CN111909261B

CN111909261B - New coronavirus RBD specific monoclonal antibody and application

Info

Publication number: CN111909261B
Application number: CN202010839228.5A
Authority: CN
Inventors: 胡超; 王应明; 韩晓建; 高凤霞; 申美莹; 李罗; 金艾顺
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2020-08-19
Filing date: 2020-08-19
Publication date: 2022-10-04
Anticipated expiration: 2040-08-19
Also published as: CN114920833B; CN111909261A; CN115925903A; CN114920833A

Abstract

The invention belongs to the technical field of monoclonal antibodies, and particularly discloses a new coronavirus RBD specific monoclonal antibody and application of the new coronavirus RBD specific monoclonal antibody. The invention has important scientific significance and application prospect for the prevention and clinical treatment of diseases caused by the novel coronavirus SARS-CoV-2 and the research and development of diagnostic reagents.

Description

New coronavirus RBD specific monoclonal antibody and application

Technical Field

The invention belongs to the technical field of monoclonal antibodies, and particularly relates to a novel coronavirus RBD specific monoclonal antibody and application thereof.

Background

Antibodies are immunoglobulin molecules composed of four polypeptide chains, including two heavy chains (H chains) and two light chains (L chains). The H chain consists of a heavy chain variable region (VH) and a heavy chain constant region consisting of three regions, CH1, CH2 and CH 3. The L chain consists of an L variable region (VL) and a light chain constant region consisting of a CL region. VH and VL can be further divided into hypervariable regions called Complementarity Determining Regions (CDRs) and conserved regions called Framework Regions (FR) that alternate.

The research shows that: the novel coronavirus (SARS-CoV-2) has four major structural proteins, spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein), respectively, wherein the S protein has two subunits: s1 and S2, receptor Binding Sites (RBDs) are located on the S1 subunit, and their primary function is to recognize host cell surface receptors, mediating fusion with host cells.

At present, specific medicine targeted treatment is not available for new pathogen COVID-19, and the research and development of vaccines need a new day. The plasma of a patient who is cured and discharged recently contains high-concentration specific antigen neutralizing antibodies, and after the plasma is input into the body of the patient, the plasma can neutralize new corona pathogens and mediate effective immune response, so that the plasma in the recovery period is expected to provide an effective treatment means for treating the patient infected with new corona viruses, the death rate is reduced, and the life safety of the patient is guaranteed.

Chinese patent application publication No. CN111303280A discloses a fully human monoclonal antibody against SARS-CoV-2 with high neutralizing activity, which provides a fully human monoclonal antibody with a recognition region of S1 non-RBD region, but the obtained fully human monoclonal antibody has limited virus blocking effect because the new coronavirus invades into the host cell and binds to ACE2 of the host cell through RBD, and the obtained antibody cDNA is obtained by labeling plasma cells, but compared with plasma cells, the memory B cells react rapidly after being activated, so the memory B cells can induce a humoral immune response faster and stronger than the primary response, and the humoral immune response induced by the plasma cells is limited.

Disclosure of Invention

The invention aims to provide a novel coronavirus RBD specific monoclonal antibody which is directed against RBD and can trigger stronger humoral immune response and application thereof.

