WO2008098484A1 - Protéines l1 recombinantes du papillomavirus et leurs utilisations - Google Patents

Protéines l1 recombinantes du papillomavirus et leurs utilisations Download PDF

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WO2008098484A1
WO2008098484A1 PCT/CN2008/000314 CN2008000314W WO2008098484A1 WO 2008098484 A1 WO2008098484 A1 WO 2008098484A1 CN 2008000314 W CN2008000314 W CN 2008000314W WO 2008098484 A1 WO2008098484 A1 WO 2008098484A1
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hpv
protein
amino acid
acid sequence
multimer
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Runlin Z. Ma
Xiaojiang Chen
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Ma Runlin Z
Xiaojiang Chen
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A61K2039/645—Dendrimers; Multiple antigen peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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 the field of prevention and treatment of human papillomavirus infection.
  • the present invention relates to an amino acid sequence of a recombinant human papillomavirus L1' capsid protein, a nucleotide sequence encoding the amino acid sequence, and a vector and transformant comprising the same, and the present invention also relates to the amino acid
  • the use of a sequence of HPV L1 protein multimers for the preparation of vaccines, pharmaceutical compositions, diagnostic antigens or antibodies. Background technique
  • the papillomavirus ( ⁇ Zoma w'r s) is a large class of DNA viruses that infect humans and other animals. Among them, human papilloma virus (HPV) is associated with various diseases of humans, including benign sputum and cancer.
  • the papillomavirus contains two structural proteins (or capsid proteins) that protect the viral chromosomes, L1 and L2, respectively.
  • 360 L1 protein molecules or 72 L1 pentamers form the outer shell of the virus particles;
  • L2 is located inside the L1 ⁇ shell, also known as the underwear shell protein, usually each L2 molecule is associated with a L1 pentamer. Therefore, L1 and L2 are important candidate immunogens.
  • prokaryotic expression Due to the host specificity of the papillomavirus during its long-term evolution, the virus is difficult to propagate and expand by conventional cell culture, and the viral capsid protein must be produced by genetic recombination. So far, there are two basic options for expressing target genes by gene recombination scale: prokaryotic expression and eukaryotic expression. Among them, prokaryotic expression has the advantages of high yield, low cost, relatively simple system and relatively mature technology relative to eukaryotic expression.
  • prokaryotic expression techniques also have drawbacks: The expression of biological macromolecules with large molecular weight is relatively difficult, especially in the biological activities of many genes in higher animals and humans.
  • the root cause of the difficulty lies in the prokaryotic expression of the host is a lower prokaryotic organism such as Escherichia coli.
  • the processing, modification elements and modification processes of the expressed product polypeptide are significantly different from those of higher organisms.
  • it is easy. The misfolding of the secondary or higher structure of the target protein molecule results in an incorrect molecular conformation, thereby affecting or losing the biological activity of the expressed product.
  • Human papillomavirus is an obligate parasitic virus in humans and other higher mammals. It does not have a gene expression system. The expression of all genes depends entirely on the protein expression, translation, processing and modification systems of its host-higher eukaryotes. Therefore, when a gene such as the L1 capsid protein is expressed in a prokaryote, the biological activity is often lost due to an error in molecular folding and conformation. For example, when an unmodified L1 protein is expressed in E. coli, the expression product often forms a water-insoluble inclusion body and precipitates. The precipitated L1 protein is currently unable to restore its biological activity, i.e., it cannot restore the soluble protein with the correct conformation of the constituents.
  • One of the ways to solve the above problems is to carefully analyze the sequence of the target gene expression product on the basis of in-depth research, and to modify the target gene without modifying or changing the three-dimensional structure of the protein molecule, so that it can Soluble expression is achieved in prokaryotic cells such as E. coli.
  • the expressed L1 protein molecule can form a correct molecular conformation and is soluble in water.
  • the soluble L1 molecule is capable of automated molecular polymerization due to the three-dimensional conformation, forming a multimer of L1 protein molecules.
  • One such polymerization mode is the formation of a five-mer of the L1 protein molecule. '
  • papillomaviruses that are parasitic on human or other mammalian epithelial cells multiply before the host cells age and die.
