WO2021072607A1 - 用于检测hcv抗体的试剂盒以及方法 - Google Patents

用于检测hcv抗体的试剂盒以及方法 Download PDF

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WO2021072607A1
WO2021072607A1 PCT/CN2019/111102 CN2019111102W WO2021072607A1 WO 2021072607 A1 WO2021072607 A1 WO 2021072607A1 CN 2019111102 W CN2019111102 W CN 2019111102W WO 2021072607 A1 WO2021072607 A1 WO 2021072607A1
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hcv
antigen
fusion protein
protein
enzyme
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PCT/CN2019/111102
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English (en)
French (fr)
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李可
江明
何建文
张裕平
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深圳迈瑞生物医疗电子股份有限公司
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Priority to PCT/CN2019/111102 priority Critical patent/WO2021072607A1/zh
Priority to CN201980101071.1A priority patent/CN114502956A/zh
Priority to EP19949154.9A priority patent/EP4047369A4/en
Publication of WO2021072607A1 publication Critical patent/WO2021072607A1/zh

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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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • 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
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • 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
    • G01N2333/186—Hepatitis C; Hepatitis NANB
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the embodiments of the present application relate to the field of Hepatitis C Virus (HCV) antibody detection, and specifically relate to HCV antibody immunoassay, especially enzyme markers used in the double antigen sandwich method or capture method.
  • HCV Hepatitis C Virus
  • Hepatitis C is a disease caused by Hepatitis C Virus (HCV) and mainly spread through blood. Chronic HCV infection can lead to chronic inflammation and necrosis of the liver and liver fibrosis. Some patients can develop cirrhosis or even hepatocellular carcinoma (HCC), which is extremely harmful to the health and life of patients, and has become a serious social and public health problem. Hepatitis C is a global epidemic and is the leading cause of end-stage liver disease in Europe, America, Japan and other countries. In the United States, the number of people infected with HCV is four times that of people living with HIV. By 2010, more people will die from HCV infection. The number of people who died from HIV infection. According to statistics from the World Health Organization, the global HCV infection rate is about 3%. It is estimated that about 170 million people are infected with HCV, and about 35,000 new cases of hepatitis C occur every year.
  • HCV Hepatitis C Virus
  • HCV immunoassay methods include enzyme-linked immunosorbent assay, chemiluminescence immunoassay, gold labeling method, fluorescence immunoassay and so on.
  • enzyme immunoassay techniques such as enzyme-linked immunosorbent assay and enzymatic chemiluminescence immunoassay are common HCV immunoassay methods that use labeled enzymes as reporter molecules.
  • enzyme immunoassay The conjugate of the labeling enzyme and the antigen substance in the conjugate, the antibody to be tested in the sample and the antigen substance coated on the solid support form a sandwich structure. By analyzing the labeling enzyme in the sandwich structure, In order to obtain qualitative or quantitative detection results of HCV.
  • the conjugate of the labeling enzyme and the antigen substance in the enzyme immunoassay, the test antibody present in the sample, and the anti-human IgG and anti-human IgM antibodies coated on the solid support are formed
  • the qualitative or quantitative detection result of HCV can be obtained by analyzing the labeling enzyme in the complex.
  • a chemical activation cross-linking method is used to link the labeling enzyme with the antigen substance to form a cross-linked or conjugate.
  • this connection process has the disadvantage of complicated operation and difficult to control; in addition, this connection method makes the molecular molar ratio of the enzyme and the antigen non-uniform, resulting in the product being a mixture of enzyme and antigen cross-linked products with different molecular molar ratios.
  • the embodiment of the present application provides an immunoassay kit for hepatitis C virus HCV antibody, which includes a fusion protein of a labeling enzyme and an HCV antigenic protein.
  • antigenic protein or “antigenic substance” refers to a protein that is immunoreactive and can be used for HCV immunological detection; it can be one HCV antigen or a fragment thereof, or two (Species) of fusion antigens of the above HCV antigens or fragments thereof (or chimeric proteins of two (species) or more of HCV antigens or fragments thereof).
  • the antigenic protein contains one or more of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen, or contains HCV core antigen, HCV NS3 antigen, HCV NS4 antigen And HCV NS5 antigen of two or more fusion antigens.
  • Core, NS3, NS4, and NS5 are the structure and membrane proteins of hepatitis C virus. A large number of studies have shown that Core, NS3, NS4, and NS5 not only have high immunogenicity but also can be used as hepatitis C virus diagnostic antigens.
  • HCV antigen refers to a substance that has immunoreactivity and can be used for HCV immunodetection, which is selected from the conserved proteins of HCV or fragments thereof.
  • exemplary HCV antigen may be HCV core antigen, HCV NS3 antigen, HCV NS4 antigen or HCV NS5 antigen.
  • the HCV antigen may exist in the form of one or more copies.
  • the HCV antigenic protein of the present application contains HCV core antigen.
  • the HCV antigenic protein of the present application contains HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen.
  • the antigenic protein is a chimeric protein or fusion antigen containing an HCV core antigen and one or more additional HCV antigens.
  • the additional HCV antigen may be an HCV antigen with high immunoreactivity, for example, one or more of HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen.
  • the HCV antigenic protein of the present application is a chimeric protein containing HCV core antigen and HCV NS3, or a chimeric protein containing HCV core antigen, HCV NS3 antigen, and HCV NS4, or is a chimeric protein containing HCV Core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen chimeric protein.
  • the antigenic protein may contain two or more HCV antigens, these HCV antigens may be present in any order.
  • the antigenic protein of the present application may sequentially contain HCV core antigen and HCV NS3 antigen from N-terminal to C-terminal; or, may contain HCV NS3 antigen and HCV-core antigen in sequence from N-terminal to C-terminal.
  • the HCV antigenic protein of the present application contains HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen in order from N-terminal to C-terminal.
  • the antigenic protein of the present application contains HCV NS3 antigen, HCV NS4 antigen, HCV core antigen, and HCV NS5 antigen in order from N-terminus to C-terminus.
  • the antigenic protein of the present application contains HCV NS3 antigen, HCV core antigen, HCV NS4 antigen, and HCV NS5 antigen in order from N-terminus to C-terminus.
  • the HCV antigens can be directly connected or can be connected through a Linker, as long as the HCV antigens are connected while ensuring that their respective structures and activities are not affected.
  • a flexible linker such as flexible (Gly 4 Ser) n , GGGS, GGSGGGSG, etc., is used.
  • enzyme and “enzyme for labeling” can be used interchangeably and refer to enzymes used in enzyme immunoassays.
  • it may be an enzyme used in an enzyme-linked immunosorbent method and an enzymatic chemiluminescence immunoassay method.
  • the enzyme of the present application may be alkaline phosphatase (EC 3.1.3.1), which, for example, can catalyze the hydrolysis of nitrophenyl phosphate (PNP), sodium ⁇ -glycerophosphate, naphthyl phosphate, 3-( 2-Spiraladamantane)-4-methoxy-4-(3-phosphooxy)-phenyl-1,2-dioxetane (AMPPD) and other chromogenic substrates and chemistry containing phosphate groups Luminescent substrate.
  • PNP nitrophenyl phosphate
  • AMPPD 3-( 2-Spiraladamantane)-4-methoxy-4-(3-phosphooxy)-phenyl-1,2-dioxetane
  • the alkaline phosphatase in the embodiments of the present application may be naturally occurring, artificially synthesized or produced through genetic engineering.
  • the alkaline phosphatase in the embodiments of the present application may be modified, such as an alkaline phosphatase treated with surface glycosylation or deglycosylation.
  • alkaline phosphatase As for the source of alkaline phosphatase, the examples of the present application are not particularly limited, as long as the enzyme immunoassay can be implemented.
  • Exemplary alkaline phosphatase can be derived from bacteria, such as Escherichia coli; mammals, such as cattle (such as Genebank: AF052227.1 (source https://www.ncbi.nlm.nih.gov/)) or humans (such as Genebank: M12551.1); shrimp; but not limited to this.
  • the enzyme of the present application may be horseradish peroxidase (EC 1.11.1.7), which uses iron porphyrin as a prosthetic group, which can catalyze the polymerization of phenol, aniline and their substitutes in the presence of hydrogen peroxide , Widely distributed in the plant kingdom, the highest content in horseradish.
  • horseradish peroxidase EC 1.11.1.7
  • iron porphyrin as a prosthetic group, which can catalyze the polymerization of phenol, aniline and their substitutes in the presence of hydrogen peroxide , Widely distributed in the plant kingdom, the highest content in horseradish.
  • the horseradish peroxidase in the examples of this application may be naturally occurring, artificially synthesized or produced through genetic engineering.
  • the horseradish peroxidase in the examples of the present application may be modified.
  • the enzymes of the present application also include mutants thereof. Compared with the wild type, the mutants of the enzymes of the embodiments of the present application have sequence homology of greater than 80%, optionally greater than 85%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%.
  • An exemplary alkaline phosphatase mutant may be GeneBank: M29670.1 (source https://www.ncbi.nlm.nih.gov/), but the examples of the application are not limited thereto.
  • An exemplary horseradish peroxidase mutant may be GnenBank: XM_018585035.1, but the embodiments of the present application are not limited thereto.
  • sequence homology To determine sequence homology, the wild-type sequence is used as a reference sequence, and the sequence to be tested is compared with the reference sequence. Then, a sequence comparison algorithm is used to calculate the sequence homology of the test sequence relative to the reference sequence.
