WO2018098878A1 - 登革病毒重组抗原及其制备方法和应用 - Google Patents

登革病毒重组抗原及其制备方法和应用 Download PDF

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WO2018098878A1
WO2018098878A1 PCT/CN2016/112374 CN2016112374W WO2018098878A1 WO 2018098878 A1 WO2018098878 A1 WO 2018098878A1 CN 2016112374 W CN2016112374 W CN 2016112374W WO 2018098878 A1 WO2018098878 A1 WO 2018098878A1
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dengue virus
recombinant antigen
detection line
pad
tris
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PCT/CN2016/112374
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English (en)
French (fr)
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朱碧银
杨耿周
宗雪
刘春艳
魏钟杰
罗沛
黄晓明
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菲鹏生物股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24131—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • 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
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue

Definitions

  • the invention relates to the field of virus detection, in particular to a dengue virus recombinant antigen and a preparation method and application thereof.
  • Dengue fever is an acute mosquito-borne disease caused by four serotypes of dengue virus, DEN-1, DEN-2, DEN-3 and DEN-4. Each serotype can cause dengue hemorrhagic fever with high dengue fever and mortality. And dengue shock syndrome. Dengue fever is widely prevalent in more than 100 countries and regions in the global tropical and subtropical regions of Africa, America, Southeast Asia and the Western Pacific. At present, China is mainly popular in Guangdong, Guangxi, Hainan and Taiwan. Its main vector is Aedes aegypti and white. Aedes mosquitoes.
  • the World Health Organization estimates that there are about 50 to 100 million cases of dengue fever worldwide each year, and it causes 25 to 500,000 cases of dengue hemorrhagic fever and 24,000 deaths.
  • the outbreak of dengue fever is on the verge of rapid development, and the rapid diagnosis of suspected cases of dengue fever is directly related to the early detection of the epidemic.
  • Dengue virus is the pathogen of dengue fever, a major member of the Flavivirus family of flaviviruses. It is a single-stranded positive-stranded RNA molecule with a envelope of about 11 kb.
  • the 5' end of the genome has a hat structure, and the 3' end is CUOH, and has no poly A structure.
  • Analysis of the cDNA sequence revealed that the 5' and 3' ends of the dengue virus RNA each have a non-coding region with only one open reading frame (ORF) in the middle, and about 96% of the nucleotide sequence of the genome is within the framework.
  • ORF open reading frame
  • the ORF sequence encodes three structural proteins of the virus (core protein c, membrane-bound protein precursor PrM and envelope protein E) and seven non-structural proteins (Ns).
  • the coding sequence of the entire genome is: 5'-C-PrM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5-3'. According to immunological studies, only C, PrM, E, NS1 and NS3 are both reactive and immunogenic.
  • the dengue virus E protein is the largest structural protein and major envelope glycoprotein of dengue virus, and is also the main virulence protein of dengue virus, and also has a type-specific, genus-specific antigenic determinant. It is also a key protein for dengue virus to adsorb target cells, cause disease and induce immune protection. It plays an important role in the pathogenesis of dengue virus and the anti-infective immunity of the body. The analysis of its structure and function has always become The focus of dengue research at home and abroad.
  • the E protein is a dengue virus VAP (virus attachment protein), a ligand for a viral receptor on the surface of a host cell, which mediates the specific binding of the virus to the target cell.
  • VAP virus attachment protein
  • the E protein on the surface of the mature virion is in the form of a homodimer.
  • the E protein acts as a VAP-mediated viral particle and target cell binding, the cytoplasmic endocytosis pathway Upon entry into the cell, the pH of the internal environment in which the phagosome is located is decreased, and the conformation of the E protein is rearranged from a homodimer to a trimer structure. The conformational change causes fusion of the host cell membrane and the viral membrane, causing the viral genome to enter the cytosol, causing infection.
  • dengue fever The clinical manifestations of dengue fever are complex and diverse, rapid spread, high incidence, and susceptible to infection, which seriously affect people's quality of life and physical health. At present, there is still no vaccine and special drug treatment for dengue infection. Therefore, specific, sensitive and rapid diagnosis methods are of great significance for the prevalence and prevention of dengue fever.
  • a dengue virus recombinant antigen comprising:
  • a method for preparing a dengue virus recombinant antigen as described above comprising the steps of:
  • Step 1 providing a gene expression vector for expressing the dengue virus recombinant antigen
  • Step 2 transforming the gene expression vector into a prokaryotic expression bacterium
  • Step 3 inducing expression of the prokaryotic expression strain transformed with the gene expression vector, and collecting and lysing after completion of expression, and retaining the first precipitate after centrifugation;
  • Step four washing the first precipitate with a buffer and then centrifuging again, retaining the second precipitate, and re-dissolving the second precipitate using the complex solution to obtain a crude product solution;
  • Step 5 purifying the crude product solution through a Ni-NTA affinity column, and performing protein renaturation to obtain the dengue virus recombinant antigen.
  • the buffer comprises 20 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, and a mass percentage of 0.01 Triton X-100, the pH of the buffer is 8;
  • the complex solution included 6 M urea and 20 mM Tris, and the pH of the complex solution was 8.
  • the equilibration solution comprises 20 mM Tris-HCl, 200 mM NaCl, and 6 M urea, and the pH of the equilibrium solution is 8.
  • the eluate included 20 mM Tris-HCl, 200 mM NaCl, 6 M urea, and 250 mM imidazole, and the pH of the eluate was 8.
  • the protein renaturation operation is: dialysis renaturation at 4 ° C with a first refolding buffer, a second refolding buffer, and a third refolding buffer, respectively;
  • the first refolding buffer comprises 4M urea, 20 mM Tris-HCl and 150 mM NaCl, the first refolding buffer has a pH of 8;
  • the second refolding buffer comprises 2M urea, 20 mM Tris-HCl and 150 mM NaCl, and the pH of the second refolding buffer is 8;
  • the third refolding buffer included 1 M urea, 20 mM Tris-HCl, and 150 mM NaCl, and the third refolding buffer had a pH of 8.
  • the above-mentioned dengue virus recombinant antigen is used in the field of preparing dengue virus detection reagents or preparing dengue virus detection equipment.
  • a dengue virus test strip containing the above-described dengue virus recombinant antigen A dengue virus test strip containing the above-described dengue virus recombinant antigen.
  • a support sheet, a sample pad, a gold standard pad, a nitrocellulose membrane, an absorption pad, a T1 detection line, a T2 detection line, and a quality control line are included;
  • the sample pad, the gold standard pad, the nitrocellulose membrane, and the absorption pad are sequentially disposed on the support sheet from one end to the other end of the support sheet, the sample pad and the gold standard
  • the pads partially overlap, the gold standard pad partially overlapping the nitrocellulose membrane, and the nitrocellulose membrane partially overlaps the absorption pad;
  • the T1 detection line, the T2 detection line, and the quality control line are all disposed on the nitrocellulose membrane, and the T1 detection line is disposed at an end of the nitrocellulose membrane near the gold standard pad.
