Specific antibodies binding to the coronavirus sars-cov-2 and sars-cov spike protein rbd and uses thereof

Abstract

This invention provides a specific antibody that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD, and its application. The amino acid sequences of the antibody's heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the amino acid sequences of the antibody's light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. This antibody can specifically bind to SARS-CoV-2 and the SARS-CoV RBD, thereby preventing the infection of cells by SARS-CoV and other viruses of the genus Sarbevirus, ultimately achieving a protective effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a specific antibody that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD and its application. Background Technology

[0002] SARS-CoV and SARS-CoV-2 belong to the Sarbecovirus subgenus of the Betacoronavirus genus. Both are highly pathogenic coronaviruses that can infect humans. Their viral particles primarily contain three structural proteins: spike protein, membrane protein, and envelope protein. The spike protein is a 180-200 kDa transmembrane glycoprotein that can be cleaved into S1 and S2 subunits by certain proteases on the target cell membrane. The S1 subunit contains an N-terminal domain (NTD) and a C-terminal receptor-binding domain (RBD). The RBD mediates the binding of SARS-CoV and SARS-CoV-2 to the human ACE2 receptor.

[0003] Neutralizing antibody therapy is an important means of preventing and treating diseases caused by various pathogens. In terms of the prevention and control of SARS-CoV infection and the prevention and control of future similar coronavirus infections, the development of new neutralizing antibodies against Sarbecovirus, including SARS-CoV, is of great clinical significance. Summary of the Invention

[0004] Based on this, the present invention provides a specific antibody that binds to the spike protein RBD of coronavirus SARS-CoV-2 and SARS-CoV and its application. The antibody can specifically bind to the RBD structural epitopes on the spike protein of SARS-CoV-2 and SARS-CoV, thereby preventing SARS-CoV and other Sarbecoviruses from infecting cells and ultimately achieving a protective effect.

[0005] The above-mentioned objective can be achieved through the following technical solution:

[0006] The first objective of this invention is to provide a specific antibody that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD, wherein the amino acid sequence of the heavy chain CDR1 of the antibody is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.3; and the amino acid sequence of the light chain CDR1 of the antibody is shown in SEQ ID NO.4, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.6.

[0007] Preferably, the heavy chain variable region of the antibody is as shown in SEQ ID NO.7.

[0008] Preferably, the light chain variable region of the antibody is as shown in SEQ ID NO.8.

[0009] Preferably, the light chain constant region of the antibody is as shown in SEQ ID NO.9.

[0010] Preferably, the heavy chain constant region of the antibody is as shown in SEQ ID NO.10.

[0011] Preferably, the sequence of the constant region of the antibody is the sequence of the IgG1 constant region.

[0012] Preferably, the species source of the constant region of the antibody is human.

[0013] The heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.8 also contain a framework region (FR), and the amino acid sequences of the four FRs do not directly participate in the binding reaction.

[0014] The antibody provided by this invention can specifically bind to SARS-CoV-2 and the SARS-CoV spike protein RBD.

[0015] A second objective of this invention is to provide the use of the above-mentioned specific antibodies that bind to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD in the preparation of SARS-CoV or SARS-CoV-2 detection products or in the preparation of anti-SARS-CoV or SARS-CoV-2 drugs.

[0016] A third objective of this invention is to provide a detection reagent, detection kit, or drug comprising the aforementioned specific antibody that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD.

[0017] A fourth object of the present invention is to provide a nucleic acid containing a nucleic acid sequence for encoding a specific antibody that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD.

[0018] A fifth objective of this invention is to provide a recombinant expression vector comprising the aforementioned nucleic acid.

[0019] Preferably, the recombinant expression vector is an antibody expression vector.

[0020] Preferably, the recombinant expression vector is AbVec2.0-IGHG1 or AbVec1.1-IGKC.

[0021] A sixth object of the present invention is to provide a host cell comprising the above-described specific antibody that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD, or the above-described nucleic acid, or the above-described recombinant expression vector.

[0022] Preferably, the host cell is a Freestyle 293-F cell or an Expi293F cell.

[0023] A seventh objective of the present invention is to provide a method for preparing the above-mentioned specific antibodies binding to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD, comprising constructing the above-mentioned host cells, culturing them, and collecting the specific antibodies binding to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD.

[0024] Preferably, the host cell is a Freestyle 293-F cell or an Expi293F cell.

