WO2021179371A1 - 新冠病毒n-s优势表位融合蛋白、制备方法、应用，及表达蛋白、微生物、应用，试剂盒 - Google Patents

新冠病毒n-s优势表位融合蛋白、制备方法、应用，及表达蛋白、微生物、应用，试剂盒 Download PDF

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WO2021179371A1
WO2021179371A1 PCT/CN2020/083016 CN2020083016W WO2021179371A1 WO 2021179371 A1 WO2021179371 A1 WO 2021179371A1 CN 2020083016 W CN2020083016 W CN 2020083016W WO 2021179371 A1 WO2021179371 A1 WO 2021179371A1
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protein
dominant epitope
antigen
coronavirus
new coronavirus
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PCT/CN2020/083016
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English (en)
French (fr)
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陈庆全
李丽满
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河北精硕生物科技有限公司
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • 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
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the invention belongs to the technical field of genetic engineering, and relates to the preparation and application of a fusion protein, specifically a novel coronavirus N-S dominant epitope fusion protein, preparation method, application, expression protein, microorganism, application, and kit.
  • Coronavirus is a large virus family, known to cause colds and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • 2019-nCoV acute respiratory disease 2019-nCoV acute respiratory disease
  • 2019-nCoV 2019 represents the year when it first appeared
  • n represents novel (New)
  • CoV stands for coronavirus (coronavirus)).
  • Coronaviruses belong to the genus Coronavirus in the family Coronaviridae (Coronaviridae).
  • the new type of coronavirus belongs to the beta genus of coronaviruses, which have an envelope, round or oval particles, often pleomorphic, with a diameter of 60 to 140 nm. Although its clinical symptoms after infection are similar to those of SARS virus, its genetic characteristics are significantly different from SARSr-CoV and MERSr-CoV. Current research shows that the new coronavirus is more than 85% homologous to the bat SARS-like coronavirus (bat-SL-CoVZC45). The two ends of the new coronavirus genome contain an untranslated region, respectively, 5'-UTR and 3'-UTR.
  • ORF1a and ORF1b which are mainly used It encodes non-structural proteins such as enzymes related to viral replication and transcription, and about 1/4 of the gene at the 3'end is used to encode surface spike (S) protein, membrane (membrane, M) protein, and small envelope ( Small envelope membrane (E) protein and nucleocapsid (N) protein and other major structural proteins.
  • the detection of virus-infected persons includes nucleic acid detection against virus genes and immune detection against virus-specific antibodies produced by the body.
  • the clinical laboratory testing process if the specific nucleic acid sequence of the new coronavirus is detected in the patient's sample, it should indicate that the patient may be infected with the new coronavirus.
  • the current tests for the new coronavirus infection are all nucleoside tests.
  • nucleic acid testing is limited by unfavorable factors such as difficulty in sampling, complicated operations, and long testing time, which will cause some infected people to miss the test. The case where the test was negative, but the patient was diagnosed later, had a certain impact on the control of the epidemic).
  • nucleic acid detection Compared with nucleic acid detection, after the body is infected with the virus, it can produce specific antibodies against multiple antigen components of the virus. Therefore, specific antibody detection is an important indicator for the diagnosis and epidemiological investigation of viral infectious diseases.
  • immunological testing has the advantages of simple operation, short testing time, testing can be completed within 10 minutes, and sample collection can be standardized, it has become an important supplement to nucleic acid testing for viral infections. Infected persons with negative nucleic acid testing are detected. Reduce missed inspections. Therefore, the development of a device that uses immunoassays to detect patients with the new coronavirus can speed up the diagnosis of patients with the new coronavirus, which is of great significance for the prevention and control of the epidemic.
  • the purpose of the present invention is to provide a novel coronavirus (i.e. 2019-nCoV) N-S predominant epitope fusion protein to use the above fusion protein for immune detection to speed up the diagnosis of people infected with the new coronavirus and reduce the missed detection rate;
  • a novel coronavirus i.e. 2019-nCoV N-S predominant epitope fusion protein
  • the other objective of the present invention is to provide the preparation method and application of the above-mentioned fusion protein, corresponding expression protein, microorganism, application, and kit.
  • a novel coronavirus N-S dominant epitope fusion protein its amino acid sequence is as follows:
  • nucleic acid sequence is as follows:
  • the second purpose of the present invention is to provide a method for preparing the above-mentioned fusion protein. Firstly, the S protein dominant epitope segment antigen and the N protein dominant epitope segment antigen of the novel coronavirus are constructed into a fusion expression antigen; Then through genetic optimization, rare codons are removed to obtain the optimized gene sequence, and finally a highly expressed soluble and highly active antigen is obtained.
  • the S protein dominant epitope segment antigen and the N protein dominant epitope segment antigen of the novel coronavirus are screened by epitope analysis and prediction respectively.
  • the method of constructing a fusion expressing antigen is to link the N protein dominant epitope segment antigen and the S protein dominant epitope segment antigen encoding genes by using the same-tailed enzymatic method with complementary sticky ends Together for fusion expression.
  • Structural proteins are the main candidate antigens for coronavirus serological antibody detection. Among them, N and S proteins are the most reported, while M and E proteins are less reported.
  • the N protein as the most abundant and conserved protein in the structural protein of coronavirus, often appears in the serum of patients in the early stage of infection, and induces the body to produce a large number of specific IgG and IgM antibodies. Therefore, it has become the first choice for detecting virus antibodies in the early stage of infection.
  • the S protein specific antibody titer is lower than the N protein three weeks before the disease, but the detection rate of the N protein IgG antibody is close to the detection rate of the N protein IgG antibody in the later period. There is complementarity between the two.
  • the combination of N protein and S protein is used as an antigen for SARS-CoV-2 antibody detection, which can further improve the sensitivity of detection and reduce missed detection than using any antigen alone.
  • the third objective of the present invention is to provide an expression vector or microorganism or cell, wherein the expression vector or microorganism or cell is prepared by expressing the fusion protein of the NS dominant epitope of the new coronavirus; the expressed microorganism is a bacterium, and The bacteria is Escherichia coli.
  • the fourth objective of the present invention is to provide an application of the above-mentioned fusion protein, including the fusion protein of the NS dominant epitope of the new coronavirus, expression vectors, and microorganisms in the preparation of reagents for the detection of new coronavirus infections and/or the preparation of anti-new coronaviruses Application of antibody reagents.
