司）相连接，转入 ER2566 细菌； 筛选阳性菌落， 提取质粒， 经 Λ Ι/ Ι酶切鉴定得到插 入目的片段的阳性表达克隆 pTO-T7-HPV-52N40C-Ll。
取 1 的 ρΤΟ- Τ7- HPV- 52N40C- L1质粒 ( 0. 15mg/ml )转化 40 μ L 以氯化钙法制备的感受态大肠杆菌 ER2566 (购自 Invi trogen公司）， 将其涂布于含卡那霉素（终浓度 100 mg/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 37 静置培养 10-12小时至 单菌落清晰可辨。挑取单菌落至含 4mL液体 LB培养基 (含卡那霉 素） 的试管， 在 37 180转 /分钟下振荡培养 10小时， 从中取 lmL菌液于 -7 O 保存。 实施例 2: HPV52N40C-L1蛋白的大量表达
从 -70 X： 取 出 实 施例 1 制 备的 携 带 重组质粒 PTO-T7-HPV52N40C- L1的大肠杆菌菌液， 接种入 50ml含卡那霉 素的 LB液体培养基中， 在 180rpm, 37 *€下培养大约 12小时； 然 后转接入 10瓶 500ml含卡那霉素的 LB培养基中 （每瓶接入 5ml 菌液） ， 在 180rpm, 37 *€下培养过夜， 作为种子液。
采用上海保兴生物公司生产的 50L发酵罐进行大规模培养。 校正发酵罐 PH电极， 将 30L LB培养基装入发酵罐， 原位 121 灭菌 30min; 校正溶氧电极， 以灭菌后未通气前为零点， 以发酵 时通气后未接种前初始搅拌速度 l OOrpm时为 100%。
补料准备： 配制 30%的酪蛋白水解物（ 30g溶至 100ml ) , 50% 的葡萄糖（50g溶至 100ml), 121X灭菌 20min。
次日将 10瓶种子液共 5L接入发酵罐中， 设定温度 pH 值 7.0, 手动调节搅拌速度及通气量， 维持溶氧在 40%以上。
流加补料：将 50%的葡萄糖和 30%的酪蛋白水解物按溶质质量 比 2: 1的比例混合。
流加速度如下 (以 25mL/min为 100%) ：
第一小时： 5%;
第二小时： 10%;
第三小时： 20%;
第四小时： 40%;
第五小时及以后： 60%。
当细菌浓度达到 0D6。。为 10左右时， 将培养温度降至 25*€， 加入 4g IPTG诱导培养 I2小时。终浓度为大约 40(OD6。。）， 下罐， 离心收集菌体。 获得表达了 HPV52N40C-L1 蛋白的菌体， 重大约 2.5kgo 实施例 3: 纯度约 70%的 HPV52N40C- L1蛋白的获得
按 lg菌体对应 10mL裂解液（20mMTris緩冲液，pH7.2, 300mM NaCl)的比例重悬菌体。 采用 APV均质机（An Invensys Group产 品）以 600bar压力破碎菌体 5次。 使用 JA- 14转头， 以 13500rpm (30000g)离心菌体破碎液 15min， 留取上清（即， 破菌上清） 。 通过 10 % SDS-聚丙烯酰胺凝胶电泳检测上清， 此时上清中 HPV52N40C-L1蛋白的纯度约为 10% (参见图 1, 泳道 1 ) 。
采用 CENTRASETTE 5切向流装置 （ PALL产品）对上清进行透 析， 所用膜包截留分子量为 30kDa, 透析液为 lOmM磷酸盐緩冲液 pH6.0, 透析体积为三倍上清体积， 运行压力为 0.5psi, 流速为 500mL/min, 切向流速为 200ml7min。
充分透析后， 使用 JA-10转头 （Beckman J25高速离心机） ， 以 9500rpm (12000g)离心 20min,收获沉淀（即，无盐沉淀产物）。 用 1/10上清体积的 lOmM磷酸盐緩冲液 pH7.0, 10mM DTT, 300mM NaCl重悬沉淀， 搅拌 30min, 然后使用 JA- 14转头（ Beckman J25 高速离心机） 以 13500rpm (30000g)离心 20min，并收获上清和沉 淀（即， 重溶后沉淀）。 使用 0.22 μιη孔径滤膜对上清进行过滤， 所获得的样品 （即， 重溶后上清）用于进行阳离子交换色谱纯化 (如实施例 4 所述） 。 取 150 μΙ 过滤后样品， 加入 30μΙ 6Χ Loading Buffer ( 12% (w/v) SDS , 0.6% (w/v)溴酚蓝， 0· 3M Tris-HCl ρΗ 6.8, 60% (ν/ν)甘油， 5% (ν/ν) β -巯基乙醇） , 混匀并于 80*Ό水浴 lOmin; 然后取 ΙΟμΙ^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。 电泳结果见图 1。 结果显示， HPV52N40C- L1蛋白在经过 沉淀、 重溶的步骤之后， 得到了纯化和富集， 其纯度从之前的约 10%提高到了约 70% (见图 1， 泳道 1和 3) 。 实施例 4: HPV52N40C-L1蛋白的色傳纯化
