Specific antibody binding to coronavirus SARS-CoV-2 and SARS-CoV spike protein and application thereof

Abstract

The invention discloses a specific antibody capable of combining with a coronavirus SARS-CoV-2 and an SARS-CoV spike protein (SARS-CoV spike protein), and an application of the specific antibody capable of combining with the coronavirus SARS-CoV-2 and the SARS-CoV spike protein. The sequences of the CDRs of the antibody disclosed by the invention are as shown in SEQ ID NO. 1 to SEQ ID NO. 6. The antibody provided by the invention comprises CDRs with a specific sequence, can be specifically combined with SARS-CoV-2 and SARS-CoV spike protein, and has a relatively strong binding reaction with spike protein of another highly pathogenic coronavirus MERS-CoV at the same time. The antibody provided by the invention is strong in targeting property and high in binding activity, and the antibody prepared by the preparation method provided by the invention is high in purity and can be prepared on a large scale. On the whole, the antibody disclosed by the invention can be used for developing and optimizing an SARS-CoV and SARS-CoV-2 infection detection method.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a specific antibody that binds to the spike proteins of coronaviruses SARS-CoV-2 and SARS-CoV, and a preparation method and application thereof. Background Art

[0002] SARS-CoV and SARS-CoV-2 belong to the genus Sarbecovirus, and are both highly pathogenic coronaviruses that can infect humans. They mainly encode four structural proteins, namely spike protein, membrane protein, envelope protein, and nucleocapsid protein. The S protein is a homotrimer that protrudes from the virion and widely decorates the virus surface like a crown. It is highly glycosylated, belongs to the type I membrane protein family, and is anchored to the virus membrane, mediating the fusion of the virus membrane and the host cell membrane. In the natural state, both the pre-fusion and post-fusion conformations of the S protein can be traced on the reconstituted virions. The SARS-CoV-2 S protein consists of 1200 amino acids and can be cleaved into two functional subunits S1 and S2 by a furin-like protease, which are responsible for mediating attachment to the host cell and membrane fusion, respectively. Due to the indispensable function of the S protein, it is an attractive target for inhibition by neutralizing antibodies (nAbs). Most COVID-19 antibodies can prevent the S1 subunit of the SARS-CoV-2 spike (S) protein from binding to the human host receptor. However, the emergence of SARS-CoV-2 immune escape variants, which have frequent mutations in the S1 subunit, may render current antibodies ineffective. In contrast, the relatively conserved S2 subunit of the S protein can induce antibodies with a broader neutralizing capacity against various SARS-CoV-2 variants.

[0003] SARS-CoV and SARS-CoV-2 are currently reported Sarbecovirus that can infect humans, and COVID-19 has brought a significant burden to the world. The spike protein is one of the structural proteins of coronaviruses, and the S protein on the virus surface is a key factor involved in infection. Similar to other coronaviruses, the S protein of SARS-CoV-2 mediates receptor recognition, cell adhesion, and fusion during the virus infection process. The trimer of the S protein located on the virus envelope surface is the basic unit for the S protein to bind to the receptor. The S1 domain contains the RBD, which is mainly responsible for the binding of the virus to the receptor, while the S2 domain mainly contains the HR domain, including HR1 and HR2, which is closely related to virus fusion. However, most current therapeutic antibodies focus on the non-conserved RBD domain of the S1 subunit, and targeting the neutralizing epitope on the more conserved S2 subunit may provide a good option for the development of neutralizing antibodies. Summary of the Invention

[0004] Based on this, the object of the present invention includes providing a specific antibody that binds to the spike proteins of coronaviruses SARS-CoV-2 and SARS-CoV. This antibody can specifically bind to the spike proteins of SARS-CoV-2 and SARS-CoV, and is used for the development of detection methods for SARS-CoV and SARS-CoV-2 infections.

