CN112505330A

CN112505330A - Novel coronavirus detection kit based on fusion protein of nucleocapsid protein

Info

Publication number: CN112505330A
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: 2021-03-16
Anticipated expiration: 2040-11-09
Also published as: CN112505330B

Abstract

The invention provides a kit for detecting a novel coronavirus based on a fusion protein of a nucleocapsid protein, which comprises an antigen for detecting the novel coronavirus, wherein the antigen for detecting the novel coronavirus comprises the fusion protein based on the nucleocapsid protein. Above-mentioned kit that novel coronavirus detected based on fusion protein of nucleocapsid protein obtains the fusion protein that contains the nucleocapsid protein of novel coronavirus through the fusion technique, makes the fusion protein have the natural spatial structure characteristic of novel coronavirus nucleocapsid protein, and sensitivity is high, the specificity is strong when being used for novel coronavirus to detect, utilizes this fusion protein based on nucleocapsid protein to carry out novel coronavirus and detects the relevance ratio that can effectively improve novel coronavirus, ensures the timely definite diagnosis of novel coronavirus pneumonia, can effectively avoid the diffusion of epidemic situation.

Description

Novel coronavirus detection kit based on fusion protein of nucleocapsid protein

Technical Field

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

Background

The new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is a new infectious Disease mainly caused by pulmonary diseases due to infection of a new coronavirus (SARS-CoV-2, also known as 2019-nCoV), and can cause damage to the digestive system and nervous system, and death of severe patients can be caused. At present, no specific therapeutic drug exists for 2019-nCoV, the vaccine is also in a clinical test stage, and early diagnosis, timely receiving and treatment and isolation of patients are crucial to effective control of epidemic situations. The large-scale investigation of the infection of the new coronavirus mainly adopts nucleic acid detection, which becomes a gold standard for confirmation of diagnosis of suspected cases due to 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 aerosol pollution. The traditional antigen-antibody reaction is an important supplement of clinical laboratory detection, and the utilization of antigen to detect antibodies in serum of infected persons is an important means for rapid screening and nucleic acid aided diagnosis. At present, many companies have developed detection kits for clinical diagnosis of COVID-19 by serological diagnostic methods, such as colloidal gold method and traditional ELISA method, and screening conserved antigen or combined antigen with dominant epitope in SARS-CoV-2 is the key for the success of serological diagnosis. The SARS-CoV-2 gene is about 29.8kb in size, and the genome is annotated to contain 14 Open Reading Frames (ORFs), which encode 27-28 proteins in total. The nucleocapsid protein (N protein) is a main structural protein of SARS-CoV-2, the sequence table is shown in SEQ ID No.4, it is located in the interior of virus, it is the antagonist of Interferon (Interferon, IFN) and virus-coded RNA interference inhibiting factor, it is related to the replication of virus, the N protein is relatively conserved among beta coronavirus, the synthetic quantity is numerous, it has very strong antigenicity, it plays an important role in inducing host immune response and even pathogenesis, it is often used as the antigen site for coronavirus diagnosis. However, the existing method for detecting SARS-CoV-2 based on N protein as antigen has low detection rate, and influences the diagnosis efficiency of COVID-19 and the control of epidemic situation.

Disclosure of Invention

Therefore, it is necessary to provide a novel coronavirus detection kit 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 the epidemic situation are influenced.

The kit 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.

In one embodiment thereof, the nucleocapsid protein-based fusion protein is a fusion protein produced by fusion expression of the amino acid sequence or a partial amino acid sequence of the nucleocapsid protein with the amino acid sequence or a partial amino acid sequence of disulfide oxidoreductase a; or, the amino acid sequence or partial amino acid sequence of the nucleocapsid protein is fused and expressed with the amino acid sequence or partial amino acid sequence of the 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 mutated sequence of the amino acid sequence shown as SEQ ID No. 1;

the amino acid sequence of the disulfide oxidoreductase C is the amino acid sequence shown as SEQ ID No.2 or the 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 mutant sequence of the amino acid sequence shown as SEQ ID No. 3.

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

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

In one embodiment, the kit is an enzyme-linked immunosorbent assay 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 10-20 mu g/mL, and the coating volume is 80-120 mu L.

In one embodiment, the kit comprises the following components:

the kit comprises an antigen ELISA plate, positive antibody titer human serum, a positive antibody titer enzyme labeling reagent, a positive antibody titer sample diluent, a color development agent A liquid, a color development agent B liquid, a concentrated washing liquid, a stop solution, a sealing plate membrane and a negative control product.

In one embodiment, the kit comprises the following components in the specification and quantity:

in one embodiment, the positive antibody titer enzyme-labeled reagent is an anti-human IgG antibody labeled with horseradish peroxidase;

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 the aqueous solution of the fusion protein on the ELISA plate, placing the ELISA plate at room temperature for 1.5-2.5 h, and washing the plate by using PBST buffer solution; and (3) sealing the plate with 10% calf serum at 4 ℃ for 10-15 h, and washing the plate with PBST buffer solution to obtain the antigen ELISA plate.

Above-mentioned kit that novel coronavirus detected based on fusion protein of nucleocapsid protein obtains the fusion protein that contains the nucleocapsid protein of novel coronavirus through the fusion technique, makes the fusion protein have the natural spatial structure characteristic of novel coronavirus nucleocapsid protein, and sensitivity is high, the specificity is strong when being used for novel coronavirus to detect, utilizes this fusion protein based on nucleocapsid protein to carry out novel coronavirus and detects the relevance ratio that can effectively improve novel coronavirus, ensures the timely definite diagnosis of novel coronavirus pneumonia, can effectively avoid the diffusion of epidemic situation.

