CN111909263B

CN111909263B - New coronavirus RBD specific monoclonal antibody and application

Info

Publication number: CN111909263B
Application number: CN202010839864.8A
Authority: CN
Inventors: 韩晓建; 王应明; 胡超; 李婷婷; 王建为; 李胜龙; 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: 2022-10-11
Anticipated expiration: 2040-08-19
Also published as: CN115925896A; CN117736313A; WO2022037616A1; CN117003862A; CN111909263A; CN114920832A; CN116813760A; CN114920832B

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 invention patent application with application publication number CN111303280A discloses a highly neutralizing anti-SARS-CoV-2 fully human monoclonal antibody, which provides a fully human 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 is combined with ACE2 of the host cell through the RBD, and the obtained antibody cDNA is obtained by marking plasma cells, but compared with the plasma cells, the antibody cDNA reacts rapidly after memory B cells are activated, so the memory B cells can initiate a faster and stronger humoral immune response than the primary reaction, and the humoral immune response initiated 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 001). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 3; the light chain amino acid sequence may also be shown as SEQ ID NO:4 (mAb 2-CQTS 002). 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 (mAb 3-CQTS 003). 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 (mAb 4-CQTS 004). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 9; the light chain amino acid sequence may also be shown as SEQ ID NO:10 (mAb 5-CQTS 005). 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 006). 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 007). 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 (monoclonal antibody 8-CQTS 008). 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 009). 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 (monoclonal antibody 10-CQTS 010). 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 011). 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 012). 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 (monoclonal antibody 13-CQTS 013). 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 (mAb 14-CQTS 014). 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 015). 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 (mAb 16-CQTS 016). 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 017). 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 (monoclonal antibody 18-CQTS 018). The heavy chain amino acid sequence can also be shown as SEQ ID NO 37; the light chain amino acid sequence may also be as shown in SEQ ID NO:38 (mAb 19-CQTS 019). 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 020).

The invention also provides the application of the monoclonal antibody with the RBD specificity of the new coronavirus in the preparation of reagents or vaccines or medicaments for detecting or diagnosing SARS-CoV-2, wherein the medicaments comprise the monoclonal antibody with the RBD specificity of the new coronavirus as described in any one of claims 1-20 and pharmaceutically acceptable excipients, diluents or carriers; 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) binding 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 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 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 diagram of cell sorting by flow cytometry analysis of memory B cells;

FIG. 2 is a cell sorting graph of RBD specific memory B cells analyzed by flow cytometry;

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 results of an experiment on the specificity of RBD.

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 obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection 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 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 CQTS001	SEQ ID NO:1	SEQ ID NO:2
Example 2 CQTS002	SEQ ID NO:3	SEQ ID NO:4
			Example 3 CQTS003	SEQ ID NO:5	SEQ ID NO:6
Example 4 CQTS004	SEQ ID NO:7	SEQ ID NO:8
			Example 5 CQTS005	SEQ ID NO:9	SEQ ID NO:10
Example 6 CQTS006	SEQ ID NO:11	SEQ ID NO:12
			Example 7 CQTS007	SEQ ID NO:13	SEQ ID NO:14
Example 8 CQTS008	SEQ ID NO:15	SEQ ID NO:16
			Example 9 CQTS009	SEQ ID NO:17	SEQ ID NO:18
Example 10 CQTS010	SEQ ID NO:19	SEQ ID NO:20
			Example 11 CQTS011	SEQ ID NO:21	SEQ ID NO:22
Example 12 CQTS012	SEQ ID NO:23	SEQ ID NO:24
			Example 13 CQTS013	SEQ ID NO:25	SEQ ID NO:26
Example 14 CQTS014	SEQ ID NO:27	SEQ ID NO:28
			Example 15 CQTS015	SEQ ID NO:29	SEQ ID NO:30
Example 16 CQTS016	SEQ ID NO:31	SEQ ID NO:32
			Example 17 CQTS017	SEQ ID NO:33	SEQ ID NO:34
Example 18 CQTS018	SEQ ID NO:35	SEQ ID NO:36
			Example 19 CQTS019	SEQ ID NO:37	SEQ ID NO:38
Example 20 CQTS020	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 from peripheral blood of a new coronary pneumonia rehabilitation patient through separation, then mRNA of the RBD specific memory B cells is obtained, an antibody variable region gene expression box is constructed through RT-PCR and nested PCR, then 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 new coronary pneumonia rehabilitation patients, separating to obtain PBMCs, and freezing and storing the PBMCs in a refrigerator at the temperature of 80 ℃ below zero for later use.

