WO2022179349A1 - 新型冠状病毒抗原及其应用 - Google Patents

新型冠状病毒抗原及其应用 Download PDF

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WO2022179349A1
WO2022179349A1 PCT/CN2022/072677 CN2022072677W WO2022179349A1 WO 2022179349 A1 WO2022179349 A1 WO 2022179349A1 CN 2022072677 W CN2022072677 W CN 2022072677W WO 2022179349 A1 WO2022179349 A1 WO 2022179349A1
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polypeptide
antibody
feminine
detection
conjugated
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PCT/CN2022/072677
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French (fr)
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林晓涛
钱纯亘
刘笔锋
陈鹏
林军
李一荣
胡鹍辉
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深圳市亚辉龙生物科技股份有限公司
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Publication of WO2022179349A1 publication Critical patent/WO2022179349A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • 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
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—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
    • 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 present invention relates to the field of biomedicine, in particular, to a novel coronavirus antigen and application thereof.
  • Coronaviruses are a group of highly diverse, enveloped, positive-sense, single-stranded RNA viruses that cause varying degrees of respiratory tract severity in a variety of animals, Intestinal, hepatic and neurological diseases, including humans. Coronaviruses are divided into four genera: alphacoronaviruses, betacoronaviruses ( ⁇ CoV), gammacoronaviruses, and deltacoronaviruses. In the past 12 years, two novel betacoronaviruses have emerged, severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV), which cause severe human disease.
  • SARS-CoV severe acute respiratory syndrome
  • MERS-CoV Middle East respiratory syndrome
  • Novel coronavirus SARS-CoV-2
  • the novel coronavirus is a new strain of coronavirus that has never been found in humans before. Its homology is more than 80% with acute respiratory syndrome (SARS-CoV), the structural protein Nucleocapsid protein homology between the two is 94.1%, and the Spike protein homology between the two is 84.1%.
  • SARS-CoV acute respiratory syndrome
  • the human body After being infected with the new coronavirus, the human body will produce IgM antibodies against the virus in about 7 days, and IgG antibodies against the virus in about 14 days.
  • the antibody produced is a protective antibody that promotes the body's recovery and self-healing from viral infections.
  • the detection of new coronavirus IgM antibodies can be used to assist in the diagnosis of new coronavirus diseases, and is an effective supplement to the new coronavirus nucleic acid diagnostic reagents.
  • the key to the development of new coronavirus IgM and IgG detection kits lies in the acquisition of effective antigens for the new coronavirus.
  • the new coronavirus structural proteins include spike protein (Spike protein, transmembrane protein, homotrimeric structure, such as "crown” distributed on the surface of the virus, which is the core component that mediates virus infection of cells), nucleocapsid protein ( Nucleoprotein, which is the most abundantly expressed component of the structural proteins of the new coronavirus), envelope protein (Envelope Protein) and membrane protein (Membrane Protein), among which, according to the abundance and spatial position of the protein, the main diagnostic tools for IgM and IgG are are nucleocapsid proteins and spike proteins.
  • the nucleocapsid protein is highly conserved, and because it is encapsulated in the virus, the body produces antibodies against it, especially IgM antibodies, relatively late. Therefore, the diagnostic antigens used in the diagnosis of novel coronavirus antibodies, especially IgM antibodies, are mainly related fragments of the spike protein.
  • the prior art usually recombinantly expresses the full length of the S1 subunit of the novel coronavirus, but using the recombinant protein as the detection antigen, the signal value generated is low. However, if the RBD domain of the C-terminal of the new coronavirus S1 subunit is expressed alone, it is easy to cause missed detection.
  • the present invention transforms the new crown spike protein by connecting the main structural domains with Linker, and uses it as a diagnostic antigen, so as to achieve the purpose of not only improving the detection signal value, but also ensuring the detection rate.
  • the present invention relates to a polypeptide whose amino acid sequence is shown in SEQ ID NO: 1.
  • the N-terminus of the above-mentioned polypeptide is further connected with a signal peptide.
  • amino acid sequence of the signal peptide is shown in SEQ ID NO:2.
  • nucleic acid encoding a polypeptide as described above.
  • a third aspect of the present invention relates to a vector containing a nucleic acid as described above.
  • a host cell containing a nucleic acid as described above, or a vector as described above.
  • the fifth aspect of the present invention relates to a new coronary pneumonia detection kit, which contains the above-mentioned polypeptide, anti-antibody and solid support;
  • the anti-antibody is an antibody against the immunoglobulin of the species from which the test sample is derived;
  • the AviTag tag in the polypeptide is biotinylated in advance, and the biotinylated AviTag tag is conjugated to the solid support.
  • the solid support is a test tube, an EP tube, a multi-well plate, a well of a micro-reaction plate, a bead or a small disc.
  • the anti-antibody is conjugated with a signal substance.
  • a new coronary pneumonia detection test strip including a sample pad, a binding pad, a reaction membrane and an absorption pad, and the reaction membrane is provided with a detection area and a quality inspection area;
  • the binding pad is coated with an anti-antibody, and the anti-antibody is conjugated with a signal substance; the anti-antibody is an antibody against the immunoglobulin of the test sample-derived species;
  • the detection region is conjugated with the polypeptide as described above; the AviTag tag in the polypeptide is biotinylated in advance, and the biotinylated AviTag tag is conjugated to the detection region.
  • the immunoglobulin is at least any one of IgG, IgM or IgA.
  • the signal substance is a metal particle, a fluorescent label, a chromophore label, an electron-dense label, a chemiluminescence label, a radiolabel, or an enzyme.