Furthermore, the kit for detecting the novel coronavirus based on the fusion protein of the nucleocapsid protein is characterized in that the fusion protein based on the nucleocapsid protein is soluble protein, the soluble protein can exist in a supernatant form, 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, the kit for detecting the novel coronavirus based on the fusion protein of the nucleocapsid protein performs fusion expression on the nucleocapsid protein of the novel coronavirus and disulfide oxidoreductase A or disulfide oxidoreductase C, makes full use of the biological characteristics of disulfide isomerase and molecular chaperone of the disulfide oxidoreductase C, effectively promotes the fusion protein to exist in an supernatant form in a recombinant expression process, further improves the detection rate of the novel coronavirus antibody, and lays a material foundation for a serological detection kit.

Drawings

FIG. 1 is a diagram showing the result of the nucleotide molecular weight gel electrophoresis for SARS-CoV-2N protein of example 1 of the present invention;

FIG. 2 is a diagram showing the results of the identification of expression forms of DsbC-N fusion proteins in a prokaryotic system in example 1 of the present invention;

FIG. 3 is a diagram showing the result of purity identification of the DsbC-N fusion protein after purification in example 1 of the present invention;

FIG. 4 is a diagram showing the results of the DsbC-N fusion protein of the present invention in ELISA experiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

By 8/20/2020, the number of people infected with SARS-CoV-2 in the world exceeds 2000 ten thousand, and in early detection, nucleic acid detection plays a great role, thus achieving the purposes of early diagnosis and early isolation. With the analysis of the genome sequence of the new coronavirus, the expression sequences of the structural proteins are sequenced, and the recombinant diagnostic antigen begins to play a role. When the virus infects organisms, IgM antibodies appear earliest in serum antibodies of the organisms and are diagnostic indexes of acute infection, but the IgM concentration is low, the maintenance time is only about one week, and the affinity is low; the later production of IgG antibody can indicate the middle and later period of infection or the production of infection before, and the IgG antibody has high concentration, long maintaining time and high affinity. At present, diagnostic methods for detecting SARS-CoV-2 IgM and IgG antibodies have been developed by several biological companies, and an excellent detection means is provided for the auxiliary diagnosis of novel coronavirus infection and epidemic disease 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 for COVID-19 detection, and most of the present clinical new coronavirus detection kits adopt N protein antigen or spike protein (S protein) antigen or a combined method to detect serum antibody. However, the use of N protein antigen or spike protein (S protein) antigen or a combination method to detect serum antibodies still needs to be further improved in sensitivity and specificity to improve the detection rate of SARS-CoV-2.

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

The kit 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 is used for obtaining the fusion protein containing the nucleocapsid protein of the novel coronavirus through a fusion technology, so that the fusion protein has the natural spatial structure characteristic of the nucleocapsid protein of the novel coronavirus and improves the property of the fusion protein, the kit is high in sensitivity and specificity when used for detecting the novel coronavirus, and is more suitable for detecting the novel coronavirus so as to improve the detection rate of the novel coronavirus, ensure the timely diagnosis of the novel coronavirus pneumonia, and effectively avoid the spread of epidemic situation.

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

As an alternative embodiment, the nucleocapsid protein-based fusion protein is a fusion protein produced by fusion expression of the amino acid sequence or a partial amino acid sequence of the nucleocapsid protein with the amino acid sequence or a partial amino acid sequence of disulfide oxidoreductase a (dsba); or, the amino acid sequence or partial amino acid sequence of the nucleocapsid protein is fused and expressed with 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 usually represents a correct space folding mode of the proteins and keeps the natural space structure of the proteins, in order to ensure the natural conception of the fusion and purification process of the new coronavirus nucleocapsid protein, the invention performs fusion expression on the novel coronavirus nucleocapsid protein and disulfide oxidoreductase A or disulfide oxidoreductase C, fully utilizes the biological characteristics of disulfide bond isomerase and molecular chaperone of the disulfide oxidoreductase A and the disulfide oxidoreductase C, improves the solubility of the fusion proteins, promotes the supernatant form of the fusion proteins in the process of recombinant expression, 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 in SEQ ID No.1 or a mutated sequence of the nucleotide sequence shown in SEQ ID No. 1; the nucleotide sequence of the disulfide oxidoreductase C is the nucleotide sequence shown in SEQ ID No.2 or the mutant sequence of the nucleotide sequence shown in SEQ ID No. 2; the nucleotide sequence of the 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 nucleotide sequence of the nucleocapsid protein can be mutated as required to improve the characteristics of the fusion protein after the fusion expression, wherein the selectable mutation mode comprises the addition, deletion or substitution of one or more nucleotides in the nucleotide sequence; similarly, disulfide oxidoreductase a nucleotide sequence or disulfide oxidoreductase C nucleotide sequence may be mutated as desired to improve the characteristics of the fusion protein after expression of the fusion, and alternative mutations may include addition, deletion or substitution of 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 via a linker to the C-terminus of the nucleotide sequence of disulfide oxidoreductase a; the amino acid sequence of the nucleocapsid protein is linked to the C-terminus of the amino acid sequence of disulfide oxidoreductase C, either directly or via a linker. For example, the nucleocapsid protein may be linked to disulfide oxidoreductase a or disulfide oxidoreductase C by a soft linker having the nucleotide sequence GGGGSGGGGS.

