Monoclonal antibody 6g21h against sars-cov-2 virus and preparation method and application thereof

Abstract

The application discloses a kind of anti-SARS-CoV-2 virus monoclonal antibody 6G21H and its preparation method and application, the monoclonal antibody has the following shown heavy light chain CDR1, CDR2 and CDR3 region: heavy chain CDR1 region: DSPSDDYG;Heavy chain CDR2 region: ISYNGINK;Heavy chain CDR3 region: AKDSGPYYDIMIGYYDLNY;Light chain CDR1 region: QGISNY;Light chain CDR2 region: AAS;Light chain CDR3 region: QQYNSYPFT.The application belongs to the field of biotechnology, the monoclonal antibody provided by the application is designed to SAR-CoV-2 virus S protein, and rapid, specific detection of SAR-CoV-2 virus can be realized, easy to operate, and the stability and accuracy of detection result are good.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a monoclonal antibody against SARS-CoV-2 virus and a preparation method thereof. BACKGROUND

[0002] The novel coronavirus is named SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) by the International Committee on Taxonomy of Viruses. Common signs of human infection with the novel coronavirus include respiratory symptoms, fever, cough, shortness of breath and difficulty breathing, and severe cases can lead to death.

[0003] Currently, there is no rapid and effective treatment method for diseases caused by the novel coronavirus. Timely and rapid detection of the novel coronavirus is crucial for the timely treatment of infected persons. Currently known detection methods include RT-PCR method, antigen detection method, serum antibody detection method, etc. Among them, the antigen detection method detects the corresponding antigen target of SARS-CoV-2 by using a monoclonal antibody, has the advantages of rapidity, simple operation and easy use, and has important significance for timely and rapid detection of the novel coronavirus.

[0004] The novel coronavirus includes four major structural proteins: spike protein (also known as S protein), nucleocapsid protein (also known as N protein), membrane protein (also known as M protein), and envelope protein (also known as E protein). Monoclonal antibody technology has developed rapidly in recent years, and existing technologies have reported monoclonal antibodies related to the novel coronavirus. For example: CN 111704666A discloses a pair of monoclonal antibodies against the N protein of the novel coronavirus and their applications, and the monoclonal antibodies are prepared by immunizing mice with purified recombinant N protein of the novel coronavirus (SARS-CoV-2). The S protein of the novel coronavirus is a key site for viral entry into host cells and an important target site for neutralizing antibodies in the host.

[0005] The existing technology has not found a detection method related to the S protein of SARS-CoV-2 virus, therefore, it is of great significance to provide a monoclonal antibody against SARS-CoV-2 virus against Spike protein and a preparation method thereof. SUMMARY

[0006] To solve the problems in the prior art, the present application provides a monoclonal antibody against the S protein of SARS-CoV-2 virus and a corresponding preparation method and application, which can realize rapid and specific detection of SARS-CoV-2 virus, is easy to operate, and has good stability and accuracy of detection results.

[0007] The objects of the present application will be further illustrated by the following detailed description.

[0008] In one aspect, the present application provides a humanized monoclonal antibody against SARS-CoV-2 virus or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to SARS-CoV-2 virus, wherein the monoclonal antibody has heavy and light chain CDR1, CDR2 and CDR3 regions as shown below

[0009] Heavy chain CDR1 region: DSPSDDYG; SEQ ID NO. 5

[0010] Heavy chain CDR2 region: ISYNGINK; SEQ ID NO. 6

[0011] Heavy chain CDR3 region: AKDSGPYYDIMIGYYDLNY; SEQ ID NO. 7

[0012] Light chain CDR1 region: QGISNY; SEQ ID NO. 8

[0013] Light chain CDR2 region: AAS;

[0014] Light chain CDR3 region: QQYNSYPFT SEQ ID NO. 9.

[0015] Further, the heavy chain variable region amino acid sequence of the monoclonal antibody is shown as SEQ ID NO: 1: QVQLVESGGGVVSLVDSLCASDSPSDDYGIHWVRQAPGKGLEWVAFISYNGINKYYVDSVKGRFTISRDYSKNTLYLQMQSLRAEDTAVYYCAKDSGPYYDIMIGYYDLNYWGPGTLVTVSS SEQ ID NO: 1.

[0016] Further, the light chain variable region amino acid sequence of the monoclonal antibody is shown as SEQ ID NO: 2: DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASILQSGVPSKFTGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPFTFGPGTTVDIKR SEQ ID NO: 2.

[0017] In another aspect, the present application provides a nucleotide sequence encoding the above-mentioned monoclonal antibody or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to SARS-CoV-2.

