CN113527474B

CN113527474B - Monoclonal antibody of anti-novel coronavirus N protein and application thereof

Info

Publication number: CN113527474B
Application number: CN202110267506.9A
Authority: CN
Inventors: 蒋大鹏; 熊向华
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2023-11-07
Anticipated expiration: 2041-03-12
Also published as: CN113527474A

Abstract

The invention relates to a monoclonal antibody of anti-novel coronavirus N protein and application thereof, belonging to the technical field of biology. The monoclonal antibody has a heavy chain complementarity determining region CDR3 shown in SEQ NO.9 and a light chain complementarity determining region CDR3 shown in SEQ NO. 15. The affinity of the monoclonal antibody is 9.21×10 ^‑10 mol/L, the level of pM is reached, and the effect of the N protein prepared by combining insect cells is equivalent to that of the N protein prepared by combining escherichia coli; the monoclonal antibody lays a foundation for new coronavirus detection and infection mechanism research.

Description

Monoclonal antibody of anti-novel coronavirus N protein and application thereof

Technical Field

The invention relates to a monoclonal antibody of anti-novel coronavirus N protein and application thereof, belonging to the technical field of biology.

Background

The novel coronavirus (severe actue respiratory syndrome coronavirus, SARS-CoV-2) is a enveloped positive-strand RNA virus belonging to the genus coronavirus beta, and the pneumonia caused by its infection is designated as COVID-19 by the world health organization. The nucleocapsid protein (Nucleocapsid protein), N protein, is one of the major structural proteins of the new coronavirus, and the largest proportion of the structural proteins of the virus consists of an N-terminal domain (NTD) and a C-terminal domain (CTD). The N protein and the viral genome RNA are combined to form a viral nucleocapsid, and the viral nucleocapsid participates in regulating the transcription and replication of the viral RNA and is closely related to pathogenicity. Through the whole genome data analysis of the disclosed novel coronaviruses, the N protein is found to have little variation in the novel coronaviruses in different areas, the variation among the coronaviruses is small, and the core structural region is conserved. The conservation and high antigenicity of the N protein has led to its common use as a diagnostic antigen for coronaviruses, and research into the N protein has also contributed to a more thorough understanding of new coronaviruses. At present, the source of the new coronaviruses is not yet elucidated, and whether cross-species transmission exists is still to be clarified. The development of new coronavirus infection mechanisms, epidemiological investigation and clinical diagnostic methods all require the use of monoclonal antibodies.

Disclosure of Invention

In view of the above, the present invention aims to provide a monoclonal antibody against a novel coronavirus N protein, which has a high affinity, and uses thereof.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

according to the invention, an N protein expressed and purified in escherichia coli is used as an antigen to immunize a BALB/c mouse, positive cells are screened by a hybridoma technology, cells with high supernatant titer are selected to prepare monoclonal antibodies by a mouse ascites induction method, antibody affinity and binding domains are detected, an antibody light-heavy chain variable region is amplified, and the Complementarity Determining Region (CDR) sequence is analyzed.

A monoclonal antibody against a novel coronavirus N protein, said monoclonal antibody having a heavy chain complementarity determining region CDR3 as shown in SEQ No.9 and a light chain complementarity determining region CDR3 as shown in SEQ No. 15.

Further, the monoclonal antibody has a heavy chain complementarity determining region CDR1 shown in SEQ No.5, a heavy chain complementarity determining region CDR2 shown in SEQ No.7, a light chain complementarity determining region CDR1 shown in SEQ No.11 and a light chain complementarity determining region CDR2 shown in SEQ No. 13.

Further, the heavy chain of the monoclonal antibody has an amino acid sequence shown in SEQ NO. 3; the light chain of the monoclonal antibody has an amino acid sequence shown in SEQ NO. 4.

Further, the nucleotide sequence encoding the CDR3 of the heavy chain complementarity determining region is shown in SEQ NO.10, and the nucleotide sequence encoding the CDR3 of the light chain complementarity determining region is shown in SEQ NO. 16.

