CN115260305A - anti-SARS-CoV-2 RBD monoclonal antibody and application - Google Patents

Abstract

The invention relates to an anti-SARS-CoV-2 RBD monoclonal antibody and application thereof. The monoclonal antibody targeting SARS-CoV-2RBD provided by the invention is different from the existing commercial monoclonal antibody, and can be specifically combined with highly conserved epitope positioned in SARS-CoV-2RBD³⁵⁰VYAWN³⁵⁴The epitope is highly conserved among different SARS-CoV-2 strains and is not influenced by the conformation change of the S protein RBD in an 'upward' and 'downward' manner; experiments prove that the anti-SARS-CoV-2 RB provided by the applicationThe D monoclonal antibody is an effective neutralizing antibody, can be used for developing medicaments for preventing and/or treating diseases caused by SARS-CoV-2 infection, and has good clinical application value. The monoclonal antibody of the invention has clear identification epitope, can effectively identify SARS-CoV-2 or SARS-CoV-2S protein in various conformational forms, has high affinity, strong specificity and good stability, and provides a new tool for preventing, detecting and treating SARS-CoV-2.

Description

anti-SARS-CoV-2 RBD monoclonal antibody and application

Technical Field

The invention belongs to the technical field of immunology, and particularly relates to an anti-SARS-CoV-2 RBD monoclonal antibody and an application thereof.

Background

The ongoing 2019 pandemic of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a huge disruption to global health and socioeconomic performance, necessitating the urgent development of safe and effective prophylactic methods and therapies. The spike (S) protein is the major surface antigen of SARS-CoV-2. The Receptor Binding Domain (RBD) of the S protein binds to the host receptor angiotensin converting enzyme 2 (ACE 2), allowing viral entry, and plays a crucial role in viral infection. Thus, antibodies targeting RBD can neutralize SARS-CoV-2 by blocking ACE2 binding, making RBD the main target for development of neutralizing antibodies.

Various SARS-CoV-2 virus vaccines (novel coronavirus vaccines) are now approved for sale, and various candidate vaccines for the novel coronavirus are in development stage and enter clinical trials. A number of antibodies targeting SARS-CoV-2RBD have also now been discovered. However, the rapidly spreading variant new coronavirus may cause antibody escape, vaccine efficacy to decline, more antibodies to neutralize the new variant required, and development of vaccines and monoclonal antibodies against the new variant strain of coronavirus to cope with the new variant strain.

Disclosure of Invention

The present invention aims to provide a monoclonal antibody against SARS-CoV-2RBD, which antibody is capable of specifically recognizing an epitope in the SARS-CoV-2 receptor binding domain³⁵⁰VYAWN³⁵⁴。

The second objective of the invention is to provide a nucleic acid molecule containing the gene encoding the monoclonal antibody, and an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium containing the nucleic acid molecule.

The third purpose of the invention is to provide the application of the monoclonal antibody, the nucleic acid molecule, the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacterium.

The fourth purpose of the invention is to provide a pharmaceutical composition containing the monoclonal antibody.

In order to achieve the purpose, the invention adopts the following technical scheme:

an anti-SARS-CoV-2 RBD monoclonal antibody comprising VHCDR1, VHCDR2 and VHCDR3 having amino acid sequences shown in SEQ ID Nos. 1 to 3, and VLCDR1, VLCDR2 and VLCDR3 having amino acid sequences shown in SEQ ID Nos. 4 to 6.

Preferably, the monoclonal antibody comprises a heavy chain variable region having an amino acid sequence shown as SEQ ID NO.7 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 8.

It is obvious to those skilled in the art that, based on the amino acid sequences of the heavy and light chain variable regions of the monoclonal antibody specifically disclosed in the present invention, modifications such as addition, deletion, substitution, etc. of one or more amino acids can be performed by conventional protein engineering methods to obtain a conservative variant or a fragment thereof, while still maintaining the specific binding to SARS-CoV-2 RBD.

Preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 type and the light chain constant region is of the Kappa type.

Preferably, the monoclonal antibody specifically binds to an epitope located on SARS-CoV-2RBD, the amino acid sequence of the epitope is shown as SEQ ID NO.9 or SEQ ID NO. 10.

An epitope of SARS-CoV-2RBD, the amino acid sequence of the epitope is shown as SEQ ID NO.9 or SEQ ID NO. 10; or the epitope is a truncated peptide of SEQ ID NO.9 containing VYAWN core sequence.