  • the seventh aspect of the present invention relates to the application of the above-mentioned polypeptide in the preparation of a SARS-CoV-2 antibody detection test strip or kit.
  • the test sample of the detection is selected from biological tissue or its lavage fluid, cell, body fluid, further selected from blood, serum, plasma, anticoagulation, cell culture supernatant, Saliva, semen, amniotic fluid, villi, tissue or tissue lysate, throat swabs, nasal swabs, eye conjunctival swabs, stool specimens, feces, urine, bronchial lavage fluid, bronchoalveolar lavage fluid, sputum.
  • the NTD domain and RBD domain of S1 protein are connected in series through two repeating linker sequences, so that these two domains have better spatial extensibility to recognize antibodies; in addition, Avi- tag.
  • AviTag-tagged proteins allow recombinant proteins to be immobilized through immobilized sites, reducing the effect of steric hindrance caused by the coating process. As a result, the polypeptide can achieve high sensitivity and specificity, and is less likely to be missed.
  • FIG. 1 is a schematic structural diagram of a polypeptide provided in an embodiment of the present invention.
  • Figure 2 is a vector map of 2019-nCoV-spike-pcDNA3.1 in an embodiment of the present invention
  • Fig. 3 is a graph showing the electrophoresis result of recombinantly prepared 2019-nCoV Spike antigen in an embodiment of the present invention.
  • polypeptide or "protein” refers to a molecule comprising at least two amino acid residues linked by peptide bonds to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as "peptides”.
  • nucleic acid refers to polymers of nucleotides (eg, ribonucleotides or deoxyribonucleotides), and include naturally occurring (adenosine, guanidine, cytosine, uracil and thymidine), non-naturally occurring and modified nucleic acids.
  • the term is not limited by the length of the multimer (eg, the number of monomers).
  • Nucleic acids can be single-stranded or double-stranded, and will typically contain 5'-3' phosphodiester linkages, although in some cases nucleotide analogs may have other linkages.
  • Monomers are generally referred to as nucleotides.
  • non-natural nucleotide or “modified nucleotide” refers to a nucleotide that contains a modified nitrogenous base, sugar or phosphate group, or incorporates a non-natural moiety into its structure.
  • non-natural nucleotides include dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.
  • kit refers to any article of manufacture (eg, a package or container) that includes at least one device for specifically amplifying, capturing, labeling/transforming, or detecting a target nucleic acid as described herein, as described herein Sequence solid support.
  • the kit may further include instructions for use, supplemental reagents and/or components or assemblies for use in the methods described herein or steps thereof.
  • detection and similar terms are used in this application to refer broadly to the process or discovery or determination of the presence or absence of something, as well as the extent, quantity or level, or probability of occurrence. It is to be understood that the expressions “detection of the presence or absence”, “detection of the presence or absence” and related expressions include both qualitative and quantitative detection. For example, quantitative detection includes determining the level, quantity, or amount of nucleic acid sequences associated with SARS-CoV-2 in a sample.
  • the present invention relates to a polypeptide whose amino acid sequence is shown in SEQ ID NO: 1.
  • the polypeptide includes the following parts: S1-NTD—Linker—Avi-tag—Linker—S1-RBD.
  • the NTD domain and RBD domain of S1 protein are connected in series through two repeating linker sequences, so that these two domains have better spatial extensibility to recognize antibodies;
  • Avi- tag, AviTag tag protein is a 15 amino acid short peptide with a single biotinylated lysine site, completely different from known natural biotinylated sequences. After fusion expression, it can be biotinylated by biotin ligase.
  • biotin ligase In order to purify recombinant proteins, low-affinity monomer avidin or avidin derivatives are used. In addition to protein separation and purification, it is also used for protein interaction. effect research.
  • the AviTag-tagged protein enables the recombinant protein to be immobilized through the immobilized site, thereby reducing the influence of steric hindrance caused by the coating process.
  • the polypeptide can achieve high sensitivity and specificity, and is less likely to be missed.
  • a signal peptide is also attached to the N-terminus of the polypeptide.
  • amino acid sequence of the signal peptide is shown in SEQ ID NO:2.
  • the present invention also relates to a nucleic acid encoding a polypeptide as described above.
  • the nucleic acid can be DNA or RNA.
  • the present invention also relates to vectors containing nucleic acids as described above.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • the vectors of the present invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (such as transcription termination signals, or multiple adenylation signals and poly U sequences, etc.).
  • the present invention also relates to a host cell containing a nucleic acid as described above, or a vector as described above.
  • the host cell does not comprise a plant cell capable of developing into a whole plant, nor does it comprise an animal cell capable of developing into a whole animal (eg, germ cells, totipotent stem cells such as germ stem cells and embryonic stem cells, fertilization egg).
  • a plant cell capable of developing into a whole plant nor does it comprise an animal cell capable of developing into a whole animal (eg, germ cells, totipotent stem cells such as germ stem cells and embryonic stem cells, fertilization egg).
  • the host cell is an animal cell.
  • the host cell is a mammalian cell.
  • the host cell is a rodent cell, eg, rat, mouse, hamster.
  • the host cell is a primate cell, preferably a human.
  • the host cells are primary cells, such as tumor cells, hepatocytes, cardiomyocytes, neurons, endothelial cells, stem cells, and the like.
  • the host cell is a cell line
  • mice derived from mice:
  • McCoy BALB/3T3, 3T6, A9, AtT-20, Clone M-3, I-10, Y-1, WEHI-3b, ES-D3, F9;
  • the present invention also relates to a new coronary pneumonia detection kit, which contains the above-mentioned polypeptide, anti-antibody and solid support;
  • the anti-antibody is an antibody against the immunoglobulin of the species from which the test sample is derived;
  • the AviTag tag in the polypeptide is biotinylated in advance, and the biotinylated AviTag tag is conjugated to the solid support.