  • the T2 detection line Provided at an end of the nitrocellulose membrane near the absorption pad, the quality control line is located between the T1 detection line and the T2 detection line;
  • the gold label pad is coated with a colloidal gold-labeled mouse IgG and a colloidal gold-labeled dengue virus recombinant antigen
  • the T1 detection line is an anti-human IgG antibody
  • the T2 detection line is an anti-human IgM antibody.
  • the quality control line is a goat anti-mouse IgG antibody.
  • a dengue virus detection kit comprising the above-described dengue virus test strip.
  • the dengue virus detecting test paper is disposed in the housing, and the housing is provided with a sampling hole and a viewing window, the loading hole and the sample pad
  • the T1 detection line, the T2 detection line, and the quality control line are disposed corresponding to the observation window.
  • the test kit prepared by the dengue virus recombinant antigen can accurately detect four serotypes of dengue virus-infected samples. Therefore, the dengue virus recombinant antigen can be used for dengue detection.
  • FIG. 1 is a schematic structural view of a dengue virus detection kit according to an embodiment
  • FIG. 2 is a schematic front view showing the structure of a dengue virus test strip of the dengue virus detecting kit shown in FIG. 1;
  • FIG. 3 is a schematic cross-sectional structural view of a dengue virus test strip of the dengue virus detection kit shown in FIG. 1;
  • Example 4 is a SDS-PAGE electrophoresis pattern of the first precipitate obtained in Example 1.
  • the invention discloses a dengue virus recombinant antigen, comprising:
  • the test kit prepared by the dengue virus recombinant antigen can accurately detect four serotypes of dengue virus-infected samples. Therefore, the dengue virus recombinant antigen can be used for dengue detection.
  • the invention also discloses a preparation method of the above dengue virus recombinant antigen, comprising the following steps:
  • Step one providing a gene expression vector for expressing the dengue virus recombinant antigen.
  • the gene expression vector is prepared as follows: First, the sequence of the dengue I to IV virus strain gene is collected in Genbank, and the genetic data of the dengue type I to IV virus strain is established, and analyzed by computer software to determine the most The active segments were detected, and the gene sequences of the most detectable segments of the four serotypes of dengue were directly ligated, artificially synthesized, and the upstream primers were introduced into the BamHI site, and the downstream primers were introduced into the EcoRI site and EcoRI.
  • the coding sequence of 6 His amino acids and the stop code TAA are preceded by the site.
  • the specific amino acid sequence is shown in SEQ ID No. 1.
  • PCR amplification is performed using the upstream and downstream primers, and the amplified PCR fragment is recovered.
  • Molecular biological extraction and recovery kits used in the present invention were purchased from Shanghai Huasheng Bioengineering Co., Ltd.), and then digested with BamHI and EcoRI (the various molecular biological enzymes used in the present invention were purchased from Dalian). Bao Bioengineering Co., Ltd.), which was ligated into the expression vector pET-24a (Novagen, Cat. No. 69864-3) after digestion with BamHI and EcoRI, and the resulting recombinant plasmid p ET-24a-DENI ⁇ IV is the desired gene expression vector.
  • Step 2 The gene expression vector obtained in the first step is transformed into the prokaryotic expression bacteria.
  • the prokaryotic expression strain can be Escherichia coli. Specifically, the prokaryotic expression strain was BL21 (DE3) (purchased from Invitrogen, USA).
  • Step 3 Inducing expression of the prokaryotic expression strain transformed with the gene expression vector obtained in the second step, and after the expression is completed, the bacteria are lysed, and the first precipitate is retained after centrifugation.
  • Step 4 The first precipitate obtained in the third step of the buffer is washed with a buffer, and then centrifuged again, the second precipitate is retained, and the second precipitate is redissolved using the complex solution to obtain a crude product solution.
  • the buffer included 20 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, and a mass percent of Triton X-100 of 0.01 with a pH of 8.
  • the complex solution included 6 M urea and 20 mM Tris, and the pH of the complex solution was 8.
  • Step 5 Purify the crude product solution obtained in the fourth step through a Ni-NTA affinity column, and then perform protein renaturation to obtain a dengue virus recombinant antigen.
  • the equilibration solution includes 20 mM Tris-HCl, 200 mM NaCl, and 6 M urea, the pH of the equilibration solution is 8, and the eluate includes 20 mM Tris-HCl, 200 mM NaCl, 6 M urea and 250 mM imidazole, the pH of the eluent was 8.
  • the renaturation of the protein was carried out by dialysis renaturation at 4 ° C with a first refolding buffer, a second refolding buffer and a third refolding buffer, respectively.
  • the first refolding buffer included 4 M urea, 20 mM Tris-HCl, and 150 mM NaCl, and the pH of the first refolding buffer was 8.
  • the second refolding buffer included 2 M urea, 20 mM Tris-HCl, and 150 mM NaCl, and the pH of the second refolding buffer was 8.
  • the third refolding buffer included 1 M urea, 20 mM Tris-HCl, and 150 mM NaCl, and the third refolding buffer had a pH of 8.
  • the dengue virus recombinant antigen obtained after dialysis and renaturation was dialyzed into a preservation solution (20 mM Tris-HCl, 150 mM NaCl, pH 8.0), and a dialysis sample was collected, and the protein concentration was measured and placed at -20 ° C until use.
  • the dengue virus recombinant antigen can be applied to the field of preparing dengue virus detection reagents or preparing dengue virus detection equipment.
  • a dengue virus detection kit including dengue virus detection
  • the test paper 100 and the casing 200, and the dengue virus test strip 100 are disposed in the casing 200.
  • the dengue virus test strip contains the above-mentioned dengue virus recombinant antigen.
  • the dengue virus test strip 100 includes a support sheet 110, a sample pad 120, a gold pad 130, a nitrocellulose membrane 140, an absorption pad 150, a T1 detection line 160, a T2 detection line 180, and a quality control line. 170.
  • the sample pad 120, the gold standard pad 130, the nitrocellulose membrane 140, and the absorption pad 150 are sequentially disposed on the support sheet 110 from one end of the support sheet 110 to the other end, and the sample pad 120 partially overlaps the gold standard pad 130, and the gold standard pad 130 is overlapped. Partially overlapping with the nitrocellulose membrane 140, the nitrocellulose membrane 140 partially overlaps the absorbent pad 150.
  • the T1 detecting line 160, the T2 detecting line 180, and the quality control line 170 are disposed on the nitrocellulose membrane 140.
  • the T1 detecting line 160 is disposed at one end of the nitrocellulose membrane 140 near the gold standard pad 130, and the T2 detecting line 180 is set at the nitrocellulose.
  • the plain film 140 is adjacent to one end of the absorption pad 150, and the quality control line 170 is located between the T1 detection line 160 and the T2 detection line 180.
  • the gold standard pad 130 is coated with the colloidal gold-labeled mouse IgG and the colloidal gold-labeled dengue virus recombinant antigen, the T1 detection line 160 is an anti-human IgG antibody, and the T2 detection line 180 is an anti-human IgM antibody, and the quality control line 170 It is a goat anti-mouse IgG antibody.
  • the housing 200 is provided with a sample loading hole 210 and a viewing window 220.
  • the sample insertion hole 210 is disposed corresponding to the sample pad 120, and the T1 detection line 160, the T2 detection line 180, and the quality control line 170 are disposed corresponding to the observation window 220.
