CN111187354B - Novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit - Google Patents
Novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention provides a novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit, and relates to the field of biotechnology. The novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit provided by the invention detects SARS-CoV-2IgM/IgG antibody by respectively adopting a colloidal gold capture method, a colloidal gold indirect method and a colloidal gold double-antigen sandwich method. The antigen is a high-activity recombinant antigen expressed by fusion of N protein fragments and S protein dominant epitopes, and the indirect labeling of colloidal gold is adopted, so that the steric hindrance is reduced, and the activity, the reaction consistency and the accuracy of the antigen are improved. The sample pad is pretreated, so that the interference of complex components of the blood sample on the reaction is greatly reduced. The kit does not need an instrument for detection, is simple, convenient and flexible to operate, can directly and manually interpret the result, is short in detection time, can interpret the result in only 15min, and is accurate in detection result.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit.
Background
Since the outbreak of pneumonia epidemic infected by novel coronavirus (SARS-CoV-2), no immunological detection reagent for the novel coronavirus exists in the market. At present, the clinical diagnosis is to detect SARS-CoV-2 specific nucleic acid sequence in the blood of patients and suspected patients by using common real-time quantitative fluorescent PCR nucleic acid detection reagent, although the method plays an important role in the investigation, the method is limited by fussy operation, long time consumption, need of centralized inspection, and the like, and meanwhile, the detection result is greatly influenced by the RNA extraction effect and has high false negative. The detection result of the method is also influenced by the quality of the PCR reagent and aerosol pollution. At present, the prior art cannot meet the detection requirements of examination and diagnosis of a large number of suspected patients, asymptomatic infectors and the like which are rapidly growing at present.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a SARS-CoV-2 recombinant antigen.
It is a second object of the present invention to provide a biological material related to a SARS-CoV-2 recombinant antigen.
The third purpose of the invention is to provide the application of SARS-CoV-2 recombinant antigen or biological material.
The fourth purpose of the invention is to provide a test paper for detecting SARS-CoV-2 antibody by a colloidal gold capture method.
The fifth purpose of the invention is to provide a test paper for detecting SARS-CoV-2 antibody by a colloidal gold indirect method.
The sixth purpose of the invention is to provide a test paper for detecting SARS-CoV-2 antibody by colloidal gold double-antigen sandwich method.
The seventh purpose of the invention is to provide a preparation method of the test paper for detecting SARS-CoV-2 antibody by the colloidal gold double-antigen sandwich method.
The eighth object of the present invention is to provide a SARS-CoV-2 antibody detection kit.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a SARS-CoV-2 recombinant antigen comprising an N protein fragment and optionally an S protein dominant epitope fragment;
the amino acid sequence of the N protein fragment is shown as SEQ ID NO.1 or SEQ ID NO. 2;
the amino acid sequence of the S protein dominant epitope fragment is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.
Further, the protein comprises an N protein fragment, a connecting peptide and an S protein dominant epitope fragment which are connected in sequence;
preferably, the amino acid sequence structure of the connecting peptide is Gn1SGn2S, wherein n1 and/or n2 is an integer of 2 to 10, preferably an integer of 2 to 5.
Further, the recombinant antigen is a protein consisting of an amino acid sequence shown in SEQ ID NO. 7.
A biological material associated with a recombinant antigen, the biological material being any one of:
(a) nucleic acid molecules encoding the recombinant antigens;
(b) an expression cassette comprising the nucleic acid molecule of (a);
(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);
(d) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);
(e) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).
The recombinant antigen or biological material is applied to the detection of SARS-CoV-2 antibody or the preparation of SARS-CoV-2 antibody detection products;
preferably, the SARS-CoV-2 antibody is an IgM or IgG;
preferably, the method for detecting SARS-CoV-2 antibody comprises a colloidal gold capture method, a colloidal gold indirect method, a colloidal gold double-antigen sandwich method or ELISA;
preferably, the product comprises a test strip or kit.
A test paper for detecting SARS-CoV-2 antibody by colloidal gold capture method comprises a base plate, a sample pad, a combination pad, a coating film and an absorption pad, wherein the sample pad, the combination pad, the coating film and the absorption pad are sequentially fixed on the base plate along the flowing direction of a liquid sample to be detected; the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with a mouse anti-human IgM/IgG antibody, and the quality control area is loaded with a goat anti-rabbit polyclonal antibody; nCOVAg01 includes a recombinant antigen of the invention.
A test paper for detecting SARS-CoV-2 antibody by colloidal gold indirect method comprises a base plate, a sample pad, a combination pad, a coating film and an absorption pad, wherein the sample pad, the combination pad, the coating film and the absorption pad are fixed on the base plate in sequence along the flowing direction of a liquid sample to be detected; the binding pad is loaded with gold-labeled protein, and the gold-labeled protein comprises a mouse anti-human IgG monoclonal antibody or a mouse anti-human IgM antibody; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with nCOVAg02, and the quality control area is loaded with goat anti-rabbit polyclonal antibody; nCOVAg02 includes a recombinant antigen of the invention.
A test paper for detecting SARS-CoV-2 antibody by colloidal gold double-antigen sandwich method comprises a base plate, a sample pad, a combination pad, a coating film and an absorption pad, wherein the sample pad, the combination pad, the coating film and the absorption pad are sequentially fixed on the base plate along the flowing direction of a liquid sample to be detected; the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with nCOVAg02, and the quality control area is loaded with goat anti-rabbit polyclonal antibody; nCOVAg01 and nCOVAg02 each independently comprise the recombinant antigen described above.