1) HPV52N40C-L1的阳离子交换色谱纯化
仪器系统： GE Healthcare公司 （原 Amershan Pharmacia公 司）生产的 AKTA explorer 100型制备型液相色傳系统。
层析介质： SP Sepharose 4 Fast Flow ( GE Healthcare公 司） 。
柱体积： 5.5cmx20cm。
緩冲液： 20mM磷酸緩冲液 pH 8.0, lOmM DTT
20mM磷酸緩冲液 pH 8.0, lOmM DTT, 2M NaCl。 流速： 25 mL/mii
检测器波长： 280nm。
样品为实施例 3中获得的经 0.22μιη孔径滤膜过滤的、 纯度 约为 70%的 HPV52N40C-L1蛋白溶液（ 3L) 。
洗脱程序为： 500mM NaCl洗脱杂蛋白， lOOOmM NaCl洗脱目 的蛋白， 收集 lOOOmMNaCl洗脱产物， 共获得 HPV52N40C- L1纯化 样品 900mL。
2) HPV52N40C-L1的 CHT- II (羟基磷灰石色谱） 纯化
仪器系统： GE Healthcare公司 （原 Amershan Pharmacia公 司）生产的 AKTA explorer 100型制备型液相色傳系统。
层析介质： CHT- 11(购自 Bio- RAD)
柱体积： 5.5cm 20cm
緩冲液： 20mM磷酸盐緩冲液 pH8.0, 10mM DTT
20mM磷酸盐緩冲液 pH8.0, lOmM DTT, 2M NaCl。 流速： 20mlVmiii。
检测器波长： 280nm。
样品为： 前一步骤获得的 lOOOmM NaCl洗脱产物，用 20mM磷 酸盐緩冲液 ρΗ 8.0, lOmM DTT将 NaCl浓度稀释至 500mM。
洗脱程序为： 500mM NaCl洗脱杂蛋白， lOOOmM NaCl洗脱目 的蛋白， 收集 lOOOmM NaCl 洗脱产物， 共获得经纯化的 HPV52N40C-L1蛋白样品 800mL。
取经本实施例的方法纯化的 HPV52N40C- L1样品 150 μί, 加 入 30 L 6X Loading Buffer, 混匀并于 80 水浴 lOmin; 然后 取 ΙΟμΙ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。 电泳结果见图 2。 结果显 示， 经过上述纯化步骤后， HPV52N40C- L1 蛋白的浓度约为 0.7mg/ml, 且纯度大于 98%。 实施例 5: HPV52N40C-L1病毒样颗粒的组装
仪器系统为 PALL生产的 CENTRASETTE5切向流系统；膜包截 留分子量为 30kDa; 样品为实施例 4 所得的纯度大于 98%的 HPV52N40C-L1, 800ml。
样品的浓缩： 调节切向流系统的切向流速为 50mL/min, 浓缩 样品至总体积为 600mL。
样品的复性：以 10L复性緩冲液（20mMPB (磷酸钠緩冲液） pH 6.0, 2mM CaCl2, 2mM MgCl2, 0.5M NaCl, 0.003% Tween-80)充 分交换样品緩冲液。 切向流装置的运行压力为 0.5psi, 切向流速 度为 10mL/min。 在复性緩冲液交换完后， 用储存緩冲液（ 20mM PB (磷酸钠緩冲液） pH6.5, 0.5MNaCl)进行交换， 交换体积为 20L。 切向流装置的运行压力为 0.5psi,切向流速度为 25mL/min。 交换 完毕后，使用 PALL 0.22μΐη滤器无菌过滤样品，得到 HPV52N40C-L1 病毒样颗粒， 将其置于 4 保存备用。 实施例 6: HPV52N40C-L1 VLP的形态学检测
1) HPV52N40C-L1病毒样颗粒的透射电镜观察
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。 将实施例 5所得 HPV52N40C- L1病毒样颗粒用 2%磷钨酸 pH7.0负染， 固定于喷炭的铜网上， 进行观察。 电镜结 果见图 3, 其中可见大量半径为 25nm左右的病毒样颗粒， 大小均 匀， 呈现为空心形态。