[0005] The above object of the invention can be achieved by the following technical solutions:

[0006] The first object of the present invention is to provide a specific antibody that binds to the spike proteins of coronaviruses SARS-CoV-2 and SARS-CoV. The heavy chain CDR1 of the antibody contains the amino acid sequence shown in SEQ ID NO.1, the heavy chain CDR2 contains the amino acid sequence shown in SEQ ID NO.2, and the heavy chain CDR3 contains the amino acid sequence shown in SEQ ID NO.3;

[0007] The light chain CDR1 of the antibody contains the amino acid sequence shown in SEQ ID NO.4, the light chain CDR2 contains the amino acid sequence shown in SEQ ID NO.5, and the light chain CDR3 contains the amino acid sequence shown in SEQ ID NO.6.

[0008] Preferably, the heavy chain variable region of the antibody is shown in SEQ ID NO.7.

[0009] Preferably, the light chain variable region of the antibody is shown in SEQ ID NO.8.

[0010] Preferably, the light chain constant region of the antibody is as shown in SEQ ID NO.9.

[0011] Preferably, the heavy chain constant region of the antibody is as shown in SEQ ID NO.10.

[0012] Preferably, the sequence of the constant region of the antibody is the sequence of the IgG1 constant region.

[0013] Preferably, the species origin of the constant region of the antibody is human.

[0014] In the heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.8 above, framework regions (FR) are also included, and the amino acid sequences of the 4 FRs do not directly participate in the binding reaction.

[0015] The antibody provided by the present invention can specifically bind to the spike proteins of SARS-CoV-2 and SARS-CoV, and the binding epitope is highly conserved in SARS-CoV-2 and SARS-CoV.

[0016] The second object of the present invention is to provide the use of the specific antibody that binds to the spike proteins of coronavirus SARS-CoV-2 and SARS-CoV in the detection of SARS-CoV or SARS-CoV-2 or the preparation of anti-SARS-CoV or anti-SARS-CoV-2 drugs.

[0017] The third object of the present invention is to provide a detection reagent, a detection kit or a drug, comprising the specific antibody that binds to the spike proteins of coronavirus SARS-CoV-2 and SARS-CoV.

[0018] The fourth object of the present invention is to provide a nucleic acid containing a nucleic acid sequence for encoding the specific antibody that binds to the spike proteins of coronavirus SARS-CoV-2 and SARS-CoV.

[0019] The fifth object of the present invention is to provide a recombinant expression vector comprising the above nucleic acid.

[0020] Preferably, the recombinant expression vector is an antibody expression vector.

[0021] Preferably, the recombinant expression vector is AbVec2.0-IGHG1 or AbVec1.1-IGKC.

[0022] The sixth object of the present invention is to provide a host cell comprising the above specific antibody, or the above nucleic acid, or the above recombinant expression vector.

[0023] Preferably, the host cell is 293T cell or Expi293F cell.

[0024] The seventh object of the present invention is to provide a method for preparing the specific antibody that binds to the spike proteins of coronavirus SARS-CoV-2 and SARS-CoV, including constructing the above-mentioned host cell, culturing, and collecting the specific antibody that binds to the spike proteins of coronavirus SARS-CoV-2 and SARS-CoV.

[0025] Preferably, the host cell is 293T cell or Expi293F cell.

[0026] Compared with the traditional technology, the above technical solutions provided by the present invention have at least the following beneficial effects:

[0027] The antibody provided by the present invention contains CDRs with specific sequences, can specifically bind to the spike proteins of coronavirus SARS-CoV-2 and SARS-CoV, and is used for the development of detection methods for SARS-CoV and SARS-CoV-2 infections. The antibody provided by the present invention has strong targeting and high binding activity, can specifically bind to the spike proteins of SARS-CoV-2 and SARS-CoV, and also has a strong binding reaction with the spike protein of another highly pathogenic coronavirus MERS-CoV. Overall, the antibody of the present invention can be used for the development of detection methods for SARS-CoV and SARS-CoV-2 infections. Using the preparation method provided by the present invention to prepare the antibody, the purity is high and it can be prepared in large quantities. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 It is a graph showing the detection result of the binding activity of the antibody in Example 2 of the present invention to the spike protein of SARS-CoV-2.