Optionally, the fusion protein based on the nucleocapsid protein is produced by expression in a prokaryotic expression system, and the preferred expression strain is escherichia coli.

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

A method for preparing an antigen for detecting a novel coronavirus comprises the following steps:

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

cutting the nucleocapsid protein gene segment from the cloning vector by using endonuclease SalI and endonuclease Nhe I;

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

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

According to the invention, a specific primer aiming at nucleocapsid protein nucleotide is designed and synthesized according to a nucleic acid sequence of a novel coronavirus nucleocapsid protein reported by GenBank: MN908947.3 and multiple cloning sites of vectors pET-DsbA and pET-DsbC, and a termination codon TAA with escherichia coli bias is introduced into 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 cutting sites GCTAG of endonuclease SalI and GCTAG of endonuclease Nhe I (bold in the sequence) are respectively introduced from the tail ends of the primer and the downstream primer so as to cut the nucleocapsid protein gene fragment from the cloning vector by the endonuclease SalI and the endonuclease Nhe I.

Optionally, the invention uses pUC18-SARS-CoV-2 nucleocapsid protein nucleotide sequence as template, under the action of heat shock TaqDNA polymerase, the SARS-CoV-2 nucleocapsid protein nucleotide sequence is amplified by upstream primer and downstream primer by conventional PCR method.

Alternatively, the amplification conditions of the PCR method are as follows:

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

After verifying the molecular weight of the PCR amplification product by 1% agarose electrophoresis, carrying out gel recovery on the PCR amplification product, carrying out double enzyme digestion on the recovered amplification product and an expression vector pET-DsbA or pET-DsbC by SalI and Nhe I, recovering the product after enzyme digestion by 1% agarose electrophoresis gel cutting, cloning and connecting the N protein gene fragment in the product (comprising the N protein gene fragment and the expression plasmid pET-DsbA or pET-DsbC) recovered by enzyme digestion under the action of T4DNA ligase to the expression plasmid pET-DsbA or pET-DsbC, transforming the cloned and connected product to an escherichia coli competent cell BL21(DE3), and screening and sequencing a positive cloned product.

Inoculating DsbA-N fusion protein or DsbC-N fusion protein engineering bacteria with correct sequencing into LB culture medium containing 50ng/ml kanamycin, placing the mixture in a shaking table, shaking for culture and activation at 37 ℃, transferring the mixture into the LB culture medium of the same system according to the proportion of 1:50 the next day, shaking for culture at 37 ℃ until the OD600 of the bacterial liquid reaches 0.4h, adding IPTG to enable the final concentration to be 0.3mmoL, and continuing shaking for culture and induction at 37 ℃ for 5 h. And (3) centrifugally collecting thalli from the induced bacterial liquid at the temperature of 4 ℃ and at the rpm of 5000, adding 1 XPBS buffer solution into the precipitated thalli, uniformly mixing, carrying out ultrasonic disruption in an ice breeding state, carrying out centrifugation at the speed of 12000rpm for 30min at the temperature of 4 ℃, collecting supernatant and precipitate, and identifying the expression form of the DsbA-N fusion protein or the DsbC-N fusion protein in a prokaryotic system by 12% SDS-PAGE.

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

affinity purification and/or gel purification of the nucleocapsid protein-based fusion protein.

For example, the DsbA-N fusion protein and the DsbC-N fusion protein have 6 XHIS tags at the N-terminus, and the collected supernatant and precipitate generated after fusion expression can be subjected to affinity chromatography using a chelating sepharose, which is convenient for purification and has high purity of the purified fusion protein.

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

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

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

As an optional implementation mode, the kit is an enzyme-linked immunosorbent kit, and comprises an antigen ELISA plate, wherein the antigen ELISA plate is an ELISA plate coated with fusion protein, the coating concentration of the fusion protein on the ELISA plate is 10-20 mu g/mL, and the coating volume is 80-120 mu L. Preferably, the concentration of the fusion protein coated 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 amounts of the components of the kit are as follows:

optionally, the positive antibody titer enzyme-labeled reagent is an anti-human IgG antibody labeled by horseradish peroxidase; 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; concentrating the washing solution into an aqueous solution containing 10% of surfactant; the developer A contains peroxide not less than 0.3 g/L; the developer 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).

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

Example 1

1. Material

The plasmid pET-DsbC, the expression strain BL21(DE3) was preserved by the applicant; SARS-CoV-2N protein expression sequence nucleic acid vector pUC18-SARS-CoV-2N was offered by Shenchu Qi teacher of Meilan science and technology company of Beijing Wan; the plasmid extraction kit and the DNA gel recovery kit are purchased from Tiangen Biotech company; endonuclease, T4DNA ligase, available from NEB; the kappa fidelity DNA polymerase and dNTPs are available from Beijing GenBank; affinity resins are available from friendship, midrange biotechnology; goat anti-human IgG and TMB developing solution is available from Solebao biology company; the novel coronavirus IgG antibody detection kit (enzyme-linked immunosorbent assay) of the control sample is a Beijing Hua Dagibibiai biotechnology company, and SARS-CoV-2 complete virus lysate is coated on an enzyme label lath; 30 clinical confirmed COVID-19 patient sera, 50 healthy human sera were collected by the biological company, and completed by the ELISA test entrustment company; ELISA experiments were performed strictly in accordance with biosafety-related regulations, following the routine rules of laboratory procedures.