  • Two examples of algorithms suitable for determining sequence homology are the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25: 3389-3402, and Altschul et al. (1990) ) J. Mol. Biol. 215:403-410.
  • the software used to perform BLAST analysis is publicly available through NCBI.
  • the enzyme can be fused to any position of the antigenic protein.
  • the enzyme can be fused to the N-terminus of the HCV antigenic protein; or, the enzyme can be fused to the C-terminus of the antigenic protein; or, the enzyme can be fused between any two adjacent antigens in the antigenic protein.
  • the fusion protein of HCV antigenic protein and enzyme contains enzyme, HCV core antigen, HCV NS3 antigen and HCV NS4 antigen in order from N-terminal to C-terminal.
  • the fusion protein of HCV antigenic protein and enzyme contains enzyme, HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen in order from N-terminal to C-terminal.
  • the fusion protein of HCV antigenic protein and enzyme contains HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, HCV NS5 antigen and enzyme in order from N-terminal to C-terminal.
  • the fusion protein of HCV antigenic protein and enzyme contains HCV core antigen, HCV NS3 antigen, enzyme, HCV NS4 antigen, and HCV NS5 antigen in order from N-terminus to C-terminus.
  • the fusion protein of the present application contains alkaline phosphatase, HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen; or HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, HCV NS5 antigen and alkaline phosphatase; or HCV core antigen, HCV NS3 antigen, alkaline phosphatase, HCV NS4 antigen and HCV NS5 antigen; or horseradish peroxidase, HCV core antigen, HCV NS3 antigen , HCV NS4 antigen and HCV NS5 antigen; or HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, HCV NS5 antigen and horseradish peroxidase; or HCV core antigen, HCV NS3 antigen, horseradish peroxidase, HCV NS3 antigen, horseradish peroxidase, HCV NS
  • the immunoassay kit for hepatitis C virus HCV antibody may include a fusion protein of a labeling enzyme and HCV core antigen, a fusion protein of a labeling enzyme and HCV NS3 antigen, and a labeling enzyme and HCV NS4 antigen. At least one of the fusion protein and the fusion protein of the labeling enzyme and the HCV NS5 antigen.
  • the immunoassay kit includes a fusion protein of a labeling enzyme and HCV core antigen, a fusion protein of a labeling enzyme and HCV NS3 antigen, a fusion protein of a labeling enzyme and HCV NS4 antigen, and a labeling enzyme Fusion protein with HCV NS5 antigen.
  • the immunoassay kit may include a fusion protein of alkaline phosphatase and HCV core antigen, a fusion protein of alkaline phosphatase and HCV NS3 antigen, a fusion protein of alkaline phosphatase and HCV NS4 antigen, and alkali At least one of the fusion proteins of sexual phosphatase and HCV NS5 antigen, or a fusion protein of horseradish peroxidase and HCV core antigen, a fusion protein of horseradish peroxidase and HCV NS3 antigen, horseradish At least one of the fusion protein of peroxidase and HCV NS4 antigen and the fusion protein of horseradish peroxidase and HCV NS5 antigen.
  • the enzyme can be directly connected to the antigenic protein or can be connected through a Linker, as long as the enzyme and the antigenic protein are connected while ensuring that their respective structures and activities are not affected.
  • a flexible linker such as flexible (Gly 4 Ser) n , GGGS, GGSGGGSG, etc., is used.
  • fusion protein refers to a fusion protein of an enzyme and an antigenic protein.
  • it can be expressed as enzyme+core+NS3.
  • it refers to a fusion protein consisting of enzyme, HCV core antigen, and HCV NS3 antigen in sequence from N-terminal to C-terminal.
  • the fusion protein of enzyme and HCV antigenic protein can be prepared by conventional recombinant expression technology.
  • the recombinant expression technology can be prokaryotic expression technology, such as E. coli expression technology, and eukaryotic expression technology, such as yeast expression technology and insect cell expression technology.
  • kits of the embodiments of the present application may also include other reagents or components for measuring HCV antibodies based on the double antigen sandwich method or capture method, for example, a solid support coated with HCV antigenic protein.
  • HCV antigenic protein coated on the solid support and the HCV antigenic protein in the fusion protein can bind to the same HCV antibody in the sample) or coated with anti-human IgM antibodies and anti-human IgG antibodies
  • the kit includes a solid phase support coated with a fusion antigen containing HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen; or a solid phase coated with HCV core antigen Support, solid support coated with HCV NS3 antigen, solid support coated with HCV NS4 antigen, and solid support coated with HCV NS5 antigen.
  • the embodiments of the present application also relate to the application of the fusion protein of enzyme and HCV antigenic protein in preparing an immunoassay kit for detecting HCV.
  • the immunoassay kit includes:
  • the first reagent which contains a solid-phase coating, and the solid-phase coating is coated with a first ligand, and the first ligand can bind to the HCV antibody in the sample;
  • the second reagent which contains an enzyme label
  • the enzyme label is a second ligand fused with an enzyme, wherein the second ligand is an HCV antigenic protein and can bind to the first ligand HCV antibody.
  • HCV is detected based on a double antigen sandwich method or a capture method.
  • an immunoassay kit for detecting HCV including:
  • the first reagent which contains a solid-phase coating, and the solid-phase coating is a solid-phase support coated with an HCV antigenic protein (first ligand);
  • the second reagent which contains an enzyme marker
  • the enzyme marker is an enzyme-fused HCV antigenic protein (second ligand), wherein the HCV antigenic protein coated on the solid support and the The HCV antigenic protein in the fusion protein can bind to the same HCV antibody in the sample;
  • the instruction manual describes the detection based on the double antigen sandwich method.
  • the HCV antigenic protein coated on the solid support in the first reagent and the HCV antigenic protein fused in the fusion protein in the second reagent are of the same type, but the source or multiple antigens
  • the connection mode or connection sequence between them can be the same or different.
  • the HCV antigenic protein coated on the solid support can be a fusion antigen of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen that are sequentially linked, and the HCV fused in the fusion protein in the second reagent
  • the antigenic protein may be a fusion antigen of HCV core antigen, HCV NS4 antigen, HCV NS5 antigen, and HCV NS3 antigen that are sequentially connected.
  • the HCV antigenic protein coated on the solid support can be the HCV antigenic protein as described above, or it can be connected in other ways, such as chemically connected.
  • HCV antibodies refer to antibodies produced in a subject after HCV infection, such as Anti-HCV IgG antibodies and Anti-HCV IgM antibodies.
  • an immunoassay kit for detecting HCV including:
  • the first reagent contains a solid-phase coating, and the solid-phase coating is a solid support coated with an anti-human IgM antibody and an anti-human IgG antibody (first ligand);
  • a second reagent which contains an enzyme label, and the enzyme label is a fusion protein of an enzyme and an HCV antigenic protein (second ligand);
  • the manual which describes the detection based on the capture method.
  • solid support refers to a solid surface to which antigens or antibodies can be attached.
  • solid support used in the examples of this application, and commercial solid supports and any solid support that can be used for immunoassays can be used in this application.
  • Exemplary solid supports can be magnetic beads (such as superparamagnetic microspheres), enzyme-labeled plates, plastic plates, plastic tubes, latex beads, agarose beads, glass, nitrocellulose membrane, nylon membrane, silicon dioxide Board or microchip, but the embodiment of the present application is not limited thereto.
  • the solid-phase coating may be present in a conventional diluent containing protein and surfactant and having buffering capacity.
  • the enzyme marker may be present in a conventional diluent containing protein and surfactant and having buffering capacity.
  • the fusion protein may be present in the second reagent at a concentration of, for example, about 50 ng/mL to 150 ng/mL.
  • the kit of the present application may further include a third reagent, which contains a blocking agent and a surfactant.
  • a blocking agent is selected from one or more of the following items: skimmed milk powder, BSA, gelatin, serum, casein, ovalbumin, animal IgG, surfactants (e.g., Tween- 20, Tween-80, TritonX-100, etc.).
  • the kit of the present application may further include a fourth reagent, which contains a reducing agent.
  • a reducing agent is selected from one or more of the following items: DTT, ⁇ -mercaptoethanol.
  • the blocking agent and the surfactant can be dissolved in a conventional diluent with buffering capacity; the reducing agent can be dissolved in a conventional diluent with buffering capacity.
  • the kit may further include a reaction substrate of the labeling enzyme.
  • the reaction substrate is 3-(2-spiradamantane)-4-methoxy-4-( 3-phosphooxy)-phenyl-1,2-dioxetane.
  • the embodiment of the present application also relates to a method for detecting HCV antibodies produced after infection of hepatitis C virus HCV in a sample with a fusion protein of an enzyme and an HCV antigenic protein, which includes the following steps:
  • the enzyme label is a fusion protein of a labeling enzyme and an HCV antigenic protein
  • a chemiluminescence substrate is added to the cleaned sandwich composite, and the number of photons generated by the reaction is detected to obtain the chemiluminescence signal value of the sample.
  • the first ligand is an HCV antigenic protein, or is an anti-human IgG antibody and an anti-human IgM antibody.
  • a fusion protein of a labeling enzyme and an HCV antigenic protein is obtained through recombinant expression, and is applied to an HCV immunoassay kit based on a dual antigen sandwich or capture method mode. It should be noted:
  • the fusion protein of the labeling enzyme and the HCV antigenic protein in the kit has a uniform molar ratio of the labeling enzyme to the HCV antigenic protein , To avoid the problem of large differences in kit production and test results between batches.