  • another object of the present invention is to provide an anti-new coronavirus antibody detection kit or a new coronavirus infection detection kit, an anti-new coronavirus antibody detection kit or a new coronavirus infection detection kit
  • the card has the above-mentioned fusion protein of the dominant epitope of the new coronavirus NS.
  • the present invention has the following beneficial effects compared with the prior art:
  • the new coronavirus NS dominant epitope fusion protein of the present invention uses the combination of the new coronavirus N protein and S protein as the antigen for SARS-CoV-2 antibody detection, which can be more sensitive than using any antigen alone. , Which reduces the missed detection rate;
  • the new coronavirus NS dominant epitope fusion protein of the present invention covers both the dominant epitope segment antigen of the new coronavirus N protein and the dominant epitope segment antigen of the S protein, and is soluble expression, high yield and high purity , Good activity and significantly improved specificity;
  • the novel coronavirus antibody detection kit of the present invention is a total antibody detection kit, which detects total antibodies including IgG and IgM. The detection only takes ten minutes, and the detection sensitivity is also significantly improved.
  • the present invention firstly analyzes and predicts the epitope, respectively screens and determines the antigen of the dominant epitope segment of the new coronavirus N protein and the dominant epitope segment of the S protein;
  • the N protein dominant epitope segment antigen is constructed into a fusion expression antigen; the optimized gene sequence is obtained by removing rare codons through genetic optimization, and finally a highly expressed soluble and highly active antigen is obtained.
  • the sensitivity and specificity are significantly better than the insoluble expressed antigen.
  • This antigen is suitable for the development of serological antibody detection reagents, for the auxiliary diagnosis of patients with new coronavirus infection, and the screening of high-risk groups and suspected patients who are in close contact with the patient.
  • the sensitivity and specificity of the N-S dominant epitope fusion protein of the present invention are both 100% in the detection of the total antibody of the novel coronavirus.
  • the N-S dominant epitope fusion protein of the new coronavirus provided by the present invention is suitable for the diagnosis and detection of patients with the new coronavirus.
  • Figure 1 is an SDS-PAGE electrophoresis diagram of the expressed N-S dominant epitope fusion protein in Example 3 of the present invention
  • Figure 2 is a schematic diagram of the results of the latex method detection in Example 4 of the present invention.
  • the antibody detection in the prior art can use cracked virus particles as the antigen, but because SARS-CoV-2 culture must be carried out in the BSL3 laboratory, and there is a high risk of infection, it is necessary to use artificially expressed antigens instead of the whole virus As a detection antigen.
  • Previous literature reports show that N protein is of moderate size and is not a glycoprotein and is easy to express efficiently. However, because S protein is too large and is a glycoprotein, it is difficult to efficiently express full-length S protein in bacteria. Therefore, it is necessary in the present invention. Select sensitive, specific, and easy-to-express segments as diagnostic antigens.
  • the B-cell epitope analysis and signal peptide analysis functions of the BIOSUN software were used to analyze the N protein amino acid sequence, and the antigenicity was selected according to the strength of the hydrophobicity and the signal peptide.
  • the good 20-220aa segment (denoted as N1), 230-300aa segment (denoted as N2) and 335-419aa segment (denoted as N3) are used as the segment antigens for screening the dominant epitope of N protein in this example .
  • the gene sequences of the N1 to N3 segments are as follows:
  • the N1 gene sequence is:
  • the N2 gene sequence is:
  • the N3 gene sequence is:
  • N1-N3 the coding genes of N1-N3 segments were artificially modified and optimized, and prokaryotic expression vectors pET30-N1, pET30-N2 and pET30-N3 were constructed according to conventional molecular biology methods.
  • the optimized nucleic acid sequences of N1-N3 are:
  • the N1 optimized nucleic acid sequence is:
  • the N2 optimized nucleic acid sequence is:
  • the N3 optimized nucleic acid sequence is:
  • the spare sample is purified by the Ni column. After equilibrium, the processed sample is slowly loaded. When the absorbance value starts to rise, the passing solution is collected. After re-equilibration, elute with a solution containing 25mM imidazole and 25mM Tris-HCl (25mM Tris-HCl contains 250mM imidazole, the pH of the solution is 8.5), collect the protein peaks, and analyze the purified product by electrophoresis to verify the purified target protein In 250mM imidazole eluent.
  • the predominant epitope segment antigens of N protein obtained after purification were respectively subjected to SDS-PAGE electrophoresis, transferred to membrane, and blocked with 5% skimmed milk powder at room temperature for 1 hour.
  • 10 portions of serum from patients with new coronary pneumonia and 10 portions of serum from healthy blood donors diluted with blocking solution (the volume ratio of serum to blocking solution is 1:50) were incubated overnight at 4°C.
  • the membrane was incubated with a secondary antibody labeled with horseradish peroxidase for 1 hour. Then the membrane was washed 3 times with TBST at room temperature, and the positive band was displayed by the ECL method.
  • N1 epitope antigen and 8 samples showed specific bands
  • N2 epitope antigen and 6 samples showed specific bands
  • N3 epitope antigen and 2 samples showed specific bands.
  • Bands In 10 healthy blood donor sera, N1 and N3 epitope antigens did not show non-specific bands, and N2 epitope antigens and 1 sample showed non-specific bands.
  • the results show that among the three expressed N protein segment antigens, the N1 predominant epitope segment antigen has the best antigenic activity and specificity.
  • the B-cell epitope analysis and signal peptide analysis functions of the BIOSUN software are used to analyze the S protein amino acid sequence, and combined with the functional domain distribution, select 65- with good antigenicity.
  • the 290aa segment (denoted as S1), the 340-510aa segment (denoted as S2) and the 520-681aa segment (denoted as S3) three dominant epitope segment antigens were used to screen the dominant epitope of S protein in this example The segment antigen.
  • the S1 gene sequence is:
  • the S2 gene sequence is:
  • the S3 gene sequence is a
  • the coding genes of each segment were artificially modified and optimized, and the prokaryotic expression vectors pET30-S1, pET30-S2 and pET30-S3 were constructed according to conventional molecular biology methods, and the sequencing was correctly recombined
  • the expression plasmid was transformed into BL21 competent cells, and the induced expression and antigen purification were performed according to the method of Example 1 to obtain purified antigen.