  • This dengue virus detection kit uses a capture method to detect whether a dengue virus-specific IgG antibody and IgM antibody are contained in a test material.
  • the sample contains a dengue virus-specific IgG antibody and an IgM antibody
  • the IgG antibody and the IgM antibody first bind to the colloidal gold-labeled dengue virus recombinant antigen to form an antigen-antibody complex, and the antigen-antibody complex due to capillary action
  • the nitrocellulose membrane 140 moves forward and reaches the T1 detection line 160 and the T2 detection line 180, the antigen-antibody complex binds to the corresponding IgG antibody and IgM antibody, thereby enriching the T1 detection line 160 and the T2 detection line.
  • a red precipitation line is formed.
  • the colloidal gold-labeled murine IgG is captured by the goat anti-mouse IgG antibody through the T1 detection line 160 and the T2 detection line 180, and is enriched on the quality control line 170 to form a red precipitation line.
  • a positive result was obtained when both the T1 detection line 160 and the T2 detection line 180 and the QC line 170 had red precipitate lines.
  • the product does not contain dengue virus-specific IgG and IgM antibodies, and the colloidal gold-labeled dengue virus recombinant antigen does not bind to antibodies on T1 detection line 160 and T2 detection line 180, colloidal gold-labeled dengue virus recombination.
  • the anti-principle was captured by the goat anti-mouse IgG antibody through T1 detection lines 160 and T2, and enriched on the quality control line 170 to form a red precipitation line, at which time a negative result was judged.
  • the structure of the dengue virus detection kit is not limited to the above description.
  • the above dengue virus recombinant antigen can be used in other dengue virus detection kits or devices in addition to the above detection kit. It will be understood by those skilled in the art that the dengue virus recombinant antigen of the present embodiment is directly or indirectly combined with other signal groups (such as magnetic microspheres, horseradish peroxidase, etc.), or the dengue virus recombinant antigen of the present embodiment.
  • As a coating antigen eg ELISA
  • it can be used in other forms of dengue virus detection reagents or devices. Therefore, the dengue virus recombinant antigen prepared in the present embodiment can be widely applied to the preparation of dengue virus detection reagents or equipment.
  • dengue virus test strip 100 can be used alone.
  • the preparation process of the dengue virus detection kit is greatly simplified, and the dengue virus recombinant antigen is applied to the dengue virus detection kit, which is higher than the international similar products. Sensitivity and greater specificity.
  • the genes of dengue I to IV strains were collected in Genbank, and the genetic data of dengue I to IV strains were established, and analyzed by computer software to determine the most detectable segment, dengue fever.
  • the gene sequences of the most detectable segments of the four serotypes were directly ligated, artificially synthesized, and the upstream primers were introduced into the BamHI site, the downstream primers were introduced into the EcoRI site, and the EcoRI site was preceded by six Hiss.
  • the coding sequence of the amino acid and the stop codon TAA, the specific amino acid sequence is shown in SEQ ID No. 1.
  • PCR amplification is performed using the upstream and downstream primers, and the obtained PCR fragment is recovered (the molecular organism used in the present invention)
  • extraction and recovery kits were purchased from Shanghai Huasheng Bioengineering Co., Ltd., they were digested with BamHI and EcoRI (used by the present invention).
  • the various molecular biology enzymes were purchased from Dalian Bao Bioengineering Co., Ltd., and ligated into the expression vector pET-24a (Novagen, Cat. No. 69864-3) after digestion with BamHI and EcoRI to obtain recombinant plasmid pET.
  • -24a-DENI to IV which is a recombinant plasmid of the labeled antigen of the present invention, and the expressed protein is named DEN040.
  • the above-described constructed recombinant plasmid pET-24a-DENI-IV was transformed into host expression strain BL21 (DE3) (purchased from Invitrogen, USA) to obtain an expression plasmid of the recombinant plasmid, which was used.
  • Buffer formulation 20 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, 0.01 wt% Triton X-100, pH 8.0.
  • the first precipitate was subjected to SDS-PAGE electrophoresis to obtain Figure 4.
  • Figure 4 most of the protein of interest is expressed in inclusion bodies, and the precipitate is retained for subsequent processing.
  • the precipitate was washed 3 times with buffer, centrifuged at 12000 rpm ⁇ 15 min, and the second precipitate was removed from the supernatant.
  • the second precipitate was dissolved with a complex solution (6 M urea, 20 mM Tris, pH 8.0), sonicated for 2 min, and placed in a 4 degree environment for 30 min. Then, ultrasonication for 2 min, 13500 rpm ⁇ 30 min, the supernatant was taken, and Ni affinity was performed.
  • Formulation of the equilibration solution 20 mM Tris-HCl, 200 mM NaCl, 6 M urea, pH 8.0.
  • the ATKA purifier chromatograph purchased from GE Healthcare
  • the Ni-NTA column is well balanced. After that, the column to be applied to the column is combined. After the combination, the column is washed with 10 column volumes of the balance solution, and then eluted with the eluent to collect the elution peak, and the protein concentration is determined. .
  • the target protein obtained by affinity purification of the above Ni-NTA was diluted to a concentration of 1 mg/mL with an equilibration solution, and then respectively subjected to a refolding buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0) containing 4 M, 2 M, 1 M urea. The dialysis was renatured at 4 degrees, and finally the target protein was dialyzed into a buffer (20 mM Tris-HCl, Dialysis samples were collected in 150 mM NaCl, pH 8.0), and the protein concentration was determined and placed at -20 °C.
  • a refolding buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0
  • PBS buffer containing 6% methanol and 0.01 M pH 7.2 was used as coating buffer, filtered through a 0.22 ⁇ m membrane, and stored at 4 ° C for one week. 1000mL 6% methanol in 0.01M pH 7.2
  • PBS buffer formulation NaCl 8g, KCl 0.2g, Na 2 HPO 4 ⁇ 12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 60mL, double distilled deionized water to a volume of 1000mL.
  • nitrocellulose membrane Dilute anti-human IgG antibody (produced by Fei Peng Biotechnology Co., Ltd., product number BA-PAB-MU0003) to 1-2 mg/mL with coating buffer, adjust the machine, and scribe the T1 detection line.
  • the T1 detection line is close to the gold standard pad end, about 5mm away from the gold standard pad end;
  • the anti-human IgM antibody is produced with the coating buffer (produced by Fei Peng Biotechnology Co., Ltd., the item number BA- PAB-MU0005) diluted to 1 ⁇ 2mg / mL, adjust the machine, the line is T2 detection line, which is the dengue virus IgM antibody detection line, T2 detection line is located above the T1 detection line, about 3 ⁇ 5mm from the T1 detection line; Dilute goat anti-mouse IgG antibody (produced by Fei Peng Biotechnology Co., Ltd., product number BA-PAB-MU0001) to 1 ⁇ 5mg/mL with coating buffer, adjust the machine, and scribe the quality control line.
  • the quality control line is close to absorption. Pad, about 3mm from the absorption pad, and 5 ⁇ 8mm from the T2 detection line, evenly. Dry at 37 ° C, packaged for use.
