CN111925444A

CN111925444A - New coronavirus RBD specific monoclonal antibody and application

Info

Publication number: CN111925444A
Application number: CN202010839863.3A
Authority: CN
Inventors: 王建为; 李胜龙; 胡超; 王应明; 李婷婷; 韩晓建; 龙顺花; 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: 2020-11-13
Anticipated expiration: 2040-08-19
Also published as: CN115925897A; CN111925444B; CN115340602A; WO2022037033A1

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) consisting of three regions, CH1, CH2, and CH3, and a heavy chain constant region. 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 known as Complementarity Determining Regions (CDRs) and conserved regions known as 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 drug-specific treatment is not available for new pathogen COVID-19, and the development of vaccines requires a current 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 invasion of new coronavirus into host cells is bound to ACE2 of host cells 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 plasma cells is limited.

Disclosure of Invention

In order to achieve the aim, the invention provides a new coronavirus RBD specific monoclonal antibody, 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 147). 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 165). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 5; the light chain amino acid sequence can also be shown as SEQ ID NO:6 (monoclonal antibody 3-CQTS 166). 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 167). 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 168). 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 169). 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 (mAb 7-CQTS 170). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 15; the light chain amino acid sequence can also be shown as SEQ ID NO:16 (mAb 8-CQTS 171). The heavy chain amino acid sequence can also be shown as SEQ ID NO 17; the light chain amino acid sequence can also be shown as SEQ ID NO:18 (mAb 9-CQTS 172). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 19; the light chain amino acid sequence can also be shown as SEQ ID NO:20 (mAb 10-CQTS 173). 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 174). 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 (mAb 12-CQTS 175). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 25; the light chain amino acid sequence can also be shown as SEQ ID NO:26 (mAb 13-CQTS 177). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 27; the light chain amino acid sequence can also be shown as SEQ ID NO:28 (monoclonal antibody 14-CQTS 178). 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 (mAb 15-CQTS 179). 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 antibody 16-CQTS 180). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 33; the light chain amino acid sequence can also be shown as SEQ ID NO:34 (monoclonal antibody 17-CQTS 185). 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 186). 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 (monoclonal antibody 19-CQTS 187). The heavy chain amino acid sequence can also be shown as SEQ ID NO: 39; the light chain amino acid sequence can also be shown as SEQ ID NO:40 (monoclonal antibody 20-CQTS 188).

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 is used as any one of the following (b1) - (b 4): (b1) binds to novel coronavirus SARS-CoV-2; (b2) detecting binding of the novel coronavirus SARS-CoV-2; (b3) binds to the S protein of the novel coronavirus SARS-CoV-2; (b4) the S protein of the novel coronavirus SARS-CoV-2 is detected.

Preferably, the RBD-specific monoclonal antibody of the novel 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 a non-RBD region of S1, the monoclonal antibody provided by the invention is combined with RBD, thereby providing wider application value for screening antibody drugs, 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 diagram of cell sorting by flow cytometry analysis of RBD-specific memory B cells;

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

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

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 a novel coronavirus RBD, the heavy chain amino acid sequence of which 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 example can be used to prepare a nucleic acid molecule, or an expression cassette, a recombinant vector, a recombinant bacterium, or a transgenic cell line comprising the nucleic acid molecule, or 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 (b1) - (b 4): (b1) binds to novel coronavirus SARS-CoV-2; (b2) detecting binding of the novel coronavirus SARS-CoV-2; (b3) binds to the S protein of the novel coronavirus SARS-CoV-2; (b4) the S protein of the novel coronavirus SARS-CoV-2 is detected.

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 CQTS147	SEQ ID NO:1	SEQ ID NO:2
Example 2 CQTS165	SEQ ID NO:3	SEQ ID NO:4
			Example 3 CQTS166	SEQ ID NO:5	SEQ ID NO:6
Example 4 CQTS167	SEQ ID NO:7	SEQ ID NO:8
			Example 5 CQTS168	SEQ ID NO:9	SEQ ID NO:10
Example 6 CQTS169	SEQ ID NO:11	SEQ ID NO:12
			Example 7 CQTS170	SEQ ID NO:13	SEQ ID NO:14
Example 8 CQTS171	SEQ ID NO:15	SEQ ID NO:16
			Example 9 CQTS172	SEQ ID NO:17	SEQ ID NO:18
Example 10 CQTS173	SEQ ID NO:19	SEQ ID NO:20
			Example 11 CQTS174	SEQ ID NO:21	SEQ ID NO:22
Example 12 CQTS175	SEQ ID NO:23	SEQ ID NO:24
			Example 13 CQTS177	SEQ ID NO:25	SEQ ID NO:26
Example 14 CQTS178	SEQ ID NO:27	SEQ ID NO:28
			Example 15 CQTS179	SEQ ID NO:29	SEQ ID NO:30
Example 16 CQTS180	SEQ ID NO:31	SEQ ID NO:32
			Example 17 CQTS185	SEQ ID NO:33	SEQ ID NO:34
Example 18 CQTS186	SEQ ID NO:35	SEQ ID NO:36
			Example 19 CQTS187	SEQ ID NO:37	SEQ ID NO:38
Example 20 CQTS188	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 cassette is constructed through RT-PCR and nested PCR, the antibody variable region gene expression cassette is transferred into 293T cells to express an antibody, supernatant is collected, 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-80 ℃ for later use.

