WO2008145021A1 - A truncated l1 protein of human papillomavirus 6 - Google Patents

A truncated l1 protein of human papillomavirus 6 Download PDF

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WO2008145021A1
WO2008145021A1 PCT/CN2008/001050 CN2008001050W WO2008145021A1 WO 2008145021 A1 WO2008145021 A1 WO 2008145021A1 CN 2008001050 W CN2008001050 W CN 2008001050W WO 2008145021 A1 WO2008145021 A1 WO 2008145021A1
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protein
hpv
hpv6
leu
vaccine
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PCT/CN2008/001050
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English (en)
French (fr)
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Shaowei Li
Huirong Pan
Bo Liu
Jun Zhang
Ji Miao
Ningshao Xia
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Xiamen University
Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.
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Application filed by Xiamen University, Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. filed Critical Xiamen University
Priority to EP08757381.2A priority Critical patent/EP2154149B1/en
Priority to US12/601,972 priority patent/US8748127B2/en
Priority to BRPI0810951A priority patent/BRPI0810951B8/pt
Priority to DK08757381.2T priority patent/DK2154149T3/da
Publication of WO2008145021A1 publication Critical patent/WO2008145021A1/zh
Priority to US14/248,063 priority patent/US9745351B2/en

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    • 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
    • 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
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12P21/00—Preparation of peptides or proteins
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
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    • C12N2796/00—Viruses not covered by groups C12N2710/00 - C12N2795/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • the present invention relates to a truncated human papillomavirus type 6 L1 protein, and a virus-like particle composed thereof, a vaccine containing the same, and a medicament for preventing genital warts or
  • Human papillomavirus HPV Human papillomavirus HPV (Human Papi l omavi rus) belongs to the genus Papovavir idae of the papillomavirus family and is a non-enveloped DNA virus.
  • the viral genome is a double-strand closed-loop DNA measuring approximately 7. 2 ⁇ 8 kb with 8 open boxes.
  • the genome can be divided into three regions according to their functions: 1 early region (E), about 4.5 kb, encoding El, E2, E4 ⁇ E7 6 non-structural proteins related to viral replication, transcription and transformation; 2 late region (L), about 2. 5 kb, encoding the major capsid protein L1 and the minor capsid protein
  • the virus particles are 45 ⁇ 55nm in diameter, the nucleocapsid is icosahedral, and there are 72 shell particles, which are composed of L1 and
  • HPV has more than 90 subtypes, which cause skin and mucous membrane lesions in the human population. According to its relationship with tumorigenesis, it can be divided into three groups: 1 low or no carcinogenic risk group, including HPV6, 11, 39, 41, 42, 43; 2 moderate carcinogenic risk group, including HPV31, 33, 35, 51 , 52; 3 high cancer risk group, including
  • HPV such as HPV6, 11
  • HPV6 HPV6
  • the anal genital mucosa is third only to chlamydia and trichomoniasis, and is a common sexually transmitted disease.
  • the lesions caused by HPV6, 11 accounted for about 90% of the total.
  • the peak of HPV infection in the female reproductive tract is between 15 and 25 years old, and is related to the sexual behavior of infected people.
  • HPV L1 protein is the major capsid protein with a molecular weight of 55 ⁇ 60kDa, which is the main target protein of HPV vaccine.
  • the HPV L1 protein expressed in various expression systems can form a virus-like particle (V rus-Like Part ic le, VLP) having a morphological structure similar to that of a natural virus particle without the aid of L2 protein.
  • This type of virus particle is an icosahedral symmetrical structure composed of 72 pentameric L1 proteins. It retains the natural epitope of the viral particle and is highly immunogenic, inducing neutralizing antibodies against the same type of HPV virus.
  • VLP vaccine has become the main direction of HPV vaccine development.
  • HPV 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 expression systems can be divided into eukaryotic expression systems and prokaryotic expression systems.
  • eukaryotic expression systems include a poxvirus expression system, an insect baculovirus expression system, and a yeast expression system.
  • the HPV L1 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 purified VLPs, which provides great convenience for purification work.
  • Due to the low expression level of the eukaryotic expression system and the high cultivation cost it has brought great difficulties to large-scale industrial production.
  • HPV vaccine Gardas il® uses Saccharomyces cerevisiae The system has a low expression level and high production cost, so the price of the product is high, which affects its wide application.
  • HPVL1 protein by the E. coli expression system in prokaryotic expression systems has been reported. For example, it has been reported to express HPV16 L1 protein by Escherichia coli (Banks, L., G. Mat lashewski, et al. (1987). J Gen Virol 68 (Pt 12): 3081-9). However, most of the HPV L1 proteins expressed by E. coli have lost their natural conformation and cannot produce protective antibodies against HPV. Alternatively, the above proteins may be obtained by HPB VLP (Kel sall, SR and JK Kul ski (1995). J Virol Methods 53 (1): 75-90) by inclusion body purification, renaturation, etc., but in the renaturation process. The medium protein loss is large, the yield is low, and it is difficult to apply it in mass production.
  • HPV L1 protein can also be expressed in E. coli in a proper conformation and soluble in the lysed supernatant of the cell, but the expression is low, and the amount of the heteroprotein in the supernatant is large and large, the purpose of purification is to be purified. 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 Cr ipe, et al. (1997). J Vi rol 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 inventors have unexpectedly discovered that a truncated HPV6L1 protein capable of inducing a neutralizing antibody against HPV6 can be obtained in an E. coli expression system, the truncated
  • HPV6L1 protein is purified to yield a high yield, at least 50% purity HPVL1 protein.
  • the inactivated HPVL1 protein is further processed to obtain viroid-like particles which are capable of inducing a protective antibody against HPV6, and the present invention has been completed based on the above invention.
  • a first aspect of the invention relates to a HPV6 L1 protein having a N-terminal truncation of 2, 3, 4, or 5 amino acids (compared to the wild-type HPV6 L1 protein).
  • the truncated protein has the sequence 1, 2, 3, or 4, preferably sequence 1.
  • a further aspect of the invention relates to a polynucleotide encoding a truncated protein of the invention and a vector comprising the polynucleotide.
  • a further aspect of the invention relates to a cell comprising the above vector.
  • the invention further relates to compositions comprising the above-described truncated proteins or polynucleotides or vectors or cells.
  • a further aspect of the invention relates to a HPV6 virus particle, wherein the virus particle comprises an HPV6 L1 protein having a N-terminally truncated 2, 3, 4, or 5 amino acids, for example, having sequences 1, 2, 3, Or a HPV6 L1 protein of 4, or an HPV6 L1 protein truncated by a N-terminus of 2, 3, 4, or 5 amino acids, for example, HPV6 L1 protein having sequence 1, 2, 3, or 4.
  • a further aspect of the invention relates to a method for obtaining an HPV6 L1 protein comprising expressing a truncated HPV6 L1 gene fragment in an E. coli expression system, and then purifying the cleavage supernatant containing the truncated protein.
  • the method of obtaining the HPV6 L1 protein comprises a) expressing a truncated HPV6 L1 gene fragment in an E. coli expression system, - b) expressing E. coli expressing the truncated HPV6 L1 protein at a salt concentration of 100 mM - Broken in 600 mM, the supernatant was separated,
  • the invention also relates to a method of obtaining an HPV L1 protein, such as the HPV6 L1 protein of the invention, comprising
  • the present invention also relates to a vaccine for preventing condyloma acuminata or HPV infection comprising the HPV6 L1 protein-like virus particle of the present invention.
  • the vaccine further comprises at least one selected from the group consisting of HPV18L1 protein virus particles, HPV11L1 protein virus particles, HPV16L1 protein virus particles, HPV31L1 protein virus particles, HPV33L1 protein virus particles, HPV45L1 protein virus particles, HPV52L1 protein virus. Granules, viroid-like particles of HPV58L1 protein-like viral particles.
  • the vaccine typically also contains an excipient or carrier for the vaccine.
  • the vaccine comprises: HPV6 virus particles and HPV11 virus particles, in particular, a protein shield having the amino acid sequence of SEQ ID NO: 4 or HPV6 virus particles formed from the protein, and containing SEQ ID NO: The protein shield of the amino acid sequence shown in 7 or the HPV11 virus particle formed by the protein. More preferably, the vaccine further comprises HPV class 16 virus particles and scorpion 8 virus particles, in particular a protein having the amino acid sequence of SEQ ID NO: 8 or a HPV class 16 virus particle formed by the protein stalk, and containing SEQ ID NO : 9 A protein of the amino acid sequence or HPV18 virus particle formed by the protein.
  • the vaccine comprises: having SEQ ID NO: a protein of the amino acid sequence shown by 4 or an HPV6 virus particle formed of the protein, a protein having the amino acid sequence of SEQ ID NO: 7 or a HPV11 virus particle formed of the protein, having SEQ ID NO A protein of the amino acid sequence of 8 or an HPV16 type virus particle formed of the protein, and a protein having the amino acid sequence of SEQ ID NO: 9 or an HPV18 type virus particle formed of the protein.
  • the invention further relates to the use of the HPV6 L1 protein of the invention or a viroid thereof in the manufacture of a vaccine for the prevention of condyloma acuminata or HPV infection.
  • the present invention also relates to a method of preventing condyloma acuminata or HPV infection comprising administering a prophylactically effective amount of the HPV6 L1 protein vaccine of the present invention to a human or animal in need of prevention of condyloma acuminata or HPV infection.
  • the invention further relates to a method of obtaining HPV6 L1 protein-like viral particles, comprising:
  • the invention further relates to a method of preparing a vaccine for preventing condyloma acuminata or HPV infection comprising the step of treating said viroid-like particle with one or more selected from the group consisting of HPV 11, 16, 18, 31, 33, 45, 52 , and the HPV-type virus-like particles of 58 and the vaccine are mixed with a carrier or an excipient.
  • a method of preparing a vaccine for preventing condyloma acuminata or HPV infection comprising the step of treating said viroid-like particle with one or more selected from the group consisting of HPV 11, 16, 18, 31, 33, 45, 52 , and the HPV-type virus-like particles of 58 and the vaccine are mixed with a carrier or an excipient.
  • E. coli expression system means composed of Escherichia coli (strain) and a vector, wherein Escherichia coli (strain) is derived from the market, and is exemplified herein but not limited to: GI698, ER2566, BL21 ( DE3), B834 (DE3), BLR (DE3).
  • carrier means that a certain protein can be encoded.
  • the vector can be transformed, transduced or transfected into a host cell such that the genetic shield element it carries is expressed in the host cell.
  • vectors include: plasmids; phage; cosmid and the like.
  • truncated HPV6 L1 protein gene fragment refers to the removal of a nucleotide encoding one or more amino acids at the 5' or 3' end of the wild-type HPV6 L1 protein gene (cDNA), wherein the wild type
  • the full-length sequence of the HPV6 L1 protein gene is exemplified by, but not limited to, the following sequences in the NCBI database: : AF067042. 1, AF092932. 1 , L41216. 1 , X00203.
  • Truncate HPV6 L1 protein refers to a protein after removal of one or more amino acids at the N-terminus and/or C-terminus of wild-type HPV6 L1 protein, examples of which are wild-type HPV6 L1 proteins, but are not limited to AF067042 in the NCBI database. 1 , AF092932. 1, L41216. 1, X00203. 1 and so on encoded full-length L1 protein.
  • the term "vaccine excipient or carrier” means selected from one or more, including but not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents are exemplified by, but not limited to, phosphate buffers
  • surfactants include cationic, anionic or nonionic surfactants.
  • Tween- 80 examples of adjuvants are, but are not limited to, aluminum hydroxide, Freund's complete adjuvant.
  • the ionic strength enhancer is exemplified by, but not limited to, sodium chloride.
  • 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 HPV6L1 protein of the present invention is preferably obtained by: disrupting Escherichia coli expressing the truncated HPV6L1 protein in a buffer having a salt concentration of 100 - 600 mM, preferably 200 - 500 mM, and isolating the disrupted solution to obtain
  • the supernatant, water or low concentration salt (usually lower than the salt concentration for crushing) reduces the salt concentration in the resulting supernatant to a salt concentration of 100 mM 0M, and separates the supernatant with a salt concentration as low as 1 OOMm - 0
  • the precipitate is precipitated; the precipitate is redissolved in a solution containing a reducing agent and a salt concentration of 150 - 2000 mM, preferably 200 mM or more, and separated to obtain a truncated HPV6L1 protein having a purity of at least 50%, preferably at least 70%, more preferably at least 80%. Solution.
  • the buffer means a solution which can maintain a stable pH within a certain range, including but not limited to, a Tri buffer, a phosphate buffer , HEPES buffer, MOPS buffer, etc.
  • the prokaryotic host cell disruption comprises, but is not limited to, one or more methods of homogenizer disruption, homogenizer disruption, sonication, milling, high pressure extrusion, lysozyme treatment;
  • salts used include, but are not limited to, neutral salts, particularly alkali metal salts, ammonium salts, hydrochloride salts, sulfate salts, sulfate salts, hydrocarbons. a salt, a phosphate or a hydrogen phosphate, in particular one or more of NaCl, KC 1, NH 4 CK (NH 4 ) 2 S0 4 . NaCl is preferred.
