CN115850457A

CN115850457A - Antibody of targeting coronavirus RBD protein

Info

Publication number: CN115850457A
Application number: CN202211272858.4A
Authority: CN
Inventors: 刘霄卉; 任建廷; 魏化伟
Original assignee: Jiangsu Dongkang Biomedical Technology Co ltd
Current assignee: Jiangsu Dongkang Biomedical Technology Co ltd
Priority date: 2022-10-18
Filing date: 2022-10-18
Publication date: 2023-03-28

Abstract

The invention provides an antibody targeting coronavirus RBD protein, which comprises a heavy chain variable region containing three CDRs and a light chain variable region containing three CDRs; wherein, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 2. 3 and 4, the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown as SEQ ID NO: 10. 11, 12. The invention also provides polynucleotide molecules encoding the antibodies of the invention, expression vectors, host cells, methods and uses for expressing the antibodies of the invention. The invention also provides conjugates, compositions and derivatives comprising the antibodies of the invention.

Description

Antibody of targeting coronavirus RBD protein

Technical Field

The invention belongs to the fields of cellular immunology and molecular biology, and relates to an antibody of a targeted coronavirus RBD protein.

Background

Coronavirus particles are irregular in shape and about 60-220nm in diameter. The virion is surrounded by a fat membrane, the surface of which has three glycoproteins: spike glycoprotein (S, spike Protein, which is the receptor binding site, cytolytic and major antigenic site); small Envelope glycoprotein (E, envelope Protein, smaller, envelope-bound Protein); membrane glycoproteins (M, membrane proteins, responsible for transmembrane transport of nutrients, budding release of nascent viruses and formation of viral envelope). A few species also have hemagglutinin glycoproteins (HE proteins). The nucleic acid of coronavirus is non-segmented single-stranded (+) RNA, has the length of 27-31kb, is the longest RNA nucleic acid chain in RNA virus, and has important structural characteristics specific to positive-strand RNA: namely, the 5 'end of the RNA chain is provided with a methylated cap, and the 3' end is provided with a polyA tail structure. This structure is very similar to eukaryotic mRNA and is an important structural basis for the genomic RNA itself to function as a translation template, and the RNA-DNA-RNA transcription process is omitted. The rate of recombination between the RNA of coronaviruses and RNA is very high, and it is this high rate of recombination that causes the virus to mutate.

Coronaviridae only infect vertebrates and are associated with many diseases in humans and animals. Since the first international discussion of coronaviruses in germany was held in 1980, it has been increasingly appreciated by medical, veterinary and molecular biologists. Such viruses have tropism for the gastrointestinal tract, respiratory tract and nervous system. Coronavirus infection in children is not common.

The novel coronavirus infection (Corona Virus Disease 2019, COVID-19) is an acute respiratory infectious Disease caused by the novel coronavirus (SARS-CoV-2), has a large-scale outbreak and rapid spread in various countries all over the world, is currently a global serious public health event, and is one of the epidemic diseases with the largest number of fatalities in human history.

Disclosure of Invention

The invention aims to provide an antibody targeting coronavirus RBD protein.

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

the invention provides a specific antibody or an antigen-binding fragment thereof for resisting RBD protein, which comprises a first variable region and a second variable region, wherein the first variable region is an antibody heavy chain variable region, and the amino acid sequences of antigen complementarity determining regions CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 is shown in the specification; wherein the second variable region is an antibody light chain variable region, and the amino acid sequences of the antigen complementarity determining regions CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO:10, SEQ ID NO: shown at 12.

The term "antibody" as described herein is used in the broadest sense and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, single chain antibodies, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanized, human and synthetic.

The term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that comprises one or more CDRs or any other antibody fragment that binds an antigen but does not comprise the entire native antibody structure. Examples of antigen binding fragments include, but are not limited to, diabodies, fabs, fab ', F (ab') 2, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2, bispecific dsFv (dsFv) s, disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (diabodies), diabodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding fragment is capable of binding to the same antigen as the parent antibody.

