CN112505330B

CN112505330B - Kit for detecting novel coronavirus based on fusion protein of nucleocapsid protein

Info

Publication number: CN112505330B
Application number: CN202011242706.0A
Authority: CN
Inventors: 侯江厚; 孙卫国; 张灵霞; 黄国红; 杨奕梅; 李夏南; 翟斐
Original assignee: Kunming Maternal And Child Health Hospital; 8th Medical Center of PLA General Hospital
Current assignee: Kunming Maternal And Child Health Hospital; 8th Medical Center of PLA General Hospital
Priority date: 2020-11-09
Filing date: 2020-11-09
Publication date: 2024-03-29
Anticipated expiration: 2040-11-09
Also published as: CN112505330A

Abstract

The invention provides a kit for detecting novel coronaviruses based on fusion proteins of nucleocapsid proteins, which comprises antigens for detecting novel coronaviruses, wherein the antigens for detecting novel coronaviruses comprise the fusion proteins based on nucleocapsid proteins. The kit for detecting the novel coronavirus based on the fusion protein of the nucleocapsid protein can obtain the fusion protein of the nucleocapsid protein containing the novel coronavirus by a fusion technology, so that the fusion protein has the natural spatial structure characteristics of the nucleocapsid protein of the novel coronavirus, has high sensitivity and strong specificity when being used for detecting the novel coronavirus, can effectively improve the detection rate of the novel coronavirus by utilizing the fusion protein based on the nucleocapsid protein to detect the novel coronavirus, ensures the timely definite diagnosis of the pneumonia of the novel coronavirus, and can effectively avoid the spread of epidemic situation.

Description

Kit for detecting novel coronavirus based on fusion protein of nucleocapsid protein

Technical Field

The invention relates to the field of biological medicine detection, in particular to a novel coronavirus detection kit based on fusion protein of nucleocapsid protein.

Background

The novel coronavirus pneumonia (Corona Virus Disease 2019, covd-19) is a new infectious disease mainly caused by lung lesions and caused by infection of novel coronavirus (SARS-CoV-2, also known as 2019-nCoV), and can cause damage to the digestive system and the nervous system, and serious cases can cause death. At present, no specific therapeutic drug exists for 2019-nCoV, and the vaccine is also in a clinical test stage, so that early diagnosis, timely recovery and isolation of patients are important for effectively controlling epidemic situation. The large amount of detection of the new coronavirus infection mainly adopts nucleic acid detection, and the nucleic acid detection becomes a gold standard for the diagnosis of suspected cases due to the strong sensitivity, but the technology has high requirements on experimental places and personnel, is complex to operate, is greatly influenced by environmental conditions, and is easy to cause false positive due to the pollution of aerosol. The traditional antigen-antibody reaction is an important supplement to clinical laboratory detection, and the use of antigens to detect antibodies in the serum of an infected person is an important means for rapid screening and nucleic acid-assisted diagnosis. Many companies have developed test kits for clinical diagnosis of covd-19 by serodiagnosis methods, such as colloidal gold and traditional ELISA, and screening of antigens or combined antigens conserved in SARS-CoV-2 with dominant epitopes is critical for serodiagnosis success. The SARS-CoV-2 gene is approximately 29.8kb in size and the genome is annotated as containing 14 open reading frames (Opening reading frame, ORF) encoding a total of 27-28 proteins. Nucleocapsid protein (N protein) is a main structural protein of SARS-CoV-2, the sequence table is shown as SEQ ID No.4, the nucleocapsid protein is positioned in virus, is an antagonist of Interferon (IFN) and an RNA interference inhibitor coded by virus, is related to the replication of virus, has relatively conservative relative to beta coronavirus, has a plurality of synthesis, has strong antigenicity, plays an important role in inducing host immune response and even pathogenesis, and is often used as an antigenic site for diagnosis of coronavirus. However, the existing method for detecting SARS-CoV-2 by directly using N protein as antigen has low detection rate, and affects the diagnosis efficiency of COVID-19 and epidemic situation control.

Disclosure of Invention

Based on the above, it is necessary to provide a kit for detecting a novel coronavirus based on a fusion protein of a nucleocapsid protein, aiming at the problems that the detection rate of the existing method for detecting the novel coronavirus directly based on the N protein as an antigen is low, and the diagnosis efficiency of the novel coronavirus pneumonia and the control of epidemic situation are affected.

The invention provides a kit for detecting novel coronaviruses based on fusion proteins of nucleocapsid proteins, which comprises antigens for detecting novel coronaviruses, wherein the antigens for detecting novel coronaviruses comprise the fusion proteins based on nucleocapsid proteins.

In one embodiment, the fusion protein based on nucleocapsid protein is generated by fusion expression of the amino acid sequence or partial amino acid sequence of nucleocapsid protein and the amino acid sequence or partial amino acid sequence of disulfide oxidoreductase A; or, the fusion protein formed by fusion expression of the amino acid sequence or partial amino acid sequence of nucleocapsid protein and the amino acid sequence or partial amino acid sequence of disulfide oxidoreductase C.

In one embodiment, the amino acid sequence of disulfide oxidoreductase a is the amino acid sequence shown as SEQ ID No.1 or a mutant sequence of the amino acid sequence shown as SEQ ID No. 1;

The amino acid sequence of the disulfide oxidoreductase C is an amino acid sequence shown as SEQ ID No.2 or a mutant sequence of the amino acid sequence shown as SEQ ID No. 2;

the amino acid sequence of the nucleocapsid protein is the amino acid sequence shown as SEQ ID No.3 or the mutation sequence of the amino acid sequence shown as SEQ ID No. 3.

In one embodiment, the amino acid sequence of the nucleocapsid protein is linked directly or through a linker to the C-terminus of the amino acid sequence of disulfide oxidoreductase a; the amino acid sequence of the nucleocapsid protein is linked directly or through a linker to the C-terminus of the amino acid sequence of the disulfide oxidoreductase C.

In one embodiment, the kit is a colloidal gold kit, the fusion protein is marked by colloidal gold and then sprayed on a gold-labeled pad, the particle size of the colloidal gold is 18-28 nm, and the spraying amount of the fusion protein marked by the gold on the gold-labeled pad is 1.4-2.0 mu L/cm.

In one embodiment, the kit is an ELISA kit, the kit comprises an antigen ELISA plate, the antigen ELISA plate is an ELISA plate coated with the fusion protein, the coating concentration of the fusion protein on the ELISA plate is 10 mu g/mL-20 mu g/mL, and the coating volume is 80 mu L-120 mu L.

In one embodiment thereof, the kit comprises the following components:

the kit comprises an antigen ELISA plate, positive antibody titer human serum, a positive antibody titer ELISA reagent, a positive antibody titer sample diluent, a color reagent A solution, a color reagent B solution, a concentrated washing solution, a stop solution, a sealing plate film and a negative control.

In one embodiment, the kit comprises the following components in the following number of specifications:

antigen elisa plate 8 x 12 wells, 1 plate;

positive antibody titer human serum, added with stabilizer and preservative 20U/mL, 0.1 mL/branch, 1 branch;

15 mL/bottle of positive antibody titer enzyme-labeled reagent, 1 bottle;

50/mL/bottle of positive antibody titer sample dilution, 1 bottle;

color reagent A solution 6 mL/bottle, 1 bottle;

color reagent B liquid 6 mL/bottle, 1 bottle;

concentrated wash (20 x) 50 mL/bottle, 1 bottle;

stop solution 6 mL/bottle, 1 bottle;

3 sealing plate films;

negative control 0.5 mL ×1 bottles.

In one embodiment, the positive antibody titer enzyme-labeled reagent is a horseradish peroxidase-labeled anti-human IgG antibody;

the positive antibody titer serum is calibrated human serum containing anti-novel coronavirus;

the positive antibody titer sample diluent is a buffer solution containing protein;

the concentrated washing liquid is an aqueous solution containing 10% of surfactant;

the color-developing agent A contains peroxide not less than 0.3 g/L;

the color-developing agent B contains TMB not less than 0.2 g/L;

the stop solution contains sulfuric acid (the concentration is not higher than 2.0 mol/L).

