CN113912710B

CN113912710B - Monoclonal antibody for resisting novel coronavirus N protein and application thereof

Info

Publication number: CN113912710B
Application number: CN202111361909.6A
Authority: CN
Inventors: 魏文涛; 上官科科
Original assignee: Sure Biotech (hangzhou) Ltd
Current assignee: Sure Biotech (hangzhou) Ltd
Priority date: 2021-11-17
Filing date: 2021-11-17
Publication date: 2023-05-26
Anticipated expiration: 2041-11-17
Also published as: CN113912710A

Abstract

The invention provides a monoclonal antibody for resisting novel coronavirus N protein and application thereof. The monoclonal antibodies include a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDRH1 with an amino acid sequence shown as SEQ ID NO. 1, CDRH2 with an amino acid sequence shown as SEQ ID NO. 2 and CDRH3 with an amino acid sequence shown as SEQ ID NO. 3. The light chain variable region comprises a CDRL1 shown in SEQ ID NO. 4, a CDRL2 shown in SEQ ID NO. 5 and a CDRL3 shown in SEQ ID NO. 6. The invention also provides a nucleic acid molecule for encoding the monoclonal antibody, application of the monoclonal antibody in preparing a reagent for detecting the novel coronavirus N protein, and a kit for detecting the novel coronavirus comprising the monoclonal antibody. The monoclonal antibody has higher sensitivity and specificity, and can be applied to immunological detection.

Description

Monoclonal antibody for resisting novel coronavirus N protein and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a monoclonal antibody for resisting novel coronavirus N protein and application thereof.

Background

New coronavirus pneumonia caused by new coronaviruses is a public health problem of current global interest. On day 11 of 2 months 2020, this novel coronavirus was designated by the International Commission on the classification of viruses as SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, also known as 2019-nCoV (2019 novel coronavirus)), and on the same day, the world health organization designated the pneumonia infected with this virus as COVID-19.SARS-CoV-2 and Severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) are animal derived zoonotics. Although coronaviruses are often associated with acute respiratory infections in humans, their ability to infect a variety of host species makes them complex pathogens. The covd-19 pandemic caused by the novel coronavirus (SARS-CoV-2) is abused worldwide and causes millions of hospitalizations and deaths.

Thus, there is a need to establish a method for rapid screening and detection of novel coronaviruses for quarantine or treatment of infected individuals. Currently known detection methods include a real-time fluorescent quantitative PCR method, a serum antibody detection method, an antigen detection method, and the like. Wherein, the antigen detection method is to detect the corresponding antigen target of SARS-CoV-2 by monoclonal antibody, and is an accurate, rapid, simple and easy-to-use diagnosis method. Monoclonal antibodies, in turn, play an important role as core raw materials for antigen detection.

Disclosure of Invention

According to one aspect of the present application, there is provided a monoclonal antibody against a novel coronavirus N protein comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDRH1 with an amino acid sequence shown as SEQ ID NO. 1, CDRH2 with an amino acid sequence shown as SEQ ID NO. 2 and CDRH3 with an amino acid sequence shown as SEQ ID NO. 3. The light chain variable region comprises a CDRL1 shown in SEQ ID NO. 4, a CDRL2 shown in SEQ ID NO. 5 and a CDRL3 shown in SEQ ID NO. 6.

In some embodiments, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 7 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 8.

According to another aspect of the present application, a nucleic acid molecule is provided. The nucleic acid molecule comprising a nucleotide sequence encoding a monoclonal antibody against a novel coronavirus N protein according to claim 1 or 2.

In some embodiments, the nucleotide sequences encoding CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are shown as SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11, respectively, and the nucleotide sequences encoding CDRL1, CDRL2 and CDRL3 of the light chain variable region are shown as SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, respectively.

In some embodiments, the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID NO. 15 and the nucleotide sequence encoding the light chain variable region is set forth in SEQ ID NO. 16.