In order to achieve the aim, the invention provides a new monoclonal antibody specific to coronavirus RBD, and particularly, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 1; the light chain amino acid sequence is shown as SEQ ID NO:2 (monoclonal antibody 1-CQTS 091). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 3; the light chain amino acid sequence can also be shown as SEQ ID NO:4 (monoclonal antibody 2-CQTS 092). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 5; the light chain amino acid sequence may also be shown as SEQ ID NO:6 (mAb 3-CQTS 093). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 7; the light chain amino acid sequence can also be shown as SEQ ID NO:8 (monoclonal antibody 4-CQTS 094). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 9; the light chain amino acid sequence can also be shown as SEQ ID NO:10 (monoclonal antibody 5-CQTS 097). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 11; the light chain amino acid sequence can also be shown as SEQ ID NO:12 (monoclonal antibody 6-CQTS 098). The heavy chain amino acid sequence can also be shown as SEQ ID NO 13; the light chain amino acid sequence can also be shown as SEQ ID NO:14 (monoclonal antibody 7-CQTS 099). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 15; the light chain amino acid sequence may also be shown as SEQ ID NO:16 (mAb 8-CQTS 100). The heavy chain amino acid sequence can also be shown as SEQ ID NO 17; the light chain amino acid sequence may also be shown as SEQ ID NO:18 (mAb 9-CQTS 101). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 19; the light chain amino acid sequence may also be shown as SEQ ID NO:20 (mAb 10-CQTS 102). The heavy chain amino acid sequence can also be shown as SEQ ID NO 21; the light chain amino acid sequence can also be shown as SEQ ID NO:22 (monoclonal antibody 11-CQTS 103). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 23; the light chain amino acid sequence can also be shown as SEQ ID NO:24 (monoclonal antibody 12-CQTS 104). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 25; the light chain amino acid sequence may also be shown as SEQ ID NO:26 (mAb 13-CQTS 105). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 27; the light chain amino acid sequence may also be shown as SEQ ID NO:28 (mAb 14-CQTS 106). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 29; the light chain amino acid sequence can also be shown as SEQ ID NO:30 (monoclonal antibody 15-CQTS 107). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 31; the light chain amino acid sequence can also be shown as SEQ ID NO:32 (monoclonal antibodies 16-CQTS 108). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 33; the light chain amino acid sequence may also be shown as SEQ ID NO:34 (mAb 17-CQTS 109). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 35; the light chain amino acid sequence can also be shown as SEQ ID NO:36 (mAb 18-CQTS 110). The heavy chain amino acid sequence can also be shown as SEQ ID NO 37; the light chain amino acid sequence can also be shown as SEQ ID NO:38 (mAb 19-CQTS 111). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 39; the light chain amino acid sequence may also be shown as SEQ ID NO:40 (mAb 20-CQTS 112).

The invention also provides the application of the monoclonal antibody with the RBD specificity of the new coronavirus in the preparation of a reagent or a vaccine or a medicament for detecting or diagnosing SARS-CoV-2, wherein the medicament comprises the monoclonal antibody with the RBD specificity of the new coronavirus and a pharmaceutically acceptable excipient, diluent or carrier; also provides a nucleic acid molecule for encoding the new coronavirus RBD specific monoclonal antibody; also provides an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule; also provides the application of the expression cassette, the recombinant vector, the recombinant bacterium or the transgenic cell line in the preparation of products.

The invention also provides a product which comprises the new coronavirus RBD specific monoclonal antibody; the product application is any one of the following (b 1) to (b 4): (b 1) binds to novel coronavirus SARS-CoV-2; (b 2) detecting binding of the novel coronavirus SARS-CoV-2; (b 3) binding to the S protein of the novel coronavirus SARS-CoV-2; (b 4) detecting the S protein of the novel coronavirus SARS-CoV-2.

Preferably, the RBD-specific monoclonal antibody of the new coronavirus is obtained by sorting RBD-specific memory B cells and obtaining antibody variable region cDNA from mRNA of the RBD-specific memory B cells.

The principle and the beneficial effects of the invention are as follows:

(1) The monoclonal antibody provided by the invention has RBD specificity, and compared with a monoclonal antibody aiming at an S1 non-RBD region, the monoclonal antibody provided by the invention is combined with RBD, thereby providing wider application value for screening antibody medicines, diagnosing, preventing and treating new coronary pneumonia.

(2) The monoclonal antibody provided by the invention is obtained by sorting RBD specific memory B cells, and compared with the prior art of sorting plasma cells, the monoclonal antibody prepared by the invention can initiate stronger humoral immune response. In addition, the invention only aims at RBD specific memory B cells to carry out subsequent RT-PCR, nested PCR and antibody function analysis, thereby greatly improving the specific binding capacity of the monoclonal antibody and the RBD.

Drawings

FIG. 1 is a cell sorting graph of RBD specific memory B cells analyzed by flow cytometry;

FIG. 2 is a diagram of cell sorting by flow cytometry analysis of RBD-specific memory B cells;

FIG. 3 is a gel electrophoresis diagram of a PCR product of a single-cell antibody gene;

FIG. 4 is a photograph of agarose gel electrophoresis following PCR amplification of an antibody gene expression cassette containing the CMV promoter, WPRE-gamma or WPRE-kappa element;

FIG. 5 is a graph showing the result of RBD-specific detection.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This example provides a monoclonal antibody specific to the novel coronavirus RBD, the heavy chain amino acid sequence is shown in SEQ ID NO 1; the light chain amino acid sequence is shown as SEQ ID NO. 2.