In one embodiment, the preparation method of the antigen ELISA plate comprises the steps of coating an aqueous solution of fusion protein on the ELISA plate, standing for 1.5-2.5 h at room temperature, and then washing the plate with PBST buffer solution; and sealing with 10% calf serum at 4 ℃ for 10-15 hours, and washing the plate with PBST buffer solution to obtain the antigen ELISA plate.

The kit for detecting the novel coronavirus based on the fusion protein of the nucleocapsid protein can obtain the fusion protein of the nucleocapsid protein containing the novel coronavirus by a fusion technology, so that the fusion protein has the natural spatial structure characteristics of the nucleocapsid protein of the novel coronavirus, has high sensitivity and strong specificity when being used for detecting the novel coronavirus, can effectively improve the detection rate of the novel coronavirus by utilizing the fusion protein based on the nucleocapsid protein to detect the novel coronavirus, ensures the timely definite diagnosis of the pneumonia of the novel coronavirus, and can effectively avoid the spread of epidemic situation.

Furthermore, the novel coronavirus detection kit based on the fusion protein of the nucleocapsid protein is a soluble protein, the soluble protein can exist in the form of supernatant, and the detection rate of the novel coronavirus antibody can be effectively improved when the kit is used for serological detection, so that the detection accuracy is ensured.

Furthermore, in the kit for detecting the novel coronavirus based on the fusion protein of the nucleocapsid protein, the nucleocapsid protein of the novel coronavirus and the disulfide oxidoreductase A or the disulfide oxidoreductase C are fused and expressed, the biological characteristics of disulfide isomerase and molecular chaperones of the disulfide oxidoreductase C are fully utilized, the fusion protein is effectively promoted to exist in the form of supernatant in the recombination expression process, the detection rate of the novel coronavirus antibody is further improved, and a material foundation is laid for a serological detection kit.

Drawings

FIG. 1 is a graph showing the results of molecular weight gel electrophoresis identification of SARS-CoV-2N protein nucleotide according to example 1 of the present invention;

FIG. 2 is a graph showing the results of the identification of the expression pattern of the DsbC-N fusion protein of example 1 in a prokaryotic system;

FIG. 3 is a graph showing the results of purity identification of DsbC-N fusion proteins purified in example 1 of the present invention;

FIG. 4 is a graph showing the results of ELISA experiments using the DsbC-N fusion proteins of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following detailed description. It should be understood that the detailed description is presented merely to illustrate the invention, and is not intended to limit the invention.

In early detection, the nucleic acid detection plays a great role, and the purposes of early diagnosis and early isolation are achieved. Along with the analysis of the genome sequence of the novel coronavirus, the expressed sequence of each structural protein is sequenced and the recombinant diagnostic antigen begins to function. When the virus infects the organism, igM antibody appears earliest in serum antibody of the organism, is the diagnostic index of acute phase infection, but IgM concentration is low, the maintenance time is only about one week and the affinity is low; the IgG antibody is produced later, can indicate the late middle-period of infection or the generation of past infection, and has high concentration, long maintenance time and high affinity. At present, a plurality of biological companies develop diagnostic methods for detecting SARS-CoV-2 IgM and IgG antibodies, and a good detection means is provided for the auxiliary diagnosis of novel coronavirus infection and epidemic investigation. The specificity of antibody detection is closely related to the conservation of antigenic sites, the N protein of SARS-CoV-2 is the main antigenic site of the detection of COVID-19, and the current clinical novel coronavirus detection kit mostly adopts N protein antigen or spike protein (S protein) antigen or a combination method to detect serum antibodies. However, the detection of serum antibodies using N protein antigen or spike protein (S protein) antigen or a combination method has yet to be further improved in sensitivity and specificity to increase the detection rate of SARS-CoV-2.

In order to improve the sensitivity and the specificity of N protein antigen in serology diagnosis and simultaneously improve the expression quantity of N protein in prokaryotic recombination, the invention adopts a method of fusion expression of N protein and DsbA or DsbC, and because in prokaryotic recombination, soluble expression often represents the correct space folding mode of recombinant protein, in order to ensure the empty epitope of detected antigen, the invention utilizes the biological characteristics of DsbA and DsbC protein chaperones and disulfide bond isomerase to obtain the supernatant expression of SARS-CoV-2N protein, and well ensures the detection rate in serology diagnosis.

The kit for detecting the novel coronavirus based on the fusion protein of the nucleocapsid protein comprises an antigen for detecting the novel coronavirus, and the antigen for detecting the novel coronavirus comprises the fusion protein based on the nucleocapsid protein. The antigen for detecting the novel coronavirus in the kit can obtain the fusion protein containing the nucleocapsid protein of the novel coronavirus through a fusion technology, so that the fusion protein has the natural spatial structural characteristics of the nucleocapsid protein of the novel coronavirus and simultaneously improves the property of the fusion protein.

Preferably, the fusion protein based on nucleocapsid protein is a soluble protein which can exist in the form of supernatant, and can effectively improve the detection rate of novel coronavirus antibody when used for serological detection and ensure the detection accuracy.

As an alternative embodiment, the fusion protein based on nucleocapsid protein is a fusion protein produced by fusion expression of the amino acid sequence or partial amino acid sequence of nucleocapsid protein with the amino acid sequence or partial amino acid sequence of disulfide oxidoreductase a (DsbA); or, the fusion protein formed by fusion expression of the amino acid sequence or partial amino acid sequence of nucleocapsid protein and the amino acid sequence or partial amino acid sequence of disulfide oxidoreductase C (DsbC). The formation of disulfide bonds in proteins is an important process of prokaryotes and eukaryotes, the soluble expression of recombinant fusion proteins often represents the correct spatial folding mode of the proteins and maintains the natural spatial structure of the proteins, in order to ensure the natural conception of the fusion purification process of novel coronavirus nucleocapsid proteins, the invention fuses and expresses the nucleocapsid proteins of novel coronaviruses with disulfide oxidoreductase A or disulfide oxidoreductase C, fully utilizes the biological characteristics of disulfide bond isomerase and molecular chaperones of the disulfide oxidoreductase A and the disulfide oxidoreductase C, improves the solubility of the fusion proteins, promotes the existence of the fusion proteins in the form of supernatant in the recombinant expression process, further improves the detection rate of novel coronavirus antibodies and lays a material foundation for a serological detection kit.

As an alternative embodiment, the nucleotide sequence of disulfide oxidoreductase A is the nucleotide sequence shown as SEQ ID No.1 or a mutant sequence of the nucleotide sequence shown as SEQ ID No. 1; the nucleotide sequence of the disulfide oxidoreductase C is a nucleotide sequence shown as SEQ ID No.2 or a mutant sequence of the nucleotide sequence shown as SEQ ID No. 2; the nucleotide sequence of nucleocapsid protein is the nucleotide sequence shown as SEQ ID No.3 or the mutant sequence of the nucleotide sequence shown as SEQ ID No. 3.

Wherein, in the actual fusion expression process of the nucleocapsid protein and the disulfide oxidoreductase A or the disulfide oxidoreductase C, the nucleocapsid protein nucleotide sequence can be mutated according to the need to improve the characteristics of the fusion protein after fusion expression, wherein, the optional mutation mode comprises adding, deleting or replacing one or a plurality of nucleotides in the nucleotide sequence; similarly, the disulfide oxidoreductase A nucleotide sequence or disulfide oxidoreductase C nucleotide sequence may be mutated as desired to improve the characteristics of the fusion protein following fusion expression, again optionally by adding, deleting or substituting one or more nucleotides in the nucleotide sequence. For example, the nucleotide sequence of disulfide oxidoreductase A is shown in SEQ ID No.1, and one of its mutant sequences is shown in SEQ ID No. 7.