  • chloroauric acid Dissolve chloroauric acid in double distilled deionized water, prepare a 1% solution, and set at 4 ° C for use, valid for four months. 1000mL 1% chloroauric acid solution formula: 10g chloroauric acid: double distilled deionized water to constant volume to 1000mL.
  • 30.1M potassium carbonate prepared by double-distilled deionized water, filtered through a 0.22 ⁇ m membrane, and placed at 4 degrees for use, valid for four months. 1000mL 0.1M potassium carbonate solution formula: 13.8g potassium carbonate; double distilled deionized water to a volume of 1000mL.
  • washing preservation solution preparation 2% bovine serum albumin (BSA), 0.05% sodium azide (NaN3), 0.01M pH7.2 PBS solution, 0.22 ⁇ membrane filtration, set at 4 ° C for use, valid for four months.
  • 1000 mL labeled wash preservation solution formulation 20 g BSA, 0.5 g NaN3, 0.01 M pH 7.2 PBS solution to a volume of 1000 mL.
  • the pH of the colloidal gold was adjusted to 8.2 with 0.1 M potassium carbonate, and the dengue virus recombinant antigen prepared in Example 1 was added to 8-10 ⁇ g of antibody/mL colloidal gold, mixed by a magnetic stirrer for 30 min, and BSA was added to the end with stirring. The concentration was 1% and allowed to stand for 1 hour. After centrifugation at 13,000 rpm, 4 ° C for 30 min, the supernatant was discarded, and the precipitate was washed twice with the label washing and preservation solution. The pellet was resuspended with a mark of one-tenth of the initial colloidal gold volume of the washing solution, and stored at 4 ° C for one week.
  • the gold standard pad was immersed in the blocking solution for 30 min and then dried at 37 °C.
  • the colloidal gold-labeled dengue virus recombinant antigen and the colloidal gold-labeled mouse IgG are uniformly mixed in an appropriate ratio, and then the mixed gel is mixed.
  • the gold-labeled dengue virus recombinant antigen and the colloidal gold-labeled mouse IgG were evenly spread on a gold standard pad, 20 cm 2 per ml of solution, freeze-dried, packaged, and stored at 4 ° C for use.
  • the sample pad was immersed in the blocking solution for 30 min, dried at 37 ° C, packaged, and placed at 4 ° C for use.
  • An absorbent pad (available from Millipore Corporation), a nitrocellulose membrane, a gold standard pad, and a sample pad were placed on a non-absorbent support sheet and cut into strips 3 mm wide. Each pack of ten strips is added with a desiccant and vacuum-packed to obtain a dengue virus test strip.
  • a kit for rapidly detecting dengue virus antibody IgG/IgM comprises: a dengue virus test strip prepared in Example 2, a shell and a sample diluent.
  • the dengue virus test strip can be placed in the housing.
  • the sample diluent was an 8% NaCl solution.
  • Preparation method 80g NaCl, add distilled water to make up to 1000mL.
  • the dengue virus antibody IgG/IgM was detected by colloidal gold method.