  • the optimized nucleic acid sequence of S1-S3 is as follows:
  • the activity identification was performed according to the WB method of Example 1.
  • the results showed that in 10 sera from patients with new coronary pneumonia, S1 epitope antigen and 5 samples showed specific bands, S2 epitope antigen and 3 samples showed specific bands, and S3 epitope antigen and 7 samples showed specific bands. Bands: In 10 healthy blood donor sera, S1, S2 and S3 did not show non-specific bands. The results show that among the three S protein segment antigens expressed in this example, the S3 predominant epitope segment antigen has the best antigenic activity and specificity.
  • nucleic acid sequence of the aforementioned novel coronavirus N-S dominant epitope fusion protein is as follows:
  • the prokaryotic expression vector pET30-N-S according to conventional molecular biology methods, transform the correctly sequenced recombinant expression plasmid into BL21 competent cells, and perform induction expression and antigen purification according to the method of Example 1 to obtain purified antigen.
  • the results show that the N-S dominant epitope fusion protein is a soluble antigen expressed, and the purity of the antigen after purification is as high as 99.1%. The result is shown in Figure 1.
  • the activity identification was performed according to the WB method of Example 1.
  • the results show that this example and 10 samples of patients with new coronary pneumonitis showed specific bands, while 10 samples of healthy blood donors did not show non-specific bands.
  • the results show that the N-S dominant epitope fusion protein has very good antigenic activity and specificity.
  • the kit of this embodiment adopts the fluorescence immunochromatography technology and the principle of immunology in the prior art to detect the concentration of the novel coronavirus IgM antibody in the sample.
  • the structure of the kit is the same as that of the kit in the prior art, and both have
  • the test card, and the test card of this example is coated with the fusion protein of the NS dominant epitope of the new coronavirus of Example 3, mouse anti-human IgM antibody and goat anti-mouse IgG.
  • the specific detection principle is: when testing, the sample is added to the sample hole of the kit or test card, the anti-new coronavirus IgM and the fluorescently labeled mouse anti-human IgM antibody in the sample undergo an immune reaction to form an immunoconjugate, and the conjugate layer When it is analyzed to the T line of the test card, it is captured by the fusion protein of the new coronavirus NS dominant epitope of the T line. When the sample is chromatographed to the C line, the fluorescently labeled mouse anti-human IgM antibody is captured by the goat anti-mouse IgG of the C line as a quality control String. The intensity of the fluorescent signal at the T line is positively correlated with the concentration of the anti-coronavirus IgM antibody in the sample. Therefore, the concentration of the anti-coronavirus IgM antibody in the sample can be detected with a suitable dry fluorescent instrument.
  • This kit needs to be used horizontally at room temperature.
  • Each box has a new coronavirus IgM antibody detection card, 1 sample pipette per person, and 1 bottle of sample diluent.
  • Whole blood sample Use a sample pipette to add 2 drops of whole blood vertically to the sample hole, then add 1-2 drops of sample diluent, 10 minutes after dilution, judge the result with a suitable instrument.
  • 2Serum/plasma sample use a sample pipette to drop 1 drop vertically into the sample hole, and then add 1-2 drops of sample diluent, 10 minutes after dilution, use an adapted instrument to judge the result.
  • This test card tested the serum samples of 50 patients with positive nucleic acid test for novel coronavirus infection, and the results showed that 45 cases were positive, with a sensitivity of 90%; out of 50 healthy control serum samples, 50 cases were negative and specific The sex is 100%.
  • a red line appears on each of the quality control line (C line) and the test line (T line). Indicates the presence of new coronavirus antibodies in the sample.
  • C line quality control line
  • T line test line
  • the structure of the kit is the same as that of the kit in the prior art, and both include a nitrocellulose membrane, a color latex-labeled anti-human IgM antibody release pad and other reagents.
  • the N-S dominant epitope fusion protein of the new coronavirus in Example 3 is immobilized on the nitrocellulose membrane.
  • the principle of capture method is used to detect new coronavirus IgM antibodies in human serum/plasma/whole blood. During the detection process, the blood sample is added to the sample hole of the kit. The sample is first mixed with the colored latex-labeled anti-human IgM on the release pad, and then chromatographed on the nitrocellulose membrane.
  • the sample contains new coronavirus IgM antibodies
  • these antibodies first bind to the colored latex-labeled anti-human IgM, so that when the mixture is chromatographed on a nitrocellulose membrane, it will be detected with the fusion protein of the new coronavirus NS dominant epitope immobilized
  • the line (T line) is captured to form a colored latex-labeled anti-human IgM-antibody-antigen immune complex, so a red line appears on the T line, which is a positive result. If there is no new coronavirus IgM antibody in the blood of the subject, a red line will not be formed on the test line (T line), which is a negative result.
  • the quality control line (line C) on the reagent card is coated with goat anti-mouse antibody. Under normal circumstances, red lines should appear on the quality control line during the test to prove that the reagent card is working properly.
  • Each box contains a new coronavirus IgM test card, 1 sample pipette per person, and 1 bottle of sample diluent. There are two modes when adding samples, namely:
  • 2Serum/plasma sample Use a sample pipette to drop 1 drop vertically into the sample hole, then add 1-2 drops of sample diluent, and judge the result directly after 10 minutes of dilution. .
  • This kit judges the result within 10 minutes after adding the sample, and the observation result is invalid after 20 minutes.
  • This test card tested the serum samples of 50 patients with positive nucleic acid test for novel coronavirus infection, 45 of which were positive, with a sensitivity of 90%; 50 of the 50 healthy control serum samples were negative with a specificity of 100% .
  • This kit adopts dry fluorescence immunochromatography technology and the principle of double antigen sandwich method to detect the concentration of the new coronavirus IgM/IgG total antibody in the sample.
  • the new coronavirus test card in this kit has the N-S dominant epitope fusion protein of the new coronavirus of Example 3.
  • the new coronavirus IgM/IgG antibody and the fluorescently labeled mouse anti-human new coronavirus antigen in the sample undergo an immune reaction to form an immune conjugate, and the conjugate is chromatographed to the test card In the T line, it is captured by the fusion protein of the NS dominant epitope of the new coronavirus on the T line.