2) HPV52N40C-L1病毒样颗粒的三维结构重建 使用冷冻电镜三维结构重建实验 ( Wolf M, Garcea RL， Gr igorieff N. et al. Proc Nat l Acad Sci U S A. (2010)， 107 (14)： 6298-303 )来重建 HPV52N40C-L1病毒样颗粒的三 维结构。 简而言之， 在 HPV52N40C-L1 VLP的冷冻电镜图像中 （图 4A )分别选取 400个大小均一、 直径超过 50nm的颗粒进行计算机 重叠和结构重建， 从而获取 HPV52N40C-L1 VLP的三维结构。 所获 得的三维结构如图 4B所示， 其中 HPV52N40C- LI VLP的分辨率为 22 A。 结果显示， HPV52N40C- LI VLP是由 72个壳粒（形态亚单位， 五聚体）形成的 T=7的二十面体结构（h=l， k=2 ) 。 与一般的符合 准等价原理的二十面体病毒衣壳不同， HPV52N40C- LI VLP结构中所 有的组成亚单位均为五聚体， 而未见六聚体， 且 VLP的最外围直径 为约 60nm。这与之前报道的天然 HPV病毒颗粒及真核表达系统（例 如， 痘病毒表达系统）来源的 HPV VLP的三维结构类似。
3 ) HPV52N40C-L1病毒样颗粒的动态光散射观察
使用的仪器为美国 Protein Solut ions公司生产的 DynaPro MS/X型动态光散射仪 (含温度控制器）,使用的算法为 Regu lat ion 算法。 样品为实施例 5所得 HPV52N40C- L1 病毒样颗粒。 样品经 0. 22 μ ιη 滤膜过滤后进行测量。 测量结果见图 5。 结果显示， HPV52N40C-L1 VLP的水化分子动力学半径为 24. 39nm。 实施例 7: HPV52N40C-L1 VLP的免疫原性测定
HPV52假病毒中和细胞模型的建立
由于 HPV难以在体外进行培养， 而且 HPV宿主特异性强， 难 以在人以外的宿主上繁殖， 缺乏合适的动物模型， 因此， 为了能 对 HPV疫苗的免疫保护性进行快速评估， 需要建立有效的体外中 和实验模型。 假病毒 ( pseudovir ions )体外感染模型：利用 HPV VLP可非 特异性包装核酸的特性，通过在细胞内表达 HPV的 L1和 L2蛋白， 通过包裹细胞内的游离体病毒 DNA或外源导入的报告质粒， 组成 HPV 假病毒（Yeager, M. D, As te-Amezaga, M. et a l (2000) Vi rology (278) 570 - 7)。 具体方法包括重组病毒表达系统法及 多质粒共转染方法。 本实施例示例性采用多质粒共转染方法。
另外， 还使用常规方法针对 HPV系统进行了如下的改进： 优 化用于 293FT细胞的磷酸钙转染方法， 以获得高达 90 %以上的转 染效率， 从而有利于进行较大规模的生产； 对表达 HPV结构蛋白 的表达质粒进行密码子优化， 以在哺乳动物细胞中高效表达 HPV L1和 L2基因， 从而有利于高效组装假病毒。
HPV假病毒的构建方法如下：
用 CsCl 密度梯度离心方法分别纯化质粒 p52Llh (携带编码 HPV52 L1蛋白（NCBI数据库，登录号 Q05138)的核苷酸序列的 pAAV 载体）、 质粒 p52L2h (携带编码 HPV52 L2蛋白（NCBI数据库， 登 录号 P36763)的核苷酸序列的 pAAV载体）及带有绿色荧光蛋白基 因的质粒 pN31-EGFP ( pN31-EGFP和上述的 pAAV载体均由 NIH的 John T. Schi l ler教授馈赠） 。 利用 CsCl密度梯度离心来纯化质 粒的方法是本领域公知的， 参见《分子克隆： 第三版》 。
使用磷酸钙转染法，用纯化后的 p52Llh、 p52L2h、 pN31-EGFP 各 4(^g 共转染培育于 10cm 细胞培养皿中的 293FT 细胞 ( Invi trogen ) 。 磷酸 转染法是本领域公知的， 参见《分子克 隆： 第三版》 。 简言之， 将 p52Llh、 p52L2h、 pN31- EGFP各 40μ8 加入 lmL的 HEPES溶液（每 50mL去离子水含有 pH = 7. 3的 1M Hepes 125 L, 4 *€储存）和 lmL的 0. 5mol /L CaCl2溶液的混合溶液中， 混匀， 然后逐滴加入 2mL 2xHeBS溶液（ 0. 28M NaCl (16. 36g) , 0.05M HEPES (11.9g) , 1.5mM Na2HP04 (0.213g)， 溶解于 lOOOmL 去离子水， pH = 6.96, - 70 储存） 中， 室温下静置 lmin; 然后 将混合液加入培养有 293FT细胞的 10cm细胞培养亚中，培养 6hr; 然后弃去原培养液， 加入 10mL 新鲜的完全培养液（Invitrogen 公司）。 转染 48hr后， 弃去培养基， 用 PBS清洗细胞 2次； 然后 将细胞刮下收集细胞， 进行细胞计数， 并且每 108个细胞用 ImL 裂解液（0.25% Brij58, 9.5mM MgCl2) 重悬。 裂解后， 以 5000g 离心 10min， 收集上清， 加入 5MNaCl (终浓度为 850mM) , 从而 获得假病毒液， 将其分装为小份后置于- 20C保存。 抗体中和滴度的测定