  • the commercial mainstream product SD Dengue IgG/IgM kit was compared with the dengue virus detection kit obtained in Example 3 (referred to as FP dengue IgG/IgM kit), and 97 dengue-positive specimens and 478 clinical samples were simultaneously tested. Negative specimens, these 97 positive specimens were tested by commercialized dengue virus typing multiplex PCR kit for RT-PCR typing, of which 37 were type II dengue virus infections and 23 were type I dengue virus infections. 15 were type III dengue virus infections and 22 were type IV dengue virus infections. The results showed that FP Dengue IgG/IgM kit detected 96 of the above 97 positive samples, while SD Dengue IgG/IgM kit detected 94 positive samples. For 478 clinically negative samples, FP kits were detected. 18 copies of false positives, 23 samples of false positives were detected in the SD kit, as shown in Table 1.
  • Example 3 The above results indicate that the dengue virus detection kit obtained in Example 3 is superior to the SD kit in terms of sensitivity and specificity, and can be completely used for rapid diagnosis of dengue virus.

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Abstract

本发明公开了一种登革病毒重组抗原及其制备方法和应用，登革病毒重组抗原包括：(a)、如SEQ ID No.1所示的氨基酸序列组成的多肽；(b)、与SEQ ID No.1所示的氨基酸序列组成的多肽具有至少98％同源性的多肽；或(c)、如SEQ ID No.1所示的氨基酸序列组成的多肽，其中一个或多个氨基酸被缺失、替代或增加得到的多肽。经过试验验证，这种登革病毒重组抗原制得的检测试剂盒能够准确的检测出四种血清型登革病毒感染的样本，因此，这种登革病毒重组抗原可以用于登革热检测。

Description

登革病毒重组抗原及其制备方法和应用 技术领域
本发明涉及病毒检测领域，特别是涉及一种登革病毒重组抗原及其制备方法和应用。
背景技术
登革热是由DEN-1、DEN-2、DEN-3和DEN-4四种血清型登革病毒引起的急性蚊媒传染病，各血清型均可引起登革热和致死率很高的登革出血热以及登革休克综合症。登革热广泛流行于全球热带和亚热带的非洲、美洲、东南亚和西太平洋区的100多个国家和地区，目前我国主要流行于广东、广西、海南和台湾地区，其主要传播媒介是埃及伊蚊和白蚊伊蚊。世界卫生组织估计全球每年登革热发病人数约5千万到1亿，并导致25到50万登革出血热病例和2.4万人死亡。登革热爆发疫情来势急，扩散快，登革热疑似病例的尽快确诊直接关系到及早发现疫情。
登革病毒是登革热的病原体，黄病毒科黄病毒属主要成员，是有包膜的单股正链RNA分子，基因组长约11kb。其基因组的5’端有帽子结构，3’端为CUOH，无多聚A结构。cDNA序列分析表明，登革病毒RNA的5’端和3’端各有一段非编码区，中间仅有一个开放阅读框(ORF)，基因组约96％的核苷酸序列在此框架内。该ORF序列编码病毒的3种结构蛋白(核心蛋白c、膜结合蛋白前体PrM和包膜蛋白E)和7种非结构蛋白(Ns)。整个基因组的编码顺序为：5’-C-PrM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5-3’。经免疫学研究发现，只有C、PrM、E、NS1和NS3既具有反应原性，又具有免疫原性。
登革病毒E蛋白是登革病毒最大的结构蛋白和主要的包膜糖蛋白，也是登革病毒的主要毒力蛋白，而且还带有型特异、属特异抗原决定簇。它也是登革病毒吸附靶细胞、致病及诱导免疫保护的关键性蛋白，在登革病毒的致病机理及诱发机体的抗感染免疫中发挥重要的作用，对其结构和功能的分析一直成为 国内外登革研究的重点。E蛋白为登革病毒VAP(病毒表面特异性吸附蛋白,virus attachment protein),即宿主细胞表面病毒受体的配体,介导病毒特异性地与靶细胞结合。在中性和微碱性的条件下,成熟病毒体表面的E蛋白是以同源二聚体的形式存在,当E蛋白作为VAP介导病毒颗粒和靶细胞结合，以胞质内吞的途径入胞,吞噬体所处的内环境的pH值有所下降,E蛋白构象重排,由同源二聚体变为三聚体结构。构象的变化引起宿主细胞膜和病毒膜的融合,使得病毒基因组进入胞浆，引起感染。
登革热临床表现复杂多样、传播迅速、发病率高、人群易感染，严重影响了人们的生活质量和身体健康。而目前对登革感染仍缺乏疫苗及特效药物治疗，因此，特异、敏感、快速的诊断方法对登革热的流行和预防控制有重要意义。
发明内容
基于此，有必要提供一种能够用于登革热检测的登革病毒重组抗原及其制备方法和应用。
一种登革病毒重组抗原，包括：
(a)、如SEQ ID No.1所示的氨基酸序列组成的多肽；
(b)、与SEQ ID No.1所示的氨基酸序列组成的多肽具有至少98％同源性的多肽；或
(c)、如SEQ ID No.1所示的氨基酸序列组成的多肽，其中一个或多个氨基酸被缺失、替代或增加得到的多肽。
一种上述的登革病毒重组抗原的制备方法，包括如下步骤：
步骤一、提供基因表达载体，所述基因表达载体用于表达所述登革病毒重组抗原；
步骤二、将所述基因表达载体转化到原核表达菌中；
步骤三、对转化了所述基因表达载体的所述原核表达菌进行诱导表达，表达完成后收菌裂解，离心后保留第一沉淀；
步骤四、用缓冲液清洗所述第一沉淀后再次离心，保留第二沉淀，使用复溶液将所述第二沉淀再溶解，得到粗产物溶液；以及
步骤五、对所述粗产物溶液过Ni-NTA亲和柱纯化，再进行蛋白复性，得到所述登革病毒重组抗原。
在一个实施例中，所述缓冲液包括20mM的Tris-HCl、1mM的EDTA、50mM的NaCl和质量百分数为0.01的Triton X-100，所述缓冲液的pH为8；
所述复溶液包括6M的尿素和20mM的Tris，所述复溶液的pH为8。
在一个实施例中，所述对所述粗产物溶液过Ni-NTA亲和柱纯化的操作中，平衡液包括20mM的Tris-HCl、200mM的NaCl和6M的尿素，所述平衡液的pH为8，洗脱液包括20mM的Tris-HCl、200mM的NaCl、6M的尿素和250mM的咪唑，所述洗脱液的pH为8。
在一个实施例中，所述进行蛋白复性的操作为：分别用第一复性缓冲液、第二复性缓冲液和第三复性缓冲液在4℃下进行透析复性；
所述第一复性缓冲液包括4M的尿素、20mM的Tris-HCl和150mM的NaCl，所述第一复性缓冲液的pH为8；
所述第二复性缓冲液包括2M的尿素、20mM的Tris-HCl和150mM的NaCl，所述第二复性缓冲液的pH为8；
所述第三复性缓冲液包括1M的尿素、20mM的Tris-HCl和150mM的NaCl，所述第三复性缓冲液的pH为8。
上述的登革病毒重组抗原在制备登革病毒检测试剂领域或制备登革病毒检测设备领域的应用。
一种登革病毒检测试纸，含有上述的登革病毒重组抗原。
在一个实施例中，包括支撑薄片、样品垫、金标垫、硝酸纤维素膜、吸收垫、T1检测线、T2检测线及质控线；