  • kits of the embodiments of the present application show better discrimination between negative and positive samples, and have a better sample coincidence rate.
  • the kit containing the fusion protein of the embodiments of the present application avoids the destruction of antigen activity or enzyme activity caused by the chemical activation cross-linking method.
  • the embodiments of the present application save the steps of chemical cross-linking reaction, making the operation more convenient and faster.
  • the fusion protein of enzyme and HCV antigenic protein in the examples of this application can be produced by recombinant expression technology.
  • An exemplary recombinant expression method may include the following steps:
  • the nucleic acid molecule encodes the above-mentioned fusion protein; and the expression vector is used to prepare the fusion protein of the enzyme and the antigenic protein of HCV by recombinant expression, and the expression vector contains the above-mentioned fusion protein that is operably linked to the expression control sequence.
  • the nucleic acid molecule in addition contains genetic elements for maintenance and reproduction in the respective host cell, such as an origin of replication and/or a selectable marker gene.
  • Step 1 Confirm the target gene.
  • the gene sequence of linker is GGA GGA GGA GGA TCA GGA GGA GGA GGA TCA GGA GGA GGA GGA TCA, and its corresponding amino acid sequence
  • the protein sequence (Gly 4 Ser) 3 add a His tag to the C-terminal of the fusion protein for purification, and initially determine the target gene sequence. Then through codon optimization, the final target gene sequence suitable for E. coli expression strain BL21 (DE3) was determined.
  • the second step vector construction. Select the appropriate restriction site, and integrate the target gene into the appropriate expression vector pET28a.
  • the third step transformation and screening of positive clone strains. Transform the expression vector containing the target gene into E. coli by electroporation or heat shock transformation. According to the reporter gene on the expression vector, appropriate antibiotics are added to screen out positive strains.
  • the fourth step protein expression and purification.
  • HCV antigen nucleic acid sequence information Core, GeneBank; AJ222676.1; NS3, GeneBank: EU84764.1; NS4, GeneBank: Z84363.1; NS5, GeneBank: S70341.1 (source https://www.ncbi.nlm.nih .gov/).
  • the first reagent Ra (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 + (0.05 * (1 +
  • a diluent with buffering capacity contains BSA, Tween-20 and skimmed milk powder.
  • a diluent with buffering capacity and contains DDT is a diluent with buffering capacity and contains DDT.
  • the first step add the sample, the fourth reagent and the first reagent to the reaction tube, and incubate at 37°C for 10 minutes to make the HCV antigen coated on the solid phase of the magnetic beads and the Anti-HCV IgG and Anti-HCV IgM in the sample
  • the antibody is fully bound; after the incubation is completed, the magnetic bead solid phase is placed in a magnetic field to be attracted, and the material bound to the magnetic bead solid phase is retained, while other unbound materials are washed and removed.
  • Step 2 Add the third reagent and the second reagent to the reaction tube, and mix; incubate at 37°C for 10 minutes, the HCV antigen on the enzyme label and the Anti-HCV IgG and Anti-anti-captured on the magnetic beads
  • the HCV IgM antibody binds to form a sandwich complex. After the incubation in the reaction tube is completed, the complex is attracted by the magnetic field, and other unbound substances are washed and removed.
  • the third step adding the chemiluminescence substrate to the reaction tube to generate chemiluminescence.
  • the number of photons produced by the reaction is measured by a photomultiplier tube to obtain the chemiluminescence signal value of the sample.
  • COI Cutoff index
  • RLU chemiluminescence signal value
  • COI ⁇ 1 indicates that the measurement sample is a positive sample
  • COI ⁇ 1 indicates that the measurement sample is Negative samples.
  • the threshold (cutoff value) is the cutoff value for judging whether the test result is positive or negative.
  • the negative coincidence rate refers to the ratio of the number of samples judged to be negative using the test method of the embodiment of this application to the negative samples actually participating in the evaluation
  • the positive coincidence rate refers to the test using the embodiment of this application
  • the method obtains the proportion of the number of samples judged to be positive to the positive samples actually participating in the evaluation; the true negative and positive results of the samples come from the diagnosis results of the hospital.
  • Combination 1 Using the E. coli expression system described above, the Core+NS3+NS4+NS5 chimeric protein (or fusion antigen) is used as the HCV antigenic protein to be fused with alkaline phosphatase to prepare the second reagent Rb, wherein The alkaline phosphatase (Genebank: AF052227.1 (source https://www.ncbi.nlm.nih.gov/)) in the fusion protein of the second reagent Rb is fused to the N of the Core+NS3+NS4+NS5 chimeric protein The end is prepared according to the above-mentioned "Reagent Preparation Method".
  • Combination 2 The chemically linked alkaline phosphatase and Core+NS3+NS4+NS5 chimeric protein conjugate is used to prepare the second reagent Rb, and the rest is the same as Combination 1.
  • a reagent combination 3 the coupling of chemically linked alkaline phosphatase with anti-human IgG and anti-human IgM was prepared The second reagent Rb was prepared with the compound, and the rest was the same as the combination 1.
  • the above-mentioned combination 3 was used to test the above-mentioned 500 cases of HCV negative samples and 100 cases of positive samples.
  • the specific steps of "indirect detection method” are as follows:
  • the first step add the sample, the fourth reagent and the first reagent to the reaction tube, and incubate at 37°C for 10 minutes to make the HCV antigen coated on the solid phase of the magnetic beads and the Anti-HCV IgG and Anti-HCV IgM in the sample
  • the antibody is fully bound; after the incubation is completed, the magnetic bead solid phase is placed in a magnetic field to be attracted, and the material bound to the magnetic bead solid phase is retained, while other unbound materials are washed and removed.
  • Step 2 Add the third reagent and the second reagent to the reaction tube and mix; incubate at 37°C for 10 minutes, and the anti-human IgG and anti-human IgM secondary antibodies on the enzyme label will be captured on the magnetic beads.
  • Anti-HCV IgG and Anti-HCV IgM antibodies combine to form a sandwich complex. After the incubation in the reaction tube is completed, the complex is attracted by the magnetic field, and other unbound substances are washed and removed.
  • the third step adding the chemiluminescence substrate to the reaction tube to generate chemiluminescence.
  • the number of photons produced by the reaction is measured by a photomultiplier tube to obtain the chemiluminescence signal value of the sample.
  • Combination 4 Using the E. coli expression system described above, Core+NS3+NS4+NS5 is used as the antigenic protein to be fused with horseradish peroxidase to prepare the second reagent Rb.
  • the fusion protein of the second reagent Rb is spicy Root peroxidase (GeneBank: KU504630.1 (source https://www.ncbi.nlm.nih.gov/)) is located at the N-terminus of the antigenic protein and is prepared according to the above "reagent preparation method”.
  • Combination 5 The second reagent Rb is prepared with a chemically linked horseradish peroxidase and Core+NS3+NS4+NS5 chimeric protein conjugate, and the rest is the same as Combination 4.
  • reagent combination 6 was prepared: using chemically linked horseradish peroxidase and anti-human IgG and anti-human IgM
  • the second reagent Rb was prepared from the conjugate, and the rest was the same as the combination 4.
  • the above-mentioned combination 6 was used to test the above-mentioned 500 cases of HCV negative samples and 100 cases of positive samples. The results are shown in Table 1.
  • the indirect method test (combination 6) can better distinguish between negative and positive samples, indicating that the Core+NS3+NS4+NS5 chimeric protein used in the examples of this application has good activity.
  • Combination 1 Using the E. coli expression system described above, the Core+NS3+NS4+NS5 chimeric protein is used as the antigenic protein to be fused with alkaline phosphatase to configure the second reagent Rb, the fusion protein of the second reagent Rb Alkaline phosphatase is fused to the N-terminus of the Core+NS3+NS4+NS5 chimeric protein, and is prepared according to the above-mentioned "reagent preparation method".
  • Combination 7 In the fusion protein of the second reagent Rb, alkaline phosphatase is fused to the C-terminus of the Core+NS3+NS4+NS5 chimeric protein, and the rest is the same as Combination 1.
  • Combination 8 In the fusion protein of the second reagent Rb, alkaline phosphatase is fused between NS3 and NS4 of the Core+NS3+NS4+NS5 chimeric protein, and the rest is the same as Combination 1.
  • Combination 4 Using the E. coli expression system described above, the Core+NS3+NS4+NS5 chimeric protein is used as the antigenic protein to be fused with horseradish peroxidase to configure the second reagent Rb and the fusion of the second reagent Rb The horseradish peroxidase in the protein was fused to the N-terminus of the Core+NS3+NS4+NS5 chimeric protein, which was prepared according to the above "reagent preparation method".
  • Combination 9 In the fusion protein of the second reagent Rb, horseradish peroxidase is fused to the C-terminus of the Core+NS3+NS4+NS5 chimeric protein, and the rest is the same as Combination 4.
  • Combination 10 In the fusion protein of the second reagent Rb, horseradish peroxidase is fused between NS3 and NS4 of the Core+NS3+NS4+NS5 chimeric protein, and the rest is the same as Combination 4.
  • Combination 1 The antigenic protein is Core+NS3+NS4+NS5 from the N-terminal to the C-terminal.
  • the alkaline phosphatase in the fusion protein is fused to the N-terminal of the antigenic protein, which is prepared according to the above "reagent preparation method".
  • Combination 11 The antigenic protein is NS3+NS4+core+NS5 from N-terminus to C-terminus, and the rest is the same as Combination 1.