将 293FT 细胞 （ Invitrogen) 铺于 96 孔细胞培养板中 (1.5x107孔） 。 5hr 后如下进行中和实验： 将待测的血清样品 (含待测抗体）分别用 10 % DMEM进行连续倍比稀释，然后取 5(^L 各个经稀释的样品分别与 50μί稀释于 10%DMEM中的如上制备的 假病毒液混合 (moi=0.1) ； 在 4 下孵育 lh后， 将混合物分别 加入预铺有 293FT细胞的 96孔细胞培养板中，并在 37*€培养 72h; 然后先通过荧光观察确定各样品大概的抗体滴度， 再用流式细胞 仪 (EPICS XL, 美国 Beckman Coulter公司）检测各孔细胞的感 染率， 计算血清的准确抗体滴度。 感染率为待测细胞样品在阳性 区中的细胞数量百分率减去未感染的对照细胞样品在阳性区中的 数量百分率。
感染抑制率 = ( 1 -加入血清的孔的感染率 /未加入血清的孔 的感染率） χΐοο%。
阳性区的定义为： 通过流式细胞仪测定的 GFP信号高于对照 细胞至少 10倍的细胞区。 抗体中和滴度的定义为： 达到高于 50 %感染抑制率的最大稀 释倍数。 经 50倍稀释后仍能达到 50 %以上感染抑制率的抗体被 视为具有中和能力。
HPV52 VLP用于免疫动物的免疫保护性评价
使用兔子来评价本发明的 HPV52 VLP的免疫保护性。 免疫用 动物为 6 ~ 8 周龄普通级雌性兔（购自广西省疾病预防与控制中 心） 。 将实施例 5所制备的 HPV52N40C- L1 病毒样颗粒（浓度为 0. lmg/ml ) 与等体积的完全弗氏佐剂 （用于初次免疫）或等体积 的不完全弗氏佐剂 （用于加强免疫） 混合均匀。 免疫程序为： 0 周时初次免疫； 4和 10周时各加强一次。 免疫方式为肌肉注射， 初次免疫剂量为 100μ8/只， 加强免疫剂量为 50μ8/只。
初次免疫后， 每周抽取外周静脉血， 分离血清， 保存待检。 然后根据上述方法检测兔子血清中针对 HPV52假病毒的抗体中和 滴度。
检测结果如图 6所示。图 6显示了 HPV52N40C-L1病毒样颗粒 接种兔后不同阶段的血清中和抗体滴度， 图中箭头所示为免疫时 间。 从图中可以看出， 在初次免疫一个月后， 血清总抗体滴度即 有明显上升； 在经过一次加强免疫后， 中和抗体的滴度即能达到 105的高水平。 这表明， 根据实施例 1-5 所述方法获得的 HPV52N40C-L1 VLP具有强的免疫原性， 能够在动物体内诱导高滴 度的针对 HPV52的中和抗体，可用作预防 HPV52感染的有效疫苗。 除了使用弗氏佐剂外， 该疫苗也可使用本领域公知的其他佐剂， 例如氢氧化铝或磷酸铝佐剂。 实施例 8: HPV52N27C— Ll、 HPV52N35C— Ll、 HPV52N38C-LK HPV52N42C-L1蛋白和病毒样颗粒的制备以及形态学观察
基本上依据实施例 1-4描述的方法， 制备和纯化 N端分别截 短了 27 个、 35 个、 38 个和 42 个氨基酸的 HPV52N27C- L1、 HPV52N35C-LU HPV52N38C-L1 和 HPV52N42C- L1 (其氨基酸序列 分别为 SEQ ID N0: 1、 7、 10、 和 13 ) 。 所获得的这 4种蛋白质的 纯度都达到 98 %以上 （见图 7 ) 。
基本上依据实施例 5描述的方法，将经纯化的 HPV52N27C-L1、 HPV52N35C-LU HPV52N38C-L1和 HPV52N42C-L1蛋白分别组装成 病毒样颗粒， 分别称为 HPV52N27C- LI 、 HPV52N35C-L1 、 HPV52N38C-L1和 HPV52N42C- L1病毒样颗粒。
基本上依据实施例 6 描述的方法， 分别对 HPV52N27C-L1、 HPV52N35C-LU HPV52N38C-L1和 HPV52N42C- L1病毒样颗粒进行 透射电镜观察和动态光散射观察。 结果示于图 8和图 9。 图 8显 示，这些截短蛋白可形成大量半径为 25nm左右的病毒样颗粒，颗 粒大小与理论大小相符， 且均匀一致。 图 9显示， HPV52N27C- Ll、 HPV52N35C-LU HPV52N38C-L1和 HPV52N42C- L1病毒样颗粒的水 化分子动力学半径均为 25nm左右， 颗粒组装百分比均为 100%。
另外， 通过使用实施例 7描述的方法可证实， 本发明所获得 的 HPV52N27C- Ll、 HPV52N35C- Ll、 HPV52N38C- L1和 HPV52N42C-L1 病毒样颗粒同样具有良好的免疫原性 , 可在动物体内诱导高滴度 的中和抗体， 从而可用作预防 HPV感染的疫苗。 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