Further, the structure of nCOVAg01 is a tag protein-recombinant antigen-linker peptide-hydrophilic protein-linker peptide-protein purification tag;
preferably, nCOVAg02 has the structure of a protein purification tag-recombinant antigen-linker peptide-protein purification tag or a recombinant antigen-linker peptide-protein purification tag;
preferably, the protein purification tag is His;
preferably, the tag protein is any one of a Trx tag, a Flag tag, MBP (maltose binding protein), GST (glutathione mercaptotransferase), an Avi tag, a SUMO tag, a c-Myc tag and an HA tag;
preferably, the amino acid sequence of the hydrophilic protein is shown as SEQ ID NO. 8;
preferably, nCOVAg01 is a protein consisting of the amino acid sequence shown in SEQ ID NO. 9;
preferably, nCOVAg02 is a protein consisting of the amino acid sequence shown in SEQ ID NO. 10;
preferably, the sample pad of the test paper is soaked in a solution containing 0.5-1.5v/v% Tween20 and 0.4-0.6w/v% PVA, and is loaded with anti-RBC antibody;
preferably, the liquid sample to be tested comprises whole blood, serum or plasma.
The preparation method of the test paper of the colloidal gold double-antigen sandwich method comprises the following steps of sequentially fixing a sample pad, a combination pad, a coating film and a water absorption pad on a bottom plate along the flowing direction of a liquid sample to be detected; the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with nCOVAg02, and the quality control area is loaded with goat anti-rabbit polyclonal antibody;
preferably, the preparation method of the gold-labeled protein of the conjugate pad comprises the following steps: uniformly mixing the colloidal gold solution and the mouse anti-tag antibody for reaction to obtain a mouse anti-tag antibody-colloidal gold cross-linked complex, and uniformly mixing the complex with nCOVAg01 for reaction to obtain a labeled antigen 1; uniformly mixing the colloidal gold solution and the rabbit IgG for reaction to obtain a labeled antibody 2; mixing the labeled antigen 1 and the labeled antibody 2 according to a molar ratio of 2-4:7 to obtain gold labeled protein;
preferably, the particle size of the colloidal gold is 20-30 nm;
preferably, the binding pad is loaded with 0.6-1.5 μ g of gold-labeled protein per square centimeter of the binding pad;
preferably, the coating film is loaded with 1.5-2.0 μ g nCOVAg02 per cm of detection area;
preferably, the coating film is loaded with 1.3-2.0 mu g of goat anti-rabbit polyclonal antibody per centimeter of quality control area;
preferably, the sample pad is loaded with 1.0-2.0 μ g of anti-RBC antibody per square centimeter;
preferably, the sample pad is soaked with a solution containing 0.5-1.5v/v% Tween20 and 0.4-0.6w/v% PVA.
A detection kit for SARS-CoV-2 antibody, comprising the above detection test paper.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a recombinant antigen of SARS-CoV-2, the recombinant antigen comprises N protein fragment and optional S protein dominant epitope fragment, wherein, the amino acid sequence of the N protein fragment is shown in SEQ ID NO.1 or SEQ ID NO. 2; the amino acid sequence of the S protein dominant epitope fragment is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6. The fragment selected by the recombinant antigen can be the dominant epitope of the conserved domain protein (N protein) of SARS-CoV-2, the scheme can improve the reaction sensitivity with the antibody to be detected, reduce the omission of the antibody and is suitable for both IgG and IgM antibodies; the recombinant antigen can also be a recombinant protein obtained by fusion expression of dominant epitopes of the N protein and the spinous process protein (S protein), and when the recombinant antigen contains the N protein fragment and the S protein fragment, the reaction accuracy is higher. The recombinant antigen is suitable for various immunological atopic detection methods, such as a colloidal gold capture method, a colloidal gold indirect method and a colloidal gold double-antigen sandwich method, and the prepared detection test paper can be used for clinical detection of SARS-CoV-2IgM/IgG antibody, does not need an instrument, is simple and flexible to operate, can directly and manually interpret the result, has short detection time, can interpret the result only in 15min, and has accurate detection result. The recombinant antigen provided by the invention has wide application prospect, and the prepared product overcomes the problems of complicated operation, long time consumption and higher false negative rate of nucleic acid detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is an SDS-PAGE result of nCOVAg01 in example 3 of the present invention;
FIG. 2 is an SDS-PAGE result of nCOVAg02 in example 3 of the present invention;
FIG. 3 is a schematic structural view of a test paper in example 4 of the present invention;
FIG. 4 shows the color standard for stability detection in example 5 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
A SARS-CoV-2 recombination antigen comprises N protein fragment and optional S protein dominant epitope fragment, wherein, the amino acid sequence of the N protein fragment is shown in SEQ ID NO.1 or SEQ ID NO. 2; the amino acid sequence of the S protein dominant epitope fragment is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.