2. Method of producing a composite material

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 and referring to the carrier pET-DsbC multiple cloning site, the specific primer aiming at the N protein nucleic acid is designed and synthesized, and the 5 end of the downstream primer is introduced with the escherichia coli biased termination codon TAA. The upstream and downstream primer design sequences are as follows:

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

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

The ends of the upstream and downstream primers are respectively introduced with SalI and Nhe I cutting sites (indicated by oblique lines).

The vector pUC18-SARS-CoV-2N nucleic acid is used as template, under the action of heat shock TaqDNA polymerase, SARS-CoV-2N nucleic acid is amplified by conventional PCR method through primers P1 and P2, and the conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 98 ℃ for 20s, annealing at 68 ℃ for 20s, and extension at 72 ℃ for 80s for 30 cycles, and extension at 72 ℃ for 5 min. The molecular weight of the amplified product is verified by 1% agarose electrophoresis, and the result is shown in FIG. 1, wherein, the serial number 1 is the nucleic acid sequence of SARS-CoV-2N protein of the amplified product of the invention; m is DL 2000. Clear and single bands exist at the target molecular weight of about 1260bp, the SARS-CoV-2N protein has the total length of 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 amplification product, carrying out double enzyme digestion on the recovered product and an expression vector pET-DsbC by Sal I and Nhe I, carrying out gel cutting through 1% agarose electrophoresis to recover the product after enzyme digestion, cloning the recovered fragment into an expression plasmid pET-DsbC under the action of T4DNA ligase, transforming the ligation product into an escherichia coli competent cell BL21(DE3), 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 with correct sequencing into an LB culture medium with kanamycin concentration of 50ng/mL, placing the LB culture medium in a shaking table to be shake-cultured and activated at 37 ℃, transferring the engineering bacteria into the LB culture medium of the same system according to the proportion of 1:50 the next day, carrying out shake culture at 37 ℃ until the OD600 of the bacterial liquid reaches 0.4h, adding IPTG to enable the final concentration to be 0.3mmoL, and continuing shake culture and induction for 5h at 37 ℃. Centrifuging the induced bacteria liquid at 5000rpm at 4 deg.C to collect thallus, adding 1 × PBS buffer solution into the precipitated thallus, mixing, performing ultrasonication under ice culture state, centrifuging at 12000rpm at 4 deg.C for 30min, and collecting supernatant and precipitate.

The expression form of the fusion protein DsbC-N in a prokaryotic system is identified by 12 percent SDS-PAGE, and the result is shown in figure 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 the ultrasonic disruption of the expression thallus; 4 is the sediment after the expression thallus is subjected to ultrasonic disruption.

As can be seen from FIG. 2, the DsbC-N fusion protein has high expression in prokaryotic system, the fusion protein accounts for more than 30% of the total protein of the thallus, after the ultrasonic disruption, the DsbC-N fusion protein mainly exists in the ultrasonic precipitation in the form of inclusion body, wherein about 30% of the DsbC-N fusion protein exists in the form of supernatant, the expression of the supernatant of the protein usually represents the natural state of the protein, so the supernatant after the ultrasonic disruption is collected in the subsequent steps for preparing affinity purification.

Affinity purification of DsbC-N fusion proteins

Carrying 6 XHIS label at the N end of the DsbC-N fusion protein, purifying the expressed ultrasonic supernatant protein by affinity chromatography, taking the centrifugal supernatant at 4 ℃, passing through a nickel ion affinity column at the speed of 2mL/min, balancing 4 column volumes by 1 XPBS, eluting the hybrid protein by 100mmoL imidazole for 3 column volumes, eluting the target fusion protein by 200mmoL imidazole, and collecting the target protein.

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

As can be seen from FIG. 3, the purity of the fusion protein can reach more than 92% after affinity purification, and the high-purity fusion protein is a guarantee for obtaining high specificity by carrying out a serology experiment subsequently.

Further detecting, the obtained fusion protein has a relative molecular weight of 68 × 10³。

2.5DsbC-N fusion protein serum antibody ELISA experiment

After the purified DsbC-N fusion protein is fully dialyzed by water, the concentration is determined by adopting a BCA method, the recombinant protein is diluted to 15 mu g/mL by using coating liquid, each hole of an ELISA plate is coated with 100 mu L, the solution is placed for 2h at room temperature, and the plate is washed by PBST buffer solution for 4 times; sealing 10% calf serum in a refrigerator at 4 ℃ overnight, washing the plate with PBST for 5 times the next day, diluting the serum of the sample to be detected and the serum of the healthy human at a ratio of 1:50, adding 100 mu L of the serum into each hole, incubating the diluted sample in an incubator at 37 ℃ for 1h, washing the plate for 5 times, drying the plate, incubating the diluted HRP-labeled goat anti-human IgG at the ratio of 1:1000 for 1h at 37 ℃, washing the plate with PBST for 5 times, developing TMB, detecting the A value at the wavelength of A450nm by an enzyme-labeling instrument, and detecting the result as shown in figure 4.

30 parts of positive serum subjected to nucleic acid detection and 50 parts of serum of healthy people to be detected are contrastively detected by using a contrast sample novel coronavirus IgG antibody detection kit, and the experimental method is strictly carried out according to the kit specification. The result shows that 1 case of false negative occurs, and the detection rate is 96 percent; false positives occurred in 2 cases with a specificity of 96%.

As can be seen from FIG. 4, the positive serum of SARS-CoV-2 infected persons has significant difference in A450 value in ELISA test compared with the healthy human serum. 30 of the samples define 29 positive serum detection cases of the new coronary pneumonia, the detection rate is 96%, and the coincidence rate with the existing IgG detection kit of a control sample is 100%; 1 sample of 50 healthy human control sera was detected, the detection rate was 2%, and the specificity was 98%. In this example, cut-off is set as the mean A value +3 times standard deviation of the human serum test of healthy individuals.

