Neutralizing antibody ZJ-9 binding coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD and its application

Abstract

The present invention provides an anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-9 and its preparation method and application. The heavy chain CDR1 of the neutralizing antibody ZJ-9 comprises the amino acid fragment shown in SEQ ID NO.1, the heavy chain CDR2 comprises the amino acid fragment shown in SEQ ID NO.2, and the heavy chain CDR3 comprises the amino acid fragment shown in SEQ ID NO.3; the light chain CDR1 of the neutralizing antibody ZJ-9 comprises the amino acid fragment shown in SEQ ID NO.4, the light chain CDR2 comprises the amino acid fragment shown in SEQ ID NO.5, and the light chain CDR3 comprises the amino acid fragment shown in SEQ ID NO.6. The neutralizing antibody ZJ-9 is able to specifically bind to the conserved epitopes on SARS-CoV-2 and SARS-CoV RBD, thereby broadly preventing the infection of cells by viruses of the genus Sabeivirus, and ultimately achieving a protective effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a neutralizing antibody ZJ-9 that binds to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitopes, and a preparation method and application thereof. Background Art

[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. The surface of their virus particles mainly has three structural proteins, spike protein, membrane protein, and envelope protein. Among them, the S protein is a 180-200kDa transmembrane glycoprotein that can be cleaved into S1 and S2 subunits by some proteases on the target cell membrane. The S1 subunit contains the N-terminal domain (NTD) and the C-terminal receptor binding domain (RBD). RBD mediates the binding of SARS-CoV and SARS-CoV-2 to the human ACE2 receptor.

[0003] Neutralizing antibody ZJ-9 therapy is an important means for preventing and treating diseases caused by infections with various pathogens. In terms of the prevention and control of SARS-CoV-2 infection and the prevention and control of similar coronavirus infections in the future, the development of new neutralizing antibodies ZJ-9 against Sarbecovirus including SARS-CoV-2 and SARS-CoV is of great clinical significance. Summary of the invention

[0004] Based on this, the purpose of the present invention includes providing a neutralizing antibody ZJ-9 that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD. The neutralizing antibody ZJ-9 can specifically bind to the conserved epitopes of the RBD domain on the SARS-CoV-2 and SARS-CoV spike proteins, thereby broadly preventing SARS-CoV-2, SARS-CoV, and Sarbecovirus from infecting cells, and ultimately achieving a protective effect.

[0005] The above-mentioned invention object can be achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a neutralizing antibody ZJ-9 that binds to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD, wherein 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;

[0007] 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.

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

[0009] Preferably, the light chain variable region of the antibody is as 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] The heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.8 also include a framework region (FR), and the amino acid sequences of the four FRs are not directly involved in the binding reaction.

[0015] The antibodies provided by the present invention can specifically bind to SARS-CoV-2 and SARS-CoV spike protein RBD, 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 a recombinant protein, which includes one or more of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3, the heavy chain variable region and the light chain variable region defined in the first object.

[0017] The third object of the present invention is to provide a detection reagent, a detection kit or a drug, comprising the neutralizing antibody ZJ-9 provided in the first object or the recombinant protein provided in the second object.

[0018] The fourth object of the present invention is to provide a nucleic acid containing a nucleic acid sequence for encoding the neutralizing antibody ZJ-9 that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD epitope.

[0019] The fifth object of the present invention is to provide a recombinant expression vector comprising the above-mentioned 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-mentioned specific antibody, or the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector.

[0023] Preferably, the host cell is a Freestyle 293-F cell or an Expi293F cell.

[0024] The seventh purpose of the present invention is to provide a method for preparing the neutralizing antibody ZJ-9 that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD, comprising constructing the above-mentioned host cell, culturing, and collecting the neutralizing antibody ZJ-9 that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD.

[0025] Preferably, the host cell is a Freestyle 293-F cell or an Expi293F cell.