  • the fluorescently labeled mouse anti-human novel coronavirus antigen is captured by the goat anti-mouse IgG on the C line as a quality control line.
  • the intensity of the fluorescent signal at the T line is positively correlated with the concentration of anti-coronavirus antibodies in the sample. Therefore, the concentration of total antibodies against the novel coronavirus IgM/IgG in the sample can be detected with a suitable dry fluorescent instrument.
  • Each box contains a new coronavirus IgM test card, 1 sample pipette per person, and 1 bottle of sample diluent. There are two modes when adding samples, namely:
  • Whole blood sample Use a sample pipette to add 2 drops of whole blood vertically to the sample hole, and then add 1-2 drops of sample diluent, 10 minutes after sample addition, use a suitable instrument to judge the result.
  • 2Serum/plasma sample use a sample pipette to drop 1 drop vertically into the sample hole, and then add 1-2 drops of sample diluent, 10 minutes after sample addition, use a suitable instrument to judge the result.
  • this test card After testing the serum samples of 50 patients with positive nucleic acid test for novel coronavirus infection, this test card showed that 50 cases were positive, with a sensitivity of 100%; of the 50 healthy control serum samples, 50 cases were negative with a specificity of 100% .
  • a red line appears on each of the quality control line (C line) and the test line (T line). Indicates the presence of total antibodies to the new coronavirus IgM/IgG in the sample.
  • C line quality control line
  • T line test line
  • the kit is composed of nitrocellulose membrane, color latex-labeled anti-human novel coronavirus antigen release pad and other reagents. Among them, a novel coronavirus-specific N-S dominant epitope fusion protein is immobilized on the nitrocellulose membrane.
  • the detection principle is: the use of latex immunochromatography technology and the principle of capture method to detect the total antibody of novel coronavirus IgM/IgG in human serum/plasma/whole blood.
  • the blood sample is added to the sample hole of the kit. The sample is first mixed with the colored latex-labeled anti-human novel coronavirus antigen on the release pad, and then chromatographed on the nitrocellulose membrane.
  • the sample contains the total antibodies of the novel coronavirus IgM/IgG, these antibodies first bind to the anti-human novel coronavirus antigen labeled with colored latex, so that when the mixture is chromatographed on the nitrocellulose membrane, the novel coronavirus antigen will be immobilized
  • the detection line (T line) is captured to form a colored latex-labeled anti-human antigen-antibody-antigen immune complex, so a red line appears on the T line, which is a positive result. If there is no total antibody to the new coronavirus IgM/IgG in the blood of the subject, a red line will not be formed on the test line (T line), which is a negative result.