  • the virus initiates the expression of the L1 capsid protein gene in the host cell while initiating the replication of its own genomic DNA.
  • the expressed L1 protein automatically forms a pentameric structure, and then these pentamers themselves are further automatically polymerized, and finally 72
  • the pentameric forms a wild virus particle, and each virus particle stores a set of DNA molecules of the virus inside.
  • the human body recognizes any invading virus mainly by the capsid protein molecules that recognize the surface of the virus, with no exceptions.
  • the surface of the pentameric molecule of the human papillomavirus L1 capsid protein constitutes the antigen recognition epitope (ie, the epitope) of the human body.
  • the body After the virus is identified, the body produces specific antibodies to neutralize the virus, fighting the infection and harm of the virus.
  • we expressed and purified the capsid protein of the virus by bioengineering and produced the vaccine using the purified L1 protein as an antigen.
  • the recombinant L1 protein pentamer itself has a complete antigenic epitope and can therefore be used as an antigen to prepare a vaccine.
  • some of the L1 pentamers can be automatically polymerized to form virus-like particles (VLP) that are exactly the same size as wild virus particles.
  • VLP virus-like particles
  • prokaryotic expression of human papillomavirus antigens is a water-insoluble inclusion body, and it is necessary to genetically modify the human papillomavirus L1 capsid protein expressed in the prokaryotic system to be soluble in water. , thereby reducing industrial costs.
  • the recombinant amino acid sequence of the human papillomavirus capsid protein L1 of the present invention which is an amino acid sequence upstream of the N-terminal conserved sequence VYLPP or VYVPP of the amino acid sequence of the wild-type HPV L1 protein, which is composed of G and S.
  • a short peptide having a number of amino acids of 2 to 10 while forming a truncated sequence relative to the wild type by introducing a stop codon for protein translation at the 5th amino acid downstream of the C-terminal conserved sequence LGRKFL. Since the DNA sequences encoding different HPV L1 proteins are highly conserved in the above-described modified regions, the above modification strategy is applicable to all known HPV types.
  • the HPV type is selected from the group consisting of HPV6, HPV1K HPV16, HPV18.
  • the Genebank accession numbers corresponding to the amino acid sequences of the wild type L1 proteins of the above different HPVs are: HPV6-NC-000904, HPV11-NC_001525, HPV16-AF402678, HPV18-AY262282:
  • HPV26-NC—001583 HPV31-NC—001527 , HPV33-NC_001528 , HPV35-NC_001529 : HPV39-NC_001535 ; HPV42-NC_00153 , HPV45-NC_001590 , HPV51 -NC_001533 : HPV52-NC_001592 HPV53-NC_001593 , HPV56-NC — 001594 , HPV58 -NC_001443 : HPV59-NC 001635, HPV66- NC 001695, HPV73-X94165, HPV82-AF29396L
  • HPV6 corresponding amino acid sequence is shown in SEQ ID NO: 1
  • HPV11 corresponding amino acid sequence is shown in SEQ ID NO: 2
  • HPV16 corresponds The amino acid sequence corresponding to SEQ ID NO: 3
  • the amino acid sequence corresponding to HPV18 is shown in SEQ ID NO: 4
  • the amino acid sequence corresponding to HPV 26 is shown in SEQ ID NO: 5
  • the amino acid sequence corresponding to HPV31 is SEQ ID NO: As shown in FIG.
  • the corresponding amino acid sequence of HPV33 is shown in SEQ ID NO: 7
  • the amino acid sequence corresponding to HPV35 is shown in SEQ ID NO: 8
  • the amino acid sequence corresponding to HPV39 is shown in SEQ ID NO: 9
  • HPV42 corresponds to The amino acid sequence is shown in SEQ ID NO: 10
  • the corresponding amino acid sequence of HPV45 is shown in SEQ ID NO: 11
  • the amino acid sequence corresponding to HPV 51 is shown in SEQ ID NO: 12
  • the amino acid sequence corresponding to HPV 52 is SEQ ID NO:
  • the amino acid sequence corresponding to HPV 53 is shown in SEQ ID NO: 14
  • the amino acid sequence corresponding to HPV 56 is shown in SEQ ID NO: 15
  • the amino acid sequence corresponding to HPV58 is shown in SEQ ID NO: 16, HPV.