Further, the specific antibody or antigen-binding fragment further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR4, and light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are respectively as shown in SEQ ID NO: 5. 6, 7 and 8; the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are respectively shown in SEQ ID NO: 13. 14, 15, 16.

Further, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: shown at 9.

Further, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 17.

Further, the specific antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region.

The term "variable" as used herein means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the light and heavy chain variable regions that become Complementarity Determining Regions (CDRs) or hypervariable regions.

The present invention provides a substance as described in any one of:

1) An isolated polynucleotide molecule encoding a specific antibody or antigen-binding fragment thereof as described above, or an expression vector comprising the same.

The term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. The vector may be used to transform, transduce or transfect a host cell so that it expresses the carried genetic element in the host cell. Examples of vectors include plasmids; phagemid; cosmids and artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda or M13 bacteriophages; and animal viruses. Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV 40).

2) An antibody derivative of a specific antibody or antigen-binding fragment thereof directed against an RBD protein, said antibody derivative comprising a complex of said specific antibody or antigen-binding fragment thereof directly or indirectly coupled to a detectable label.

The amino acid sequence derivative of the present invention is a sequence different from the natural amino acid sequence due to deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues or a combination thereof, and is a derivative having the same biological activity as the natural amino acid sequence or having improved structural stability (e.g., heat resistance, pH resistance, etc.).

The amino acid sequence derivatives of the invention have at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the native amino acid sequence.

3) An engineered host cell comprising a specific antibody or antigen-binding fragment thereof as described above, or a polynucleotide molecule as described above or an expression vector comprising the same, or a population of host cells comprising the same.

The term "host cell" as used herein refers to a host cell into which a vector comprising a polynucleotide sequence encoding an antibody can be introduced for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors of the invention are prokaryotes, yeast or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example of the family Enterobacteriaceae (Enterobacteriaceae), such as the genus Escherichia (Escherichia), for example Escherichia coli; enterobacter (Enterobacter); erwinia (Erwinia); klebsiella (Klebsiella); proteus (Proteus); salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); serratia species (Serratia), such as Serratia marcescens (Serratia marcescens); and Shigella (Shigella), and bacillus (bacillus), such as bacillus subtilis and bacillus licheniformis (b.licheniformis); pseudomonas (Pseudomonas), such as Pseudomonas aeruginosa (p. Aeruginosa); and Streptomyces (Streptomyces).

4) A composition comprising an engineered host cell as described above or a population of host cells comprising the same.

Further, the composition comprises pharmaceutically acceptable adjuvants.

5) A product for detecting an RBD protein of a coronavirus, said product comprising a specific antibody or antigen-binding fragment thereof as defined above, a polynucleotide molecule or an expression vector comprising the same as defined above, or an antibody derivative as defined above.

Further, the product comprises a test paper, a chip or a kit.

6) A product for detecting coronavirus infection, said product comprising a specific antibody or antigen-binding fragment thereof as defined above, a polynucleotide molecule or an expression vector comprising the same, or an antibody derivative as defined above.

Further, the product comprises a test paper, a chip or a kit.

Further, the expression vector comprises a plasmid conventional expression vector, a lentivirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a piggyBac expression vector or a Sleeping Beauty transposition expression vector.

Further, the detectable label includes a fluorescent pigment such as fluorescein, rhodamine, texas red, phycoerythrin, phycocyanin, allophycocyanin, polymethacrylene-chlorophyll protein, avidin such as biotin, avidin, streptavidin, yolk avidin, avidin-like, paramagnetic atoms, radioactive isotopes such as radioactive iodine, radioactive cesium, radioactive iridium, radioactive cobalt, enzyme labels such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, β -galactosidase, lysozyme, malate dehydrogenase.

Further, the modified host cell includes prokaryotic cells such as including bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia, eukaryotic cells such as mammalian cells, insect cells, plant cells, yeast cells.

Further, the bacteria include Escherichia coli, bacillus subtilis, salmonella typhimurium, pseudomonas, streptomyces, and Staphylococcus.

Further, the mammalian cells include fibroblasts, lymphocytes, epithelial cells, and myeloblasts.