According to a further aspect of the present application there is provided the use of a monoclonal antibody as described above against a novel coronavirus N protein in the preparation of a reagent for detecting an anti-novel coronavirus N protein. The monoclonal antibody of the novel coronavirus N protein is used for immunological detection of novel coronaviruses.

According to yet another aspect of the present application, a kit for detecting a novel coronavirus is provided. The kit comprises the monoclonal antibody for resisting the novel coronavirus N protein.

Detailed Description

As used in this application and in the claims, the terms "a," "an," "the," and/or "the" are not specific to the singular, but may include the plural, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that the steps and elements are explicitly identified, and they do not constitute an exclusive list, as other steps or elements may be included in a method or apparatus.

2019-nCoV (SARS-CoV-2) has four major structural proteins: spike protein (also known as S protein), nucleocapsid protein (nucleocapsid protein, also known as N protein), membrane protein (also known as M protein) and envelope protein (also known as E protein).

The core protein of 2019-nCoV is the N protein. As one of the most important proteins within the viral nucleocapsid, the N protein is mainly responsible for the replicative function of RNA. N protein and viral genome RNA are intertwined to form a viral nucleocapsid, which plays an important role in the synthesis process of viral RNA. Meanwhile, N protein is relatively conserved, the proportion of the N protein in structural proteins of viruses is the largest, and the organism can generate high-level antibodies against the N protein in early infection.

Thus, the corresponding monoclonal antibody is prepared by using the N protein of 2019-nCoV as an antigen. Specifically, a prokaryotic expressed N protein is utilized to immunize a Balb/c mouse, spleen cells of the mouse are fused with myeloma cells, hybridoma cells with high specificity are obtained through specific high-throughput screening, a large amount of mouse ascites is obtained through culture and re-immunization, and then monoclonal antibodies with high titer, high purity, high sensitivity and high specificity, which are used for resisting novel coronavirus N proteins, are obtained through multi-step separation and purification. The anti-novel coronavirus monoclonal antibody can be used for immunological detection such as ELISA, immunochromatography, immunoblotting, immunofluorescence and the like, and provides a required raw material for immunological detection reagents (for example, an N protein detection immunoassay test strip). The detection kit developed by taking the antibody as a raw material has good application value.

The monoclonal antibody may include a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDRH1 with an amino acid sequence shown as SEQ ID NO. 1, CDRH2 with an amino acid sequence shown as SEQ ID NO. 2 and CDRH3 with an amino acid sequence shown as SEQ ID NO. 3. The light chain variable region comprises a CDRL1 shown in SEQ ID NO. 4, a CDRL2 shown in SEQ ID NO. 5 and a CDRL3 shown in SEQ ID NO. 6.

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

The present application also provides a nucleic acid molecule. The nucleic acid molecule may comprise a nucleotide sequence encoding a monoclonal antibody against the novel coronavirus N protein described above. In some embodiments, the nucleotide sequences encoding CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are set forth in SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11, respectively. The nucleotide sequences of CDRL1, CDRL2 and CDRL3 which code for the light chain variable region are shown in SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 respectively. In some embodiments, the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID NO. 15 and the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID NO. 16.

The monoclonal antibody against the novel coronavirus N protein can be applied to a reagent for detecting the novel coronavirus N protein, for example, an N protein immune test strip. In some embodiments, monoclonal antibodies directed against novel coronavirus N proteins can be used for immunological detection of novel coronaviruses, e.g., ELISA, immunochromatography, immunoblotting, immunofluorescence, and the like.

The application also provides a kit for detecting the novel coronavirus. The kit can comprise the monoclonal antibody against the novel coronavirus N protein. The detection kit developed by taking the monoclonal antibody for resisting the novel coronavirus N protein as a raw material has good application value, high sensitivity and specificity, and can detect patients suffering from the novel coronavirus at an early stage.