The fragment selected by the recombinant antigen can be the dominant epitope of the conserved domain protein (N protein) of SARS-CoV-2, the scheme can improve the reaction sensitivity with the antibody to be detected, reduce the omission of the antibody and is suitable for both IgG and IgM antibodies; the recombinant antigen can also be a recombinant protein obtained by fusion expression of dominant epitopes of the N protein and the spinous process protein (S protein), and when the recombinant antigen contains the N protein fragment and the S protein fragment, the reaction accuracy is higher. The recombinant antigen is suitable for various immunological atopic detection methods, such as a colloidal gold double-antigen sandwich method, a colloidal gold capture method, a colloidal gold indirect method and the like, and has wide application prospect. It will be appreciated that the amino acid sequence of the recombinant antigen may include the form: SEQ ID NO.1, or SEQ ID NO.2, or SEQ ID NO.1 and SEQ ID NO.3, or SEQ ID NO.1 and SEQ ID NO.4, or SEQ ID NO.1 and SEQ ID NO.5, or SEQ ID NO.1 and SEQ ID NO.6, or SEQ ID NO.2 and SEQ ID NO.3, and so forth.
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA(SEQ ID NO.1)。
MKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTAAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA(SEQ ID NO.2)。
SKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYH(SEQ ID NO.3)。
ITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLT(SEQ ID NO.4)。
MFVFLVLLPLVSSQCVNLTTRTQLPPA(SEQ ID NO.5)。
AGICASYQTQTNSPRRARSVASQSIIAYT(SEQ ID NO.6)。
In a preferred embodiment, the recombinant antigen comprises an N protein fragment, a connecting peptide and an S protein dominant epitope fragment connected in sequence. The connecting peptide can increase antigen flexibility, so that the antigen epitope is exposed more fully. Preferably, the amino acid sequence structure of the connecting peptide is Gn1SGn2S, wherein n1 and/or n2 is an integer of 2 to 10, preferably an integer of 2 to 5. N1 and n2 represent the number of G, and the amino acid sequence structure of the connecting peptide is n 1G (glycine) -S (serine) -n 2G-S, wherein n1 and n2 may be the same or different.
In a preferred embodiment, the recombinant antigen is a protein consisting of the amino acid sequence shown in SEQ ID NO. 7.
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQAASGGGGSGGGSSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYH(SEQ ID NO.7)。
The invention also protects the biological material related to the recombinant antigen provided by the invention, and specifically any one of the following materials:
(a) a nucleic acid molecule encoding a recombinant antigen of the invention;
(b) an expression cassette comprising the nucleic acid molecule of (a);
(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);
(d) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);
(e) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).
The invention also protects the application of the recombinant antigen and the related biological material in the detection of SARS-CoV-2 antibody or the preparation of SARS-CoV-2 antibody detection products. The recombinant antigen provided by the invention can generate specific immunological reaction with SARS-CoV-2 antibody, and can be used for detecting SARS-CoV-2 antibody so as to diagnose SARS-CoV-2 infection, and biological materials related to the recombinant antigen, such as nucleic acid molecules, recombinant vectors or recombinant host cells, and the like are all biological modules containing information related to the recombinant antigen, can be used for producing and preparing the recombinant antigen, can quickly obtain the recombinant antigen, and can be used for detecting the SARS-CoV-2 antibody or preparing products. For SARS-CoV-2 antibody, IgM and IgG can be detected, and the means for detection may be, for example, a colloidal gold capture method, a colloidal gold indirect method, a colloidal gold double-antigen sandwich method, or ELISA, etc. The colloidal gold method can realize on-site rapid and high-flux rapid detection of suspected patient investigation, and has the advantages of short detection time, simple operation, high sensitivity, strong specificity and good stability. The specific product form can be test paper or kit, etc.
The invention also protects a product for detecting SARS-CoV-2 antibody, the antigen used in the method is the recombinant antigen provided by the invention.
The SARS-CoV-2 antibody detecting test paper includes base plate, sample pad, combining pad, coating film and absorbing pad, and the sample pad, the combining pad, the coating film and the absorbing pad are successively fixed on the base plate along the flow direction of the liquid sample to be detected. According to different detection principles, the detection test paper can be divided into a colloidal gold capture method detection test paper, a colloidal gold indirect method detection test paper and a colloidal gold double-antigen sandwich method detection test paper, and specifically, specific information of each detection test paper is as follows:
test paper for colloidal gold capture: the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with a mouse anti-human IgM/IgG antibody, and the quality control area is loaded with a goat anti-rabbit polyclonal antibody; nCOVAg01 includes a recombinant antigen of the invention.
According to the detection test paper, when a liquid sample to be detected is added into a sample pad to form IgM/IgG-gold-labeled protein, the IgM/IgG-gold-labeled protein flows to a detection line along with chromatography, a mouse anti-human IgM/IgG antibody coated at the detection line is captured to form mouse anti-human IgM/IgG antibody-IgM/IgG-gold-labeled protein, and a positive result is red spots. When the negative sample is added into the sample pad, IgM/IgG-gold labeled protein cannot be formed, and the negative result is not colored.
Test paper for detecting by a colloidal gold indirect method: the binding pad is loaded with gold-labeled protein, and the gold-labeled protein comprises a mouse anti-human IgG monoclonal antibody or a mouse anti-human IgM antibody; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with nCOVAg02, and the quality control area is loaded with goat anti-rabbit polyclonal antibody; nCOVAg02 includes a recombinant antigen of the invention.