S2, firstly removing Dead cells of PBMC obtained by S1 by using 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 for the living 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 at-80 ℃ in a 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 ranging from 1 to 2. mu.g/mL, preferably at a concentration of 1.5. mu.g/mL for CD19 in this example. 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 B cell surface receptor produced by Biolegend, and the concentration range when staining is 1-2 μ g/mL, and the concentration when staining mIg-D is 1.5 μ g/mL is preferred in the embodiment; S-RBD is a novel coronavirus produced by sinobiological, is a protein receptor domain, and is stained at a concentration ranging from 1 to 2. mu.g/mL, and the concentration of S-RBD staining is preferably 1.5. mu.g/mL in this example.

Cell sorting of PBMC by CD19, mIg-G, mIg-D and S-RBD for RBD-specific memory B cells by flow cytometry cell sorting of PBMC with S-RBD-specific memory B cells cell sorting profiles 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 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 cell markers mIg-G positive and mIg-D negative, and memory B cells expressing RBD-specific IgG, and then a CD19 cell population was divided from the cell population using a flow cytometer, and mIg-G was divided 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 cytometry sorter.

S3, sorting to obtain mRNA of single RBD specific memory B cell, and obtaining antibody variable region cDNA by RT-PCR amplification. 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 table one and primer sequence table two), which effectively improves the amplification rate of antibody genes, and the experimental results are shown in fig. 3.

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

S3 and S4 were performed in total by the following six sections: (1) extracting mRNA of RBD specific memory B cells; (2) single cell mRNA Reverse Transcription (RT); (3) adding a G tail (TDT); (4) first round PCR (1st PCR); (5) second round PCR (2nd PCR); (6) BCR-ORF PCR amplification constructs gene expression cassettes; (7) CMV, WPRE-gamma/kappa/l fragment amplification and CMV, BCR-Vgamma/kappa/l ((6) products), 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^TMmRNA DIRECT^TMThe single cell mRNA extraction is carried out by a Purification Kit (Thermo Fisherscientific), and the method specifically comprises the following steps:

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;

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;

preparing: adding the microspheres into a 50mL centrifuge tube according to 9. mu.l/hole lysine Buffer, adding the 500. mu.l microsphere suspension, and uniformly blowing by using a gun;

fourthly, 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;

moistening and washing: pasting a film on a 96-hole plate, then rinsing the periphery of the tube wall for 2 cycles;

sixthly, incubation: standing at room temperature for 5min to fully release and combine mRNA of the RBD specific memory B cells 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;

seventhly, washing by 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;

and (8) washing with 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-H₂O	4.5μl
5×primerscript Buffer	2.0μl
		2.5mM dNTP	2.0μl
RNase Inhibitor	1μl
		Sample	beads
PrimerScriptⅡRTase	0.5μl
		Total volume	10μl

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

after the reaction is finished, 600 Xg is instantaneously separatedThe core 96-well plate was placed on DynaMag^TMOn a 96-side Magnet magnetic plate, the supernatant was aspirated off by a discharge gun, and then a previously prepared TDT reaction solution was added at 10. mu.l/well, 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
		H₂O	6.4μl
5×TdT buffer	2.0μl
		10mM dGTP	0.5μl
0.1％BSA	1.0μl
		Sample	beads
TdT	0.1μl
		Total volume	10μl

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^TMOn a 96-side Magnet magnetic plate, the supernatant was aspirated off by a pipette, and then a first PCR (1st 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:

name of reagent	Volume of
		H₂O	1.9μl
2×GC Buffer	5μl
		2.5mM dNTP	1μl
FP:AP3-dC(10μM)	0.5μl
		RP1:Cg-1st(10μM)	0.5μl
RP2:Ck-1st(10μM)	0.5μl
		RP3:CI-RT(10μM)	0.5μl
PrimesTAR	0.1μl
		sample	beads
Total volume	10μl

Based on the PCR principle, the experimental reaction conditions of 1st PCR are as follows: firstly, pre-denaturation is carried out for 3min at 95 ℃; ② denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 5sec, extension at 72 ℃ for 1min, 30-35cycles, preferably 30cycles in the embodiment; ③ extension for 5min at 72 ℃ and preservation at 4 ℃.

(5) Second round PCR (2nd 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
		H₂O	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: firstly, pre-denaturation is carried out for 3min at 95 ℃; ② denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 5s, extension at 72 ℃ for 1min, 30-35cycles, preferably 35cycles in the embodiment; extending for 5min at 72 deg.C, 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: firstly, pre-denaturation is carried out for 3min at 95 ℃; ② denaturation at 95 ℃ for 15sec, annealing at 56 ℃ for 15sec, extension at 72 ℃ for 1min, 30 cycles; ③ extension for 5min at 72 ℃ and preservation at 12 ℃.