The embodiment also provides the application of the new coronavirus RBD specific monoclonal antibody in the preparation of a reagent or a medicament for detecting or diagnosing SARS-CoV-2.

In practical production, the RBD-specific monoclonal antibody obtained in this embodiment can be used to prepare a nucleic acid molecule, or prepare an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule, or prepare a pharmaceutical composition comprising the above-mentioned novel coronavirus RBD-specific monoclonal antibody and a pharmaceutically acceptable excipient, diluent or carrier.

In application, the related product prepared by the RBD specific monoclonal antibody obtained in the embodiment can have the following application of any one of (b 1) to (b 4): (b 1) binds to novel coronavirus SARS-CoV-2; (b 2) detecting binding of the novel coronavirus SARS-CoV-2; (b 3) binding to the S protein of the novel coronavirus SARS-CoV-2; (b 4) detecting the S protein of the novel coronavirus SARS-CoV-2.

Examples 2 to 20

Examples 2-20 differ from example 1 in that: the amino acid sequences of the RBD-specific monoclonal antibodies were varied, and the amino acid sequences of examples 2-20 are shown in the following table:

experimental group	Heavy chain amino acid sequence	Light chain amino acid sequence
			Example 1 CQTS091	SEQ ID NO:1	SEQ ID NO:2
Example 2 CQTS092	SEQ ID NO:3	SEQ ID NO:4
			Example 3 CQTS093	SEQ ID NO:5	SEQ ID NO:6
Example 4 CQTS094	SEQ ID NO:7	SEQ ID NO:8
			Example 5 CQTS097	SEQ ID NO:9	SEQ ID NO:10
Example 6 CQTS098	SEQ ID NO:11	SEQ ID NO:12
			Example 7 CQTS099	SEQ ID NO:13	SEQ ID NO:14
Example 8 CQTS100	SEQ ID NO:15	SEQ ID NO:16
			Example 9 CQTS101	SEQ ID NO:17	SEQ ID NO:18
Example 10 CQTS102	SEQ ID NO:19	SEQ ID NO:20
			Example 11 CQTS103	SEQ ID NO:21	SEQ ID NO:22
Example 12 CQTS104	SEQ ID NO:23	SEQ ID NO:24
			Example 13 CQTS105	SEQ ID NO:25	SEQ ID NO:26
Example 14 CQTS106	SEQ ID NO:27	SEQ ID NO:28
			Example 15 CQTS107	SEQ ID NO:29	SEQ ID NO:30
Example 16 CQTS108	SEQ ID NO:31	SEQ ID NO:32
			Example 17 CQTS109	SEQ ID NO:33	SEQ ID NO:34
Example 18 CQTS110	SEQ ID NO:35	SEQ ID NO:36
			Example 19 CQTS111	SEQ ID NO:37	SEQ ID NO:38
Example 20 CQTS112	SEQ ID NO:39	SEQ ID NO:40

The RBD-specific monoclonal antibodies provided in examples 1-20 above were obtained by the following method: firstly, single RBD specific memory B cells are obtained by separating peripheral blood of a new coronary pneumonia rehabilitation patient, then mRNA of the RBD specific memory B cells is obtained, then an antibody variable region gene expression box is constructed through RT-PCR and nested PCR, the antibody variable region gene expression box is transduced into 293T cells to express an antibody, supernatant is collected, the RBD specificity of the supernatant is detected through an ELISA method, and RBD specific monoclonal antibodies are obtained through screening.

The method specifically comprises the following steps:

s1, collecting peripheral blood of a plurality of patients with the new coronary pneumonia, separating to obtain PBMC, and freezing and storing in a refrigerator at the temperature of-80 ℃ for later use.

S2, firstly removing Dead cells of PBMC obtained in the S1 by adopting Dead cell Dye (Dead Dye), then adopting CD19, mIg-G, mIg-D and S-RBD to stain and mark the memory B cells with high specificity and binding capacity on the live RBD in the PBMC, and screening out the memory B cells specific to the RBD; specific memory B cells were sorted using a flow cytometric sorter onto 96-well plates, one specific memory B cell per well, and frozen in a-80 ℃ freezer for use.