  • Reducing agents used include, but are not limited to, DTT, 2-mercaptoethanol. The amount used includes but is not limited to l OmM-l OOmMo
  • the truncated HPV6L1 protein-like virus particles of the present invention are obtained as follows: A truncated HPVL1 protein solution having a purity of at least 50% as described above is further separated by, for example, chromatographic chromatography to obtain a purified truncated HPV6L1 protein solution. The reducing agent in the purified truncated HPV6L1 protein solution was removed to obtain a virus-like particle of truncated HPV6L1. Ways to remove the reducing agent include, but are not limited to, techniques known in the art, such as dialysis, ultrafiltration or chromatography.
  • the truncated HPVL1 protein of the invention preferably has the sequence 1.
  • the vaccine of the present invention may be in a form acceptable to the patient including, but not limited to, oral or injection, preferably injection.
  • the vaccine of the present invention is preferably used in a unit dosage form, wherein the amount of the truncated HPV6L1 protein virus particles in the unit dosage form is 5 g - 8 (mg, preferably 2 0 ⁇ & -
  • the expression systems used in the preparation of 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 often a simple purification process can be used to obtain VLPs with the correct conformation.
  • the baculovirus expression system and the yeast expression system used in the eukaryotic expression system have the defects of low expression and high cultivation cost, which brings 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 the E. coli expression system 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 implement VLPs with correct conformations from inclusion bodies in large-scale production and can only be limited to small-scale laboratory studies.
  • HPV L1 can also be expressed in the correct conformational soluble form in E. coli lysed supernatant, the expression level is low, and it is quite difficult to purify HPV L1 protein from a wide variety of soluble proteins in E. coli lysate supernatant, often requiring fusion. Purification by means of expression and affinity chromatography, etc., often requires expensive enzymes and is difficult to industrially produce.
  • the present invention expresses the N-terminally truncated HPV6 L1 protein in an E. coli expression system, and selectively precipitates the HPV6 L1 protein expressed in the E. coli lysate supernatant by mild means, and further reconstitutes the HPV6 L1 with a salt buffer.
  • the protein allows the purity of the HPV6 L1 protein to be significantly improved and the reconstituted protein can be directly purified by ion exchange chromatography and hydrophobic exchange chromatography to obtain pure protein.
  • the purified truncated HPV6 L1 truncated protein can be assembled into viroid-like particles by the above procedure, which has good immunogenicity and can induce high titers against HP V 6
  • Neutralizing antibodies which prevent HPV6 infection in humans, are a good form of vaccine.
  • the truncated HPV6 L1 protein used in the present invention is easy to express in an E. coli expression system while retaining the antigenicity and particle assembly ability of the full-length HPV6 L1 protein, and the purification method used does not require the use of expensive enzymes, and the cost low.
  • the conformation of the target protein in the purification process has not undergone a severe denaturing renaturation process, and the loss is small, and it can be applied to large-scale industrial production.
  • Figure 1 shows the results of SDS polyacrylamide gel electrophoresis of HPV6N3C-L1 protein in steps a) - d) of the method of the present invention.
  • 1 cleavage supernatant; 2, HPV6N3C-L1 after tangential flow precipitation; 3, HPV6N3C-L1 resuspended by resuspension.
  • the results showed that the HPV6N3C-L1 protein had a purity of about 70% after the step of reconstitution by precipitation.
  • Figure 2 shows the results of SDS polyacrylamide gel electrophoresis of HPV6N3C-L1 obtained in step d) after further purification by the step e) of the present invention.
  • the purified HPV6N3C-L1 is loaded with the obtained HPV6N3C-L1 by the step e) of the present invention; 2, the HPV6N3C-L1 purified by the step e) of the present invention is loaded with 20 ⁇ 1: .
  • the knot shows that the purity of HPV6N3C-L1 purified by step e) is about 98%.
  • Figure 3 shows the results of transmission electron microscopy (50,000 times) of the HPV6N3C-L1 virus particles obtained in step f).
  • a large number of viroid-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 uniform.
  • Figure 4 shows the results of dynamic light scattering observation of the HPV6N3C-L1 virus particles obtained in step f). The results showed that the HPV6N3C-L1 virus particle had a hydration molecular dynamic radius of 24.70 nm and a particle assembly percentage of 100%.
  • FIG. 5 shows the serum at different stages after inoculation of HPV6N3C-L1 virus particles
  • Figure 6 shows serum neutralizing antibody titers at different stages after inoculation of HPV6N3C-L1 virus particles.
  • the arrows in the figure show the immunization time. After the initial free one week, neutralizing antibody titers increased rapidly, after the first vaccination, the neutralizing antibody titer that is able to achieve a higher level of 106.
  • Figure 7 is a graph showing the changes in the titer of HPV6 and HPV11 neutralizing antibodies in serum at different times after HPV6/11 bivalent vaccination in Example 5.
  • the immunization program is 0, 2W (weeks). After the first immunization, the HPV6, HPV11 neutralizing antibody titer increased significantly, and after a booster immunization, the antibody titer reached 1 X 10 4 -1 X 10 5 .
  • Figure 8 shows the changes in serum titer of HPV6, HPV11, HPV16, and HPV18 in serum at different times after HPV6/11/16/18 tetravalent vaccination in the examples.
  • the immunization program is 0, 2W (weeks). After the first immunization, the HPV6, HPV11, HPV16, HPV18 neutralizing antibody titers increased significantly, and after a booster immunization, the antibody titer reached 1 X 10 5 -1 10 6 .
  • Figure 9 shows HPV6 L1 protein HPV6N2C-L1, HPV6N4C-L HPV6N5C-L1 with N, 2, or 5 amino acids at the N-terminus in steps a)-e) of the present invention (the amino acid sequences are respectively See SDS polyacrylamide gel electrophoresis results for SEQ ID NO: 2, 3, 4).
  • Figure 10 shows HPV6 L1 protein HPV6N2C-L1, HPV6N4C-L with N, 2, or 5 amino acids cut at the N-terminus, respectively, via steps a) to f) ⁇ ⁇ 6 ⁇ 0; -1 ⁇ virus particles observed by transmission electron microscopy (50,000 times). 1, transmission electron microscopic observation of HPV6N2C-L1 virus particles obtained by steps a) -f )
  • Figure 11 shows that the N-ends obtained by steps a) -f) are truncated by 2, respectively.
  • Dynamic light scattering observations of HPV6N5C-L1 virus particles 1, the dynamic light scattering observation result of the HPV6N2C-L1 virus particle obtained by the steps a) -f); 2, the dynamic light scattering observation result of the HPV6N4C-L1 virus particle obtained by the steps a) -f); Dynamic light scattering observation of HPV6N5C-L1 virus particles obtained by a) -f); the results showed that the hydration molecular dynamics radius of HPV6N2C-Ll, HPV6N4C-Ll, HPV6N5C-L1 virus particles was about 25 nm, and the percentage of particle assembly was painting.
  • VllVOVOlVO VVDDVIOVOO DVDV03V1V0 VIOOIOIDVI 1011IOIDVV DIVVIODOOI T 96 tL 9 ⁇ JL iL y ⁇ 9 ⁇ VVOVVIVOVO ODVDDOOYYY raim worker
  • DOOVDVDVOl I00XV1V030 01V90VDVIV I10IOVDDV1 IV1IDVVOVI 1D030DDD0I
  • VDVDOIDVV1 OIOOOIOIOO V11V000V3V OOOOVDOOOI 00V0V13D00 ⁇
  • V3V00IVD90 OIVIOOllIO 3VVDV0VV3V ⁇ vom ⁇ IDIDIODIOV 01D30I1V30 ⁇ n mvvvDvvi DOIVOVDOV1 10I00100VV 111VIOOOVD VIVVOIV1VO OVDIOIOOVV ⁇ 8 ⁇
  • OSOlOO/800ZN3/X3d 1261 ATTACCTGTC AAAAGCCCAC TCCTGAAAAG CAAAAGCCAG ATCCCTATAA GAACCTTAGT
  • Example 1 Expression of truncated HPV6 L1 protein with sequence 1
  • HPV-6 L1 gene The full length of HPV-6 L1 gene was synthesized by Shanghai Boya Company.
  • the synthesized gene fragment has a full length of 1503 bp and its sequence is sequence 5.
  • a template for the truncated HPV6 L1 protein of the present invention was prepared based on the full length fragment of the artificially synthesized HPV-6L1 gene.
  • the HPV6L1 full-length gene fragment synthesized in the previous step was used as a template for the re-PCR reaction.
  • 6N3F 5' - CAT ATg GCT AGC GAC AGC ACA GTA TA - 3, (SEQ ID NO: 10) is a forward primer, which introduces a restriction endonuclease Ndel site at the 5th end, and the Ndel site sequence is CAT ATG, ATG is the initiation codon in the E. coli system;
  • 6CR 5 ⁇ -GTC GAC TTA CCT TTT AGT TTT GGC GC -3' (SEQ ID NO: 11) is a reverse primer with a 5, end introduced restriction Endonuclease Sa il site.
  • the PCR reaction was carried out in a PCR thermocycler (Biometra T3) under the following conditions:
  • a DNA fragment of about 1.5 kb in size was amplified.
  • the PCR product was ligated with a commercially available pMD18-T vector (manufactured by TAKARA Co., Ltd.), and identified by Ndel/Sa ll digestion to obtain a positive clone pMD 18-T-HPV6N3C-L1 inserted into the truncated HPV6L1 gene.
  • the nucleotide sequence of interest inserted into the pMD 18-T-HPV6N3C-L1 plasmid was sequence 6, and the encoded amino acid sequence was the sequence.
  • the corresponding protein of this sequence is HPV6 L1 protein with N-terminally truncated 3 amino acids and C-terminal untruncated, named HPV6N3C-L
  • the above pMD18-T-HPV6N3C-L1 plasmid was digested with Ndel/Sa l l to obtain a truncated HPV6N3C-L1 gene fragment.
  • the fragment was ligated to the prokaryotic expression vector pTrxFus (purchased from Invi trogen) which was digested with Ndel/Sal l. Since the fusion protein has been cleaved, the target amino acid is followed by the starting amino acid Met without other fusion polypeptide. . Ndel/Sa l 1 digestion and identification of the inserted L1 protein gene
  • the positive expression of the segment was cloned pTRX-HPV-6N3C-L1.
  • the pTRX-HPV-6N3C-L1 plasmid (0.15 mg/ml) of ⁇ was transformed into 40 ⁇ L of competent Escherichia coli GI698 (purchased from Invi trogen) prepared by calcium chloride method and coated on ampicillin. (final concentration 100 mg/ml, the same as below) resistant solid CAA medium (6 g Na 2 HP0 4 , 3 g KH 2 P0 4 , 0.5 g NaCl, lg NH 4 C1, 20 g casein hydrolysate, 0. 095 g MgCl 2 , 1.
  • the E. coli freeze-dried strain carrying the recombinant plasmid pTRX - HPV6N3C-L1 was removed from -70TC, and placed in ampicillin-resistant 50 ml IMC liquid medium, 200 rpm, 30 n, cultured for about 8 hours, and then transferred to 10 bottles. In 500 ml of medium, 5 ml of bacterial solution was added to each vial, 200 rpm, 30 ⁇ , and shake flasks were cultured overnight.
  • the casein hydrolysate was prepared at a concentration of 30% (30 g dissolved to 100 ml), glucose 50% (50 g dissolved to l OOffll), and sterilized at 121 20 for 20 minutes.
  • the feed was fed, and 50% glucose and 30% casein hydrolysate were mixed at a solute mass ratio of 2 :1.
  • the flow acceleration is as follows:
  • the culture temperature was lowered to 25 X when the concentration reached OD600 of about 10: the culture was induced by adding 4 g of tryptophan for 4 hours.
  • the final concentration is about 40 (OD600), and the centrifuge collects about 2. 5kg.
  • IMC medium is as follows (1 liter):
  • Example 2 Obtaining HPV6N3C-L1 with a purity of about 70%
  • the cells were resuspended in a ratio of 10 mL of lysate (20 mM Tr i s buffer pH 7.2, 300 mM NaCl), and the cells were disrupted 5 times with an APV homogenizer (An Invensys Group product) at a pressure of 600 bar.
  • the JA-14 rotor was centrifuged at 13500 rpm (30000 g) for 15 min, and the supernatant was taken and detected by 10% SDS-polyacrylamide gel electrophoresis. At this time, the purity of HPV6N3C-L1 in the supernatant was about 10%.
  • the supernatant was dialyzed using a CENTRASETTE 5 tangential flow device (PALL product) with a membrane molecular weight cutoff of 30 kDa, a dialysate of 1 OmM phosphate buffer pH 6.0 buffer, and a dialysis volume of three times the supernatant volume.
  • the running pressure was 0.5 ps i
  • the flow rate was 500 mL/niin
  • the tangential flow rate was 200 mL/min.
  • ⁇ - 10 rpm Beckman J25 high speed centrifuge
  • 9500 rpm (12000 g) harvested by centrifugation at 20 min, using 1/10 supernatant
  • Instrument System GE Healthcare Original AKTA explorer 100 preparative LC system from Amershan Pharmacia.
  • Buffer 20 mM phosphate buffer pH 7.0, lOmM DTT
  • the sample is 3L pure about 70% HPV6N3C-L1 solution
  • the elution procedure was as follows: 200 mM NaCl was used to elute the protein, 500 mM NaCl was used to elute the protein of interest, and 500 mM NaCl was collected to obtain a purified sample of HPV6N3C-L1 900 mL.