[0025] Compared with traditional technologies, the technical solution provided by this invention has at least the following beneficial effects:

[0026] The specific antibody provided in this application contains specific CDR sequences that can specifically bind to the SARS-CoV-2 and SARS-CoV spike protein RBD, thereby preventing the infection of cells by Sarbecoviruses such as SARS-CoV, ultimately achieving a protective effect. Furthermore, the specific antibody provided in this application exhibits high neutralizing activity. Overall, this application provides a new candidate solution for the prevention and treatment of Sarbecovirus infection. Attached Figure Description

[0027] Figure 1 This is a graph showing the detection results of the binding activity of the specific antibody in Example 2 of the present invention.

[0028] Figure 2This is a diagram showing the results of the neutralization experiment of specific antibodies against pseudoviruses such as SARS-CoV in Example 2 of the present invention. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the invention. The purpose of providing these embodiments and examples is to enable a more thorough and complete understanding of the disclosure of the present invention. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for descriptive purposes only and is not intended to be limiting of the invention.

[0031] the term

[0032] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0033] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0034] In this invention, terms such as "multiple," "various," and "multiple times" are used, and unless otherwise specified, they refer to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0035] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0036] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this invention, solve the technical problem of this invention, and achieve the expected technical effect of this invention.

[0037] In this document, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments and should be understood not to limit the scope of protection of this invention. If multiple "preferred" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "preferred" term shall be independent.

[0038] In this invention, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0039] In this invention, "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent.

[0040] In this invention, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on the quantity.

[0041] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0042] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptively based on the description in this application.

[0043] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0044] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheeted and linked by three CDRs forming a linking loop, and in some cases, partially β-sheeted structures. The CDRs in each chain are tightly packed 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)]. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0045] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (called κ and λ) based on the amino acid sequence of their constant regions. Immunoglobulins can be classified into different types based on the amino acid sequence of their heavy chain constant regions, primarily five classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant regions of the heavy chains corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0046] Generally, 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 variable regions (CDRs). These regions are divided into four frame 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 a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0047] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0048] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained using standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0049] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0050] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0051] The following are some specific examples.

[0052] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0053] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0054] In some embodiments of the present invention, the host cell is a Freestyle 293-F cell or an Expi293F cell.

[0055] Example 1: Construction and expression of expression vectors for anti-SARS-CoV-2 and SARS-CoV-specific antibodies With purification

[0056] The anti-SARS-CoV-2 and SARS-CoV-specific antibodies prepared in this embodiment have heavy chain variable regions including VHCDR1 as shown in SEQ ID NO.1, VHCDR2 as shown in SEQ ID NO.2, and VHCDR3 as shown in SEQ ID NO.3, and light chain variable regions including VLCDR1 as shown in SEQ ID NO.4, VLCDR2 as shown in SEQ ID NO.5, and VLCDR3 as shown in SEQ ID NO.6.

[0057] 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.

[0058] SEQ ID NO.1: TGNYRWN.

[0059] SEQ ID NO. 2: YIYYSGDITYNPSLTS.

[0060] SEQ ID NO.3:DDYYGNPFAY。

[0061] SEQ ID NO.4:KASHDVSTSVA。

[0062] SEQ ID NO.5:WASTRHT。

[0063] SEQ ID NO.6:QQYYSIPPT。

[0064] SEQ ID NO.7: DVQLQESGPGLVKPSQTVSLTCTVTGISITTGNYRWNWIRQFPGNKLEWIGYIYYSGDITYNPSLTSRTTITRDTSKNQFFLEMNSLTAEDTATYYCARDDYYGNPFAYWGQGTLVTVSA。

[0065] SEQ ID NO.8: DIVMTQSHKFMSTSVGDRVSITCKASHDVSTSVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISTVQAEDLALYYCQQYYSIPPTFGGGTKLEIR。

[0066] SEQ ID NO.9: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0067] SEQ ID NO.10: STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0068] The method for preparing anti-SARS-CoV-2 and SARS-CoV-specific antibodies in this embodiment includes the following steps:

[0069] I. Nucleotide sequence fragments containing the heavy chain variable region (SEQ ID NO. 7) and light chain variable region (SEQ ID NO. 8) encoding anti-SARS-CoV-2 and SARS-CoV-specific antibodies were integrated into vectors AbVec2.0-IGHG1 and AbVec1.1-IGKC containing human IgG1 antibody heavy chain and light chain constant region sequence fragments, respectively. (The reference used for the vectors is: 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 H. J Immunol Methods. 2008 Jan 1;329(1-2):112-24. Epub 2007 Oct 31. 10.1016 / j.jim.2007.09.017 PubMed) In 17996249), recombinant expression vectors capable of expressing the heavy and light chains of the target antibody were obtained.