  • the quality control line (line C) on the reagent card is coated with goat anti-mouse antibody. Under normal circumstances, red lines should appear on the quality control line during the test to prove that the reagent card is working properly.
  • kits needs to be used horizontally at room temperature.
  • Each kit has a new coronavirus IgM/IgG total antibody detection card, 1 sample pipette per person, and 1 bottle of sample diluent.
  • 2Serum/plasma sample use a sample pipette to drop 1 drop vertically into the sample hole, then add 1-2 drops of sample diluent, and directly judge the result 10 minutes after sample addition.
  • This reagent card tested the serum samples of 50 patients with positive nucleic acid test for novel coronavirus infection, and the results showed that 50 were positive with a sensitivity of 100%; 50 of the 50 healthy control serum samples were negative with a specificity of 100 %.

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Abstract

一种新冠病毒N-S优势表位融合蛋白、制备方法、应用，及表达蛋白、微生物、应用和试剂盒，属于基因工程技术领域。其中新型冠状病毒N-S优势表位融合蛋白为新型冠状病毒N蛋白和S蛋白的优势表位融合蛋白，首先由新型冠状病毒的S蛋白优势表位区段抗原与N蛋白优势表位区段抗原构建成一个融合表达抗原；然后通过基因优化去除稀有密码子获得优化后基因序列，最终获得高效表达的可溶性高活性抗原。所述新型冠状病毒N-S优势表位融合蛋白为可溶性表达、产量高、纯度高、活性好，适用于新型冠状病毒患者的确诊检测。

Description

新冠病毒N-S优势表位融合蛋白、制备方法、应用，及表达蛋白、微生物、应用，试剂盒 技术领域
本发明属于基因工程技术领域，涉及一种融合蛋白的制备及应用，具体地说是一种新冠病毒N-S优势表位融合蛋白、制备方法、应用，及表达蛋白、微生物、应用，试剂盒。
背景技术
[根据细则9.2改正16.06.2020]　
冠状病毒是一个大型病毒家族，已知可引起感冒以及中东呼吸综合征(MERS)和严重急性呼吸综合征(SARS)等较严重疾病。而2019年出现的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。2020年1月31日，世界卫生组织建议将当前新型冠状病毒肺炎暂命名为“2019-nCoV acute respiratory disease”，把病毒暂命名为“2019-nCoV”(2019代表首次出现的年份，n表示novel(新)，CoV表示coronavirus(冠状病毒))。2月7日，中国国家卫健委发出通知，决定将“新型冠状病毒感染的肺炎”暂命名为“新型冠状病毒肺炎”，简称“新冠肺炎”，英文名称为“novel coronavirus pneumonia”，简称“NCP”。2月11日，世界卫生组织发布了新型冠状病毒感染引起的疾病的正式名称“COVID-19”，其中“CO”代表“corona(冠状物)”，“VI”代表“virus(病毒)”，“D”代表“disease(疾病)”。同日，国际病毒分类委员会(ICTV)将新型冠状病毒命名为SARS-CoV-2，并强调了新病毒与SARS病毒的相似性。
冠状病毒在系统分类上属冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)。而新型冠状病毒则属于β属的冠状病毒，其具有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60～140nm。虽然其感染后的临床症状与SARS病毒相似，其基因特征与SARSr-CoV和MERSr-CoV有明显区别。目前研究显示新型冠状病毒与蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)同源性达85％以上。新型冠状病毒基因组的两端分别包含一个非翻译区，分别为5'-UTR、3'-UTR，其中5'端约3/4包含2个大的重叠的开放读框ORF1a和ORF1b，主要用于编码与病毒复制转录有关的酶类等非结构蛋白，而3'端约1/4的基因用于编码表面棘突(spike，S)蛋白、膜(membrane，M)蛋白、小包膜(small envelope membrane，E)蛋白及核衣壳(nucleocapsid，N)蛋白等主要结构蛋白。
目前临床上，对于病毒感染者的检测包括针对病毒基因的核酸检测，以及针对机体产生的病毒特异性抗体的免疫检测。在临床实验室检测过程中，如果在患者样本中检测到新型冠状病毒的特异核酸序列，应提示该患者可能被新型冠状病毒感染，目前对于新冠病毒感染的检测采用的均是核苷检测。但是由于核酸检测受到取样困难、操作复杂、检测时间长等不 利因素的限制，会造成部分感染者漏检(例如，我国利用核苷检测对新冠肺炎疑似患者检测的过程中出现了多例核苷检测呈阴性，但后期患者确诊的例子，对疫情的控制造成了一定的影响)。相对核酸检测而言，机体在感染病毒后,能够产生针对病毒多种抗原成分的特异性抗体,因此特异性抗体检测是病毒性传染病诊断和流行病学调查的重要指标。此外，由于免疫学检测具有操作简便、检测时间短、最短可在10分钟内完成检测、样本采集可以标准化等优点，而成为病毒感染核酸检测的重要补充，检出核酸检测阴性中的感染者，减少漏检。因此，研发出利用免疫检测法对新型冠状病毒患者的检测装置，能够加快对新冠病毒感染者的确诊，对于疫情的防控具有重要意义。
发明内容
本发明的目的，是要提供一种新冠病毒(即2019-nCoV)N-S优势表位融合蛋白，以利用上述融合蛋白进行免疫检测来加快新冠病毒感染者的确诊，同时降低漏检率；