  • the solid support is a test tube, an EP tube, a multi-well plate, a well of a microplate, a bead, or a small disk.
  • the term "particles" may be spherical, nearly spherical, cubic, polyhedral or irregular in shape.
  • the diameter of the microspheres is preferably 10 nm ⁇ 1 mm, such as 100 nm, 500 nm, 1 ⁇ m, 10 ⁇ m, 100 ⁇ m, 500 ⁇ m; preferably 400 nm ⁇ 10 ⁇ m.
  • the fine particles are preferably magnetic fine particles containing a magnetic substance as a component.
  • the magnetic substance can be a metal (elemental metal or alloy), a non-metal, or a composite formed by a metal and a non-metal.
  • Metals such as iron, AlNiCo metal, etc.; non-metals such as ferrite non-metals (preferably Fe 2 O 3 or Fe 3 O 4 magnetic nanoparticles); composites formed by metals and non-metals such as NdFeB rubber magnets composite material.
  • the multi-well plate is preferably an ELISA plate, which can contain 8, 16, 32, 48, 64, 96 or more wells.
  • the anti-antibody is conjugated to a signal substance.
  • the COVID-19 detection kit may further include sample pretreatment reagents (such as sample purification and enrichment reagents, lysing solution, etc.), washing solution (such as PBS, etc.), buffer, and signal substances for display.
  • sample pretreatment reagents such as sample purification and enrichment reagents, lysing solution, etc.
  • washing solution such as PBS, etc.
  • buffer such as PBS, etc.
  • signal substances for display such as PBS, etc.
  • Color reagent if the signal substance is horseradish peroxidase, the color reagent is ECL), etc.
  • the present invention also relates to a new coronary pneumonia detection test strip, including a sample pad, a binding pad, a reaction membrane and an absorption pad, and the reaction membrane is provided with a detection area and a quality inspection area;
  • the binding pad is coated with an anti-antibody, and the anti-antibody is conjugated with a signal substance; the anti-antibody is an antibody against the immunoglobulin of the test sample-derived species;
  • the detection region is conjugated with the polypeptide as described above; the AviTag tag in the polypeptide is biotinylated in advance, and the biotinylated AviTag tag is conjugated to the detection region.
  • the immunoglobulin is at least any one of IgG, IgM, or IgA.
  • the species source of the anti-antibody is different from the species source of the tested sample.
  • the following species sources can be selected: rat, mouse, dog, goat, sheep, horse, donkey, rabbit ,chicken.
  • the signal substance is a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radiolabel, or an enzyme.
  • the signal substance is colloidal gold, fluorescein, fluorescent microspheres, acridine esters, horseradish peroxidase, alkaline phosphatase, or beta-galactosidase.
  • colloidal gold fluorescein, fluorescent microspheres, acridine esters, horseradish peroxidase, alkaline phosphatase, or beta-galactosidase.
  • Enzymes that produce a detectable signal eg, by colorimetry, fluorescence, and luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase.
  • Chromophores such as fluorophores, quantum dots, fluorescent microspheres, luminescent compounds and dyes.
  • a detectable group such as its molecular size is sufficient to induce a modification detectable in its physical and/or chemical properties; such detection can be achieved by optical methods (eg diffraction, surface plasmon resonance, surface variation and contact variation angle) or Physical methods (such as atomic force spectroscopy and tunneling) are implemented.
  • optical methods eg diffraction, surface plasmon resonance, surface variation and contact variation angle
  • Physical methods such as atomic force spectroscopy and tunneling
  • Electron dense substances such as radioactive molecules (eg 32 P, 35 S or 125 I).
  • the present invention also relates to the application of the above-mentioned polypeptide in the preparation of a test strip or kit for SARS-CoV-2 antibody detection.
  • the polypeptide in the test strip or kit, is conjugated to the test strip or the solid phase support in the kit through an AviTag-tagged protein.
  • the detected sample is selected from biological tissue or its lavage fluid, cells, body fluids, further selected from blood, serum, plasma, anticoagulation, cell culture supernatant, saliva, semen, Amniotic fluid, villi, tissue or tissue lysate, throat swab, nasal swab, conjunctival swab, fecal specimen, stool, urine, bronchial lavage, bronchoalveolar lavage, sputum.
  • the subjects for the above purposes may refer to patients or animals suspected of carrying SARS-CoV-2, especially mammals, such as bats and civet cats; preferably primates, more preferably humans.
  • Using the recombinant antigen of the present invention to prepare a novel coronavirus antibody detection kit or test strip can effectively improve the sensitivity and detection rate of the reagent.
  • the present invention also relates to a method for detecting SARS-CoV-2 antibodies, comprising:
  • a new coronavirus S1 expression clone was constructed, wherein the linker sequence is PKSCDKTHTCPPCPAPELLGG, that is, the hinge region sequence of human IgG1, which can maintain the extensibility of the target protein in the spatial structure.
  • the sequence of Avi-tag is GLNDIFEAQKIEWHE, which can be modified by directional biotinylation.
  • the secretory signal peptide is the new coronavirus (Uniprot: P0DTC2) S1 amino acid sequence aa1-12
  • S1-NTD is the new coronavirus spike protein (Uniprot: P0DTC2) amino acid sequence aa13-303
  • S1-RBD is the new coronavirus spike protein ( Uniprot: PODTC2) amino acid sequence aa319-541 (223).