[0070] II. Cell transfection, antibody expression and purification

[0071] 1. Cell transfection and antibody expression

[0072] The Gibco Expi293F expression system was used, and transfection was performed according to the instructions. The steps are briefly described as follows:

[0073] 1) Mix 30 μg of DNA from two recombinant expression vectors expressing the heavy and light chains of the target antibody, respectively, with the transfection reagent ExpiFectamine™ and incubate at room temperature for 20 minutes to allow them to form a stable complex.

[0074] 2) Then add to 25.5 mL of the solution, which has been adjusted to a concentration of 2.9 × 10⁻⁶. 6 In Expi 293F cell culture medium at cells / mL;

[0075] 3) Incubate at 37℃, 8% (v / v) CO2, and 125 rpm for 20 hours in a shaker;

[0076] 4) Add transfection enhancer 1 and transfection enhancer 2 provided with the Expi293F expression system;

[0077] 5) Continue to incubate at 37℃, 8% (v / v) CO2, and 125 rpm for 4 days.

[0078] 2. Purification

[0079] Collect the culture prepared in step 1, centrifuge at 3000 rpm for 15 minutes to collect the cell culture supernatant, and purify the antibody using Genscript Protein A magnetic beads. The purification steps are briefly described as follows:

[0080] 1) Mix Protein A magnetic beads with cell culture supernatant and incubate on a shaker at room temperature for 4 hours;

[0081] 2) Adsorb the magnetic beads onto the magnetic rack, discard the cell supernatant, and wash the magnetic beads 5 times with 1×PBS containing 0.1% (v / v) Tween 20 at pH 7.0;

[0082] 3) Elute with 0.1M glycine in elution buffer (pH 2.0);

[0083] 4) Equilibrate with 1M Tris buffer (pH 8.5);

[0084] 5) Take the equilibrated target antibody for desalting and DPBS solvent replacement;

[0085] 6) The purified antibody is stored in a -80°C freezer.

[0086] Example 2: Functional analysis of anti-SARS-CoV-2 and SARS-CoV-specific antibodies

[0087] 1. Detection of the binding activity of the antibody prepared in Example 1 to the antigen

[0088] The ability of the antibody prepared in Example 1 to bind to the spike protein S of SARS-CoV-2, the spike protein S of SARS-CoV, the RBD domain of SARS-CoV-2, and the RBD domain of SARS-CoV was determined by ELISA.

[0089] The steps are briefly described as follows:

[0090] (1) 25 ng of SARS-CoV-2 wild-type (WT) spike protein S (trimer S6P), SARS-CoV spike protein S (trimer S2P), SARS-CoV-2 RBD protein and SARS-CoV RBD protein per well were coated onto ELISA plates using 0.1 M Na2HPO4 (pH 9.0) as the coating solution and incubated overnight at 4 °C.

[0091] (2) Use DPBS containing 10% (v / v) fetal bovine serum as blocking solution and block at 37 °C for 2 hours; add serially diluted antibody prepared in Example 1 and incubate at 37 °C for 2 hours;

[0092] (3) Add HRP-conjugated Goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) diluted 1:10000 as a secondary antibody and incubate at 37 ℃ for 1 hour;

[0093] (4) After developing the color with TMB single-component colorimetric solution, terminate the reaction with Solarbio's ELISA stop solution and measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0094] result:

[0095] The results of the binding activity assays of the anti-SARS-CoV-2 and SARS-CoV-specific antibodies prepared in Example 1 with the antigens are shown in the figure. Figure 1 ,Depend on Figure 1 It can be seen that the antibody's binding activity against the SARS-CoV-2 S protein is EC50. 50 = 0.003 μg / mL, the binding activity against the S protein of SARS-CoV is EC 50 = 0.0015 μg / ml, the binding activity of the RBD protein against SARS-CoV-2 is EC 50 =0.0106 μg / mL, RBD protein binding activity against SARS-CoV is EC 50=0.830 μg / mL.