[0018] Further, the nucleotide sequence comprises a nucleotide sequence encoding an amino acid shown in SEQ ID NO: 1; and / or

[0019] The gene comprises a nucleotide sequence encoding an amino acid shown in SEQ ID NO: 2.

[0020] Further, the nucleotide sequence encoding an amino acid shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3, and the nucleotide sequence encoding an amino acid shown in SEQ ID NO: 2 is shown in SEQ ID NO: 4.

[0021] The sequence of SEQ ID NO: 3 is shown as follows:

[0022] caggtgcagctggtggagtctgggggaggcgtggtcagcctggtcgactctctgtgcgcgtctgattcaccttcagatgactatggcattcactg ggtccgccaggctccaggcaaggggctggagtgggtggcttttatatcgtataatggaattaataaatactacgtagactccgtgaagggccgattcaccatctccagagactattccaagaacaccctgtatctgcaaatgcagagcctgagagccgaggacacggctgtgtattactgtgcgaaagat tcggggccctattacgatattatgattggttattacgacttgaactactggggcccgggaaccctggtcaccgtctcctca.

[0023] The sequence of SEQ ID NO: 4 is shown as follows:

[0024] gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggcattagtaattatt tagcctggtttcagcagaaaccagggaaagcccctaagtccctgatctatgctgcatccattttgcaaagtggggtcccatcaaagttcaccggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacaatataacagttacccattcactttcggccctgggaccacagtggatatcaaacga.

[0025] In yet another aspect, the present application provides a vector containing the above-mentioned nucleotide sequence.

[0026] In yet another aspect, the present application provides a cell containing the above-mentioned nucleotide sequence or containing the above-mentioned vector.

[0027] In yet another aspect, the present application provides a method for producing the above-mentioned anti-SARS-CoV-2 virus monoclonal antibody or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to the SARS-CoV-2 virus, the method comprising culturing genetically engineered cells containing a nucleotide sequence encoding the heavy and light chains of the anti-SARS-CoV-2 monoclonal antibody or the above-mentioned vector or directly culturing the above-mentioned cells, collecting, and purifying the anti-SARS-CoV-2 monoclonal antibody.

[0028] In yet another aspect, the present application provides the use of the above-mentioned monoclonal antibody in the treatment and prevention of COVID-19.

[0029] In yet another aspect, the present application provides a new coronavirus drug comprising a safe and effective amount of the above-mentioned monoclonal antibody and a pharmaceutically acceptable excipient.

[0030] In yet another aspect, the present application provides a kit comprising the above-mentioned antibody or antigen-binding fragment thereof. Further, the kit is used for detecting SARS-CoV-2 or the S protein of SARS-CoV-2.

[0031] In yet another aspect, the present application provides a detection reagent comprising the above-mentioned antibody or antigen-binding fragment thereof. Further, the detection reagent is used for detecting SARS-CoV-2 or the S protein of SARS-CoV-2.

[0032] In another aspect, the present application provides the use of the above-mentioned antibody or antigen-binding fragment thereof as a detection reagent for enzyme-linked immunosorbent assay (ELISA), Western Blot, flow cytometry (FACS), immunohistochemistry (IHC) detection or immunoprecipitation PCR for detecting S protein of SARS-CoV-2.

[0033] In the above-mentioned immunological detection, the antibody or antigen-binding fragment thereof can be connected to a conjugate including horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin, fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose, etc. conjugate by chemical coupling, electrostatic adsorption or hydrophobic adsorption, alone or in combination.

[0034] In the technical solution of the present application, the detection reagent can be used for non-diagnostic or therapeutic purpose detection.

[0035] Compared with the prior art, the beneficial effects of the present application include:

[0036] The monoclonal antibody of SARS-CoV-2 virus S protein provided by the present application is a fully human monoclonal antibody without other species components, and no toxic and side effects such as anti-mouse antibody and anti-antibody occur in the human body, has better biocompatibility and safety, can realize rapid and specific detection of SARS-CoV-2 virus, is easy to operate, and has good stability and accuracy of detection results.

[0037] The present application also provides a preparation method of the monoclonal antibody of SARS-CoV-2 virus S protein, and the monoclonal antibody of SARS-CoV-2 virus S protein obtained by the phage library related technology has good quality stability. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Schematic diagram of ELISA detection of novel coronavirus. DETAILED DESCRIPTION

[0039] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below, but it should not be understood as limiting the scope of the present application.