(7) Amplification of CMV, WPRE-gamma/kappa/l fragments 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 3 min; denaturation at 95 ℃ for 15sec, annealing at 50 ℃ for 15sec, extension at 72 ℃ for 1.5min, 10 cycles; extending for 5min at 72 deg.C, and storing at 12 deg.C.

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

The experimental system is shown in table 7 below:

PCR amplification procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15sec, annealing at 58 ℃ for 15sec, extension at 72 ℃ for 1.5min, 30 cycles; extending for 5min at 72 deg.C, and storing at 12 deg.C.

After amplification, agarose gel electrophoresis is adopted, 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 band 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 30 min; 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 in S3 and S4 are described in the primer sequence Listing I below:

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

s5, the variable region gene expression cassette of the antibody obtained from S4 is transduced into 293T cells to express the antibody within 48 hours, the supernatant is collected, the RBD specificity of the supernatant is detected by an ELISA method, and the RBD specificity fully human monoclonal antibody is screened.

(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) Formulation of PBST (0.05% Tween 20, Cat # TB 220): 1L of PBS was added with 0.5mL of Tween 20;

PBST machine washed plates (Thermoscientific wellwash versa) or hand washed (plates that were machine washed were still manually photographed/centrifuged using a microplate centrifuge (MPC-P25) for 1min to make the plates invisible to water and air bubbles).

And (3) sealing: mu.l of 5% BSA (BioFroxx, Cat. NO:4240GR100) (formulated in PBST) were added to the washed plates and incubated for 1h at 37 ℃ 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 1.95 ng/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 30 min. 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 30 min.

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 (450mm) values were measured using (Thermoscientific Muttiskan GO) for 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60 min. 50mg PNPP powder (Thermo, Prod #34045) +40mL ddH₂O +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 merely a preferred embodiment of this invention, which is intended to be illustrative, not limiting; 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 1; the light chain amino acid sequence is shown as SEQ ID NO. 2.

2. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO. 3; the light chain amino acid sequence can also be shown as SEQ ID NO. 4.

3. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO. 5; the light chain amino acid sequence can also be shown as SEQ ID NO 6.

4. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO. 7; the light chain amino acid sequence can also be shown as SEQ ID NO. 8.

5. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further as set forth in SEQ ID No. 9; the light chain amino acid sequence can also be shown as SEQ ID NO. 10.

6. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO. 11; the light chain amino acid sequence can also be shown as SEQ ID NO 12.

7. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 13; the light chain amino acid sequence can also be shown as SEQ ID NO. 14.

8. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO. 15; the light chain amino acid sequence can also be shown as SEQ ID NO 16.

9. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 17; the light chain amino acid sequence can also be shown as SEQ ID NO. 18.

10. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 19; the light chain amino acid sequence can also be shown as SEQ ID NO: 20.

11. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 21; the light chain amino acid sequence can also be shown as SEQ ID NO. 22.

12. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO. 23; the light chain amino acid sequence can also be shown as SEQ ID NO. 24.

13. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 25; the light chain amino acid sequence can also be shown as SEQ ID NO. 26.

14. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 27; the light chain amino acid sequence can also be shown as SEQ ID NO 28.

15. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO. 29; the light chain amino acid sequence can also be shown as SEQ ID NO 30.

16. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 31; the light chain amino acid sequence can also be shown as SEQ ID NO: 32.

17. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 33; the light chain amino acid sequence can also be shown as SEQ ID NO. 34.

18. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 35; the light chain amino acid sequence can also be shown as SEQ ID NO: 36.

19. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 37; the light chain amino acid sequence can also be shown as SEQ ID NO 38.

20. The monoclonal antibody specific to the novel coronavirus RBD according to claim 1, wherein the heavy chain amino acid sequence is further represented by SEQ ID NO 39; the light chain amino acid sequence can also be shown as SEQ ID NO: 40.

21. The monoclonal antibody specific for RBD of the novel coronavirus according to any one of claims 1-20, wherein the antibody variable region cDNA is obtained from the mRNA of RBD-specific memory B cells by sorting the RBD-specific memory B cells.

22. Use of the monoclonal antibody specific for the novel coronavirus RBD according to any one of claims 1-20 in the preparation of a reagent or medicament for the detection or diagnosis of SARS-CoV-2, wherein the medicament comprises the monoclonal antibody specific for the novel coronavirus RBD according to any one of claims 1-20 and a pharmaceutically acceptable excipient, diluent or carrier.

23. A nucleic acid molecule encoding the novel coronavirus RBD-specific monoclonal antibody of any one of claims 1-20.

24. An expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of claim 23.

25. Use of an expression cassette, recombinant vector, recombinant strain or transgenic cell line according to claim 24 for the preparation of a product for any of the following uses (b1) - (b 4): (b1) binds to novel coronavirus SARS-CoV-2; (b2) detecting binding of the novel coronavirus SARS-CoV-2; (b3) binds to the S protein of the novel coronavirus SARS-CoV-2; (b4) the S protein of the novel coronavirus SARS-CoV-2 is detected.