  • amino acid sequence is shown in SEQ ID NO: 17, the amino acid sequence corresponding to HPV 66 is shown in SEQ ID NO: 18, the amino acid sequence corresponding to HPV 73 is shown in SEQ ID NO: 19, and the amino acid sequence corresponding to HPV 82 is SEQ ID NO: NO: 20 is shown.
  • the invention also relates to a nucleic acid molecule comprising a nucleotide sequence encoding the above modified amino acid sequence.
  • a common feature of the nucleotide sequence encoding the above amino acid sequence is that the expressed product has the same immunological properties as the wild-type HPV L1 protein.
  • the nucleic acid molecule includes, but is not limited to, the following form: A fusion sequence comprising a nucleotide sequence encoding the above modified amino acid sequence and a nucleic acid sequence encoding an HPV prophase protein (e.g., E6/E7).
  • the nucleic acid molecule is a nucleotide sequence encoding the above modified amino acid sequence.
  • the present invention also relates to an expression vector comprising the above nucleotide sequence obtained by cloning the above nucleotide sequence into a ligation vector comprising a suitable promoter and other suitable transcriptional expression regulatory elements, wherein the vector for ligation
  • the vector for ligation include commercially available plasmids, bacteriophage and cosmids.
  • the vector for ligation is selected from the group consisting of pGEX-4T-pGEX-4T-2, pGEX-4T-3, pGEX-6P-1, pGEX-6P-2, pGEX-6P-3, P ET-28a> pcDNA3 .1 and other commercially available expression plasmids.
  • the molecular cloning method used is described in Molecular Cloning (Third Edition, Science Press, August 2002).
  • the present invention also relates to a transformant comprising the above expression vector obtained by transferring the above expression vector into a host cell, the host cell being Escherichia coli.
  • the present invention further relates to a HPV L1 protein multimer obtained by inducing expression of the above transformant, the primary structure of the monomer consisting of the recombinant amino acid modification sequence of the present invention, the secondary structure and the high-order structure thereof It is determined by its primary structure.
  • Multimers of the invention include, but are not limited to, the following forms: dimers, pentamers, dodecamers or heptadecomers.
  • the multimer is a pentameric or heptameric.
  • the present invention also provides a method of producing the above HPV L1 protein multimer, which comprises the steps of: (1) fermenting the transformant as described above and expressing the HPV L1 protein;
  • the present invention discloses the use of the above recombinant HPV L1 protein multimer in the preparation of a vaccine for preventing HPV infection.
  • the vaccine of the present invention is a monovalent or combination prophylactic vaccine consisting of any one or more HPV-type recombinant HPV L1 protein multimers.
  • the vaccine of the present invention may also be in the form of a combination protein comprising the HPV L1 protein multimer + L2 protein.
  • the immunological component of the above vaccine also includes a physiologically acceptable carrier including, but not limited to, a simple low concentration salt solution capable of maintaining the integrity of the HPV L1 protein multimer, such as 10 mM NaCl, O.lmM EDTA; or greater
  • the carrier includes slow-moving macromolecules such as proteins, polysaccharides, polylactoic acid, polyglyceric acids, complex amino acids, and inactivated virions.
  • Pharmacologically acceptable salts can also be used in the complex of HPV L1 protein multimers.
  • the immune component also includes liquids such as water, physiological saline, glycerin, alcohol, and other substances such as emulsifiers, pH buffers, and the like.
  • the human body When the combined protein of the above recombinant HPV L1 protein multimer or HPV L1 protein multimer + L2 protein is inoculated into the human body in an immunologically effective dose, the human body can be induced to produce an immune response against these recombinant proteins. This specific immune response in the human body can help the body prevent the invasion of human papillomavirus or neutralize the invaded human papillomavirus.
  • Vaccines prepared from recombinant HPV L1 protein multimers or HPV L1 protein multimer + L2 protein combinations can be administered to humans in a variety of ways, including intravenous, intramuscular, and subcutaneous injections.
  • the vaccine of the present invention is used in a range that is effective to elicit an immunological response to the L1 protein.
