CN111995675A

CN111995675A - Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof

Info

Publication number: CN111995675A
Application number: CN202010922039.4A
Authority: CN
Inventors: 董金华; 李海梅; 陈丽梅
Original assignee: Weifang Medical University
Current assignee: Weifang Medical University
Priority date: 2020-05-15
Filing date: 2020-09-04
Publication date: 2020-11-27
Anticipated expiration: 2040-09-04
Also published as: CN111961133A; CN111995677A; CN112010965B; CN112010966B; CN111995678A; CN112010965A; CN111961133B; CN111995677B; CN111995676A; CN111995678B; CN111995675B; CN111995676B; CN112010966A

Abstract

The invention discloses a monoclonal antibody against new coronavirus SARS-CoV-2 spinous process protein RBD region and its application, the monoclonal antibody is specifically combined with new coronavirus SARS-CoV-2 spinous process glycoprotein RBD region, including complementarity determining regions CDRH1, CDRH2, CDRH3 of heavy chain variable region and complementarity determining regions CDRL1, CDRL2, CDRL3 of light chain variable region; the monoclonal antibody aiming at the spinous process protein RBD region of the SARS-CoV-2 of the new coronavirus has high titer and strong specificity, can be efficiently expressed, can be specifically combined with the spinous process protein RBD region on the surface of the SARS-CoV-2 of the new coronavirus, can be used for detecting the SARS-CoV-2 of the new coronavirus, neutralizes and weakens certain toxicity of the new coronavirus, and plays a role in preventing or/and treating pneumonia of the new coronavirus.

Description

Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof

Technical Field

The invention relates to the technical field of biotechnology, in particular to cellular immunology and molecular biology technologies, and specifically relates to a monoclonal antibody aiming at a novel coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof.

Background

The novel coronavirus SARS-CoV-2 is a single-stranded positive-strand RNA virus whose genome encodes 4 structural proteins: spinous process protein (S), small protein (E), matrix protein (M), and nucleocapsid protein (N). The S protein is a type I fusion protein, forms a trimer on the surface of virus particles, and consists of two subunits, namely S1 subunit and S2 subunit: s1 is responsible for receptor binding and S2 is responsible for membrane fusion. The Receptor Binding Domain (RBD) in S1 interacts with the human SARS-CoV receptor angiotensin converting enzyme II (ACE2) molecule, while S2 contains essential elements required for the membrane fusion process to achieve fusion of virus and cell, i.e., SARS-CoV-2 uses angiotensin converting enzyme 2(ACE2) as a receptor to enter target cells, and thus, the S protein determines the infectivity and its transmissibility in the host. And because the S protein is a main antigen for inducing protective immune response, the research and development of the vaccine mainly take the S protein as a target. It is therefore crucial to closely monitor the antigenic changes in the S protein of the spreading new coronavirus SARS-CoV-2.

Antibodies are important glycoprotein molecules in the mammalian immune system. The molecular structure is composed of two Heavy chains (Heavy chain) and two Light chains (Light chain), wherein the Heavy chain is divided into a Variable region of the Heavy chain (VH) and three Constant regions (Constant region of the Heavy chain; CH1, CH2, CH3), and the Light chain is composed of a Variable region (VL) and a Constant region (CL). The variable region has a function of specifically binding to an antigen, and is different from individual antibody, while the constant region of an antibody is determined by the species and subtype of the antibody. The heavy chain variable region of an antibody comprises three Complementarity determining regions (CDRH 1, CDRH2, CDRH3) and the light chain variable region also comprises three Complementarity determining regions (CDRL 1, CDRL2, and CDRL 3), which are also known as hypervariable regions and directly bind to an epitope.