Among them, 30 positive samples were missed, and it was presumed that the patient from whom the sample was derived might be in the "window stage", and although nucleic acid detection was positive, viral antibodies in blood could not be detected in the blood of the patient, or serum was accumulated or degraded during long-term storage and not captured by antigen.

The fusion protein of soluble SARS-CoV-2N protein obtained by the invention is used for serological diagnosis, and has strong sensitivity and high specificity.

2.6 preparation of novel coronavirus detection kit based on DsbC-N fusion protein

Kit products were prepared in the specification amounts as shown in table 1.

TABLE 1 Specification amounts of the kit Components

Composition of matter	Number of specifications
		Antigen enzyme label plate	8 x 12 wells, 1 plate
Positive antibody titer human serum, added with stabilizer and antiseptic	20U/mL, 0.1 mL/piece, 1 piece
		Enzyme labeling reagent with positive antibody titer	15 mL/bottle, 1 bottle
Positive antibody titerSample diluent	50 mL/bottle, 1 bottle
		Color developing agent A liquid	6 mL/bottle, 1 bottle
Developer B liquid	6 mL/bottle, 1 bottle
		Concentrated washing solution (20X)	50 mL/bottle, 1 bottle
Stopping liquid	6 mL/bottle, 1 bottle
		Sealing plate film	3 are provided with
Negative control	0.5mL × 1 bottle

2.6.1 test principle of kit

The kit quantitatively detects a novel coronavirus (SARS-CoV-2) IgG antibody in human serum or plasma by applying an indirect method principle (ELISA), pre-coats a novel coronavirus DsbC-N fusion protein antigen combined with a sample related antibody on an enzyme-labeled lath, adds a sample to be detected for incubation, combines the IgG antibody in the sample with the IgG antibody, washes a plate to remove substances which are not combined with a coating antigen, adds an enzyme-labeled reagent for secondary incubation, forms a coating antigen-IgG antibody antihuman IgG enzyme-linked complex when the novel coronavirus (SARS-CoV-2) IgG antibody exists in the sample, adds a color developing agent after washing the plate again, catalyzes a reaction by HRP connected on the complex to generate a blue product, and turns yellow after terminating the reaction; no color was developed if no SARS-CoV-2 virus IgG antibody was present in the sample. The OD value is measured on a microplate reader or an enzyme immunoassay system, and the presence or absence and the OD value are determined based on the OD value, thereby measuring the content of the novel coronavirus (SARS-CoV-2) IgG antibody.

2.6.2 storage conditions and Effect of the kit

The kit is stored at 2-8 ℃ and has the validity period of 1 year. The kit was equilibrated to room temperature (about 30min) prior to use. The liquid reagent is gently shaken and uniformly mixed before the experiment, and is immediately sealed and placed back to 2-8 ℃ for storage after use. The unused enzyme label plate bar is sealed by a self-sealing bag together with a drying agent and stored at the temperature of 2-8 ℃.

2.6.3 applicable instrument for reagent kit

Sample injector, incubator, plate washer, enzyme-linked analyzer with wavelength of 450nm and 490 nm.

2.6.4 sample requirements of the kit

The sample types are human serum and blood plasma.

Sample collection the process of collecting and testing the patient's blood samples must be performed according to the guidelines for laboratory testing of pneumonia of new coronavirus infection (third edition) issued by the national health and wellness agency. Sample storage, namely separating a sample in time for detection after blood sample collection, wherein if the sample storage cannot be detected in time, the sample storage is executed according to the requirements of national health commission (technical guidelines for laboratory detection of pneumonia infected by novel coronavirus (third edition)).

Sample safety all samples were considered potentially infectious items and were performed strictly in accordance with national relevant standards and guidelines.

Before use, the sample is balanced for more than 30min at room temperature, and is unfrozen and uniformly mixed before the sample is frozen for experiment.

2.6.5 test method of kit

Preparation of reagents all reagents should be equilibrated at room temperature (10-30 ℃) for 30min before use, and it should be confirmed that the surface moisture had been dried out before use.

The coated plate can be directly used. The aluminum foil bags must be opened after the coated plates have equilibrated to room temperature to prevent the strips from absorbing water vapor from the air. Please replace the remaining slats immediately into the desiccant containing aluminum bag (or plastic bag) and seal.

Negative and positive controls can be used directly and mixed well before use.

The enzyme working solution can be directly used and is fully and uniformly mixed before use. And (3) concentrating the washing solution by 20 times, sucking a required amount of washing solution from the bottle by using a clean straw, and diluting the washing solution into the washing solution by using purified water at a ratio of 1:19 for later use. For example, the concentrated solution of ImL was diluted with 19mL of purified water.

Buffer after dilution: can be stabilized at 2-8 deg.C for at most one week. If 20 times of concentrated washing solution appears crystallization, heating to 379C before dilution, fully dissolving and mixing uniformly.

The medium substrate liquid A and the medium substrate liquid B can be directly used. Because the substrate liquid is sensitive to light, the bottle cap is covered immediately after use and the substrate liquid is stored in the dark as much as possible. The stop solution can be used directly.

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

2.6.6 test procedure

1. Preparing liquid: the concentrated washing solution was diluted 20 times with distilled or deionized water.

2. Numbering: the samples are numbered in sequence corresponding to the ELISA plates, and each plate is provided with a reference substance hole and a blank control 1 hole (the blank control hole can be omitted in the dual-wavelength detection).