  • Combination 12 The antigenic protein is NS3+core+NS4+NS5 from N-terminus to C-terminus, and the rest is the same as Combination 1.
  • Example 1 use combinations 1, 11, and 12 to test the 500 HCV negative samples and 100 positive samples confirmed in Example 1 according to the above-mentioned "dual antigen sandwich detection method". The results are shown in Table 5 below. .
  • Combination 1 Using the E. coli expression system described above, the Core+NS3+NS4+NS5 chimeric protein is used as the antigenic protein to be fused with wild-type alkaline phosphatase to prepare the second reagent Rb, the wild-type base in the fusion protein The phosphatase is fused to the N-terminus of the antigenic protein and prepared according to the above-mentioned "reagent preparation method".
  • Combination 13 Use mutant alkaline phosphatase (GeneBank: M29670.1 (source https://www.ncbi.nlm.nih.gov/)), and the rest is the same as Combination 1.
  • yeast expression system was further used to prepare the fusion protein of alkaline phosphatase and Core+NS3+NS4+NS5, and the combination 14 was prepared according to the "reagent preparation method".
  • Step 1 Confirm the target gene.
  • the corresponding amino acid sequence is the protein sequence (Gly 4 Ser) 3 and add a His tag to the C-terminus of the fusion protein for purification, and preliminarily determine the target gene sequence.
  • the final target gene sequence suitable for Pichia pastoris yeast (X-33) was determined.
  • the number of amino acids of the linker short peptide is greater than 8, especially greater than 10.
  • the second step vector construction. Select the appropriate restriction site, and integrate the target gene into the appropriate expression vector pPICZ ⁇ A.
  • the third step transformation and screening of positive clone strains.
  • the expression vector containing the target gene is transferred into Pichia pastoris cells.
  • the reporter gene on the expression vector add appropriate antibiotics to screen out positive strains,
  • the fourth step protein expression and purification.

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Abstract

本申请提供了一种用于检测HCV的试剂盒，包括标记用酶与HCV抗原性蛋白的融合蛋白，其中所述抗原性蛋白含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原中的一种或多种，或其中的两种以上的融合抗原。本申请还提供了一种相应的检测样本中感染丙型肝炎病毒HCV后产生的抗体的方法。

Description

用于检测HCV抗体的试剂盒以及方法 技术领域
本申请实施例涉及丙型肝炎病毒(Hepatitis C Virus,HCV)抗体检测领域，具体涉及HCV抗体免疫检测、尤其是双抗原夹心法或捕获法中所使用的酶标记物。
背景技术
丙型肝炎是一种由丙型肝炎病毒(Hepatitis C Virus,HCV)引起并主要经血液传播的疾病。HCV慢性感染可导致肝脏慢性炎症坏死和肝纤维化，部分患者可发展为肝硬化甚至肝细胞癌(HCC)，对患者的健康和生命危害极大，已成为严重的社会和公共卫生问题。丙型肝炎呈全球性流行，是欧美及日本等国家终末期肝病的最主要原因，在美国，HCV感染者人数是艾滋病毒感染者的4倍，到2010年，死于HCV感染的人数要多于死于艾滋病毒感染的人数。据世界卫生组织统计，全球HCV的感染率约为3％，估计约1.7亿人感染了HCV，每年新发丙型肝炎病例约3.5万例。
目前，针对丙型肝炎尚无有效的疫苗，所以控制丙型肝炎只能从传染源和传播途径入手。因此，早期准确诊断和发现HCV感染者是防控丙型肝炎传染源、阻断传播途径最为有效的手段，而其中免疫检测是丙型肝炎病毒的诊断和管理的主要方法之一。
HCV免疫检测方法有酶联免疫吸附法、化学发光免疫分析法、金标法、荧光免疫法等。其中，诸如酶联免疫吸附法和酶促化学发光免疫分析法的酶免疫测定技术，是利用标记用酶作为报告分子的常见HCV免疫检测方法，在双抗原夹心法的反应模式下，酶免疫测定中的标记用酶与抗原物质的偶联物、样本中存在的待测的抗体以及包被在固相支持物上的抗原物质形成夹心结构，通过对该夹心结构中的标记用酶进行分析，从而获得HCV的定性或定量检测结果。在捕获法的反应模式下，酶免疫测定中的标记用酶与抗原物质的偶联物、样本中存在的待测抗体以及包被在固相支持物上的抗人IgG和抗人IgM抗体形成复合物，通过对该复合物中的标记用酶进行分析，从而获得HCV的定性或定量检测结果。
通常，基于双抗原夹心法或捕获法的HCV酶免疫测定中，采用化学活化交联方法将标记用酶与抗原物质相连形成交联物或者说偶联物。然而，该连接过程具有操作复杂且难控制的缺点；另外，该这样的连接方式使得酶和抗原的分子摩尔比不均一，导致产物是不同分子摩尔比的酶和抗原交联物的混合物。因此，使用化学活化交联方法所制备的酶和抗原交联物的混合物应用于丙型肝炎病毒抗体的检测，导致了丙肝检测试剂盒生产控制难、检测结果批间差异大等问题。
发明内容
为了解决上述问题，本申请实施例提供了一种用于丙型肝炎病毒HCV抗体的免疫检测试剂盒，包括标记用酶与HCV抗原性蛋白的融合蛋白。
如本申请实施例所使用的，“抗原性蛋白”或“抗原物质”是指具有免疫反应性且可用于HCV免疫学检测的蛋白；其可以为一个HCV抗原或其片段，还可以为两个(种)以上HCV抗原或其片段的融合抗原(或者说两个(种)以上HCV抗原或其片段的嵌合蛋白)。
在本申请实施例中，所述抗原性蛋白含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原中的一种或更多种，或含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原中的两种以上的融合抗原。Core、NS3、NS4、NS5是丙型肝炎病毒的结构和膜蛋白，大量研究表明Core、NS3、NS4、NS5不仅有高免疫原性而且能够作为丙型肝炎病毒诊断性抗原。
如本申请实施例所使用的，“HCV抗原”是指具有免疫反应性且能够用于HCV免疫检测的物质，其选自HCV的保守性蛋白或其片段。在示例性的HCV抗原可以是HCV core抗原、HCV NS3抗原、HCV NS4抗原或HCV NS5抗原。在本申请实施例中，HCV抗原可以一个或多个拷贝的形式存在。
在一些实施方式中，本申请的HCV抗原性蛋白含有HCV core抗原。示例性地，本申请的HCV抗原性蛋白含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原。
在一些实施方式中，所述抗原性蛋白为含有HCV core抗原以及一种或更多种额外的HCV抗原的嵌合蛋白或者说融合抗原。其中，额外的HCV 抗原可以是具有高免疫反应性的HCV抗原，例如，HCV NS3抗原、HCV NS4抗原和HCV NS5抗原中的一种或更多种。