At present, no specific therapeutic drug or vaccine aiming at the new coronavirus is available on the market, and the anti-new coronavirus drugs mainly comprise small-molecule drugs of Reiciclovir, chloroquine and hydroxychloroquine, lopinavir and ritonavir and three drugs of lopinavir, ritonavir and interferon, but have no obvious therapeutic effect and some even serious toxic and side effects. On the other hand, antibody drugs play an important role in the treatment of infectious diseases, autoimmune diseases, tumors and the like, and the development of monoclonal antibodies against the novel coronavirus SARS-CoV-2 is of great practical significance under the conditions that the SARS-CoV-2 vaccine which is currently faced is difficult to develop and has a long period and the great side effect of the traditional drugs is not obvious.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a monoclonal antibody aiming at the RBD region of the spike protein of the novel coronavirus SARS-CoV-2 and application thereof.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention discloses a monoclonal antibody aiming at a new coronavirus SARS-CoV-2 spinous process protein RBD region, which is specifically combined with the new coronavirus SARS-CoV-2 spinous process glycoprotein RBD region and comprises complementarity determining regions CDRH1, CDRH2, CDRH3 of a heavy chain variable region and complementarity determining regions CDRL1, CDRL2 and CDRL3 of a light chain variable region; the amino acid sequences of the complementarity determining regions CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4; the amino acid sequences of the complementarity determining regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: 8.

a preferred monoclonal antibody aiming at the RBD region of the spinous process protein of the novel coronavirus SARS-CoV-2 is named C3, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO: 1, the amino acid sequence of the light chain variable region of the monoclonal antibody is SEQ ID NO: 5.

the invention also includes an isolated nucleic acid molecule encoding a monoclonal antibody as described in any one of the above.

The invention also includes an expression vector comprising a nucleic acid molecule as described above, which expression vector comprises, in addition to the nucleic acid molecule as described above, an expression control sequence operably linked to the sequence of said nucleic acid molecule.

An expression vector refers to a nucleic acid vehicle into which a polynucleotide encoding the C3 antibody can be inserted and the C3 antibody expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed within the host cell. Types of vectors include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. In principle, any vector may be used as long as it is replicable and stable in the host. In addition to the origin of replication, the expression vector may contain a marker gene and other translational regulatory elements.

The invention also includes a host cell comprising a nucleic acid molecule as described above or an expression vector as described above.

The host cell expressing the C3 antibody can be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, Streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells or Bowes melanoma cells.

The invention also includes a method for detecting the level of the novel coronavirus SARS-CoV-2 for non-diagnostic purposes, comprising the steps of:

extracting a sample containing new coronavirus SARS-CoV-2;

contacting the sample obtained in the step I with the monoclonal antibody C3;

and thirdly, detecting the immunoreaction of the sample and the monoclonal antibody.

The invention also includes the application of the monoclonal antibody aiming at the new coronavirus SARS-CoV-2 spinous process protein RBD zone in the preparation of new coronavirus SARS-CoV-2 detection products.

The detection product includes, but is not limited to, a detection reagent, a kit, a chip or a test paper. Any assay product capable of detecting SARS-CoV-2 comprising a binding molecule as described above is included within the scope of the invention.

The invention also includes the application of the monoclonal antibody aiming at the new coronavirus SARS-CoV-2 spinous process protein RBD region in the preparation of the medicine for inhibiting the new coronavirus SARS-CoV-2 antibody.

The invention also includes the application of the monoclonal antibody aiming at the spinous process protein RBD region of the new coronavirus SARS-CoV-2 in the preparation of an antibody pharmaceutical preparation for preventing or treating pneumonia caused by the new coronavirus SARS-CoV-2.

The terms "new coronavirus SARS-CoV-2" and "SARS-CoV-2 virus", "new coronavirus", "SARS-CoV-2" and "new coronavirus SARS-CoV-2" used in the present invention can be used interchangeably.

Compared with the prior art, the invention has the following advantages:

the monoclonal antibody aiming at the spinous process protein RBD region of the SARS-CoV-2 of the new coronavirus has high titer and strong specificity, can be efficiently expressed, can be specifically combined with the spinous process protein RBD region on the surface of the SARS-CoV-2 of the new coronavirus, can be used for detecting the SARS-CoV-2 of the new coronavirus, neutralizing and weakening certain toxicity of the new coronavirus, and plays a role in preventing or/and treating pneumonia of the new coronavirus.

The phage display technology inserts exogenous DNA into the gene of phage coding coat protein, so that the expression product corresponding to the exogenous DNA fragment is fused in the coat protein of the phage to form fusion protein, and the fusion protein is displayed on the surface of the phage. Has the following remarkable advantages: direct physical connection between the genotype and the phenotype is established, so that the screening is simple, convenient and efficient. The invention screens the antibody which can be combined with the S protein of the new coronavirus SARS-CoV-2 from the synthetic antibody library Tomlinson I + J phage display antibody library, and the antibodies have important application value in the aspects of detecting the new coronavirus and weakening the virus toxicity.