所述样品垫、所述金标垫、所述硝酸纤维素膜和所述吸收垫从所述支撑薄片的一端向另一端依次设置在所述支撑薄片上，所述样品垫与所述金标垫部分重叠，所述金标垫与所述硝酸纤维素膜部分重叠，所述硝酸纤维素膜与所述吸收垫部分重叠；
所述T1检测线、所述T2检测线和所述质控线均设在所述硝酸纤维素膜上，所述T1检测线设在所述硝酸纤维素膜靠近所述金标垫的一端，所述T2检测线 设在所述硝酸纤维素膜靠近所述吸收垫的一端，所述质控线位于所述T1检测线和所述T2检测线中间；
所述金标垫上涂覆有胶体金标记的鼠IgG和胶体金标记的所述登革病毒重组抗原，所述T1检测线为抗人IgG抗体，所述T2检测线为抗人IgM抗体，所述质控线为羊抗鼠IgG抗体。
一种登革病毒检测试剂盒，包括上述的登革病毒检测试纸。
在一个实施例中，还包括壳体，所述登革病毒检测试纸设置在所述壳体内，并且所述壳体上设有加样孔和观察窗，所述加样孔与所述样品垫对应设置，所述T1检测线、所述T2检测线和所述质控线与所述观察窗对应设置。
经过试验验证，这种登革病毒重组抗原制得的检测试剂盒能够准确的检测出四种血清型登革病毒感染的样本，因此，这种登革病毒重组抗原可以用于登革热检测。
附图说明
图1为一实施方式的登革病毒检测试剂盒的结构示意图；
图2为如图1所示的登革病毒检测试剂盒的登革病毒检测试纸的正面结构示意图；
图3为如图1所示的登革病毒检测试剂盒的登革病毒检测试纸的剖面结构示意图；
图4为实施例1中得到的第一沉淀的SDS-PAGE电泳图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施的限制。
本发明公开了一种登革病毒重组抗原，包括：
(a)、如SEQ ID No.1所示的氨基酸序列组成的多肽；
(b)、与SEQ ID No.1所示的氨基酸序列组成的多肽具有至少98％同源性的多肽；或
(c)、如SEQ ID No.1所示的氨基酸序列组成的多肽，其中一个或多个氨基酸被缺失、替代或增加得到的多肽。
经过试验验证，这种登革病毒重组抗原制得的检测试剂盒能够准确的检测出四种血清型登革病毒感染的样本，因此，这种登革病毒重组抗原可以用于登革热检测。
本发明还公开了上述登革病毒重组抗原的制备方法，包括如下步骤：
步骤一、提供基因表达载体，基因表达载体用于表达上述登革病毒重组抗原。
基因表达载体的制备方法如下：首先在Genbank中搜集登革I～IV型病毒株基因的序列，建立登革I～IV型病毒株的基因数据，并且利用计算机软件对其进行分析，确定最具有检测活性的区段，将登革热4个血清型的最具检测活性的区段的基因序列直接串联起来后，进行人工合成，并在其上游引物引入BamHI位点，下游引物引入EcoRI位点且EcoRI位点之前带有6个His氨基酸的编码序列以及终止密码TAA，具体的氨基酸序列见SEQ ID No.1，合成目的片段后，用上下游引物进行PCR扩增，扩增获得的PCR片段经回收(本发明所使用的分子生物学提取和回收试剂盒均购自上海华舜生物工程有限公司)之后，用BamHI和EcoRI酶切(本发明所采用的各种分子生物学用酶均购自大连宝生物工程有限公司)，连接到用BamHI和EcoRI酶切之后的表达载体pET-24a(Novagen公司，货号：69864-3)中，得到的重组质粒pET-24a-DENI～IV即为所需的基因表达载体。
步骤二、将步骤一得到的基因表达载体转化到原核表达菌中。
原核表达菌可以为大肠杆菌。具体的，原核表达菌为BL21(DE3)(购自美国Invitrogen公司)。
步骤三、对步骤二得到的转化了基因表达载体的原核表达菌进行诱导表达，表达完成后收菌裂解，离心后保留第一沉淀。
步骤三具体为：将转化了基因表达载体的原核表达菌置于37℃、200rpm摇床过夜培养至菌体浓度为OD600＝2.814，加入IPTG(终浓度为0.5mM)进行诱导，190rpm，37度诱导约4h，收菌进行超声裂解，离心分离并保留第一沉淀。
步骤四、用缓冲液清洗步骤三得到的第一沉淀后再次离心，保留第二沉淀，使用复溶液将第二沉淀再溶解，得到粗产物溶液。
缓冲液包括20mM的Tris-HCl、1mM的EDTA、50mM的NaCl和质量百分数为0.01的Triton X-100，缓冲液的pH为8。
复溶液包括6M的尿素和20mM的Tris，复溶液的pH为8。
步骤五、对步骤四得到的粗产物溶液过Ni-NTA亲和柱纯化，再进行蛋白复性，得到登革病毒重组抗原。
对粗产物溶液过Ni-NTA亲和柱纯化的操作中，平衡液包括20mM的Tris-HCl、200mM的NaCl和6M的尿素，平衡液的pH为8，洗脱液包括20mM的Tris-HCl、200mM的NaCl、6M的尿素和250mM的咪唑，洗脱液的pH为8。
进行蛋白复性的操作为：分别用第一复性缓冲液、第二复性缓冲液和第三复性缓冲液在4℃下进行透析复性。
第一复性缓冲液包括4M的尿素、20mM的Tris-HCl和150mM的NaCl，所述第一复性缓冲液的pH为8。
第二复性缓冲液包括2M的尿素、20mM的Tris-HCl和150mM的NaCl，所述第二复性缓冲液的pH为8。
第三复性缓冲液包括1M的尿素、20mM的Tris-HCl和150mM的NaCl，所述第三复性缓冲液的pH为8。
透析复性后得到的登革病毒重组抗原透析至保存液(20mM Tris-HCl，150mM NaCl，pH 8.0)中，收集透析样品，测定蛋白浓度，置于-20℃备用。
这种登革病毒重组抗原可以应用于制备登革病毒检测试剂领域或制备登革病毒检测设备领域。
接下来，以上述登革病毒重组抗原应用于制备登革病毒检测试剂盒为例进行简单介绍。
如图1～图3所示的一实施方式的登革病毒检测试剂盒，包括登革病毒检测 试纸100和壳体200，登革病毒检测试纸100设置在壳体200内。
登革病毒检测试纸中含有上述登革病毒重组抗原。
结合图2和图3，登革病毒检测试纸100包括支撑薄片110、样品垫120、金标垫130、硝酸纤维素膜140、吸收垫150、T1检测线160、T2检测线180及质控线170。
样品垫120、金标垫130、硝酸纤维素膜140、吸收垫150从支撑薄片110的一端向另一端依次设置在支撑薄片110上，样品垫120与金标垫130部分重叠，金标垫130与硝酸纤维素膜140部分重叠，硝酸纤维素膜140与吸收垫150部分重叠。
T1检测线160、T2检测线180及质控线170设在硝酸纤维素膜140上，T1检测线160设在硝酸纤维素膜140靠近金标垫130的一端，T2检测线180设在硝酸纤维素膜140靠近吸收垫150的一端，质控线170位于T1检测线160和T2检测线180中间。
金标垫130上涂覆有胶体金标记的鼠IgG和胶体金标记的上述登革病毒重组抗原，T1检测线160为抗人IgG抗体，T2检测线180为抗人IgM抗体，质控线170为羊抗鼠IgG抗体。
结合图1，壳体200上设有加样孔210和观察窗220。加样孔210与样品垫120对应设置，T1检测线160、T2检测线180及质控线170与观察窗220对应设置。
这种登革病毒检测试剂盒利用捕获法来检测被检材料中的是否含有登革病毒特异性的IgG抗体和IgM抗体。检测时，若样品中含有登革病毒特异性的IgG抗体和IgM抗体，IgG抗体和IgM抗体先和胶体金标记的登革病毒重组抗原结合形成抗原抗体复合物，由于毛细管作用，抗原抗体复合物沿硝酸纤维素膜140向前泳动，到达T1检测线160和T2检测线180时，抗原抗体复合物会与相应的IgG抗体和IgM抗体结合，从而富集在T1检测线160和T2检测线180上，形成红色沉淀线。胶体金标记的鼠IgG则通过T1检测线160和T2检测线180，被羊抗鼠IgG抗体捕获，富集在质控线170上，形成红色沉淀线。当T1检测线160和T2检测线180与质控线170上同时有红色沉淀线时判为阳性结果。若样 品中不含有登革病毒特异性的IgG抗体和IgM抗体，胶体金标记的登革病毒重组抗原不会与T1检测线160和T2检测线180上的抗体结合，胶体金标记的登革病毒重组抗原则通过T1检测线160和T2被羊抗鼠IgG抗体捕获，并富集在质控线170上形成红色沉淀线，此时判为阴性结果。