According to the detection test paper, when a liquid sample to be detected is added into a sample pad to form a specific IgM/IgG antibody-mouse anti-human IgM/IgG-colloidal gold compound, the compound is electrophoresed to a detection line along with chromatography, a recombinant antigen nCOVAg02 coated at the detection line is captured to form a recombinant antigen nCOVAg 02-specific IgM/IgG antibody-mouse anti-human IgM/IgG-colloidal gold compound, and a positive result shows red spots.
Test paper for colloidal gold double-antigen sandwich method: the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with nCOVAg02, and the quality control area is loaded with goat anti-rabbit polyclonal antibody; nCOVAg01 and nCOVAg02 each independently comprise a recombinant antigen of the invention.
The detection test paper adopts a colloidal gold double-antigen sandwich method for detection, the sequences of recombination antigens used by nCOVAg01 and nCOVAg02 can be the same or different, the recombination antigen provided by the invention is used as a detection antigen, when a positive liquid sample to be detected is added into a sample pad, a specific IgM/IgG antibody-gold-labeled protein compound is formed by chromatography to a binding pad, the detection area is captured by nCOVAg02 coated on the detection area along with the migration of the chromatography to the detection area, and a positive result is a red spot.
The three test paper provided by the invention fill up the blank of the domestic immunological method for detecting SARS-CoV-2 coronavirus diagnosis, and the SARS-CoV-2IgM/IgG antibody is detected by respectively adopting a colloidal gold capture method, a colloidal gold indirect method and a colloidal gold double-antigen sandwich method, so that the test paper has the advantages of no need of instruments, simple and flexible operation, direct manual interpretation of the result, short detection time, interpretation of the result only within 15min, accurate detection result and capability of overcoming the problems of complicated operation, long time consumption and high false negative rate of nucleic acid detection.
In a preferred embodiment, in the colloidal gold double antigen sandwich test strip: the grain size of the colloidal gold is 20 nm; 1.0 mu g of gold-labeled antigen is loaded on each square centimeter of the bonding pad; 2.0 mu g nCOVAg02 is loaded on each centimeter of detection area on the coating film; 1.5 mu g of goat anti-rabbit polyclonal antibody is loaded on each centimeter of quality control area on the coating film; the sample pad is loaded with 1.5 mu g of anti-RBC antibody in square centimeter and is used for removing red blood cells in whole blood; the sample pad is soaked by a solution containing 0.5-1.5v/v% Tween20 and 0.4-0.6w/v% PVA, so that the interference of the complex components of the blood sample on the reaction is greatly reduced.
In a preferred embodiment, nCOVAg01 has the structure tag protein-recombinant antigen-linker peptide-hydrophilic protein-linker peptide-protein purification tag. The mouse anti-tag protein antibody is used as the connecting protein of the colloidal gold and the recombinant antigen to realize the indirect marking of the recombinant antigen, and the antibody, the connecting peptide and the hydrophilic protein improve the antigen reactivity of the recombinant antigen, so that the antigen epitope is exposed more fully. Specifically, the tag protein may be any one of a Trx tag, a Flag tag, MBP (maltose binding protein), GST (glutathione mercaptotransferase), an Avi tag, a SUMO tag, a c-Myc tag, and an HA tag. The amino acid sequence of the hydrophilic protein can be SEQ ID No. 8. The protein purification tag may be His or the like.
STQQNTGGPQTT(SEQ ID NO.8)。
In a more preferred embodiment, nCOVAg01 is a protein consisting of the amino acid sequence set forth in SEQ ID NO. 9.
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAMGHMSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQAASGGGGSGGGSSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGGSGGSTQQNTGGPQTTGGGSGGSLEHHHHHH(SEQ ID NO.9)。
In a preferred embodiment, nCOVAg02 has the structure of a protein purification tag-recombinant antigen-linker peptide-protein purification tag or a recombinant antigen-linker peptide-protein purification tag. The protein purification tag is beneficial to the purification of the recombinant antigen, and when more than one protein purification tag is used, the purification yield of the recombinant antigen is more beneficial. The protein purification tag may be a His tag or the like.
In a more preferred embodiment, nCOVAg02 is a protein consisting of the amino acid sequence set forth in SEQ ID No. 10.
MGSSHHHHHHSSGLVPRGSHMSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQAASGGGGSGGGSSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGSLEHHHHHH(SEQ ID NO.10)。
In alternative embodiments, the liquid sample to be tested comprises whole blood, serum or plasma.
The invention also protects the preparation method of the test paper for detecting SARS-CoV-2 antibody by the colloidal gold double-antigen sandwich method, along the flowing direction of the liquid sample to be detected, the sample pad, the combination pad, the envelope membrane and the water absorption pad are fixed on the bottom plate in sequence; the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, wherein the detection area is loaded with nCOVAg02, and the quality control area is loaded with goat anti-rabbit polyclonal antibody.
In a preferred embodiment, the method for preparing the gold-labeled protein of the conjugate pad comprises: uniformly mixing the colloidal gold solution and the mouse anti-tag antibody for reaction to obtain a mouse anti-tag antibody-colloidal gold cross-linked complex, and uniformly mixing the complex with nCOVAg01 for reaction to obtain a labeled antigen 1; uniformly mixing the colloidal gold solution and the rabbit IgG for reaction to obtain a labeled antibody 2; and mixing the labeled antigen 1 and the labeled antigen 2 according to the molar ratio of 2-4:7 to obtain the gold labeled protein.