S2, firstly removing Dead cells of PBMC obtained in the S1 by adopting a Dead cell Dye (Dead Dye), then adopting CD19, mIg-G, mIg-D and S-RBD to stain and mark memory B cells with high specificity and binding capacity on the living RBD in the PBMC, and screening out 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, 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 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 sorting by flow cytometry the cell sorting profiles for B cells with specific memory for 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 CD19, mIg-G, mIg-D and S-RBD in this example is as follows: PBMC were washed with Dead cell Dye (Dead Dye)) B cell marker CD19, memory B cell markers migg-G positive and migg-D negative, and memory B cells expressing RBD-specific IgG, then dividing the population of CD19 cells from the population using a flow cytometer, and then dividing migg-G from the population of CD19 positive cells ⁺ 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 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), and the method specifically 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 by vortex, sucking enough microspheres according to 2 mu l/hole, placing on a magnet block, standing for 30s, discarding supernatant, and resuspending by using 500 mu l lysine Buffer;

(3) preparation: 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;

(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 discharge gun;

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

(6) and (3) 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;

(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 μ 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 (mix 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

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

At the end of the reaction, the 96-well plate was centrifuged at 600 Xg instantaneously 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:

name of reagent	Volume of
		H ₂ O	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: (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) extension is carried out for 5min at 72 ℃ in a circulating way, and the product is stored at 4 ℃.

(5) Second round PCR (2 nd PCR) (10. Mu.l system) (see primer sequence Listing one and primer sequence Listing two): 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; extension is carried out for 5min at 72 ℃ in a circulating way, and the product is stored at 4 ℃.

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

(6) Amplification and construction of antibody expression cassette (BCR-ORF): PCR amplification promoter region (CMV promoter), WPRE-gamma (antibody gamma chain) and WPRE-kappa (antibody kappa chain) the PCR amplification system is 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) CMV, WPRE-gamma/kappa/l fragment amplification and CMV, BCR-Vgamma/kappa/l, WPRE-gamma/kappa/l Overlap PCR (Overlap PCR) pre-connection, the experimental system is shown in the following table 6.

The PCR amplification conditions were: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15sec, annealing at 50 ℃ for 15sec, extension 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) BCR-gamma ORF, BCR-kappa ORF and BCR-l PCR amplification: 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, extension 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, 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 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:

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 the supernatant, detecting the RBD specificity of the supernatant by using 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 were measured using (Thermoscientific Muttiskan GO) for 5min,10 min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60 min. 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.

The results are shown in FIG. 5, and FIG. 5 shows that the OD value greater than 0.1 is 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 1; the light chain amino acid sequence is shown as SEQ ID NO. 2.

2. 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. 7; the light chain amino acid sequence is shown as SEQ ID NO. 8.

3. The monoclonal antibody specific for RBD of the novel coronavirus according to any one of claims 1-2, 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.

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

5. Nucleic acid molecule encoding the novel coronavirus RBD-specific monoclonal antibody of any one of claims 1-2.

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

7. The use of the expression cassette, recombinant vector, recombinant strain or transgenic cell line of claim 6 in the preparation of a product for any of the following uses (b 1) to (b 4): (b 1) binds 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.