在示例性的实施方式中，本申请的HCV抗原性蛋白为含有HCV core抗原和HCV NS3的嵌合蛋白，或者为含有HCV core抗原、HCV NS3抗原和HCV NS4的嵌合蛋白，或者为含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原的嵌合蛋白。
在抗原性蛋白含有两种以上的HCV抗原的情况下，这些HCV抗原可以任意的顺序存在。在示例性的实施方式中，本申请的抗原性蛋白可自N端至C端顺序含有HCV core抗原和HCV NS3抗原；或者，可自N端至C端顺序含有HCV NS3抗原HCV core抗原。
在具体的实施方式中，本申请的HCV抗原性蛋白自N端至C端顺序含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原。
在具体的实施方式中，本申请的抗原性蛋白自N端至C端顺序含有HCV NS3抗原、HCV NS4抗原、HCV core抗原和HCV NS5抗原。
在具体的实施方式中，本申请的抗原性蛋白自N端至C端顺序含有HCV NS3抗原、HCV core抗原、HCV NS4抗原和HCV NS5抗原。
在本申请实施例中，各HCV抗原可以直接相连或可通过Linker相连，只要在连接HCV抗原的同时保证它们各自的结构以及活性不受影响即可。在本申请实施例中，采用柔性linker，例如柔性的(Gly 4Ser) n、GGGS、GGSGGGSG等。
在本申请实施例中，“酶”、“标记用酶”可以互换使用，是指用于酶免疫测定中的酶。例如，可以是用于酶联免疫吸附法和酶促化学发光免疫分析法中的酶。
在一些实施方式中，本申请的酶可以是碱性磷酸酶(EC 3.1.3.1)，其例如可以催化水解硝基苯磷酸盐(PNP)、β-甘油磷酸钠、磷酸萘酯、3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷(AMPPD)等含磷酸根基团的显色底物和化学发光底物。
本申请实施例的碱性磷酸酶可以是自然存在的、人工合成的或通过基因工程产生的。此外，本申请实施例的碱性磷酸酶可以是经修饰的，如表面糖基化处理或去糖基处理的碱性磷酸酶。
对于碱性磷酸酶的来源，本申请实施例没有特别的限制，只要能够实 现酶免疫测定即可。示例性的碱性磷酸酶可以来源于细菌，如大肠杆菌；哺乳动物，如牛(如Genebank：AF052227.1(来源https://www.ncbi.nlm.nih.gov/))或人(如Genebank：M12551.1)；虾；但不限于此。
在一些实施方式中，本申请的酶可以是辣根过氧化物酶(EC 1.11.1.7)，其以铁卟啉为辅基，在过氧化氢存在时能催化苯酚、苯胺及其取代物聚合，广泛分布于植物界中，在辣根中含量最高。
本申请实施例的辣根过氧化物酶可以是自然存在的、人工合成的或通过基因工程产生的。此外，本申请实施例的辣根过氧化物酶可以是经修饰的。
在一些实施方式中，本申请的酶还包括其突变体。与野生型相比，本申请实施例的酶的突变体具有大于80％，可选地大于85％、大于90％、大于95％、大于98％或大于99％的序列同源性。示例性的碱性磷酸酶突变体可以是GeneBank：M29670.1(来源https://www.ncbi.nlm.nih.gov/)，但本申请实施例不限于此。示例性的辣根过氧化酶突变体可以是GnenBank：XM_018585035.1，但本申请实施例不限于此。
序列同源性的确定，以野生型序列充当参考序列，将待测试序列与参考序列进行比较。然后，使用序列比较算法计算测试序列相对于参考序列的序列同源。适用于确定序列同源性的算法的两个实例是BLAST和BLAST2.0算法，其分别描述于Altschul et al.(1977)Nuc.Acids Res.25:3389-3402，和Altschul et al.(1990)J.Mol.Biol.215:403-410中。用于进行BLAST分析的软件可通过NCBI公开获得。
在本申请实施例中，酶可以融合至抗原性蛋白的任意位置。酶可以融合在HCV抗原性蛋白的N端；或者，酶可以融合在抗原性蛋白的C端；又或者，酶可以融合在抗原性蛋白中的任意两个相邻抗原之间。
在一些实施方式中，HCV抗原性蛋白与酶的融合蛋白自N端至C端顺序含有酶、HCV core抗原、HCV NS3抗原和HCV NS4抗原。
在一些实施方式中，HCV抗原性蛋白与酶的融合蛋白自N端至C端顺序含有酶、HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原。
在一些实施方式中，HCV抗原性蛋白与酶的融合蛋白自N端至C端 顺序含有HCV core抗原、HCV NS3抗原、HCV NS4抗原、HCV NS5抗原和酶。
在一些实施方式中，HCV抗原性蛋白与酶的融合蛋白自N端至C端顺序含有HCV core抗原、HCV NS3抗原、酶、HCV NS4抗原和HCV NS5抗原。
在具体的实施方式中，本申请的融合蛋白自N端至C端依次含有碱性磷酸酶、HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原；或HCV core抗原、HCV NS3抗原、HCV NS4抗原、HCV NS5抗原和碱性磷酸酶；或HCV core抗原、HCV NS3抗原、碱性磷酸酶、HCV NS4抗原和HCV NS5抗原；或辣根过氧化物酶、HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原；或HCV core抗原、HCV NS3抗原、HCV NS4抗原、HCV NS5抗原和辣根过氧化物酶；或HCV core抗原、HCV NS3抗原、辣根过氧化物酶、HCV NS4抗原和HCV NS5抗原。
在一些实施方式中，用于丙型肝炎病毒HCV抗体的免疫检测试剂盒可以包含标记用酶与HCV core抗原的融合蛋白、标记用酶与HCV NS3抗原的融合蛋白、标记用酶与HCV NS4抗原的融合蛋白以及标记用酶与HCV NS5抗原的融合蛋白中的至少一种融合蛋白。在具体的实施方式中，所述免疫检测试剂盒包括标记用酶与HCV core抗原的融合蛋白、标记用酶与HCV NS3抗原的融合蛋白、标记用酶与HCV NS4抗原的融合蛋白以及标记用酶与HCV NS5抗原的融合蛋白。
在具体的实施方式中，免疫检测试剂盒可以包括碱性磷酸酶与HCV core抗原的融合蛋白、碱性磷酸酶与HCV NS3抗原的融合蛋白、碱性磷酸酶与HCV NS4抗原的融合蛋白以及碱性磷酸酶与HCV NS5抗原的融合蛋白中的至少一种融合蛋白，或者包括辣根过氧化物酶与HCV core抗原的融合蛋白、辣根过氧化物酶与HCV NS3抗原的融合蛋白、辣根过氧化物酶与HCV NS4抗原的融合蛋白以及辣根过氧化物酶与HCV NS5抗原的融合蛋白中的至少一种融合蛋白。
在本申请实施例中，酶可以与抗原性蛋白直接相连或可通过Linker相连，只要在连接酶与抗原性蛋白的同时保证它们各自的结构以及活性不受影响即可。在本申请实施例中，采用柔性linker，例如柔性的(Gly 4Ser) n、GGGS、GGSGGGSG等。
在本申请实施例中，“融合蛋白”是指酶与抗原性蛋白的融合蛋白。例如，可表示为酶+core+NS3，在这种情况下，是指自N端至C端依次为酶、HCV core抗原和HCV NS3抗原的融合蛋白。
酶与HCV抗原性蛋白的融合蛋白可通过常规的重组表达技术制备得到。在本申请实施例中，重组表达技术可以是原核表达技术，如大肠杆菌表达技术等；和真核表达技术，如酵母表达技术和昆虫细胞表达技术等。
本领域技术人员能够理解，本申请实施例的试剂盒还可以包括基于双抗原夹心法或捕获法测定HCV抗体的其它试剂或组分，例如，含包被有HCV抗原性蛋白的固相支持物(包被在所述固相支持物上的HCV抗原性蛋白和所述融合蛋白中的HCV抗原性蛋白能与样本中的同一HCV抗体结合)或包被有抗人IgM抗体和抗人IgG抗体的固相支持物；用于绘制标准曲线的校准品；用于质量控制的质控品；用于发生化学发光反应的底物液；和/或清洗缓冲液以及样本稀释液等。
在一些实施方式中，所示试剂盒包括包被有包含HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原的融合抗原的固相支持物；或者包括包被有HCV core抗原的固相支持物、包被有HCV NS3抗原的固相支持物、包被有HCV NS4抗原的固相支持物和包被有HCV NS5抗原的固相支持物。
另一方面，本申请实施例还涉及酶与HCV抗原性蛋白的融合蛋白在制备用于检测HCV的免疫检测试剂盒中的应用。所述免疫检测试剂盒包括：
第一试剂，其含有固相包被物，所述固相包被物上包被有第一配体，该第一配体能结合样本中的HCV抗体；
第二试剂，其含有酶标记物，所述酶标记物为融合有酶的第二配体，其中，所述第二配体为HCV抗原性蛋白并且能结合所述第一配体所结合的HCV抗体。
在具体的实施方式中，基于双抗原夹心法或捕获法来检测HCV。
在本申请实施例的一个变型中，提供了一种用于检测HCV的免疫检测试剂盒，包括：
第一试剂，其含有固相包被物，所述固相包被物为包被有HCV抗原性蛋白(第一配体)的固相支持物；
第二试剂，其含有酶标记物，所述酶标记物为融合有酶的HCV抗原性 蛋白(第二配体)，其中，包被在所述固相支持物上的HCV抗原性蛋白和所述融合蛋白中的HCV抗原性蛋白能与样本中的同一HCV抗体结合；和
说明书，其记载了基于双抗原夹心法进行检测。