此外，在其他实施方式中，登革病毒检测试剂盒的结构不限于上文描述。上述登革病毒重组抗原除应用在上述检测试剂盒外，还可以用于其他的登革病毒检测试剂盒或设备中。本领域技术人员可以理解，将本实施方式的登革病毒重组抗原直接或间接结合其他信号基团(如磁性微球、辣根过氧化酶等)，或将本实施方式的登革病毒重组抗原作为包被抗原(例如ELISA)，则可用于其他形式的登革病毒检测试剂或设备。故本实施方式所制备得到的登革病毒重组抗原可广泛适用于制备登革病毒检测试剂或设备。
特别需要指出的是，上述登革病毒检测试纸100可以单独使用。
通过使用上述登革病毒重组抗原，大大简化了登革病毒检测试剂盒的制备工艺，且将该登革病毒重组抗原应用于登革病毒检测试剂盒中，其表现了比国际同类产品更高的灵敏度和更强的特异性。
以下为具体实施例。
具体实施例中采用药物和仪器均为本领域常规选择。
实施例1、登革病毒重组抗原的制备
1、登革病毒重组抗原表达质粒的构建
首先在Genbank中搜集登革I～IV型病毒株基因的序列，建立登革I～IV型病毒株的基因数据，并且利用计算机软件对其进行分析，确定最具有检测活性的区段，将登革热4个血清型的最具检测活性的区段的基因序列直接串联起来后，进行人工合成，并在其上游引物引入BamHI位点，下游引物引入EcoRI位点且EcoRI位点之前带有6个His氨基酸的编码序列以及终止密码TAA，具体的氨基酸序列见SEQ ID No.1，合成目的片段后，用上下游引物进行PCR扩增，扩增获得的PCR片段经回收(本发明所使用的分子生物学提取和回收试剂盒均购自上海华舜生物工程有限公司)之后，用BamHI和EcoRI酶切(本发明所采用 的各种分子生物学用酶均购自大连宝生物工程有限公司)，连接到用BamHI和EcoRI酶切之后的表达载体pET-24a(Novagen公司，货号：69864-3)中，得到重组质粒pET-24a-DENI～IV，即本发明的标记抗原的重组质粒，表达的蛋白命名为DEN040。将上述构建好的重组质粒pET-24a-DENI～IV转化至宿主表达菌BL21(DE3)(购自美国Invitrogen公司)，得到重组质粒的表达菌株，备用。
2、登革病毒重组抗原的原核表达
缓冲液的配制：20mM Tris-HCl，1mM EDTA，50mM NaCl，0.01wt％Triton X-100，pH8.0。
将上述宿主表达菌置于37度200rpm摇床过夜培养至菌体浓度为OD600＝2.814，加入IPTG(终浓度为0.5mM)进行诱导，190rpm，37度诱导约4h，收菌进行超声裂解，离心分离上清和第一沉淀。
第一沉淀经SDS-PAGE电泳，得到图4。由图4可以看出，大部分目的蛋白为包涵体表达，保留沉淀进行后续处理。
沉淀用缓冲液洗杂3次，12000rpm×15min离心，去上清保留第二沉淀，第二沉淀用复溶液(6M尿素，20mM Tris，pH8.0)溶解，超声2min，4度环境放置30min后，再超声2min，13500rpm×30min，取上清，待进行Ni亲和。
3、登革病毒重组抗原的纯化和复性
平衡液的配制：20mM Tris-HCl，200mMNaCl，6M尿素，pH8.0。
洗脱液的配制：20mM Tris-HCl，200mMNaCl，6M尿素，再加入250mM咪唑，pH8.0。
配制好平衡液和洗脱液以后，利用ATKA purifier层析仪(购自GE医疗集团)，先用平衡液清洗平衡Ni-NTA亲和柱(Qiagen公司，货号30210)，Ni-NTA柱平衡好之后，将待上柱样品上柱结合，结合完之后，再用10倍柱体积的平衡液清洗柱子，然后再用洗脱液进行逐步洗脱，收集洗脱峰，测定蛋白浓度，待复性。
将上述Ni-NTA亲和纯化得到的目的蛋白用平衡液稀释浓度为1mg/mL，再分别用含有4M、2M、1M尿素的复性缓冲液(20mM Tris-HCl，150mM NaCl，pH 8.0)于4度进行透析复性，最终将目的蛋白透析至缓冲液(20mM Tris-HCl， 150mM NaCl，pH 8.0)中，收集透析样品，测定蛋白浓度，置于-20度备用。
实施例2、登革病毒检测试纸的制备
1、硝酸纤维素膜的制备
包被缓冲液的配制：含6％甲醇、0.01M pH7.2的PBS缓冲液为包被缓冲液，0.22μm膜滤过，置4℃备用，有效期一周。1000mL 6％甲醇的0.01M pH 7.2PBS缓冲液配方：NaCl 8g、KCl 0.2g、Na2HPO4·12H2O 2.9g、KH2PO4 0.2g、甲醇60mL，双蒸去离子水定容至1000mL。
硝酸纤维素膜的制备：用包被缓冲液将抗人IgG抗体(菲鹏生物股份有限公司生产，货号BA-PAB-MU0003)稀释到1～2mg/mL，调整机器，划线为T1检测线，即为登革病毒IgG抗体检测线，T1检测线靠近金标垫端，距金标垫端约5mm；用包被缓冲液将抗人IgM抗体(菲鹏生物股份有限公司生产，货号BA-PAB-MU0005)稀释到1～2mg/mL，调整机器，划线为T2检测线，即为登革病毒IgM抗体检测线，T2检测线位于T1检测线上面，距T1检测线约3～5mm；用包被缓冲液将羊抗鼠IgG抗体(菲鹏生物股份有限公司生产，货号BA-PAB-MU0001)稀释到1～5mg/mL，调整机器，划线为质控线，质控线靠近吸收垫，距吸收垫约3mm，与T2检测线距离5～8mm，均匀。37℃烘干，封装备用。
2、胶体金标记的鼠IgG和胶体金标记的登革病毒重组抗原的制备。
(1)溶液的配制。
①氯金酸的配制：用双蒸去离子水溶解氯金酸，配成1％溶液，置4℃备用，有效期四个月。1000mL 1％氯金酸溶液配方：10g氯金酸：双蒸去离子水定容至1000mL。
②柠檬酸三钠的配制：用双蒸去离子水溶解柠檬酸钠，配成1％溶液，0.22μm膜滤过，置4度备用，有效期容至1000mL。
③0.1M碳酸钾的配制：用双蒸去离子水配制，0.22μm膜滤过，置4度备用，有效期四个月。1000mL0.1M碳酸钾溶液配方：13.8g碳酸钾；双蒸去离子水定容至1000mL。
④标记洗涤保存液的配制：2％牛血清白蛋白(BSA)，0.05％叠氮钠(NaN3)，0.01M pH7.2PBS溶液，0.22μ膜滤过，置4℃备用，有效期四个月。1000mL标记洗涤保存液配方：20g BSA，0.5g NaN3、0.01M pH7.2PBS溶液定容至1000mL。
(2)胶体金的制备。
用双蒸去离子水将1％氯金酸稀释成0.01％，置电炉煮沸，按每100mL 0.01％氯金酸加入2mL 1％柠檬酸三钠，继续煮沸，直到液体呈亮红色即停止加热，冷却至室温后补足失水。制备好的胶体金外观应纯净、透亮、无沉淀和漂浮物，有效期一周。
(3)胶体金标记的登革病毒重组抗原的制备。
用0.1M碳酸钾调胶体金的pH值至8.2，按8～10μg抗体/mL胶体金加入实施例1中制备得到的登革病毒重组抗原，磁力搅拌器混匀30min，搅拌下加入BSA至终浓度为1％静置1小时。13000rpm、4℃离心30min，弃上清，沉淀用标记洗涤保存液洗涤两次，用十分之一初始胶体金体积的标记洗涤保存液将沉淀重悬，置4℃备用，有效期一周。
(4)胶体金标记鼠IgG的制备。
用0.1M碳酸钾调胶体金的pH值至8.2，按8～10μg抗体/mL胶体金加入鼠IgG(菲鹏生物股份有限公司，货号BA-PAB-MU0006)，磁力搅拌器混匀30min，搅拌下加入BSA至终浓度为1％静置1小时。13000rpm、4℃离心30min，弃上清，沉淀用标记洗涤保存液洗涤两次，用十分之一初始胶体金体积的标记洗涤保存液将沉淀重悬，置4℃备用，有效期一周。