  • the specific amino acid sequence for constructing the expression clone is as follows:
  • the bacteria containing the expression plasmids into LB medium, cultivate at 37°C and 200rpm for 16-20h, collect the bacteria by centrifugation, and use an endotoxin-free plasmid extraction kit to prepare a large number of recombinant plasmids.
  • Mammalian cells HEK293F were thawed and cultured at a cell density of 1.5-2.5 ⁇ 10 6 cells/ml before transfection. During transfection, slowly drop the transfection working solution into the mammalian cells to be transfected, after mixing evenly, incubate at 37°C, 120rpm, 5% CO2 for 48-50h, add feed solution and continue to culture for 48-50h , stop the culture and collect the supernatant by centrifugation, which is the 2019-nCoV-spike antigen expression product.
  • Magnetic bead coating process Connect the prepared 2019-nCoV-spike recombinant antigen to SA magnetic microspheres.
  • the specific implementation steps are: take 10 mg of SA magnetic beads, add 0.05-0.1 mg of 2019-nCoV-spike recombinant antigen, and add 0.05 to 0.1 mg of 2019-nCoV-spike recombinant antigen.
  • the reaction was continuously mixed for 30 min at 37°C to bind the antigen to the SA magnetic beads through the biotinylated AviTag tag.
  • the SA magnetic beads were washed with a phosphate buffer solution containing 0.05-0.1% Tween-20 to remove free Antigen, magnetic microparticles conjugated with 2019-nCoV-spike recombinant antigen were stored in phosphate buffered solution containing 0.5% BSA, 0.05 Tween-20.
  • Acridine labeling process Dissolve 1 mg of mouse anti-human IgG antibody (6.67 nmol, manufacturer Feipeng) in 1 mL of 50 mM PBS buffer (pH 8.0), add 10 ul of 10 mmol/L acridine ester dissolved in DMSO solvent, and react at 25°C 1h, use AKTA purification equipment of GE company, select 5ml G25 pre-packed purification column (GE company), use 50mM PBS (pH 8.0) buffer as elution buffer, remove free acridine ester and reaction by-product NHS, etc., Purified and isolated mouse anti-human IgG antibody acridine label solution.

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Abstract

提供新型冠状病毒抗原多肽及其应用，所述多肽包括如下部分：S1-NTD-Linker-Avi-tag-Linker-S1-RBD，其中通过两个重复的linker序列把S1蛋白的NTD结构域和RBD结构域串联起来，使得这两个结构域具备更好的空间伸展性，以识别抗体；两个linker序列之间引入AviTag标签蛋白使得重组蛋白可以通过固定的位点被固相化，降低包被过程所带来的空间位阻的影响。该多肽能够达到很高的灵敏度和特异性，并且不易发生漏检。

Description

新型冠状病毒抗原及其应用 技术领域
本发明涉及生物医学领域，具体而言，涉及一种新型冠状病毒抗原及其应用。
背景技术
冠状病毒(CoVs；冠状病毒科亚科，冠状病毒科，病毒目)是一组高度多样性，有包膜的，正义链，单链RNA病毒，在多种动物中引起不同严重程度的呼吸道，肠道，肝和神经系统疾病，包括人类。冠状病毒分为四个属：α冠状病毒，β冠状病毒(βCoV)，γ冠状病毒和δ冠状病毒。在过去的12年中，出现了两种新型的β-冠状病毒，即严重的急性呼吸道综合症(SARS-CoV)和中东呼吸道综合症(MERS-CoV)，这两种病毒会引起严重的人类疾病。