Further, the nucleotide sequence for encoding the CDR1 of the heavy chain complementarity determining region is shown as SEQ NO.6, and the nucleotide sequence for encoding the CDR2 of the heavy chain complementarity determining region is shown as SEQ NO. 8; the nucleotide sequence for coding the CDR1 of the light chain complementarity determining region is shown as SEQ NO.12, and the nucleotide sequence for coding the CDR2 of the light chain complementarity determining region is shown as SEQ NO. 14.

Further, the nucleotide sequence for encoding the heavy chain is shown as SEQ NO. 1; the nucleotide sequence for coding the light chain is shown as SEQ NO. 2.

Use of a monoclonal antibody against a novel coronavirus N protein for detection of a novel coronavirus.

Advantageous effects

The affinity of the monoclonal antibody is 9.21×10 ^-10 mol/L, the level of pM is reached, and the effect of the N protein prepared by combining insect cells is equivalent to that of the N protein prepared by combining escherichia coli; the high-affinity murine monoclonal antibody against the novel coronavirus N protein lays a foundation for novel coronavirus detection and infection mechanism research.

Drawings

FIG. 1 shows the result of polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the N protein of example 1 before purification;

FIG. 2 shows the result of SDS-PAGE analysis of the purified N protein of example 1;

FIG. 3 shows the results of the mouse tail blood titer determination in example 2;

FIG. 4 shows the result of SDS-PAGE analysis of the purified 3H12 antibody of example 5;

FIG. 5 shows the results of the specific identification of 3H12 antibodies in example 6;

FIG. 6 is a graph showing the identification of the 3H12 antibody binding domain in example 7;

FIG. 7 shows the results of the affinity test for the 3H12 antibody of example 8.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

The material sources in the following examples are as follows:

SPF-grade female BALB/c mice; sp2/0 mouse myeloma cells (stored in laboratory); pET22b-N, pET22b-NTD and pET22b-CTD plasmids (preserved in laboratory) containing target genes; coli BL21 (DE 3) competent cells (Beijing full gold Biotechnology Co., ltd.); freund's complete adjuvant, freund's incomplete adjuvant (Sigma Co.); DMEM medium (Gibco); fetal bovine serum (Gibco company); a mixture of green streptomycin (Gibco Co.); antibody subtype identification kit (Beijing bloolone immunotechnology Co., ltd.); ultrapure RNA extraction kit (Jiangsu kang is century biotechnology Co., ltd.); TA/Blunt-Zero Cloning Kit (Nanjinouzan Biotechnology Co., ltd.); hitrp Protein GHP affinity chromatography column (GE); PD10 desalting column (GE company); goat anti-mouse secondary antibody (Beijing boolone immune technologies Co., ltd.) with HRP label; 96-well ELISA plates (Corning Co.); BCA protein assay kit (Shanghai elegance biosciences limited).

Example 1

Protein purification:

e.coli BL21 (DE 3) competent cells were transformed with pET22b-N, pET b-NTD and pET22b-CTD plasmids containing the target gene, respectively, clones were picked up and inoculated into 200mL of LB (Amp) medium, cultured at 37℃and 200rpm to mid-log phase, isopropyl-beta-D-thiogalactoside (IPTG) with a final concentration of 0.5mM was added to induce overnight at 20℃and 200rpm, cells were collected by centrifugation at 12000rpm for 20min at 4℃and 20mL of binding buffer (20 mmol/L PB,100mmol/L NaCl,20mmol/L imidazole, pH 7.2) was added to resuspend, cells were sonicated in an ice-water bath, supernatant was collected by centrifugation at 20min at 12000rpm at 4℃and filtered through a 0.45 μm filter membrane, and loaded onto Ni which had been equilibrated with binding buffer ²⁺ And (3) carrying out affinity chromatography column, eluting the target protein by using an elution buffer (20 mmol/L PB,500mmol/L NaCl,500mmol/L imidazole, pH 7.2) in a linear gradient manner, and collecting elution components in each stage for polyacrylamide gel electrophoresis (SDS-PAGE) analysis to obtain the purified N protein.

SDS-PAGE of the purified pre-proteins is shown in FIG. 1, adjacent lanes reveal specific protein expression bands with a molecular weight of about 50kDa, consistent with the expected size of the N protein, compared to the negative control (-). As shown in FIG. 2, the purity of the purified N protein is more than 90%, and the method of the embodiment realizes the high expression of the N protein in escherichia coli.