[0026] Compared with the conventional technology, the above technical solution provided by the present invention has at least the following beneficial effects:

[0027] The neutralizing antibody ZJ-9 provided in the present application, which comprises CDRs of specific sequences, can specifically bind to the conserved epitopes on the RBD domains of the spike proteins of SARS-CoV-2 and SARS-CoV, thereby preventing the infection of cells by Sarbecovirus, and finally achieving a protective effect. In addition, the neutralizing antibody ZJ-9 provided in the present application has a broad spectrum and can be applied to the infection prevention and control of representative viruses in a variety of Sarbecoviruses. At the same time, the neutralizing antibody ZJ-9 provided in the present application has a high neutralizing activity. On the whole, the present application provides a new candidate for the prevention and treatment of Sarbecovirus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0029] Figure 1 Figure 2 is a graph showing the binding activity test results of the monoclonal neutralizing antibody ZJ-9 in Example 2 of the present invention.

[0030] Figure 2 The graph is a test result of the neutralization experiment of the monoclonal neutralizing antibody ZJ-9 against SARS-CoV-2 and representative Sarbecovirus strain pseudovirus in Example 2 of the present invention; DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure 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 embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present invention. In addition, in the following description, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0033] the term

[0034] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0035] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the 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 that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of 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, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0036] In the present invention, "plurality", "multiple", "multiple times", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0037] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0038] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0039] Herein, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. If multiple "preferred" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "preferred" item is independent.

[0040] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0041] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0042] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they 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 enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0043] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0045] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 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 a heavy chain by a covalent disulfide bond, while the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. 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 end; 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.

[0046] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region 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, Volume I, 647-669 pages (1991)]. The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0047] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are 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, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0048] 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). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains 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 region.

[0049] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0050] In the present invention, antibodies include murine, 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 parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are from different animal species, such as a chimeric antibody having a variable region from a monoclonal antibody from a mouse, and a constant region from a human immunoglobulin (see, for example, U.S. Pat. No. 4,816,567 and U.S. Pat. No. 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 complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0051] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0052] In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0053] The following are some specific embodiments.

[0054] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0055] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0056] In some embodiments of the present invention, the host cell is a Freestyle 293-F cell or an Expi293F cell.

[0057] Example 1. Construction and expression of expression vector of anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-9 Reproduction and purification

[0058] The anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-9 prepared in this example has a heavy chain variable region comprising 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 a light chain variable region comprising 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.

[0059] 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] SEQ ID NO.1: TYGVH,

[0061] SEQ ID NO.2: VIWSDGRTEYNAAFKS,

[0062] SEQ ID NO.3: KNGNYDWYFDV,

[0063] SEQ ID NO.4: RASENIYSNLA,

[0064] SEQ ID NO.5: DATNLAD,

[0065] SEQ ID NO.6: QHFWGTPLT,

[0066] SEQ ID NO.7:

[0067] QVQLKQSGPGLVQPSQSLSITCTASGYSSTTYGVHWVRQSPGKDLEWLGVIWSDGRTE YNAAFKSRLSISKDSSKSQAFFKMNSLQTNDTAIYYCARKNGNYDWYFDVWGAGTTVTVS S,

[0068] SEQ ID NO.8:

[0069] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYDATNLADGV PSRFSGSGSGTQFSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELK,

[0070] SEQ ID NO.9:

[0071] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,

[0072] SEQ ID NO.10:

[0073] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0074] The preparation method of the anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-9 in this example includes the following steps:

[0075] 1. The nucleotide sequence fragments containing the heavy chain variable region (SEQ ID NO.7) and light chain variable region (SEQ ID NO.8) encoding the anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-9 were respectively integrated into AbVec2.0-IGHG1 and AbVec1.1-IGKC containing the heavy and light chain constant region sequence fragments of human IgG1 antibody (reference for vector use: 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 17996249), and obtained recombinant expression vectors capable of expressing the heavy chain and light chain of the target antibody respectively.