Examples

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Construction of recombinant plasmids

The inactivated SARS-CoV-2 strain (from the disease prevention and control center of Zhejiang province) was taken, RNA of the virus was extracted, reverse transcribed into cDNA template, and a fragment encoding N protein of 1257bp SARS-CoV-2 strain was PCR amplified using primers with BamH I and EcoR I restriction enzyme sites (forward primer: GGATCCATGTCTGATAATGGACCCCAAA (SEQ ID NO: 17) and reverse primer: GAATTCGGCCTGAGTTGAGTCAGCACTG (SEQ ID NO: 18)) according to the sequence of N protein of SARS-CoV-2 (Genebank accession number: 43740575). The PCR conditions were: pre-denatured at 94℃for 5 min, 94℃for 30 sec, 60℃for 30 sec, 72℃for 1 min, 35 cycles, 72℃for 10 min.

After obtaining the PCR product, the PCR product was ligated into pMD18T vector (purchased from TAKARA), and the ligation product was transformed into DH5a competent cells by hot-shock method, and clones were sequenced to complete correct nucleotide sequence of N protein. After the cleavage verification, the gene fragment of the N protein was cleaved with BamHI and EcoRI after the sequencing was correct, and ligated into pET28a vector (purchased from Novagen Co.) to obtain pET28a-NP recombinant plasmid. And performing enzyme digestion and sequencing verification again. The pET28a-NP plasmid with correct enzyme digestion and sequencing verification is transformed into escherichia coli BL21 (DE 3) strain to induce expression.

Preparation of N protein of SARS-CoV-2

BL21 (DE 3) containing pET28a-NP was cloned in 10mL of LB and 0.5% glucose medium containing 50. Mu.g/mL of ampicillin and chloramphenicol, cultured overnight at 37℃at 200rpm, and transferred 1:100 magnification to 500mL of the above medium the next day.

IPTG (Isopropyl. Beta. -D-Thiogalactoside, isopropyl Thiogalactoside) was added at a final concentration of 0.5mM when the cells were cultured at 37℃and 200rpm to an OD600 of about 0.6, and the cells were collected after induction at 16℃for 16 hours.

The cells were placed in 500mL fungus harvesting bottles, and after culturing at 4000rpm and 4℃for 10min, the supernatant was discarded.

15mL of a ligation buffer (20 mM sodium phosphate, 0.5M sodium chloride, 20mM imidazole, pH 7.4) was added, the cells were suspended by shaking, and the solution was sonicated (70 w, sonicated 90 times, 10s each time) at 4℃on ice (steps were performed on ice) and the pellet was removed by centrifugation after the solution was cleared.

The N protein was then collected by column chromatography with reference to GE company Glutathione Ni Sepharose, 6fast flow4b, as follows:

(1) Clarifying and filtering a sample: the prepared cell suspension supernatant was clarified using a 50ml syringe and a 0.22 μm filter;

(2) Adopting a protein chromatographic column to capture and purify on AKTA;

(3) Performing system flushing, flushing an A1 pump of AKTA by using balance liquid, and flushing a B1 pump by using eluent;

(4) Setting the flow rate of the system to be 0.1ml/min, selecting a corresponding column position No. 1 connexin chromatographic column, balancing AKTA and the column by using a balancing solution, and carrying out ultraviolet zeroing after balancing;

(5) Starting loading, and transferring the A1 pump into a loading centrifuge tube for loading;

(6) After the sample loading is finished, transferring the A1 pump into a balance buffer solution, flushing the balance buffer solution until the detection wavelength is stable, distributing and eluting, and collecting an eluent;

(7) Washing with balancing solution A, and finally washing with 20% ethanol, and preserving.

Finally, eluting the recombinant 6 XHis-tagged N protein from the column by using an elution buffer, analyzing and purifying the protein by using SDS-PAGE, and observing by using a Coomassie brilliant blue staining method to finally obtain the high-purity SARS-CoV-2 nucleocapsid protein N protein.

Preparation of monoclonal antibody Mouse anti-SARS-COV-2mAb4 (NP-Ab)

Female Balb/c mice 6-8 weeks old and healthy were selected for immunization according to a pre-specified immunization protocol. The N protein obtained in the above steps is used as an immunogen to immunize Balb/c mice, and spleen lymphocytes of the mice which are successfully immunized are extracted. Lymphocytes were fused with mouse myeloma cell SP2/0 by cell fusion techniques. After two rounds of subcloning and screening, hybridoma cell strains which stably secrete monoclonal antibodies against novel coronavirus N proteins are obtained, so that monoclonal antibodies against novel coronavirus N proteins are obtained.