  • Buffer lOmM phosphate buffer ⁇ 7 ⁇ 0, 10 mM DTT, 0.5 M NaCl.
  • the sample is: SP Sepharose 4 Fast Flow 500 mM NaCl elution product
  • the elution procedure is: direct collection of the breakthrough containing the protein of interest
  • the breakthrough product was collected to obtain a purified HPV6N3C-L1 sample of 1000 mL. Take 150 ⁇ L of HPV6N3C-L1 sample purified by the method of this example, add 6 ⁇ Loading Buffer 30 ⁇ mixed hook, and take ⁇ in 10% SDS-polyacrylamide gel for 120min at 120V after lOmin in 80 water bath. The electrophoresis bands were then visualized by Coomassie brilliant blue. The electrophoresis results are shown in Figure 2. From the electrophoresis results, the target protein concentration was about 0.7 mg/ml, and the SDS-PAGE staining purity was greater than 98%.
  • Example 4 Assembly of HPV6N3C-L1 viroid particles
  • the instrument system was a CENTRASETTE5 tangential flow system produced by PALL; the membrane molecular weight cutoff was 30 kDa; and the sample was 1000 ml of HPV6N3C-L1 obtained in Example 3.
  • the tangential flow rate of the conditioning system is 50 mL/min, and the sample is concentrated to a total volume of 800 mL.
  • the sample buffer was fully exchanged with 10 L of refolding buffer (20 mM PB pH 6.0, 2 mM CaC12, 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.
  • the storage buffer (20 L PBS: 20 mM PB pH 6.5, 0.5 M NaCl) was used.
  • Exchange, exchange volume is 20L.
  • the operating pressure was 0.5 psi and the tangential flow rate was 25 mL/min.
  • the sample is sterile filtered using a PALL 0.20 ⁇ filter to obtain HPV6N3C-L1 virus particles.
  • the instrument is a l OOkV transmission electron microscope produced by Japan Electronics Co., Ltd., and the magnification is
  • Example 4 has a large number of viroid-like particles having a radius of about 25 nm, and has a uniform size and a hollow form.
  • the instrument is a DynaPro MS/X dynamic light scattering instrument (including temperature controller) produced by Prote in Solut ions, USA, using the algorithm as Regulat ion algorithm.
  • the sample was the sample obtained in Example 4.
  • the sample was filtered through a 0.22 ⁇ m filter and measured.
  • the measurement results are shown in Figure 4.
  • the results show that the hydration molecular dynamics radius of HPV6N3C-L1 VLP is 25.46 nm.
  • HPV Since HPV is difficult to culture in vitro, and HPV hosts are highly specific, it is difficult to propagate on hosts other than humans, and there is a lack of suitable animal models. Therefore, in order to be able to
  • HPV VLP in vitro infection model of pseudovir ions:
  • the HPV VLP can be used to non-specifically package nucleic acid characteristics by expressing HPV L1 and L2 proteins in cells, by wrapping intracellular free viral DNA or exogenously introduced reporter plasmids.
  • Composition of HPV pseudoviruses. (Yeager, M. D, As te-Amezaga, M. et a l (2000) Virology ( 278 ) 570 - 7)
  • Specific methods include recombinant viral expression system and multi-plasmid co-transfection
  • the multi-plasmid co-transfection method used in the present invention and the following improvements are made for the HPV system: An optimized calcium phosphate transfection method for 293FT cells is established, and transfection efficiency of up to 90% can be obtained, which is advantageous for carrying out Larger scale production. An expression plasmid for the codon-optimized HPV structural protein, which efficiently expresses the HPV L1 and L2 genes in mammalian cells, facilitates efficient assembly of pseudoviruses.
  • the plasmid p6L1h carrying the HPV6L1 gene, the plasmid p6L2h carrying the HPV6L2 gene, and the plasmid PN31-EGFP carrying the green fluorescent protein gene were separately purified by CsCl density gradient centrifugation.
  • the method for purifying plasmids by CsCl density gradient centrifugation is described in Molecular Cloning: Third Edition. Briefly: The plasmid was transformed into E. coli DH5ot, and a single colony was picked and inoculated into 500 mL of LB medium, and cultured in a shake flask for 16 hours.
  • a closed loop DNA band was collected using a syringe needle and repeatedly extracted 4 times with an equal volume of isoamyl alcohol.
  • a DNA pellet was collected by adding 3 volumes of water and 8 volumes of absolute ethanol, 20000 g, 4 rpm for 30 min. Wash 75% alcohol once and dissolve the DNA pellet with 1 mL of TE. Determine the concentration of the DNA solution and store it in small portions at -20.
  • Orphaned 293FT cells (Invitrogen) were cultured in 10 cm cells.
  • the medium was discarded, washed twice with PBS, the cells were scraped off and the cells were collected, counted, and resuspended per 10 8 cells with 1 mL of lysate (0.25% Brij58, 9.5 mM MgCl 2 ). After the completion of the lysis, 5000 g was centrifuged for 10 min, the supernatant was collected, and 5 M NaCl (final concentration: 850 mM) was added, which was a pseudovirus solution, and the mixture was dispensed into small portions and then stored.
  • lysate 0.25% Brij58, 9.5 mM MgCl 2
  • the infection rate is the percentage of the number of cells in the positive zone of the cell sample minus the percentage of the number of uninfected control cell samples in the positive zone.
  • Antibody neutralization titers are defined as: Maximum dilution factor that achieves greater than 50% inhibition of infection. Monoclonal antibody or more that can achieve 50% infection inhibition rate after 50-fold dilution Resistance is considered to have neutralizing capabilities.
  • Rabbit Normal, female, 6-8 weeks old, purchased from the Guangxi Provincial Center for Disease Control and Prevention, and raised at the center.
  • the HPV6N3C-L1 viroid particle prepared in Example 4 was firstly mixed with an equal amount of Freund's complete adjuvant, and the booster immunization was prepared by mixing with an equal amount of Freund's incomplete adjuvant.
  • the immunological method was intramuscular injection, and the initial immunization dose was l OOug/only, and then strengthen each time in 4, 10 weeks, the booster dose is 50ug/only. After autoimmunization, peripheral venous blood is taken weekly, serum is separated, and stored for examination.
  • Example 4 Normal, female, 6-8 weeks old, purchased from the Guangxi Provincial Center for Disease Control and Prevention, and raised at the center.
  • the HPV6N3C-L1 viroid particle prepared in Example 4 was firstly mixed with an equal amount of Freund's complete adjuvant, and the booster immunization was prepared by mixing with an equal amount of Freund's incomplete adjuvant.
  • the immunological method was intramuscular injection, and the initial immunization dose was Lmg/only, then strengthen each time at 4, 10, 18 weeks, the booster dose is
  • peripheral venous blood is taken weekly, serum is separated, and stored for examination.
  • the neutralization titer of the above antiserum was evaluated by the above pseudovirus neutralizing cell model experiment, as shown in Figs.
  • the results showed that the HPV6N3C-L1 virus-like particles obtained in Example 4 of the present invention (in addition to the Freund's adjuvant used in the experiment, may also be a commercial or self-made aluminum hydroxide or aluminum phosphate adjuvant) mixed and formulated into a vaccine. It has good immunogenicity and can induce high titers of neutralizing antibodies in animals, which can be used as a vaccine against HPV infection.
  • HPV6N5C L1 and HPV11N4C-L1 virus particles were prepared in a manner similar to that described in Examples 1-4 of the present invention.
  • the mixture was mixed at a ratio of 1:2 (weight ratio) so that the concentration of the above two types of virus-like particles after mixing was 40, 80 g/ml. After that, for the first time, add an equal volume of Freund's complete adjuvant and mix it. Immunization is prepared by mixing with an equal volume of Freund's incomplete adjuvant.
  • the immunization method is intramuscular injection, and the initial immunization dose is
  • the booster dose was 2 weeks, and the booster dose was 20 ⁇ 8 /HP for HPV6N5C-L1 virus particles and 40 ⁇ ⁇ /only for HPV11N4C-L1 virus particles.
  • peripheral venous blood was drawn weekly, serum was separated, and the neutralizing antibody titer against HPV6, HPV11 pseudoviral particles in the immunized mice was separately measured as described in Example 5.
  • the results of the test are shown in Fig. 7.
  • the results show that the HPV6 and HPV11 bivalent vaccines prepared by mixing the HPV6N5C-L1 and HPV11N4C-L1 virus particles obtained by the methods described in Examples 1-4 of the present invention have good immunogenicity.
  • HPV16N5C-L1, HPV11N4C-L1 two kinds of virus particles are mixed with commercial or homemade aluminum hydroxide or aluminum phosphate adjuvant).
  • amino acid sequence corresponding to the above HPV11N4C-Iil viroid particle is (SEQ ID NO: 7):
  • Immunized animals 4 out of 4 weeks old SPF BALB/c mice.
  • the virus-like particles of HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1, and HPV18N65C-L1 were prepared in a similar manner as described in Examples 1 - 4 of the present invention.
  • the above four kinds of virus particles: HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1, HPV18N65C-L1 are mixed in a ratio of 1:2:2:1 (weight ratio), so that the concentration of the above four kinds of virus particles after mixing is 40, 80, 80, 40 g/inl.
  • HPV6N5C-L1 HPV18N65C-L1 virus particles were 20 ⁇ g/mouse
  • HPV11N4C-L1 HPV16N30C-L1 virus particles were 40 g/mouse each.
  • peripheral venous blood was taken weekly, serum was separated, and neutralizing antibody titers against HPV6, HPV11, HPV16, and HPV18 pseudoviral particles were detected in the immunized mice as described in Example 5.
  • the test results are shown in FIG. 8.
  • HPV6, HPV11, HPV11N5C-L1, HPV11N30C-L1, HPV16N30C-L1, HPV18N65C-L1 virus particles obtained by the methods described in Examples 1-4 of the present invention are mixed.
  • the HPV16, ⁇ 8 tetravalent vaccine has good immunogenicity and can induce high titers in animals.
  • the neutralizing antibody of HPV6, HPV11, HPV16, HPV18 can be used as an effective vaccine for preventing HPV6/HPV11/HPV16/HPV18 infection (in addition to the Freund's adjuvant used in the examples, the vaccine can also be prepared by the HPV6N5C prepared by the present invention.
  • HPV6N5C prepared by the present invention.
  • HPV11N4C-L1 HPV16N30C-L1, HPV18N65C-L1
  • the L1 amino acid sequence of the above HPV6N5C-L1 viroid particle is shown in SEQ ID NO: 4.
  • the L1 amino acid sequence of the above HPV16N30C-L1 virus particle is (SEQ ID NO: 8):
  • Tyr lie Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr
  • Tyr lie Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr
  • a truncated HPV6L1 protein having the sequence 2, 3, 4 was prepared, and the above truncated proteins were purified to obtain a protein having a purity of more than 98%, and assembled into a class having a radius of about 25 nm. Virus particles.
  • the results are shown in Figures 9, 10 and 11.