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

4. Sample adding: after dilution, the sample is 100 mu l/hole, and at least two holes are parallel (the sample to be detected can also be detected by gradient dilution).

5. Washing the plate by hand, namely adding 300uL of washing liquid into each hole, standing for 5-10 seconds, discarding the washing liquid, repeatedly washing for 5 times, and patting the plate dry; and (4) operating the plate washing machine, namely adding 300-350uL washing liquid into each hole, washing at an interval of 5.10 seconds for each time, repeatedly washing for 5 times, and then beating to dry.

6. Adding enzyme working solution to incubate, adding 100uL of enzyme working solution to each well, sticking sealing glue, and incubating at 37 ℃ for 20 minutes.

7. Washing the plate by hand, namely adding 300uL of washing liquid into each hole, standing for 5-10 seconds, discarding the washing liquid, repeatedly washing for 5 times, and patting the plate dry; the plate washer is operated, 300-350uL. washing solution is added into each hole, the washing interval is 5.10 seconds for each time, and the plate washer is dried after washing is repeated for 5 times.

8. And (3) performing color reaction, adding the substrate solution A50 uL and the substrate solution BS50uL into each hole, patting the mixture evenly, and placing the mixture in 37C for 10 minutes in a dark place for color development.

9. Stopping reaction, after color development is finished, adding stop solution 5OuL into each hole, and lightly beating and mixing

10. The results were read, and after the reaction was terminated, the results were measured within 10 minutes, and the A value of each well was measured after the well was set at a wavelength of 490nm at 450nm in a microplate reader.

Quality control, each test should satisfy positive control OD value >0.50, negative control OD value S0.10; otherwise, the test result is regarded as invalid.

2.6.7 determination of results

When the OD value of the sample is larger than the critical value, the sample is judged to be positive by the novel coronavirus (SARS-CoV-2) lgG antibody, and is judged to be negative by being smaller than the critical value, the re-detection is recommended near the critical value, and if the sample is positive by the re-detection, the sample is judged to be positive, otherwise, the sample is judged to be negative. And (4) applying other methods to detect the sample with weak positive so as to eliminate false positive.

[ Positive judgment value ]

Critical value (cutoff value) calculation:

cut off value of 0.10+ negative control OD mean value

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

The detection of the index in normal population should be negative, if positive, it may be the infection of new type coronavirus (SARS-CoV-2), and should be confirmed by combining clinical symptoms and other diagnostic methods.

2.6.8 interpretation of test results

Taking the SARS-CoV-2 antibody content of the standard serum (0.63U/ml-20.00U/ml) and the corresponding absorbance A value as a curve, solving a four-parameter regression equation, substituting the absorbance A value of the sample to be detected into the regression equation, and multiplying the value by the dilution coefficient of the sample to be detected to obtain the anti-SARS-CoV-2-IgG titer (U/ml) of the sample to be detected. 1U/ml corresponds to 1 IU/ml.

Example (c): taking the content of SARS-CoV-2-IgG as independent variable (X) and the corresponding absorbance as dependent variable (Y), solving a four-parameter regression equation 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

2.6.9 notes

1. The product is only used for in vitro diagnosis, and the operation should be strictly carried out according to the instruction. The sealing plate membrane can not be reused, and the enzyme labeling plates with different batch numbers, the enzyme labeling reagent and the reference substance can not be used in a mixed way and can not be used in a mixed way with reagents of other manufacturers.

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

3. Before use, the reagent is balanced to room temperature (for 30min), the reagent is gently shaken and uniformly mixed before the experiment, and the mixture is immediately placed back to 2-8 ℃. And sealing the unused microporous plate strips and the drying agent together by using a self-sealing bag at 2-8 ℃ for storage. The expired reagent is not used.

4. The liquid is added by using a sample injector, and the accuracy of the sample injector is frequently calibrated. When different samples or different reagent components are added, the suction head and the sample adding groove of the sample adding device are required to be replaced so as to prevent cross contamination.

5. When washing, each hole needs to be filled with washing liquid to prevent the free enzyme in the hole from being unable to be washed. After washing the plate, the next step must be carried out immediately, and the microplate must not be dried. Long interruptions of the experimental procedure were avoided to ensure uniform experimental conditions per well.

6. The result determination must be based on the reading of the microplate reader. When reading the result, the bottom of the ELISA plate should be wiped dry, and no air bubbles can exist in the holes. Rather than touching the outer wall of the bottom of the hole, the fingerprint or scratch may affect the reading of the plate hole.

7. The reference substance of the product is inactivated, HBsAg, anti-HIV, anti-HCV and anti-TP are all negative, but the infectivity of microorganisms such as potential viruses cannot be guaranteed, and the negative and positive control and the sample for detection are strictly operated according to the related rules of biological safety, follow the conventional rules of laboratory operation, and are used and processed according to the sample with potential biological infectivity. All samples, waste fluids and waste materials should be treated as infectious agents. The stop solution is sulfuric acid, and the use of the stop solution needs to be safe.

8. When developing color, firstly adding the developer A liquid and then adding the developing liquid B liquid to avoid too low development.

9. Each experiment needs to be provided with a positive standard substance, and the experimental result must be obtained by using a current standard curve, otherwise, the quantitative result error is possibly overlarge.

10. Abnormal points in the standard curve may cause deviations in the results of the whole plate experiment, and therefore a two-well assay reference is suggested to improve the accuracy of the experiment.