Animal immunization experiments were performed using the expressed proteins described above.

The animal immunity experiment comprises the following specific steps:

1. Balb/C mice with consistent weight and age-averaged values were randomly divided into 3 groups (3 groups were divided according to the dose of the N protein of the immunizing antigen obtained in the above steps, respectively: 50 ug/mouse for the immunizing antigen contained in the group A mice, 100 ug/mouse for the immunizing antigen contained in the group B mice, 150 ug/mouse for the immunizing antigen contained in the group C mice, and a control group for each group of mice).

2. Before the start of the experiment, mice were each collected with preimmune serum, blood was taken through eyeballs, a proper amount of blood was taken to ensure the normal state of the mice), and the collected serum was stored at-80 ℃.

3. The preparation mode of the aluminum adjuvant (aluminum hydroxide adjuvant) group comprises the following steps: prior to immunization, each antigen was diluted to the corresponding dose (50 ug/mouse, 100 ug/mouse, 150 ug/mouse) in 75 μl PBS, respectively, and mixed with alum adjuvant (i.e., aluminum hydroxide adjuvant) (1 mg/mouse) at a volume antigen: adjuvant=3:1 (i.e., 25ul of adjuvant was added to 75ul of immunogen dilution); shaking the adjuvant evenly before using, and slowly dripping the injected adjuvant (25 ul) into the immunogen solution; after the adjuvant and the immunogen dilution were thoroughly mixed, the two were thoroughly mixed for 30 minutes. Allowing the adjuvant to effectively adsorb the antigen; the subsequent experiments were performed according to the experimental procedure of immunized animals.

4. Group without aluminum adjuvant: the antigen was diluted to the corresponding dose, i.e., 50 ug/mouse, 100 ug/mouse, 150 ug/mouse, respectively, in 100 μl of PBS, and the immunogen was 100 μl of the diluted antigen, followed by the following experimental procedure for immunized animals.

5. Subcutaneous injections at 2 week intervals: the experiment is designed into a 3-time immunization mode, but partial mouse supernatant is obtained by taking blood from eyeballs after 7 days of each immunization injection and centrifuging, serum antibody titer is detected first, the heart takes blood to obtain maximum blood after 7 days of the last immunization, and the supernatant is obtained by centrifuging and stored at-80 ℃. The test data after 2 immunizations are shown in table 1 below.

Table 12 serum antibody titer test data after immunization

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The immune spleen cells are fused with myeloma cell line SP2/0 cells, the fused cells are screened by a HAT selection medium (the HAT selection medium contains hypoxanthine, aminopterin and thymine), and ELISA positive screening and subcloning are carried out on the fused cells; the positive monoclonal selected is taken, ascites antibody generated by hybridoma cells is purified by Protein A/G antibody purification columns, ELISA titer of the purified ascites antibody is more than 1:128,000, purity is more than 90%, and the result is determined by detecting the binding activity of the recognition N Protein by ELISA (enzyme-linked immunosorbent assay) described below.

ELISA detection of binding Activity of recognition N protein

1. IgG antibody titer detection mode

(1) And (3) coating a bottom plate: the antigen used was diluted to 3ug/ml with coating dilution, 100 ul of the prepared coating was added to each well, and the mixture was placed in a refrigerator at 4℃for 24 hours.

(2) After 24h, taking out from the refrigerator, balancing at 37 ℃ for 30min, and then discarding the liquid in the holes; washing the wells with washing liquid for 3 times and 3min each time.

(3) Closing the enzyme-labeled reaction hole: 200ul of 5% calf serum is added into each hole, the holes are sealed for 90min at 37 ℃, and after the sealing is finished, the holes are washed 3 times by washing liquid, and each time is 3min.