新型冠状病毒(SARS-CoV-2)，于1月10日完成病毒核酸检测，并将该基因组信息共享到了virologic.org网站和GenBank上。新型冠状病毒是以前从未在人体中发现的冠状病毒新毒株。其同源性与急性呼吸道综合症(SARS-CoV)达到80％以上，其中的两者的结构蛋白Nucleocapsid protein同源性为94.1％，两者的Spike protein同源性为84.1％。感染新型冠状病毒后，人体会在约7天左右会产生针对病毒的IgM抗体，约14天左右会产生病毒的IgG抗体。产生的抗体是一种保护性的抗体，能促进人体从病毒感染中恢复和自愈。
新型冠状病毒IgM抗体的检测，同时结合新型冠状病毒IgG抗体的检测，可以用于辅助诊断新型冠状病毒疾病，是新冠核酸诊断试剂的有效补充。新冠IgM和IgG检测试剂盒的开发，关键在于新型冠状病毒有效抗原的获得。新冠冠状病毒结构蛋白有刺突蛋白(Spike protein，跨膜蛋白，同源三聚体结构，如“冠状”分布于病毒表面，是介导病毒感染细胞的核心组分)、核衣壳蛋白(Nucleoprotein，是新冠病毒结构蛋白中表达丰度最高的组分)、包膜蛋白(Envelope Protein)和膜蛋白(Membrane Protein)，其中根据蛋白的丰度和空间位置，用于IgM和IgG诊断的主要是核衣壳蛋白以及刺突蛋白。但由于核衣壳 蛋白的保守性较强，且由于被包裹在病毒内，机体针对其产生的抗体特别是IgM抗体比较晚。因此，新冠抗体诊断试剂特别是IgM抗体诊断，所使用的诊断抗原主要是刺突蛋白的相关片段。
现有技术通常重组表达新型冠状病毒S1亚基全长，但使用该重组蛋白作为检测抗原，所产生的信号值偏低。而如果单独表达新型冠状病毒S1亚基C-端的RBD结构域，则容易造成漏检。
发明内容
本发明是通过Linker连接主要结构域而改造新冠刺突蛋白，并用之作为诊断抗原，达到既改善检测信号值，也保证检出率的目的。
本发明涉及一种多肽，其氨基酸序列如SEQ ID NO:1所示。
可选的，如上所述的多肽，其N端还连接有信号肽。
可选的，如上所述的多肽，所述信号肽的氨基酸序列如SEQ ID NO:2所示。
根据本发明的第二方面，涉及编码如上所述的多肽的核酸。
根据本发明的第三方面，涉及含有如上所述核酸的载体。
根据本发明的第四方面，涉及宿主细胞，其含有如上所述的核酸，或如上所述的载体。
根据本发明的第五方面，涉及新冠肺炎检测试剂盒，其含有如上所述的多肽、抗抗体以及固相支持物；
所述抗抗体为抗受试样品来源种属免疫球蛋白的抗体；
所述多肽中的AviTag标签预先被生物素化，并通过该生物素化的AviTag标签缀合于所述固相支持物上。
可选的，如上所述的试剂盒，所述固相支持物为试管、EP管、多孔板、微量反应板凹孔、小珠或小圆片。
可选的，如上所述的试剂盒，所述抗抗体与信号物质缀合。
根据本发明的第六方面，涉及新冠肺炎检测试纸条，包括样品垫、结合垫、反应膜和吸收垫，所述反应膜上设置有检测区和质检区；
所述结合垫包被有抗抗体，且所述抗抗体与信号物质缀合；所述抗抗体为 抗受试样品来源种属免疫球蛋白的抗体；
所述检测区缀合有如上所述的多肽；所述多肽中的AviTag标签预先被生物素化，并通过该生物素化的AviTag标签缀合于所述检测区。
可选的，如上所述的试剂盒，或如上所述的试纸条，所述免疫球蛋白为IgG、IgM或IgA中的至少任意一种。
可选的，如上所述的试剂盒，或如上所述的试纸条，所述信号物质是金属粒子、荧光标记、发色团标记、电子致密标记、化学发光标记、放射性标记或酶。
根据本发明的第七方面，涉及如上所述的多肽在制备SARS-CoV-2抗体检测试纸条或试剂盒中的应用。
可选的，如上所述的应用，所述检测的受试样品选自生物组织或其灌洗液、细胞、体液，进一步选自血液、血清、血浆、抗凝血、细胞培养上清、唾液、精液、羊水、绒毛、组织或组织裂解液、咽拭子、鼻拭子、眼结膜拭子、粪便标本、粪便、尿液、支气管灌洗液、肺泡灌洗液、痰液。
本发明的有益效果为：
通过两个重复的linker序列把S1蛋白的NTD结构域和RBD结构域串联起来，使得这两个结构域具备更好的空间伸展性，以识别抗体；此外，两个linker序列之间引入Avi-tag。AviTag标签蛋白使得重组蛋白可以通过固定的位点被固相化，降低包被过程所带来的空间位阻的影响。由此，该多肽能够达到很高的灵敏度和特异性，并且不易发生漏检。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明一个实施例中所提供多肽的结构示意图；
图2为本发明一个实施例中2019-nCoV-spike-pcDNA3.1的载体图谱；
图3为本发明一个实施例中重组制备的2019-nCoV Spike抗原的电泳结果图。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
术语“多肽”或“蛋白质”是指包含至少两个由肽键连接以形成多肽的氨基酸残基的分子。少于50个氨基酸的小多肽可以称作“肽”。
术语“核酸”、“多核苷酸”和“寡核苷酸”是指核苷酸(例如，核糖核苷酸或脱氧核糖核苷酸)的多聚体，并且包括天然存在的(腺苷、胍、胞嘧啶、尿嘧啶和胸苷)、非天然存在的和修饰的核酸。该术语不受多聚体长度(例如，单体数目)的限制。核酸可以是单链或双链的，并且将通常含有5'-3'磷酸二酯键，尽管在一些情况下，核苷酸类似物可能具有其他键。单体一般被称为核苷酸。术语“非天然核苷酸”或“修饰的核苷酸”是指含有修饰的含氮碱基、糖或磷酸基团，或在其结构中掺入非天然部分的核苷酸。非天然核苷酸的实例包括双脱氧核苷酸，生物素化的，氨基化的，脱氨基的，烷基化的，苄化的和荧光团标记的核苷酸。术语“试剂盒”是指包括至少一个设备的任何制品(例如，包装或容器)，其包括如本文所述的用于特异性扩增、捕获、标记/转化或检测如本文所述的靶核酸序列的固相支持体。试剂盒可进一步包括在本文中描述的方法或其步骤中使用的使用说明书、补充试剂和/或组分或组件。