1. 一种截短的 HPV52 L1蛋白或其变体，其与野生型 HPV52 L1 蛋白相比， N端截短了 27-42个氨基酸， 例如 27个、 28个、 29 个、 30个、 31个、 32个、 33个、 34个、 35个、 36个、 37个、 38个、 39个、 40个、 41个或 42个氨基酸；
例如， 与野生型 HPV52 L1蛋白相比， 该截短的 HPV52 L1蛋 白 N端截短了 27-42个氨基酸， 例如 27个、 35个、 38个、 40个 或 42个氨基酸；
例如， 该截短的 HPV52 L1蛋白具有 SEQ ID NO: 1、 SEQ ID NO:
2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 5、 SEQ ID NO: 6、 SEQ ID NO: 7、 SEQ ID NO: 8、 SEQ ID NO: 9、 SEQ ID NO: 10、 SEQ ID NO: 11、 SEQ ID NO: 12或 SEQ ID NO: 13所示的氨 基酸序列， 例如具有 SEQ ID NO: 1、 SEQ ID NO: 7、 SEQ ID NO: 10、 SEQ ID NO: 12或者 SEQ ID NO: 13所示的氨基酸序列， 更 优选地具有 SEQ ID NO: 12所示的氨基酸序列。
2. 一种分离的核酸， 其编码权利要求 1 的截短的 HPV52 L1 蛋白或其变体。
3. 包含权利要求 2的分离的核酸的载体。
4. 包含权利要求 2的分离的核酸和 /或权利要求 3的载体的 宿主细胞。
5. 一种 HPV52病毒样颗粒， 其含有权利要求 1的截短蛋白或 其变体， 或者由权利要求 1的截短蛋白或其变体组成。
6. 一种组合物， 其包含权利要求 1 的截短的 HPV52 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 HPV52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 HPV52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 HPV52病毒样颗粒以预防 HPV感染或宫颈癌有 效量存在；
优选地， 所述药物组合物或疫苗还包含至少一种选自下列的 病毒样颗粒： HPV6 L1蛋白病毒样颗粒， HPV11 L1蛋白病毒样颗 粒， HPV16 L1蛋白病毒样颗粒， HPV18 L1蛋白病毒样颗粒， HPV31 L1蛋白病毒样颗粒， HPV33 L1蛋白病毒样颗粒， HPV45 L1蛋白 病毒样颗粒和 HPV58 L1蛋白病毒样颗粒。
8. 获得截短的 HPV52 L1 蛋白的方法， 其包括利用大肠杆菌 表达系统表达权利要求 1的截短的 HPV52 L1蛋白， 然后将含有该 蛋白的裂解上清进行纯化处理，
优选地， 所述方法包括步骤：
a)在大肠杆菌中表达所述截短蛋白，
b)将表达所述截短蛋白的大肠杆菌在盐浓度为 l OOmM- 600mM 的溶液中破碎， 分离得到上清液，
c)用水或低盐溶液将 b )获得的上清液的盐浓度降至 l OOmM 或以下， 并收集沉淀，
d)将 c )获得的沉淀在 150mM - 250mM盐溶液中重新溶解， 同 时加入还原剂， 分离得到溶液， 该溶液中含纯度至少 50 %的截短 的 HPV52 L1蛋白。
9. 制备权利要求 5的 HPV52病毒样颗粒的方法，其包括步骤： a)将纯度至少 50 %的权利要求 1的截短的 HPV52 L1蛋白通 过色谱层析进行纯化，
b)将步骤 a)中得到的截短蛋白去除还原剂。
10. 一种制备疫苗的方法， 其包括将权利要求 5的 HPV52病毒 样颗粒或通过权利要求 9的方法获得的 HPV52病毒样颗粒与药学 可接受的载体和 /或赋形剂混合， 任选地还混合一种或多种选自 HPV6 , 11 , 16 , 18 , 31 , 33 , 45和 58的 HPV型别的病毒样颗粒。
11. 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 HPV52病毒样颗粒， 或通过权 利要求 9的方法获得的 HPV52病毒样颗粒， 或权利要求 7的药物 组合物或疫苗，或通过权利要求 10的方法获得的疫苗施用给受试 者，
优选地， 所述 HPV感染是 HPV52感染；
优选地， 所述由 HPV感染所导致的疾病是宫颈癌；
优选地， 所述受试者是哺乳动物， 例如人。
12. 权利要求 1 的截短的 HPV52 L1蛋白或其变体， 或权利要 求 5的 HPV52病毒样颗粒， 或通过权利要求 8的方法获得的截短 的 HPV52 L1蛋白，或通过权利要求 9的方法获得的 HPV52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 HPV52感染；