As an alternative embodiment, the nucleotide sequence of the nucleocapsid protein is linked directly or through a linker to the C-terminus of the nucleotide sequence of disulfide oxidoreductase a; the amino acid sequence of the nucleocapsid protein is linked directly or via a linker to the C-terminus of the amino acid sequence of disulfide oxidoreductase C. For example, nucleocapsid proteins can be linked to disulfide oxidoreductase a or disulfide oxidoreductase C by a soft linker of nucleotide sequence GGGGSGGGGS.

Alternatively, fusion proteins based on nucleocapsid proteins are produced by expression in a prokaryotic expression system, the preferred expression strain being E.coli.

Alternatively, the preparation method of the antigen for novel coronavirus detection used in the kit of the present invention may be prepared by, but not limited to, the following method.

A method for preparing an antigen for use in novel coronavirus detection comprising the steps of:

synthesizing nucleocapsid protein gene fragment according to the nucleotide sequence shown in SEQ ID No. 3;

cleaving the nucleocapsid protein gene fragment from the cloning vector by means of the endonuclease SalI and the endonuclease NheI;

the nucleocapsid protein gene fragment is cloned to a vector PET-DsbA or PET-DsbC, and is transformed into a prokaryotic expression system to induce expression to generate fusion protein based on nucleocapsid protein.

Further alternatively, the nucleotide sequence of the upstream primer for synthesizing the nucleocapsid protein gene fragment is shown in SEQ ID No. 5; the nucleotide sequence of the downstream primer for synthesizing the nucleocapsid protein gene fragment is shown in SEQ ID No. 6.

According to the nucleic acid sequence of novel coronavirus nucleocapsid protein reported by GenBank: MN908947.3, specific primers aiming at nucleocapsid protein nucleotide are designed and synthesized by referring to the multiple cloning sites of vectors pET-DsbA and pET-DsbC, and a stop codon TAA of escherichia coli bias is introduced at the 5 end of a downstream primer. Specifically, the nucleotide sequence of the upstream primer is 5-ACGCGTCGACTCTGATAATGGACC-3; the nucleotide sequence of the downstream primer is 5-CTAGCTAGCTTAGGCCTGAGTTGAGTCAGC-3, and the cleavage sites GCTAG of the endonuclease SalI and GCTAG (bold portions in the sequence) of the endonuclease NheI are introduced from the ends of the primer and the downstream primer, respectively, so that the nucleocapsid protein gene fragment is excised from the cloning vector by the cleavage enzymes SalI and NheI.

Alternatively, the invention uses the nucleotide sequence of the nucleocapsid protein of the vector pUC18-SARS-CoV-2 as a template, and uses an upstream primer and a downstream primer to amplify the nucleotide sequence of the nucleocapsid protein of SARS-CoV-2 by a conventional PCR method under the action of heat shock TaqDNA polymerase.

Alternatively, the PCR method amplification conditions are as follows:

the pre-denaturation at 94℃for 5 min, denaturation at 98℃for 20 s, annealing at 68℃for 20 s and elongation at 72℃for 80 s were performed for 30 cycles, and elongation at 72℃for 5 min.

After the molecular weight of the PCR amplified product is verified by 1% agarose electrophoresis, the PCR amplified product is recovered by gel, the recovered amplified product and the expression vector pET-DsbA or pET-DsbC are subjected to double enzyme digestion by SalI and NheI, the enzyme-digested product is recovered by 1% agarose electrophoresis gel digestion, under the action of T4DNA ligase, the enzyme-digested recovered product (comprising N protein gene fragment and N protein gene fragment in the expression plasmid pET-DsbA or pET-DsbC) is cloned and connected to the expression plasmid pET-DsbA or pET-DsbC, the cloned and connected product is transformed into escherichia coli competent cells BL21 (DE 3), and positive cloned products are screened for sequencing.

Inoculating the DsbA-N fusion protein or DsbC-N fusion protein engineering bacteria which are sequenced correctly into LB culture medium containing 50 ng/ml kanamycin, placing the LB culture medium into a shaking table, shaking and culturing at 37 ℃ for activation, transferring the LB culture medium into the same system according to the ratio of 1:50 in the next day, shaking and culturing at 37 ℃ until bacterial liquid OD600 reaches 0.4 h, adding IPTG to enable the final concentration to be 0.3 mmoL, and continuing shaking and culturing at 37 ℃ for induction of 5 h. After induction, the bacterial liquid is centrifugally collected at 5000 rpm under the condition of 4 ℃, the precipitated bacterial liquid is added into 1 XPBS buffer solution for uniform mixing, ultrasonic crushing is carried out under the ice-culturing state, centrifugation is carried out at 12000 rpm for 30 min under the condition of 4 ℃, the supernatant and the precipitate are collected, and the expression form of DsbA-N fusion protein or DsbC-N fusion protein in a prokaryotic system is identified by 12% SDS-PAGE.

As an alternative embodiment, the preparation method further comprises the following steps:

affinity purification and/or gel purification of fusion proteins based on nucleocapsid proteins.

For example, the DsbA-N fusion protein and the DsbC-N fusion protein have 6×HIS tags at the N-terminal, and affinity chromatography can be performed on the collected supernatant and precipitate generated after fusion expression by chelating sephrose, so that the purification is convenient and the purity of the purified fusion protein is higher.

Purifying the expressed ultrasonic supernatant protein by utilizing affinity chromatography, taking the centrifugal supernatant at the temperature of 4 ℃ and passing through a nickel ion affinity column at the speed of 2 mL/min, balancing 4 column volumes by using 1 XPBS, eluting 3 column volumes of the hybrid protein by using 100 mmoL imidazole, eluting the target fusion protein by using 200mmoL imidazole, collecting the target protein, and measuring the purity of the purified fusion protein by using 15% SDS-PAGE.

As an alternative implementation mode, the kit is a colloidal gold kit, the fusion protein is sprayed on the gold-labeled pad after being labeled by the colloidal gold, the particle size of the colloidal gold is 18-28 nm, and the spraying amount of the fusion protein labeled by the gold on the gold-labeled pad is 1.4-2.0 mu L/cm.

Specifically, the composition, preparation method and use method of the colloidal gold kit can refer to the traditional colloidal gold kit.

As an alternative implementation mode, the kit is an ELISA kit, the kit comprises an antigen ELISA plate, the antigen ELISA plate is an ELISA plate coated with fusion protein, the coating concentration of the fusion protein on the ELISA plate is 10 mu g/mL-20 mu g/mL, and the coating volume is 80 mu L-120 mu L. Preferably, the coating concentration of the fusion protein on the ELISA plate is 15 mu g/mL, and the coating volume is 100 mu L.

As an alternative embodiment, the kit comprises the following components:

Further, the specification number of the components of the kit is as follows:

antigen elisa plate 8 x 12 wells, 1 plate;

15 mL/bottle of positive antibody titer enzyme-labeled reagent, 1 bottle;

50/mL/bottle of positive antibody titer sample dilution, 1 bottle;

color reagent A solution 6 mL/bottle, 1 bottle;

Color reagent B liquid 6 mL/bottle, 1 bottle;

concentrated wash (20 x) 50 mL/bottle, 1 bottle;

stop solution 6 mL/bottle, 1 bottle;

3 sealing plate films;

negative control 0.5 mL ×1 bottles.

Wherein, optionally, the positive antibody titer enzyme-labeled reagent is horseradish peroxidase-labeled anti-human IgG antibody; the positive antibody titer serum is a calibrated human serum containing anti-novel coronavirus; the positive antibody titer sample diluent is a buffer solution containing protein; concentrating the washing liquid into an aqueous solution containing 10% of surfactant; the color reagent A contains peroxide not less than 0.3 g/L; the color reagent B contains TMB not less than 0.2 g/L; the stop solution contains sulfuric acid (concentration is not higher than 2.0 mol/L).