  • the specific dose of the complex used to enhance the immune response varies depending on the L1 complex. In general, the amount of L1 multimer is between 1-500 ⁇ ⁇ / ⁇ ⁇ body weight.
  • the above dosage ranges do not exclude the possibility of higher or lower doses.
  • the specific dosage will depend on whether other drug doses are accompanied, or on the individual's pharmacokinetics, drug accumulation, and metabolic rate.
  • the present invention also discloses the use of the above recombinant HPV L1 protein multimer in the preparation of a pharmaceutical composition for preventing HPV infection.
  • the pharmaceutical composition includes, but is not limited to, the following forms: A pharmaceutical composition comprising a recombinant HPV L1 protein multimer and HPV ⁇ 6/ ⁇ 7 protein, and other pharmaceutically acceptable excipients.
  • the invention further discloses the use of the above recombinant HPV L1 protein multimer for the preparation of an HPV virus immunodiagnostic antigen or antibody.
  • the above recombinant HPV L1 protein multimers can be used to generate antibodies with specific affinity for HPV L1 protein, which can be used to directly detect the presence or absence of a particular HPV type associated with a particular pathological stage in a biological sample.
  • the HPV L1 protein multimer can also be used in immunoassays to detect the presence of antibodies or antigens of the HPV virus in biological samples.
  • Polyclonal or monoclonal antibodies, including single chain antibodies that specifically bind to the L1 protein can be produced by those skilled in the art using well known techniques. These antibodies, which are induced by specific antigens (e.g., different subtypes of HPV), have specificity for recognizing specific HPV subtypes, such as HPV16 and HPV18.
  • Methods for detecting L1 protein by immunoassay using antibody reaction include ELISA, 'Western blot, radioimmunoassay, immunohistochemistry, immunoprecipitation, and the like. Both antibodies and L1 multimers can be used as detection markers, such as enzyme-linked immunosorbent assays, radiolabels, fluorescent or chemiluminescent.
  • the antibody or L1 multimer can be immobilized on a solid support (such as glass or plastic slides, tissue culture plates, multiwell plates, test tubes, exchange columns, exchange column packing, proteins), particles (such as microspheres, including but not limited to Latex, polystyrene, glass spheres, or films (such as acetate or nylon membranes).
  • the biological sample can be any sample suspected of containing an HPV virus, such as a tissue biopsy, smear, tissue cut (such as skin, uterus, reproductive epithelial cells, larynx, upper respiratory tract, conjunctiva, or oral tissue).
  • tissue biopsy such as a tissue biopsy, smear, tissue cut (such as skin, uterus, reproductive epithelial cells, larynx, upper respiratory tract, conjunctiva, or oral tissue).
  • the recombinant HPV L1 capsid protein expressed in the prokaryotic system achieves correct spatial folding, is soluble in water, and solves the current HPV L1 capsid expressed in the prokaryotic system. Proteins are a problem of water-insoluble inclusion bodies (no biological activity), breaking the bottleneck of producing HPV L1 capsid proteins using prokaryotic expression systems.
  • the invention makes the industrial production of HPV L1 capsid protein by prokaryotic expression system a reality, and has the advantages of more stable product quality, higher yield, lower cost and convenient quality control than the currently used eukaryotic expression system, and has great economics. Benefits and social effects.
  • Figure 1 shows SDS-polyacrylamide gel electrophoresis of HPV16 L1 capsid protein obtained after recombinant transformation.
  • Lane 1 indicates marker, lane 2 and lane 3 indicate protein supernatant, and lane 4 and lane 5 indicate resin adsorption.
  • Protein, Lane 6, Lane 7 and Lane 8 represent the resin after digestion, Lane 9 represents the eluate of the protein of interest;
  • Figure 2 is a liquid chromatogram of the HPV16 L1 capsid protein obtained after recombinant engineering;
  • Figure 3 is a liquid chromatogram of the HPV6 L1 capsid protein obtained after recombinant transformation
  • Figure 4 is a liquid chromatogram of the HPV11L1 capsid protein obtained after recombinant transformation
  • Figure 5 is a liquid chromatogram of the HPV18L1 capsid protein obtained after recombinant transformation
  • Figure 6 is a liquid chromatogram of the HPV33L1 capsid protein obtained after recombinant transformation
  • Figure 7 is a liquid chromatogram of the HPV35L1 capsid protein obtained after recombinant transformation
  • Figure 8 is a liquid chromatogram of the HPV58L1 capsid protein obtained after recombinant transformation.