The application of the epitope is shown as any one of the following:

1) The application in preparing SARS-CoV-2 antiserum or monoclonal antibody;

2) The application in preparing SARS-CoV-2 vaccine;

3) The application in preparing SARS-CoV-2 immunity detection reagent or reagent box.

A nucleic acid molecule comprising a gene sequence encoding the monoclonal antibody described above. Preferably, the gene sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.11, and the gene sequence of the light chain variable region is shown in SEQ ID NO. 12.

The antibody nucleic acid molecule can be obtained by using genetic engineering recombination technology or chemical synthesis method. It is obvious to those skilled in the art that the variable sequences of the nucleotide sequences of the heavy chain variable regions and/or the nucleotide sequences of the light chain variable regions obtained after the above-mentioned nucleic acid molecules provided by the present invention are mutated by one or more nucleotide additions, deletions, substitutions, modifications, etc., and the single-chain antibodies or chimeric monoclonal antibodies or modified monoclonal antibodies or antibody fragments in other forms consisting of the encoded amino acid sequences still retain the ability to specifically bind to SARS-CoV-2 RBD.

An expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium containing the nucleic acid molecule.

Specifically, the recombinant vector is selected from prokaryotic or eukaryotic expression vectors, and further, the recombinant vector is selected from bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors. The expression system is a bacterial, yeast, filamentous fungus, mammalian cell, insect cell, plant cell or cell-free expression system.

The application of the monoclonal antibody, the nucleic acid molecule or the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacterium is any one of the following:

1) The application in preparing the medicine for preventing and/or treating the diseases caused by SARS-CoV-2 infection;

2) The application in preparing SARS-CoV-2 immunity detection reagent or reagent box.

A pharmaceutical composition comprising the monoclonal antibody described above and a pharmaceutically acceptable carrier, diluent or excipient.

The invention has the following beneficial effects:

the present invention provides monoclonal antibodies against SARS-CoV-2RBD which specifically bind to an epitope located on SARS-CoV-2RBD³⁵⁰VYAWN³⁵⁴The epitope is highly conserved among different SARS-CoV-2 strains and is not influenced by the conformation change of the S protein RBD in an 'upward' and 'downward' manner; and tests prove that the monoclonal antibody for resisting SARS-CoV-2RBD provided by the application is an effective neutralizing antibody, can be used for developing medicaments for preventing and/or treating diseases caused by SARS-CoV-2 infection, and has good clinical application value. The monoclonal antibody of the invention has clear identification epitope, can effectively identify SARS-CoV-2 or SARS-CoV-2S protein with various conformational forms, has excellent affinity, high specificity and good stability, and provides a new tool for preventing, detecting and treating SARS-CoV-2.

The invention provides variable region amino acid sequences of heavy chains and light chains of monoclonal antibodies targeting SARS-CoV-2RBD, on the basis, the monoclonal antibodies of the invention can be obtained by adopting a conventional antibody engineering method, and further, the active fragments or conservative variants thereof can be obtained by adopting modification of addition, deletion, substitution and the like of one or more amino acids, thereby laying a foundation for further improving the specificity and the affinity of the antibodies.

The invention also provides a newly found epitope positioned on SARS-CoV-2RBD, which is highly conserved among different SARS-CoV-2 strains, is not influenced by the 'upward' and 'downward' conformational changes of S protein RBD, has stronger immunoreactivity and immunogenicity, and provides important antigen targets for SARS-CoV-2 vaccine design, antibody development and antibody detection kit development.

Drawings

FIG. 1 shows the reaction of a positive-reacting polypeptide (SEQ ID NO. 9) with antisera;

in the figure, S1-S6 are swine serum obtained by RBD-based antigen design immunization; M1-M6 are mouse serum obtained by RBD-based antigen design immunization; NC is a non-RBD region polypeptide as a negative control.

FIG. 2 is the serum titer of immunized mice;

in the figure, 1-5 are numbers of 5 Balb/c mice immunized by the positive reaction polypeptide (SEQ ID NO. 9); SMCC-BSA was a negative control.

FIG. 3 is a graph showing the result of IFA identification of the reactivity of the monoclonal antibody of the present invention;

in the figure, 15G9 is anti-SARS-CoV-2 RBD monoclonal antibody, alexa Fluor488 is Alexa Fluor 488-labeled goat anti-mouse IgG; DAPI is a nuclear dye, indicating the nucleus.

FIG. 4 is a diagram showing the Western-blotting identification result of the monoclonal antibody (15G 9) of the present invention;

in the figure, M represents the protein Marker, SARS-CoV-2, SARS-CoV and MERS-CoV represent the S protein of the corresponding virus in the corresponding lane, respectively.