(4) Adding a sample to be detected: diluting the sample according to the required proportion, adding the diluted sample into enzyme-labeled reaction holes, placing 100 mu l of each hole at 37 ℃ for 90min; washing the wells with washing liquid for 3 times and 3min each time.

(5) Adding enzyme-labeled antibody: adding secondary antibody with proper concentration according to the specification; 100 μl of wash was applied to each well at 37deg.C for 90 min.

(6) Adding a substrate solution: the substrate is added in an amount of 100 mu l per hole, and the mixture is placed at 37 ℃ and protected from light for 15-30 min.

(7) Terminating the reaction: the reaction was stopped by adding 50. Mu.l of stop solution to each well, and the experimental results were measured within 20 min.

Detection of binding Activity of monoclonal antibody MOUSE ANTI-SARS-COV-2MAB4 (NP-AB) to recognize N protein

(1) Cell fusion and clone screening data

3 rounds of fusion are carried out, and the numbers of the mice are A1, B2 and C0 in sequence; selecting 26 positive clones for subcloning by fusion screening of the A1 mice, and finally completing 17 cell strains; and (3) performing fusion screening, namely selecting 160 positive clones with OD450 value more than 2.2, performing multiple ratio dilution detection titer, and performing secondary and tertiary subclone screening. 6 cell lines, designated A1-1 to A1-6, were obtained. And B2 mice are subjected to fusion screening, 76 positive holes are selected for subcloning, 120 positive holes with OD450 value more than 2.1 are selected for multiple ratio dilution detection titer, and then secondary and third subcloning screening is performed, so that 4 cell strains are finally completed, and the cell strains are respectively named as B2-1 to B2-4. The C0 mice were fused and positive wells were not screened.

(2) Ascites preparation and test data

A total of 10 hybridoma cell lines, 3F 1 mice per complete cell line, were prepared for a total of 10 ascites fluid, all of which were assayed for potency data as shown in table 2 below:

TABLE 2 ascites antibody assay potency data

(3) Investigation of antibody purification conditions and detection data

The above ascites was purified by 3.3% n-octanoic acid-thiamine precipitation to give 10 antibodies in total, and the potency detection data for all antibodies are shown in table 3 below:

TABLE 3 potency detection data for all antibodies

Dilution concentration of antibody	A1-1	A1-2	A1-3	A1-4	A1-5	A1-6	B2-1	B2-2	B2-3	B2-4
											20ug/ml	3.153	3.391	3.163	3.288	2.887	3.058	3.116	3.25	3.183	3.082
4ug/ml	3.204	3.204	3.028	3.107	3.153	3.153	3.238	3.238	3.183	3.014
											800ug/ml	3.375	3.074	2.709	3.484	2.914	3.05	3.066	3.007	3.09	2.591
160ng/ml	2.364	3.028	2.798	3.426	2.413	2.576	3.344	2.48	3.074	2.765
											32ng/ml	1.561	2.047	1.978	2.197	1.682	1.569	2.648	1.528	2.412	2.129
6.4ng/ml	1.309	1.151	0.633	1.785	0.581	0.496	1.865	1.744	1.603	0.797
											1.28ng/ml	0.606	0.461	0.194	1.3	0.249	0.23	1.252	0.616	0.775	0.392
PBS	0.084	0.046	0.072	0.051	0.067	0.063	0.052	0.113	0.044	0.118

According to Table 3 above, antibody A1-1 was designated as Mouse anti-SARS-COV-2Mab4 (NP-Ab) antibody, which has high titer, high purity, high sensitivity and high specificity. The monoclonal antibody Mouse anti-SARS-COV-2Mab4 (NP-Ab) can be used for ELISA, immunochromatography, immunoblotting, immunofluorescence and other immunological detection.