Further, the mammalian cells include HEK293 cells.

The present invention provides a method of producing a specific antibody or antigen-binding fragment thereof as hereinbefore described, the method comprising the steps of:

a) Providing a polynucleotide molecule as hereinbefore described or an expression vector comprising the same;

b) Transforming a host cell with the expression vector of step a);

c) Culturing the host cell obtained in step b) under suitable conditions;

d) Separating and purifying from the culture solution of the host cell to obtain the specific antibody or the antigen-binding fragment thereof.

The present invention provides any one of the following methods, the method comprising:

(1) A method for detecting RBD or fragments thereof in a test sample at a non-diagnostic and non-therapeutic destination, said method comprising the steps of: contacting a sample to be tested with the specific antibody or antigen-binding fragment thereof described above, or contacting a sample to be tested with the antibody derivative described above, and detecting the formation of a complex between the RBD or fragment thereof and the specific antibody or antigen-binding fragment thereof described above, or the antibody derivative described above.

(2) A method of making an engineered host cell as described above, or a population of host cells comprising the same, the method comprising the steps of: introducing into a host cell the specific antibody or antigen-binding fragment thereof as described above, or the polynucleotide molecule as described above or an expression vector comprising the same.

(3) A method of specifically inhibiting RBD, the method comprising the steps of: the specific antibody or antigen-binding fragment thereof, or the polynucleotide molecule or expression vector comprising the same, is introduced into a cell of an organism, and the RBD activity is inhibited by expression of the specific antibody or antigen-binding fragment thereof.

The term "RBD" in the present invention refers to the cellular Receptor Binding Domain (RBD) of the S protein domain of coronavirus. Coronaviruses generally include spike (S), small envelope protein (E), membrane vesicle (M), and nucleoprotein (N). Binding of the spike glycoprotein (S protein) to host cell receptors mediates viral entry and determines viral tissue or host tropism. The S protein, which recognizes host cell receptors and mediates membrane fusion, is critical for entry of viral particles into cells and is a key factor for viral infection of host cells. The RBD of the S protein domain is directly involved in the recognition of host receptors, and the amino acid variation of the region can cause the change of species tropism and infection characteristics of viruses.

The invention provides any one of the following applications, including:

(1) Use of a specific antibody or antigen-binding fragment thereof as hereinbefore described, a polynucleotide molecule or expression vector comprising the same as hereinbefore described, an antibody derivative as hereinbefore described, or a population of engineered host cells or host cells comprising the same as hereinbefore described for the detection of an RBD protein or fragment thereof.

(2) Use of a specific antibody or antigen-binding fragment thereof as hereinbefore described, a polynucleotide molecule or expression vector comprising the same as hereinbefore described, an antibody derivative as hereinbefore described, or an engineered host cell or population of host cells comprising the same as hereinbefore described in the manufacture of a product for the detection of coronavirus infection.

The term "coronavirus infection" refers to an infection with a coronavirus, including 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV. The symptoms caused by the coronavirus include fever, dry cough, fatigue, expectoration, headache, hemoptysis and diarrhea, and serious patients are accompanied with complications such as respiratory failure, acute respiratory distress syndrome, shock, organ failure and the like.

(3) Use of a specific antibody or antigen-binding fragment thereof as hereinbefore described, a polynucleotide molecule as hereinbefore described or an expression vector comprising the same, an engineered host cell as hereinbefore described or a population of host cells comprising the same for the preparation of a pharmaceutical composition for the diagnosis of a coronavirus related disease.

The term "coronavirus" or "CoV" refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV-2 refers to a coronavirus that was identified as emerging. SARS-CoV-2 is also known as 2019-nCoV. It binds to the human host cell receptor angiotensin converting enzyme 2 (ACE 2) via the viral spike protein.

(4) Use of a specific antibody or antigen-binding fragment thereof as hereinbefore described, a polynucleotide molecule or an expression vector comprising the same as hereinbefore described, or an engineered host cell or a population of host cells comprising the same as hereinbefore described in the manufacture of a medicament for the prophylaxis or treatment of a coronavirus-related disease.