Specifically, the preferred concentration range of the Dead Dye staining in this embodiment is 1-2 μ g/mL, and the preferred concentration range of the Dead Dye staining in this embodiment is 1.5 μ g/mL; CD19 is a B cell marker produced by Biolegend and is stained at a concentration in the range of 1-2. Mu.g/mL, and in this example, CD19 is preferably stained at a concentration of 1.5. Mu.g/mL. mIg-G is a B cell surface receptor produced by Biolegend, and the concentration range of the mIg-G during staining is 1-2 mu G/mL, and the concentration of the mIg-G during staining is 1.5 mu G/mL in the embodiment; mIg-D is a B cell surface receptor produced by Biolegend, and the concentration range of the staining is 1-2 mug/mL, and the concentration of 1.5 mug/mL is preferable when the mIg-D is stained in the embodiment; S-RBD is a novel coronavirus produced by sinobiological, which is a protein receptor domain, and the concentration range when staining is 1-2. Mu.g/mL, and the concentration range when staining S-RBD is 1.5. Mu.g/mL is preferable in this example.

Cell sorting of PBMC by CD19, mIg-G, mIg-D and S-RBD for RBD-specific memory B cell sorting by flow cytometry the cell sorting profiles for B cells with memory specific to S-RBD are shown in FIGS. 1 and 2, where

Batch ID

0428, 0505, 0522, 0528 in FIG. 2 are screening batches. The principle of screening RBD-specific memory B cells by using CD19, mIg-G, mIg-D and S-RBD in this example is as follows: PBMC were stained with Dead cell stain (Dead Dye), B cell marker CD19, memory B cellsMarkers mIg-G positive and mIg-D negative and memory B cells expressing RBD-specific IgG were stained, and CD19 cell population was then fractionated from the cell population using a flow cytometer, and mIg-G was then fractionated from the CD19 positive cell population ⁺ mIg-D ^- Cell population from mIg-G ⁺ mIg-D ^- Dividing the cell group into RBD positive memory B cells, and sorting the RBD positive memory B cells by a flow cytometric sorter.

S3, sorting to obtain mRNA of a single RBD specific memory B cell, and performing RT-PCR amplification to obtain antibody variable region cDNA. Specifically, when RT-PCR is used to amplify antibody variable region cDNA, the primer front segment of the primer designed in this example is designed with a universal Leader (see primer sequence listing i and primer sequence listing ii), which effectively improves the amplification rate of antibody gene, and the experimental result is shown in fig. 3.

S4, amplifying the antibody variable region cDNA obtained from the S1-S3 by adopting nested PCR to construct an antibody variable region gene expression cassette.

S3 and S4 are performed by the following six sections in total: (1) extracting mRNA of RBD specific memory B cells; (2) single cell mRNA Reverse Transcription (RT); (3) adding G tail (TDT); (4) first round PCR (1 st PCR); (5) second round PCR (2 nd PCR); (6) PCR amplification of BCR-ORF to construct a gene expression cassette; (7) CMV, WPRE-gamma/kappa/l fragment amplification and CMV, BCR-V gamma/kappa/l ((6) product), WPRE-gamma/kappa/l Overlap PCR (Overlap PCR) pre-connection; (8) BCR-gamma ORF, BCR-kappa ORF and BCR-lPCR amplification.

The preparation and reaction conditions of each part of reaction liquid are as follows:

(1) Using Dynabeads ^TM mRNA DIRECT ^TM The single-cell mRNA extraction is carried out by a Purification Kit (Thermo Fisherscientific), which comprises the following steps:

(1) centrifuging: taking out the 96-well plate sorted with single RBD specific memory B cells from a refrigerator at-80 ℃, and centrifuging the plate at 600 Xg for 30s to enable the cells to be centrifuged at the bottom of the well;

(2) cleaning: taking out a Dynabeads oligo (dT) 25 microsphere bottle, uniformly mixing the Dynabeads oligo (dT) 25 microsphere bottle by vortex, sucking enough microspheres according to 2 mu l/hole, placing the microspheres on a magnet block, standing for 30s, discarding supernatant, and resuspending the microspheres by using 500 mu l of lysine Buffer;

(3) preparation: adding the mixture into a 50mL centrifuge tube according to 9. Mu.l/hole lysine Buffer, adding the 500. Mu.l microsphere suspension into the centrifuge tube, and uniformly blowing by using a gun;

(4) subpackaging: subpackaging the microspheres by using an eight-connecting tube, and then adding the microspheres into a cell plate according to 9 mu l/hole by using a row gun;

(5) rinsing: pasting a membrane on a 96-well plate, and then rinsing the periphery of the tube wall for 2 cycles;