FIG. 5 is a graph showing the results of the determination of the neutralizing ability of the monoclonal antibody of the present invention;

in the figure, 15G9 is an anti-SARS-CoV-2 RBD monoclonal antibody, NC is a monoclonal antibody against another virus, and this is used as a negative control.

FIG. 6 is a diagram showing the results of identifying epitope minimum motifs;

in the figure, the left side shows the reaction result of the N-terminal truncated polypeptide; the right side is the C-terminal truncated polypeptide reaction results.

FIG. 7 is a spatial distribution of epitopes.

In the figure, the circles indicate the epitopes.

Detailed Description

The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; the instruments and equipment involved in the following examples are conventional instruments and equipment unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.

Example 1B-cell epitope peptide for serum analysis of animals immunized with antigens based on RBD structural design

1. Design of antigens

The antigen is engineered on the basis of SARS-CoV-2RBD by a strategy similar to that reported previously (A Universal Design of Betaconovir Vaccines against drugs COVID-19, MERS, and SARS, dai, L, etc.), and the engineered antigen is in a stable RBD-dimer form without introducing any foreign sequence, thereby retaining the antigen efficacy and improving the neutralizing antibody titer.

2. Obtaining animal serum

The mice and pigs were immunized intramuscularly with RBD-based designed antigen (5 μ g per mouse, 50 μ g per pig), respectively. After the initial injection, all animals were boosted 2 times at day 14 and day 28 after the initial immunization. Blood samples were collected 14 days after the last booster and used for B cell epitope peptide analysis experiments.

5363 method for analyzing cell epitope peptide of 3.B

According to the reference sequence of SARS-CoV-2S protein (access number: YP _ 009724390), 22 overlapping peptide fragments covering the whole RBD and having a length of 20 amino acids were synthesized, and the adjacent peptide fragments overlapped by 5 amino acids.

And respectively coating the peptide segments on a 96-hole enzyme label plate, carrying out indirect enzyme-linked immunosorbent assay (ELISA) detection on the 96-hole enzyme label plate and the obtained animal serum, and determining the positive reaction peptide segments, wherein the amino acid sequence of the positive reaction peptide segments is shown as SEQ ID NO.9, and the reaction of the positive reaction polypeptide and the immune animal serum is shown in figure 1.

The indirect ELISA procedure was as follows:

(1) Diluting the peptide fragment into coating solution with the concentration of 2.5 mu g/mL by CBS solution, coating the ELISA plate with the coating solution at 100 mu l/hole, and sealing overnight at 4 ℃;

(2) Discarding the coating solution, washing the plate with PBST, sealing with 5% skimmed milk, and sealing at 4 deg.C overnight;

(3) Will be measured at a rate of 1:100 diluted mouse serum samples (primary antibody) and a 1: adding 1000 diluted pig serum samples (primary antibody) into an enzyme label plate in sequence, incubating at 50 mu l/hole for 30min at 37 ℃;

(4) Discarding the primary antibody, washing the plate by PBST, cleaning and drying;

(5) Adding diluted HRP-labeled goat anti-mouse IgG (secondary antibody) or goat anti-pig IgG (secondary antibody) into the reaction well, incubating at the temperature of 50 mu l/well for 30min at 37 ℃;

(6) Discarding the secondary antibody, washing with PBST, and patting dry;

(7) Adding 100 mul of TMB color developing solution prepared in situ into each hole, and reacting for 5min in a dark room;

(8) Add 50. Mu.l 2M H per well₂SO₄Terminating the reaction;

(9) Microplate reader for reading OD of each well₄₅₀The value is obtained.

EXAMPLE 2 preparation of monoclonal antibodies

1. Preparation of immunogen-polypeptide coupled Carrier protein BSA (bovine serum Albumin)

Coupling was performed using a water-soluble amino-mercapto crosslinker, sulfo-SMCC. Sulfo-SMCC has two reactive groups, sulfo-NHS ester and maleimide, and can react between primary amino and sulfhydryl. Firstly, under the condition of pH7-9, sulfo-SMCC reacts with primary amine groups of carrier protein BSA to form stable amide bonds, so as to obtain activated carrier protein BSA. Next, the activated BSA was dialyzed against PBS (pH 7.2-7.4) and the dialyzate was changed at least three times at 6-hour intervals. The dialyzed solution was collected and adjusted to a protein concentration of 5mg/ml with PBS. Finally, under the condition of pH 6.5-7.5, the activated BSA reacts with the sulfydryl of the positive reaction peptide segment SEQ ID NO.9 to form a stable thioether bond, and a conjugate of the immunogenic carrier protein BSA and the positive reaction peptide segment is formed for antibody production.