本发明其它的发明目的，是要提供上述融合蛋白的制备方法、应用，相应表达蛋白、微生物、应用，试剂盒。
为了实现上述目的，本发明所采用的技术方案如下：
一种新冠病毒N-S优势表位融合蛋白，其氨基酸序列如下：
Figure PCTCN2020083016-appb-000001
作为对本发明的限定：其核酸序列如下：
Figure PCTCN2020083016-appb-000002
Figure PCTCN2020083016-appb-000003
本发明的第二个目的，是要提供上述融合蛋白的一种制备方法，首先将新型冠状病毒的S蛋白优势表位区段抗原与N蛋白优势表位区段抗原构建成一个融合表达抗原；然后通过基因优化去除稀有密码子获得优化后基因序列，最终获得高效表达的可溶性高活性抗原。
作为对上述方法的限定：所述新型冠状病毒的S蛋白优势表位区段抗原与N蛋白优势表位区段抗原分别通过表位分析预测进行筛选。
作为对上述方法的另一种限定：建成融合表达抗原的方法为：利用具有互补粘性末端的同尾酶法将N蛋白优势表位区段抗原和S蛋白优势表位区段抗原的编码基因连接在一起进行融合表达。
结构蛋白是冠状病毒血清学抗体检测的主要候选抗原，其中报道最多的是N和S蛋白，而M和E蛋白的报道较少。而N蛋白作为冠状病毒结构蛋白中含量最丰富和保守的蛋白质，常在感染早期病人血清中出现，诱导机体产生大量特异性IgG和IgM抗体，因此成为感染早期检测病毒抗体的首选抗原。与N蛋白相比，一方面，S蛋白特异性抗体滴度在患病前三周低于N蛋白，但是后期则与N蛋白IgG抗体的检出率接近，两者之间存在互补性，因此本发明中以N蛋白和S蛋白组合作为抗原用于SARS-CoV-2的抗体检测，可以比单独使用任何一种抗原进一步提高检测的灵敏度，减少漏检。
本发明的第三个目的，是提供一种表达载体或微生物或细胞，其中表达载体或微生物或细胞均通过表达新冠病毒N-S优势表位融合蛋白制得；所述表达的微生物为细菌，所述细菌为大肠杆菌。
本发明的第四个目的，是提供上述融合蛋白的一种应用，包括新冠病毒N-S优势表位融合蛋白、表达载体、微生物在制备检测新型冠状病毒感染试剂中和/或制备检测抗新型冠状病毒抗体试剂中的应用。
另外，本发明还有一个目的，是提供一种抗新型冠状病毒抗体检测试剂盒或一种新型 冠状病毒感染检测试剂盒，抗新型冠状病毒抗体检测试剂盒或新型冠状病毒感染检测试剂盒的检测卡内均具有上述新冠病毒N-S优势表位融合蛋白。
由于采用了上述的技术方案，本发明与现有技术相比，所取得的有益效果如下：
(1)本发明的新冠病毒N-S优势表位融合蛋白以新型冠状病毒的N蛋白和S蛋白组合作为抗原用于SARS-CoV-2的抗体检测，可以比单独使用任何一种抗原检测的灵敏度高，降低了漏检率；
(2)本发明的新冠病毒N-S优势表位融合蛋白同时涵盖了新型冠状病毒N蛋白的优势表位区段抗原和S蛋白的优势表位区段抗原，并且为可溶性表达，产量高、纯度高、活性好、特异性显著提高；本发明的新型冠状病毒抗体检测试剂盒为总抗体检测试剂盒，检测包含IgG和IgM在内的总抗体，检测只需十分钟，检测灵敏度也显著提高。
本发明首先通过表位分析预测，分别筛选确定新型冠状病毒N蛋白优势表位区段抗原和S蛋白优势表位区段抗原；然后将难表达的S蛋白优势表位区段抗原与易表达的N蛋白优势表位区段抗原构建成一个融合表达抗原；通过基因优化去除稀有密码子获得优化后基因序列，最终获得高效表达的可溶性高活性抗原，灵敏度和特异性显著优于非可溶性表达抗原，该抗原适合用于研制血清学抗体检测试剂，用于新型冠状病毒感染患者的辅助诊断，以及与病人密切接触的高危人群及疑似病人的筛查。利用本发明的N-S优势表位融合蛋白在新型冠状病毒总抗体检测中，灵敏度和特异性均为100％。
本发明所提供的新冠病毒N-S优势表位融合蛋白适用于新型冠状病毒患者的确诊检测。
附图说明
图1为表达的本发明实施例3中N-S优势表位融合蛋白的SDS-PAGE电泳图；
图2为本发明实施例4中乳胶法检测的结果模式图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明。如未特别指明，实施例中所用的技术手段为本领域技术人员熟知的常规手段。
现有技术中抗体检测可以采用裂解的病毒颗粒作为抗原，但是由于SARS-CoV-2培养必须在BSL3实验室中进行，且存在很高的感染危险，因此有必要采用人工表达的抗原代替全病毒作为检测用抗原。先前的文献报道显示N蛋白大小适中，不属于糖蛋白，易于进行高效表达，而由于S蛋白太大，且为糖蛋白，难以在细菌中高效表达全长S蛋白，因此，本发明中有必要选择选择敏感、特异、易表达的区段作为诊断用抗原。
实施例1 N蛋白优势表位区段抗原筛选确定
根据Genebank中公布的新型冠状病毒N蛋白全长氨基酸序列，用BIOSUN软件B-细胞表位分析和信号肽分析功能分析N蛋白氨基酸序列，根据疏水性的强弱及信号肽情况，分别选取抗原性好的20-220aa区段(记为N1)、230-300aa区段(记为N2)和335-419aa区段(记为N3)用作本实施例中筛选N蛋白优势表位的区段抗原。
其中N1至N3区段的基因序列分别为：
N1基因序列为：
Figure PCTCN2020083016-appb-000004
N2基因序列为：
Figure PCTCN2020083016-appb-000005
N3基因序列为：
Figure PCTCN2020083016-appb-000006
为了获得高效表达的目的蛋白，对N1-N3各区段的编码基因进行人工改造和优化，并按照常规分子生物学方法分别构建原核表达载体pET30-N1、pET30-N2和pET30-N3。N1-N3优化后的核酸序列分别为：
N1优化核酸序列为：
Figure PCTCN2020083016-appb-000007
N2优化核酸序列为：
Figure PCTCN2020083016-appb-000008
N3优化核酸序列为：
Figure PCTCN2020083016-appb-000009
按照顺序进行以下操作：
(1)将测序正确的重组表达质粒转化入BL21感受态细胞，挑取单菌落于3ml含氨苄西林钠的LB液体培养基中，37℃振荡培养过夜，次日接种于250ml新鲜的LB液体培养基中，37℃、160r/min培养4h至对数生长期，加150μl 1M的IPTG诱导液，15℃诱导12-14h(本实施例诱导了12.5h)。
(2)4℃，6000r/min离心10min收集诱导后的菌体；用25mM Tris-HCl(PH8.5)重悬菌体，然后对菌体进行冰浴超声。
(3)冰浴超声后的菌体于4℃，12000r/min离心10min后收集上清，过滤得到备用样品。
(4)Ni柱纯化备用样品，平衡后，将处理好的样品缓慢上样，待吸光度值开始上升时，收集穿过液。再次平衡后，用含25mM咪唑、25mM Tris-HCl(25mM Tris-HCl中含250mM咪唑，溶液的pH为8.5)的溶液洗脱，分别收集蛋白峰，电泳分析纯化产物，验证纯化后的目的蛋白在250mM咪唑洗脱液中。