优选地， 所述由 HPV感染所导致的疾病是宫颈癌。
1 3. 权利要求 1 的截短的 HPV52 L1蛋白或其变体， 或权利要 求 5的 HPV52病毒样颗粒， 或通过权利要求 8的方法获得的截短 的 HPV52 L1蛋白，或通过权利要求 9的方法获得的 HPV52病毒样 颗粒， 其用于预防 HPV感染或由 HPV感染所导致的疾病，
优选地， 所述 HPV感染是 HPV52感染；
优选地， 所述由 HPV感染所导致的疾病是宫颈癌。
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Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103044531B (zh) * 2012-09-29 2014-07-30 重庆原伦生物科技有限公司 耐甲氧西林金黄色葡萄球菌(mrsa)疫苗重组蛋白抗原hi2的纯化方法
CN106701796B (zh) * 2015-08-12 2021-11-16 北京康乐卫士生物技术股份有限公司 52型重组人乳头瘤病毒病毒样颗粒及其制备方法
CN105985934A (zh) * 2015-08-18 2016-10-05 北京康乐卫士生物技术股份有限公司 一种二价、三价或多价hpv假病毒及其应用
CN106831961B (zh) * 2015-12-04 2019-11-05 厦门大学 一种人乳头瘤病毒58型l1蛋白的突变体
CN106831959B (zh) * 2015-12-04 2019-11-05 厦门大学 一种人乳头瘤病毒33型l1蛋白的突变体
CN107557347B (zh) * 2016-06-30 2022-03-29 中国科学院上海巴斯德研究所 肠道病毒71型的新型病毒样颗粒、其制备方法及应用
AU2020317321B2 (en) * 2019-07-19 2023-07-27 Sinocelltech Ltd. Polyvalent immunogenicity composition for human papillomavirus
WO2021013078A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒52型l1蛋白
CN114539365B (zh) * 2020-11-26 2023-12-01 中国医学科学院基础医学研究所 一种改造的人乳头瘤病毒52型l1蛋白及其用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6551597B1 (en) * 1999-03-18 2003-04-22 President & Fellows Of Harvard College Vaccine compositions for human papillomavirus
CN1934131A (zh) * 2004-03-24 2007-03-21 默克公司 Hpv52 l1在酵母中的优化表达
CN101153280A (zh) * 2006-09-29 2008-04-02 厦门大学 从原核生物中纯化人乳头瘤病毒晚期蛋白l1的方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1293093C (zh) 2002-08-30 2007-01-03 马润林 乳头瘤病毒衣壳蛋白的原核制备和应用
US9364529B2 (en) 2007-04-29 2016-06-14 Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. Truncated L1 protein of human papillomavirus type 18
US9428555B2 (en) 2007-04-29 2016-08-30 Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. Truncated L1 protein of Human Papillomavirus type 16
CN101343315B (zh) 2007-05-29 2012-06-13 厦门大学 截短的人乳头瘤病毒6型l1蛋白
DK2907821T3 (en) 2007-05-29 2017-04-10 Univ Xiamen Process for Preparation of an N-Terminal Truncated L1 Protein of Human Papillomavirus (HPV)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6551597B1 (en) * 1999-03-18 2003-04-22 President & Fellows Of Harvard College Vaccine compositions for human papillomavirus