Preferably, the preparation method of the antigen ELISA plate comprises the steps of coating an aqueous solution of fusion protein on the ELISA plate, standing for 1.5-2.5 h at room temperature, and then washing the plate with PBST buffer solution; and sealing with 10% calf serum at 4 ℃ for 10-15 hours, and washing the plate with PBST buffer solution to prepare the antigen ELISA plate.

Example 1

1. Material

Plasmid pET-DsbC and expression strain BL21 (DE 3) are stored by the applicant; the nucleic acid vector pUC 18-SARS-CoV-2N of SARS-CoV-2N protein expression sequence is given away by Shen Chunji teacher of Beijing Wan Fang Mei lan technology company; plasmid extraction kits and DNA gel recovery kits were purchased from tenna biotechnology company; endonucleases, T4 DNA ligase, purchased from NEB company; kapa fidelity DNA polymerase and dntps are available from beijing-like micro gene corporation; the affinity resin can be used by friendship in-between biotechnology company; sheep anti-human IgG, TMB chromogenic solution was available from soribao biosystems; the novel coronavirus IgG antibody detection kit (enzyme-linked immunosorbent assay) of the control sample is Beijing Hua Daji biological technology company, and SARS-CoV-2 whole virus lysate is coated on the enzyme-labeled strip; 30 cases of clinically definite diagnosis of serum of a patient with COVID-19, 50 cases of serum of healthy people are collected by biological companies, and ELISA experiments are completed by the company commissioned; ELISA experiments were run strictly in accordance with biosafety-related regulations, following the usual regulations of laboratory procedures.

2. Method of

2.1 Preparation of SARS-CoV-2N protein nucleotide amplification primer

According to the SARS-CoV-2N protein nucleic acid sequence reported by GenBank: MN908947.3, referring to the vector pET-DsbC multiple cloning site, a specific primer aiming at N protein nucleic acid is designed and synthesized, and a stop codon TAA of colibacillus bias is introduced at the 5 end of the downstream primer. The design sequences of the upstream and downstream primers are as follows:

the nucleotide sequence of the upstream primer is P1:5-ACGCGTCGACTCTGATAATGGACC-3;

the nucleotide sequence of the downstream primer is P2:5-CTAGCTAGCTTAGGCCTGAGTTGAGTCAGC-3.

The upstream and downstream primer ends were introduced with SalI and NheI cleavage sites, respectively (diagonal lines).

The vector pUC 18-SARS-CoV-2N nucleic acid is used as a template, and under the action of heat shock TaqDNA polymerase, the SARS-CoV-2N nucleic acid is amplified by the conventional PCR method through the primers P1 and P2 under the following conditions: the pre-denaturation at 94℃for 5 min, denaturation at 98℃for 20 s, annealing at 68℃for 20 s and elongation at 72℃for 80 s were performed for 30 cycles, and elongation at 72℃for 5 min. The molecular weight of the amplified product is verified by 1% agarose electrophoresis, and the result is shown in figure 1, wherein the sequence number 1 is the SARS-CoV-2N protein nucleic acid sequence of the amplified product; m is DL2000. A clear single band exists at the target molecular weight of about 1260bp, the total length of SARS-CoV-2N protein is 420 amino acids, the corresponding molecular weight is 1260bp, and the experimental result accords with the theoretical value.

2.2 preparation of pET-DsbC-N protein fusion expression plasmid

And (3) carrying out gel recovery on the PCR amplified product, carrying out double enzyme digestion on the recovered product and an expression vector pET-DsbC by SalI and NheI, recovering the enzyme-digested product by 1% agarose electrophoresis gel cutting, cloning the recovered fragment into the expression plasmid pET-DsbC under the action of T4DNA ligase, converting the ligation product into an escherichia coli competent cell BL21 (DE 3), and screening positive clones for sequencing.

2.3 Prokaryotic system expression of DsbC-N fusion proteins

Inoculating the pET-DsbC-N fusion protein engineering bacteria which are sequenced correctly into LB culture medium with 50 ng/mL kanamycin concentration, placing the LB culture medium into a shaking table, shaking and culturing to activate the strain at 37 ℃, transferring the strain into LB culture medium of the same system according to the proportion of 1:50 in the next day, shaking and culturing the strain at 37 ℃ until the strain OD600 reaches 0.4 h, adding IPTG to enable the final concentration to be 0.3 mmoL, and continuing shaking and culturing at 37 ℃ to induce 5 h. After induction, the bacterial liquid is centrifugally collected at 5000 rpm under the condition of 4 ℃, the precipitated bacterial liquid is added into 1 XPBS buffer solution for uniform mixing, ultrasonic crushing is carried out under the ice culture state, and the bacterial liquid is centrifugally collected at 12000 rpm for 30 min under the condition of 4 ℃, and the supernatant and the precipitate are collected.

The expression of the fusion protein DsbC-N in the prokaryotic system was identified by 12% SDS-PAGE, and the results are shown in FIG. 2, wherein M is a low molecular weight protein standard; 1 is no induction control; 2 is expressed whole mycoprotein; 3 is the supernatant after ultrasonic disruption of the expression cells; 4 is the sediment after ultrasonic disruption of the expression cells.

As can be seen from FIG. 2, the DsbC-N fusion protein is highly expressed in the prokaryotic system, the fusion protein accounts for more than 30% of the total protein of the bacterial cells, and after ultrasonic disruption, the DsbC-N fusion protein is found to exist in ultrasonic precipitation mainly in the form of inclusion bodies, wherein about 30% of the DsbC-N fusion protein exists in the form of supernatant, and the expression of the supernatant of the protein often represents the natural state of the protein, so that the supernatant after ultrasonic disruption is collected in a subsequent step for affinity purification.

2.4 Affinity purification of DsbC-N fusion proteins

The N end of DsbC-N fusion protein is provided with a 6 XHIS label, the expressed ultrasonic supernatant protein is purified by utilizing affinity chromatography, centrifugal supernatant is taken to pass through a nickel ion affinity column at the speed of 2 mL/min at the temperature of 4 ℃, after 4 column volumes are balanced by 1 XPBS, 3 column volumes of hybrid protein are eluted by 100 mmoL imidazole, the target fusion protein is eluted by 200 mmoL imidazole, and the target protein is collected.

The purified fusion protein was subjected to purity measurement by 15% SDS-PAGE, and the results are shown in FIG. 3, wherein M is a low molecular weight protein standard; 1. 2, 3 are purified DsbC-N fusion proteins.

As can be seen from FIG. 3, after affinity purification, the purity of the fusion protein can reach over 92%, and the high purity of the fusion protein is ensured by carrying out subsequent serological experiments to obtain high specificity.

Further detection, the obtained fusion protein has a relative molecular weight of 68×10 ³ 。

2.5 ELISA experiment of serum antibody of DsbC-N fusion protein

After the purified DsbC-N fusion protein is fully dialyzed by water, the concentration is measured by adopting a BCA method, the recombinant protein is diluted to 15 mug/mL by coating liquid, 100 mug of each hole of an ELISA plate is coated, the ELISA plate is placed for 2 hours at room temperature, and the PBST buffer solution is washed for 4 times; 10% calf serum is sealed overnight in a refrigerator at 4 ℃ and washed with PBST for 5 times the next day, the serum of a sample to be tested and the serum of a healthy person are diluted by 1:50 times, 100 mu L of the serum of each hole is added, the mixture is placed in a 37 ℃ incubator to be incubated for 1 h, the washed plates are dried by beating, the goat anti-human IgG marked by HRP diluted by 1:1000 times is incubated for 1 h at 37 ℃, the PBST is washed for 5 times, TMB is developed, and the enzyme-labeled instrument detects the A value at the wavelength of A450 nm, and the detection result is shown in figure 4.