In a more preferred embodiment, nCOVAg01 is a protein composed of the amino acid sequence of SEQ ID No.9, and is prepared by labeling a mouse anti-Trx antibody to colloidal gold to obtain a mouse anti-Trx antibody-colloidal gold complex, and labeling nCOVAg01 to the mouse anti-Trx antibody-colloidal gold complex to obtain a labeled antigen 1; uniformly mixing the colloidal gold solution and the rabbit IgG for reaction to obtain a labeled antibody 2; and mixing the labeled antigen 1 and the labeled antibody 2 according to the molar ratio of 2-4:7 to obtain the gold-labeled protein.
In a preferred embodiment, the test strip is prepared to meet the following requirements: the grain size of the colloidal gold is 20 nm; 1.0 mu g of gold-labeled antigen is loaded on each square centimeter of the bonding pad; 2.0 mu g nCOVAg02 is loaded on each centimeter of detection area on the coating film; 1.5 mu g of goat anti-rabbit polyclonal antibody is loaded on each centimeter of quality control area on the coating film; the sample pad is loaded with 1.5 mu g of anti-RBC antibody in square centimeter and is used for removing red blood cells in whole blood; the sample pad was soaked with a solution containing 0.5-1.5v/v% Tween20 and 0.4-0.6w/v% PVA for promoting the release of colloidal gold.
The invention finally protects a detection kit for SARS-CoV-2 antibody, comprising the detection test paper of the invention.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1 antigen sequence design
Recombinant expression antigens, nCoVAg01 and nCoVAg02, were designed based on the known SARS-CoV-2 coronavirus NC-045512.2 complete gene sequence. Wherein the nCOVAg01 antigen structure is Trx + N + S + hydrophilic sequence + His, and the nCOVAg02 antigen structure is His + N + S + His. Specifically, the nCOVAg01 antigen is a protein consisting of an amino acid sequence shown in SEQ ID NO. 9; nCOVAg02 is a protein composed of the amino acid sequence shown in SEQ ID NO. 10.
Example 2 antigen construction
Three sequences of N01(SEQ ID NO.11), S01(SEQ ID NO.12) and S02(SEQ ID NO.13) are synthesized by whole genes for constructing antigen.
N01-pUC plasmid is subjected to double enzyme digestion of Nco I and Nhe I to obtain a fragment 1, S01-pUC plasmid is subjected to double enzyme digestion of Nhe I and XhoI to obtain a fragment 2, the fragment 1 and the fragment 2 are connected with pET30a-Trx vector skeletons (Nco I and XhoI) to transform escherichia coli DH5 alpha, positive clones are screened, and the nCoVAg01 plasmid is obtained through sequencing and identification.
Carrying out double digestion on Nde I and Nhe I of N01-pUC plasmid to obtain a fragment 3, carrying out double digestion on Nhe I and XhoI of S02-pUC plasmid to obtain a fragment 4, connecting pET28a vector frameworks (Nde I and XhoI) of the fragment 3 and the fragment 4, transforming Escherichia coli DH5 alpha, screening positive clones, and sequencing to obtain nCoVAg02 plasmid.
Example 3 antigen expression and purification
The nCOVAg01 and nCOVAg02 plasmids transform an escherichia coli expression bacterium BL21(DE3), IPTG induced expression is carried out, and two target proteins are finally obtained. Both antigens are expressed in the supernatant in E.coli, and the supernatant is collected for protein purification after the homogenate of the thalli is broken.
Purification of recombinant antigen using Ni-NTA affinity column, as described in the Ni-NTA SefiniseTM Resin Kit, generally comprises the following steps: adding the Escherichia coli supernatant into an affinity column, binding the target protein and Ni ions, remaining in the affinity column, and allowing the hybrid protein to flow through; passing 20mM Tris-HCl (pH8.0) +150mM NaCl +50mM imidazole through a column to remove non-specifically adsorbed heteroproteins; then, the target protein is eluted with 20mM Tris-HCl (pH8.0) +150mM NaCl +200mM imidazole to obtain protein solutions, namely nCOVAg01 and nCOVAg02 antigen protein. The purified target protein is subjected to SDS-PAGE, and the protein purity is detected to be more than or equal to 80 percent, and the results are respectively shown in figure 1 and figure 2.
EXAMPLE 4 preparation of the kit
The main components of the kit are as follows:
| numbering | Component name | Storage conditions |
| 1 | Test paper card | 4～30℃ |
| 2 | Auxiliary liquid | 4～30℃ |
| 3 | Disposable blood collection tube | 4～30℃ |
| 4 | Non-drying adhesive paper for experimental recording | 4～30℃ |
| 5 | Description | At room temperature |
The nitrocellulose membrane and the coating solution are purchased from Hipposhu scientific Co., Ltd. The novel coronavirus multiple proteins, rabbit IgG and goat anti-rabbit polyclonal antibody are all produced by Beijing New Biotechnology Co.
Preparation method of nitrocellulose membrane
The nCoVAg02 antigen and the goat anti-rabbit polyclonal antibody are respectively diluted to 5-15ug/ml by using an envelope buffer solution (10mM PBS (pH7.2)) according to a certain proportion, are uniformly enveloped on different positions of an NC membrane, and the NC membrane is placed in a drying room (the temperature is 24 +/-4 ℃ and the humidity is less than 25 percent) and is dried (1-2 h). And (3) storage: and filling the dried NC film into an aluminum foil bag for sealing, adding a drying agent, vacuumizing, packaging and storing at 4-30 ℃.
