CN112500494A

CN112500494A - Antigen for detecting novel coronavirus and preparation method thereof

Info

Publication number: CN112500494A
Application number: CN202011242676.3A
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: CN112500494B

Abstract

The invention provides an antigen for detecting a novel coronavirus and a preparation method thereof. The antigen for detecting the novel coronavirus and the preparation method thereof 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 structure characteristic of the nucleocapsid protein of the novel coronavirus and simultaneously improves the property of the fusion protein, the antigen is high in sensitivity and strong in 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.

Description

Antigen for detecting novel coronavirus and preparation method thereof

Technical Field

The invention relates to the field of biomedical detection, in particular to an antigen for detecting novel coronavirus and a preparation method thereof.

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, is located in the interior of virus, is an antagonist of Interferon (Interferon, IFN) and a virus-coded RNA interference inhibitor, is related to the replication of virus, is relatively conserved among beta coronaviruses, has a large synthetic quantity and strong antigenicity, plays an important role in inducing host immune response and even pathogenesis, and is often used as an antigenic 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 an antigen for detecting a novel coronavirus and a preparation method thereof, aiming at the problems that the detection rate of the existing method for detecting SARS-CoV-2 directly based on N protein as an antigen is low, and the diagnosis efficiency of COVID-19 and the control of an epidemic situation are influenced.

The invention provides an antigen for detecting a novel coronavirus, which comprises a fusion protein based on a nucleocapsid protein.

In one embodiment thereof, the nucleocapsid protein-based fusion protein is a soluble 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 nucleotide sequence of disulfide oxidoreductase a is the nucleotide sequence shown as SEQ ID No.1 or a mutated sequence of the nucleotide sequence shown as SEQ ID No. 1;

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

In one embodiment thereof, the nucleotide sequence of the nucleocapsid protein is linked directly or via a linker to the C-terminus of the nucleotide 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 nucleocapsid protein-based fusion protein is produced by expression in a prokaryotic expression system.

The invention also provides a preparation method of the antigen for detecting the novel coronavirus, which 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 a 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.

In one embodiment, 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.

In one embodiment, the preparation method further comprises the following steps:

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

In one embodiment, the prokaryotic expression system is E.coli.

Above-mentioned antigen for novel coronavirus detects and preparation method thereof obtains the fusion protein that contains the nucleocapsid protein of novel coronavirus through the fusion technique, makes the fusion protein improve its nature when having novel coronavirus nucleocapsid protein natural space structure characteristic, and sensitivity is high, the specificity is strong when being used for novel coronavirus to detect, more suitably is used for novel coronavirus to detect in order to improve the relevance ratio of novel coronavirus, ensures the timely definite diagnosis of novel coronavirus pneumonia, can effectively avoid the diffusion of epidemic situation.

Furthermore, according to the antigen for detecting the novel coronavirus and the preparation method thereof, the fusion protein based on the nucleocapsid protein is soluble protein, the soluble protein can exist in the form of supernatant, the detection rate of the novel coronavirus antibody can be effectively improved when the antigen is used for serological detection, and the detection accuracy is ensured.

Furthermore, the antigen for detecting the novel coronavirus and the preparation method thereof are characterized in that the nucleocapsid protein of the novel coronavirus and the disulfide oxidoreductase A or the disulfide oxidoreductase C are subjected to fusion expression, the biological characteristics of disulfide isomerase and molecular chaperone of the disulfide oxidoreductase C are fully utilized, the solubility of the fusion protein is improved, the fusion protein is promoted to exist in an supernatant form in a recombination expression process, the detection rate of the novel coronavirus antibody is further improved, and a material basis is laid 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 invention provides an antigen for detecting a novel coronavirus, which comprises a fusion protein based on a nucleocapsid protein. This an antigen for novel coronavirus detects obtains the fusion protein that contains the nucleocapsid protein of novel coronavirus through the fusion technique, makes the fusion protein improve its nature when having 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, more suitably is used for novel coronavirus to detect in order to improve the relevance ratio of novel coronavirus, ensures the timely definite diagnosis of novel coronavirus pneumonia, can effectively avoid the diffusion 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.