[0030] Figure 2 It is a graph showing the detection result of the binding activity of the antibody in Example 2 of the present invention to the spike protein of SARS-CoV.

[0031] Figure 3This is the graph showing the binding activity of the antibody to the MERS-CoV spike protein in Example 2 of the present invention. Detailed implementation manners

[0032] The present invention will be further described in detail below with reference to the accompanying drawings, implementation manners and examples. It should be understood that these implementation manners and examples are only used to illustrate the present invention and not to limit the scope of the present invention. The purpose of providing these implementation manners and examples is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the implementation manners and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing the implementation manners and examples, and are not intended to limit the present invention.

[0034] Terms

[0035] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0036] The term "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include any two related listed items, any more related listed items, or the combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are used to connect at least three items, it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, D, that is, includes the combination of any two or any three of A, B, C, D, and also includes the four-item combination of A, B, C, D (that is, the technical solution connected by "logical AND").

[0037] In the present invention, terms such as "multiple", "diverse", "multiple times", etc., unless otherwise specified, mean greater than or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0038] As used herein, "combinations thereof", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more of the listed items.

[0039] In this article, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of the present invention, solve the technical problems of the present invention, and achieve the expected technical effects of the present invention.

[0040] In this article, "preferred", "better", "more preferable", "it is advisable" are only used to describe the implementation modes or examples with better effects. It should be understood that they do not constitute a limitation on the protection scope of the present invention. If "preferred" appears multiple times in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction relationship, each "preferred" is independent.

[0041] In the present invention, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be understood as a limitation on the protection scope of the present invention.

[0042] In the present invention, "optionally", "optional", "optional" mean optional, that is, it refers to any one of the two parallel options of "having" or "not having". If "optional" appears multiple times in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction relationship, each "optional" is independent.

[0043] In the present invention, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive listing description, and it should be understood that they do not constitute a closed limitation on quantity.

[0044] In the present invention, for the technical features described in an open-ended manner, it includes the closed technical solutions composed of the listed features, and also includes the open-ended technical solutions containing the listed features.

[0045] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. Unless it conflicts with the inventive purpose and / or technical solution of the present application, the cited documents related to the present invention are incorporated by reference in their entirety and for all purposes. When referring to cited documents in the present invention, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When referring to cited documents in the present invention, examples and preferred modes of the relevant technical features cited can also be incorporated as references into the present application, provided that the present invention can be implemented. It should be understood that when the cited content conflicts with the description in the present application, the present application shall prevail or be amended adaptively according to the description in the present application.

[0046] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a plurality of constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0047] As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, and it forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions of the variable regions are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which generally assume a β-sheet configuration and are connected by three CDRs forming connecting loops, and in some cases can form a partial β-sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody [see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)]. The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell cytotoxicity.

[0048] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (designated kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions, with five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are designated alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known to those skilled in the art.

[0049] In general, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called complementarity-determining regions (CDRs), which divide the segment into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures that are brought into proximity in the spatial structure by the beta-sheets formed by the intervening FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. Which amino acids constitute the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.

[0050] The present invention includes not only intact antibodies but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives, and analogs of the said antibodies.

[0051] In the present invention, antibodies include murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which include human and non-human portions, can be obtained by standard DNA recombinant techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are from different animal species, such as a chimeric antibody having the variable region of a monoclonal antibody from a mouse and the constant region of a human immunoglobulin (see, for example, U.S. Patent Nos. 4,816,567 and 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species that has one or more complementarity-determining regions (CDRs) derived from a non-human species and framework regions derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared by DNA recombinant techniques well known in the art.

[0052] In the present invention, an antibody can be monospecific, bispecific, trispecific, or more multispecific.