(5) Use of a specific antibody or antigen-binding fragment thereof as hereinbefore described, a polynucleotide molecule as hereinbefore described or an expression vector comprising the same, an engineered host cell as hereinbefore described or a population of host cells comprising the same for the preparation of a pharmaceutical composition for the diagnosis of a coronavirus infection.

(6) Use of a specific antibody or antigen-binding fragment thereof as hereinbefore described, a polynucleotide molecule or expression vector comprising the same as hereinbefore described, or an engineered host cell or population of host cells comprising the same as hereinbefore described in the manufacture of a medicament for the prophylaxis or treatment of a coronavirus infection.

Drawings

FIG. 1 is an electrophoretogram of detection specific antibody 7D 9;

FIG. 2 is an HPLC chart for detecting specific antibody 7D 9;

FIG. 3 is a graph showing the binding activity of the specific antibody 7D9 detected by ELISA.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. It should be understood that the following examples are only illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out under conventional conditions.

EXAMPLE 1 screening of monoclonal antibodies

1. Synthesis of recombinant neo-corona RBD protein

Synthesizing an RBD protein sequence of the SARS-CoV-2 virus, and constructing into a pEM5.1 vector; extracting plasmids for transfection; transfecting to HEK293 cells, and culturing the cells for 7 days; and (3) harvesting the supernatant, purifying by using a Ni column, and concentrating and replacing a buffer solution to obtain the recombinant new crown RBD protein, wherein the sequence of the recombinant new crown RBD protein is derived from Uniprot P0DTC2. The sequence is shown as SEQ ID NO:1 is shown.

2. Immunization

The first immunization is carried out by 100 mug each of Freund's complete adjuvant through intraperitoneal injection, the total dose is 0.5 ml/mouse, and the second immunization is carried out at intervals of 3 weeks; taking Freund incomplete adjuvant at a dose of 50 μ g/0.5 ml/piece for the second time, and performing the third immunization at an interval of 2 weeks; cell fusion was prepared 10 days after the third injection;

taking feeder cells, and pressing 10 ⁵ Perforamen, plating 10 the day before fusion ⁵ Each well is 100 mu l; mice immunized spleen cells were fused with prepared myeloma cells using the fusion agent PEG, and plated on 96-cell culture plates to which feeder cells had been added at 100. Mu.l/well.

3. Screening and cloning of hybridoma cells

Screening positive holes by an ELISA detection method, and laying recombinant expression RBD protein overnight; washing the plate, adding skimmed milk powder, sealing, and standing at 37 deg.C for 1 hr; washing the plate, adding 100 mul of culture solution supernatant of 96 holes, and incubating for 1h at 37 ℃; washing the plate, adding a goat anti-mouse secondary antibody marked by HRP, and incubating for 30min at 37 ℃; washing the plate, adding a developing solution, developing for 10min, adding a stop solution, and reading the numerical value of OD 450; screening high expression cell strain for subcloning culture.

4. Sequencing

The cells were harvested, total RNA was extracted using Trizol, and cDNA was generated by reverse transcription using oligo (dT) 20 as a primer. Then, specific primers are used for PCR amplification of the heavy chain variable region gene and the light chain variable region gene respectively. And (3) after the PCR product is subjected to electrophoretic purification, inserting the PCR product into a vector through TA cloning, converting, and selecting positive clones for sequencing.

5. Results

The monoclonal antibody 7D9 against SARS-CoV-2 was selected and its sequence is shown in Table 1.

TABLE 1 sequences of monoclonal antibody 7D9

Example 2 functional study of monoclonal antibody 7D9

1. Expression and purification of monoclonal antibodies

1) The selected sequence is chemically synthesized and cloned into a eukaryotic expression vector.

2) And amplifying and extracting the plasmid.

3) Plasmids encoding the antibodies were transiently transfected into mammalian cells HEK293.

4) Collecting the supernatant, and purifying by affinity chromatography to obtain the monoclonal antibody.