The N-terminal domain (NTD) and the C-terminal domain (CTD) of the N protein are expressed by the same method (shown in figure 1), and the purity of the purified target protein is more than 85% (shown in figure 2).

Example 2

Animal immunization:

5 SPF-class 6-8 week old female BALB/c mice were selected, numbered 1-5 (Mouse 1, mouse2, mouse3, mouse4, mouse 5). Primary immunization Each mouse was subcutaneously injected with 50. Mu.g of purified N protein as described in example 1. Specifically: diluting 300 mug of purified N protein described in example 1 with Phosphate Buffer (PBS), adding an equal volume of Freund's complete adjuvant, fully mixing until a stable water-in-oil emulsion is formed, selecting 5 points on the back of each mouse, and performing subcutaneous injection at 0.2 mL/point; 21d and 42d 50 μg purified N protein as described in example 1/mouse each was boosted 1 time (Freund's incomplete adjuvant); the tail vein of the 49d mice was bled and serum titers were determined by indirect ELISA.

The blood titers of 5 mice after 3 booster immunizations were determined by indirect ELISA and the results are shown in FIG. 3. And selecting the No.5 mouse with the best immune effect according to the result to carry out a hybridoma cell fusion experiment.

Example 3

Cell fusion:

mice number 5 with the highest serum titers after three immunizations were selected for intraperitoneal injection with 50 μg of purified N protein as described in example 1 for impact immunization, and cell fusion was performed after 3 d. On the day of fusion, mice were sacrificed at cervical scission and immersed in 75% (volume percent) alcohol for 5min; separating mouse spleen under aseptic condition, grinding spleen thoroughly, sieving with cell sieve, washing with small amount of serum-free DMEM, adding into 50mL centrifuge tube, centrifuging at 1500r/min for 5min, discarding supernatant, resuspending cell precipitate with DMEM, repeatedly washing for 3 times, and adjusting the number of spleen cells to 10 with DMEM ⁸ About one/mL; washing myeloma Sp2/0 cells in logarithmic phase with DMEM for 2 times to adjust cell number to 10 ⁷ About one/mL (splenocytes: sp2/0 cells=10:1); adding spleen cells and Sp2/0 cells into a 50mL centrifuge tube, uniformly mixing, centrifuging at 1500r/min for 5min, carefully discarding the supernatant, and flicking the bottom of the centrifuge tube to loosen the cells; placing the centrifuge tube in warm water at 37 ℃, slowly adding 1mL of polyethylene glycol (PEG) 1500 within 1min, gently shaking while adding, and standing for 90s after adding; 1mL of DMDM was added uniformly over 1min, followed by 4mL of DMDM,800, over 2minCentrifuging for 3min at r/min; carefully discarding the supernatant, lightly suspending the cell pellet with 20mL of fetal calf serum, adding the prepared thymus cells, and uniformly mixing; simultaneously preparing 30mL of semi-solid culture medium subjected to high-pressure sterilization, pouring the semi-solid culture medium into a centrifuge tube containing thymus cells, and uniformly mixing the semi-solid culture medium upside down; evenly poured into 25 cell culture dishes, transferred to 37 ℃ and 5% (volume percent) CO ₂ Culturing in an incubator to obtain fused cells.

Screening and cloning of positive cell lines

Screening and culturing the fused cells by using a semi-solid culture medium for 2 weeks, picking up monoclonal on a cell culture dish, inoculating the monoclonal on a 96-well cell culture plate (thymus cells are added in advance in the plate, 100 mu L/hole) and picking up 4 plates altogether, and culturing in an incubator; after 3d, hybridoma cells were positively screened for 2 rounds by indirect ELISA. And (3) performing 3 times of cloning on positive hybridoma cells obtained after 2 rounds of screening after amplification culture, and finally freezing and storing stable positive cell strains.

After passage, expansion culture and cloning screening of 68 positive cells, 8 positive cell strains capable of stably secreting antibodies are finally obtained.

After 2 weeks, total cell fusion rate of 4 96-well cell culture plates was calculated to be 100%. ELISA screening gave 68 positive cell lines with a positive rate of 17.7% as shown in Table 1.