[0040] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will now be described in detail below in conjunction with specific examples, which should be understood as merely illustrating the present application but not limiting the scope of the present application. In the examples, each of the original reagent materials can be commercially available, and the experimental methods not specified with specific conditions are the conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0041] As used herein, the term "antibody" refers to a molecule comprising at least one antigen binding site that immunospecifically binds to a specific target antigen target. Thus, the term "antibody" includes, but is not limited to, full-length antibodies and / or variants thereof, fragments thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, and antibody mimetics or specified fragments or portions thereof that mimic the structure and / or function of an antibody, including single-chain antibodies and fragments thereof. Binding of an antibody to a target can elicit a variety of effects, such as, but not limited to, modulating, decreasing, increasing, antagonizing, agonizing, mitigating, slowing, blocking, inhibiting, abrogating, and / or interfering with at least one target activity or binding, or receptor activity or binding, in vitro, in situ, and / or in vivo. Thus, antibodies of the present disclosure encompass antibody fragments that are capable of binding to a biomolecule (e.g., an antigen or receptor) or portion thereof, including, but not limited to, Fab, Fab' and F(ab')2, pFc', Fd, single domain antibodies (sdAbs), variable fragments (Fv), single-chain variable fragments (scFv), or disulfide-linked Fv (sdFv); bifunctional antibodies or divalent bifunctional antibodies; linear antibodies; single-chain antibody molecules; multispecific antibodies formed from antibody fragments. An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0042] As used herein, the term "monoclonal antibody" refers to an antibody that is derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

[0043] As used herein, the term HCDR has the same meaning as heavy chain complementarity determining region, and LCDR has the same meaning as light chain complementarity determining region.

[0044] As used herein, a monoclonal antibody includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, that exhibit the desired biological activity.

[0045] As used herein, the term "SAR-COV-2" also known as "novel coronavirus" refers to the newly occurring virus causing novel coronavirus infection (COVID-19).

[0046] As used herein, S protein refers to the spike protein on the coronavirus, SARS-CoV-2 recognizes ACE2 on the surface of human cells through the spike protein on the surface of the virus and invades the host cells. By blocking the S protein on the surface of the coronavirus SARS-CoV-2, the virus can effectively inhibit the adhesion of the target cell receptor, preventing the virus from invading the cell.

[0047] The term "humanized antibody" or "humanization" as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The humanized antibodies of the present application can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis or by in vivo somatic mutation).

[0048] The term "antigen-binding fragment" and the like as used herein includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The term "antigen-binding fragment" of an antibody as used herein refers to one or more fragments of an antibody that retain the ability to bind to the S protein of SAR-COV-2.

[0049] The present application will be further described in detail in conjunction with the accompanying drawings and examples.

[0050] In the present application, the components, reagents and kits involved are all conventional commercially available products, or can be obtained by conventional technical means in the art, such as the S protein of the novel coronavirus purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0051] Example 1 Preparation of monoclonal antibody against SARS-CoV-2 virus

[0052] The present example provides a method for preparing the monoclonal antibody against SARS-CoV-2 virus, comprising the following steps:

[0053] 1) Extract the RNA of PBMC of recovered COVID-19 patients, reverse transcribe into cDNA, amplify the antibody gene by PCR technology, connect M13 phage particle carrier, electroshock transform TG1 host bacteria, and construct phage antibody library.

[0054] 2) Identify specific phages: elute the specific phages combined, infect bacteria, culture and extract bacterial bodies containing exogenous gene fragments, and identify the monoclonal antibody against SARS-CoV-2 virus by DNA sequence; the steps of identifying specific phages include: 96-well plate ELISA screening, analyzing positive clones and performing sequencing analysis.

[0055] The sequencing results are as follows:

[0056] This example provides a monoclonal antibody 6G21H against SARS-CoV-2 virus, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 1, and the underlined parts are CDR1, CDR2 and CDR3 respectively:

[0057] QVQLVESGGGVVSLVDSLCAS DSPSDDYG IHWVRQAPGKGLEWVAF ISYNGINK YYVDSV KGRFTISRDYSKNTLYLQMQSLRAEDTAVYYC AKDSGPYYDIMIGYYDLNY WGPGTLVTV SS(SEQ ID NO:1)。

[0058] The amino acid sequence of the light chain variable region is shown as SEQ ID NO: 2, and the underlined parts are CDR1, CDR2 and CDR3 respectively:

[0059] DIQMTQSPSSLSASVGDRVTITCRAS QGISNY LAWFQQKPGKAPKSLIY AAS ILQSGVPSKFT GSGSGTDFTLTISSLQPEDFATYYC QQYNSYPFT FGPGTTVDIKR(SEQ ID NO:2)。