将纯化后所获得的N蛋白优势表位区段抗原分别按常规进行SDS-PAGE电泳，转膜，5％脱脂奶粉室温封闭1h。分别用封闭液稀释的10份新冠肺炎患者血清和10份健康献血员血清(血清与封闭液的体积比为1:50)于4℃孵育过夜。TBST室温洗膜3次后，辣根过氧化物酶标记的二抗孵育膜1h。然后TBST室温洗膜3次，以ECL法显示阳性条带。
结果显示在10份新冠肺炎患者血清中，N1表位抗原与8份样本呈现特异性条带，N2表位抗原与6份样本呈现特异性条带，N3表位抗原与2份样本呈现特异性条带；在10份健康献血员血清中，N1和N3表位抗原未呈现非特异性条带，N2表位抗原与1份样本呈现非特异性条带。结果说明在所表达的三种N蛋白区段抗原中，N1优势表位区段抗原具有最佳 的抗原活性和特异性。
实施例2 S蛋白优势表位区段抗原筛选确定
根据Genebank中公布的新型冠状病毒S蛋白全长氨基酸序列，采用BIOSUN软件B-细胞表位分析和信号肽分析功能分析S蛋白氨基酸序列，并结合功能结构域分布，分别选取抗原性好的65-290aa区段(记为S1)、340-510aa区段(记为S2)和520-681aa区段(记为S3)三个优势表位区段抗原用作本实施例中筛选S蛋白优势表位的区段抗原。
S1基因序列为：
Figure PCTCN2020083016-appb-000010
S2基因序列为：
Figure PCTCN2020083016-appb-000011
S3基因序列为
Figure PCTCN2020083016-appb-000012
而为了获得高效表达的目的蛋白，对各区段的编码基因进行了人工改造和优化，并按照常规分子生物学方法构建原核表达载体pET30-S1、pET30-S2和pET30-S3，将测序正确的重组表达质粒转化入BL21感受态细胞，按照实施例1的方法进行诱导表达和抗原纯化，获得纯化后抗原。S1-S3优化后的核酸序列如下：
S1优化核酸序列
Figure PCTCN2020083016-appb-000013
Figure PCTCN2020083016-appb-000014
S2优化核酸序列
Figure PCTCN2020083016-appb-000015
S3优化核酸序列
Figure PCTCN2020083016-appb-000016
然后，再按照实施例1的WB方法进行活性鉴定。结果显示在10份新冠肺炎患者血清中，S1表位抗原与5份样本呈现特异性条带，S2表位抗原与3份样本呈现特异性条带，S3表位抗原与7份样本呈现特异性条带；在10份健康献血员血清中，S1、S2和S3均未呈现非特异性条带。结果说明本实施例所表达的三种S蛋白区段抗原中，S3优势表位区段抗原具有最佳的抗原活性和特异性。
实施例3 新型冠状病毒的N-S优势表位融合蛋白的制备
本实施例中利用具有互补粘性末端的同尾酶法将N1优势表位区段抗原和S3优势表位区段抗原的编码基因连接在一起进行融合表达，得到新冠病毒N-S优势表位融合蛋白，其氨基酸序 列如下所示：
Figure PCTCN2020083016-appb-000017
上述新冠病毒N-S优势表位融合蛋白的核酸序列如下所示：
Figure PCTCN2020083016-appb-000018
然后按照常规分子生物学方法构建原核表达载体pET30-N-S，将测序正确的重组表达质粒转化入BL21感受态细胞，按照实施例1的方法进行诱导表达和抗原纯化，获得纯化后抗原。结果显示N-S优势表位融合蛋白为可溶性表达抗原，纯化后抗原纯度高达99.1％。结果如图1所示。
然后，再按照实施例1的WB方法进行活性鉴定。结果显示本实施例与10份新冠肺 炎患者血清均呈现特异性条带，而与10份健康献血员血清未呈现非特异性条带。结果说明N-S优势表位融合蛋白具有非常好的抗原活性和特异性。
实施例4 新型冠状病毒抗体检测试剂盒
一、采用荧光免疫层析法的新型冠状病毒IgM抗体检测试剂盒
本实施例的试剂盒采用现有技术中的荧光免疫层析技术及免疫学法原理检测样本中新型冠状病毒IgM抗体的浓度，试剂盒的结构与现有技术中试剂盒的结构相同，均具有检测卡，同时本实施例的检测卡包被有实施例3的新冠病毒N-S优势表位融合蛋白、鼠抗人IgM抗体和羊抗鼠IgG。具体检测原理为：检测时，将样本加入到试剂盒或检测卡的加样孔中，样本中抗新型冠状病毒IgM和荧光标记鼠抗人IgM抗体发生免疫反应而形成免疫结合物，结合物层析至检测卡T线时，被T线的新冠病毒N-S优势表位融合蛋白捕获，样本层析至C线时，荧光标记鼠抗人IgM抗体被C线的羊抗鼠IgG捕获，作为质控线。T线处荧光信号强度与样品中抗新型冠状病毒IgM抗体浓度正相关，因此，用适用的干式荧光仪器便可检测出样本中抗新型冠状病毒IgM抗体的浓度。
本试剂盒需要在室温下水平使用，每盒中具有新型冠状病毒IgM抗体检测卡、每人份1支加样吸管、1瓶样品稀释液。加样时具有两种模式，分别为：
①全血样品：用加样吸管往加样孔垂直滴加2滴全血，再加入1-2滴样品稀释液，稀释后10分钟，用适配仪器判断结果。
②血清/血浆样品：用加样吸管往加样孔内垂直滴加1滴，再加入1-2滴样品稀释液，稀释后10分钟，用适配仪器判断结果。
无论上述哪种检测模式，在加样后，20分钟后观察的结果均为无效结果。
本检测卡对于50例新型冠状病毒感染核酸检测阳性患者的血清样本进行检测后，结果显示检测45出例为阳性，灵敏度为90％；50例健康对照血清样本中，50例检测为阴性，特异性为100％。
二、采用乳胶法的新型冠状病毒IgM抗体检测试剂盒
试剂盒检测结果的解释
阳性：质控线(C线)和检测线(T线)各有一条红色线条出现。表明样本中存在新型冠状病毒抗体。
阴性：只有质控线(C线)有一条红色线条，而检测线(T线)无红色线条出现，表示样本中没有新型冠状病毒抗体或新型冠状病毒抗体低于检出水平。
无效：质控线(C线)无红色线条出现，表示失效。可能是由于操作不正确或试剂盒失效，应重试。
本试剂盒结构与现有技术中试剂盒的结构相同，均包括硝酸纤维素膜与彩色乳胶标记的抗人IgM抗体的释放垫及其他试剂。于硝酸纤维素膜上固定有实施例3中的新冠病毒N-S优势表位融合蛋白。应用乳胶免疫层析技术，采用捕获法的原理检测人血清/血浆/全血中的新型冠状病毒IgM抗体。检测过程中，血液标本加到试剂盒的加样孔中，样品首先和释放垫上的彩色乳胶标记的抗人IgM混合，接着再往硝酸纤维素膜上层析。如果样本中含有新型冠状病毒IgM抗体，这些抗体首先与彩色乳胶标记的抗人IgM的结合，这样混合物往硝酸纤维膜上层析时，便会被固定有新冠病毒N-S优势表位融合蛋白的检测线(T线)捕获而形成彩色乳胶标记抗人IgM-抗体-抗原的免疫复合物，因而在T线出现一条红色线条，为阳性结果。如果被检者血液中没有新型冠状病毒IgM抗体存在，则不会在检测线(T线)上形成红色线条，为阴性结果。在试剂卡上的质控线(C线)包被有羊抗鼠抗体，正常情况下在检测时质控线上都应有红色线条出现，以证明试剂卡工作正常。
本试剂盒需要在室温下水平使用，每盒中具有新型冠状病毒IgM检测卡、每人份1支加样吸管、1瓶样品稀释液。加样时具有两种模式，分别为：