[0053] In the present invention, the antibodies of the present invention further include their conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids in the amino acid sequence of the antibody of the present invention with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0054] The following are some specific examples.

[0055] The embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, the guidance given in the present invention is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0056] In the following specific examples, for the measurement parameters of the raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0057] In some embodiments of the present invention, the host cell is 293T cell or Expi293F cell.

[0058] Example 1: Construction, expression and purification of the expression vector of monoclonal antibody

[0059] For the monoclonal antibody prepared in this example, its heavy chain variable region contains the heavy chain CDR1 shown in SEQ ID NO.1, the heavy chain CDR2 shown in SEQ ID NO.2, and the heavy chain CDR3 shown in SEQ ID NO.3, and its light chain variable region contains the light chain CDR1 shown in SEQ ID NO.4, the light chain CDR2 shown in SEQ ID NO.5, and the light chain CDR3 shown in SEQ ID NO.6. Specifically, its heavy chain variable region is shown in SEQ ID NO.7, its light chain variable region is shown in SEQ ID NO.8, its light chain constant region is shown in SEQ ID NO.9, and its heavy chain constant region is shown in SEQ ID NO.10.

[0060] The preparation method of the monoclonal antibody in this example includes the following steps:

[0061] 1. Referring to the invention patent published by the inventor, "A method for preparing high-titer Epstein-Barr virus, a method and application of Epstein-Barr virus immortalized memory B cells", patent number: 20190975849.3, positive cell clones binding to coronavirus SARS-CoV-2 and SARS-CoV spike protein were screened, and RNA was extracted from the positive cell clones. The reference used was Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann HJ Immunol Methods. 2008 Jan 1; 329 (1-2): 112-24. Epub 2007 Oct 31.10.1016 / j.jim.2007.09.017 PubMed The method in 17996249 performs PCR amplification on the heavy chain and light chain of the antibody respectively to obtain the variable region sequences of the heavy chain and light chain of the antibody.

[0062] 2. The nucleotide sequences encoding the antibody heavy chain (SEQ ID NO.7) and light chain variable regions (SEQ ID NO.8) were respectively integrated into AbVec2.0-IGHG1 and AbVec1.1-IGKC containing the heavy and light chain constant region sequences of human IgG1 antibodies (the vectors were purchased from the addgene official website, with the catalog numbers: #80795 and #80796, respectively). The restriction cloning sites used for AbVec2.0-IGHG1 were AgeI and SalI, and the restriction cloning sites used for AbVec1.1-IGKC were AgeI and BsiWI. After restriction cleavage and ligation, recombinant expression vectors that can express the heavy chain and light chain of the target antibody, respectively, were obtained.

[0063] 3. Cell transfection, monoclonal antibody expression and purification

[0064] 1. Transfection

[0065] The Expi293F expression system (Cat. No. A14635) from Gibco was used and transfection was performed according to the instructions. The steps are briefly described as follows:

[0066] A total of 30 μg of two recombinant expression vector DNAs expressing the heavy and light chains of the antibody (15 μg each of the heavy and light chains) were mixed with 80 μL of the matching transfection reagent ExpiFectamine TMMix and let stand at room temperature for 20 minutes to form a stable complex;

[0067] Subsequently, add it to 25.5 mL of Expi293F cell culture medium with an adjusted concentration of 2.9×10 6 cells / mL;

[0068] Culture in a shaker at 37 °C, 8% (v / v) CO2, and 125 rpm for 20 hours;

[0069] Add transfection enhancer 1 (150 μL) and transfection enhancer 2 (1.5 mL) provided by the Expi293F expression system;

[0070] Continue to culture in a shaker at 37 °C, 8% (v / v) CO2, and 125 rpm for 4 days.

[0071] 2. Purification

[0072] Centrifuge at 3000 rpm for 15 minutes to collect the supernatant, and purify the antibody using Protein A magnetic beads from Genscript.