Drawings

FIG. 1 shows the results of enzyme-linked immunoassay for the antigen binding performance of phages obtained by panning an antibody library;

FIG. 2 shows the results of ELISA assay for the antigen specificity of monoclonal antibody C3;

FIG. 3 shows the results of ELISA assay for the antigen specificity of monoclonal antibody A3;

FIG. 4 is a schematic diagram showing the principle of detection of new coronavirus using panning antibodies C3 and A3;

FIG. 5 is the results of detection of viral S protein by the C3 antibody in combination with phage-displayed A3.

Detailed Description

The invention aims to provide a monoclonal antibody aiming at a new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof, which are realized by the following technical scheme:

the present invention aims at providing monoclonal antibody against new coronavirus SARS-CoV-2 spike protein RBD region and its application, and the present invention is further illustrated by the following examples. The examples of the invention are intended to be illustrative and not limiting, and simple modifications thereof in accordance with the principles of the invention are intended to be within the scope of the claims. The invention is further described with reference to specific examples.

Example 1

Amplification of a phage display antibody library:

mu.L of E.coli TG-1 (MRC HGMP resources center, UK) containing Tomlinson I + J phage display antibody library was inoculated into 25mL of 2YT medium (1.6% Tryptone, 1% Yeast Extract, 0.5% NaCl) containing 100. mu.g/mL ampicillin and 1% glucose, and cultured at 37 ℃ to OD₆₀₀At 0.4, add 10⁹cfu (Colony Formation Unit) KM13 helper phage (MRC HGMP resources center, UK), 1 hour after infection at 37 ℃, 3000g was centrifuged for 30 minutes, the supernatant was discarded, and ammonia containing 100. mu.g/ml was usedBenzyl penicillin, 50. mu.g/mL kanamycin and 0.1% glucose in 2YT medium 50mL (1.6% Tryptone, 1% Yeast Extract, 0.5% NaCl) suspension of the thalli, 30 ℃, 250rpm speed shake bacteria for 16 hours, the next day 5000g, 30 minutes centrifugal culture fluid, separation and recovery of supernatant 40mL, in the supernatant adding 10mL PEG/NaCl solution, mixing evenly, then on ice for 30 minutes, 5000g, 30 minutes centrifugal, discard the supernatant, adding 2mL sterile PBS solution to dissolve the precipitate, as phage display antibody library solution, using Escherichia coli to titrate the phage display antibody library, the concentration of the prepared antibody library is 10¹²cfu/mL。

Second, panning of phage display antibody library

mu.L of a PBS solution containing 10. mu.g/ml of SARS-CoV-2 virus S protein (Nanjing Kingkumquat Biotech Co., Ltd.) was added to each of 10 wells of a 96-well microplate, overnight incubation was performed at 4 ℃ and the antigen solution was discarded the next time, 200. mu.L of a PBS solution containing 2% skim milk powder was added to each well, incubation was performed at 25 ℃ for 2 hours for blocking, and after 3 times of PBST washing, 100. mu.L of a phage solution (R0; each well containing 10. mu.L of the antigen solution) was added to each well⁹cfu phage) were incubated at room temperature for 2 hours, and after washing with PBST, phage bound to viral S protein were eluted by adding 100 μ L trypsin per well.

Culturing TG-1 E.coli to OD₆₀₀Taking 4mL of bacterial liquid, adding 500 mu L of dissolved phage solution into the bacterial liquid, infecting for 30 minutes at 37 ℃, centrifuging for 20 minutes at 5000g, discarding the supernatant, suspending the bacterial cells by using a 2YT culture medium containing 100 mu g/mL ampicillin, 50 mu g/mL kanamycin and 0.1% glucose, and shaking the bacterial cells for 16 hours at 30 ℃ and 250 rpm; centrifuging the culture solution for 30 minutes at 5000g the next day, separating and recovering the supernatant, adding 1/5 volumes of PEG/NaCl solution into the supernatant solution, uniformly mixing, placing on ice for 30 minutes, centrifuging for 30 minutes at 5000g, discarding the supernatant, and adding 200 μ L of sterilized PBS solution to serve as phage solution (R1) after the first enrichment; repeating the steps to respectively obtain phage solutions R2 and R3; and performing enzyme-linked immunosorbent assay, and verifying the binding specificity and binding performance of the phage display antibody library and the S protein obtained in the panning process.