[0076] II. Cell transfection, expression and purification of neutralizing antibody ZJ-9 against coronavirus SARS-CoV-2 and SARS-CoV 1. Cell transfection and expression of monoclonal neutralizing antibody ZJ-9

[0077] The Expi293F expression system of Gibco was used, and the transfection operation was performed according to the instructions. The steps are briefly described as follows:

[0078] A total of 30 μg of two recombinant expression vector DNAs expressing the heavy chain and light chain of the monoclonal neutralizing antibody ZJ-9 were mixed with the transfection reagent ExpiFectamineTM and placed at room temperature for 20 minutes to form a stable complex;

[0079] Then add 25.5 mL to adjust the concentration to 2.9×10 6 cells / mL in Expi 293F cell culture medium;

[0080] Incubate at 37°C, 8% (v / v) CO2, 125 rpm shaker for 20 h;

[0081] Add transfection enhancer 1 and transfection enhancer 2 provided by the Expi293F expression system;

[0082] The cells were then cultured in a 37°C, 8% (v / v) CO2, 125 rpm shaker for 4 days.

[0083] 2. Purification

[0084] The culture prepared in step 1 was collected, and the cell culture supernatant was collected by centrifugation at 3000 rpm for 15 minutes, and the antibody was purified using Genscript's Protein A magnetic beads. The purification steps are briefly described as follows:

[0085] Mix the Protein A magnetic beads with the cell culture supernatant and combine them on a shaker at room temperature for 4 hours;

[0086] The magnetic beads were adsorbed on a magnetic stand, the cell supernatant was discarded, and the beads were washed five times with 1× PBS containing 0.1% (v / v) Tween 20 at pH 7.0;

[0087] Elution was performed with 0.1 M glycine in Elution buffer at pH 2.0;

[0088] Equilibrated with pH 8.5 1M Tris buffer;

[0089] The equilibrated monoclonal neutralizing antibody ZJ-9 was desalted and replaced with DPBS solvent.

[0090] The purified neutralizing antibody ZJ-9 was stored in a -80°C refrigerator.

[0091] Example 2: Functional analysis of the anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-9

[0092] 1. Detection of binding activity between human anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-9 prepared in Example 1 and antigen

[0093] The ELISA method was used to determine the ability of the monoclonal neutralizing antibody ZJ-9 to bind to the spike protein S of SARS-CoV-2, the spike protein S of SARS-CoV, and the RBD domains of SARS-CoV-2 and SARS-CoV.

[0094] The steps are briefly described as follows:

[0095] (1) 25 ng of SARS-CoV-2 wild type (WT) spike protein S (trimeric S6P), SARS-CoV spike protein S (trimeric S2P), and SARS-CoV-2 and SARS-CoV RBD proteins were coated on ELISA plates using 0.1 M Na2HPO4 (pH 9.0) as the coating solution at 4°C overnight.

[0096] (2) 10% (v / v) calf serum in DPBS was used as a blocking solution and the mixture was blocked at 37° C. for 2 hours; the neutralizing antibody ZJ-9 prepared in Example 1 was added after serial gradient dilution and incubated at 37° C. for 2 hours;

[0097] (3) Add 1:10000 diluted HRP-conjugated Goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) as secondary antibody and incubate at 37°C for 1 hour;

[0098] (4) After color development with TMB single-component color development solution, the reaction was terminated with ELISA stop solution from Solarbio, and the absorbance value at 450 nm was detected with an ELISA reader.

[0099] result:

[0100] The results of the binding activity test of the monoclonal neutralizing antibody ZJ-9 with the antigen are shown in Figure 1 ,Depend on Figure 1 It can be seen that the binding activity of monoclonal neutralizing antibody ZJ-9 against SARS-CoV-2S protein is EC 50 =0.0023μg / mL, the binding activity against SARS-CoV S protein is EC 50 =0.003μg / ml, for SARS-CoV-2, SARS-CoV RBD protein EC 50 They were 0.0043 μg / mL and 0.017 μg / mL respectively.