(6) and (3) incubation: standing at room temperature for 5min to fully release mRNA of RBD specific memory B cells and combine the mRNA to the microspheres, and after the incubation is finished, performing 600 Xg instantaneous centrifugation to enable the microspheres to be centrifuged at the bottom of the hole. Place 96-well plates in DynaMag ^TM -96side Magnet magnetic plate, pipette off supernatant;

(7) wash A: adding Washing Buffer A according to 8 mul/hole, walking the plate back and forth for 7-8 times to fully wash the microspheres, and discarding the supernatant;

(8) wash B: wash Buffer B was added at 8. Mu.l/well, the plate was walked back and forth 7-8 times to wash the microspheres thoroughly, the supernatant was discarded, and then the pre-prepared Reverse Transcription (RT) reaction was added at 10. Mu.l/well. The reagent preparation and reaction conditions are described in the following (2).

(2) Reverse Transcription (RT) (10. Mu.l system)

The reagents required for formulation are shown in table 1 below:

name of reagent	Volume of
		DEPC-H ₂ O	4.5μl
5×primerscript Buffer	2.0μl
		2.5mM dNTP	2.0μl
RNase Inhibitor	1μl
		Sample	beads
PrimerScriptⅡRTase	0.5μl
		Total volume	10μl

The reaction conditions are as follows: 42 ℃ for 60min (mixing every 20 min);

after the reaction was completed, the 96-well plate was instantaneously centrifuged at 600 Xg, and then the 96-well plate was placed on DynaMag ^TM On a 96-side Magnet magnetic plate, the supernatant was aspirated off by a pipette, and then 10. Mu.l/well of the previously prepared TDT reaction solution was added, and the reagent preparation and reaction conditions were as described in (3) below.

(3) Add G tail (TDT) (10. Mu.l system)

The reagents required for formulation are shown in table 2 below:

name of reagent	Volume of
		H ₂ O	6.4μl
5×TdT buffer	2.0μl
		10mM dGTP	0.5μl
0.1％BSA	1.0μl
		Sample	beads
TdT	0.1μl
		Total volume	10μl

The reaction conditions are as follows: 37 ℃ for 40min (mix every 20 min).

After the reaction, the reaction mixture was centrifuged at 600 Xg in a 96-well plate and then placed in DynaMag ^TM On a 96-side Magnet magnetic plate, the supernatant was aspirated off by a pipette, and then a first PCR (1 st PCR) reaction solution prepared in advance was added at 10. Mu.l/well, and the reagent preparation and reaction conditions were as described in (4) below.

(4) 1st PCR (10. Mu.l System) (see primer sequence Listing)

The reagents required for formulation are shown in table 3 below:

based on the PCR principle, the experimental reaction conditions of 1st PCR are as follows: (1) pre-denaturation at 95 ℃ for 3min; (2) denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 5sec, elongation at 72 ℃ for 1min,30-35cycles, preferably 30cycles in this example; (3) circulating at 72 deg.C for external extension for 5min, and storing at 4 deg.C.

(5) Second round PCR (2 nd PCR) (10. Mu.l system) (see primer sequence Listing I and primer sequence Listing II)

The reagents required for formulation are shown in table 4 below:

name of reagent	Volume of
		H ₂ O	1.5μl
2×GC Buffer	5μl
		2.5mM dNTP	1μl
FP:MAC-AP3/AP3(10μM)	0.5μl
		RP:Cg-nest/K20/CI-nest(10μM)	0.5μl
PrimesTAR	0.5μl
		sample	1μl
Total volume	10μl

Based on the PCR principle, the experimental reaction conditions of 2nd PCR are as follows: (1) pre-denaturation at 95 ℃ for 3min; (2) denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 5s, elongation at 72 ℃ for 1min,30-35cycles, preferably 35cycles in this example; circulating at 72 deg.C for external extension for 5min, and storing at 4 deg.C.

After the PCR is finished: mu.l of each well was subjected to 1.5% agarose gel electrophoresis. The cell pore paired with the Gamma chain and Kappa chain or Lamada chain was sequenced.

(6) Amplification and construction of antibody expression cassettes (BCR-ORF)

PCR amplification promoter region (CMV promoter), WPRE-gamma (antibody gamma chain) and WPRE-kappa (antibody kappa chain) with the following PCR amplification system shown in Table 5 below:

the PCR amplification conditions were: (1) pre-denaturation at 95 ℃ for 3min; (2) denaturation at 95 ℃ for 15sec, annealing at 56 ℃ for 15sec, elongation at 72 ℃ for 1min,30cycles; (3) extension is carried out for 5min at 72 ℃ in a circulating way, and the product is stored at 12 ℃.