Enzyme linked immunosorbent assayThe operation of the test is as follows: 100. mu.L of PBS containing a neocoronavirus S protein solution (2. mu.g/mL) or bovine serum albumin BSA (2. mu.g/mL) was added to a 96-well plate, overnight at 4 ℃, the antigen solution was discarded the next day, 200. mu.L of a solution containing 2% skim milk powder was added, and the plate was incubated at 25 ℃ for 2 hours and then blocked. The microplate was washed 3 times with PBS containing 0.1% Tween 20, and diluted R0, R2 and R3 phage solutions (10) were added⁹cfu/well), incubation at 25 ℃ for 1 hour, washing the microplate with PBST solution, addition of HRP-labeled mouse anti-M13 antibody, incubation for 1 hour, washing the plate with PBST, addition of HRP substrate TMBZ (prepared with sodium acetate solution pH6.0, containing 1/10000 diluted 30% H₂O₂) And after color development, measuring the absorbance at 450nm and 630nm by using an enzyme-labeling instrument, drawing a histogram, and comparing the steps to obtain the binding performance of the phage antibody with the S protein and the BSA.

The results of the enzyme-linked immunosorbent assay are shown in fig. 1, and when the binding capacities of the phage libraries R0, R2 and R3 obtained in the phage panning process and the S protein are compared, it is found that the binding capacity of the phage solution R2 obtained in the second panning process and the S protein is significantly increased, and the binding performance to BSA is very weak and unchanged, which indicates that the antibodies against the new coronavirus S protein in the constructed phage display antibody library are enriched.

Thirdly, screening of monoclonal antibody

Culturing TG-1 E.coli to OD₆₀₀Taking 100 mu L of elutriation sieve R2 phage antibody library dissolved out phage solution for infecting 200 mu L of escherichia coli bacterial solution, incubating for 30 minutes at 37 ℃, coating the bacterial solution on a 2YT culture medium plate containing 100 mu g/mL ampicillin, 50 mu g/mL kanamycin and 1% glucose, and culturing overnight at 37 ℃; the next day, 96 colonies were picked and inoculated onto 96-well plates, and cultured at 37 ℃ to OD₆₀₀To 0.4, M13 phage was added to each well, centrifuged at 5000g for 20 minutes after infection, the supernatant was removed, 200. mu.L of 2YT medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose was added to each well, the cells were suspended, and cultured at 30 ℃ and 250rpm for 16 hours; centrifuging the culture solution at 5000g for 30 min the next day, separating and recovering supernatant, performing enzyme-linked immunosorbent assay, and verifying each single gramBinding specificity and binding Performance of the monoclonal antibody to the S protein.

The enzyme-linked immunosorbent assay was performed as follows: 100 μ L of PBS solution containing virus S protein (1 μ g/mL) was added to a 96-well plate, overnight at 4 ℃, the antigen solution was discarded the next day, 200 μ L of a solution containing 2% skim milk powder was added, incubation was performed at 25 ℃ for 2 hours, and the plate was blocked. Washing the ELISA plate with PBS solution containing 0.1% Tween 20 for 3 times, adding phage solution, incubating at 25 deg.C for 1 hr, washing the ELISA plate with PBST solution, adding HRP-labeled mouse anti-M13 antibody, incubating for 1 hr, washing the plate with PBST, adding HRP substrate TMBZ (prepared with sodium acetate solution of pH6.0, containing 1/10000 diluted 30% H)₂O₂) After the development, absorbance at 450nm was measured with a microplate reader, a histogram was drawn, and the binding properties of the phage antibody prepared by each clone to S protein and bovine serum albumin were compared.

According to the experimental results, 6 microwells are dark in color, the absorbance of the microwells is 1.40, 1.30, 1.35, 1.05, 1.10 and 0.80 respectively, after the thallus corresponding to the microwells is subjected to amplification culture, plasmids are extracted from the antibodies in the microwells with the absorbance of 1.1 and subjected to gene sequencing, the antibodies are named as monoclonal antibodies C3 according to the positions (C rows and 3 columns) of the microwells in the experiment, the plasmids are extracted from the antibodies in the microwells with the absorbance of 1.3 and subjected to gene sequencing, the antibodies are named as monoclonal antibodies A3 according to the positions (A rows and 3 columns) of the microwells in the experiment, and the antibodies are compared with the antibody sequences registered in an antibody gene library, so that the antibodies are novel antibodies, and the same sequences as the antibody genes are not found. The details of the amino acid sequence of the antibody are as follows.