And (3) comparing and detecting 30 positive serum subjected to nucleic acid detection and 50 healthy serum to be detected by using a novel coronavirus IgG antibody detection kit of a control sample, wherein the experimental method is strictly carried out according to the specification of the kit. The result shows that 1 example of false negative appears, and the detection rate is 96%; 2 cases with false positives occurred with a specificity of 96%.

As can be seen from FIG. 4, the A450 values in ELISA experiments were significantly different from the positive serum of SARS-CoV-2 infected person compared to the serum of healthy person. Wherein 30 cases of the kit show that the number of the new coronatine seropositive detection cases is 29 cases, the detection rate is 96%, and the coincidence rate with the existing IgG detection kit of the control sample is 100%; the serum of 50 healthy people is detected for 1 case by contrast, the detection rate is 2%, and the specificity is 98%. The present example sets the cut-off value to be +3 standard deviations from the average A value of serum detection for healthy persons.

Among 30 positive samples, one missed sample is assumed that the patient from the sample may be in a 'window period', and although the nucleic acid detection is positive, the virus antibodies in the blood of the patient cannot be detected, or the serum is accumulated or degraded in the long-term preservation process, and is not captured by the antigen.

The fusion protein of soluble SARS-CoV-2N protein is used in serodiagnosis, and has high sensitivity and high specificity.

2.6 Kit for preparing novel coronavirus detection based on DsbC-N fusion protein

Kit products were prepared in the amounts specified in table 1.

Table 1 specification and quantity of the constituent components of the kit

Composition of components	Number of specifications
		Antigen ELISA plate	8 x 12 wells, 1 plate
Human serum with positive antibody titer, stabilizer and antiseptic	20 U/mL,0.1 mL/branch, 1 branch
		Positive antibody titer enzyme-labeled reagent	15 mL/bottle, 1 bottle
Positive antibody titer sample diluent	50 mL/bottle, 1 bottle
		Color reagent A liquid	6 mL/bottle, 1 bottle
Color reagent B liquid	6 mL/bottle, 1 bottle
		Concentrated washing liquid (20 x)	50 mL/bottle, 1 bottle
Stop solution	6 mL/bottle, 1 bottle
		Sealing plate film	3 pieces of
Negative control	0.5 mL x 1 bottle

2.6.1 Principle of testing a kit

The kit quantitatively detects novel coronavirus (SARS-CoV-2) IgG antibody in human serum or plasma by using an indirect method principle (ELISA), pre-coating novel coronavirus DsbC-N fusion protein antigen combined with sample related antibody on an enzyme-labeled strip, adding a sample to be detected for incubation, combining the IgG antibody in the sample, washing a plate to remove substances which are not combined with the coating antigen, adding an enzyme-labeled reagent for secondary incubation, when the novel coronavirus (SARS-CoV-2) IgG antibody exists in the sample, forming a complex of coating antigen-IgG antibody anti-human IgG enzyme-linked matter, washing the plate again, adding a color developing agent, reacting with HRP catalyst connected to the complex to generate a blue product, turning yellow after stopping the reaction, and not developing color if the sample does not contain the SARS-CoV-2 virus IgG antibody. The OD value is measured on an enzyme-labeled instrument or an enzyme-linked immunosorbent assay system, and the presence or absence of the novel coronavirus (SARS-CoV-2) IgG antibody is determined according to the OD value and the OD value.

2.6.2 Storage conditions and expiration date of the kit

The kit is stored at 2-8deg.C, and has a shelf life of 1 year. The kit was equilibrated to room temperature (about 30 min) prior to use. The liquid reagent is gently vibrated and evenly mixed before the experiment, and the liquid reagent is immediately sealed and put back to 2-8 ℃ for preservation after use. The unused ELISA plate strips are sealed with a desiccant by a self-sealing bag for storage at 2-8 ℃.

2.6.3 Suitable instrument for kit

Sample feeder, incubator, plate washer and enzyme label instrument with wavelength 450nm and 490 nm.

2.6.4 Sample requirement of kit

Sample type is human serum and plasma.

Sample collection the collection and testing of a patient's blood sample must be performed in accordance with the national health committee (third edition, the new laboratory guidelines for the detection of coronavirus infection for pneumonia). Sample preservation, namely, sample is timely separated for detection after blood sample collection, and if the sample preservation cannot be timely detected, the sample preservation is required to be executed according to the requirements of the national health committee (the third edition of the laboratory detection technical guidelines for novel coronavirus infection).

Sample safety-all samples are considered potentially infectious items, performed strictly according to national relevant standards and guidelines.

Before use, the sample is balanced for more than 30 minutes at room temperature, and before a frozen sample experiment, the sample is required to be thawed and mixed uniformly.

2.6.5 Method for testing kit

Reagent preparation all reagents should be equilibrated at room temperature (10-30 ℃) for 30min before use, confirming that the surface moisture was dry before use.

The coating plate can be directly used. The overwrap aluminum profile must be opened after the coated panel has equilibrated to room temperature to prevent the panels from absorbing water vapor in the air. The remaining strips were immediately replaced with a desiccant-containing aluminum bag (or plastic bag) and sealed.

The negative and positive control can be directly used, and are fully and uniformly mixed before use.

The enzyme working solution can be directly used and fully and uniformly mixed before use. The washing liquid is concentrated by 20 times, the required amount is sucked from the bottle by a clean suction pipe, and the washing liquid is diluted into washing liquid for standby by purified water in a ratio of 1:19. For example, the ImL concentrated washing solution is diluted with 19mL of purified water.

Buffer after dilution: the composition can be stable at 2-8deg.C for one week at most. If crystallization occurs in the 20-time concentrated washing solution, the washing solution is heated to 379 ℃ before dilution, and is fully dissolved and uniformly mixed.

The intermediate substrate solutions A, B can be used as such. Because the substrate liquid is sensitive to light, the bottle cap should be covered immediately after use and stored in a dark place as much as possible. The stopping solution can be directly used.

The sealing glue is directly used. The sealing glue is used only once to avoid cross contamination.

2.6.6 Detection program

1. Preparing liquid: the concentrated wash was diluted 20-fold with distilled or deionized water.

2. Numbering: the samples are numbered corresponding to the ELISA plates in sequence, and each plate is provided with a reference sample hole and a blank control hole (a blank control hole can be omitted in dual-wavelength detection).

3. Positive standard, sample dilution: 20 times, 40 times, 80 times, 160 times, 320 times dilution.

4. Sample adding: at least two wells are parallel (the sample to be tested can also be tested by gradient dilution) after dilution of 100 μl/well.

5. Washing the plate by hand, namely adding 300uL of washing liquid into each hole, standing for 5-10 seconds, discarding, repeatedly washing for 5 times, and beating to dry; the plate washer is operated by adding 300-350uL of washing liquid into each hole, washing for 5.10 seconds each time, repeatedly washing for 5 times, and then beating to dry.

6. The enzyme working solution is added to each well for incubation, 100uL of the enzyme working solution is added, sealing glue is attached, and the mixture is incubated for 20 minutes at 37 ℃.

7. Washing the plate by hand, namely adding 300uL of washing liquid into each hole, standing for 5-10 seconds, discarding, repeatedly washing for 5 times, and beating to dry; the plate washer is operated by adding 300-350uL of washing liquid into each hole, washing for 5.10 seconds each time, repeatedly washing for 5 times, and then beating to dry.

8. And (3) performing color reaction, namely adding substrate solution A50 uL and substrate solution BS50uL into each hole, tapping and mixing uniformly, and placing at 37 ℃ to perform color development for 10 minutes in a dark place.

9. Terminating the reaction, adding a terminating solution 5OuL into each hole after the color development is finished, and tapping and mixing

10. The results were read, and after termination of the reaction, the results were measured within 10 minutes, and after placing the results on an ELISA apparatus at 450nm and 490nm, the A value of each well was measured.