(7) Amplification of CMV, WPRE-gamma/kappa/l fragment and pre-ligation of CMV, BCR-Vgamma/kappa/l, WPRE-gamma/kappa/l Overlap PCR (Overlap PCR)

The experimental system is shown in table 6 below:

the PCR amplification conditions were: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15sec, annealing at 50 ℃ for 15sec, elongation at 72 ℃ for 1.5min,10cycles; extension is carried out for 5min at 72 ℃ in a circulating way, and the product is stored at 12 ℃.

(8) PCR amplification of BCR-gamma ORF, BCR-kappa ORF, BCR-l

The experimental system is shown in table 7 below:

PCR amplification procedure: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15sec, annealing at 58 ℃ for 15sec, elongation at 72 ℃ for 1.5min,30cycles; extension is carried out for 5min at 72 ℃ in a circulating way, and the product is stored at 12 ℃.

After amplification, agarose gel electrophoresis is adopted, and whether the size of the obtained antibody variable region gene is correct or not is analyzed by gel imaging, the experimental result is shown in figure 4, the Marker is in the middle position, and the strip is in the position of 5000 bp.

BCR-gamma ORF and BCR-kappa/ORF ethanol precipitation: placing 30 μ l of PCR products of BCR-gamma ORF and BCR-kappa ORF in 8 connecting tubes respectively, adding 120 μ l of anhydrous ethanol and 6 μ l of sodium acetate solution, mixing well, and standing at-80 deg.C for 30min;10000rpm, centrifuging for 20min, discarding the supernatant, sequentially rinsing with 200 μ l of 70% ethanol and anhydrous ethanol once respectively, fully volatilizing the ethanol at 56 deg.C, adding 40 μ l of sterile water, oscillating to fully dissolve the precipitate, and detecting the concentration of antibody variable region gene.

The Leader primers used for S3 and S4 are referred to as the following primer sequence I:

the J-region primers used for S3 and S4 are described in the following primer sequence Listing:

primer ID	sequence
		IGHJ_01	GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCAGGGTGCCCTGGCCCCAGT
IGHJ_02	GATGGGCCCTTGGTGGAGGGTGAGGAGACAGTGACCAGGGTGCCACGGCCCCAGA
		IGHJ_03	GATGGGCCCTTGGTGGAGGGTGAAGAGACGGTGACCATTGTCCCTTGGCCCCAGA
IGHJ_04	GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCGTGGTCCCTTGCCCCCAGA
		IGKJ_01	GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC
IGKJ_02	GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC
		IGKJ_03	GATGGTGCAGCCACAGTTCGTTTGATATCCACTTTGGTCCCAGGGCCGAAAGTGA
IGKJ_04	GATGGTGCAGCCACAGTTCGTTTGATCTCCACCTTGGTCCCTCCGCCGAAAGTGA
		IGKJ_05	GATGGTGCAGCCACAGTTCGTTTAATCTCCAGTCGTGTCCCTTGGCCGAAGGTGA
IGLJ_01	GGGGCAGCCTTGGGCTGACCTAGGACGGTGACCTTGGTCCCAGTTCCGAAGACAT
		IGLJ_02	GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTTGGTCCCTCCGCCGAATACCA
IGLJ_03	GGGGCAGCCTTGGGCTGACCTAAAATGATCAGCTGGGTTCCTCCACCAAATACAA
		IGLJ_04	GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCGGTCCCCTCACCAAACACCC
IGLJ_05	GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCCGTCCCCTCACCAAACACCC
		IGLJ_06	GGGGCAGCCTTGGGCTGACCGAGGACGGTCACCTTGGTGCCACTGCCGAACACAT
IGLJ_07	GGGGCAGCCTTGGGCTGACCGAGGACGGTCAGCTGGGTGCCTCCTCCGAACACAG
		IGLJ_08	GGGGCAGCCTTGGGCTGACCGAGGGCGGTCAGCTGGGTGCCTCCTCCGAACACAG

and S5, transducing the antibody variable region gene expression cassette obtained in the S4 into 293T cells for 48 hours to express the antibody, collecting supernatant, detecting the RBD specificity of the supernatant by an ELISA method, and screening the RBD-specific fully human monoclonal antibody.