2. Animal immunization

(1) Adding Freund's complete adjuvant into immunogen, emulsifying for first immunization;

(2) 5 female BALB/c mice of 4-8 weeks old are immunized by a method of subcutaneous multipoint injection at the back, and the immunization dose is 20 mu g/mouse;

(3) Emulsifying the immune antigen with Freund's incomplete adjuvant every 2 weeks, and performing 2 times of booster immunization on BALB/c mice by the same method and dosage;

(4) After 2 weeks, tail vein blood collection was performed to determine the titer of specific antibody against SARS-CoV-2, and mice with higher titer (FIG. 2) were selected, and BALB/c mice were subjected to super-strong immunization with immunogen without adjuvant by tail vein injection 4 days before cell fusion, and the immunization dose was 40. Mu.g/mouse.

3. Cell fusion and monoclonal antibody preparation

Adopting a polyethylene glycol method, and mixing splenocytes of an immunized mouse and myeloma cells SP2/0 of the mouse according to the cell number of 8:1, and screening the fused cells by using HAT selective medium; 12 days after the fusion, SARS-CoV-2S protein and positive reaction peptide fragment are respectively used as coating antigen, and positive hybridoma cells are primarily screened by indirect ELISA method;

the indirect ELISA procedure was as in example 1, with the primary antibody and the secondary antibody being different, with the primary antibody being 1: hybridoma supernatant at 100 dilutions, secondary antibody HRP-labeled goat anti-mouse IgG (secondary antibody), and other procedures were as in example 1.

4. Subcloning of hybridoma cells by limiting dilution method

Diluting the above-mentioned positive hybridoma cells to about 1.5cells/ml with 1640/10 complete medium, adding 100. Mu.l per well to a 96-well plate pre-plated with 100. Mu.l of feeder cells, placing at 37 ℃,5% CO₂Cultured in an incubator6 to 8 days; further screening positive hybridoma cells by an indirect ELISA method; subcloning for 2-3 times until obtaining hybridoma cell strain secreting monoclonal antibody of anti SARS-CoV-2S protein and positive polypeptide SEQ ID NO.9, obtaining target hybridoma cell, expanding and culturing the screened positive monoclonal antibody with cell number of 1-2X 10⁶Freezing and storing in a tube.

5. Stability identification of monoclonal hybridoma cell strain

Continuously culturing the established monoclonal hybridoma cell strain for 3 months and repeatedly freezing and storing by liquid nitrogen for resuscitation so as to identify the stability of the hybridoma cell; the results show that the monoclonal hybridoma cell strain has good stability.

6. In vivo induced ascites method for preparing monoclonal antibody

Selecting female Balb/c mice, injecting 500 μ l sterilized paraffin into abdominal cavity, injecting obtained monoclonal hybridoma cells into abdominal cavity one week later, the injection amount is 2 × 10⁵And (3) taking out ascites after the belly of the mouse expands, centrifuging, taking out supernatant, and purifying the ascites by using a saturated ammonium sulfate method.

EXAMPLE 3 purification and characterization of antibodies

1. The saturated ammonium sulfate precipitation method is used for purifying the antibody and the operation method is as follows:

(1) 5ml of monoclonal antibody ascites is taken, 5ml of PBS buffer solution is added, 2.5ml of saturated ammonium sulfate solution is added dropwise to obtain 20% ammonium sulfate solution, the ammonium sulfate solution is added and stirred, and after the mixture is fully and uniformly mixed, the mixture is kept stand for 30min.

(2) 8000r/min, centrifuging for 20min, and discarding the precipitate to remove fibrin.

(3) Adding 12.5ml saturated ammonium sulfate solution into the supernatant, mixing well, standing for 30min.

(4) 8000r/min, centrifuging for 20min, and discarding the supernatant.

(5) Dissolving the precipitate in 10ml PBS buffer solution, adding 5ml saturated ammonium sulfate solution to obtain 33% ammonium sulfate solution, mixing, and standing for 30min.

(6) 8000r/min, centrifuging for 20min, and discarding supernatant to remove albumin.

(7) Repeat 5,2-3 times.

(8) The precipitate was dissolved in 5ml of PBS buffer, and the solution was placed in a dialysis bag, dialyzed against PBS buffer at 4 ℃ and changed 4 times.