本领域技术人员能够理解，第一试剂中包被在固相支持物上的HCV抗原性蛋白和第二试剂中融合蛋白中融合的HCV抗原性蛋白的类型是相同的，但是来源或者多种抗原间的连接方式或连接顺序可以相同或不同。例如，包被在固相支持物上的HCV抗原性蛋白可以为依次连接的HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原的融合抗原，而第二试剂中融合蛋白中融合的HCV抗原性蛋白可以为依次连接的HCV core抗原、HCV NS4抗原、HCV NS5抗原和HCV NS3抗原的融合抗原。
此外，在本申请实施例中，固相支持物上包被的HCV抗原性蛋白可以是如上所描述的HCV抗原性蛋白，也可以是其他方式连接、例如化学连接的HCV抗原性蛋白。
在本申请实施例中，HCV抗体是指HCV感染后受试者体内产生的抗体，例如Anti-HCV IgG抗体和Anti-HCV IgM抗体。
在本申请实施例的另一个变型中，提供了一种用于检测HCV的免疫检测试剂盒，包括：
第一试剂，其含有固相包被物，所述固相包被物为包被有抗人IgM抗体和抗人IgG抗体(第一配体)的固相支持物；
第二试剂，其含有酶标记物，所述酶标记物为酶与HCV抗原性蛋白(第二配体)的融合蛋白；和
说明书，其记载了基于捕获法进行检测。
如本申请实施例所使用的，“固相支持物”是指可以附着抗原或抗体的固体表面。对用于本申请实施例的固相支持物没有特别的限制，商品化的固相支持物及任何可用于免疫检测的固相支持物均可用于本申请。示例性的固相支持物可以是磁珠(如超顺磁性微球)、酶标板、塑料板、塑料管、乳胶珠、琼脂糖珠、玻璃、硝酸纤维素膜、尼龙膜、二氧化硅板或微芯片，但本申请实施例不限于此。
在本申请实施例中，固相包被物可存在于含有蛋白及表面活性剂且具有缓冲能力的常规稀释液中。
在本申请实施例中，酶标记物可存在于含有蛋白及表面活性剂且具有 缓冲能力的常规稀释液中。
在本申请实施例中，融合蛋白例如可以约50ng/mL～150ng/mL的浓度存在于第二试剂中。
在具体的实施方式中，本申请的试剂盒还可以包括第三试剂，其含有封闭剂及表面活性剂。例如，所述封闭剂选自由以下项组成的组中的一种或更多种：脱脂奶粉、BSA、明胶、血清、酪蛋白、卵清蛋白、动物IgG、表面活性剂(例如：吐温-20，吐温-80，TritonX-100等)。
在具体的实施方式中，本申请的试剂盒还可以包括第四试剂，其含有还原剂。例如，所述还原剂选自由以下项组成的组中的一种或更多种：DTT、β-巯基乙醇。
在本申请实施例中，封闭剂及表面活性剂可溶于具有缓冲能力的常规稀释液中；还原剂可溶于具有缓冲能力的常规稀释液中。
在一些实施方式中，所述试剂盒还可以包括所述标记用酶的反应底物，优选地所述反应底物为3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷。
除非特殊说明，本申请实施例中的用语“第一”、“第二”、“第三”和“第四”等仅用于区分多个相似的要素，而不意在表示要素之间重要性或次序等方面的任何差异。
本申请实施例还涉及酶与HCV抗原性蛋白的融合蛋白用于检测样本中感染丙型肝炎病毒HCV后产生的HCV抗体的方法，包括以下步骤：
将样本和包被有第一配体的固相支持物混合，使得固相支持物上包被的第一配体与样本中的HCV抗体充分结合；
将上述混合物进行清洗，除去未结合的物质；
在上述经清洗的混合物中加入具有第二配体的酶标记物进行混匀，使得该酶标记物中的第二配体与所述固相支持物上结合的HCV抗体结合，形成夹心复合物，其中所述酶标记物为标记用酶与HCV抗原性蛋白的融合蛋白；
对上述夹心复合物进行清洗，除去未结合的物质；
在上述经清洗的夹心复合物中加入化学发光底物，检测反应所产生的光子数，以得到样本的化学发光信号值。
在本申请实施例中，所述第一配体为HCV抗原性蛋白，或者为抗人IgG 抗体和抗人IgM抗体。
本申请实施例通过重组表达得到了标记用酶与HCV抗原性蛋白的融合蛋白，并将其应用于基于双抗原夹心或捕获法模式的HCV免疫检测试剂盒中。需要说明的是：
通过用这样的融合蛋白代替由化学活化交联产生的酶与抗原物质的连接物，试剂盒中标记用酶与HCV抗原性蛋白的融合蛋白具有均一的标记用酶与HCV抗原性蛋白的摩尔比，避免了试剂盒生产及检测结果批间差异大的问题。
另一方面，与常规的双抗原夹心法或捕获法试剂盒相比，本申请实施例的试剂盒表现出更好阴阳性样本的区分度，并且具有更好样本符合率。
再一方面，含有本申请实施例的融合蛋白的试剂盒避免了化学活化交联方法所导致的对抗原活性或酶活性的破坏。
又一方面，本申请实施例节省了需要进行化学交联反应的步骤，使得操作更加方便、快捷。
具体实施方式
下面对本申请实施方式中的技术方式进行清楚、完整地描述，显然，所描述的实施方式仅仅是本申请的一部分实施方式，而不是全部的实施方式。基于本申请中的实施方式，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式，都属于本申请保护的范围。
如前所述，本申请实施例中酶与HCV抗原性蛋白的融合蛋白可通过重组表达技术生产。
示例性的重组表达方法可包含以下步骤：
i)将核酸分子或表达载体转染或转化的宿主细胞；
ii)在表达融合蛋白的条件下，培养所述宿主细胞；和
iii)分离所述融合蛋白。
在上述步骤中，核酸分子编码上述融合蛋白；而表达载体用于通过重组表达制备酶与HCV的抗原性蛋白的融合蛋白，该表达载体包含可操作地连接到表达控制序列上的编码上述融合蛋白的核酸分子，另外包含用于在各自宿主细胞中维持和繁殖的遗传元件，如复制起点和/或选择标记基因。
融合蛋白的制备
利用大肠杆菌表达系统生产融合蛋白：
第一步：目的基因确认。根据两个待融合蛋白的基因序列以及氨基酸序列，在两者之间添加合适的linker，例如linker的基因序列为GGA GGA GGA GGA TCA GGA GGA GGA GGA TCA GGA GGA GGA GGA TCA，其对应的氨基酸序列为蛋白序列(Gly 4Ser) 3，并在融合蛋白的C端添加His标签以便纯化，初步确定目的基因序列。再通过密码子优化，确定最终的适合大肠杆菌表达菌株BL21(DE3)的目的基因序列。
第二步：载体构建。选择合适的酶切位点，将目的基因整合到合适的表达载体pET28a中。
第三步：转化及阳性克隆菌株的筛选。通过电转化或者热激转化，将含有目的基因的表达载体转入到大肠杆菌中。根据表达载体上的报告基因，添加合适的抗生素，筛选出阳性菌株。
第四步：蛋白的表达以及纯化。
HCV抗原核酸序列信息：Core，GeneBank；AJ222676.1；NS3，GeneBank：EU84764.1；NS4，GeneBank：Z84363.1；NS5，GeneBank：S70341.1(来源https://www.ncbi.nlm.nih.gov/)。
试剂配制方法
第一试剂Ra：
使用移液器或量筒量取3.5mL“包被Core+NS3+NS4+NS5嵌合蛋白的磁珠”加入至一个磁珠包被管置换上清，即将其磁分离后吸走上清然后加入等体积(3.5mL)的磁珠包被物稀释液，并混合均匀；将磁珠混匀后加入到盛有96.5mL磁珠包被物稀释液的配液瓶中；搅拌直至磁珠悬浮液完全混匀，制得第一试剂Ra，其中包被Core+NS3+NS4+NS5嵌合蛋白的磁珠的浓度为0.7mg/mL；磁珠包被物稀释液为具有缓冲能力的常规稀释液，且含有蛋白及表面活性剂。
第二试剂Rb：
使用合适的量筒量取99mL的示踪剂稀释液加入到配液瓶中，用移液器量取1mL的“酶与HCV抗原性蛋白的融合蛋白”或“经化学连接的酶与HCV抗原性蛋白的偶联物”，或“经化学连接的酶与抗人IgG和抗人IgM 的偶联物”作为酶标记物加入到示踪剂稀释液中；用搅拌器搅拌溶液，使其充分溶解、混匀；然后使用合适的0.22μm孔径过滤器将配制好的溶液进行过滤，收集过滤液，制得示踪剂Rb，其中上述酶标记物的浓度为100ng/mL；示踪剂稀释液为具有缓冲能力的常规稀释液，且含有蛋白及表面活性剂。
第三试剂Rc：
具有缓冲能力的稀释液，且含有BSA、吐温-20和脱脂奶粉。
第四试剂Rd：
具有缓冲能力的稀释液，且含有DDT。
双抗原夹心检测方法
第一步：将样本、第四试剂和第一试剂加入反应管中，在37℃孵育10分钟，使得磁珠固相上包被的HCV抗原与样本中的Anti-HCV IgG和Anti-HCV IgM抗体充分结合；孵育完成后，磁珠固相置于磁场内被吸住，结合在磁珠固相上的物质被保留，而其他未结合的物质被清洗除去。
第二步：将第三试剂和第二试剂添加到该反应管，进行混匀；在37℃孵育10分钟，酶标记物上的HCV抗原与磁珠上被捕获的Anti-HCV IgG和Anti-HCV IgM抗体结合，形成夹心复合物。在反应管内孵育完成后，该复合物被磁场吸住，而其他未结合的物质被清洗除去。
第三步：将化学发光底物添加到反应管内，产生化学发光。再通过光电倍增管对反应所产生的光子数进行测量，以得到样本的化学发光信号值。
在本申请实施例中，COI(Cutoff index)为测定样本的化学发光信号值(RLU)与阈值(Cutoff value)的比值，其中COI≥1表示测定样本为阳性样本，COI＜1表示测定样本为阴性样本。对定性检测方法而言，阈值(截断值)为判断测试结果为阳性或阴性的分界值。
在本申请实施例中，阴性符合率是指使用本申请实施例的测试方法得到判断为阴性的样本个数占实际参与评估的阴性样本的比例，阳性符合率是指使用本申请实施例的测试方法得到判断为阳性的样本个数占实际参与评估的阳性样本的比例；样本的真实阴阳性结果来自医院诊断结果。
实施例1
为比较本申请实施例的酶与HCV抗原性蛋白的融合蛋白相对于通过化学交联产生的酶与HCV抗原性蛋白的偶联物的HCV检测效果，制备以下三种试剂组合。
