CN113912710A

CN113912710A - Monoclonal antibody for resisting novel coronavirus N protein and application thereof

Info

Publication number: CN113912710A
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: 2022-01-11
Anticipated expiration: 2041-11-17
Also published as: CN113912710B

Abstract

The invention provides a monoclonal antibody for resisting novel coronavirus N protein and application thereof. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises a CDRH1 with the amino acid sequence shown as SEQ ID NO. 1, a CDRH2 with the amino acid sequence shown as SEQ ID NO. 2 and a CDRH3 with the 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 coding the monoclonal antibody, application of the monoclonal antibody in preparing a reagent for detecting the N protein of the novel coronavirus and a kit for detecting the novel coronavirus containing 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 coronavirus is a public health problem currently of global concern. On day 11/2 of 2020, the new coronavirus was named SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, also known as 2019-nCoV (2019 new coronavirus)) by the international committee for classification of viruses, and on the same day, the world health organization named covi-19 for pneumonia infected by the virus. SARS-CoV-2 is a zoonotic pathogen of animal origin, as well as Severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV). Although coronaviruses are commonly associated with acute respiratory infections in humans, their ability to infect a variety of host species makes them complex pathogens. The great popularity of COVID-19 caused by the novel coronavirus (SARS-CoV-2) is devastating worldwide and causes millions of hospitalizations and deaths.

Therefore, there is a need to establish a method for rapid screening and detection of novel coronaviruses to isolate or treat infected persons. Currently known detection methods include a real-time fluorescence 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. The monoclonal antibody plays an important role as a core raw material of an antigen detection method.

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 a CDRH1 with the amino acid sequence shown as SEQ ID NO. 1, a CDRH2 with the amino acid sequence shown as SEQ ID NO. 2 and a CDRH3 with the 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 heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 7 and the light chain variable region has the amino acid sequence shown in SEQ ID NO. 8.

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

In some embodiments, the nucleotide sequences of CDRH1, CDRH2, and CDRH3 encoding the heavy chain variable region are shown in SEQ ID NOs 9, 10, and 11, respectively, and the nucleotide sequences of CDRL1, CDRL2, and CDRL3 encoding the light chain variable region are shown in SEQ ID NOs 12, 13, and 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 the above monoclonal antibody against the N protein of the novel coronavirus for the preparation of a reagent for the detection of the N protein of the novel coronavirus. The monoclonal antibody against the novel coronavirus N protein is used for immunological detection of the novel coronavirus.

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 the appended claims, the terms "a," "an," "the," and/or "the" are not intended to be inclusive in the singular, but rather are intended to be inclusive in the plural unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that steps and elements are included which are explicitly identified, that the steps and elements do not form an exclusive list, and that a method or apparatus may include other steps or elements.

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

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 primarily responsible for the replication function of RNA. The N protein and the virus genome RNA are intertwined to form the virus nucleocapsid, which plays an important role in the synthesis process of the virus RNA. Meanwhile, the N protein is relatively conserved and accounts for the largest proportion in the structural proteins of the virus, and an organism can generate high-level antibodies for resisting the N protein in the early infection stage.

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

The monoclonal antibody may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises a CDRH1 with the amino acid sequence shown as SEQ ID NO. 1, a CDRH2 with the amino acid sequence shown as SEQ ID NO. 2 and a CDRH3 with the 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 of CDRH1, CDRH2 and CDRH3 encoding the heavy chain variable region are shown in SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, respectively. The nucleotide sequences of CDRL1, CDRL2 and CDRL3 encoding 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, such as an immune test strip for detecting the N protein. In some embodiments, monoclonal antibodies against the N protein of the novel coronavirus may be used for immunological detection of the novel coronavirus, e.g., ELISA, immunochromatography, immunoblotting, immunofluorescence, and the like.