[0073] The purification steps are briefly described as follows:

[0074] Mix 500 μL of Protein A magnetic beads with 30 mL of cell supernatant and incubate in a shaker at room temperature for 4 hours at a rotation speed of 210 RPM;

[0075] Use a magnetic stand to adsorb the magnetic beads, discard the cell supernatant, and wash the magnetic beads 5 times with 10 mL of 1×PBS containing 0.1% (v / v) Tween 20 at pH 7.0;

[0076] Elute with 2.5 mL of Elution buffer containing 0.1 M glycine at pH 2.0;

[0077] Equilibrate to pH 7.0 with 1 M Tris buffer at pH 8.5;

[0078] Take the equilibrated monoclonal antibody and perform desalting treatment and DPBS solvent replacement using a PD-10 Desalting Column (Cytiva, catalog number 17085101). After washing the desalting column with 25 mL of DPBS, pass 2.5 mL of the equilibrated antibody solution through it, and then elute with 3.5 mL of DPBS to obtain a monoclonal antibody solution with the solvent replaced by DPBS.

[0079] Store the purified antibody in a -80 °C refrigerator.

[0080] A monoclonal antibody is thus obtained, with its heavy-chain variable region as shown in SEQ ID NO.7, light-chain variable region as shown in SEQ ID NO.8, light-chain constant region as shown in SEQ ID NO.9, and heavy-chain constant region as shown in SEQ ID NO.10.

[0081] Example 2: Functional analysis of the monoclonal antibody

[0082] 1. Detection of the binding activity of specific antibodies that bind to the spike proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV to the antigen

[0083] The binding ability of the monoclonal antibody obtained in Example 1 to the spike proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV was determined by ELISA.

[0084] The steps are briefly described as follows:

[0085] (1) Coat 25 ng per well of the spike protein of SARS-CoV-2 (amino acid sequence as shown in SEQ ID NO.11), the spike protein of SARS-CoV (amino acid sequence as shown in SEQ ID NO.12), and the spike protein of MERS-CoV (amino acid sequence as shown in SEQ ID NO.13) on an ELISA microplate. Use DPBS (Dulbecco's phosphate buffered saline) as the coating solution and incubate overnight at 4°C.

[0086] (2) Use DPBS containing 10% (v / v) calf serum as the blocking solution and block at 37°C for 2 hours. Add the test monoclonal antibody prepared in Example 1 after serial dilution (10 -4 、10 -2 、10 0 、10 2 ) and incubate at 37°C for 2 hours.

[0087] (3) Add 1:40000 diluted HRP-conjugated Goat anti-human IgG(H+L) antibody (Jackson ImmunoResearch) as the secondary antibody and incubate at 37°C for 1 hour.

[0088] (4) After developing color with TMB single-component chromogenic solution, terminate the reaction with 2M sulfuric acid and detect the absorbance A450 value with an ELISA reader.

[0089] Results:

[0090] The results of the binding activity detection of the monoclonal antibody to the antigen are shown in Figure 1, the binding activity against the spike protein of SARS-CoV-2 is EC50 = 0.0053 μg / mL( Figure 1 ); the binding activity against the spike protein of SARS-CoV is EC50 = 0.0082 μg / mL( Figure 2 ); the binding activity against the spike protein of MERS-CoV is EC50 = 0.0045 μg / mL( Figure 3 ).

[0091] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0092] The above-described examples only represent several embodiments of the present invention, which are convenient for understanding the technical solutions of the present invention specifically and in detail, but should not be construed as limiting the protection scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of this invention patent should be determined by the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

[0093] SEQ ID NO.1

[0094] SGYTFTN

[0095] SEQ ID NO.2

[0096] LEWMGIINPSGGGTRYS

[0097] SEQ ID NO.3

[0098] VGTQPGD

[0099] SEQ ID NO.4

[0100] LSILNN SEQ ID NO.5

[0101] PRLLIYAASRRPSEQ ID NO.6

[0102] YCSSPPTFSEQ ID NO.7

[0103] QLVQSGAEVKKPGASVRISCKSSGYTFTNYYMNWVRQAPGQGLEWMGIINPSGGGTRYSQKFQGRVTMTRDTSTNTDFMELSILRSEDTAVYYCARVGTQPGDYWGQGTLVTVSSEQ ID NO.8