[0096] 2. Detection of neutralizing activity of the antibody prepared in Example 1 against pseudoviruses of representative strains of Sarbecovirus.

[0097] Antibody neutralizing activity was detected using representative pseudoviruses of the Sarbecovirus genus: SARS-CoV, Bat-CoVRaTG13, and Pangolin-CoV-2020. The steps are briefly described as follows:

[0098] 1) 20 μg of each of the plasmids pCDNA3.1-SARS-CoV-S, pCDNA3.1-Bat-CoV RaTG13-S, and pCDNA3.1-Pangolin-CoV-2020-S (using pCDNA3.1 as a vector, the S protein encoding genes of SARS-CoV, Bat-CoV RaTG13, and Pangolin-CoV-2020 were inserted into them to obtain recombinant plasmids) were transfected into 293T cells. 24 hours later, 293T cells were infected with modified VSV. After 48 hours, the cell supernatant was collected to prepare pseudoviruses corresponding to SARS-CoV, Bat-CoV RaTG13, and Pangolin-CoV-2020.

[0099] 2) After mixing the pseudovirus and serially diluted antibodies, incubate at 37 °C for 1 hour, then add it to the digested 293T-ACE2 cells and continue to incubate in 96-well white plates at 37 °C for 24 hours.

[0100] 3) After discarding the cell plate, add the chromogenic substrate of luciferase (Steady-Glo Luciferase asssy), incubate at room temperature for 3 minutes, and detect the fluorescence value;

[0101] 4) The inhibition rate was calculated by comparing the readings of cell wells containing only pseudovirus, and the pseudovirus neutralizing activity (in IC50) was also calculated. 50 express).

[0102] result:

[0103] The results of the detection of neutralizing activity of the anti-SARS-CoV-2 and SARS-CoV-specific antibodies prepared in Example 1 against representative strains of Sarbecovirus pseudovirus are shown in the figure. Figure 2 ,Depend on Figure 2It is known that this antibody can neutralize a variety of Sarbecoviruses and has good neutralizing activity (SARS-CoV, 0.2084 μg / mL; Bat-CoV RaTG13, 0.0850 μg / mL; Pangolin-CoV-2020, 0.1041 μg / mL).

[0104] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, 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, they should be considered to be within the scope of this specification.

[0105] The above-described embodiments are merely illustrative of several implementation methods of the present invention, facilitating a detailed and specific understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Furthermore, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A specific antibody that binds to the SARS-CoV-2 coronavirus spike protein RBD, characterized in that, Including heavy chains and light chains; The amino acid sequences of the heavy chain CDR1 of the antibody are shown in SEQ ID NO.1, the heavy chain CDR2 are shown in SEQ ID NO.2, and the heavy chain CDR3 are shown in SEQ ID NO.3; the amino acid sequences of the light chain CDR1 of the antibody are shown in SEQ ID NO.4, the light chain CDR2 are shown in SEQ ID NO.5, and the light chain CDR3 are shown in SEQ ID NO.

6.

2. The specific antibody against coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD according to claim 1, characterized in that, The specific antibody satisfies one or more of the following conditions: 1) The heavy chain variable region of the antibody is shown in SEQ ID NO.7; 2) The variable region of the light chain of the antibody is shown in SEQ ID NO.

8.

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

10.

5. The use of the specific antibody according to any one of claims 1 to 4, which binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD, in the preparation of SARS-CoV or SARS-CoV-2 detection products or in the preparation of anti-SARS-CoV or SARS-CoV-2 drugs.

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

7. A nucleic acid, characterized in that, Includes a nucleic acid sequence encoding a specific antibody that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD as described in any one of claims 1 to 4.

8. A recombinant expression vector, characterized in that, It comprises the nucleic acid of claim 7; optionally, the recombinant expression vector is an antibody expression vector; further optionally, the recombinant expression vector is an AbVec2.0-IGHG1 and / or AbVec1.1-IGKC expression vector.

9. A host cell, characterized in that, The present invention comprises a specific antibody that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD as described in any one of claims 1 to 4, or a nucleic acid as described in claim 7, or a recombinant expression vector as described in claim 8; optionally, the host cell is a Freestyle 293-F cell or an Expi293F cell.

10. A method for preparing a specific antibody binding to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD as described in any one of claims 1 to 4, characterized in that, This includes constructing the host cell as described in claim 9, culturing it, and collecting specific antibodies that bind to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD.

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