The application also provides a kit for detecting the novel coronavirus. The kit may comprise the above monoclonal antibody against the N protein of the novel coronavirus. The detection kit developed by taking the monoclonal antibody 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 procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Construction of recombinant plasmids

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

After obtaining the PCR product, the PCR product was ligated to pMD18T vector (purchased from TAKARA), and then the ligation product was transformed into DH5a competent cells by heat shock method, and the nucleotide sequence of N protein was selected by sequencing to select a clone having the perfect nucleotide sequence. After enzyme digestion verification, the gene fragment of the N protein was digested with BamH I and EcoR I after sequencing was correctly, and ligated into pET28a vector (purchased from Novagen) to obtain pET28a-NP recombinant plasmid. And carrying out enzyme digestion and sequencing verification again. The correct pET28a-NP plasmid verified by enzyme digestion and sequencing is transformed into an Escherichia coli BL21(DE3) strain for induction expression.

Preparation of N protein of SARS-CoV-2

BL21(DE3) clone containing pET28a-NP was cultured overnight (200rpm) at 37 ℃ in 10mL of LB and 0.5% glucose medium containing 50. mu.g/mL of ampicillin and chloramphenicol, and the next day was transferred to 500mL of the above medium at 1:100 magnification.

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

The cells were placed in a 500 mL-volume strain receiving flask, cultured at 4000rpm at 4 ℃ for 10min, and the supernatant was discarded.

15mL of a ligation buffer (20mM sodium phosphate, 0.5M sodium chloride, 20mM imidazole, pH7.4) was added, the cells were suspended with shaking, placed on ice (all subsequent steps were performed on ice), sonicated (70w, 90 cycles with sonication, 10s each) at 4 ℃ until the solution cleared, and the pellet was removed by centrifugation.

The N protein was then collected on a column using a guide with reference to GE Glutathione Ni Sepharose 6Fast Flow4B as follows:

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

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

(3) carrying out system flushing, flushing an A1 pump of AKTA by using a balancing solution, and flushing a B1 pump by using an 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 adjusting ultraviolet to zero after balancing is finished;

(5) starting loading, transferring an A1 pump to a loading centrifugal tube for loading;

(6) after the sample loading is finished, transferring the A1 pump into the equilibrium buffer solution, flushing the equilibrium solution until the detection wavelength is stable, then distributing and eluting, and collecting the eluent;

(7) washing with balance solution A, washing with 20% ethanol, and storing.

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

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

Healthy female Balb/c mice of 6-8 weeks of age were selected for immunization injection according to a pre-specified immunization schedule. And (3) taking the N protein obtained in the step as an immunogen to immunize Balb/c mice, and extracting mouse spleen lymphocytes successfully immunized. Lymphocytes were fused with mouse myeloma cells SP2/0 by cell fusion techniques. After two rounds of subcloning and screening, hybridoma cell strains which stably secrete the monoclonal antibody resisting the novel coronavirus N protein are obtained, so that the monoclonal antibody resisting the novel coronavirus N protein is obtained.

Animal immunization experiments were performed using the aforementioned expressed proteins.

The animal immunization experiment comprises the following specific steps:

1. Balb/C mice with consistent body weight and week age mean were randomly divided into 3 groups (3 groups were divided based on the dose of the immunizing antigen N protein obtained in the above procedure, respectively: 50 ug/mouse of the immunizing antigen contained in the A group of mice, 100 ug/mouse of the immunizing antigen contained in the B group of mice, 150 ug/mouse of the immunizing antigen contained in the C group of mice, each group of mice having a control group).

2. Before the experiment, the mice collect serum before immunization, blood is taken through eyeballs, and proper amount of blood is taken to ensure the normal state of the mice) as negative control, and the collected serum is stored at-80 ℃.