[0104] IVLTQSPGTLSLSPGERATLSCRASLSILNNYLAWYQQRPGQAPRLLIYAASRRPTDIPDRFSGSGSGTDFTLTISRLEPEDFAMYYCQQYCSSPPTFTFGPGTKVEISEQ ID NO.9

[0105] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO.10

[0106] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.11

[0107]

[0108]

[0109] SEQ ID NO.13

[0110] MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRT

[0111] YSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAA

[0112] ANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCIL

[0113] EPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDE

[0114] ILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAA

[0115] FYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSV

[0116] VEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNC

[0117] YSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINK

[0118] CSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAM

[0119] TEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVG

[0120] VRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISST

[0121] MSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP

[0122] RSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYV

[0123] CNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLT

[0124] LLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYK

[0125] VLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQK

[0126] LIANKFNQALGAMQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQR

[0127] LDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCG

[0128] QGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTR

[0129] IVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVS

[0130] TSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWLGFIA

[0131] GLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYDLEPHKVHVH。

Claims

1. A specific antibody that binds to the spike protein S2 of coronavirus SARS-CoV-2 and SARS-CoV, characterized in that, comprising a heavy chain and a light chain; the heavy chain CDR1 of the antibody comprises the amino acid sequence shown in SEQ ID NO.1, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO.2, and the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO.3; the light chain CDR1 of the antibody comprises the amino acid sequence shown in SEQ ID NO.4, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO.5, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO.

6.

2. The specific antibody that binds to the spike protein S2 of coronavirus SARS-CoV-2 and SARS-CoV as claimed in claim 1, characterized in that, the variable region of the heavy chain of the antibody is as shown in SEQ ID NO.7; and / or, the variable region of the light chain of the antibody is as shown in SEQ ID NO.

8.

3. The specific antibody that binds to the spike protein S2 of coronavirus SARS-CoV-2 and SARS-CoV according to claim 1, characterized in that, the sequence of the constant region of the antibody is the sequence of the IgG1 constant region; or / and, the species origin of the constant region of the antibody is human.

4. The specific antibody that binds to the spike protein S2 of coronavirus SARS-CoV-2 and SARS-CoV according to claim 1, characterized in that, the light chain constant region of the antibody is as shown in SEQ ID NO.9; or / and, the heavy chain constant region of the antibody is as shown in SEQ ID NO.

10.

5. Use of the specific antibody according to any one of claims 1 to 4, which binds to the spike protein S2 of coronavirus SARS-CoV-2 and SARS-CoV, in the detection of SARS-CoV or SARS-CoV-2 or the preparation of drugs against SARS-CoV or SARS-CoV-2.

6. A detection reagent, detection kit or drug, characterized in that, comprising the specific antibody according to any one of claims 1 to 4, which binds to the spike protein S2 of coronavirus SARS-CoV-2 and SARS-CoV.

7. A nucleic acid, characterized in that, comprising a nucleic acid sequence encoding the specific antibody according to any one of claims 1 to 4, which binds to the spike protein S2 of coronavirus SARS-CoV-2 and SARS-CoV.

8. A recombinant expression vector, characterized in that, comprising the nucleic acid according to claim 7.

9. A host cell, characterized in that, comprising the specific antibody according to any one of claims 1 to 4, or the nucleic acid according to claim 7, or the recombinant expression vector according to claim 8.

10. A method for preparing a specific antibody that binds the spike protein S2 of coronavirus SARS-CoV-2 and SARS-CoV as described in any one of claims 1 to 4, characterized in that, including constructing the host cell according to claim 9, culturing, and collecting the specific antibody that binds to the spike protein S2 of coronavirus SARS-CoV-2 and SARS-CoV.

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