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Description

截短的人乳头瘤病毒 6型 L1蛋白 技术领域
本发明涉及一种截短的人乳头瘤病毒 6型 L1蛋白，及由其组 成的类病毒颗粒， 含该类病毒颗粒的疫苗及其在预防尖锐湿疣或
HPV (特别是 HPV6 )感染中的用途。 背景技术
人乳头瘤病毒 HPV (Human Papi l l omavi rus)属乳头多瘤空泡 病毒科（Papovavir idae)乳头瘤病毒属， 为无包膜 DNA病毒。 病毒 基因组为双链闭环 DNA,大小约为 7. 2 ~ 8kb,具有 8个开放框。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 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、 表达调控元件。 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 90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又可分为 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、 52; ③高度癌风险组， 包括
HPV16、 18、 45、 56。
根据流行病学调查肛生殖器粘膜的 HPV例如 HPV6, 11的感染 仅次于衣原体和滴虫病而居于第三位，是一种常见的性传播疾病。 而其中由 HPV6 , 11 引起的病变占了总数的 90 %左右。 在美国， 女性生殖道 HPV感染的高峰在 15 - 25岁，并与感染者的性行为关
氺 系密切。 在我国， 女性 HPV的感染率的高峰期在 20 - 29岁之间， 感染率为 1606. 1八 0万。 35岁以上的妇女 HPV感染率逐渐降低。 但是由于 HPV感染大多是亚临床感染， 感染率难以准确估计， 但 美国 CDC的估计一生中累 i十 HPV感染风险大约 10 %。此外由于采 集大样本男性标本困难，且男性感染 HP V造成后果没有女性严重， 所以关于男性感染的资料较少。 不过据估计， 男性的感染率应当 接近女性。 而在美国， 可见的尖锐湿疣见于 1%的性活动期成年男 性。 因此， 开发安全有效的 HPV6, 11疫苗是预防性传播疾病有效 的有效手段。
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与天然病毒颗粒相似的类病毒颗粒 ( Vi rus-Like Part ic le, VLP ) 。 该种类病毒颗粒为二十面体立 体对称结构， 由 72个 L1蛋白的五聚体組成。 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 Na t l Acad Sc i U S A 89 (24) : 12180-4. ) 并且， 类病毒颗粒并不带有病毒 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 HPV L1蛋白天 然构象破坏少， 能自发的形成 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 HPV疫苗 Gardas i l® 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。
原核表达系统中利用大肠杆菌表达系统表达 HPVL1蛋白已有 报道。例如有报道利用大肠杆菌表达 HPV16 L1蛋白（ Banks, L.， G. Mat lashewski, et al. (1987) . J Gen Virol 68 ( Pt 12) : 3081-9 ) 。 但是由于大肠杆菌所表达的 HPV L1蛋白大多失去其天 然构象， 不能产生针对 HPV的保护抗体。或者上述蛋白虽然通过包 含体纯化， 复性等步骤也可得到 HPV VLP ( Kel sa l l, S. R. and J. K. Kul ski (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， 难以在大规模生产上应用。
HPV L1蛋白虽然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， 但是表达量较低， 而且上清中杂蛋白 种类多且量大， 要从中纯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. Cr ipe, et a l. (1997) . J Vi rol 71 (4) : 2988-95. ) ， 但融和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， 本领域仍然需要具有低成本， 能够诱导产生针对 HPV保 护性抗体的 HPVL1蛋白及由其组成的类病毒颗粒，从而使大规模工 业化生产尖锐湿疣疫苗成为可能。 发明内容
本发明的目的是提供一种新的 HPV6 L1蛋白， 及由其组成的类 病毒颗粒及含该类病毒颗粒的疫苗。
本发明人经研究出人意料地发现， 在大肠杆菌表达系统能得 到可以诱导针对 HPV6的中和抗体的截短 HPV6L1蛋白， 该截短的
HPV6L1蛋白经纯化后得到高产率， 纯度至少 50 %的 HPVL1 蛋白。
氺 钝化后的 HPVL1蛋白经进一步处理得到可诱导针对 HPV6保护性抗 体的类病毒颗粒， 本发明基于以上发明现已完成。
因此，本发明第一方面涉及一种（与野生型 HPV6 L1蛋白相比） N 端截短了 2个、 3个、 4个、 或 5个氨基酸的 HPV6 L1蛋白。 优选该截 短蛋白具有序列 1、 2、 3、 或 4， 优选序列 1。
本发明再一方面涉及编码本发明截短蛋白的多核苷酸以及含 有该多核苷酸的载体。
本发明再一方面涉及包含上述载体的细胞。
本发明还涉及包含上述截短蛋白或多核苷酸或载体或细胞的 组合物。
本发明再一方面涉及一种 HPV6类病毒颗粒， 其中该类病毒颗 粒包含 N端截短了 2个、 3个、 4个、 或 5个氨基酸的 HPV6 L1蛋白例 如具有序列 1、 2、 3、 或 4的 HPV6 L1蛋白， 或者由 N端截短了 2个、 3个、 4个、 或 5个氨基酸的 HPV6 L1蛋白例如具有序列 1、 2、 3、 或 4的 HPV6 L1蛋白组成或形成。
本发明再一方面涉及一种获得 HPV6 L1蛋白的方法， 其包括在 大肠杆菌表达系统中表达截短的 HPV6 L1基因片段， 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 HPV6 L1蛋白的方法包括 a)在大肠杆菌表达系统中表达截短的 HPV6 L1基因片段，— b)将表达了截短 HPV6 L1 蛋白的大肠杆菌在盐浓度 100mM-600mM中破碎， 分离得到上清液，
c)用水或低盐溶液将 b )上清液中盐浓度降至 l OOmM或以下， 最低至 0， 收集沉淀，
d)将 c ) 中沉淀在 150mM - 2500mM盐溶液中重新溶解， 同时 加入还原剂，分离得到溶液，其中含纯度至少 50 %的截短 HPV6 L1 蛋白。 更一般性地， 本发明还涉及一种获得 HPV L1蛋白例如本发明 HPV6 L1蛋白的方法， 其包括
a)在大肠杆菌表达系统中表达编码 HPV L1蛋白的 HPV L1基 因，
b)将表达了 HPV L1蛋白的大肠杆菌在盐浓度 100mM-600inM中 破碎， 分离得到上清液，
c)用水或低盐溶液将 b )上清液中盐浓度降至 l OOmM或以下， 最低至 0， 收集沉淀，
d)将 c )中沉淀在 150mM - 2500mM盐溶液中重新溶解， 同时加 入还原剂， 分离得到溶液， 其中含纯度至少 50 %的 HPV L1蛋白。
本发明还涉及一种预防尖锐湿疣或 HPV感染的疫苗， 其包含本 发明的 HPV6 L1蛋白类病毒颗粒。优选该疫苗还包含至少一种选自 HPV18L1蛋白类病毒颗粒， HPV11L1蛋白类病毒颗粒， HPV16L1蛋白 类病毒颗粒， HPV31L1蛋白类病毒颗粒， HPV33L1蛋白类病毒颗粒， HPV45L1蛋白类病毒颗粒， HPV52L1蛋白类病毒颗粒， HPV58L1蛋白 类病毒颗粒的类病毒颗粒。 该疫苗通常还包含疫苗用赋形剂或载 体。
优选地， 所述疫苗含有： HPV6类病毒颗粒和 HPV11类病毒颗 粒，特别是， 含有具有 SEQ ID NO: 4所示氨基酸序列的蛋白盾或者 由该蛋白质形成的 HPV6 类病毒颗粒， 和含有具有 SEQ ID NO: 7 所示氨基酸序列的蛋白盾或者由该蛋白质形成的 HPV11类病毒颗 粒。 更优选所述疫苗还含有 HPV16类病毒颗粒和 ΗΡΠ8类病毒颗 粒， 特别是含有具有 SEQ ID NO: 8所示氨基酸序列的蛋白质或者 由该蛋白盾形成的 HPV16类病毒颗粒， 和含有具有 SEQ ID NO: 9 所示氨基酸序列的蛋白质或者由该蛋白质形成的 HPV18类病毒颗 粒。
在一个特别优选的实施方案中，所述疫苗包含：含有具有 SEQ ID NO: 4所示氨基酸序列的蛋白质或者由该蛋白质形成的 HPV6类 病毒颗粒， 含有具有 SEQ ID NO: 7所示氨基酸序列的蛋白质或者由 该蛋白质形成的 HPV11类病毒颗粒， 含有具有 SEQ ID NO: 8所示氨 基酸序列的蛋白质或者由该蛋白质形成的 HPV16类病毒颗粒，和含 有具有 SEQ ID NO: 9所示氨基酸序列的蛋白质或者由该蛋白质形成 的 HPV18类病毒颗粒。
本发明进一步涉及本发明 HPV6 L1蛋白或其类病毒颗粒在制备 用于预防尖锐湿疣或 HPV感染疫苗中的用途。
本发明还涉及一种预防尖锐湿疣或 HPV感染的方法， 其包括将 含预防有效量的本发明 HPV6 L1蛋白疫苗给予需预防尖锐湿疣或 HPV感染的人或动物。
本发明还涉及一种获得 HPV6 L1蛋白类病毒颗粒的方法， 其 包括：
e)将纯度至少 50 %的截短 HPV6 L1蛋白进一步通过色谱层 析纯化，
0将 e)步骤中得到的 HPV6 L1蛋白去除还原剂。
本发明还涉及一种制备用于预防尖锐湿疣或 HPV感染疫苗的 方法， 其包括将上述类病毒颗粒与任选的一种或多种选自 HPV11, 16, 18， 31, 33, 45, 52,和 58的 HPV型别的类病毒颗粒及疫 苗用栽体或者赋形剂混合。 本发明中相关术语的说明及解释
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， 其中大肠杆菌 （菌株） 来源于市场上可得到的， 在此举例但不限于: GI698, ER2566, BL21 (DE3)， B834 (DE3) , BLR (DE3)。
根据本发明， 术语 "载体"一词指的是， 可将某编码蛋白的
氺 多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。 载 体可以通过转化， 转导或者转染宿主细胞， 使其携带的遗传物盾 元件在宿主细胞中获得表达。 举例来说， 载体包括： 质粒； 噬菌 体； 柯斯质粒等等。
根据本发明， 术语 "截短的 HPV6 L1蛋白基因片段" 是指在 野生型 HPV6 L1蛋白基因 （ cDNA )的 5' 端或 3' 端去掉编码一个 或者多个氨基酸的核苷酸， 其中野生型 HPV6 L1蛋白基因全长序 列举例但不限于 NCBI 数据库中如下序列： ： AF067042. 1， AF092932. 1 , L41216. 1 , X00203. 1等。
"截短的 HPV6 L1蛋白" 是指在野生型 HPV6 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 HPV6 L1 蛋白的例子有但不限于 NCBI 数据库中 AF067042. 1 , AF092932. 1， L41216. 1， X00203. 1等所编码的全长 L1蛋白。
根据本发明， 术语 "疫苗用赋形剂或载体 "是指选自一种或 多种， 包括但不限于： pH调节剂， 表面活性剂， 佐剂， 离子强度 增强剂。 例如， pH调节剂举例但不限于磷酸盐緩冲液， 表面活性 剂包括阳离子， 阴离子或者非离子型表面活性剂。 举例但不限 于： Tween- 80。 佐剂举例但不限于氢氧化铝， 氟氏完全佐剂。 离子 强度增强剂举例但不限于氯化钠。
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 HPV6L1蛋白优选如下获得： 将 表达有截短 HPV6L1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，分离破碎溶液，得到上清液， 用水或低浓度盐 （通常低于破碎用的盐浓度） 降低所得上清液中 盐浓度至盐浓度 l OOmM 0M， 分离盐浓度低至 l OOMm- 0的上清液
氺 中的沉淀； 将沉淀在含还原剂及盐浓度 150 - 2000mM优选 200mM 以上的溶液中重新溶解，,分离， 得到纯度至少为 50 %， 优选至少 70 % , 更优选至少 80 %的截短 HPV6L1蛋白溶液。
根据本发明， 在本发明获得的截短 HPV6L1蛋白的方法中， 緩冲液是指可在一定范围内维持 pH 值稳定的溶液， 包括但不限 于， Tr i s緩冲液， 磷酸盐緩冲液， HEPES緩沖液， MOPS緩冲液等 等。
根据本发明，所述原核宿主细胞破碎包括但不限于通过匀浆 器破碎、 均质机破碎、 超声波处理、 研磨、 高压挤压、 溶菌酶处 理中的一项或者多项方法来实现；
根据本发明， 在本发明获得的截短 HPV6L1蛋白的方法中， 所用的盐包括但不限于是中性盐， 特别是碱金属盐、 铵盐、 盐酸 盐、硫酸盐，硫酸盐，碳 氢盐，磷酸盐或磷酸氢盐，特别是 NaCl、 KC 1、 NH4CK (NH4) 2S04中的一种或几种。 优选 NaCl。 所用的还原 剂包括但不限于 DTT， 2 -巯基乙醇。 所用量包括但不限于 l OmM-l OOmMo
根据本发明， 本发明的截短 HPV6L1蛋白类病毒颗粒如下获 得： 将上述纯度至少 50 %的截短 HPVL1蛋白溶液通过例如色谱层 析进一步分离，得到纯化的截短 HPV6L1蛋白溶液。去除纯化的截 短 HPV6L1蛋白溶液中的还原剂，得到截短 HPV6L1的类病毒颗粒。 去除还原剂的方式包括但不限于本领域已知技术， 例如， 透析， 超滤或者层析等。