3. The formulation of the aluminum adjuvant (aluminum hydroxide adjuvant) group is as follows: before immunization, each antigen was diluted individually to the corresponding dose (50 ug/mouse, 100 ug/mouse, 150 ug/mouse) in 75 μ L PBS and mixed with alum adjuvant (i.e. aluminum hydroxide adjuvant) (1 mg/mouse) in a volume of antigen: adjuvant 3:1 (i.e. 25ul adjuvant was added to 75ul immunogen dilution); shaking the adjuvant before use, and slowly dripping the injection adjuvant (25ul) into the immunogen solution; after the adjuvant and the immunogen diluent were mixed thoroughly, both were mixed thoroughly for 30 minutes. Allowing the adjuvant to effectively adsorb the antigen; the subsequent steps are carried out according to the experimental operation of the immune animals.

4. Group without aluminum adjuvant: the antigen is respectively diluted to corresponding doses in 100 mu L PBS, namely 50 ug/mouse, 100 ug/mouse and 150 ug/mouse, and the immunogen is 100 mu L diluted antigen, and then the test is carried out according to the experimental operation of the immune animal.

5. Subcutaneous injections were performed at 2 week intervals: the experiment is designed in a 3-time immunization mode, but eyeball blood is taken 7 days after each immunization injection, partial mouse supernatant is obtained by a centrifugation method, the serum antibody titer is firstly detected, the heart blood is taken 7 days after the last immunization, the maximum blood volume is taken, and the supernatant is obtained by centrifugation and stored at-80 ℃. The data of the assay after 2 immunizations are shown in table 1 below.

Table 12 serum antibody titer test data after immunization

Fusing immune splenocytes with myeloma cell line SP2/0, screening fused cells by HAT selection medium (HAT selection medium contains hypoxanthine, aminopterin and thymidine), and performing ELISA positive screening and subcloning on the fused cells; and (3) taking ascites from the screened positive monoclonal, purifying the ascites antibody generated by the hybridoma cells by using a Protein A/G antibody purification column, wherein the ELISA titer of the purified ascites antibody is more than 1:128,000, the purity is more than 90%, and the result is determined by the detection and recognition of the binding activity of the N Protein through enzyme-linked reaction ELISA described in the following step.

Enzyme-linked immunosorbent assay (ELISA) for detecting and identifying binding activity of N protein

1. IgG antibody titer detection mode

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

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

(3) And (3) sealing the enzyme-labeled reaction hole: adding 200ul of 5% calf serum into each well, sealing at 37 deg.C for 90min, and washing with washing solution for 3 times (each time for 3 min).

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

(5) Adding an enzyme-labeled antibody: adding a secondary antibody with a proper concentration according to the instruction; the wells were washed at 37 ℃ for 90min with 100. mu.l/well as before.

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

(7) And (3) terminating the reaction: the reaction was stopped by adding 50. mu.l of stop solution to each well and the results were measured within 20 min.

Detection of the binding Activity of monoclonal antibody MOUSE ANTI-SARS-COV-2MAB4(NP-AB) for recognizing N protein

(1) Cell fusion and clonal screening data

3 times of fusion are carried out, and the mouse numbers are A1, B2 and C0 in sequence; a1 mouse fusion screening picks up 26 positive clones for subcloning, and finally 17 cell strains are completely obtained; through fusion screening, 160 positive clones with OD450 value greater than 2.2 are selected for detecting titer by multiple dilution, and then the second and third subclone screening is carried out. 6 cell lines were obtained and designated A1-1 to A1-6, respectively. B2 mouse fusion screening selects 76 positive holes for subcloning, selects 120 positive holes with OD450 value more than 2.1 for detecting titer by multiple dilution, and performs secondary and third subcloning screening to complete 4 cell strains, which are respectively named as B2-1 to B2-4. C0 mice were fused and positive wells were not screened.