The heavy chain variable region sequence of the C3 antibody is SEQ ID NO: 1, CDRH1 sequence of SEQ ID NO: 2; the CDRH2 sequence is SEQ ID NO: 3; the CDRH3 sequence is SEQ ID NO: 4;

the variable region sequence of the light chain of the C3 antibody is SEQ ID NO: 5, CDRL1 sequence is SEQ ID NO: 6; the CDRL2 sequence is SEQ ID NO: 7; the CDRL3 sequence is SEQ ID NO: 8.

the heavy chain variable region sequence of the A3 antibody is SEQ ID NO: 9, CDRH1 sequence as SEQ ID NO: 10; the CDRH2 sequence is SEQ ID NO: 11; the CDRH3 sequence is SEQ ID NO: 12;

the variable region sequence of the light chain of the A3 antibody is SEQ ID NO: 13, CDRL1 sequence of SEQ ID NO: 14; the CDRL2 sequence is SEQ ID NO: 15; the CDRL3 sequence is SEQ ID NO: 16.

the sequence referred to by C3 has the following specific information:

SEQ ID NO：1：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIASSGYYTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDADSFDYWGQGTLVTVSS；

SEQ ID NO：2：GFTFSSYA；

SEQ ID NO：3：IASSGYYT；

SEQ ID NO：4：AKDADSFDY；

SEQ ID NO：5：

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSAPSTFGQGTKVEIKR；

SEQ ID NO：6：QSISSY；

SEQ ID NO：7：AAS；

SEQ ID NO：8：QQAYSAPST；

the sequence referred to by A3 has the following specific information:

SEQ ID NO：9：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYITDDGANTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYATFDYWGQGTLVTVSS；

SEQ ID NO：10：

GFTFSSYA；

SEQ ID NO：11：

ITDDGANT；

SEQ ID NO：12：

AKSYATFDY；

SEQ ID NO：13：

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAAYYPTTFGQGTKVEIKR；

SEQ ID NO：14：

QSISSY；

SEQ ID NO：15：

NAS；

SEQ ID NO：16：

QQAAYYPTT。

fourth, antigen specificity of monoclonal antibody

Adding 100 mu L of new coronavirus S protein, S-RBD protein and BSA protein solution with the concentration of 1 mu g/mL into a 96-hole enzyme label plate, staying overnight at 4 ℃, discarding the protein solution the next day, adding 200 mu L of 2% skimmed milk powder solution, incubating for 2 hours at 25 ℃, and sealing the enzyme label plate; after washing the microplate 3 times with PBS containing 0.1% Tween 20, 100. mu.L of diluted phage display antibody solution was added to each well, incubated at 25 ℃ for 1 hour, the microplate was washed with PBST solution, HRP-labeled mouse anti-M13 antibody was added, and after incubation for 1 hour, the microplate was washed with PBST, and HRP substrate TMBZ (prepared with sodium acetate solution pH6.0, containing 1/10000 diluted 30% H) was added₂O₂) After the development, absorbance at 450nm was measured with a microplate reader, a histogram was plotted, and the binding properties of the phage antibody produced by each clone and the envelope protein were compared.

The results of ELISA using monoclonal antibody C3 are shown in FIG. 2, in which C3 binds to S protein and S-RBD protein, and C3 antibody was compared with coated BSA as an antibody recognizing RBD region of S protein, indicating that the binding of C3 antibody to S protein is specific.

The results of ELISA using monoclonal antibody A3 are shown in FIG. 3, in which A3 binds to S protein, but binds to S-RBD protein only weakly and mainly to the region other than the RBD region of S protein, and A3 antibody shows specificity in binding to S protein when compared to coated BSA.