5) As a result, the expression amount of the purified antibody was 189mg/L.

2. Detection of physicochemical Properties of monoclonal antibodies

2.1 gel electrophoresis detection of purity of monoclonal antibodies

1) The instruments and equipment are shown in Table 2

TABLE 2 Instrument Equipment

Name (R)	Manufacturer of the product	Model number
			Chemiluminescence imager	Tanon	Tanon-5200
Electrophoresis apparatus	BIO-RAD	poweerpac basic
			Electrophoresis tank	BIO-RAD	DYC-Mini4

2) The main reagents are shown in Table 3

TABLE 3 major reagents

Name (R)	Manufacturer of the product	Specification of	Goods number
				1M Tris-HCl buffer	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	60 ml/bottle	20200911
1.5M Tris-HCl buffer	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	100 ml/bottle	20200911
				10％SDS	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	10 ml/bottle	20200911
FastStain	Gene Universal	1000 ml/bottle	21DA
				30% glue solution (29	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	500 ml/bottle	20210414
Rainbow 180 broad-spectrum protein Marker	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	250μl(50T)	1202F021

3) Sample preparation

Mixing 20 μ l sample with 5 μ l 5 × reduction buffer, heating at 95 deg.C for 5min, and cooling;

mu.l of the sample was mixed well with 5. Mu.l of 5 Xnon-reducing buffer.

4) Electrophoresis

Preparing gel, adding a proper amount of electrophoresis buffer solution, adding the sample, and performing electrophoresis.

5) Dyeing and bleaching

After electrophoresis, putting the gel into a proper amount of Coomassie brilliant blue staining solution, and staining for 1 hour or more at room temperature; pouring out the staining solution, adding appropriate amount of Coomassie brilliant blue staining decolorization solution, and decolorizing at room temperature for 4-24 hr. After completion of decolorization, ddH was used ₂ O soaking, comparing with the undyed gel according to Marker protein, cutting off the gel of the needed protein component, and collecting. The protein to be purified is then separated from the gel.

6) Results

The results are shown in FIG. 1, the bands from left to right are marker and reduction band; the detection purity of the monoclonal antibody is more than 95 percent.

2.2HPLC detection of purity of monoclonal antibodies

1) The instruments and equipment are shown in Table 4

TABLE 4 Instrument Equipment

2) The main reagents are shown in Table 5

TABLE 5 major reagents

Name(s)	Manufacturer of the product	Specification of	Goods number
				Dipotassium hydrogen phosphate trihydrate	Sinopharm Group Chemical Reagent Co., Ltd.	500 g/bottle	10017592
Potassium dihydrogen phosphate	Sinopharm Group Chemical Reagent Co., Ltd.	500 g/bottle	10017692
				Potassium chloride	Sinopharm Group Chemical Reagent Co., Ltd.	500 g/bottle	10016392

3) Fluid phase system

Dipotassium phosphate trihydrate, potassium dihydrogen phosphate and potassium chloride are added into about 900ml of purified water, stirred and dissolved, the volume is adjusted to 1L, and the pH value is determined to be between 6.2 +/-0.1 by measuring with a pH meter. Filtering with 0.22 μm filter membrane, and storing at room temperature.

4) Sample preparation

System applicability sample: MIL62 Standard diluted to 2mg/ml with mobile phase

And (3) testing the sample: the sample to be tested was diluted to 2mg/ml with mobile phase.

5) The chromatographic conditions are shown in Table 6

TABLE 6 chromatographic conditions

6) Results

As shown in FIG. 2, the purity of monoclonal antibody was greater than 95%.

3. Detection of binding Activity of monoclonal antibodies

1) Coating: diluting the antigen RBD protein to 2 μ g/ml with coating solution, mixing well, adding 96-well coated plate, 100 μ l/well, sealing with membrane, and standing at 4 deg.C overnight.

2) The plate washer washes 3 times, no liquid can remain on the plate in the last time, and the liquid on the surface of the plate is patted dry by using absorbent paper.