CN1934131A (zh) * 2004-03-24 2007-03-21 默克公司 Hpv52 l1在酵母中的优化表达
CN101153280A (zh) * 2006-09-29 2008-04-02 厦门大学 从原核生物中纯化人乳头瘤病毒晚期蛋白l1的方法

Non-Patent Citations (22)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1995, MACK PUBLISHING COMPANY
"The Molecular Cloning Experiment Guide"
BAKER TS; NEWCOMB WW; OLSON NH. ET AL., BIOPHYS J., vol. 60, no. 6, 1991, pages 1445 - 1456
BANKS, L.; G. MATLASHEWSKI ET AL., J GEN VIROL, vol. 68, 1987, pages 3081 - 9
BOYLE, P.; J. FERLAY, ANN ONCOL, vol. 16, 2005, pages 481 - 8
BRUMMELL ET AL., BIOCHEM., vol. 32, 1993, pages 1180 - 1187
BUCK CB; CHENG N; THOMPSON CD. ET AL., J VIROL., vol. 82, no. 11, 2008, pages 5190 - 7
BURKS ET AL., PROC. NATL ACAD. SET USA, vol. 94, 1997, pages 412 - 417
DATABASE GENBANK 29 June 2005 (2005-06-29), XP003031798, Database accession no. NC-001592 *
E. MEYERS; W. 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.
HAGENSEE ME; OLSON NH; BAKER TS ET AL., J VIROL., vol. 68, no. 7, 1994, pages 4503 - 4505
KELSALL, S. R.; J. K. KULSKI, J VIROL METHODS, vol. 53, no. 1, 1995, pages 75 - 90
KIRNBAUER, R.; F. BOOY ET AL., PROC NATL ACAD SCI USA, vol. 89, no. 24, pages 12180 - 4
KOBAYASHI ET AL., PROTEIN ENG., vol. 12, no. 10, 1999, pages 879 - 884
LI, M.; T. P. CRIPE ET AL., J VIROL, vol. 71, no. 4, 1997, pages 2988 - 95
NEEDLEMAN ET AL., J. MOL. BIOL., 1970, pages 443 - 453
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 444 - 453
SAMBROOK J ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP2589604A4
WOLF M; GARCEA RL; GRIGORIEFF N. ET AL., PROC NATL ACAD SCI U S A., vol. 107, no. 14, 2010, pages 6298 - 303
YEAGER, M. D; ASTE-AMEZAGA, M. ET AL., VIROLOGY, 2000, pages 570 - 7

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US20130230548A1 (en) 2013-09-05
DK2589604T3 (en) 2017-01-16
IN2013CN00536A (zh) 2015-07-03
EP2589604A1 (en) 2013-05-08
BR112013000031A2 (pt) 2016-05-10
US9499591B2 (en) 2016-11-22

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