Preparation method of sample pad
The treatment for removing red blood cells in whole blood is carried out on the sample pad: adding anti-RBC antibodies; promoting the release of the colloidal gold: 1% Tween20+ 0.5% PVA was added.
Preparation method of bonding pad
(a) Labeling a mouse anti-Trx antibody to 20nm of colloidal gold, adjusting the pH value of a colloidal gold solution to be 6-7, adjusting the antibody concentration to be 10-25ug/ml, sealing by 20% BSA, and suspending by using a TB9 solution to 1/10 of the original volume after centrifugation;
(b) marking nCoVAg01 antigen on a mouse anti-Trx antibody-colloidal gold compound with the diameter of 20nm, adjusting the pH value of the colloidal gold solution to 6-7 and the antigen concentration to 5-15ug/ml, and suspending the colloidal gold solution to 1/10 of the original volume by using TB9 solution after centrifugation;
(c) labeling rabbit IgG on colloidal gold with the diameter of 20nm, adjusting the pH value of the colloidal gold solution to 5-6, adjusting the antibody concentration to 10-25ug/ml, sealing with 20% BSA, and suspending with TB9 solution (Trion) to 1/10 of the original volume after centrifugation;
(d) mixing the labeled antigen and the labeled antibody rabbit IgG according to the ratio of 3: 7;
(e) antigen was reconstituted with DOA reconstituted in original volume 9/10.
And uniformly coating the marked colloidal gold solution on a glass cellulose membrane (1200 glass fibers) at a concentration of 42 ml/sheet, and freeze-drying or drying the gold pad by using a freeze dryer to obtain the combined pad.
5) And (3) putting the dried gold pad into an aluminum foil bag for sealing, adding a drying agent, vacuumizing, packaging and storing at 4-30 ℃.
Assembly of colloidal gold test strip
And sequentially fixing the sample pad, the combination pad, the nitrocellulose membrane and the absorption pad on the bottom plate along the flow direction of the liquid sample to be detected to obtain the detection test paper, wherein the schematic structural diagram is shown in fig. 3.
Detection method of kit
1) Numbering: and taking out the test paper, sticking the test paper to a corresponding position on the adhesive sticker recording paper, horizontally placing the test paper and making a mark.
2) Sample adding: 100 μ l of fresh whole blood (or serum, plasma) is aspirated by a sample applicator or a disposable blood collection tube, and slowly dropped onto a pad or a sample application well of a test card below the arrow of the test paper (the volume of the disposable blood collection tube is 80 μ l).
3) Interpretation: to prevent the missed detection of the samples of Wen Yang, please observe and record the results 10 minutes after the sample is added.
Interpretation of test results
1) Positive: two purple red lines appear on the test paper at the detection line and the quality control line.
2) Negative: the test paper only shows a purple red line at the position of the quality control line.
3) And (3) failure: the test paper has no purple red line or only has a purple red line on the detection line.
Example 5 kit Performance testing
The following tests were carried out according to the test method of the kit in example 4:
(1) specific experiments:
detection of negative samples: the test results of 1000 cases of common clinical blood are negative.
Detection of positive samples: the detection rate of 459 cases, namely 91.8%, of the novel coronavirus infected serum confirmed by the disease control center in Hubei province is 500.
(2) Stability test:
dividing the randomly-extracted kit to be detected produced in the same batch into 2 parts, placing 1 part in a refrigerator at 4 ℃, placing the other 1 part in a thermostat at 37 ℃ for 6 days, taking out, placing in the refrigerator at 4 ℃ for balancing overnight, and performing stability detection by using positive key quality control products.
The interpretation method comprises the following steps: the detection line shows the color strength, and the interpretation is from L0 to L10, as shown in FIG. 4.
And (4) judging a result: the sensitivity of the kit placed at 37 ℃ for 6 days is judged to be consistent with that of the kit placed at 4 ℃ for initial detection, and the more consistent the color development, the better the stability.