  • Example 1 Taking HPV16 as an example to illustrate the recombination of the amino acid sequence of HPV L1 capsid protein
  • HPV16 DNA Clinical cell sample waste that may contain wild-type HPV16 virus (the remainder of cervical exfoliated cells used for routine cell morphology examination) was purchased from Beijing Chun'an ⁇ Hospital gynecological clinic. The exfoliated cells suspended in 1.0 ml of water (about 1 ⁇ 10 5 ) exfoliated cells were treated with 1000 units of proteinase K (purchased from Huamei Biological Products Co., Ltd.) at 55 ° C for 300 minutes, and centrifuged at 100 g for 30 minutes, and the resulting supernatant was used. To detect the presence of HPV virus.
  • proteinase K purchased from Huamei Biological Products Co., Ltd.
  • HPV16 type LI protein DNA The HPV16 type L1 capsid protein gene is obtained by DNA polymerase chain reaction (PCR) from a target gene-specific oligonucleotide primer pair.
  • PCR DNA polymerase chain reaction
  • the classical PCR reaction system consisted of 20 ug DNA template, lx PCR buffer, forward and reverse specific primers (concentration of 0.2 ⁇ ), 1.5 mM magnesium ion, and 1.0 unit of TAQ DNA polymerase.
  • the reaction conditions were: denaturation at 95 ° C for 5 minutes, amplification by 36 PCR cycles (94 degrees 30 seconds per cycle, 55 degrees 30 seconds, 72 degrees 2 minutes), and the reaction product was incubated at 72 degrees for 10 minutes, then Stop the reaction.
  • the forward primer sequence and reverse primer sequence of the PCR reaction are shown in Table 1.
  • PCR amplification of amplified HPV 16 L1 cDNA fragment by TA cloning (for details, see Molecular Cloning, Third Edition, Science Press, August 2002) Cloning to a T-Easy Vector Plasmid (purchased from Promega, USA) Biological reagent company).
  • Modification of HPV16 L1 protein gene The immunogenicity and antigenicity of HPV 16 L1 protein depend on the correct three-dimensional conformation produced by its correct molecular folding.
  • the purpose of modifying the HPV16 L1 protein gene sequence is to increase the solubility of the recombinant target gene in prokaryotic expression, thereby producing a biologically active L1 protein using E C0 /.
  • the basic step of genetic modification is to design specific primers, and then use the specific primers as a template to PCR amplification of the L1 target gene fragment.
  • the engineered recombinant HPV16 L1 protein gene DNA sequence contains a BcrnH I restriction enzyme site at its 5' end, and contains a DNA codon encoding GSGGG to replace the amino acid encoding the amino acid sequence N-terminal conserved sequence VYLPP upstream amino acid. child.
  • the forward primer sequence and reverse primer sequence used for the transformation are shown in Table 1.
  • the downstream primer introduced the stop codon UAA, resulting in a decrease in LI cDNA of about 25 amino acids at the 3'-end.
  • the PCR reaction system contained 20 ug DNA template, lx PCR buffer, forward and reverse specific primers (concentration of 0.2 ⁇ ), 1.5 mM magnesium ion, and 1.0 unit of TAQ DNA polymerase.
  • the reaction conditions were: denaturation at 95 ° C for 5 minutes, amplification by 36 PCR cycles (94 degrees 30 seconds per cycle, 55 degrees 30 seconds, 72 degrees 2 minutes), and the reaction product was incubated at 72 degrees for 10 minutes, then Stop the reaction. .
  • the HPV16 LI cDNA product amplified by PCR reaction was digested with Sa HI and ⁇ restriction enzymes to form a sticky end, and the plasmid pGEX-4T-2 (purchased from Huamei Biological Products Co., Ltd.) was digested with BamHI and Xhol.
  • the PCR product and expression plasmid were ligated using T4 ligase (purchased from Promega BioReagents, USA).