And (3) quality control, wherein the OD value of positive control is more than 0.50, the OD value of negative control is S0.10, and otherwise, the test result is regarded as invalid.

2.6.7 Result determination

The OD value of the detected sample is greater than the critical value, and the detected sample should be judged as positive for the novel coronavirus (SARS-CoV-2) lgG antibody, and if the OD value is less than the critical value, the detected sample is judged as negative, the detection is recommended to be re-carried out near the critical value, and if the OD value is greater than the critical value, the detected sample should be judged as positive, otherwise, the detected sample should be judged as negative. For the sample with weak positive, other methods are applied to detect to exclude false positive.

[ Positive judgment value ]

Critical value (cutoff value) calculation:

cut off value = 0.10+ negative control OD mean

(negative control OD mean, e.g., less than 0.05 calculated as 0.05)

The index detection should be negative, if positive, the infection may be a novel coronavirus (SARS-CoV-2) infection, and should be confirmed in combination with clinical symptoms and other diagnostic methods.

2.6.8 Interpretation of test results

The SARS-CoV-2 antibody content of standard serum (0.63U/ml-20.00U/ml) and the corresponding absorbance A value are used as curves, a four-parameter regression equation is obtained, the absorbance A value of the sample to be tested is substituted into the regression equation, and then the dilution coefficient of the sample to be tested is multiplied to obtain the SARS-CoV-2-IgG titer (U/ml) of the sample to be tested. 1U/ml corresponds to 1IU/ml.

Examples: the SARS-CoV-2-IgG content is used as independent variable (X), the absorbance corresponding to the SARS-CoV-2-IgG content is used as dependent variable (Y), and the four-parameter regression equation is calculated as follows: y= -2.321/(1+ (x/8.325) 1.069) +2.322.

The data are shown in table 2.

Table 2 four parameter regression equation data

anti-SARS-CoV-2 IgG antibody positivity (U/ml)	1.563	3.125	6.25	12.50	25.00	50.00	Sample of
								Absorbance at 450nm/490nm	0.22	0.28	0.54	0.96	1.14	1.32	0.82

2.6.9 Notice matters

1. The product is only used for in vitro diagnosis, and the operation should be strictly carried out according to instructions. The sealing plate film cannot be reused, and the enzyme-labeled plates, the enzyme-labeled reagents and the reference products with different batches cannot be mixed and used with other manufacturer reagents.

2. Avoiding operation in the environment of volatile substances and hypochlorous acid disinfectant (such as 84 disinfectant).

3. Before use, the reagent is equilibrated to room temperature (30 min), the reagent is gently mixed by shaking before experiment, and the reagent is immediately returned to 2-8 ℃ after use. The unused microporous plate strip and the drying agent are sealed by a self-sealing bag for storage at the temperature of 2-8 ℃. The expiration reagent is not used.

4. The sample injector is used for adding liquid, and the accuracy of the sample injector is often checked. When different samples or different reagent components are added, the suction head and the sample adding groove of the sample adding device should be replaced so as to prevent cross contamination.

5. During washing, each hole needs to be filled with washing liquid, so that free enzyme in the hole can be prevented from being incapable of being washed. After the washing of the plate is completed, the next step must be performed immediately, and the ELISA plate cannot be dried. Long interruptions of the experimental procedure are avoided to ensure uniformity of experimental conditions per well.

6. The result determination must be based on the microplate reader reading. When the result is read, the bottom of the ELISA plate should be dried, and no bubbles can exist in the hole. Instead of touching the outer wall of the bottom of the well, fingerprints or scratches may affect the reading of the well.

7. The reference sample is inactivated, HBsAg, anti-HIV, anti-HCV and anti-TP are negative, but cannot ensure that the reference sample does not have the infectivity of microorganisms such as potential viruses, and the negative and positive control and detection samples are strictly operated according to biological safety related regulations, follow the routine regulations of laboratory operation and are used and treated according to the samples with potential biological infectivity. The samples, waste solutions and wastes used should be treated as infectious agents. The stop solution is sulfuric acid, and the safety is needed to be paid attention to when the stop solution is used.

8. When developing color, the color developing agent A liquid and the color developing agent B liquid are added firstly to avoid too low color development.

9. Each experiment needs to carry a positive standard substance, and the experimental result needs to be obtained by a current standard curve, otherwise, the error of the quantitative result may be overlarge.

10. Abnormal points in the standard curve may cause deviation of the experimental result of the whole plate, so that a double-hole detection reference substance is suggested to improve experimental accuracy.

11. When the goodness of fit of the standard curve R2 is more than or equal to 0.99, the experiment is considered to be effective, otherwise the experiment is ineffective

12. The detection method has large sample dilution multiple, so that the sample dilution affects the result. It is recommended that the sample be diluted in a gradient of not more than 5 times per step, the dilution volume not less than 0.5. 0.5 mL.

2.6.10 Limitations of the inspection method

1. According to the basic theory of antibody production after body infection, specific IgM antibody is produced earlier and has shorter duration after body infection by virus, igG antibody is produced later and has longer duration than IgM antibody. In addition, since it takes a certain period of time from the infection of a virus to the generation of a specific antibody by the organism, there is an individual difference in the intensity of the antibody, and the amount of the infectious antigen and the intensity of the antigenicity of the antigen are related. Therefore, the lgG antibody detection result, igM antibody detection result sampling time, clinical indication, appearance time and the like of the product should be comprehensively considered. Those who are positive for antibody detection should also be combined with other clinical indication comprehensive judgments.

2. The kit can only be used for the measurement of serum or plasma samples and cannot be used for other body fluid samples.

The kit prepared by the method is used for detecting novel coronavirus IgG antibodies of 30 positive serum and 50 healthy serum which are detected by nucleic acid detection, and the detection result is consistent with the ELISA experiment of the DsbC-N fusion protein serum antibody.

Example 2

1. Material

Plasmid pET-DsbA and expression strain BL21 (DE 3) are stored by the applicant; the nucleic acid vector pUC 18-SARS-CoV-2N of SARS-CoV-2N protein expression sequence is given away by Shen Chunji teacher of Beijing Wan Fang Mei lan technology company; plasmid extraction kits and DNA gel recovery kits were purchased from tenna biotechnology company; endonucleases, T4 DNA ligase, purchased from NEB company; kapa fidelity DNA polymerase and dntps are available from beijing-like micro gene corporation; the affinity resin can be used by friendship in-between biotechnology company; sheep anti-human IgG, TMB chromogenic solution was available from soribao biosystems; the novel coronavirus IgG antibody detection kit (enzyme-linked immunosorbent assay) of the control sample is Beijing Hua Daji biological technology company, and SARS-CoV-2 whole virus lysate is coated on the enzyme-labeled strip; 30 cases of clinically definite diagnosis of serum of a patient with COVID-19, 50 cases of serum of healthy people are collected by biological companies, and ELISA experiments are completed by the company commissioned; ELISA experiments were run strictly in accordance with biosafety-related regulations, following the usual regulations of laboratory procedures.

2. Method of

2.1 Preparation of SARS-CoV-2N protein nucleotide amplification primer

According to the SARS-CoV-2N protein nucleic acid sequence reported by GenBank: MN908947.3, referring to the vector pET-DsbA multiple cloning site, a specific primer aiming at N protein nucleic acid is designed and synthesized, and a stop codon TAA of escherichia coli bias is introduced at the 5-end of a downstream primer. The design sequences of the upstream and downstream primers are as follows:

The vector pUC 18-SARS-CoV-2N nucleic acid is used as a template, and under the action of heat shock TaqDNA polymerase, the SARS-CoV-2N nucleic acid is amplified by the conventional PCR method through the primers P1 and P2 under the following conditions: the pre-denaturation at 94℃for 5 min, denaturation at 98℃for 20 s, annealing at 68℃for 20 s and elongation at 72℃for 80 s were performed for 30 cycles, and elongation at 72℃for 5 min. The amplified product was checked for molecular weight by 1% agarose electrophoresis, and the detected amplified product had a molecular weight of about 1260 bp.