Test result of stability of kit
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
SEQUENCE LISTING
<110> Beijing Biotechnology Ltd
<120> novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit
<160> 13
<170> PatentIn version 3.5
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Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
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Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala
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Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly
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Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp
20 25 30
Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr
35 40 45
Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly
50 55 60
Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser
65 70 75 80
Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn
85 90 95
Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala Arg Met Ala Gly
100 105 110
Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn
115 120 125
Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln
130 135 140
Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln
145 150 155 160
Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg
165 170 175
Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile
180 185 190
Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala
195 200 205
Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val
210 215 220
Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Ala Ala Ile Lys Leu Asp
225 230 235 240
Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His
245 250 255
Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys
260 265 270
Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys
275 280 285
Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser
290 295 300
Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser Thr Gln Ala
305 310 315
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Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val
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Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val
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Tyr Tyr His
35
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Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln
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Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu
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Thr
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Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
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Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala
20 25
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Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg
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Ala Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr
20 25
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Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
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Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
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Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Ser Lys
420 425 430
Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys
435 440 445
Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr
450 455 460
His
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Ser Thr Gln Gln Asn Thr Gly Gly Pro Gln Thr Thr
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Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
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Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Met Gly His
100 105 110
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
115 120 125
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
130 135 140
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
145 150 155 160
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
165 170 175
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
180 185 190
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
195 200 205
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
210 215 220
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
225 230 235 240
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
245 250 255
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
260 265 270
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
275 280 285
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
290 295 300
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
305 310 315 320
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
325 330 335
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
340 345 350
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
355 360 365
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
370 375 380
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
385 390 395 400
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
405 410 415
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
420 425 430
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
435 440 445
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
450 455 460
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
465 470 475 480
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
485 490 495
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
500 505 510
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
515 520 525
Thr Gln Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Ser Lys
530 535 540
Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys
545 550 555 560
Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr
565 570 575
His Gly Gly Gly Gly Gly Ser Gly Gly Ser Thr Gln Gln Asn Thr Gly
580 585 590
Gly Pro Gln Thr Thr Gly Gly Gly Ser Gly Gly Ser Leu Glu His His
595 600 605
His His His His
610
<210> 10
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<400> 10
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala
20 25 30
Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln
35 40 45
Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly
50 55 60
Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly
65 70 75 80
Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr
85 90 95
Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg
100 105 110
Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp
115 120 125
Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly
130 135 140
Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn
145 150 155 160
Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala
165 170 175
Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr
180 185 190
Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser
195 200 205
Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly
210 215 220
Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala
225 230 235 240
Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly
245 250 255
Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala
260 265 270
Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr
275 280 285
Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly
290 295 300
Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His
305 310 315 320
Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly
325 330 335
Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr
340 345 350
Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp
355 360 365
Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro
370 375 380
Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln
385 390 395 400
Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro
405 410 415
Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser
420 425 430
Ser Ala Asp Ser Thr Gln Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly
435 440 445
Gly Ser Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn
450 455 460
Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu
465 470 475 480
Gly Val Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu Glu
485 490 495
His His His His His His
500
<210> 11
<211> 1283
<212> DNA
<213> Artificial sequence
<400> 11
ccatgggcca tatgagcgat aacggtccgc aaaaccagcg taacgcgccg cgtattacct 60
tcggtggtcc gagcgatagc accggtagca accaaaacgg cgaacgtagc ggtgcgcgta 120
gcaaacaacg tcgtccgcaa ggtctgccga acaacaccgc gagctggttt accgcgctga 180
cccagcacgg taaagaagac ctgaagttcc cgcgtggtca gggcgtgccg attaacacca 240
acagcagccc ggacgatcaa attggttatt accgtcgtgc gacccgtcgt atccgtggcg 300
gtgatggcaa aatgaaagat ctgagcccgc gttggtactt ctattacctg ggtaccggcc 360
cggaagcggg tctgccgtac ggtgcgaaca aggatggcat catctgggtt gcgaccgaag 420
gtgcgctgaa caccccgaaa gaccacattg gtacccgtaa cccggcgaac aacgcggcga 480
ttgttctgca gctgccgcaa ggtaccaccc tgccgaaagg tttctatgcg gagggtagcc 540
gtggtggtag ccaagcgagc agccgtagca gcagccgtag ccgtaacagc agccgtaaca 600
gcaccccggg tagcagccgt ggtaccagcc cggcgcgtat ggcgggtaac ggtggcgatg 660
cggcgctggc gctgctgctg ctggatcgtc tgaaccaact ggagagcaag atgagcggca 720
agggtcagca acaacagggt caaaccgtta ccaaaaagag cgcggcggaa gcgagcaaga 780
aaccgcgtca gaaacgtacc gcgaccaagg cgtacaacgt gacccaggcg tttggtcgtc 840
gtggtccgga acaaacccag ggcaactttg gtgaccaaga actgatccgt caaggcaccg 900
actacaaaca ctggccgcag atcgcgcaat tcgcgccgag cgcgagcgcg ttctttggta 960
tgagccgtat tggtatggaa gtgaccccga gcggtacctg gctgacctac accggtgcga 1020
tcaaactgga cgataaggac ccgaacttca aagaccaagt gatcctgctg aacaagcaca 1080
tcgacgcgta caaaaccttt ccgccgaccg agccgaagaa agacaagaag aaaaaggcgg 1140
atgaaaccca ggcgctgccg caacgtcaga aaaagcaaca gaccgtgacc ctgctgccgg 1200
cggcggatct ggacgatttc agcaaacagc tgcagcaaag catgagcagc gcggatagca 1260
cccaagcggc tagctaactc gag 1283
<210> 12
<211> 225
<212> DNA
<213> Artificial sequence
<400> 12
gctagcggcg gcggcggcag cggcggcggc agcagcaaaa cccagagcct gctgattgtg 60
aacaacgcga ccaacgtggt gattaaagtg tgcgaatttc agttttgcaa cgatccgttt 120
ctgggcgtgt attatcatgg cggcggcggc ggcagcggcg gcagcaccca gcagaacacc 180
ggcggcccgc agaccaccgg cggcggcagc ggcggcagcc tcgag 225
<210> 13
<211> 171
<212> DNA
<213> Artificial sequence
<400> 13
gctagcggcg gcggcggcag cggcggcggc agcagcaaaa cccagagcct gctgattgtg 60
aacaacgcga ccaacgtggt gattaaagtg tgcgaatttc agttttgcaa cgatccgttt 120
ctgggcgtgt attatcatgg cggcggcggc agcggcggcg gcagcctcga g 171
Claims (20)
1. A SARS-CoV-2 recombinant antigen is nCOVAg01 or nCOVAg 02;
nCOVAg01 is a protein consisting of the amino acid sequence shown in SEQ ID NO. 9;
nCOVAg02 is a protein composed of the amino acid sequence shown in SEQ ID NO. 10.