Fifthly, detecting the novel coronavirus S protein by using the Fab fragment of the C3 antibody and the A3 antibody displayed by phage

The detection principle is shown in FIG. 4, the C3 protein and A3 displayed by phage are used for detecting the S protein of the new coronavirus, the Fab fragment of the C3 antibody is coated in the hole of a 96-hole microplate, the microplate is sealed, S protein or BSA protein is added, phage display A3 antibody is added after the plate is washed, then anti-phage antibody which is marked with horse radish peroxidase is added, the plate is washed, and then substrate is added for color development, when the sample does not contain new coronavirus S protein or BSA protein, the reaction system does not develop color, when virus S protein exists, the system develops color, and the more virus S protein in the sample, the more A3 phage captured to the microplate by the virus S protein, the more anti-phage antibody captured correspondingly, and the darker the color of the enzyme-added substrate after development, and the method can be used to determine whether there is virus S1 protein in the sample, the method can also be used to detect the presence of SARS-CoV-2 virus in a sample.

The specific operation is as follows: adding the Fab fragment of the C3 antibody into the hole of the 96-hole enzyme label plate, staying overnight at 4 ℃, discarding the liquid in the hole the next time, adding 200 mu L of 2% skimmed milk powder solution, standing for two hours at room temperature, and sealing the enzyme label plate. After washing the plate, 100. mu.L of a solution containing the S protein of the novel coronavirus or a Bovine Serum Albumin (BSA) solution was added to the wells, incubated at 25 ℃ for 1 hour, the well-containing solution was removed, the plate was washed with a PBS solution (PBST solution) containing 0.1% Tween, and a phage-displayed A3 antibody solution (10) was added⁹cfu/mL), incubation for 1 hour at 25 ℃, adding an anti-phage antibody solution (1 mu g/mL) marked with horseradish peroxidase (HRP) after plate washing, incubation for 1 hour at 25 ℃, removing the solution in the wells, washing the plates for 3 times by PBST, finally adding an HRP substrate 3,3,5, 5-tetramethylbenzidine hydrochloride (TMBZ) solution for color development, measuring the absorbance of the solution in the wells at 450nm and 630nm, making a histogram, and comparing the binding capacity of the antibody with the S protein of the new coronavirus and the bovine serum albumin.

As shown in FIG. 5, the C3 antibody combined with phage-displayed A3 detected viral S protein, with the horizontal axis representing the name of the detected protein and the vertical axis representing the absorbance of the solution in the corresponding well. The absorbance of the wells containing the viral S protein ELISA in the solution of the combination of C3 and A3 was 0.64, while the absorbance of the wells with BSA added was 0.02, indicating that the combination with A3 can be used to detect the novel coronavirus S protein in solution.

Sequence listing

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1 5

Claims

1. A monoclonal antibody against the RBD region of the spike protein of the novel coronavirus SARS-CoV-2, which is characterized in that: the monoclonal antibody is specifically combined with an RBD region of spinous process glycoprotein of a novel coronavirus SARS-CoV-2, and comprises complementarity determining regions CDRH1, CDRH2 and CDRH3 of a heavy chain variable region and complementarity determining regions CDRL1, CDRL2 and CDRL3 of a light chain variable region; the amino acid sequences of the complementarity determining regions CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4; the amino acid sequences of the complementarity determining regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: 8.

2. the monoclonal antibody against the RBD region of the spike protein of the novel coronavirus SARS-CoV-2 according to claim 1, wherein: the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO: 1, the amino acid sequence of the light chain variable region of the monoclonal antibody is SEQ ID NO: 5.

3. an isolated nucleic acid molecule, wherein: the nucleic acid molecule encodes the monoclonal antibody of any one of claims 1-2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. A method for detecting the level of the novel coronavirus SARS-CoV-2 for non-diagnostic purposes, comprising: the method comprises the following steps:

extracting a sample containing new coronavirus SARS-CoV-2;

contacting the sample obtained in the step I with the monoclonal antibody of any one of claims 1-2;

7. The use of the monoclonal antibody against the spinous process protein RBD region of the novel coronavirus SARS-CoV-2 as claimed in any one of claims 1-2 in the preparation of a novel coronavirus SARS-CoV-2 detection product.

8. The use of the monoclonal antibody against the spinous process protein RBD region of the neocoronavirus SARS-CoV-2 as claimed in any one of claims 1-2 in the preparation of a medicament for inhibiting the neocoronavirus SARS-CoV-2.

9. The use of the monoclonal antibody against the spinous process protein RBD region of the neocoronavirus SARS-CoV-2 as claimed in any one of claims 1 to 2 in the preparation of an antibody pharmaceutical preparation for preventing or treating pneumonia caused by the neocoronavirus SARS-CoV-2.