11. When the goodness of fit R2 of the standard curve is more than or equal to 0.99, the experiment is regarded as effective, otherwise, the experiment is invalid

12. The detection method has larger sample dilution times, so the dilution of the sample affects the result. It is recommended that the sample be diluted with a gradient of no more than 5-fold per step, and the volume of dilution be no less than 0.5 mL.

2.6.10 test method limitations

1. According to the basic theory of antibody production after body infection, after the body is infected by virus, the specific IgM antibody is produced earlier and has shorter duration, and the IgG antibody is produced later and has longer duration than the IgM antibody. In addition, since a certain period of time is required from the infection with a virus to the production of a specific antibody in the body, individual differences exist in the strength of the antibody, and these differences are correlated with the amount of an antigen to be infected and the antigenic strength of the antigen. Therefore, the lgG antibody detection result, the IgM antibody detection result sampling time, the clinical indication and the appearance time should be considered comprehensively. The positive antibody detection should be combined with other clinical indications for comprehensive judgment.

2. The kit can be only used for measuring serum or plasma samples and can not be used for other body fluid samples.

30 parts of positive serum subjected to nucleic acid detection and 50 parts of serum of healthy people to be detected are subjected to novel coronavirus IgG antibody detection by adopting the prepared kit, and the detection result is consistent with the DsbC-N fusion protein serum antibody ELISA experiment.

Example 2

1. Material

The plasmid pET-DsbA, the expression strain BL21(DE3) was preserved by the applicant; SARS-CoV-2N protein expression sequence nucleic acid vector pUC18-SARS-CoV-2N was offered by Shenchu Qi teacher of Meilan science and technology company of Beijing Wan; the plasmid extraction kit and the DNA gel recovery kit are purchased from Tiangen Biotech company; endonuclease, T4DNA ligase, available from NEB; the kappa fidelity DNA polymerase and dNTPs are available from Beijing GenBank; affinity resins are available from friendship, midrange biotechnology; goat anti-human IgG and TMB developing solution is available from Solebao biology company; the novel coronavirus IgG antibody detection kit (enzyme-linked immunosorbent assay) of the control sample is a Beijing Hua Dagibibiai biotechnology company, and SARS-CoV-2 complete virus lysate is coated on an enzyme label lath; 30 clinical confirmed COVID-19 patient sera, 50 healthy human sera were collected by the biological company, and completed by the ELISA test entrustment company; ELISA experiments were performed strictly in accordance with biosafety-related regulations, following the routine rules of laboratory procedures.

2. Method of producing a composite material

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, and referring to carrier pET-DsbA multiple cloning site, the specific primer aiming at N protein nucleic acid is designed and synthesized, and the 5 end of downstream primer is introduced with colibacillus biased stop codon TAA. The upstream and downstream primer design sequences are as follows:

The vector pUC18-SARS-CoV-2N nucleic acid is used as template, under the action of heat shock TaqDNA polymerase, SARS-CoV-2N nucleic acid is amplified by conventional PCR method through primers P1 and P2, and the conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 98 ℃ for 20s, annealing at 68 ℃ for 20s, and extension at 72 ℃ for 80s for 30 cycles, and extension at 72 ℃ for 5 min. The molecular weight of the amplified product is verified by 1% agarose electrophoresis, and the molecular weight of the amplified product is about 1260bp by detection.

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

And (3) performing gel recovery on the PCR amplification product, performing double enzyme digestion on the recovered product and an expression vector pET-DsbA by Sal I and Nhe I, performing gel cutting through 1% agarose electrophoresis to recover the product after enzyme digestion, cloning the recovered fragment into an expression plasmid pET-DsbA under the action of T4DNA ligase, transforming the ligation product into an escherichia coli competent cell BL21(DE3), 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 with correct sequencing into an LB culture medium with kanamycin concentration of 50ng/mL, placing the LB culture medium in a shaking table to be shake-cultured and activated at 37 ℃, transferring the engineering bacteria into the LB culture medium of the same system according to the proportion of 1:50 the next day, carrying out shake culture at 37 ℃ until the OD600 of the bacterial liquid reaches 0.4h, adding IPTG to enable the final concentration to be 0.3mmoL, and continuing shake culture and induction for 5h at 37 ℃. Centrifuging the induced bacteria liquid at 5000rpm at 4 deg.C to collect thallus, adding 1 × PBS buffer solution into the precipitated thallus, mixing, performing ultrasonication under ice culture state, centrifuging at 12000rpm at 4 deg.C for 30min, and collecting supernatant and precipitate.

The expression form of the fusion protein DsbA-N in a prokaryotic system is identified by 12 percent SDS-PAGE, the detection result shows that the DsbA-N fusion protein obtains higher expression in the prokaryotic system, the fusion protein accounts for more than 30 percent of the total protein of thalli, and after ultrasonic disruption, the DsbA-N fusion protein is mainly in ultrasonic precipitation in the form of inclusion bodies, wherein about 30 percent of the DsbA-N fusion protein exists in the form of supernatant, and the expression of the supernatant of the protein usually represents the natural state of the protein, so that the supernatant after ultrasonic disruption is collected in the subsequent steps for preparing affinity purification.

Affinity purification of DsbA-N fusion proteins

Carrying 6 XHIS label at the N end of the DsbA-N fusion protein, purifying the expressed ultrasonic supernatant protein by affinity chromatography, taking the centrifugal supernatant at 4 ℃, passing through a nickel ion affinity column at the speed of 2mL/min, balancing 4 column volumes by 1 XPBS, eluting the hybrid protein by 100mmoL imidazole for 3 column volumes, eluting the target fusion protein by 200mmoL imidazole, and collecting the target protein.