组合1：利用以上所描述的大肠杆菌表达系统，使用Core+NS3+NS4+NS5嵌合蛋白(或者说融合抗原)作为HCV抗原性蛋白与碱性磷酸酶进行融合以配制第二试剂Rb，其中第二试剂Rb的融合蛋白中碱性磷酸酶(Genebank：AF052227.1(来源https://www.ncbi.nlm.nih.gov/))融合在Core+NS3+NS4+NS5嵌合蛋白的N端，按上述“试剂配制方法”制备得到。
组合2：采用经化学连接的碱性磷酸酶与Core+NS3+NS4+NS5嵌合蛋白的偶联物配制第二试剂Rb，其余与组合1相同。
接下来，使用组合1和2，按照上述“双抗原夹心检测方法”分别对来自综合医院的经确诊的500例HCV阴性样本和100例阳性样本进行测试。结果如下表1所示。
表1
试剂组合 组合1 组合2 组合3(间接法)
阴性样本1(COI) 0.11 0.12 0.11
阴性样本2(COI) 0.16 0.18 0.31
阳性样本1(COI) 2.78 1.71 2.45
阳性样本2(COI) 13.83 8.05 10.26
阳性样本3(COI) 85.50 27.48 45.73
阴性符合率(500例) 100％ 99.6％ 99％
阳性符合率(100例) 100％ 99％ 100％
由表1可知，组合1的阴性符合率为100％、阳性符合率为100％，高于使用了化学连接的常规双抗原夹心法(组合2：阴性符合率99.6％、阳性符合率99％)，这个结果表明：使用碱性磷酸酶与Core+NS3+NS4+NS5的融合蛋白能够避免常规双抗原夹心法中，使用化学键连接的方法制备过程对抗原或碱性磷酸酶活性的破坏。在本申请实施例中，碱性磷酸酶与Core+NS3+NS4+NS5的融合蛋白中的碱性磷酸酶和抗原都保持了较高的活性。
此外，为了证明本申请实施例所使用的Core+NS3+NS4+NS5嵌合蛋白的活性，制备了试剂组合3：采用经化学连接的碱性磷酸酶与抗人IgG和抗人IgM的偶联物配制第二试剂Rb，其余与组合1相同。
按照间接检测方法，使用上述组合3对上述经确诊的500例HCV阴性样本和100例阳性样本进行测试。“间接法检测方法”具体步骤如下：
第一步：将样本、第四试剂和第一试剂加入反应管中，在37℃孵育10分钟，使得磁珠固相上包被的HCV抗原与样本中的Anti-HCV IgG和Anti-HCV IgM抗体充分结合；孵育完成后，磁珠固相置于磁场内被吸住，结合在磁珠固相上的物质被保留，而其他未结合的物质被清洗除去。
第二步：将第三试剂和第二试剂添加到反应管，进行混匀；在37℃孵育10分钟，酶标记物上的抗人IgG和抗人IgM的二抗与磁珠上被捕获的Anti-HCV IgG和Anti-HCV IgM抗体结合，形成夹心复合物。在反应管内孵育完成后，该复合物被磁场吸住，而其他未结合的物质被清洗除去。
第三步：将化学发光底物添加到反应管内，产生化学发光。再通过光电倍增管对反应所产生的光子数进行测量，以得到样本的化学发光信号值。
结果如表1所示，间接法测试(组合3)能较好地区分样本的阴性和阳性，说明本申请实施例所使用的Core+NS3+NS4+NS5嵌合蛋白具有较好的活性。
实施例2
为比较本申请实施例的酶与HCV抗原性蛋白的融合蛋白相对于通过化学交联产生的酶与抗原性蛋白的偶联物的HCV检测效果，制备以下三种试剂组合：
组合4：利用以上所描述的大肠杆菌表达系统，使用Core+NS3+NS4+NS5作为抗原性蛋白与辣根过氧化物酶进行融合以配制第二试剂Rb，第二试剂Rb的融合蛋白中辣根过氧化物酶(GeneBank：KU504630.1(来源https://www.ncbi.nlm.nih.gov/))位于抗原性蛋白的N端，按上述“试剂配制方法”制备得到。
组合5：采用经化学连接的辣根过氧化物酶与Core+NS3+NS4+NS5嵌合蛋白的偶联物配制第二试剂Rb，其余与组合4相同。
接下来，使用组合4和5，按照上述“双抗原夹心检测方法”分别对实施例1中的500例HCV阴性样本和100例阳性样本进行测试。结果如下表2所示。
表2
试剂组合 组合4 组合5 组合6(间接法)
阴性样本1(COI) 0.21 0.19 0.42
阴性样本2(COI) 0.26 0.25 0.51
阳性样本1(COI) 1.53 1.24 1.39
阳性样本2(COI) 4.21 2.97 3.78
阳性样本3(COI) 25.44 15.04 24.36
阴性符合率(500例) 100％ 99.4％ 99％
阳性符合率(100例) 100％ 98％ 100％
由表2可知，组合4的阴性符合率为100％、阳性符合率为100％，高于使用了化学连接的常规双抗原夹心法(组合5：阴性符合率99.4％、阳性符合率98％)，这个结果表明：使用辣根过氧化物酶与Core+NS3+NS4+NS5的融合蛋白能够避免常规双抗原夹心法中，使用化学键连接的方法制备过程对抗原或辣根过氧化物酶活性的破坏。在本申请实施例中，辣根过氧化物酶与Core+NS3+NS4+NS5融合蛋白中的辣根过氧化物酶和抗原都保持了较高的活性。
同样地，为了证明本申请实施例所使用的Core+NS3+NS4+NS5嵌合蛋白的活性，制备了试剂组合6：采用经化学连接的辣根过氧化物酶与抗人IgG和抗人IgM的偶联物配制第二试剂Rb，其余与组合4相同。
按照上述间接检测方法，使用上述组合6对上述经确诊的500例HCV阴性样本和100例阳性样本进行测试。结果如表1所示，间接法测试(组合6)能较好地区分样本的阴性和阳性，说明本申请实施例所使用的Core+NS3+NS4+NS5嵌合蛋白具有较好的活性。
实施例3
为考察酶的融合位置对本申请实施例的融合蛋白的影响，制备以下三种试剂组合：
组合1：利用以上所描述的大肠杆菌表达系统，使用Core+NS3+NS4+NS5嵌合蛋白作为抗原性蛋白与碱性磷酸酶进行融合以配置第二试剂Rb，第二试剂Rb的融合蛋白中碱性磷酸酶融合在Core+NS3+NS4+NS5嵌合蛋白的N端，按上述“试剂配制方法”制备得到。
组合7：第二试剂Rb的融合蛋白中，碱性磷酸酶融合在Core+NS3+NS4+NS5嵌合蛋白的C端，其余与组合1相同。
组合8：第二试剂Rb的融合蛋白中，碱性磷酸酶融合在 Core+NS3+NS4+NS5嵌合蛋白的NS3与NS4之间，其余与组合1相同。
接下来，使用组合1、7和8，按照上述“双抗原夹心检测方法”分别对来自实施例1中的经确诊的500例HCV阴性样本和100例阳性样本进行测试，结果如下表3所示。
表3
试剂组合 组合1 组合7 组合8
阴性样本1(COI) 0.11 0.10 0.12
阴性样本2(COI) 0.16 0.17 0.15
阳性样本1(COI) 2.78 2.92 2.65
阳性样本2(COI) 13.83 14.32 13.17
阳性样本3(COI) 85.50 89.78 82.43
阴性符合率(500例) 100％ 100％ 100％
阳性符合率(100例) 100％ 100％ 100％
由表3可知，将碱性磷酸酶融合于抗原性蛋白的N端(组合1)、C端(组合7)和中间(组合8)时，样本的阴性符合率与阳性符合率均能达到100％。此外，虽然不愿意受到理论约束，发明人认为，在将酶融合在HCV抗原性蛋白的中间的情况下，酶的活性中心会被其他蛋白部分阻挡，因此将酶融合在HCV抗原性蛋白的中间比将酶融合在HCV抗原性蛋白的N和C端效果略微较差。
实施例4
为考察酶的融合位置对本申请实施例的融合蛋白的影响，制备以下三种试剂组合：
组合4：利用以上所描述的大肠杆菌表达系统，使用Core+NS3+NS4+NS5嵌合蛋白作为抗原性蛋白与辣根过氧化物酶进行融合以配置第二试剂Rb，第二试剂Rb的融合蛋白中辣根过氧化物酶融合在Core+NS3+NS4+NS5嵌合蛋白的N端，按上述“试剂配制方法”制备得到。
组合9：第二试剂Rb的融合蛋白中，辣根过氧化物酶融合在Core+NS3+NS4+NS5嵌合蛋白的C端，其余与组合4相同。
组合10：第二试剂Rb的融合蛋白中，辣根过氧化物酶融合在Core+NS3+NS4+NS5嵌合蛋白的NS3与NS4之间，其余与组合4相同。
接下来，使用组合4、9和10，按照上述“双抗原夹心检测方法”分别对来自实施例1中的经确诊的500例HCV阴性样本和100例阳性样本进 行测试，结果如下表4所示。
表4
试剂组合 组合4 组合9 组合10
阴性样本1(COI) 0.21 0.24 0.22
阴性样本2(COI) 0.26 0.29 0.27
阳性样本1(COI) 1.53 1.95 1.42
阳性样本2(COI) 4.21 4.50 3.93
阳性样本3(COI) 25.44 24.72 23.22
阴性符合率(500例) 100％ 100％ 100％
阳性符合率(100例) 100％ 100％ 100％
由表4可知，将辣根过氧化物酶融合于抗原性蛋白的N端(组合4)、C端(组合9)和中间(组合10)时，样本的阴性符合率与阳性符合率均能达到100％。此外，虽然不愿意受到理论约束，发明人认为，在将酶融合在HCV抗原性蛋白的中间的情况下，酶的活性中心会被其他蛋白部分阻挡，因此将酶融合在HCV抗原性蛋白的中间比将酶融合在HCV抗原性蛋白的N和C端效果略微较差。
实施例5
为考察抗原性蛋白的顺序对本申请实施例的融合蛋白的影响，制备以下三种试剂组合：
组合1：抗原性蛋白自N端至C端依次为Core+NS3+NS4+NS5，融合蛋白中碱性磷酸酶融合在抗原性蛋白的N端，按上述“试剂配制方法”制备得到。
组合11：抗原性蛋白自N端至C端依次为NS3+NS4+core+NS5，其余与组合1相同。
组合12：抗原性蛋白自N端至C端依次为NS3+core+NS4+NS5，其余与组合1相同。