3) And (3) sealing: add 5% milk powder (0.5 g milk powder in 10ml DPBS), 300. Mu.l/well, incubate 1h at 37 ℃ and wash plate 3 times as per step 2).

4) The antibody was diluted in a gradient of 100. Mu.l/well, reacted at 37 ℃ for 1h, and the plate was washed 3 times according to step 2).

5) Adding a secondary antibody: diluted with DPBS according to 1.

6) Color development: TMB was added in 100. Mu.l/well and developed in the dark at room temperature for 10min.

7) And (4) terminating: 2N H was added ₂ SO ₄ 100 μ l/well.

8) Measuring OD 450 with enzyme labeling instrument, and detecting within 10min.

9) Results

As a result, as shown in FIG. 3, the monoclonal antibody 7D9 can specifically bind to the antigen RBD protein and exhibits a concentration dependence with an EC50 of 0.03111. Mu.g/ml.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof specific for an RBD protein, comprising a first variable region and a second variable region, wherein the first variable region is an antibody heavy chain variable region having the amino acid sequences of CDR1, CDR2 and CDR3 of the antigen complementarity determining regions are set forth in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 is shown in the specification; wherein the second variable region is an antibody light chain variable region, and the amino acid sequences of the antigen complementarity determining regions CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO:10, SEQ ID NO: shown at 12.

2. The specific antibody or antigen-binding fragment of claim 1, further comprising heavy chain variable region framework regions FR1, FR2, FR3 and FR4 and light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are set forth in SEQ ID NOs: 5. 6, 7 and 8; the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are respectively shown in SEQ ID NO: 13. 14, 15 and 16;

preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:9 is shown in the figure;

preferably, the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: shown at 17.

3. A specific antibody or antigen-binding fragment as claimed in claim 1 or claim 2 wherein the specific antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region.

4. A substance according to any one of the following:

1) An isolated polynucleotide molecule or an expression vector comprising the same, wherein the polynucleotide molecule encodes the specific antibody or antigen-binding fragment thereof of claim 1 or 2;

2) An antibody derivative of a specific antibody or an antigen-binding fragment thereof against RBD protein, comprising a complex of the specific antibody or the antigen-binding fragment thereof of claim 1 or 2 coupled directly or indirectly to a detectable label;

3) An engineered host cell or a population of host cells comprising the same, wherein the engineered host cell comprises the specific antibody or antigen-binding fragment thereof of claim 1 or 2, or the polynucleotide molecule of claim 4 1) or an expression vector comprising the same;

4) A composition comprising the engineered host cell of claim 4 3) or a population of host cells comprising the same;

preferably, the composition comprises pharmaceutically acceptable adjuvants;

5) A product for detecting coronavirus RBD protein, said product comprising a specific antibody or antigen-binding fragment thereof of claim 1 or 2, a polynucleotide molecule or expression vector comprising 1) of claim 4, or an antibody derivative of 2) of claim 4;

preferably, the product comprises a test strip, chip or kit;

6) A product for detecting coronavirus infection, said product comprising a specific antibody or antigen-binding fragment thereof according to claim 1 or 2, a polynucleotide molecule or expression vector comprising 1) according to claim 4, an antibody derivative according to 2) according to claim 4;

preferably, the product comprises a dipstick, a chip or a kit.

5. The agent according to claim 4, wherein said expression vector comprises a plasmid-based regular expression vector, a lentiviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a piggyBac expression vector, or a Sleeping Beauty transposable expression vector.

6. Substance according to claim 4, characterized in that said detectable label comprises fluorochromes such as fluorescein, rhodamine, texas Red, phycoerythrin, phycocyanin, allophycocyanin, polymethacrylic flavin-chlorophyll protein, avidin such as biotin, avidin, streptavidin, vitellin, avidin-like, paramagnetic atoms, radioisotopes such as radioactive iodine, radioactive cesium, radioactive iridium, radioactive cobalt, enzyme labels such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, β -galactosidase, lysozyme, malate dehydrogenase.