[0101] 2. Neutralization activity test of human anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-9 prepared in Example 1 against pseudoviruses of representative strains of Sarbecovirus

[0102] The antibody neutralizing activity was detected using pseudoviruses SARS-CoV-2 (WT), SARS-CoV, Bat-CoVWIV1, and Bat-CoVRaTG13, representative strains of the Sarbecovirus genus.

[0103] The steps are briefly described as follows:

[0104] 20 μg of pCDNA3.1-SARS-CoV-2(WT)-S, pCDNA3.1-SARS-CoV-S, pCDNA3.1-Bat-CoVWIV1-S, and pCDNA3.1-Bat-CoVRaTG13-S plasmids were respectively transfected into 293T cells, and 24 hours later, 293T cells were infected with VSV Delta G (with luciferase reporter), and cell supernatants were collected 48 hours later to prepare pseudoviruses corresponding to SARS-CoV-2(WT), SARS-CoV, Bat-CoVWIV1, and Bat-CoV RaTG13;

[0105] The pseudovirus and serially diluted monoclonal neutralizing antibody ZJ-9 were mixed and incubated at 37°C for 1 hour, then added to the digested 293T-ACE2 cells and cultured in a 96-well white plate at 37°C for 24 hours;

[0106] After discarding the liquid from the cell plate, add the luciferase colorimetric substrate (Steady-Glo Luciferase asssy), incubate at room temperature for 3 minutes, and detect the fluorescence value;

[0107] The inhibition rate was calculated by comparing the readings of the wells with only pseudovirus, and the pseudovirus neutralizing activity (IC 50 express).

[0108] result:

[0109] The results of the neutralizing activity of the monoclonal neutralizing antibody ZJ-9 against pseudoviruses of representative strains of Sarbecovirus are shown in Figure 2 ,Depend on Figure 2 It can be seen that the neutralizing antibody ZJ-9 can broadly neutralize a variety of Sarbecovirus viruses (Sarbecovirus), and has good neutralizing activity (SARS-CoV-2 (WT), 9.1086μg / mL; SARS-CoV, 0.8352μg / mL; Bat-CoVWIV1, 0.3513μg / mL; Bat-CoV RaTG13, 0.1381μg / mL).

[0110] Table 1

[0111]

[0112] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes 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.

[0113] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solutions of the present invention in detail, but cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above-mentioned teaching content 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 on the basis of the technical solutions provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A neutralizing antibody ZJ-9 that binds to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD, characterized in that: Including heavy and light chains, 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; 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.

2. The neutralizing antibody ZJ-9 that binds to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD according to claim 1, characterized in that: The neutralizing antibody ZJ-9 meets 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 light chain variable region of the antibody is shown in SEQ ID NO.

8.

3. The neutralizing antibody ZJ-9 that binds to 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 neutralizing antibody ZJ-9 that binds to 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 as SEQ ID NO.9; or / and, the heavy chain constant region of the antibody is shown as SEQ ID NO.

10.

5. Use of the neutralizing antibody ZJ-9 that binds to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD as described in any one of claims 1 to 4 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, a detection kit or a drug, characterized in that: A neutralizing antibody ZJ-9 comprising 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 A nucleic acid sequence encoding the neutralizing antibody ZJ-9 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: Comprising the nucleic acid of claim 7.

9. The recombinant expression vector according to claim 8, characterized in that The recombinant expression vector is an antibody expression vector.

10. The recombinant expression vector according to claim 9, characterized in that: The recombinant expression vector is AbVec2.0-IGHG1 and / or AbVec1.1-IGKC expression vector.

11. A host cell, characterized in that Comprising the neutralizing antibody ZJ-9 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.

12. The host cell according to claim 11, characterized in that The host cell is a Freestyle 293-F cell or an Expi293F cell.

13. A method for preparing the neutralizing antibody ZJ-9 that binds to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD according to any one of claims 1 to 4, characterized in that: The method comprises constructing the host cell as described in claim 11, culturing, and collecting the neutralizing antibody ZJ-9 that binds to the coronavirus SARS-CoV-2 and the SARS-CoV spike protein RBD.

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