接下来，使用组合1、11和12，按照上述“双抗原夹心检测方法”分别对来自实施例1中的经确诊的500例HCV阴性样本和100例阳性样本进行测试，结果如下表5所示。
表5
试剂组合 组合1 组合11 组合12
阴性样本1(COI) 0.11 0.15 0.12
阴性样本2(COI) 0.16 0.21 0.14
阳性样本1(COI) 2.78 3.24 3.06
阳性样本2(COI) 13.83 16.77 15.23
阳性样本3(COI) 85.50 74.43 71.59
阴性符合率(500例) 100％ 100％ 100％
阳性符合率(100例) 100％ 100％ 100％
由表5可知，采用按照Core+NS3+NS4+NS5、NS3+NS4+core+NS5或NS3+core+NS4+NS5顺序融合的抗原性蛋白，样本的阴性符合率与阳性符合率均能达到100％。
实施例6
为了考察使用酶的突变体时的HCV检测效果，制备以下试剂组合：
组合1：利用以上所描述的大肠杆菌表达系统，使用Core+NS3+NS4+NS5嵌合蛋白作为抗原性蛋白与野生型碱性磷酸酶进行融合以配制第二试剂Rb，融合蛋白中野生型碱性磷酸酶融合在抗原性蛋白的N端，按上述“试剂配制方法”制备得到。
组合13：使用突变型碱性磷酸酶(GeneBank：M29670.1(来源https://www.ncbi.nlm.nih.gov/))，其余与组合1相同。
接下来，使用组合1和13，按照上述“双抗原夹心检测方法”分别对实施例1中的500例HCV阴性样本和100例阳性样本进行测试，结果如表6所示。
表6
试剂组合 组合1 组合13
阴性样本1(COI) 0.11 0.13
阴性样本2(COI) 0.16 0.15
阳性样本1(COI) 2.78 4.56
阳性样本2(COI) 13.83 24.11
阳性样本3(COI) 85.50 150.73
阴性符合率(500例) 100％ 100％
阳性符合率(100例) 100％ 100％
由表6可知，采用野生型碱性磷酸酶或突变型碱性磷酸酶，样本的阴性符合率与阳性符合率均能达到100％。此外，相比于野生型碱性磷酸酶，采用突变型碱性磷酸酶时的检测区分度更好。
实施例7
为了考察不同表达技术对本申请实施例的HCV检测效果的影响，进一步采用酵母表达系统制备碱性磷酸酶与Core+NS3+NS4+NS5的融合蛋白，并按“试剂配制方法”制备得到组合14。
酵母表达系统生产融合蛋白的具体方法如下：
第一步：目的基因确认。在两个待融合蛋白的基因序列两者之间添加能够编码linker短肽的核苷酸序列GGA GGA GGA GGA TCA GGA GGA GGA GGA TCA GGA GGA GGA GGA TCA，其对应的氨基酸序列为蛋白序列(Gly 4Ser) 3，并在融合蛋白的C端添加His标签以便纯化，初步确定目的基因序列。再通过密码子优化，确定最终的适合毕赤酵母表达菌株Pichia pastoris yeast(X-33)的目的基因序列。在本申请实施例中，linker短肽的氨基酸数量大于8个、尤其是大于10个。
第二步：载体构建。选择合适的酶切位点，将目的基因整合到合适的表达载体pPICZαA中。
第三步：转化及阳性克隆菌株的筛选。通过电转化，将含有目的基因的表达载体转入到毕赤酵母细胞中。根据表达载体上的报告基因，添加合适的抗生素，筛选出阳性菌株，
第四步：蛋白的表达以及纯化。
接下来，使用组合1和14，按照上述“双抗原夹心检测方法”分别对实施例1中的500例HCV阴性样本和100例阳性样本进行测试，结果如表7所示。
表7
试剂组合 组合1 组合14
阴性样本1(COI) 0.11 0.13
阴性样本2(COI) 0.16 0.15
阳性样本1(COI) 2.78 2.34
阳性样本2(COI) 13.83 9.56
阳性样本3(COI) 85.50 67.19
阴性符合率(500例) 100％ 99.6％
阳性符合率(100例) 100％ 100％
由表7可知，在信噪比方面，使用大肠杆菌表达系统制备融合蛋白的组合1要优于使用酵母表达系统制备融合蛋白的组合14。另外，使用大肠杆菌表达系统的组合1的阴性符合率(100％)高于使用酵母表达系统制备融合蛋白的组合14(99.6％)。

Claims (17)

  1. 一种用于检测丙型肝炎病毒HCV抗体的试剂盒，包括：
    标记用酶与HCV抗原性蛋白的融合蛋白；
    其中，所述抗原性蛋白含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原中的一种或多种，或其中的两种以上的融合抗原。
  2. 权利要求1所述的试剂盒，其中，所述HCV抗原性蛋白为含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原的融合抗原。
  3. 权利要求2所述的试剂盒，其中，所述HCV抗原性蛋白自N端至C端依次含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原。
  4. 权利要求1～3中任一项所述的试剂盒，其中，所述标记用酶融合于所述抗原性蛋白的N端或C端。
  5. 权利要求1～4中任一项所述的试剂盒，其中，所述标记用酶为碱性磷酸酶或辣根过氧化物酶。
  6. 根据权利要求5所述的试剂盒，其中，所述碱性磷酸酶融合于所述抗原性蛋白的N端或C端；或者，所述辣根过氧化物酶融合于所述抗原性蛋白的N端或C端。
  7. 根据权利要求6所述的试剂盒，其中，所述融合蛋白自N端至C端依次为碱性磷酸酶、HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原；或HCV core抗原、HCV NS3抗原、HCV NS4抗原、HCV NS5抗原和碱性磷酸酶；或者，所述融合蛋白自N端至C端依次为辣根过氧化物酶、HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原；或HCV core抗原、HCV NS3抗原、HCV NS4抗原、HCV NS5抗原和辣根过氧化物酶。
  8. 权利要求1所述的试剂盒，其中，所述标记用酶与HCV抗原性蛋白的融合蛋白包括所述标记用酶与HCV core抗原的融合蛋白、所述标记用酶与HCV NS3抗原的融合蛋白、所述标记用酶与HCV NS4抗原的融合蛋白以及所述标记用酶与HCV NS5抗原的融合蛋白中的至少一种融合蛋白。
  9. 权利要求8所述的试剂盒，其中，所述标记用酶与HCV抗原性蛋 白的融合蛋白包括所述标记用酶与HCV core抗原的融合蛋白、所述标记用酶与HCV NS3抗原的融合蛋白、所述标记用酶与HCV NS4抗原的融合蛋白以及所述标记用酶与HCV NS5抗原的融合蛋白。
  10. 根据权利要求1至9中任一项所述的试剂盒，其中，所述试剂盒还包括：
    包被有HCV抗原性蛋白的固相支持物，其中，包被在所述固相支持物上的抗原性蛋白含有HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原中的一种或多种，或其中的两种以上的融合抗原，并且包被在所述固相支持物上的HCV抗原性蛋白和所述融合蛋白中的HCV抗原性蛋白能与样本中的同一HCV抗体结合；或
    包被有抗人IgG抗体和抗人IgM抗体的固相支持物。
  11. 根据权利要求2或3或9所述的试剂盒，其中，所述试剂盒还包括：
    包被有包含HCV core抗原、HCV NS3抗原、HCV NS4抗原和HCV NS5抗原的融合抗原的固相支持物；或者
    包被有HCV core抗原的固相支持物、包被有HCV NS3抗原的固相支持物、包被有HCV NS4抗原的固相支持物和包被有HCV NS5抗原的固相支持物。
  12. 权利要求1至11中任一项所述的试剂盒，所述试剂盒进一步包括封闭剂；优选地，所述封闭剂选自由以下项组成的组中的一种或更多种：脱脂奶粉、BSA、明胶、血清、酪蛋白、卵清蛋白、动物IgG、表面活性剂。
  13. 权利要求1至12中任一项所述的试剂盒，所述试剂盒进一步包括还原剂；优选地，所述还原剂选自由以下项组成的组中的一种或更多种：DTT、β-巯基乙醇。
  14. 权利要求1至13中任一项所述的试剂盒，其中，所述试剂盒还包括所述标记用酶的反应底物，优选地所述反应底物为3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷。
  15. 根据权利要求1至14中任一项所述的试剂盒，其中，所述融合蛋白经由以下表达技术产生：原核表达技术。
  16. 一种检测样本中感染丙型肝炎病毒HCV后产生的HCV抗体的方法，包括以下步骤：
    将样本和包被有第一配体的固相支持物混合，使得固相支持物上包被的第一配体与样本中的HCV抗体充分结合；
    将上述混合物进行清洗，除去未结合的物质；
    在上述经清洗的混合物中加入具有第二配体的酶标记物进行混匀，使得该酶标记物中的第二配体与所述固相支持物上结合的HCV抗体结合，形成夹心复合物，其中所述酶标记物为标记用酶与HCV抗原性蛋白的融合蛋白；
    对上述夹心复合物进行清洗，除去未结合的物质；
    在上述经清洗的夹心复合物中加入化学发光底物，检测反应所产生的光子数，以得到样本的化学发光信号值。
  17. 根据权利要求16所述的方法，其中，所述第一配体为HCV抗原性蛋白，或者为抗人IgG抗体和抗人IgM抗体。
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