(9) 8000r/min, centrifuging for 20min, discarding precipitate to obtain supernatant as purified antibody, measuring antibody concentration, packaging, and storing at-20 deg.C.

2. Monoclonal antibody potency assay

The indirect ELISA assay was performed with reference to example 1, with a slight difference in primary antibody: diluting the purified monoclonal antibody by using 5% skim milk at a ratio of 2 times from 1; other steps are carried out according to example 1, and the ELISA detection result shows that the monoclonal antibody titer is 1: 4.096X 10⁵。

3. Subtype identification

The subtype of the Monoclonal Antibody is identified by a Mouse Monoclonal Antibody subtype identification Kit (Sigma), and the identification result shows that the Monoclonal Antibody belongs to IgG1 and the light chain type is Kappa type.

4. Identification of monoclonal antibody specificity

Diluting the monoclonal antibody according to a certain proportion, respectively adding the diluted monoclonal antibody into 293T cells transiently transfected by a eukaryotic expression vector pLVX-SARS-CoV-2-S, and determining the result by using an IFA detection method, wherein the monoclonal antibody specifically reacts with SARS-CoV-2S protein which is a cell source, but does not react with untransfected cells; as shown in FIG. 4, the result of Western-blotting assay was that the S protein specifically recognizing SARS-CoV-2 reacted with the monoclonal antibody, but not with the S proteins of SARS-CoV and MERS-CoV.

5. Neutralizing Activity of monoclonal antibodies

By substituting the virus neutralization assay (sVNT), in particular using cPass^TMThe neutralizing capacity of the monoclonal antibody is evaluated by a SARS-CoV-2 neutralizing antibody kit (Kinsrui, nanjing, china), the specific operation steps are carried out according to the product instruction, the negative control is the monoclonal antibody of the African swine fever virus, and the result is shown in figure 5.Inhibition value) = (1-sample OD value/negative control OD value) × 100%, when Inhibition value is not less than 20%, knot is formedIf the result is positive, the detected monoclonal antibody is a neutralizing antibody; when the suppression value is<At 20%, the result is negative, indicating that no neutralizing antibody was detected; as can be seen from fig. 5, the monoclonal antibody is a neutralizing antibody.

EXAMPLE 4 monoclonal antibody variable region Gene amplification and sequencing

1. Primer design

Designing heavy chain variable region primer sequence according to sequence characteristics of mouse-derived monoclonal antibody

P1：5’-AGGTSMARC TGCAGSAGTCWGG-3’；

P2：5’-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC-3’。

Design of light chain variable region primer sequences

P3：5’-ACTAGTCGACATGGAGWCAGACACACTSCTGYTATGGGT-3’；

P4：5’-CCAGCTTGGTCCCCCCTCCGAACGTGT-3’。

2. Polymerase Chain Reaction (PCR) amplification

The variable region sequences of the monoclonal antibodies are respectively obtained by a molecular cloning technology and sent to Shanghai Biotechnology Limited company for sequencing.

Total RNA from hybridoma cells was isolated using TRIzol and PrimeScript was used^TMII kit (Takara Biomedical Technology (Beijing) Co.) cDNA was synthesized. The light chain variable region (VL) and the variable region heavy chain (VH) of the monoclonal antibody were amplified separately in two separate PCR reactions using the primers described above.

The sequencing results were as follows: the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively shown in SEQ ID NO.7 and SEQ ID NO. 8. Further analyzing to obtain the amino acid sequences of the heavy chain variable region VHCDR1-3 of the monoclonal antibody as shown in SEQ ID NO.1-3 respectively; the amino acid sequences of the light chain variable region VLCDR1-3 of the monoclonal antibody are respectively shown in SEQ ID NO. 4-6.

Example 5 identification of epitope minimum motifs recognized by anti-SARS-CoV-2 RBD monoclonal antibodies

By systematically creating a truncated library from the two-terminal truncated positive polypeptide R345 (SEQ ID NO. 9), as shown in Table 1, the truncated polypeptides and the monoclonal were identified by indirect ELISAThe antibody reaction, indirect ELISA assay method was performed as described in example 1, and the results of the first antibody using the monoclonal antibody of the present invention are shown in FIG. 6, in which the results of the reaction of the N-terminally truncated polypeptide with the monoclonal antibody are shown on the left and the results of the reaction of the C-terminally truncated polypeptide with the monoclonal antibody are shown on the right, and it can be seen from FIG. 6 that the minimum epitope peptide required for epitope activity is shown "³⁵⁰VYAWN³⁵⁴”(SEQ ID NO.10)。

TABLE 1 truncated peptide amino acid sequence listing

Example 6 conservation analysis of epitopes

In order to analyze the conservation of the epitope in the currently circulating SARS-CoV-2 strain, the receptor binding site changes occurred in different SARS-CoV-2 virus strains were collected from the GISAID database, and a total of 431,752 SARS-CoV-2 virus data were collected, which revealed that the epitope was highly conserved among different SARS-CoV-2 strains, and no mutation was observed at the site of the epitope.