在本申请中使用术语“检测”和类似术语来概括地指过程或发现或确定某事物的存在或不存在，以及程度、数量或水平，或发生的概率。要理解的是，表 述“检测存在或不存在”、“存在或不存在的检测”和相关表述包括定性和定量检测。例如，定量检测包括确定样品中SARS-CoV-2有关的核酸序列的水平、数量或量。
本发明涉及一种多肽，其氨基酸序列如SEQ ID NO:1所示。
所述多肽包括如下部分：S1-NTD——Linker——Avi-tag——Linker——S1-RBD。
通过两个重复的linker序列把S1蛋白的NTD结构域和RBD结构域串联起来，使得这两个结构域具备更好的空间伸展性，以识别抗体；此外，两个linker序列之间引入Avi-tag，AviTag标签蛋白是一个15个氨基酸的短肽，具有一个单生物素化赖氨酸位点，与已知天然可生物素化序列完全不同。融合表达后，可被生物素连接酶生物素化，为了纯化重组蛋白选用低亲和性的单体抗生物素蛋白或抗生物素蛋白衍生物，除了用于蛋白质分离纯化，还用于蛋白质相互作用研究。在本发明中，AviTag标签蛋白使得重组蛋白可以通过固定的位点被固相化，降低包被过程所带来的空间位阻的影响。由此，该多肽能够达到很高的灵敏度和特异性，并且不易发生漏检。
在一些实施方式中，所述多肽的N端还连接有信号肽。
在一些实施方式中，所述信号肽的氨基酸序列如SEQ ID NO:2所示。
根据本发明的再一方面，本发明还涉及编码如上所述的多肽的核酸。
所述核酸可以是DNA或者RNA。
本发明还涉及含有如上所述核酸的载体。
术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头 多瘤空泡病毒(如SV40)。在一些实施方式中，本发明所述载体中包含基因工程中常用的调控元件，例如增强子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号，或者多腺苷酸化信号和多聚U序列等)。
根据本发明的再一方面，本发明还涉及宿主细胞，其含有如上所述的核酸，或如上所述的载体。
在一些实施方式中，所述宿主细胞不包含能够发育为完整植物的植物细胞，也不包含能够发育为完整动物的动物细胞(例如生殖细胞，具有全能性的干细胞如生殖干细胞和胚胎干细胞，受精卵)。
在一些实施方式中，所述宿主细胞为动物细胞。
在一些实施方式中，所述宿主细胞为哺乳动物细胞。
在一些实施方式中，所述宿主细胞为啮齿类动物细胞，例如大鼠、小鼠、仓鼠。
在一些实施方式中，所述宿主细胞为灵长类动物细胞，优选为人。
在一些实施方式中，所述宿主细胞为原代细胞，例如肿瘤细胞、肝细胞、心肌细胞、神经元、内皮细胞、干细胞等。
在一些实施方式中，所述宿主细胞为细胞系；
常见的细胞系例如：
来源于人的细胞系：
293、IMR-90、W1-38、A549、A431、BHL-100、BeWo、Caco-2、Chang、HCT-15、HeLa、HEp-G2、HEp-2、HT-1080、HT-29、JEG-2、MCF7、KB、Saos-2、WI-38、WISH、WS1、HUVEC、EB-3、Raji、IM-9、Daudi、H9、HL-60、Jurkat、K-562、U937、KG-1；
来源于小鼠的细胞系：
McCoy、BALB/3T3、3T6、A9、AtT-20、Clone M-3、I-10、Y-1、WEHI-3b、ES-D3、F9；
来源于仓鼠的细胞系：
BHK-21、HaK、CHO-K1；
来源于大鼠的细胞系：
AR42J、BRL3A、Clone 9、H4--Ⅱ-E-C3、GH1、GH3、IEC-6、L2、XC、LLC-WRC 256、Jensen、Rat2(TK-)、PC12、L6；
来源于其他动物的细胞系：
D-17、BT、MARC-145、CV-1、COS-1、COS-3、COS-7、Vero、B95-8、CRFK。
本发明还涉及新冠肺炎检测试剂盒，其含有如上所述的多肽、抗抗体以及固相支持物；
所述抗抗体为抗受试样品来源种属免疫球蛋白的抗体；
所述多肽中的AviTag标签预先被生物素化，并通过该生物素化的AviTag标签缀合于所述固相支持物上。
在一些实施方式中，所述固相支持物为试管、EP管、多孔板、微量反应板凹孔、小珠或小圆片。
在本发明中，术语“微粒”可以为球体、近球体、立方体、多面体或不规则形状。微球的直径优选为10nm～1mm，例如100nm、500nm、1μm、10μm、100μm、500μm；优选为400nm～10μm。
微粒优选为磁微粒，其成分中含有磁性物质。磁性物质可以为金属(金属单质或合金)、非金属，或金属与非金属所形成的复合物。金属例如铁、铝镍钴金属等；非金属例如铁氧体非金属(优选为Fe 2O 3或Fe 3O 4磁性纳米粒子)；金属与非金属所形成的复合物例如钕铁硼橡胶磁复合材料。
多孔板优选为酶标板，其含有的孔位可以为8、16、32、48、64、96或更多。
在一些实施方式中，所述抗抗体与信号物质缀合。
在一些实施方式中，所述新冠肺炎检测试剂盒中还可以包含样品预处理试剂(如样品纯化富集试剂，裂解液等)、清洗液(如PBS等)、缓冲液、针对信号物质的显色试剂(如信号物质为辣根过氧化物酶，则显色试剂为ECL)等。
本发明还涉及新冠肺炎检测试纸条，包括样品垫、结合垫、反应膜和吸收垫，所述反应膜上设置有检测区和质检区；
所述结合垫包被有抗抗体，且所述抗抗体与信号物质缀合；所述抗抗体为 抗受试样品来源种属免疫球蛋白的抗体；
所述检测区缀合有如上所述的多肽；所述多肽中的AviTag标签预先被生物素化，并通过该生物素化的AviTag标签缀合于所述检测区。
在一些实施方式中，所述免疫球蛋白为IgG、IgM或IgA中的至少任意一种。
在一些实施方式中，所述抗抗体的种属来源与被检测样品的种属来源不同，常见的可以选择以下种属来源：大鼠、小鼠、犬、山羊、绵羊、马、驴、兔、鸡。