(2) Ascites preparation and test data

A1 mouse and B2 mouse 10 hybridoma cell lines in total, each complete cell line was used to prepare 3F 1 mice, 10 ascites were prepared, and the titer data of all ascites tests are shown in the following table 2:

TABLE 2 ascites antibody test titer data

(3) Antibody purification condition exploration and detection data

The ascites fluid is purified by a 3.3% caprylic acid-thiamine precipitation method to obtain 10 antibodies in total, and the titer detection data of all the antibodies are shown in the following table 3:

TABLE 3 titer test data for all antibodies

Diluted 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 the above Table 3, the antibody A1-1 was named 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 immunological detection such as ELISA, immunochromatography, immunoblotting, immunofluorescence and the like.

To examine whether the Mouse anti-SARS-COV-2Mab4(NP-Ab) antibody specifically binds to the Nucleocapsid Protein (also referred to as N Protein) of the New crown Virus 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 Quiny Qianzhou. Whether the Mouse anti-SARS-COV-2Mab4(NP-Ab) antibody binds to SARS-CoV-NP and MERS-CoV-NP was detected by enzyme-linked immunosorbent ELISA, and the data of the results of the crossover experiment are shown in Table 4 below:

table 4 cross experimental results data

The above data show 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 to SARS-CoV-NP and MERS-CoV-NP antigens, and is specific for detecting novel coronavirus.

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

Primers for amplifying heavy chain variable region and light chain variable region genes of monoclonal antibody Mouse anti-SARS-COV-2Mab4(NP-Ab) are designed.

Taking hybridoma cell strain (about 10) of monoclonal antibody Mouse anti-SARS-COV-2Mab4(NP-Ab) in logarithmic growth phase⁷Individual cells) are extracted according to the instructions of the Trizol RNA extraction kit, total RNA is used as a template to carry out reverse transcription to synthesize a first chain of cDNA, and the amplified product is used as the template to carry out PCR amplification on the heavy chain variable region gene and the light chain variable region gene of the antibody.

The 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. The sequencing results were as follows:

the nucleotide sequence (345bp) of the heavy chain variable region encoding 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 of the heavy chain variable region (115aa) is as follows:

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: INTETVEP (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 (336bp) encoding the light chain variable region of monoclonal antibody MOUSE ANTI-SARS-COV-2MAB4(NP-AB) is as follows:

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

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

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

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

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

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).

The beneficial effects brought by the method disclosed by the application include but are not limited to: (1) the monoclonal antibody for resisting the novel coronavirus N protein has high titer, high purity, high sensitivity and high specificity; (2) the monoclonal antibody for resisting the novel coronavirus N protein can be used for immunological detection such as immunoblotting and immunofluorescence, and provides a required raw material for an immunological detection reagent (for example, an immunological test strip for detecting the N protein). It is to be noted that different embodiments may produce different advantages, and in different embodiments, any one or combination of the above advantages may be produced, or any other advantages may be obtained.

It should be understood by those skilled in the art that the above examples are only illustrative and not limiting of the present invention. Any modification, equivalent replacement, and variation made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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<213> Artificial Sequence (Artificial Sequence)

<400> 12

cagagccttg tacacattac tggaaacacc tatttacatt ggtac 45

<210> 13

<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 a novel coronavirus N protein, comprising a heavy chain variable region and a light chain variable region,

the heavy chain variable region comprises a CDRH1 with an amino acid sequence shown as SEQ ID NO. 1, a CDRH2 with an amino acid sequence shown as SEQ ID NO. 2 and a 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 according to claim 1, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO. 7 and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 8.

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

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequences of CDRH1, CDRH2 and CDRH3 encoding the heavy chain variable region are shown in SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, respectively, and the nucleotide sequences of CDRL1, CDRL2 and CDRL3 encoding the light chain variable region are shown in 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 represented by SEQ ID NO. 15 and the nucleotide sequence encoding the light chain variable region is represented by 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 the detection of an anti-novel coronavirus N protein.

7. Use according to claim 6, characterized in that the monoclonal antibodies against the N protein of the novel coronavirus are used in the immunological detection of the novel coronavirus.

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