To examine whether the Mouse anti-SARS-COV-2Mab4 (NP-Ab) antibody specifically binds to the nucleocapsid protein (nucleocapsid protein, also known as N protein) of the novel coronavirus 2019-nCoV (SARS-CoV-2), the N protein of SARS virus (SARS-CoV Nucleocapsid Protein (His Tag), cat# 40143-V08B) and the N protein of MERS virus (MERS-CoV Nucleocapsid Protein (His Tag), cat# 40068-V08B) were purchased from Beijing Yizhuzhushen corporation. Detection of whether the Mouse anti-SARS-COV-2Mab4 (NP-Ab) antibody bound to SARS-CoV-NP and MERS-CoV-NP by ELISA, the results of the cross-assay are shown in Table 4 below:

TABLE 4 Cross-test results data

The above data shows that monoclonal antibody Mouse anti-SARS-COV-2Mab4 (NP-Ab) has good specific binding capacity only to 2019-nCov-NP-2 antigen (i.e., novel coronavirus N protein), but does not bind SARS-CoV-NP and MERS-CoV-NP antigens, and is specific for detecting novel coronaviruses.

Sequence analysis of heavy chain variable region and light chain variable region of monoclonal antibody Mouse anti-SARS-COV-2Mab4 (NP-Ab)

Primers were designed to amplify the heavy chain variable region and light chain variable region genes of monoclonal antibody Mouse anti-SARS-COV-2Mab4 (NP-Ab).

Hybridoma cell strain (about 10) in logarithmic growth phase of monoclonal antibody Mouse anti-SARS-COV-2Mab4 (NP-Ab) ⁷ And (3) extracting total RNA of the cells according to the instruction of the Trizol RNA extraction kit, performing reverse transcription to synthesize a first cDNA chain by taking the total RNA as a template, and amplifying heavy chain variable region genes and light chain variable region genes of the antibody by taking the amplified product as the template through PCR.

Heavy chain variable region and light chain variable region gene fragments of monoclonal antibody Mouse anti-SARS-COV-2Mab4 (NP-Ab) were recovered and sequenced. Sequencing results were as follows:

the nucleotide sequence (345 bp) encoding the heavy chain variable region of the monoclonal antibody MOUSE ANTI-SARS-COV-2MAB4 (NP-AB) is as follows:

GTGAAGCTGCAGGAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAGGATC TCCTGCAAGGCTTCTGGTTATACCCTCACAGACTATTCAATACACTGGGTGAAGCAGGC TCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGTTGAGCCAACA TATACAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGC CTATTTGCAAATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCCTCAA CTGGGACGGAATTTGACTACTGGGGCCAGGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:15)。

the sequence of CDRH1 encoding the heavy chain variable region is: GGTTATACCCTCACAGACTATTCA (SEQ ID NO: 9).

The sequence of CDRH2 encoding the heavy chain variable region is: ATAAACACTGAGACTGTTGAGCCA (SEQ ID NO: 10).

The sequence of CDRH3 encoding the heavy chain variable region is: GCCTCAACTGGGACGGAATTTGACTAC (SEQ ID NO: 11).

The amino acid sequence (115 aa) of the heavy chain variable region is shown below:

VKLQESGPELKKPGETVRISCKASGYTLTDYSIHWVKQAPGKGLKWMGWINTETVEPTYT DDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCASTGTEFDYWGQGTTLTVSS(SEQ ID NO:7)。

the amino acid sequence of CDRH1 of the heavy chain variable region is: GYTLTDYS (SEQ ID NO: 1).

The amino acid sequence of CDRH2 of the heavy chain variable region is: INTETYEP (SEQ ID NO: 2).

The amino acid sequence of CDRH3 of the heavy chain variable region is: ASTGTEFDY (SEQ ID NO: 3).

The nucleotide sequence (336 bp) encoding the light chain variable region of the monoclonal antibody MOUSE ANTI-SARS-COV-2MAB4 (NP-AB) is shown below:

GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTC CATCTCTTGCAGATCTAGTCAGAGCCTTGTACACATTACTGGAAACACCTATTTACATT GGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCG ATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTC AAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACAC ATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:16)。

the sequence of CDRL1 encoding the light chain variable region is: CAGAGCCTTGTACACATTACTGGAAACACCTATTTACATTGGTAC (SEQ ID NO: 12).