And (3) performing purity determination on the purified fusion protein by using 15% SDS-PAGE, wherein the purity of the fusion protein after affinity purification reaches more than 92%, and the high-purity fusion protein is a guarantee for obtaining high specificity by carrying out a subsequent serology experiment.

2.5DsbA-N fusion protein serum antibody ELISA experiment

After the purified DsbA-N fusion protein is fully dialyzed by water, the concentration is determined by adopting a BCA method, the recombinant protein is diluted to 15 mu g/mL by using coating liquid, each hole of an ELISA plate is coated with 100 mu L, the solution is placed for 2h at room temperature, and the plate is washed by PBST buffer solution for 4 times; sealing 10% calf serum in a refrigerator at 4 ℃ overnight, washing the plate with PBST for 5 times the next day, diluting the serum of the sample to be detected and the serum of the healthy human with 1:50 times, adding 100 mu L of the diluted serum into each hole, placing the diluted serum in a incubator at 37 ℃ for incubation for 1h, washing the plate for 5 times, patting the plate dry, incubating the diluted HRP-labeled goat anti-human IgG with 1:1000 times at 37 ℃ for 1h, washing the plate with PBST for 5 times, developing TMB, and detecting the A value at the wavelength of A450nm by a microplate reader.

In example 2, the positive sera of SARS-CoV-2 infected persons showed significant differences in A450 values in ELISA experiments compared to the healthy human sera. 30 of the cases show that the number of seropositive detection cases of the new coronary pneumonia is 29, and the detection rate is 96%; 1 sample of 50 healthy human control sera was detected, the detection rate was 2%, and the specificity was 98%. In this example, cut-off is set as the mean A value +3 times standard deviation of the human serum test of healthy individuals.

2.6 preparation of novel coronavirus detection kit based on DsbA-N fusion protein

Kit products were prepared in the specification amounts as shown in table 3.

TABLE 3 Specification amounts of kit Components

Composition of matter	Number of specifications
		Antigen enzyme label plate	8 x 12 wells, 1 plate
Positive antibody titer human serum, added with stabilizer and antiseptic	20U/mL, 0.1 mL/piece, 1 piece
		Enzyme labeling reagent with positive antibody titer	15 mL/bottle, 1 bottle
Positive antibody titer sample diluent	50 mL/bottle, 1 bottle
		Color developing agent A liquid	6 mL/bottle, 1 bottle
Developer B liquid	6 mL/bottle, 1 bottle
		Concentrated washing solution (20X)	50 mL/bottle, 1 bottle
Stopping liquid	6 mL/bottle, 1 bottle
		Sealing plate film	3 are provided with
Negative control	0.5mL × 1 bottle

30 parts of positive serum subjected to nucleic acid detection and 50 parts of serum of healthy people to be detected are subjected to novel coronavirus IgG antibody detection by adopting the prepared kit, and the detection result is consistent with an ELISA (enzyme-linked immunosorbent assay) experiment of DsbA-N fusion protein serum antibodies.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Kunming City, women and children health care institute; the eighth medical center of the general Chinese liberated military hospital

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

<130> 2020

<160> 7

<170> PatentIn version 3.5

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<211> 567

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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

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<212> DNA

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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

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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

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<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. Kit for the detection of a novel coronavirus based on a fusion protein of a nucleocapsid protein, characterized in that it comprises an antigen for the detection of a novel coronavirus comprising a fusion protein based on a nucleocapsid protein.

2. The kit according to claim 1, wherein the nucleocapsid protein-based fusion protein is a fusion protein produced by fusion expression of an amino acid sequence or a partial amino acid sequence of a nucleocapsid protein and an amino acid sequence or a partial amino acid sequence of disulfide oxidoreductase A; or, the amino acid sequence or partial amino acid sequence of the nucleocapsid protein is fused and expressed with the amino acid sequence or partial amino acid sequence of the disulfide oxidoreductase C.

3. The kit according to claim 2, characterized in that the amino acid sequence of disulfide oxidoreductase a is the amino acid sequence shown as SEQ ID No.1 or a mutated sequence of the amino acid sequence shown as SEQ ID No. 1;

4. The kit of claim 3, wherein 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 the disulfide oxidoreductase A; the amino acid sequence of the nucleocapsid protein is linked to the C-terminus of the amino acid sequence of the disulfide oxidoreductase C either directly or via a linker.

5. The kit according to any one of claims 1 to 4, wherein the kit is a colloidal gold kit, the fusion protein is labeled by colloidal gold and then sprayed on the gold-labeled pad, the particle size of the colloidal gold is 18nm to 28nm, and the spraying amount of the gold-labeled fusion protein on the gold-labeled pad is 1.4 μ L/cm to 2.0 μ L/cm.

6. The kit according to any one of claims 1 to 4, 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 10-20 μ g/mL, and the coating volume is 80-120 μ L.

7. The kit according to claim 6, characterized in that it comprises the following components:

8. The kit of claim 7, wherein the kit components are in the following specification amounts:

9. the kit according to claim 8,

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

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;

10. The kit according to claim 6, wherein the antigen ELISA plate is prepared by coating an aqueous solution of the fusion protein on the ELISA plate, standing at room temperature for 1.5-2.5 h, and washing the plate with PBST buffer solution; and (3) sealing the plate with 10% calf serum at 4 ℃ for 10-15 h, and washing the plate with PBST buffer solution to obtain the antigen ELISA plate.