2. The biomaterial associated with the recombinant antigen of claim 1, wherein the biomaterial is any one of:
(a) a nucleic acid molecule encoding the recombinant antigen of claim 1;
(b) an expression cassette comprising the nucleic acid molecule of (a);
(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);
(d) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);
(e) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).
3. Use of the recombinant antigen of claim 1 or the biological material of claim 2 for the preparation of a SARS-CoV-2 antibody detection product.
4. Use according to claim 3, wherein the SARS-CoV-2 antibody is an IgM or IgG.
5. The use according to claim 3, wherein the method for detecting SARS-CoV-2 antibody comprises a colloidal gold capture method, a colloidal gold indirect method, a colloidal gold double antigen sandwich method or an ELISA.
6. Use according to claim 3, wherein the product comprises a dipstick or a kit.
7. A test paper for detecting SARS-CoV-2 antibody by colloidal gold capture method is characterized in that it comprises a bottom plate, a sample pad, a combination pad, a coating film and an absorption pad, wherein the sample pad, the combination pad, the coating film and the absorption pad are fixed on the bottom plate in sequence along the flowing direction of the liquid sample to be detected; the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with a mouse anti-human IgM/IgG antibody, and the quality control area is loaded with a goat anti-rabbit polyclonal antibody; nCOVAg01 is a protein composed of the amino acid sequence shown in SEQ ID NO. 9.
8. The test paper for detecting SARS-CoV-2 antibody by colloidal gold indirect method is characterized by comprising a base plate, a sample pad, a combination pad, a coating film and an absorption pad, wherein the sample pad, the combination pad, the coating film and the absorption pad are fixed on the base plate in turn along the flowing direction of a liquid sample to be detected; the binding pad is loaded with gold-labeled protein, and the gold-labeled protein comprises a mouse anti-human IgG monoclonal antibody or a mouse anti-human IgM antibody; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with nCOVAg02, and the quality control area is loaded with goat anti-rabbit polyclonal antibody; nCOVAg02 is a protein composed of the amino acid sequence shown in SEQ ID NO. 10.
9. A test paper for detecting SARS-CoV-2 antibody by colloidal gold double-antigen sandwich method is characterized in that it comprises a bottom plate, a sample pad, a combination pad, a coating film and an absorption pad, wherein the sample pad, the combination pad, the coating film and the absorption pad are fixed on the bottom plate in sequence along the flowing direction of the liquid sample to be detected; the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with nCOVAg02, and the quality control area is loaded with goat anti-rabbit polyclonal antibody; nCOVAg01 is a protein consisting of the amino acid sequence shown in SEQ ID NO.9, and nCOVAg02 is a protein consisting of the amino acid sequence shown in SEQ ID NO. 10.
10. The test strip according to any one of claims 7 to 9, wherein the sample pad of the test strip is impregnated with a solution containing 0.5 to 1.5v/v% Tween20 and 0.4 to 0.6w/v% PVA, and loaded with anti-RBC antibodies.
11. The test strip of claim 10, wherein the fluid sample to be tested comprises whole blood, serum, or plasma.
12. The method of preparing a test strip according to claim 9, wherein the sample pad, the conjugate pad, the envelope and the absorbent pad are fixed to the base plate in this order along the direction of flow of the liquid sample to be tested; the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, wherein the detection area is loaded with nCOVAg02, and the quality control area is loaded with goat anti-rabbit polyclonal antibody.
13. The method of claim 12, wherein the method of preparing the gold-labeled binding pad protein comprises: uniformly mixing the colloidal gold solution and the mouse anti-tag antibody for reaction to obtain a mouse anti-tag antibody-colloidal gold cross-linked complex, and uniformly mixing the complex with nCOVAg01 for reaction to obtain a labeled antigen 1; uniformly mixing the colloidal gold solution and the rabbit IgG for reaction to obtain a labeled antibody 2; and mixing the labeled antigen 1 and the labeled antibody 2 according to the molar ratio of 2-4:7 to obtain a gold-labeled cross-linked complex.
14. The method according to claim 13, wherein the colloidal gold has a particle size of 20 to 30 nm.
15. The method of claim 12, wherein the conjugate pad has 0.6-1.5 μ g of the gold-labeled protein per square centimeter of the conjugate pad.
16. The method of claim 12, wherein the coating film comprises 1.5-2.0 μ g nCOVAg02 per cm of the detection area.
17. The method of claim 12, wherein the coating membrane is loaded with 1.3-2.0 μ g goat anti-rabbit polyclonal antibody per cm of the quality control region.
18. The method of claim 12, wherein the sample pad is loaded with 1.0-2.0 μ g of anti-RBC antibody per square centimeter.
19. The method of claim 12, wherein the sample pad is soaked in a solution containing 0.5-1.5v/v% Tween20 and 0.4-0.6w/v% PVA.
20. A kit for detecting SARS-CoV-2 antibody, comprising the test strip according to any one of claims 7 to 11.
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