The sequence of CDRL2 encoding the light chain variable region is: AAAGTTTCC (SEQ ID NO: 13).

The sequence of CDRL3 encoding the light chain variable region is: TCTCAAAGTACACATGTTCCGCTCACG (SEQ ID NO: 14).

The amino acid sequence of the light chain variable region (112 aa) is shown below:

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHITGNTYLHWYLQKPGQSPKLLIYKVSNRFSG VPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELK(SEQ ID NO:8)。

the amino acid sequence of CDRL1 of the light chain variable region is: QSLVHITGNTY (SEQ ID NO: 4).

The amino acid sequence of CDRL2 of the light chain variable region is: KVS (SEQ ID NO: 5).

The amino acid sequence of CDRL3 of the light chain variable region is: SQSTHVPLT (SEQ ID NO: 6).

One of the benefits of the present disclosure includes, but is not limited to: (1) The monoclonal antibody of the novel coronavirus N protein has high titer, high purity, high sensitivity and high specificity; (2) The monoclonal antibody of the novel coronavirus N protein can be used for immunoblotting, immunofluorescence and other immunological detection, and provides a required raw material for immunological detection reagents (for example, an N protein detection immunoassay test strip). It should be noted that, the advantages that may be generated by different embodiments may be different, and in different embodiments, the advantages that may be generated may be any one or a combination of several of the above, or any other possible advantages that may be obtained.

It will be appreciated by those skilled in the art that the above examples are illustrative of the invention and are not to be construed as limiting the invention. Any modifications, equivalent substitutions and variations, etc., which are within the spirit and principles of the present invention, are intended to be included within the scope of the present invention.

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<211> 9

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

aaagtttcc 9

<210> 14

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

tctcaaagta cacatgttcc gctcacg 27

<210> 15

<211> 345

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gtgaagctgc aggagtctgg acctgagctg aagaagcctg gagagacagt caggatctcc 60

tgcaaggctt ctggttatac cctcacagac tattcaatac actgggtgaa gcaggctcca 120

ggaaagggtt taaagtggat gggctggata aacactgaga ctgttgagcc aacatataca 180

gatgacttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg 240

caaatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgcctc aactgggacg 300

gaatttgact actggggcca gggcaccact ctcacagtct cctca 345

<210> 16

<211> 336

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagccttgta cacattactg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

ctcacgttcg gtgctgggac caagctggag ctgaaa 336

<210> 17

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ggatccatgt ctgataatgg accccaaa 28

<210> 18

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

gaattcggcc tgagttgagt cagcactg 28

Claims

1. A monoclonal antibody against novel coronavirus N protein, which is characterized by comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises CDRH1 with an amino acid sequence shown as SEQ ID NO. 1, CDRH2 with an amino acid sequence shown as SEQ ID NO. 2 and CDRH3 with an amino acid sequence shown as SEQ ID NO. 3; and

the light chain variable region comprises a CDRL1 shown in SEQ ID NO. 4, a CDRL2 shown in SEQ ID NO. 5 and a CDRL3 shown in SEQ ID NO. 6.

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 7 and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 8.

3. A nucleic acid molecule comprising a nucleotide sequence encoding the monoclonal antibody against the novel coronavirus N protein of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequences encoding CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are shown as SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11, respectively, and the nucleotide sequences encoding CDRL1, CDRL2 and CDRL3 of the light chain variable region are shown as SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, respectively.

5. The nucleic acid molecule of claim 4, wherein the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID NO. 15 and the nucleotide sequence encoding the light chain variable region is set forth in SEQ ID NO. 16.

6. Use of a monoclonal antibody against a novel coronavirus N protein according to claim 1 or 2 for the preparation of a reagent for detecting an anti-novel coronavirus N protein.

7. The use according to claim 6, wherein said monoclonal antibody against the novel coronavirus N protein is used for immunological detection of novel coronaviruses.

8. A kit for detecting a novel coronavirus, comprising the monoclonal antibody against the novel coronavirus N protein of claim 1 or 2.