2.2 preparation of pET-DsbA-N protein fusion expression plasmid

And (3) carrying out gel recovery on the PCR amplified product, carrying out double enzyme digestion on the recovered product and an expression vector pET-DsbA by SalI and NheI, recovering the enzyme-digested product by 1% agarose electrophoresis gel cutting, cloning the recovered fragment into the expression plasmid pET-DsbA under the action of T4DNA ligase, converting the ligation product into an escherichia coli competent cell BL21 (DE 3), and screening positive clones for sequencing.

2.3 Prokaryotic system expression of DsbA-N fusion proteins

Inoculating the pET-DsbA-N fusion protein engineering bacteria which are sequenced correctly into LB culture medium with 50 ng/mL kanamycin concentration, placing the LB culture medium into a shaking table, shaking and culturing at 37 ℃ for activation, transferring the strain into LB culture medium of the same system according to the proportion of 1:50 in the next day, shaking and culturing at 37 ℃ until the bacterial liquid OD600 reaches 0.4 h, adding IPTG to enable the final concentration to be 0.3 mmoL, and continuing shaking and culturing at 37 ℃ for induction of 5 h. After induction, the bacterial liquid is centrifugally collected at 5000 rpm under the condition of 4 ℃, the precipitated bacterial liquid is added into 1 XPBS buffer solution for uniform mixing, ultrasonic crushing is carried out under the ice culture state, and the bacterial liquid is centrifugally collected at 12000 rpm for 30 min under the condition of 4 ℃, and the supernatant and the precipitate are collected.

The expression form of the fusion protein DsbA-N in a prokaryotic system is identified by 12% SDS-PAGE, and the detection result shows that the DsbA-N fusion protein is highly expressed in the prokaryotic system, the fusion protein accounts for more than 30% of the total protein of the bacterial body, after ultrasonic disruption, the DsbA-N fusion protein is found to exist in ultrasonic precipitation mainly in the form of inclusion bodies, wherein about 30% of the DsbA-N fusion protein exists in the form of supernatant, the expression of the supernatant of the protein often represents the natural state of the protein, and the supernatant after ultrasonic disruption is collected in a subsequent step for affinity purification.

2.4 Affinity purification of DsbA-N fusion proteins

The N end of the DsbA-N fusion protein is provided with a 6 XHIS label, the expressed ultrasonic supernatant is subjected to target protein purification by utilizing affinity chromatography, centrifugal supernatant is taken to pass through a nickel ion affinity column at the speed of 2 mL/min at the temperature of 4 ℃, after 4 column volumes are balanced by 1 XPBS, 3 column volumes of the hybrid protein are eluted by 100 mmoL imidazole, the target fusion protein is eluted by 200 mmoL imidazole, and the target protein is collected.

The purity of the purified fusion protein is measured by 15% SDS-PAGE, the purity of the fusion protein after affinity purification is more than 92%, and the high purity fusion protein is ensured by carrying out serological experiments subsequently.

2.5 ELISA experiment of serum antibody of DsbA-N fusion protein

After the purified DsbA-N fusion protein is fully dialyzed by water, the concentration is measured by adopting a BCA method, the recombinant protein is diluted to 15 mug/mL by coating liquid, 100 mug of each hole of an ELISA plate is coated, the plate is placed for 2 hours at room temperature, and the plate is washed by PBST buffer solution for 4 times; 10% calf serum is blocked overnight in a refrigerator at 4 ℃, PBST is used for washing the plate 5 times the next day, the serum of a sample to be tested and the serum of a healthy person are diluted by 1:50 times, 100 mu L of the serum of each hole is added, the sample is placed in a 37 ℃ incubator for incubation of 1 h, the plate is washed 5 times, the plate is patted dry, HRP-marked goat anti-human IgG diluted by 1:1000 times is incubated for 1 h at 37 ℃, the plate is washed by PBST for 5 times, TMB is developed, and an enzyme-labeled instrument detects the A value at the wavelength of A450 nm.

In example 2, the A450 values in ELISA experiments were significantly different from the positive serum of SARS-CoV-2 infected persons compared to the serum of healthy persons. The number of 30 cases of the seropositive detection cases of the new coronatine is 29 cases, and the detection rate is 96%; the serum of 50 healthy people is detected for 1 case by contrast, the detection rate is 2%, and the specificity is 98%. The present example sets the cut-off value to be +3 standard deviations from the average A value of serum detection for healthy persons.

2.6 Kit for preparing novel coronavirus detection based on DsbA-N fusion protein

Kit products were prepared in the amounts of the specifications shown in table 3.

TABLE 3 specification quantity of kit Components

And (3) detecting novel coronavirus IgG antibodies by using the prepared kit to detect 30 positive serum subjected to nucleic acid detection and 50 healthy person serum, wherein the detection result is consistent with the ELISA (enzyme-linked immunosorbent assay) experiment of the DsbA-N fusion protein serum antibodies.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

<110> kunming city, women and child healthcare institute; eighth medical center of general hospitals for Chinese people's liberation army

<120> kit for novel coronavirus detection based on fusion protein of nucleocapsid protein