[0060] The nucleotide sequence encoding the amino acid shown in SEQ ID NO: 1 of the heavy chain variable region is shown as SEQ ID NO: 3:

[0061] CAGGTGCAGCTGGTGGAGTCTGGGGGAGCGTGGTCAGCCTGGTCGACTCTCTGTGC GCCTGATTTCACCTTCAGATGACTATGGCATT CACTGGGTCCGCCAGGCTCCAGGC AAGGGGCTGGAGTGGGTGGCTTTTATATCGTATAATGGAATTAATAAATACTACG TAGACTCCGTGAAGGGCCGATTCA CCATCTCCAGAGACTATTCCAAGA AC ACCCTGTATCTGCAAATGCAGAGCCTGAGAGCCGAGGACACGGCTGTGTATTACT GTGCGA AAGATTGGGGCCCTATTACGATATTATGATTGGTTATTACGACTTGAAC TACTGGGGCCCAGGAACCCTGGTCACCCTCTCCTCA (SEQ ID NO: 3)

[0062] The nucleotide sequence encoding the amino acid set forth in SEQ ID NO: 2 for the light chain variable region is set forth in SEQ ID NO: 4:

[0063] GACATCCAGATGACCCAGTCTCCATCCTC ACTGTCTGCATCTGTAAGA GACAGAGTCACCATC ACTGTTCGGGCAGATCAGGGCATTAGTAATTATTTAGCCTGGTTTCAGCAGAA ACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCATTTTGCAAAGTGGG GTCCCATCAAAGTTCACCAGCAGTGGATCTGGGACAGATTTCACTCTCACCATC AGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAATATAACAGT TACCCATTCAC TTTAGGCCCTGGGACCACAGTGGATATCAAACGA. (SEQ ID NO: 4)

[0064] Example 2 Detection of SARS-CoV-2 Virus

[0065] Coated each well with 50 ng of SARS-CoV-2 S protein, 37°C for 3h, after PBS washing, add 200 μL 1% BSA-PBS blocking for 30 min, add convalescent serum (positive control PC), normal human serum (NC) or 50 ng of 6G21H antibody, incubate for 1h. Then add HRP labeled anti-human antibody, incubate for 1h, then add TMB to show, 1M sulfuric acid to stop showing, preferably read at 450nm on spectrophotometer. The detection results are shown in Figure 1 The monoclonal antibody against SARS-CoV-2 virus provided by the application can specifically bind to SARS-CoV-2 S protein, and has good specificity and accuracy.

[0066] The above is a further detailed description of the application in combination with specific preferred embodiments, and cannot be considered as limiting the specific implementation of the application to these descriptions. For ordinary skilled persons in the art to which the application belongs, a number of simple deductions or substitutions can be made without departing from the concept of the application, and all of them should be considered as falling within the protection scope of the application.

Claims

1. A humanized monoclonal antibody against SARS-CoV-2 virus, characterized in that, The monoclonal antibody has the following heavy and light chain CDR1, CDR2, and CDR3 regions as shown below. Heavy chain CDR1 region: DSPSDDYG, Heavy chain CDR2 region: ISYNGINK Heavy chain CDR3 region: AKDSGPYYDIMIGYYDLNY Light Chain CDR1 Zone: QGISNY Light chain CDR2 zone: AAS, Light chain CDR3 zone: QQYNSYPFT.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:

1.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

2.

4. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1-3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleic acid molecule comprises a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1; and / or The nucleic acid molecule comprises a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:

2.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:3, and the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:2 is shown in SEQ ID NO:

4.

7. A carrier, characterized in that, The vector comprises the nucleic acid molecule according to any one of claims 4-6.

8. A cell, characterized in that, The cell contains the nucleic acid molecule as described in any one of claims 4-6 or the vector as described in claim 7.

9. A detection reagent, characterized in that, The detection reagent comprises the monoclonal antibody according to any one of claims 1-3, and the detection reagent is a reagent for enzyme-linked immunosorbent assay (ELISA).

10. The use of the monoclonal antibody according to any one of claims 1-3 in the preparation of a detection reagent, characterized in that, The detection reagent is a reagent used for enzyme-linked immunosorbent assay (ELISA), and the detection reagent specifically binds to SARS-CoV-2.

11. The use according to claim 10, characterized in that, The detection reagent specifically binds to the S protein of SARS-CoV-2.

12. The use according to claim 10, characterized in that, The monoclonal antibody in the detection reagent is alone or coupled by chemical bonds, electrostatic adsorption or hydrophilic-hydrophobic adsorption to a conjugate, wherein the conjugate is selected from horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin, fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose.

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