  • the ligation product was electrotransformed and transferred to host cells of E. coli BL21 (purchased from Huamei Bioproducts Co., Ltd.) (for details of electroporation, see Molecular Cloning, Third Edition, Science Press, August 2002).
  • Prokaryotic expression and detection of L1 protein The expression plasmid carrying the HPV16 L1 gene insert was introduced into E. coli BL21 cells, and LB medium was used (for the formula, see Molecular Cloning, Third Edition, Science Press, August 2002) Published) 37-degree culture, growth was carried out for 12 hours after the strain was introduced, and induction with the expression inducer IPTG (purchased from Promega, USA) was started. The cells were harvested 9 hours later, and the cells were repeated at 800 bar using a Niro Soavi NS2006 high-pressure homogenizer. The cells were disrupted twice, and the cell breakage rate was over 90% by microscopy.
  • the L1 protein in the supernatant solution was purified by affinity chromatography: prepackaged with glutathione-agarose resin (Glutathione Sepharose 4 B from Amersham) column, 50% Glutathione Sepharose 4 B homogenate Place in the column (5-10ml homogenate per 200ml of protein serum). Wash the resin with 5-10 times the bed volume of buffer A (component: 50 mmol/L Tric-HCl, 200 mmol/L NaCl, 1 mmol/L EDTA, pH 8.0), and add the protein supernatant to the column. After mixing with the resin and allowing to act at room temperature for 20 minutes, the filtrate was discharged, and the resin column was washed with 10 column volumes of buffer A.
  • buffer A component: 50 mmol/L Tric-HCl, 200 mmol/L NaCl, 1 mmol/L EDTA, pH 8.0
  • HPV type L1 capsid protein including HPV6, HPV11, HPV18, HPV 26, HPV31, HPV33, HPV35, HPV39, HPV42) HPV45, HPV 51, HPV 52, HPV 53, HPV 56, HPV58,
  • HPV 59, HPV 66, HPV 73, and HPV 82 are the same as those in Embodiment 1, and the differences are shown in Table 1.
  • Primer pairs used for the modification of the L 1 protein gene by the primers used for the HPV type clone L1 protein gene (the forward primer does not include the Linker DNA sequence encoding the GSGGG amino acid)
  • HPV6 Forward 5'-GCATTGCCTGACTCGTCTCT-3' Forward 5'-TCCTCCTAACCCTGTATCCAAAGTTG-3'
  • HPV42 Forward 5'-ATGTGCMCGCACCMCTAC-3' Forward 5'-ATCGTAGTTATTTTTGGCGTAGGCGC-3' Reverse: 5'-CCATAGGCCTCAGCAGACAT-3' Reverse 5'-GAAATTGATCTAAATCAGTAGAAAAC-3'
  • HPV52 Forward 5'-ATGCAGGCAGTTCTCGATTA-3' Forward 5'-ACGTCGCAGGCGTAMCGTTTTCC-3'

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Abstract

L'invention concerne des protéines L1 recombinante du papillomavirus humain (HPV) obtenu par substitution du peptide lieur court pour des acides aminés en amont d'une séquence de conservation VYLPP ou VYVPP à la terminaison N de la protéine de type sauvage et la suppression simultanée des acides aminés démarrant vers l'arrière depuis le cinquième acide aminé en aval de la séquence de conservation LGRKFL à la terminaison C. Les protéines HPV L1 recombinantes exprimées dans E. coli sont solubles et forment des multimères utilisés pour la préparation de vaccins immunogènes et de compositions de diagnostic.
PCT/CN2008/000314 2007-02-14 2008-02-04 Protéines l1 recombinantes du papillomavirus et leurs utilisations WO2008098484A1 (fr)

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DE102016124171A1 (de) 2016-12-13 2018-06-14 Abviris Deutschland Gmbh Serologischer Test zur Therapiekontrolle von HPV16 positiven Karzinomen
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CN114127100B (zh) * 2019-07-19 2024-04-02 神州细胞工程有限公司 嵌合的人乳头瘤病毒39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
JP2024503452A (ja) * 2021-01-14 2024-01-25 神州細胞工程有限公司 ヒトパピローマウイルスウイルス様粒子ワクチンの安定な調製

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