根据本发明， 本发明的截短 HPVL1蛋白优选具有序列 1。
根据本发明，本发明的疫苗可采用患者可接受的形式， 包括 但不限于口服或者注射， 优选注射。
根据本发明， 本发明疫苗优选单位剂型使用， 其中单位剂型 中截短 HPV6L1蛋白类病毒颗粒的量为 5 g - 8(mg， 优选 20μ& -
氺 4 (mg。 有益效果
目前 HPV 类病毒颗粒的制备所釆用的表达系统可以分为真核 表达系统和原核表达系统。
在真核表达系统中所表达的 HPV L1蛋白天然构象破坏少， 能 自发的形成 VLP，往往只 迸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所采用的杆状病毒表达系统及酵 母表达系统均有表达量低， 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。 但在大肠杆菌表达系统中表达的 HPV L1蛋白往往 失去正确天然构象， 以包含体形式表达于沉淀中。 目前对表达于 包含体中蛋白进行复性依然是一个世界性难题。 复性困难， 效率 低下，使得从包含体中获得有正确构象的 VLP在大规模生产中难以 实施， 只能局限于小规模的实验室研究中。 虽然 HPV L1也可以正 确构象可溶性形式表达于大肠杆菌裂解上清中，但是表达量低下， 而且要从大肠杆菌裂解上清中种类繁多的可溶性蛋白纯化出 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进 行纯化， 以上手段往往需要昂贵的酶， 难以进行工业化生产。
本发明在大肠杆菌表达系统中表达 N端截短 HPV6 L1蛋白， 并 且采用温和的手段选择性沉淀表达在大肠杆菌裂解上清中的 HPV6 L1蛋白， 进一步釆用含盐緩冲液重溶 HPV6 L1蛋白， 使得在保持 HPV6 L1蛋白正确构象前提下使其纯度有了显著提高且重溶后的 目的蛋白可以直接进行离子交换层析及疏水交换层析纯化获得纯 蛋白。通过以上步骤获得纯化后的截短 HPV6 L1截短蛋白可以组装 为类病毒颗粒，具有良好的免疫原性，可以诱导高滴度的针对 HP V 6
氺 的中和抗体， 预防 HPV6 对人体的感染， 是一种良好的疫苗形式。 此外， 本发明中采用的截短 HPV6 L1蛋白在保留全长 HPV6 L1蛋白 的抗原性及颗粒组装能力的同时， 易于在大肠杆菌表达系统中表 达， 所采用的纯化方法无需使用昂贵的酶， 成本低廉。 而且目的 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， 可应用于大规模工业化生产。
在参考下列详述和附图后，本发明的这些和其它方面将是显 然的。 此处公开的所有参考文献在此均完整引用作为参考。 附图说明
图 1显示了在本发明方法步骤 a) -d)中 HPV6N3C-L1蛋白的 SDS聚丙烯酰胺凝胶电泳结果。 1 , 裂解上清； 2， 经过切向流沉淀 的 HPV6N3C- L1 ; 3 , 经重悬液重悬的 HPV6N3C-L1。 结果显示， HPV6N3C-L1蛋白在通过沉淀，复溶的步骤之后， 纯度达到了约 70 %左右。
图 2 显示了步骤 d)中所得的 HPV6N3C-L1进一步经本发明步 驟 e )纯化后的 SDS聚丙烯酰胺凝胶电泳结果。 1 , 经本发明步骤 e )纯化所得 HPV6N3C-L1 上样 Ι Ο μ Ι ; 2， 经本发明步骤 e )纯 化所得 HPV6N3C-L1上样 20 μ 1:。 结杲显示， 经过步骤 e ) 纯化的 HPV6N3C-L1蛋白纯度达到了 98 %左右。
图 3显示了步骤 f )中所得的 HPV6N3C-L1类病毒颗粒透射电 镜观察（50， 000倍） 结果。 视野中可见大量半径为 25nm左右的 类病毒颗粒， 颗粒大小与理论大小相符， 均匀一致。
图 4显示了步骤 f )中所得的 HPV6N3C-L1类病毒颗粒的动态 光散射观测结果。 结果显示， HPV6N3C-L1类病毒颗粒的水化分子 动力学半径为 24. 70nm， 颗粒组装百分比为 100%。
图 5显示了 HPV6N3C-L1类病毒颗粒接种羊后不同阶段血清
氺 中和抗体滴度。 图中箭头所示为免疫时间。 在初免一周后， 中和 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 107 - 108的较高水平。
图 6显示了 HPV6N3C-L1类病毒颗粒接种兔后不同阶段血清 中和抗体滴度。 图中箭头所示为免疫时间。 在初免一周后， 中和 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 106的较高水平。
图 7显示了实施例 5中 HPV6/11二价疫苗接种小鼠后不同时 间的血清中 HPV6， HPV11中和抗体滴度变化情况。免疫程序为 0， 2W (周）。第一次免疫后， HPV6，HPV11中和抗体滴度即有明显上升， 在经过一次加强免疫后， 抗体的滴度即能达到 1 X 104-1 X 105。
图 8显示了实施例 中 HPV6/11/16/18四价疫苗接种小鼠后 不同时间的血清中 HPV6，HPV11， HPV16,HPV18中和抗体滴度变化 情况。 免疫程序为 0， 2W ( 周 ） 。 第 一次免疫后 ， HPV6,HPV11 ,HPV16,HPV18 中和抗体滴度即有明显上升， 在经过 一次加强免疫后， 抗体的滴度即能达到 1 X 105-1 106。
图 9 显示了在本发明方法步骤 a)-e)中 N端分别截短了 2个、 4个、 或 5个氨基酸的 HPV6 L1蛋白 HPV6N2C-L1、 HPV6N4C-L HPV6N5C-L1 (其氨基酸序列分别见 SEQ ID NO: 2、 3、 4) 的 SDS 聚丙烯酰胺凝胶电泳结果。 1， 分子量 Marker; 2, 经本发明步骤 a)-e)纯化所得 HPV6N2C-L1 上样 ΙΟμ Ι; 3，经本发明步骤 a) -e ) 纯化所得 HPV6N4C-L1 上样 ΙΟμΙ; 4， 经本发明步骤 a) -e )纯化 所得 HPV6N5C-L1 上样 10 μ 1; 结果显示， Ν端截短了 2个、 4 个、 或 5 个氨基酸的 HPV6 L1蛋白 HPV6N2C-L1、 HPV6N4C-LK HPV6N5C-L1经过步骤 a) -e)处理，蛋白纯度均达到了 98%左右。
图 10显示了经过步骤 a) ~f ) 所得的 N端分别截短了 2个、 4个、 或 5个氨基酸的 HPV6 L1蛋白 HPV6N2C-L1、 HPV6N4C-L ^ ^6^0；-1^类病毒颗粒透射电镜观察（50， 000倍） 结果。 1， 经 过步骤 a ) -f ) 所得的 HPV6N2C-L1 类病毒颗粒透射电镜观察
( 50， 000倍） 结果； 2， 经过步骤 a ) -f ) 所得的 HPV6N4C-L1类 病毒颗粒透射电镜观察（ 50， 000倍） 结果； 3经过步骤 a ) -f ) 所得的 HPV6N5C-U类病毒颗粒透 ^"电镜观察（ 50， 000倍）结果； 结果显示，视野中可见大量半径为 25nm左右的类病毒颗粒，颗粒 大小与理论大小相符， 均匀一致。
图 11显示了经过步骤 a ) -f ) 所得的 N端分别截短了 2个、
4个、 或 5个氨基酸的 HPV6 L1蛋白 HPV6N2C-L1 , HPV6N4C- Ll、
HPV6N5C- L1类病毒颗粒的动态光散射观测结果。 1， 经过步骤 a ) -f ) 所得的 HPV6N2C-L1类病毒颗粒动态光散射观测结果； 2， 经 过步骤 a ) -f )所得的 HPV6N4C-L1类病毒颗粒动态光散射观测结 果； 3， 经过步骤 a ) -f ) 所得的 HPV6N5C-L1类病毒颗粒动态光 散射观测结果； 结果显示， HPV6N2C- Ll、 HPV6N4C- Ll、 HPV6N5C-L1 类病毒颗粒的水化分子动力学半径 25nm左右，颗粒组装百分比为 画。
序列 1 (SEQ ID NO: 1)：
1 MPSDSTVYVP PPNPVSKVVA TDAYVTRTNI FYHASSSRLL AVGHPYFSIK RANKTVVPKV
61 SGYQYRVFKV VLPDPN FAL PDSSLFDPTT QRLVWACTGL EVGRGQPLGV GVSGHPFLN
121 YDDVENSGSG GNPGQDN VN VGMDY QTQL CMVGCAPPLG EHWG GKQCT NTPVQAGDCP
181 PLELITSVIQ DGDMVDTGFG AMNFADLQTN KSDVPIDICG TTC YPDYLQ MAADPYGDRL
241 FFFLRKEQMF ARHFFNRAGE VGEPVPDTLI I GSGNRTSV GSSIYVNTPS GSLVSSEAQL
301 FNKPYWLQKA QGHNNGICWG NQLFVTVVDT TRSTNMTLCA SVTTSSTYTN SDYKEYMRHV
361 EEYDLQFIFQ LCSITLSAEV VAYIHTMNPS VLEDWNFGLS PPPNGTLEDT YRYVQSQAIT
421 CQKPTPEKQ PDPY NLSFW EVNLKE FSS ELDQYPLGRK FLLQSGYRGR SSIRTGVKRP
481 AVS ASAAPK RKRAKT R
序列 2 (SEQ ID NO: 2)：
1 MRPSDSTVYV PPPNPVSKVV ATDAYVTRTN IFYHASSSRL LAVGHPYFSI KRANKTVVPK
61 VSGYQYRVFK VVLPDPNKFA LPDSSLFDPT TQRLVWACTG LEVGRGQPLG VGVSGHPFLN
121 KYDDVENSGS GGNPGQDNRV NVGMDYKQTQ LCMVGCAPPL GEHWGKGKQC TNTPVQAGDC
181 PPLELITSVI QDGDMVDTGF GAMNFADLQT NKSDVPIDIC GTTC YPDYL QMAADPYGDR
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氺 ■ε ι-
0000VDV0J.0 YDOL IVI O VV9VIJLV3VI OOXVVVDDOO OIOOQ01Y11 I0Z I
000IU3VV0 013V0VV00I OiLO ODD IVVOIVVDVD VD11V1VI33 OOIVVIOVVO I V U
100IDIOIIV DV1IVOOV10 1V1IVV311I IIVUIVVDV IUVOIVIOV 0VV00101V3 T 80 I 丄 Mt WV丄;) VOVVVIVIIV 01DIIVVD3V OVlVOVDDll OIVOV1DVV1 0D01V3D101 UO I
VllVOVOlVO VVDDVIOVOO DVDV03V1V0 VIOOIOIDVI 1011IOIDVV DIVVIODOOI T 96 tL 9丄 JL iL y丄 9 丄 VVOVVIVOVO ODVDDOOYYY raim工 VIVD39VVIV VXIXOllVVD 106
VOOOVOIDIO 0 130000VV0D ODVDVVllOI VIVIVIOVIO VOOOVIOIOI
oDvvoaivvv OOIOVJ.OOOV VI1VV1VI1D 1DVIV0103D 10133VV000 OOIOOVODOO I 8 i
I3000V0VVI IIlIIiVDVO V3D0III01V VVDVVOOVVO 丄丄 LimV V UL
IVOIOOIVIV 3DDV0V3010 OOlVVVDVll IVIIVOVODI VIVVVI01V3 VI3V3D010I 199
V1VOVOIIV1 DOIIOIVOVD IVVVIVVDOV OVOOUIVDI DOUIIVVOI VIDOIOOIU 109
DOOVDVDVOl I00XV1V030 01V90VDVIV I10IOVDDV1 IV1IDVVOVI 1D030DDD0I
3V0100I390 V3VI0133V0 VIVVIDVIOI OVDVVVIDOV VVIOOOOIIV D0V030001I
10D30D3D0I 01V00II901 V301V1IVV0 vovvvovvv丄 VIIVOOIVIO OXIOXVVIIO
OOVOVV1VOO V0V00ID33V VIOOIOOIOV OOOVDUVVV VOUOIVOIV OIVIVVVIVV
VDVDOIDVV1 OIOOOIOIOO V11V000V3V OOOOVDOOOI 00V0V13D00 τοε
V3V00IVD90 OIVIOOllIO 3VVDV0VV3V 丄 vom丄 IDIDIODIOV 01D30I1V30 \ n mvvvDvvi DOIVOVDOV1 10I00100VV 111VIOOOVD VIVVOIV1VO OVDIOIOOVV Ϊ8Ι
V330I0II0X 3VVVV0VVI3 oooovvvvxv 33丄丄丄丄丄 v 丄 OOLVdlQOQl 0V30UDU3 I I I
V0VI3II0V0 0V300IVD1V IIIIVIVDVV 0DV303J.0V1 丄 ί)丄 003V0301IO T 9
IIOVVVODIV 1010DOVVIO D13DI30010 IVIV10V3V0 OVOVOOOVIO 300300I01V ΐ
: (S -OR GI DHS) 5
XHXdVVSVXS m
AVd¾3A01¾I SS¾0¾AOS01
¾AIGHlIONd !ASdN IH IA VAAHVSll I S
3ΗΛΗΜΑΗ¾Α aSfilAISSll ASVOII IS τοε
SOSdlNAAI S SOASIMOSO i n
VWOlAOdAIO 丄丄 Μΐα Λα SXNlOiaVilN OGillAS丄 ΙΊ3 181 iddoaovt)Ad 1ΝΙ0ί)3030Α HHOlddVDOA 。ΛΜ観 OOd NOOSOSNHAO I I I aA3NlddH0S AOA01d50¾0 AHIOIOVAAT ¾i)iidainss adiv^Ndad 19
OSAidAAIXN V ISdAdHO AVllHSSSVH AdINIHIAAV QlVAA3SAdN ddJAAAISQW I
: (fr m ai ims
¾3dVVSV¾SA
VdHXAOIHI S SHOMAOSbn iiiHOldAOai dsi ad丄 <πί)