7. The substance of claim 4, wherein said modified host cell comprises prokaryotic cells such as including bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia, eukaryotic cells such as mammalian cells, insect cells, plant cells, yeast cells;

preferably, the bacteria include escherichia coli, bacillus subtilis, salmonella typhimurium, pseudomonas, streptomyces, staphylococcus;

preferably, the mammalian cells include fibroblasts, lymphocytes, epithelial cells, myeloblasts;

preferably, the mammalian cells comprise HEK293 cells.

8. A method of producing a specific antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising the steps of:

a) Providing a polynucleotide molecule according to claim 4 1) or an expression vector comprising the same;

b) Transforming a host cell with the expression vector of step a);

c) Culturing the host cell obtained in step b) under suitable conditions;

d) Separating and purifying the host cell culture fluid to obtain the specific antibody or the antigen-binding fragment thereof according to claim 1 or 2.

9. Any of the following methods, wherein the method comprises:

(1) A method for detecting RBD or fragments thereof in a test sample at a non-diagnostic and non-therapeutic destination, comprising the steps of: contacting a sample to be tested with the specific antibody or antigen-binding fragment thereof according to claim 1 or 2, or with the antibody derivative according to 2) of claim 4, and detecting the formation of a complex between the RBD or fragment thereof and the specific antibody or antigen-binding fragment thereof according to claim 1 or 2, or the antibody derivative according to 2) of claim 4;

(2) A method of preparing the engineered host cell of claim 4) or a population of host cells comprising the same, comprising the steps of: introducing the specific antibody or antigen-binding fragment thereof of claim 1 or 2, or the polynucleotide molecule of claim 4 1) or an expression vector comprising the same into a host cell;

(3) A method of specifically inhibiting RBD, comprising the steps of: introducing the specific antibody or antigen-binding fragment thereof according to claim 1 or 2, or the polynucleotide molecule according to claim 4 1) or an expression vector comprising the same into a cell of an organism, and inhibiting the activity of RBD by expressing the specific antibody or antigen-binding fragment thereof according to claim 1 or 2.

10. Any one of the following applications, characterized by the following, said applications comprising:

(1) Use of the specific antibody or antigen-binding fragment thereof of claim 1 or 2, the polynucleotide molecule of 1) of claim 4 or an expression vector comprising the same, the antibody derivative of 2) of claim 4, or the engineered host cell of 3) of claim 4 or a population of host cells comprising the same for detecting an RBD protein or fragment thereof;

(2) Use of a specific antibody or antigen-binding fragment thereof according to claim 1 or 2, a polynucleotide molecule or expression vector comprising the same according to claim 4, an antibody derivative according to claim 4, 2), or an engineered host cell according to claim 4, 3), or a population of host cells comprising the same, for the preparation of a product for the detection of a coronavirus infection;

(3) Use of a specific antibody or antigen-binding fragment thereof according to claim 1 or 2, a polynucleotide molecule according to claim 1) or an expression vector comprising the same according to claim 4, an engineered host cell according to claim 3) or a population of host cells comprising the same according to claim 4 for the preparation of a pharmaceutical composition for the diagnosis of a coronavirus-related disease;

(4) Use of a specific antibody or antigen-binding fragment thereof according to claim 1 or 2, a polynucleotide molecule according to 1) of claim 4 or an expression vector comprising the same, or an engineered host cell according to 3) of claim 4 or a population of host cells comprising the same, for the manufacture of a medicament for the prevention or treatment of a coronavirus-related disease;

(5) Use of a specific antibody or antigen-binding fragment thereof according to claim 1 or 2, a polynucleotide molecule according to claim 1) or an expression vector comprising the same according to claim 4, an engineered host cell according to claim 3) or a population of host cells comprising the same according to claim 4 for the preparation of a pharmaceutical composition for the diagnosis of a coronavirus infection;

(6) Use of a specific antibody or antigen-binding fragment thereof according to claim 1 or 2, a polynucleotide molecule according to claim 1) or an expression vector comprising the same according to claim 4, or an engineered host cell according to claim 3) or a population of host cells comprising the same according to claim 4, for the manufacture of a medicament for the prevention or treatment of a coronavirus infection.