<130> 2020

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 567

<212> DNA

<213> artificial sequence

<400> 1

gcgcagtatg aagatggtaa acagtacact accctggaaa aaccggtagc tggcgcgccg 60

caagtgctgg agtttttctc tttcttctgc ccgcactgct atcagtttga agaagttctg 120

catatttctg ataatgtgaa gaaaaaactg ccggaaggcg tgaagatgac taaataccac 180

gtcaacttca tgggtggtga cctgggcaaa gatctgactc aggcatgggc tgtggcgatg 240

gcgctgggcg tggaagacaa agtgactgtt ccgctgtttg aaggcgtaca gaaaacccag 300

accattcgtt ctgcttctga tatccgcgat gtatttatca acgcaggtat taaaggtgaa 360

gagtacgacg cggcgtggaa cagcttcgtg gtgaaatctc tggtcgctca gcaggaaaaa 420

gctgcagctg acgtgcaatt gcgtggcgtt ccggcgatgt ttgttaacgg taaatatcag 480

ctgaatccgc agggtatgga taccagcaat atggatgttt ttgttcagca gtatgctgat 540

acagtgaaat atctgtccga gaaaaaa 567

<210> 2

<211> 648

<212> DNA

<213> artificial sequence

<400> 2

gatgacgcgg caattcaaca aacgttagcc aaaatgggca tcaaaagcag cgatattcag 60

cccgcgcctg tagctggcat gaagacagtt ctgactaaca gcggcgtgtt gtacatcacc 120

gatgatggta aacatatcat tcaggggcca atgtatgacg ttagtggcac ggctccggtc 180

aatgtcacca ataagatgct gttaaagcag ttgaatgcgc ttgaaaaaga gatgatcgtt 240

tataaagcgc cgcaggaaaa acacgtcatc accgtgttta ctgatattac ctgtggttac 300

tgccacaaac tgcatgagca aatggcagac tacaacgcgc tggggatcac cgtgcgttat 360

cttgctttcc cgcgccaggg gctggacagc gatgcagaga aagaaatgaa agctatctgg 420

tgtgcgaaag ataaaaacaa agcgtttgat gatgtgatgg caggtaaaag cgtcgcacca 480

gccagttgcg acgtggatat tgccgaccat tacgcacttg gcgtccagct tggcgttagc 540

ggtactccgg cagttgtgct gagcaatggc acacttgttc cgggttacca gccgccgaaa 600

gagatgaaag aattcctcga cgaacaccaa aaaatgacca gcggtaaa 648

<210> 3

<211> 1269

<212> DNA

<213> artificial sequence

<400> 3

gtcgactctg ataatggacc ccaaaatcag cgaaatgcac cccgcattac gtttggtgga 60

ccctcagatt caactggcag taaccagaat ggagaacgca gtggggcgcg atcaaaacaa 120

cgtcggcccc aaggtttacc caataatact gcgtcttggt tcaccgctct cactcaacat 180

ggcaaggaag accttaaatt ccctcgagga caaggcgttc caattaacac caatagcagt 240

ccagatgacc aaattggcta ctaccgaaga gctaccagac gaattcgtgg tggtgacggt 300

aaaatgaaag atctcagtcc aagatggtat ttctactacc taggaactgg gccagaagct 360

ggacttccct atggtgctaa caaagacggc atcatatggg ttgcaactga gggagccttg 420

aatacaccaa aagatcacat tggcacccgc aatcctgcta acaatgctgc aatcgtgcta 480

caacttcctc aaggaacaac attgccaaaa ggcttctacg cagaagggag cagaggcggc 540

agtcaagcct cttctcgttc ctcatcacgt agtcgcaaca gttcaagaaa ttcaactcca 600

ggcagcagta ggggaacttc tcctgctaga atggctggca atggcggtga tgctgctctt 660

gctttgctgc tgcttgacag attgaaccag cttgagagca aaatgtctgg taaaggccaa 720

caacaacaag gccaaactgt cactaagaaa tctgctgctg aggcttctaa gaagcctcgg 780

caaaaacgta ctgccactaa agcatacaat gtaacacaag ctttcggcag acgtggtcca 840

gaacaaaccc aaggaaattt tggggaccag gaactaatca gacaaggaac tgattacaaa 900

cattggccgc aaattgcaca atttgccccc agcgcttcag cgttcttcgg aatgtcgcgc 960

attggcatgg aagtcacacc ttcgggaacg tggttgacct acacaggtgc catcaaattg 1020

gatgacaaag atccaaattt caaagatcaa gtcattttgc tgaataagca tattgacgca 1080

tacaaaacat tcccaccaac agagcctaaa aaggacaaaa agaagaaggc tgatgaaact 1140

caagccttac cgcagagaca gaagaaacag caaactgtga ctcttcttcc tgctgcagat 1200

ttggatgatt tctccaaaca attgcaacaa tccatgagca gtgctgactc aactcaggcc 1260

taagctagc 1269

<210> 4

<211> 418

<212> PRT

<213> artificial sequence

<400> 4

Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr Phe

1 5 10 15

Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser

20 25 30

Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr

35 40 45

Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys

50 55 60

Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp

65 70 75 80

Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly Gly

85 90 95

Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu

100 105 110

Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly

115 120 125

Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His

130 135 140

Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu

145 150 155 160

Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg

165 170 175

Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn Ser

180 185 190

Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala Arg

195 200 205

Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp

210 215 220

Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln

225 230 235 240

Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

245 250 255

Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala

260 265 270

Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln

275 280 285

Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala

290 295 300

Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly

305 310 315 320

Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile

325 330 335

Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu Leu

340 345 350

Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys

355 360 365

Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln Arg

370 375 380

Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu Asp

385 390 395 400

Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser Thr

405 410 415

Gln Ala

<210> 5

<211> 24

<212> DNA

<213> artificial sequence

<400> 5

acgcgtcgac tctgataatg gacc 24

<210> 6

<211> 30

<212> DNA

<213> artificial sequence

<400> 6

ctagctagct taggcctgag ttgagtcagc 30

<210> 7

<211> 566

<212> DNA

<213> artificial sequence

<400> 7

gcgcagtatg aagatggtaa acagtacact accctggaaa aaccggtagc tggcgcgccg 60

caagtgctgg agtttttctc tttcttcagc ccgcacagct atcagtttga agaagttctg 120

catatttctg ataatgtgaa gaaaaaactg ccggaaggcg tgaagatgac taaataccac 180

gtcaacttca tgggtggtga cctgggcaaa gatctgactc aggcatgggc tgtggcgatg 240

gcgctgggcg tggaagacaa agtgactgtt ccgctgtttg aaggcgtaca gaaaacccag 300

accattcgtt ctgcttctga tatccgcgat gtatttatca acgcaggtat taaaggtgaa 360

gagtacgacg cggcgtggaa cagcttcggg tgaaatctct ggtcgctcag caggaaaaag 420

ctgcagctga cgtgcaattg cgtggcgttc cggcgatgtt tgttaacggt aaatatcagc 480

tgaatccgca gggtatggat accagcaata tggatgtttt tgttcagcag tatgctgata 540

cagtgaaata tctgtccgag aaaaaa 566

Claims

1. A kit for novel coronavirus detection based on a nucleocapsid protein fusion protein, characterized in that the kit comprises an antigen for novel coronavirus detection, the antigen for novel coronavirus detection comprises a nucleocapsid protein-based fusion protein, and the nucleocapsid protein-based fusion protein is a fusion protein generated by fusion expression of a nucleocapsid protein nucleotide sequence and a disulfide oxidoreductase a nucleotide sequence; or, the nucleotide sequence of nucleocapsid protein and the nucleotide sequence of disulfide oxidoreductase C are fused and expressed to generate fusion protein;

the nucleotide sequence of the disulfide oxidoreductase A is shown as SEQ ID No. 1;

the nucleotide sequence of the disulfide oxidoreductase C is shown as SEQ ID No. 2;

the nucleotide sequence of the nucleocapsid protein is shown as SEQ ID No. 3.

2. The kit of claim 1, wherein the nucleotide sequence of the nucleocapsid protein is linked directly or through a linker to the C-terminus of the nucleotide sequence of disulfide oxidoreductase a; the nucleotide sequence of the nucleocapsid protein is linked directly or through a linker to the C-terminus of the nucleotide sequence of the disulfide oxidoreductase C.

3. The kit according to claim 2, wherein the kit is a colloidal gold kit, the fusion protein is coated on a gold-labeled pad after being labeled by colloidal gold, the particle size of the colloidal gold is 18-28 nm, and the coating amount of the fusion protein on the gold-labeled pad after being labeled by colloidal gold is 1.4-2.0 mu L/cm.

4. A kit according to any one of claims 1 to 3, wherein the kit is an enzyme-linked immunosorbent kit, the kit comprises an antigen elisa plate, the antigen elisa plate is an elisa plate coated with the fusion protein, the coating concentration of the fusion protein on the elisa plate is 10 μg/mL to 20 μg/mL, and the coating volume is 80 μl to 120 μl.

5. The kit of claim 4, wherein the kit comprises the following components:

6. The kit of claim 5, wherein the kit comprises the following components in the number of specifications:

Antigen elisa plate 8 x 12 wells, 1 plate;

15 mL/bottle of positive antibody titer enzyme-labeled reagent, 1 bottle;

50 mL/bottle of positive antibody titer sample diluent, 1 bottle;

6 mL/bottle of developer A liquid and 1 bottle;

6 mL/bottle of color reagent B solution and 1 bottle;

concentrating 20 x 50mL of washing liquid per bottle, 1 bottle;

stop solution 6 mL/bottle, 1 bottle;

3 sealing plate films;

negative control 0.5 mL. Times.1 bottle.

7. The kit according to claim 6, wherein,

the positive antibody titer enzyme-labeled reagent is an anti-human IgG antibody marked by horseradish peroxidase;

the positive antibody titer human serum is marked anti-novel coronavirus-containing human serum;

The color reagent A liquid contains peroxide not less than 0.3 g/L;

the color reagent B liquid contains TMB not less than 0.2 g/L;

the termination solution contains sulfuric acid, and the concentration of the sulfuric acid is not higher than 2.0 mol/L.

8. The kit according to claim 4, wherein the antigen ELISA plate is prepared by coating an aqueous solution of a fusion protein on an ELISA plate, standing at room temperature for 1.5-2.5 h, and washing the plate with a PBST buffer solution; and sealing with 10% calf serum at 4 ℃ for 10-15 hours, and washing the plate with PBST buffer solution to obtain the antigen ELISA plate.