DlIVi)S0AA¾ AiaHllDNdd dSiD疆 aai ASdNWIHIAV AAHVSllI SO lOiildOiaAH m
HAHHWAHlAa SNIAISSIIA SV01IWNIS¾ • dAA丄 ΛίΠί) NOADIONNHO ίΐπίΠΑ人 (JM το ε ili)VHSSAlS OSdINAAI SS 0AS1¾NDS03 i niadAdao nz iiiHaoAdavv xooiaidAas M丄 Oiay扁 ddlAS丄 ΙΊ3Ί IS l
NI0D¾030AH HOlddVOOAW ANA¾liai)9dli oososNHAaa I I I
OAOlclOOiOA ΛΛ3ίΛ¾Α()Α0 19
SA3dAAI¾MV HI SHAdHOA VTOSSSVHA IVAA3SAdNd ddAAAlSQSW I
: (ε m ai HS) £
H¾I3VH3¾3 dVVSV3SAVd
OADIEISSi OHAOSCmdJI ¾01dA0aiflS SiOa™應 HSlNMdad¾ 03Hdld3i)DI m
IVOSOAAiAl aailONdddS lOdMOHlAS dNWiHIAVAA HVSllI SDli) dldOlGAaaA \ 9£
HHWAaSAOSfl 1AXSS1IASV OllWfilSHIJ, GKAUdlMO ΛΟΙΟΝΜΗθδν XiHMdMiH I0£
OvaSSAlSOS dXNAAI SSOA SXM0S03I I liadAdHOAS OVMildHUVil Wims HttiH I H
OSOlOO/800ZN3/X3d 1261 ATTACCTGTC AAAAGCCCAC TCCTGAAAAG CAAAAGCCAG ATCCCTATAA GAACCTTAGT
1321 TTTTGGGAGG TTAATTTAAA AGAAAAGTTT TCTAGTGAAT TGGATCAGTA TCCTTTGGGA
1381 CGCAAGTTTT TGTTACAAAG TGGATATAGG GGACGGTCCT CTATTCGTAC CGGTGTTAAG
1441 CGCCCTGCTG TTTCCAAAGC CTCTCCTGCC CCTAAACGTA AGCGCGCCAA AACTAAAAGG
1501 TAA 序列 6 (SEQ ID NO: 6) :
1 ATGCCTAGCG ACAGCACAGT ATATGTGCCT CCTCCTAACC CTGTATCCAA
51 AGTTGTTGCC ACGGATGCTT ATGTTACTCG CACCAACATA TTTTATCATG
101 CCAGCAGTTC TAGACTTCTT GCAGTGGGTC ATCCTTATTT TTCCATAAAA
151 CGGGCTAACA AAACTGTTGT GCCAAAGGTG TCAGGATATC AATACAGGGT
201 ATTTAAGGTG GTGTTACCAG ATCCTAACAA ATTTGCATTG CCTGACTCGT
251 CTCTTTTTGA TCCCACAACA CAACGTTTGG TATGGGCATG CACAGGCCTA
301 GAGGTGGGCA GGGGACAGCC ATTAGGTGTG GGTGTAAGTG GACATCCTTT
351 CCTAAATAAA TATGATGATG TTGAAAATTC AGGGAGTGGT GGTAACCCTG
401 GACAGGATAA CAGGGTTAAT GTTGGTATGG ATTATAAACA AACACAATTA
451 TGCATGGTTG GATGTGCCCC CCCTTTGGGC GAGCATTGGG GTAAAGGTAA
501 ACAGTGTACT AATACACCTG TACAGGCTGG TGACTGCCCG CCCTTAGAAC
551 TTATTACCAG TGTTATACAG GATGGCGATA TGGTTGACAC AGGCTTTGGT
601 GCTATGAATT TTGCTGATTT GCAGACCAAT AAATCAGATG TTCCTATTGA
651 TATATGTGGC ACTACATGTA AATATCCAGA TTATTTACAA ATGGCTGCAG
701 ACCCTTATGG TGATAGATTA TTTTTTTTTC TACGGAAGGA ACAAATGTTT
751 GCCAGACATT TTTTTAACAG GGCTGGCGAG GTGGGGGAAC CTGTGCCTGA
801 TACTCTTATA ATTAAGGGTA GTGGAAATCG AACGTCTCTA CCGAGTAGTA
851 TATATGTTAA CACCCCAAGC GGCTCTTTGG TGTCCTCTGA GGCACAATTG
901 TTTAATAAGC CATATTGGCT ACAAAAAGCC CAGGGACATA ACAATGGTAT
951 TTGTTGGGGT AATCAACTGT TTGTTACTGT GGTAGATACC ACACGCAGTA
1001 CCAACATGAC ATTATCTGCA TCCGTAACTA CATCTTCCAC ATACACCAAT
1051 TCTCATTATA AAGAGTACAT GCGTCATGTG GAAGAGTATG ATTTACAATT
1101 TATTTTTCAA TTATGTAGCA TTACATTGTC TGCTGAAGTA GTGGCCTATA
1151 TTCACACAAT GAATCCCTCT GTTTTGGAAG ACTGGAACTT TGGGTTATCG
1201 CCTCCCCCAA ATGGTACATT AGAAGATACC TATAGGTATG TGCAGTCACA
1251 GGCCATTACC TGTCAAAAGC CCACTCCTGA AAAGCAAAAG CCAGATCCCT
1301 ATAAGAACCT TAGTTTTTGG GAGGTTAATT TAAAAGAAAA GTTTTCTAGT
1351 GAATTGGATC AGTATCCTTT GGGACGCAAG TTTTTGTTAC AAAGTGGATA
1401 TAGGGGACGG TCCTCTATTC GTACCGGTGT TAAGCGCCCT GCTGTTTCCA
1451 AAGCCTCTGC TGCCCCTAAA CGTAAGCGCG CCAAAACTAA AAGGTAA
下面结合实施例对本发明进一步举例描述。 这些实施例是非 限制性的。
实施例 1 : 具有序列 1的截短 HPV6 L1蛋白的表达
用做模板之 HPV6 L1基因片段的制备
HPV-6 L1基因全长由上海博亚公司合成。 所合成的基因片段 全长为 1503bp， 其序列为序列 5。 在此人工合成的 HPV-6L1基因 全长片段的基础上制备本发明截短 HPV6 L1蛋白之模板。
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氺 截短 HPV6 LI基因的非融合表达载体的构建
以前一步骤所合成的 HPV6L1全长基因片段用做再次 PCR反应 的模板。 以 6N3F: 5' - CAT ATg GCT AGC GAC AGC ACA GTA TA - 3， (SEQ ID NO: 10)为正向引物， 其 5， 端引入限制性内切酶 Ndel位 点， Ndel位点序列为 CAT ATG, ATG为大肠杆菌系统中的起始密 码子； 6CR: 5^ -GTC GAC TTA CCT TTT AGT TTT GGC GC -3' (SEQ ID NO: 11)为反向引物， 其 5， 端引入限制型内切酶 Sa i l位点。
在 PCR热循环仪（Biometra T3 )按照如下条件进行 PCR反应：
Figure imgf000016_0001
扩增得到 1. 5kb左右大小特异的 DNA片段。 将该 PCR产物与 商售的 pMD 18-T载体（TAKARA公司生产）连接， 经 Ndel/Sa l l 酶切鉴定， 得到插入截短 HPV6L1 基因的阳性克隆 pMD 18-T-HPV6N3C-L1.
在上海博亚生物工程公司， 利用 M13 (+) I (-)引物， 测得 pMD 18-T-HPV6N3C-L1质粒中插入的目的核苷酸序列为序列 6，其编码 的氨基酸序列即为序列 1 :该序列对应的蛋白质为为 N端被截短 3 个氨基酸、 C端未被截短的 HPV6 L1蛋白， 命名为 HPV6N3C-L
将上述的 pMD 18- T-HPV6N3C-L1质粒， 经 Ndel/Sa l l酶切， 获得截短的 HPV6N3C-L1 基因片段。 再将该片段与经 Ndel/Sal l 酶切的 pTrxFus原核表达载体（购自 Invi trogen公司）相连接， 由于融合蛋白已被切除，在起始氨基酸 Met之后紧接着目的蛋白， 不带其他融合多肽。 Ndel/Sa l l酶切鉴定得到插入 L1蛋白基因片
- 15- 氺 段的阳性表达克隆 pTRX - HPV- 6N3C-L1。 取 Ι μ Ι^ 的 pTRX - HPV-6N3C-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 GI698 ( (购自 Invi trogen公司），涂布于氨苄（终 浓度 100 mg/ml， 下同）抗性的固体 CAA培养基（6g Na2HP04, 3g KH2P04, 0. 5g NaCl , lg NH4C1， 20g酪蛋白水解物， 0. 095g MgCl2, 1. 5g琼脂粉溶解于 900ml去离子水中， 加入 50 %甘油 20ml)， 30 静置培养 10-12小时至单菌落清晰可辨。 挑取单菌落至含 4mL 氨苄抗性的液体 IMC培养基之试管， 25 Γ 220转 /分振荡培养 10 小时， 从中取 lmL菌液于 冻干保存。
HPV6N3C-L1的大量表达
从 - 70TC中取出带有重组质粒 pTRX - HPV6N3C-L1 的大肠杆 菌冻干菌种， 接入氨苄抗性的 50ml IMC液体培养基， 200rpm, 30 n, 培养大约 8小时， 然后转接入 10瓶 500ml培养基中， 每瓶接 入 5ml菌液， 200rpm， 30Π，摇瓶培养过夜。
主要仪器， 上海保兴生物公司 50升发酵罐
校正发酵罐 ρΗ电极， 配制 30升培养基装入发酵罐， 121 灭 菌 30分钟， 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 lOOrpm时为 100%。
补料准备，酪蛋白水解物配制浓度为 30% ( 30g溶至 100ml ) , 葡萄糖 50% (50g溶至 l OOffll) , 121 Ό灭菌 20分钟。
第二天将 10瓶种子液 5升接入发酵罐中， 温度 30 C， ΡΗ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%葡萄糖和 30%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：
100%为 25ml/min
第一'〗、时： 5%; 第二小时： 10%;
第三小时： 20%;
第四小时： 40%;
第五小时以后 60%
培养至菌浓度达到 OD600大约 10左右时将培养温度降至 25 X：加入 4 g色氨酸诱导培养 4小时。终浓度大约为 40左右（ OD600 ) 下罐， 离心收集菌体大约 2. 5kg
IMC培养基配方如下（1升）：
Figure imgf000018_0001
实施例 2: 纯度约 70 %的 HPV6N3C-L1的获得
按 lg菌体对应 10mL裂解液（20mM Tr i s緩冲液 pH7. 2， 300mM NaCl)的比例重悬菌体， 釆用 APV均质机（An Invensys Group 产 品）以 600bar压力破碎菌体 5次。 JA - 14转头 13500rpm (30000g)， 离心 15min，留取上清， 通过 10 % SDS-聚丙烯酰胺凝胶电泳检测， 此时上清中 HPV6N3C- L1的纯度约为 10%。 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 l OmM磷酸緩冲液 pH 6. 0緩冲液， 透析体积为三 倍上清体积，运行时压力为 0. 5ps i， 流速为 500mL/niin，切向流速 为 200mL/min。 透析充分后， ： ΓΑ - 10转头 ( Beckman J25 高速离 心机） 9500rpm (12000g) , 20min离心收获沉淀, 用 1/10上清体
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氺 积的 10mM磷酸緩冲液 pH7.0, 10mM DTT, 300mM NaCl重悬沉淀， 搅拌 30min。 JA-14 转头 （ Beckman J25 高速离心机） ， 13500rpm(30000g)，离心 20min,离心得上清， 使用 0·22μπι孔径 滤膜过滤样品， 以该样品进行下一步的阳离子交换色讲纯化。 取 150 过滤后样品， 加入 6Χ Loading Buffer 30 μ 混匀， 于 80Ό水浴 lOmin后取 10 μ L于 10 % SDS-聚丙烯酰胺凝胶中以 120V 电压电泳 120min。 随后以考马斯亮兰染色显示电泳条带， 电泳结 果见图 1。 通过 SDS- PAGE分析可知 HPV6N3C-L1蛋白在通过沉淀、 复溶的步骤之后， 目的蛋白得到了纯化和富集， 纯度达到了约 70 %左右。 实施例 3: HPV6N3CTL1的色谱纯化
HPV6N3C-L1的阳离子交换色谱纯化
仪器系统： GE Healthcare 原 Amershan Pharmacia公司生产 的 AKTA explorer 100型制备型液相色谱系统。
层析介质： SP Sepharose 4 Fast Flow。
柱体积： 5.5cmx20cm。
緩冲液： 20mM磷酸緩冲液 pH7.0， lOmM DTT
20mM磷酸緩冲液 pH7.0 10mM DTT 2M NaCl„
流速 ·· 25 mL/min
检测器波长： 280nm
样品为 3L纯度约为 70%HPV6N3C-L1溶液
洗脱程序为： 200mM NaCl洗脱杂蛋白， 500mM NaCl洗脱目的 蛋白， 收集 500mM NaCl洗脱产物， 共获得 HPV6N3C-L1纯化样品 900mL.