Example 7 spatial localization and structural analysis of epitopes

The RBD of SARS-CoV-2S protein includes two different conformation forms of "up" and "down", and when all RBD of SARS-CoV-2S protein trimer are in "down" conformation, partial amino acid residues of RBD are concealed, so that some antibodies capable of recognizing the concealed region epitope can not recognize SARS-CoV-2 or SARS-CoV-2S protein, and can affect the monoclonal antibody binding capacity and accuracy of the immunoassay method established by using corresponding antibody. The spatial distribution and structural characteristics of the epitope (SEQ ID NO. 10) were analyzed by mapping to SARS-CoV-2S protein trimer (PDB ID:7A 95), and as a result, as shown in FIG. 7, the epitope was exposed on the surface of SARS-CoV-2S protein trimer, indicating that the epitope recognition is not affected by the S protein RBD 'up' and 'down' conformational changes, and can effectively recognize SARS-CoV-2 or SARS-CoV-2S protein in various conformational forms, ensuring the stability of immunoassay.

<110> Zheng Zhou university

<120> anti-SARS-CoV-2 RBD monoclonal antibody and application

<160> 28

<170> PatentIn version 3.5

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Gly Tyr Thr Phe Thr Asp Tyr Ala

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cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300

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<212> PRT

<213> Artificial sequence

<221> aa 345-353

<400> 24

Cys Thr Arg Phe Ala Ser Val Tyr Ala Trp

1 5 10

<210> 25

<211> 22

<212> DNA

<213> Artificial sequence

<221> heavy chain variable region upstream primer

<400> 25

aggtsmarct gcagsagtcw gg 22

<210> 26

<211> 32

<212> DNA

<213> Artificial sequence

<221> downstream primer for heavy chain variable region

<400> 26

tgaggagacg gtgaccgtgg tcccttggcc cc 32

<210> 27

<211> 39

<212> DNA

<213> Artificial sequence

<221> upstream primer for light chain variable region

<400> 27

actagtcgac atggagwcag acacactsct gytatgggt 39

<210> 28

<211> 27

<212> DNA

<213> Artificial sequence

<221> downstream primer for light chain variable region

<400> 28

ccagcttggt cccccctccg aacgtgt 27

Claims (9)

1. An anti-SARS-CoV-2 RBD monoclonal antibody, which comprises VHCDR1, VHCDR2 and VHCDR3 having amino acid sequences shown in SEQ ID Nos. 1 to 3, and VLCDR1, VLCDR2 and VLCDR3 having amino acid sequences shown in SEQ ID Nos. 4 to 6.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No.7 and a light chain variable region having an amino acid sequence as set forth in SEQ ID No. 8.

3. The monoclonal antibody of claim 1, wherein the heavy chain constant region is of the IgG1 type and the light chain constant region is of the Kappa type.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically binds to an epitope on SARS-CoV-2RBD, wherein the amino acid sequence of the epitope is as shown in SEQ ID No.9 or SEQ ID No. 10.

5. A nucleic acid molecule comprising a gene sequence encoding the monoclonal antibody of claim 1 or 2.

6. The nucleic acid molecule of claim 5, wherein the variable region of the heavy chain of the monoclonal antibody has the gene sequence shown in SEQ ID No.11 and the variable region of the light chain has the gene sequence shown in SEQ ID No. 12.

7. An expression cassette, recombinant vector, recombinant cell or recombinant bacterium comprising a nucleic acid molecule according to claim 5 or 6.

8. Use of a monoclonal antibody according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5 or 6 or an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium according to claim 7, which is any one of:

1) The application in preparing the medicine for preventing and/or treating the diseases caused by SARS-CoV-2 infection;

2) The application in preparing SARS-CoV-2 immunity detection reagent or reagent box.

9. A pharmaceutical composition comprising a monoclonal antibody of any one of claims 1-4 and a pharmaceutically acceptable carrier, diluent or excipient.

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