在一些实施方式中，所述信号物质是金属粒子、荧光标记、发色团标记、电子致密标记、化学发光标记、放射性标记或酶。
在一些实施方式中，所述信号物质是胶体金、荧光素、荧光微球、吖啶酯、辣根过氧化物酶、碱性磷酸酶或β-半乳糖苷酶。下面非限定部分列出这些标记：
·产生可检测信号的酶，如通过比色法、荧光和发光来检测，如辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶和葡萄糖-6-磷酸脱氢酶。
·发色团，如荧光、量子点、荧光微球、发光化合物和染料。
·具有能被电子显微镜或通过其电特性，如传导性、电流分析、电压测量和电阻等检测的电子密度的基团。
·可检测基团，如其分子大小足以诱导在其物理和/或化学特性上可检测的修饰；这种检测可通过光学方法(如衍射、表面胞质团共振，表面变异和接触变异角度)或物理方法(如原子力谱学和隧道效应)实现。
·电子致密物质，如放射性分子(如 32P， 35S或 125I)。
根据本发明的再一方面，还涉及如上所述的多肽在制备SARS-CoV-2抗体检测试纸条或试剂盒中的应用。
在一些实施方式中，在所述试纸条或试剂盒中，所述多肽通过AviTag标签蛋白缀合在所述试纸条上，或所述试剂盒中的固相支持物上。
在一些实施方式中，所述检测的受试样品选自生物组织或其灌洗液、细胞、体液，进一步选自血液、血清、血浆、抗凝血、细胞培养上清、唾液、精液、羊水、绒毛、组织或组织裂解液、咽拭子、鼻拭子、眼结膜拭子、粪便标本、粪便、尿液、支气管灌洗液、肺泡灌洗液、痰液。
上述用途的受试者可以指患者或怀疑携带有SARS-CoV-2的动物，特别是哺乳动物，例如蝙蝠、果子狸；优选为灵长类动物，更优选为人。
使用本发明的重组抗原，制备新型冠状病毒抗体检测试剂盒或试纸条，可有效提高试剂的灵敏度和检出率。
本发明还涉及一种检测SARS-CoV-2抗体的方法，包括：
使用如上所述的多肽、试剂盒或试纸条检测SARS-CoV-2抗体。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1 表达克隆构建
按照图1所示示意图，构建新型冠状病毒S1表达克隆，其中linker序列为PKSCDKTHTCPPCPAPELLGG，即人IgG1的铰链区序列，可以维持目的蛋白在空间结构上的伸展性。Avi-tag序列为GLNDIFEAQKIEWHE，可以进行定向的生物素化修饰。分泌信号肽为新型冠状(Uniprot：P0DTC2)S1氨基酸序列aa1-12，S1-NTD为新型冠状病毒刺突蛋白(Uniprot：P0DTC2)氨基酸序列aa13-303，S1-RBD为新型冠状病毒刺突蛋白(Uniprot：P0DTC2)氨基酸序列aa319-541(223个)。构建表达克隆的具体氨基酸序列如下所示：
Figure PCTCN2022072677-appb-000001
通过化学合成上述氨基酸后，利用“经典克隆”的方法将其构建至表达载体pcDNA3.1(+)中，获得表达克隆，将其命名为2019-nCoV-spike-pcDNA3.1，其载体图谱如图2所示。
实施例2 重组蛋白表达及纯化
接种含表达质粒的菌中至LB培养基，37℃、200rpm培养16-20h后，离心收集菌体，利用无内毒素质粒抽提试剂盒大量制备重组质粒，质粒要求A260/A280＝1.8-2.0。
取适量的PBS缓冲液，加入适量的质粒，混匀，记为A液，另取适量的PBS缓冲液，加入适量PEI转染试剂，记为B液，将B液加入A液混匀，制成转染工作液，室温静置10-20分钟，逐滴加入已准备好的细胞中，37℃,120rpm条件下悬浮培养。
复苏并培养哺乳动物细胞HEK293F，并使细胞密度在转染前为1.5～2.5×10 6个/ml。转染时，将转染工作液缓慢滴加入待转染的哺乳动物细胞中，混合均匀后，37℃、120rpm、5％CO 2培养48-50h后，添加补料液并继续培养48-50h，停止培养、离心收集上清液，此即为2019-nCoV-spike抗原表达产物。
将2019-nCoV-spike重组抗原表达产物使用0.45μm滤膜过滤，上样至预先平衡的层析柱(如HisTrap Excel)中，完成上样后再用平衡缓冲液(如：1×PBS，pH7.4)冲洗层析柱直至基线平衡，最后用洗脱缓冲液(如：1×PBS，500mM Imidazole，pH7.4)洗脱，收集洗脱液，电泳检测(图3)，将含有目的蛋白的洗脱液收集合并后透析至储存缓冲液(如：(如：1×PBS，pH7.4))中。
实施例3 活性分析
将制备的2019-nCoV-spike重组抗原偶联至SA磁性微球中，使用吖啶标记的二抗的(如鼠抗人IgG抗体、鼠抗人IgM抗体)，使用化学发光仪检测经核酸检测确认的新冠阳性样本和阴性样本，主要分析IgM抗体反应情况。结果如表1所示，本发明获得的2019-nCoV-spike重组抗原，较之S1全长重组抗原，信噪比更高；较之RBD重组抗原，检出率更高。
磁珠包被工艺：将制备的2019-nCoV-spike重组抗原连接至SA磁性微球中，具体实施步骤为：取10mg SA磁珠，加入0.05～0.1mg的2019-nCoV-spike重组抗原，在37℃下连续混合反应30min，使抗原通过生物素化的AviTag标签结合在SA磁珠上，结合完成后，用含0.05～0.1％Tween-20的磷酸盐缓冲溶液清洗SA磁珠，除去游离的抗原，将结合了2019-nCoV-spike重组抗原的磁微粒保存在含0.5％BSA、0.05Tween-20的磷酸盐缓冲溶液中。
吖啶标记工艺：将1mg鼠抗人IgG抗体(6.67nmol，厂家菲鹏)溶解于1mL50mM PBS缓冲液(pH 8.0)中，加入10ul溶解于DMSO溶剂的10mmol/L吖啶酯，于25℃反应1h，用GE公司的AKTA纯化设备，选用5ml G25预装纯化柱(GE公司)，以50mM PBS(pH 8.0)缓冲液作为洗脱缓冲液，除去游离的吖啶酯及反应副产物NHS等，纯化分离得到鼠抗人IgG抗体吖啶标记物溶液。