TABLE 1

Potency determination:

detecting positive hybridoma cell culture solution supernatant titers by adopting an indirect ELISA method: the purified N protein described in example 1 was added to a 96-well ELISA plate with a final concentration of 5. Mu.g/mL, 100. Mu.L/well, and coated for 2h at 37℃with a substrate coating buffer; washing 1 time with 300. Mu.L/well phosphate Tween buffer (PBST); 300. Mu.L/well was blocked with 5% nonfat milk powder at 37℃for 2h and washed 3 times with PBST; samples (positive hybridoma cell culture supernatant) were subjected to gradient dilution with PBST and incubated at 100. Mu.L/well 37℃for 1h; PBST wash 5 times; HRP-labeled goat diluted 1:10000 times (volume ratio)Incubation of anti-mouse secondary antibody at 100. Mu.L/well at 37℃for 1h; PBST wash 5 times; adding TMB color development liquid into 100 mu L/hole for color development; 50 μl/well of 2M H ₂ SO ₄ The reaction was terminated and the absorbance of OD450 was read by the microplate reader.

The indirect ELISA detection result shows that the titer of the culture supernatant of 8 positive hybridoma cells is shown in table 2, the titer is from 1:6400 to 1:204800, wherein the highest titer of the culture supernatant of the hybridoma cells with the number of 3H12 in the 96-well ELISA plate is more than 20 ten thousand times.

Identification of antibody subtypes:

cell subtypes are identified according to the antibody subtype identification kit instructions.

The subtype of the cell is identified by adopting a subtype identification kit, and the result shows that 6 antibody heavy chains are of IgG1 type, and 2 antibody heavy chains are of IgM type; the 5-strain antibody light chain was kappa type, and the 3-strain antibody was lambda type (as shown in Table 2). Thus, 3H12 hybridoma cells in which the cell culture supernatant titer was highest were selected for subsequent antibody preparation experiments.

TABLE 2

Example 4

The 3H12 positive hybridoma cell strain is subjected to expansion culture, cells are collected by low-speed centrifugation, total RNA of the cells is extracted according to the specification of an ultrapure RNA extraction kit and is reversely transcribed into cDNA, and variable region genes of heavy chains and light chains of antibodies are amplified by PCR (degenerate primers are shown in table 3). 1% (mass percent) agarose gel electrophoresis, then cutting gel, recovering target fragments, connecting into a T carrier, transferring into DH5 alpha competent coating LB (Amp-IPTG-X-gal) plates, picking white spots into LB (Amp) culture medium for culture, and then delivering to sequence; the target sequence is aligned and analyzed by IgBLAST software.

TABLE 3 Table 3

The sequencing result of the target sequence is as follows:

the nucleotide sequence encoding the heavy chain is as follows SEQ NO.1:

CAGGTCAAACTGCAGGAGTCAGGAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGACTTCTGGATACATCTTCACCAGCTACTGGATTCACTGGGTGAAACAGAGGTCTGGACAGGGCCTTGAGTGGATTGCAAGGCTTTATCCTAGAACTGATAATACTTACTACAGTGAGAAATTCAAGGGCAAGGCCACTCTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAACTCAACAGCCTGAAATCTGAGGACTCTGCTGTCTATTTCTGTGTAAGAAGGGATGATTACTCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCCGCAGCCAAAACGACACCCCCA；

the nucleotide sequence encoding the light chain is as follows SEQ NO.2:

GACATTGAGCTCACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGAAATTAGTGGTTACTTAAGCTGGCTTCAGCAGAAACCAGATGGAACTATTAAACGCCTGATCTACGCCGCATCCACTTTAGATTCTGGTGTCCCAAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGTCTTGAGTCTGAAGATTTTGCAGACTATTACTGTCTACAATATGGTAGTTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG；

from this, the heavy chain amino acid sequence of the 3H12 antibody is shown as SEQ NO.3:

QVKLQESGAELVRPGASVKLSCKTSGYIFTSYWIHWVKQRSGQGLEWIARLYPRTDNTYYSEKFKGKATLTADKSSSTAYMQLNSLKSEDSAVYFCVRRDDYSFAYWGQGTLVTVSAAKTTPP；

the light chain amino acid sequence of the 3H12 antibody is shown in SEQ NO.4:

DIELTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYGSYPWTFGGGTKLEIKR；

as can be seen from the IgBLAST software analysis, the number of CDR1 in the heavy chain is 8, and the amino acid sequence is SEQ NO.5: GYIFTSYW, the nucleotide sequence for encoding the CDR1 is SEQ NO.6: GGATACATCTTCACCAGCTACTGG;

as can be seen from the IgBLAST software analysis, the number of CDR2 in the heavy chain is 8, and the amino acid sequence is SEQ NO.7: LYPRTDNT, the nucleotide sequence encoding said CDR2 is SEQ NO.8: CTTTATCCTAGAACTGATAATACT;

analysis by IgBLAST software shows that the number of CDR3 in the heavy chain is 10, and the amino acid sequence is SEQ NO.9: VRRDDYSFAY the nucleotide sequence encoding said CDR3 is SEQ NO.10: AGAAGGGATGATTACTCGTTTGCTTAC;

as can be seen from the IgBLAST software analysis, the number of CDR1 in the light chain is 6, and the amino acid sequence is SEQ NO.11: QEISGY, the nucleotide sequence encoding said CDR1 is SEQ No.12: CAGGAAATTAGTGGTTAC;

as can be seen from the IgBLAST software analysis, the number of CDR2 in the light chain is 3, and the amino acid sequence is SEQ NO.13: AAS, the nucleotide sequence encoding the CDR2 is SEQ No.14: GCCGCATCC;

as can be seen from the IgBLAST software analysis, the number of CDR3 in the light chain is 9, and the amino acid sequence is SEQ NO.15: LQYGSYPWT the nucleotide sequence encoding said CDR3 is SEQ NO.16: CTACAATATGGTAGTTATCCGTGGACG.

The IgBLAST software analysis shows that the light chain variable region of the 3H12 antibody has 97.9 percent of sequence homology with the mouse germ line gene IGKV9-124 x 01; the heavy chain variable region of the 3H12 antibody had 89.1% sequence homology with the mouse germline gene IGHV1-76 x 01, and the results are shown in table 4.

TABLE 4 Table 4

Example 5

Ascites preparation and antibody purification:

female BALB/c mice of 6-8 weeks of age were ordered, and each mice was preimmunized by intraperitoneal injection of 0.5mL of Freund's incomplete adjuvant. About 1X 10 after 7d ⁶ Performing cell injection of the 3H12 positive hybridoma cells in example 3, and simultaneously injecting the Sp2/0 myeloma cells with the same number as a negative control, wherein the DMEM culture medium with the same volume is used as the negative control; and 5d, extracting ascites according to the abdominal size of the mice, centrifuging at 4 ℃ and 10000r/min for 15min, absorbing supernatant of the middle layer ascites, filtering with a 0.45 mu m filter membrane, purifying by Protein G column affinity chromatography, and desalting by a PD10 column. SDS-PAGE was used to detect antibody purification effect using Gel-pro32 software analysis of antibody purity BCA protein quantification kit protein quantification of purified antibodies.

The indirect ELISA detection result shows that the titer of the ascites supernatant of the 3H12 positive hybridoma cell reaches 1:2 multiplied by 10 ⁵ . After the 3H12 positive hybridoma cells with good growth state are subjected to expansion culture, the cells are injected into the abdominal cavity of the mice in an intraperitoneal injection mode, and ascites is extracted after 7 d. And (3) purifying the ascites Protein through a Protein G column after centrifuging the ascites, desalting and changing the liquid through a PD10 column, and detecting the expression condition of the purified Protein by SDS-PAGE. As shown in FIG. 4, the 3H12 purified antibody had specific protein bands at both 2.5kDa and 5kDa, which were sized to match the molecular weight of the light and heavy chains of the antibody; purity of 3H12 antibody protein by analysis with Gelpro32 software>95％。

Example 6

Antibody specificity identification:

the specificity of the antibodies was identified by indirect ELISA using N protein expressed and purified from E.coli in the laboratory and N protein expressed by purchased insect cells as antigens, respectively, and the samples in the titer assay of example 3 were replaced with purified 3H12 antibodies, and the remainder were the same as in the titer assay of example 3.