(A) Antigen was diluted with PBS (final concentration 2. Mu.g/mL), 10. Mu.l/well, and coated onto 384-well ELISA plates overnight at 4 ℃ or 2h at 37 ℃ (4 ℃ overnight is preferred in this example). NOTE: after the addition, the liquid is instantly centrifuged to ensure that the liquid is at the bottom.

The experimental system is shown in table 8 below:

name of reagent	Goods number	Original concentration	Final concentration	Dilution ratio
					SARS-COV-2RBD	Cat:40592-V08H	200μg/mL	2μg/mL	1:100
Goat pab to Hu IgG－ALP	Cat:ab97221	1mg/mL	2μg/mL	1:500

(B) PBST (0.05% Tween 20, cat #, TB220) was prepared: 1L of PBS was added with 0.5mL of Tween 20;

PBST machine washed plates (Thermoscient well wash versas) or hand washed (plates that were machine washed were still manually tapped/centrifuged for 1min using a microplate centrifuge (MPC-P25) to make the plates invisible to water and air bubbles).

And (3) sealing: 80 μ l of BSA (BioFroxx, cat. NO:4240GR 100) at 5% (prepared in PBST) was added to the washed plate and incubated at 37 ℃ for 1 hour in an incubator. PBST machine washing board or hand washing.

(C) Sample adding and standard substance. Wherein, the standard substance: 10 μ l/well stock concentration 1 μ g/mL, gradient dilutions 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.9ng/mL, and 1.95ng/mL. (blocking solution dilution); sample preparation: cell supernatants transfected with antibody genes. Negative control/blank wells: blocking solution 10. Mu.l/well.

Incubate at 37 ℃ for 30min. PBST machine washing board or hand washing.

(D) Secondary antibody was added at a concentration of 10. Mu.l/well, followed by incubation at 37 ℃ for 30min.

The experimental system is shown in table 9 below:

name of secondary antibody	Goods number	Original concentration	Final concentration	Dilution ratio
					goat-anti-human IgG-ALP	A18808	1.5mg/ml	0.3μg/ml	1:5000
Goat pab to Hu IgG-ALP	Ab98532	0.5mg/ml	0.25μg/ml	1:2000

PBST machine washing board or hand washing. Mu.l/well of PNPP (disodium p-nitrophenylphosphate) and OD (450 mm) values of 5min,10 min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60min were measured using (Thermoscientific Muttiskan GO). 50mg PNPP powder (Thermo, prod # 34045) +40mL ddH ₂ O +10mL of Diethylhanol amine substrate Buffer (5X), and PNPP was stored at 4 ℃ protected from light.

As shown in FIG. 5, the OD value greater than 0.1 was positive.

The foregoing is illustrative of the preferred embodiments of the present invention, which is set forth only, and not to be taken as limiting the invention; those skilled in the art will appreciate that many variations, modifications, and even equivalent variations are possible within the spirit and scope of the invention as defined in the appended claims.

Claims

1. The monoclonal antibody with the specificity of the new coronavirus RBD is characterized in that the amino acid sequence of a heavy chain is shown as SEQ ID NO. 5; the light chain amino acid sequence is shown as SEQ ID NO. 6.

2. The monoclonal antibody specific to RBD of the novel coronavirus of claim 1, wherein the antibody variable region cDNA is obtained by sorting RBD-specific memory B cells and then passing the mRNA of the RBD-specific memory B cells.

3. Use of the monoclonal antibody specific to the novel coronavirus RBD of claim 1 in the preparation of a reagent or a medicament for detecting or diagnosing SARS-CoV-2, wherein the medicament comprises the monoclonal antibody specific to the novel coronavirus RBD of claim 1, and a pharmaceutically acceptable excipient, diluent or carrier.

4. A nucleic acid molecule encoding the novel coronavirus RBD-specific monoclonal antibody of claim 1.

5. An expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of claim 4.

6. The use of the expression cassette, recombinant vector, recombinant strain or transgenic cell line of claim 5 in the preparation of a product for any of the following uses (b 1) to (b 4): (b 1) binding to novel coronavirus SARS-CoV-2; (b 2) detecting a novel coronavirus SARS-CoV-2; (b 3) binding to the S protein of the novel coronavirus SARS-CoV-2; (b 4) detecting the S protein of the novel coronavirus SARS-CoV-2.