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.

As an alternative embodiment, the nucleocapsid protein-based fusion protein is produced by expression in a prokaryotic expression system, preferably E.coli.

The second aspect of the present invention also provides a method for preparing an antigen for detecting a novel coronavirus, the method comprising the following steps:

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

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:

The N ends of the DsbA-N fusion protein and the DsbC-N fusion protein are provided with 6 XHIS labels, and a chelating sepharose can be used for affinity chromatography to collect supernatant and precipitate generated after fusion expression, so that the purification is convenient and the purity of the purified fusion protein is high.

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.

The antigen for detecting the novel coronavirus is mainly used for preparing a kit for detecting the novel coronavirus.

For example, the kit is a colloidal gold kit, the fusion protein is labeled by colloidal gold and then sprayed on a 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.

For another example, 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 fusion protein, the coating concentration of the fusion protein on the ELISA plate is 10-20 mug/mL, and the coating volume is 80-120 mug 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.

Further optionally, 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.

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 pUC 18-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;

nucleotide sequence of 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 pUC 18-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。

2.5 DsbC-N fusion protein serum antibody ELISA experiment

Enzyme linked immunosorbent assay (ELISA) is usually used for antibody detection of serum viruses of patients in the progressive stage and the convalescent stage, is a traditional laboratory detection technology, has relatively low operation requirement, can determine whether the patients are infected with SARS-CoV-2 recently or previously by using the ELISA method, and is helpful for determining the negative nucleic acid detection but clinically suspected patients.

2.4 after the purified DsbC-N fusion protein is fully dialyzed by water, a BCA method is adopted for concentration determination, recombinant protein is diluted to 15 mu g/mL by 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.

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 pUC 18-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:

nucleotide sequence of downstream primer

P2:5-CTAGCTAGCTTAGGCCTGAGTTGAGTCAGC-3。

The vector pUC 18-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.

2.5 DsbA-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.

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> antigen for detecting novel coronavirus and preparation method thereof

<130> 2020

<160> 7

<170> PatentIn version 3.5

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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

<211> 648

<212> DNA

<213> Artificial sequence

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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

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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

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<213> Artificial sequence

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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

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

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

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

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

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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

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

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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

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Gln Ala

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<213> Artificial sequence

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acgcgtcgac tctgataatg gacc 24

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<212> DNA

<213> Artificial sequence

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ctagctagct taggcctgag ttgagtcagc 30

<210> 7

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<212> DNA

<213> Artificial sequence

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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. An antigen for the detection of a novel coronavirus, wherein said antigen for the detection of a novel coronavirus comprises a nucleocapsid protein-based fusion protein.

2. The antigen for the detection of novel coronaviruses according to claim 1, wherein said nucleocapsid protein-based fusion protein is a soluble protein.

3. The antigen for the detection of a novel coronavirus according to claim 2, wherein the nucleocapsid protein-based fusion protein is a fusion protein obtained 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.

4. The antigen for the detection of a novel coronavirus according to claim 3, wherein the nucleotide sequence of disulfide oxidoreductase A is the nucleotide sequence shown in SEQ ID No.1 or a mutant sequence of the nucleotide sequence shown in SEQ ID No. 1;

5. The antigen for the detection of a novel coronavirus according to claim 4, wherein the nucleotide sequence of the nucleocapsid protein is linked to the C-terminus of the nucleotide sequence of the disulfide oxidoreductase A directly or via a linker;

6. The antigen for the detection of novel coronaviruses according to any one of claims 1 to 5, wherein the nucleocapsid protein-based fusion protein is produced by expression in a prokaryotic expression system.

7. A method for preparing an antigen for detecting a novel coronavirus, the method comprising the steps of:

8. The method for preparing the antigen for detecting the novel coronavirus according to claim 7, wherein 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.

9. The method of claim 7, further comprising the steps of:

10. The method for producing an antigen for the detection of a novel coronavirus according to claim 7 or 8, wherein the prokaryotic expression system is Escherichia coli.