As shown in FIG. 5, the 3H12 antibody was equivalent to the effect of binding to purified N protein expressed in E.coli (3H 12-N-E.coli), and the 3H12 antibody was equivalent to the effect of binding to purified N protein expressed in insect cells (3H 12-N-insert).

Example 7

Identification of antibody binding domains:

the antigen was diluted with a coating buffer solution to a final concentration of 5. Mu.g/mL using N protein, N-NTD and N-CTD, and then the antibody binding domain was identified by indirect ELISA, and the sample in the titer measurement of example 3 was replaced with the 3H12 antibody, and the other steps were the same as in the titer measurement method of example 3.

As shown in fig. 6, the t-test of two sets of data, p=0.103, for the 3H12 antibody with N protein and NTD protein, the difference between the two sets was not statistically significant (P > 0.05); the 3H12 antibody was very significantly different from the N protein and CTD protein data t-test p=0.003 (P < 0.01), indicating that the 3H12 antibody binds to the N-terminal domain (NTD) of the N protein.

Example 8

Antibody affinity constant determination

The affinity constants of the antibodies were determined using a non-competitive ELISA method. Binding curves were determined for 4 concentrations of antigen N protein (4, 2, 1, 0.5. Mu.g/mL) and 15 concentrations of 3H12 antibody (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.020, 0.010, 0.005, 0.0025, 0.00125, 0.000625. Mu.g/mL). The affinity constants of the antibodies were calculated using SPSS2.6 software and the original 9.6 software was curve fitted as shown in fig. 7.

The affinity constant of the 3H12 antibody and the N protein antigen is 9.21 multiplied by 10 calculated by SPSS software ^-10 The mol/L reaches the pmol/L level, and shows that the antibody has high affinity with the antigen.

In view of the foregoing, it will be appreciated that the invention includes but is not limited to the foregoing embodiments, any equivalent or partial modification made within the spirit and principles of the invention.

Sequence listing

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Claims

1. A monoclonal antibody against the N protein of a novel coronavirus, characterized in that: the amino acid sequence of a heavy chain complementarity determining region CDR1 of the monoclonal antibody is shown as SEQ NO.5, the amino acid sequence of a heavy chain complementarity determining region CDR2 is shown as SEQ NO.7, and the amino acid sequence of a heavy chain complementarity determining region CDR3 is shown as SEQ NO. 9; the amino acid sequence of the CDR1 of the light chain complementarity determining region is shown as SEQ NO.11, and the amino acid sequence of the CDR2 of the light chain complementarity determining region is shown as SEQ NO. 13; the amino acid sequence of CDR3 of the light chain complementarity determining region is shown in SEQ NO. 15.

2. A monoclonal antibody against the N protein of the new coronavirus according to claim 1, characterized in that: the heavy chain of the monoclonal antibody has an amino acid sequence shown in SEQ NO. 3; the light chain of the monoclonal antibody has an amino acid sequence shown in SEQ NO. 4.

3. A nucleotide sequence encoding a monoclonal antibody against a novel coronavirus N protein, characterized in that: the nucleotide sequence of the heavy chain complementarity determining region CDR1 is shown as SEQ NO.6, the nucleotide sequence of the heavy chain complementarity determining region CDR2 is shown as SEQ NO.8, and the nucleotide sequence of the heavy chain complementarity determining region CDR3 is shown as SEQ NO. 10; the nucleotide sequence for coding the CDR1 of the light chain complementarity determining region is shown as SEQ NO.12, and the nucleotide sequence for coding the CDR2 of the light chain complementarity determining region is shown as SEQ NO. 14; the nucleotide sequence encoding CDR3 of the light chain complementarity determining region is shown in SEQ NO. 16.

4. A nucleotide sequence encoding a monoclonal antibody against a novel coronavirus N protein according to claim 3, wherein: wherein the nucleotide sequence of the coding heavy chain is shown as SEQ NO. 1; the nucleotide sequence of the coded light chain is shown as SEQ NO. 2.

5. Use of a monoclonal antibody against the N protein of the new coronavirus according to claim 1 or 2, characterized in that: the monoclonal antibody is applied to preparation of a reagent for detecting the novel coronavirus.