①全血样品：用加样吸管往加样孔垂直滴加2滴全血，再加入1-2滴样品稀释液，稀释后10分钟直接判断结果。。
②血清/血浆样品：用加样吸管往加样孔内垂直滴加1滴，再加入1-2滴样品稀释液，稀释后10分钟后直接判断结果。。
本试剂盒在加样后10分钟内判断结果，20分钟后观察结果为无效结果。
乳胶法检测结果模式图如图2所示。
本检测卡对于50例新型冠状病毒感染核酸检测阳性患者的血清样本进行检测后，45例为阳性，灵敏度为90％；50例健康对照血清样本中，50例检测为阴性，特异性为100％。
三、采用荧光免疫层析法的新型冠状病毒IgM/IgG总抗体检测试剂盒
本试剂盒采用干式荧光免疫层析技术及双抗原夹心法原理检测样本中新型冠状病毒IgM/IgG总抗体的浓度。本试剂盒中的新型冠状病毒检测卡上具有实施例3的新冠病毒N-S优势表位融合蛋白。检测时，将样本加入到检测卡的加样孔中，样本中新型冠状病毒IgM/IgG抗体和荧光标记鼠抗人新型冠状病毒抗原发生免疫反应而形成免疫结合物，结合物层析至检测卡T线时，被T线的新冠病毒N-S优势表位融合蛋白捕获，样本层析至C线时，荧光标记鼠抗人 新型冠状病毒抗原被C线的羊抗鼠IgG捕获，作为质控线。T线处荧光信号强度与样品中抗新型冠状病毒抗体浓度正相关，因此，用适用的干式荧光仪器便可检测出样本中抗新型冠状病毒IgM/IgG总抗体的浓度。
本试剂盒需要在室温下水平使用，每盒中具有新型冠状病毒IgM检测卡、每人份1支加样吸管、1瓶样品稀释液。加样时具有两种模式，分别为：
①全血样品：用加样吸管往加样孔垂直滴加2滴全血，再加入1-2滴样品稀释液，加样后10分钟，用适配仪器判断结果。
②血清/血浆样品：用加样吸管往加样孔内垂直滴加1滴，再加入1-2滴样品稀释液，加样后10分钟，用适配仪器判断结果。
无论上述哪种检测模式，在加样后，20分钟后观察的结果均为无效结果。
本检测卡对于50例新型冠状病毒感染核酸检测阳性患者的血清样本检测后，显示50例为阳性，灵敏度为100％；50例健康对照血清样本中，50例检测为阴性，特异性为100％。
四、采用乳胶法的新型冠状病毒IgM/IgG总抗体检测试剂盒
检测结果的解释
阳性：质控线(C线)和检测线(T线)各有一条红色线条出现。表明样本中存在新型冠状病毒IgM/IgG总抗体。
阴性：只有质控线(C线)有一条红色线条，而检测线(T线)无红色线条出现。表示样本中没有新型冠状病毒IgM/IgG总抗体，或新型冠状病毒IgM/IgG总抗体低于检出水平。
无效：质控线(C线)无红色线条出现，表示失效。可能是由于操作不正确或试剂盒失效，应重试。
本试剂盒由硝酸纤维素膜、彩色乳胶标记的抗人新型冠状病毒抗原的释放垫及其他试剂组成。其中硝酸纤维素膜上固定有新型冠状病毒特异性N-S优势表位融合蛋白。检测原理为：应用乳胶免疫层析技术，采用捕获法的原理检测人血清/血浆/全血中的新型冠状病毒IgM/IgG总抗体。检测过程中，血液标本加到试剂盒的加样孔中，样品首先和释放垫上的彩色乳胶标记的抗人新型冠状病毒抗原混合，接着再往硝酸纤维素膜上层析。如果样本中含有新型冠状病毒IgM/IgG总抗体，这些抗体首先与彩色乳胶标记的抗人新型冠状病毒抗原的结合，这样混合物往硝酸纤维膜上层析时，便会被固定有新型冠状病毒抗原的检测线(T线)捕获而形成彩色乳胶标记抗人抗原-抗体-抗原的免疫复合物，因而在T线出现一条红色线条，为阳性结果。如果被检者血液中没有新型冠状病毒IgM/IgG总抗体存在，则不会在检测线(T 线)上形成红色线条，为阴性结果。在试剂卡上的质控线(C线)包被有羊抗鼠抗体，正常情况下在检测时质控线上都应有红色线条出现，以证明试剂卡工作正常。
本试剂盒需要在室温下水平使用，每盒中具有新型冠状病毒IgM/IgG总抗体检测试剂卡、每人份1支加样吸管、1瓶样品稀释液。加样时具有两种模式，分别为：
①全血样品：用加样吸管往加样孔，垂直滴加2滴全血，再加入1-2滴样品稀释液，在加样后10分钟直接判断结果。
②血清/血浆样品：用加样吸管往加样孔内垂直滴加1滴，再加入1-2滴样品稀释液，在加样后10分钟直接判断结果。
本试剂卡在20分钟后观察的结果均为无效结果。其本试剂卡检测结果的模式图仍为图2所示。
本试剂卡对于50例新型冠状病毒感染核酸检测阳性患者的血清样本检测后，结果显示50例为阳性，灵敏度为100％；50例健康对照血清样本中，50例检测为阴性，特异性为100％。
说明书核苷酸和氨基酸序列表
Figure PCTCN2020083016-appb-000019
Figure PCTCN2020083016-appb-000020
Figure PCTCN2020083016-appb-000021
Figure PCTCN2020083016-appb-000022
Figure PCTCN2020083016-appb-000023
Figure PCTCN2020083016-appb-000024
Figure PCTCN2020083016-appb-000025
Figure PCTCN2020083016-appb-000026

Claims (10)

  1. 如权利要求1或2所述的新冠病毒N-S优势表位融合蛋白的一种制备方法，其特征在于：首先将新型冠状病毒的S蛋白优势表位区段抗原与N蛋白优势表位区段抗原构建成一个融合表达抗原；然后通过基因优化去除稀有密码子获得优化后基因序列，最终获得高效表达的可溶性高活性抗原。
  2. 如权利要求4所述的新冠病毒N-S优势表位融合蛋白的制备方法，其特征在于：所述新型冠状病毒的S蛋白优势表位区段抗原与N蛋白优势表位区段抗原分别通过表位分析预测进行筛选。
  3. 如权利要求4或5所述的新冠病毒N-S优势表位融合蛋白的制备方法，其特征在于：建成融合表达抗原的方法为：利用具有互补粘性末端的同尾酶法将N蛋白优势表位区段抗原和S蛋白优势表位区段抗原的编码基因连接在一起进行融合表达。
  4. 一种通过表达如权利要求1或2中所述的新冠病毒N-S优势表位融合蛋白的表达载体。
  5. 一种通过表达如权利要求1或2中所述的新冠病毒N-S优势表位融合蛋白的微生物。
  6. 如权利要求8所述的微生物，其特征在于：所述微生物为细菌，所述细菌为大肠杆菌。
  7. 一种如权利要求7所述表达载体在制备检测新型冠状病毒感染试剂中和/或在制备检测抗新型冠状病毒抗体试剂中的应用。
  8. 一种如要求8或9所述的微生物在制备检测新型冠状病毒感染试剂中和/或在制备检测抗新型冠状病毒抗体试剂中的应用。
  9. 一种抗新型冠状病毒抗体检测试剂盒，其特征在于：其检测卡中具有如权利要求1或2所述的新冠病毒N-S优势表位融合蛋白，检测方法为荧光免疫层析法。
  10. 一种抗新型冠状病毒抗体检测试剂盒，其特征在于：其检测卡中具有如权利要求1或2所述的新冠病毒N-S优势表位融合蛋白，检测方法为乳胶法。
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