HPV6N3C-L1的 CHT- II (羟基磷灰石色谱） 纯化
仪器系统： GE Healthcare 原 Amershan Pharmacia公司生产
氺 的 AKTA explorer 100型制备型液相色讲系统。
层析介质： CHT-IlO^ Bio-RAD)
柱体积： 5.5cm X 20cm
緩冲液： lOmM磷酸緩冲液 ρΗ7· 0， 10mM DTT, 0.5M NaCl。
流速： 20mL/min。
检测器波长： 280nm。
样品为： SP Sepharose 4 Fast Flow 500mM NaCl洗脱产物 洗脱程序为： 直接收取含目的蛋白的穿透
收集穿透产物， 获得纯化的 HPV6N3C-L1样品 1000mL。 取经 本实施例方法纯化的 HPV6N3C- L1样品 150 μ L, 加入 6Χ Loading Buffer 30μΙ混勾， 于 80 水浴 lOmin后取 ΙΟμΙ于 10%SDS-聚 丙烯酰胺凝胶中以 120V电压电泳 120min。 随后以考马斯亮兰染 色显示电泳条带， 电泳结果见图 2。 由电泳结果可知， 目的蛋白 浓度约为 0.7mg/ml， SDS-PAGE考染纯度大于 98%。 实施例 4: HPV6N3C-L1 类病毒颗粒的组装
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 3所得 HPV6N3C-L1 1000ml。
样品的浓缩： 调节系统切向流速为 50mL/min， 浓缩样品至总 体积为 800mL„
样品的复性：以 10L复性緩冲液（ 20mM PB pH 6.0, 2mM CaC12， 2mMMgCl2, 0.5M NaCl, 0.003% Tween-80 ) 充分交换样品緩冲液。 切向流装置运行时压力为 0.5psi，切向流速度为 10mL/min，待复 性緩冲液交换完后， 改为储存緩冲液（ 20L PBS: 20mM PB pH 6.5， 0.5M NaCl )进行交换， 交换体积为 20L。 运行时压力为 0.5psi， 切向流速为 25mL/min。 待所有液体交换完毕后， 使用 PALL 0.20 μαι 滤器无菌过滤样品， 即得 HPV6N3C- L1 类病毒颗粒， 将其置
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氺 于 4 保存备用 实施例 5: HPV6N3C-L1 VLP的形态学检测及其免疫原性测定
HPV6N3C-L1 类病毒颗粒透射电镜观察
仪器为日本电子公司生产的 l OOkV 透射电镜， 放大倍数为
100, 000倍。 HPV6N3C-L1 类病毒颗粒经 2%磷钨酸 pH7. 0 负染， 固定于喷炭的铜网上进行观察。 结果如图 3， 可见实施例 4所得 样品大量半径为 25nm左右的类病毒颗粒， 大小均匀，呈现为空心 形态。
HPV6N3C-L1类病毒颗粒动态光散射观察
仪器为美国 Prote in Solut ions 公司生产的 DynaPro MS/X 型动态光散射仪（含温度控制器），使用算法为 Regulat ion算法。 样品为实施例 4所得样品。样品经 0. 22 μ m滤膜过滤后进行测量。 测量结果见图 4。 结果显示 HPV6N3C-L1 VLP的水化分子动力学半 径为 25. 46nm。
HPV6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，需要建立有效体外中和实验 模型。
假病毒（ pseudovir ions )体外感染模型：利用了 HPV VLP可非 特异包装核酸的特性，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)具体方法有重组病毒表达系统法及多质粒共转染
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氺 方法。
本发明釆用的多质粒共转染方法，并且针对 HPV系统采取了如 下的改进： 建立了对 293FT细胞的优化的磷酸钙转染方法，可获得 高达 90%以上的转染效率， 有利于进行较大规模的生产。 获得了 密码子优化的 HPV结构蛋白的表达质粒，其可在哺乳动物细胞中高 效表达 HPV L1和 L2基因， 有利于高效组装假病毒。
HPV假病毒的构建
用 CsCl密度梯度离心方法分别纯化带有 HPV6L1基因的质粒 p6Llh、 带有 HPV6L2基因的质粒 p6L2h、 及带有绿色荧光蛋白基 因的质粒 PN31-EGFP (以上质粒由 NIH的 John T. Schiller教授 馈赠） 。 CsCl密度梯度离心纯化质粒的方法参照《分子克隆： 第 三版》 。 简言之： 将质粒转化大肠杆菌 DH5ot， 挑取单菌落接种 到 500mLLB培养基中， 37 摇瓶培养 16小时。 9000g离心 5min， 收集菌体。每 lOOOmL菌液收获的菌体，依次加入 40mL溶液 I( 50mM 葡萄糖， 25mMTris-Cl (ρΗ8· 0)， 10mM EDTA (pH=8.0) )和 2mL RNase A (1μβ/μΙ) ， 40mL 溶液 II ( 0.2M NaOH, 1½SDS ) ， 48mL 溶 液 III ( 5M乙酸钟 60.0mL, 冰乙酸 11.5mL， 去离子水 28.5mL)。 在冰上放置 lOmin后， 15000g， 4"C离心 20min， 取上清与 0.6 倍体积的异丙醇混合， 15000g离心 30min， 弃去上清， 用 70%乙 醇洗沉淀 1次， 用 TE溶解沉淀， 测定 DNA含量。 在 DNA溶液中溶 入 CsCl (每克 DNA对应 l.Olg CsCl) ， 再溶入 lOOuL lOmg/mL 的溴化乙锭溶液， 用 Bepkman NVT65转子， 62000rpm， 20Ό离心
10hr。 用注射器针头收集闭环 DNA区带， 用等体积的异戊醇重复 抽提 4次。 加入 3倍体积的水和 8倍体积的无水乙醇， 20000g， 4 Ό离心 30min， 收集 DNA沉淀。 75%酒精洗涤 1次， 用 lmL TE溶 解 DNA沉淀。 测定 DNA溶液的浓度， 分装成小份储存于 -20 。
纯化后的 p6Llh、 p6L2h、 pN31-EGFP用磷酸钙方法共转染培
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氺 育于 10cm细胞培养孤中的 293FT细胞（ Invitrogen)。磷酸钙转 染方法： 将 p6Llh、 p6L2h、 pN31- EGFP各 40ug加入 ImL的 HEPES 溶液（每 50mL去离子水含有 pH = 7.3的 1M Hepes 125uL, 储 存）和 ImL的 0.5mol/L CaCl2溶液的混合溶液， 混合后逐滴加入 2mL 2 X HeBS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, -70TC储存） 中， 室温下静置 lmin后将混合液加入培养有 293FT 细胞的 10cm细胞培养亚中， 6hr后弃去原培养液， 加入 10mL完 全培养液（ invitrogen公司产品）。 转染 48hr后， 弃去培养基， 用 PBS洗 2次， 将细胞刮下收集细胞， 细胞计数， 每 108个细胞 用 ImL裂解液（0.25%Brij58， 9.5mM MgCl2 )重悬。 裂解完成后， 5000g离心 lOmin, 收集上清， 加入 5MNaCl (终浓度为 850mM) ， 即为假病毒液， 分装为小份后置于 保存。
将 293FT细胞（ Invitrogen)铺于 96孔细胞培养板中 （ 1.5 χΐθ4/孔） 。 5hr后进行中和实验， 将待测的血清样品分别用 10 %DMEM进行连续倍比稀释，然后取 50μί分别与 50 L稀释于 10 % DMEM的上文制备的假病毒液（moi=0.1 )混合。 4 孵育 lh后 分别加入预铺有 293FT细胞的 96孔细胞培养板中， 371C培养 72h 后先用荧光观察确定各样品大概的中和滴度， 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， 计算单抗或多抗血清的准确中和滴度。 感染率为细胞样品在 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 的数量百分率。
感染抑制率- (1- 阻断扎的感染率 /未阻断孔的感染率） X 画。
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倍稀释后能达到 50%以上感染抑制率的单抗或多 抗被视为具有中和能力。
HPV6 VLP疫苗免疫动物的免疫保护性评价
兔： 普通级， 雌性， 6 ~ 8 周龄， 购自广西省疾病预防与控制 中心， 并在该中心饲养。 实施例 4所制备的 HPV6N3C-L1 类病毒 颗粒初免与等量福氏完全佐剂混合， 加强免疫则与等量福氏不完 全佐剂混合进行制备， 免疫方式为肌肉注射， 初次免疫剂量为 l OOug/只，此后分别于 4，10周各加强一次，加强免疫剂量为 50ug/ 只。 自免疫后， 每周抽取外周静脉血， 分离血清， 保存待检。
羊： 普通级， 雌性， 6 ~ 8 周龄， 购自广西省疾病预防与控制 中心， 并在该中心饲养。 实施例 4所制备的 HPV6N3C-L1 类病毒 颗粒初免与等量福氏完全佐剂混合， 加强免疫则与等量福氏不完 全佐剂混合进行制备， 免疫方式为肌肉注射， 初次免疫剂量为 lmg/只， 此后分别于 4， 10, 18周各加强一次， 加强免疫剂量为
0. 5mg/只。 自免疫后， 每周抽取外周静脉血， 分离血清， 保存待 检。
以上述假病毒中和细胞模型实验评估上述抗血清的中和效 价，如图 5和 6所示。结果表明，本发明实施例 4获得的 HPV6N3C-L1 类病毒颗粒（除了实验中用到的福氏佐剂外， 也可以是商用或自 制的氢氧化铝或磷酸铝佐剂） 混合配制成为疫苗， 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， 可作为预防 HPV 感染的疫苗。
HPV6/11二价疫苗免疫小鼠的免疫保护性评价
免疫用动物为 4-5周龄 SPF级 BALB/c小鼠 4只。 按本发明 实施例 1 - 4所述类似方法制备 HPV6N5C L1和 HPV11N4C-L1类 病毒颗粒。将上述 2种类病毒颗粒： HPV6N5C-L1和 HPV11N4C-L1
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氺 按 1 : 2 (重量比）的比例混合， 使得混合后的上述两种类病毒 颗粒的浓度为 40， 80 g/ml。 之后， 对于初免， 再加入等体积 的福氏完全佐剂与之混合均勾。 加强免疫则与等体积福氏不完 全佐剂混合进行制备。 免疫方式为肌肉注射， 初次免疫剂量为
HPV6N5C-L1类病毒颗粒 10 μ g/只， HPV11N4C-L1类病毒颗粒 20
g/只。此后分别于 2周加强 次，加强免疫剂量为 HPV6N5C-L1 类病毒颗粒 20 μ 8/只， HPV11N4C-L1类病毒颗粒 40 μ §/只。 自 免疫后， 每周抽取外周静脉血， 分离血清， 按实施例 5 所述方 法分别检测免疫小鼠中针对 HPV6, HPV11假病毒颗粒的中和抗体 滴度。 检测结果如图 7 所示， 结果表明， 由本发明实施例 1-4 所述方法获得的 HPV6N5C-L1, HPV11N4C-L1 两种类病毒颗粒混 合而制备的 HPV6、 HPV11 二价疫苗具有良好的免疫原性， 可在 动物体内诱导高滴度的针对 HPV6，HPV11的中和抗体， 可作为预 防 HPV6/HPV11感染的有效疫苗（除了实施例中所采用的福氏佐 剂外，该疫苗也可由本发明所制备的 HPV16N5C-L1, HPV11N4C-L1 两种类病毒颗粒与商用或自制的氢氧化铝或磷酸铝佐剂混合而 成） 。
上述的 HPV11N4C-Iil 类病毒颗粒对应的氨基酸序列为（SEQ ID NO: 7)：
Met Ser Asp Ser Thr Val Tyr Val Pro Pro Pro Asn Pro Val Ser Lys
1 5 10 15
Val Val Ala Thr Asp Ala Tyr Val Lys Arg Thr Asn lie Phe Tyr His
20 25 30
Ala Ser Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr Tyr Ser lie
35 40 45
Lys Lys Val Asn Lys Thr Val Val Pro Lys Val Ser Gly Tyr Gin Tyr
50 55 60
Arg Val Phe Lys Val Val Leu Pro Asp Pro Asn Lys Phe Ala Leu Pro
65 70 75 80
Asp Ser Ser Leu Phe Asp Pro Thr Thr Gin Arg Leu Val Trp Ala Cys
85 90 95
Thr Gly Leu Glu Val Gly Arg Gly Gin Pro Leu Gly Val Gly Val Ser
100 105 110
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氺 Gly His Pro Leu Leu Asn Lys Tyr Asp Asp Val Glu Asn Ser Gly Gly 115 120 125
Tyr Gly Gly Asn Pro Gly Gin Asp Asn Arg Val Asn Val Gly Met Asp 130 135 140
Tyr Lys Gin Thr Gin Leu Cys Met Val Gly Cys Ala Pro Pro Leu Gly 145 150 155 160
Glu His Trp Gly Lys Gly Thr Gin Cys Ser Asn Thr Ser Val Gin Asn
165 170 175
Gly Asp Cys Pro Pro Leu Glu Leu lie Thr Ser Val lie Gin Asp Gly
180 185 190
Asp Met Val Asp Thr Gly Phe Gly Ala Met Asn Phe Ala Asp Leu Gin
195 200 205
Thr Asn Lys Ser Asp Val Pro Leu Asp lie Cys Gly Thr Val Cys Lys 210 215 220
Tyr Pro Asp Tyr Leu Gin Met Ala Ala Asp Pro Tyr Gly Asp Arg Leu 225 230 235 240
Phe Phe Tyr Leu Arg Lys Glu Gin Met Phe Ala Arg His Phe Phe Asn
245 250 255
Arg Ala Gly Thr Val Gly Glu Pro Val Pro Asp Asp Leu Leu Val Lys
260 265 270
Gly Gly Asn Asn Arg Ser Ser Val Ala Ser Ser lie Tyr Val His Thr 275 280 285
Pro Ser Gly Ser Leu Val Ser Ser Glu Ala Gin Leu Phe Asn Lys Pro 290 295 300
Tyr Trp Leu Gin Lys Ala Gin Gly His Asn Asn Gly lie Cys Trp Gly 305 310 315 320
Asn His Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met
325 330 335
Thr Leu Cys Ala Ser Val Ser Lys Ser Ala Thr Tyr Thr Asn Ser Asp
340 345 350
Tyr Lys Glu Tyr Met Arg His Val Glu Glu Phe Asp Leu Gin Phe lie 355 360 365
Phe Gin Leu Cys Ser lie Thr Leu Ser Ala Glu Val Met Ala Tyr lie 370 375 380
His Thr Met Asn Pro Ser Val Leu Glu Asp Trp Asn Phe Gly Leu Ser 385 390 395 400
Pro Pro Pro Asn Gly Thr Leu Glu Asp Thr Tyr Arg Tyr Val Gin Ser
405 410 415 Gin Ala lie Thr Cys Gin Lys Pro Thr Pro Glu Lys Glu Lys Gin Asp
420 425 430
Pro Tyr Lys Asp Met Ser Phe Trp Glu Val Asn Leu Lys Glu Lys Phe 435 440 445
Ser Ser Glu Leu Asp Gin Phe Pro Leu Gly Arg Lys Phe Leu Leu Gin
450 455 460
Ser Gly Tyr Arg Gly Arg Thr Ser Ala Arg Thr Gly lie Lys Arg Pro
465 470 475 480
Ala Val Ser Lys Pro Ser Thr Ala Pro Lys Arg Lys Arg Thr Lys Thr
485 490 495
Lys Lys
HPV6/11/16/18四价疫苗免疫小鼠的免疫保护性评价
免疫动物： 4-5周龄 SPF级 BALB/c小鼠 4只。 按本发明实施 例 1 - 4 所述类似方法制备 HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1 ,和 HPV18N65C- L1的类病毒颗粒。将上述 4种类病 毒颗 粒 ： HPV6N5C-L1, HPV11N4C-L1 ， HPV16N30C-L1 ， HPV18N65C-L1 按 1： 2： 2： 1 (重量比） 的比例混合， 使得混合 后的上述四种类病毒颗粒的浓度为 40， 80, 80 , 40 g/inl。之后， 对于初免， 再加入等体积的福氏完全佐剂与之混合均 。 加强免 疫则与等体积福氏不完全佐剂混合进行制备。 免疫方式为肌肉注 射， 初次免疫剂量为 HPV6N5C- Ll， HPV18N65C-L1 类病毒颗粒各 10 g/只， HPV11N4C-L1 , HPV16N30C-L1类病毒颗粒各 20 μ g/只。 此后分别于 2 周加强一次， 加强免疫剂量为 HPV6N5C-L1 , HPV18N65C-L1 类病毒颗粒各 20 μ g/只 ， HPV11N4C-L1， HPV16N30C-L1类病毒颗粒各 40 g/只。 自免疫后，每周抽取外周 静脉血， 分离血清， 按实施例 5所述方法分别检测免疫小鼠中针 对 HPV6，HPV11， HPV16，HPV18假病毒颗粒的中和抗体滴度。 检测 结果如图 8所示， 结果表明， 由本发明实施例 1-4所述方法获得 的 HPV6N5C- Ll， HPV11N4C-L1, HPV16N30C-L1, HPV18N65C-L1四 种类病毒颗粒混合而制备的 HPV6、 HPV11、 HPV16、 ΗΡΠ 8四价疫 苗具有良好的免疫原性， 可在动物体内诱导高滴度的针对