表1 重组抗原发光检测结果
样品情况 2019-nCoV spike抗原 S1全长抗原 RBD抗原
阳性 99189 34433 107913
阳性 81735 28357 125636
阳性 6380 2289 6185
阳性 2669 996 2842
阳性 2933 1001 4457
阳性 29058 10047 320
阳性 3696 1270 4048
阳性 3146 1017 4677
阳性 13314 4770 18687
阳性 23115 8079 27676
阳性 12033 3961 21574
阳性 4539 1513 6099
阳性 2655 1013 230
阳性 46539 16269 450
阳性 339881 111256 523942
阳性 3633 1305 7902
阳性Aveage 42157 14224 53915
阴性 299 190 138
阴性 353 290 134
阴性 342 210 139
阴性 279 200 157
阴性 295 170 122
阴性 434 175 112
阴性 818 293 244
阴性 685 180 749
阴性 673 185 324
阴性 268 193 119
阴性 267 235 162
阴性 463 273 110
阴性 287 253 107
阴性 346 260 917
阴性 371 393 448
阴性 294 335 225
阴性 354 200 324
阴性 344 875 981
阴性 628 213 129
阴性 550 523 543
阴性 486 483 419
阴性 793 258 190
阴性 309 430 297
阴性 276 290 159
阴性 285 270 252
阴性 368 203 146
阴性 335 213 180
阴性 380 253 639
阴性 379 383 337
阴性 481 438 279
阴性 279 810 191
阴性 299 453 120
阴性 809 178 181
阴性 232 310 264
阴性 306 358 782
阴性 245 288 2050
阴性 450 310 305
阴性 219 418 337
阴性 287 328 210
阴性 257 463 1998
阴性 298 235 282
阴性 267 333 184
阴性 280 378 497
阴性 241 348 218
阴性 276 200 152
阴性 471 990 207
阴性 88 370 208
阴性 86 430 281
阴性 91 268 291
阴性 138 238 401
阴性 49 215 221
阴性 144 263 271
阴性 82 193 163
阴性 45 178 129
阴性 213 240 181
阴性 254 293 166
阴性 95 188 128
阴性 103 360 376
阴性 270 278 161
阴性 115 295 166
阴性 209 820 952
阴性 170 245 157
阴性 120 193 217
阴性 213 300 289
阴性 83 208 170
阴性 146 240 315
阴性 130 278 428
阴性 153 233 143
阴性 119 275 180
阴性 324 338 403
阴性 428 463 262
阴性 152 418 248
阴性Average 301 320 333
信噪比 140 44 162
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
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. 多肽，其氨基酸序列如SEQ ID NO:1所示；
    可选的，所述的多肽N端还连接有信号肽；
    可选的，所述信号肽的氨基酸序列如SEQ ID NO:2所示。
  2. 编码权利要求1所述的多肽的核酸。
  3. 含有权利要求2所述核酸的载体。
  4. 宿主细胞，其含有权利要求2所述的核酸，或权利要求3所述的载体。
  5. 新冠肺炎检测试剂盒，其含有权利要求1所述的多肽、抗抗体以及固相支持物；
    所述抗抗体为抗受试样品来源种属免疫球蛋白的抗体；
    所述多肽中的AviTag标签预先被生物素化，并通过该生物素化的AviTag标签缀合于所述固相支持物上；
    可选的，所述固相支持物为试管、EP管、多孔板、微量反应板凹孔、小珠或小圆片；
    可选的，所述抗抗体与信号物质缀合。
  6. 新冠肺炎检测试纸条，包括样品垫、结合垫、反应膜和吸收垫，所述反应膜上设置有检测区和质检区；
    所述结合垫包被有抗抗体，且所述抗抗体与信号物质缀合；所述抗抗体为抗受试样品来源种属免疫球蛋白的抗体；
    所述检测区缀合有权利要求1所述的多肽；所述多肽中的AviTag标签预先被生物素化，并通过该生物素化的AviTag标签缀合于所述检测区。
  7. 根据权利要求5所述的试剂盒，或权利要求6所述的试纸条，所述免疫球蛋白为IgG、IgM或IgA中的至少任意一种。
  8. 根据权利要求5所述的试剂盒，或权利要求6所述的试纸条，所述信号物质是金属粒子、荧光标记、发色团标记、电子致密标记、化学发光标记、放射性标记或酶。
  9. 权利要求1所述的多肽在制备SARS-CoV-2抗体检测试纸条或试剂盒中的应用。
  10. 根据权利要求9所述的应用，所述检测的受试样品选自生物组织或其灌洗液、细胞、体液，进一步选自血液、血清、血浆、抗凝血、细胞培养上清、唾液、精液、羊水、绒毛、组织或组织裂解液、咽拭子、鼻拭子、眼结膜拭子、粪便标本、粪便、尿液、支气管灌洗液、肺泡灌洗液、痰液。
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