3、金标垫的制备
(1)封闭液的配制。
2％BSA，0.1％TritonX-100、0.05％NaN3，0.01M pH7.2PBS溶液，0.22μm膜滤过，置4度备用，有效期四个月。1000mL封闭液配方：20g BSA，0.5g NaN3、1mL TritonX-100、0.01M pH7.2PBS溶液定容至1000mL。
(2)金标垫的制备。
将金标垫浸泡于封闭液中30min后，于37℃烘干。将胶体金标记的登革病毒重组抗原和胶体金标记的鼠IgG按照适当比例混合均匀，然后将混合好的胶 体金标记的登革病毒重组抗原和胶体金标记的鼠IgG均匀的铺在金标垫上，每毫升溶液铺20平方厘米，冷冻干燥，封装，置4℃备用。
4、试纸条样品垫的制备
(1)封闭液的配制。
2％BSA，0.1％TrtionX-100、0.05％NaN3，0.01M pH7.2PBS溶液，0.22μm膜滤过，置4度备用，有效期四个月。1000mL封闭液配方：20g BSA，0.5g NaN3、1mL TrtionX-100、0.01M pH7.2PBS溶液定容至1000mL。
(2)样品垫的制备。
将样品垫浸泡于封闭液中30min后，于37℃烘干，封装，置4℃备用。
5.登革病毒检测试纸的组装
将吸收垫(购自Millipore公司)、硝酸纤维素膜、金标垫、样品垫设置在不吸水的支撑薄片上，切成3mm宽的小条。每十小条一包，加入干燥剂，真空封装，得到登革病毒检测试纸。
实施例3、登革病毒检测试剂盒
1、快速检测登革病毒抗体IgG/IgM的试剂盒包括：实施例2制作的登革病毒检测试纸、壳体和样品稀释液。
登革病毒检测试纸放置到壳体中即可。
样品稀释液为8％NaCl溶液。配制方法：80gNaCl，加蒸馏水定容至1000mL。
2、胶体金法检测登革病毒抗体IgG/IgM。
(1)直接吸取10μL采集好的人血清或血浆加入到试纸卡加样孔，等待15min后即可观察结果。
(2)结果判定：当试纸条出现肉眼可见的红色质控线，没有出现肉眼可见的红色检测线，结果判为阴性；当试纸条出现肉眼可见的红色质控线，同时也出现肉眼可见的红色检测线，结果判为阳性。检测线颜色越深说明被检测样品的登革病毒抗体IgG或IgM水平越高。当试纸条没有出现肉眼可见的紫红色质控线，不管有没有出现肉眼可见的紫红色检测线，结果都判为检测试纸条失效，应废弃。
实施例4、登革病毒检测试剂盒
以商业化的主流产品SD Dengue IgG/IgM试剂盒和实施例3得到的登革病毒检测试剂盒(简称FP dengue IgG/IgM试剂盒)做对比，同时检测97份登革阳性标本和478份临床阴性标本，这97例阳性标本再通过商品化的登革病毒分型多重PCR试剂盒进行RT-PCR分型检测，其中37份为II型登革病毒感染，23份为I型登革病毒感染、15份为III型登革病毒感染、22份为IV型登革病毒感染。结果显示，FP Dengue IgG/IgM试剂盒能检出上述97份阳性标本中的96份，而SD Dengue IgG/IgM试剂盒检出阳性标本94份，对于478份临床阴性标本，FP试剂盒检出假阳18份，SD试剂盒检出假阳23份，具体见表1。
Figure PCTCN2016112374-appb-000001
上述结果说明实施例3得到的登革病毒检测试剂盒在灵敏度和特异性方面均优于SD的试剂盒，完全可以用于登革病毒的快速诊断。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种登革病毒重组抗原，其特征在于，包括：
    (a)、如SEQ ID No.1所示的氨基酸序列组成的多肽；
    (b)、与SEQ ID No.1所示的氨基酸序列组成的多肽具有至少98％同源性的多肽；或
    (c)、如SEQ ID No.1所示的氨基酸序列组成的多肽，其中一个或多个氨基酸被缺失、替代或增加得到的多肽。
  2. 一种根据权利要求1所述的登革病毒重组抗原的制备方法，其特征在于，包括如下步骤：
    步骤一、提供基因表达载体，所述基因表达载体用于表达所述登革病毒重组抗原；
    步骤二、将所述基因表达载体转化到原核表达菌中；
    步骤三、对转化了所述基因表达载体的所述原核表达菌进行诱导表达，表达完成后收菌裂解，离心后保留第一沉淀；
    步骤四、用缓冲液清洗所述第一沉淀后再次离心，保留第二沉淀，使用复溶液将所述第二沉淀再溶解，得到粗产物溶液；以及
    步骤五、对所述粗产物溶液过Ni-NTA亲和柱纯化，再进行蛋白复性，得到所述登革病毒重组抗原。
  3. 根据权利要求2所述的制备方法，其特征在于，所述缓冲液包括20mM的Tris-HCl、1mM的EDTA、50mM的NaCl和质量百分数为0.01的Triton X-100，所述缓冲液的pH为8；
    所述复溶液包括6M的尿素和20mM的Tris，所述复溶液的pH为8。
  4. 根据权利要求2所述的制备方法，其特征在于，所述对所述粗产物溶液过Ni-NTA亲和柱纯化的操作中，平衡液包括20mM的Tris-HCl、200mM的NaCl和6M的尿素，所述平衡液的pH为8，洗脱液包括20mM的Tris-HCl、200mM的NaCl、6M的尿素和250mM的咪唑，所述洗脱液的pH为8。
  5. 根据权利要求2所述的制备方法，其特征在于，所述进行蛋白复性的操作为：分别用第一复性缓冲液、第二复性缓冲液和第三复性缓冲液在4℃下进行 透析复性；
    所述第一复性缓冲液包括4M的尿素、20mM的Tris-HCl和150mM的NaCl，所述第一复性缓冲液的pH为8；
    所述第二复性缓冲液包括2M的尿素、20mM的Tris-HCl和150mM的NaCl，所述第二复性缓冲液的pH为8；
    所述第三复性缓冲液包括1M的尿素、20mM的Tris-HCl和150mM的NaCl，所述第三复性缓冲液的pH为8。
  6. 根据权利要求1所述的登革病毒重组抗原在制备登革病毒检测试剂领域或制备登革病毒检测设备领域的应用。
  7. 一种登革病毒检测试纸，其特征在于，含有根据权利要求1所述的登革病毒重组抗原。
  8. 根据权利要求7所述的登革病毒检测试纸，其特征在于，包括支撑薄片、样品垫、金标垫、硝酸纤维素膜、吸收垫、T1检测线、T2检测线及质控线；
    所述样品垫、所述金标垫、所述硝酸纤维素膜和所述吸收垫从所述支撑薄片的一端向另一端依次设置在所述支撑薄片上，所述样品垫与所述金标垫部分重叠，所述金标垫与所述硝酸纤维素膜部分重叠，所述硝酸纤维素膜与所述吸收垫部分重叠；
    所述T1检测线、所述T2检测线和所述质控线均设在所述硝酸纤维素膜上，所述T1检测线设在所述硝酸纤维素膜靠近所述金标垫的一端，所述T2检测线设在所述硝酸纤维素膜靠近所述吸收垫的一端，所述质控线位于所述T1检测线和所述T2检测线中间；
    所述金标垫上涂覆有胶体金标记的鼠IgG和胶体金标记的所述登革病毒重组抗原，所述T1检测线为抗人IgG抗体，所述T2检测线为抗人IgM抗体，所述质控线为羊抗鼠IgG抗体。
  9. 一种登革病毒检测试剂盒，其特征在于，包括如权利要求7或8所述的登革病毒检测试纸。
  10. 根据权利要求9所述的登革病毒检测试剂盒，其特征在于，还包括壳体，所述登革病毒检测试纸设置在所述壳体内，并且所述壳体上设有加样孔和 观察窗，所述加样孔与所述样品垫对应设置，所述T1检测线、所述T2检测线和所述质控线与所述观察窗对应设置。
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