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氺 HPV6, HPV11 ， HPV16, HPV18 的 中 和抗体 ， 可作 为 预 防 HPV6/HPV11/HPV16/HPV18感染的有效疫苗（除了实施例中所采用 的福氏佐剂外， 该疫苗也可由本发明所制备的 HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1, HPV18N65C-L1 四种类病毒颗粒与 商用或自制的氢氧化铝或磷酸铝佐剂混合而成） 。
上述的 HPV6N5C-L1 类病毒颗粒的 L1氨基酸序列见 SEQ ID NO: 4。
上述的 HPV16N30C- L1类病毒颗粒的 L1氨基酸序列为（SEQ ID NO: 8)：
Met Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser
1 5 10 15
Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn lie Tyr Tyr
20 25 30
His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe Pro
35 40 45
lie Lys Lys Pro Asn Asn Asn Lys lie Leu Val Pro Lys Val Ser Gly
50 55 60
Leu Gin Tyr Arg Val Phe Arg lie His Leu Pro Asp Pro Asn Lys Phe
65 70 75 80
Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gin Arg Leu Val
85 90 95
Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gin Pro Leu Gly Val
100 105 110
Gly lie Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn
115 120 125
Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys lie
130 135 140
Ser Met Asp Tyr Lys Gin Thr Gin Leu Cys Leu He Gly Cys Lys Pro
145 150 155 160
Pro lie Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn Val Ala
165 170 175
Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu lie Asn Thr Val lie
180 185 190
Gin Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Thr
195 200 205
Thr Leu Gin Ala Asn Lys Ser Glu V&l Pro Leu Asp lie Cys Thr Ser
210 215 220
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氺 lie Cys Lys Tyr Pro Asp Tyr lie Lys Met Val Ser Glu Pro Tyr Gly
225 230 235 240
Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gin Met Phe Val Arg His
245 250 255
Leu Phe Asn Arg Ala Gly Ala Val Gly Asp Asn Val Pro Asp Asp Leu
260 265 270
Tyr lie Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr
275 280 285
Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gin lie Phe
290 295 300
Asn Lys Pro Tyr Trp Leu Gin Arg Ala Gin Gly His Asn Asn Gly lie
305 310 315 320
Cys Trp Gly Asn Gin Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
325 330 335
Thr Asn Met Ser Leu Cys Ala Ala lie Ser Thr Ser Glu Thr Thr Tyr
340 345 350
Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp
355 360 365
Leu Gin Phe lie Phe Gin Leu Cys Lys lie Thr Leu Thr Ala Asp lie
370 375 380
Met Thr Tyr lie His Ser Met Asn Ser Thr He Leu Glu Asp Trp Asn
385 390 395 400
Phe Gly Leu Gin Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg
405 410 415
Phe Val Thr Ser Gin Ala lie Ala Cys Gin Lys His Thr Pro Pro Ala
420 425 430
Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu
435 440 445
Lys Glu Lys Phe Ser Ala Asp Leu Asp Gin Phe Pro Leu Gly Arg Lys
450 455 460
Phe Leu Leu Gin Ala Gly Leu Glu Ala Lys Pro Lys Phe Thr Leu Gly
465 470 475 480
Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys
485 490 495
Arg Lys Lys Arg Lys Leu 500 上述的 HPV18N65C-L1类病毒颗粒的 LI氨基酸序列为（SEQ ID NO: 9)：
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氺 Met Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro Ser Val Ala
1 5 10 15
Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser lie Phe Tyr
20 25 30
His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro Tyr Phe Arg
35 40 45
Val Pro Ala Gly Gly Gly Asn Lys Gin Asp lie Pro Lys Val Ser Ala
50 55 60
Tyr Gin Tyr Arg Val Phe Arg Val Gin Leu Pro Asp Pro Asn Lys Phe
65 70 75 80
Gly Leu Pro Asp Thr Ser lie Tyr Asn Pro Glu Thr Gin Arg Leu Val
85 90 95
Trp Ala Cys Ala Gly Val Glu lie Gly Arg Gly Gin Pro Leu Gly Val
100 105 110
Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu Ser
115 120 125
Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val
130 135 140
Ser Val Asp Tyr Lys Gin Thr Gin Leu Cys lie Leu Gly Cys Ala Pro
145 150 155 160
Ala lie Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro
165 170 175
Leu Ser Gin Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu
180 185 190
Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser
195 200 205
Thr Leu Gin Asp Thr Lys Cys Glu Val Pro Leu Asp lie Cys Gin Ser
210 215 220
lie Cys Lys Tyr Pro Asp Tyr Leu Gin Met Ser Ala Asp Pro Tyr Gly
225 230 235 240
Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gin Leu Phe Ala Arg His
245 250 255
Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro Gin Ser Leu
260 265 270
Tyr lie Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr
275 280 285
Ser Pro Ser Pro Ser Gly Ser lie Val Thr Ser Asp Ser Gin Leu Phe
290 295 300
Asn Lys Pro Tyr Trp Leu His Lys Ala Gin Gly His Asn Asn Gly Val
305 310 315 320
Cys Trp His Asn Gin Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
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氺 Thr Asn Leu Thr lie Cys Ala Ser Thr Gin Ser Pro Val Pro Gly Gin
340 345 350
Tyr Asp Ala Thr Lys Phe Lys Gin Tyr Ser Arg His Val Glu Glu Tyr
355 360 365
Asp Leu Gin Phe lie Phe Gin Leu Cys Thr lie Thr Leu Thr Ala Asp
370 375 380
Val Met Ser Tyr lie His Ser Met Asn Ser Ser lie Leu Glu Asp Trp
385 390 395 400
Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr
405 410 415
Arg Phe Val Gin Ser Val Ala lie Ala Cys Gin Lys Asp Ala Ala Pro
420 425 430
Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp
435 440 445
Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gin Tyr Pro Leu Gly Arg
450 455 460
Lys Phe Leu Val Gin Ala Gly Leu Arg Arg Lys Pro Thr lie Gly Pro
465 470 475 480
Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ala Ser Lys Pro Ala Lys
485 490 495
Arg Val Arg Val Arg Ala Arg Lys
上述的 HPV11N4C-L1 类病毒颗粒对应的氨基酸序列显示如 上 （ SEQ ID NO: 7 ) 。 实施例 6:
依据本发明实施例 1 - 5所采用的技术， 制备具有序列 2， 3， 4 的截短 HPV6L1蛋白， 以上截短蛋白均可纯化得到纯度达到 98 %以上的蛋白，组装为半径 25nm左右的类病毒颗粒。结果见图 9、 图 10和图 11。
氺

Claims

1. N端截短了 2个、 3个、 4个、 或 5个氨基酸的 HPV6 L1 蛋白。
2. 权利要求 1的蛋白， 其具有序列 1、 2、 3、 或 4， 优选 序列 1。
3. 编码权利要求 1― 2任一项的蛋白的多核苷酸。
4. 包含权利要求 3的多核苷酸的载体。
5. 包含权利要求 4的载体的细胞。
6. 包含权利要求 1或 2的蛋白的组合物。
7. —种 HPV6 类病毒颗粒， 其中该类病毒颗粒含有权利要 求 1或 2的蛋白或者由权利要求 1或 2的蛋白形成。
8. 一种制备 HPV L1蛋白例如权利要求 1或 2的蛋白的方 法， 其包括
a)在大肠杆菌表达系统中表达编码 HPV L1的 HPV L1基因， b)将表达了 HPV L1蛋白的大肠杆菌在盐浓度 100mM-600mM 中破碎， 分离得到上清液，
c)用水或低盐溶液将 b )上清液中盐浓度降至 l OOmM或以下， 最低至 0， 收集沉淀，
d)将 c ) 中沉淀在 150mM - 2500mM盐溶液中重新溶解， 同时 加入还原剂， 分离得到溶液， 其中含纯度至少 50 %的 HPV L1蛋 白。
9. 一种用于预防尖锐湿疣或 HPV感染的疫苗，其包含： （1 ) 权利要求 7的 HPV6类病毒颗粒， （ 2 )可有可无的至少一种（例 如 2、 3、 4种）选自 11 , 16， 18 , 31， 33， 45 , 52， 和 58型别 的 HPV类病毒颗粒（例如 HPV L1类病毒颗粒） ， 及（3 )疫苗 用赋形剂或载体，
优选地， 所述疫苗含有： HPV6类病毒颗粒和 HPV11类病毒 颗粒，特别是， 含有具有 SEQ ID NO: 4所示氨基酸序列的蛋白质 的 HPV6类病毒颗粒和含有具有 SEQ ID NO: 7所示氨基酸序列的 蛋白质的 HPV11类病毒颗粒， 更优选还含有 HPV16类病毒颗粒 和 HPV18类病毒颗粒， 特别是含有具有 SEQ ID NO: 8所示氨基 酸序列的蛋白质的 HPV16 类病毒颗粒和含有具有 SEQ ID NO: 9 所示氨基酸序列的蛋白质的 HPV18类病毒颗粒。
10. 权利要求 1 - 2任一项的蛋白或权利要求 7的类病毒颗 粒在制备用于预防尖锐湿疣或 HPV感染的疫苗中的用途。
11. 一种预防尖锐湿疣或 HPV感染的方法， 其包括将含预防 有效量的权利要求 1 - 2任一项的 HPV6 L1蛋白或权利要求 7的 类病毒颗粒的疫苗或预防有效量的权利要求 9 的疫苗给予需要 预防尖锐湿疣或 HPV感染的人。
12. 获得 HPV6 L1蛋白类病毒颗粒的方法， 其包括：
e)将纯度至少 50 %的权利要求 1或 2的 HPV6 L1蛋白进 一步通过色谱层析纯化， f)将 e)步骤中得到的 HPV6 LI蛋白去除还原剂
13. 制备用于预防尖锐湿疣或 HPV感染疫苗的方法， 其包括 将权利要求 7 的类病毒颗粒与任选的一种或多种选自 HPV11, 16， 18, 31, 33, 45， 52，和 58的 HPV型别的类病毒颗粒及 疫苗用载体或者赋形剂混合。
PCT/CN2008/001050 2007-05-29 2008-05-29 A truncated l1 protein of human papillomavirus 6 WO2008145021A1 (en)

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US12/601,972 US8748127B2 (en) 2007-05-29 2008-05-29 Truncated L1 protein of human papillomavirus type 6
BRPI0810951A BRPI0810951B8 (pt) 2007-05-29 2008-05-29 proteína l1 do hpv6, polinucleotídeo, vetor, célula, partícula semelhante ao vírus (vlp) hpv6, método para produzir a proteína hpv6 l1 truncada, vacina para prevenção de condiloma acuminado ou de infecções por hpv, uso da proteína hpv6 l1 truncada, método para prevenção de condiloma acuminado ou infecções por hpv, método para obter uma vlp de uma proteína l1 do hpv6 e método para produzir uma vacina para a prevenção de condiloma acuminado ou de infecções por hpv
DK08757381.2T DK2154149T3 (da) 2007-05-29 2008-05-29 En trunkeret l1-protein af human papillomavirus 6
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