CN113831409B - anti-SAR-COV-2 antibody or antigen binding fragment thereof and application thereof - Google Patents

anti-SAR-COV-2 antibody or antigen binding fragment thereof and application thereof Download PDF

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CN113831409B
CN113831409B CN202010513187.0A CN202010513187A CN113831409B CN 113831409 B CN113831409 B CN 113831409B CN 202010513187 A CN202010513187 A CN 202010513187A CN 113831409 B CN113831409 B CN 113831409B
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万晓春
李俊鑫
何燕
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

The invention relates to an anti-SAR-COV-2 antibody or an antigen binding fragment thereof and application thereof, and specifically discloses an anti-SAR-COV-2 antibody with the amino acid sequence shown in SEQ ID No:1-3, and SEQ ID No:4-6, or having the light chain complementarity determining region set forth in SEQ ID No:7-9 and the heavy chain complementarity determining region set forth in SEQ ID No:10-12, or having the light chain complementarity determining region set forth in SEQ ID No:13-15, and the heavy chain complementarity determining regions shown in SEQ ID No:16-18, or having the light chain complementarity determining region set forth in SEQ ID No:19-21, and the heavy chain complementarity determining regions shown in SEQ ID nos: 22-24. The antibody of the invention is a humanized antibody, and has low side effect and high affinity and specificity.

Description

anti-SAR-COV-2 antibody or antigen binding fragment thereof and application thereof
Technical Field
The invention belongs to the field of immunological antibodies, and particularly relates to an anti-SAR-COV-2 (COVID-19) fully human monoclonal antibody and application thereof.
Background
Of the ten global mass-market drugs in 2018, 8 were fully human or humanized monoclonal antibody drugs. The first anti-TNFa monoclonal antibody Humira of Abwe company for treating arthritis is a fully human monoclonal antibody, which is sold for more than 100 hundred million years. Since the first monoclonal antibody drug was marketed in 1986, the monoclonal antibody drug underwent the stages of murine monoclonal antibody drug (e.g., orthoclone OKT 3), chimeric monoclonal antibody drug (Rituximab), humanized monoclonal antibody drug (Herceptin), and fully human monoclonal antibody drug (Humira). Since the human body has an anti-mouse antibody reaction (HAMA), the mouse monoclonal antibody drug and the chimeric monoclonal antibody drug are gradually eliminated, and the monoclonal antibody drugs currently occupying the market are all humanized monoclonal antibody drugs. Compared with the international advanced humanized antibody production technology, the Shenzhen has a great gap even in China, is mainly represented in the field of humanized antibody medicines, has weak innovation capability and few varieties which are independently researched and developed, has no report of original humanized monoclonal antibody medicines on the market at present, and has huge antibody medicine market occupied by foreign medicine enterprises. The situation of falling behind is changed in China, and the market of antibody medicines at home and abroad with huge consumption potential is striven for overcoming the technology of fully humanized monoclonal antibodies.
The humanized monoclonal antibody has high-specificity remarkable curative effect in the aspects of treating inflammation, cancer, influenza, especially coronavirus infection and the like. Covd-19 is an acute respiratory infectious disease caused by a SAR-COV-2 coronavirus, which caused global pandemic in 2020, and severely threatened human lives and properties. By 22 months 05 of 2020, a total of 5113375 people worldwide infect SAR-COV-2 and die 330052 people, and effective medicines and vaccines are still lacking. The new coronavirus needs to rely on specific molecular Spike protein (Spike, S protein) expressed by the virus itself to bind to receptors on human cells when invading cells to infect the cells and further expand. The humanized antibody for neutralizing the virus is a certain specific antibody generated by human B lymphocytes, and can be combined with antigens on the surface of the virus, so that the virus is prevented from adhering to target cell receptors, the virus is prevented from invading cells, and the SAR-COV-2 influenza can be effectively prevented and treated. However, no effective human specific antibodies have been developed.
Disclosure of Invention
In order to solve the above problems, the present invention provides an antibody against SAR-COV-2 or an antigen binding fragment thereof, which specifically neutralizes SAR-COV-2.
In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof directed against SAR-COV-2, which specifically binds to SAR-COV-2 surface S protein; it has three heavy chain complementarity determining regions (HCDR) and three light chain complementarity determining regions (LCDR) of any of the following groups:
1)9g8
HCDR1：GGSITTSSDY SEQ ID No：1；
HCDR2：IYYSGRT SEQ ID No：2；
HCDR3：ARRLTYYYDSSGYANWYFDL SEQ ID No：3；
LCDR1：QRFSTF SEQ ID No：4；
LCDR:2：AAS SEQ ID No：5；
LCDR3: QQSYSIPYS SEQ ID No:6, preparing a base material; or alternatively
2)6J19
HCDR1：GFTFSSYS SEQ ID No：7；
HCDR2：ISSSGTFI SEQ ID No：8；
HCDR3：ARERFVGVLDI SEQ ID No：9；
LCDR1：SSNIGRST SEQ ID No：10；
LCDR:2：SSY SEQ ID No：11；
LCDR3: AAWDDSLNGPV SEQ ID No:12; or alternatively
3)7N13
HCDR1：GFTFSSYS SEQ ID No：13；
HCDR2：ISSSSSTM SEQ ID No：14；
HCDR3：ARGVGATGELFDY SEQ ID No：15；
LCDR1：QGIGNE SEQ ID No：16；
LCDR:2：AAS SEQ ID No：17；
LCDR3: LQDYNYPRT SEQ ID No:18; or alternatively
4)8L19
HCDR1：GFTFSNYS SEQ ID No：19；
HCDR2：ISTTGTYT SEQ ID No：20；
HCDR3：ARPYYYGSGSPDY SEQ ID No：21；
LCDR1：QSISTF SEQ ID No：22；
LCDR:2：AAS SEQ ID No：23；
LCDR3: HQTYSKPWT SEQ ID No:24, a step of detecting the position of the base; or alternatively
5)6M9
HCDR1：GFTFRNYD SEQ ID No：25；
HCDR2：ISGSGIDT SEQ ID No：26；
HCDR3：VRGLAGAFDY SEQ ID No：27；
LCDR1：QSVTSGY SEQ ID No：28；
LCDR:2：GTS SEQ ID No：29；
LCDR3：QQHRSSPMYS SEQ ID No：30。
In another aspect, the invention provides an isolated antibody or antigen binding fragment thereof against SAR-COV-2 having a heavy chain variable region and a light chain variable region as shown below;
1)9g8
heavy chain variable region:
QLQLQESGPGLVKPSETLSLTCTVSGGSITTSSDYWGWIRQPPGKGLEWIGSIYYSGRTYYNPSLKSRVTISVDTSKNDFSLKLSSVTAADTAVYYCARRLTYYYDSSGYANWYFDLWGRGTLVTVSS SEQ ID No：31
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQRFSTFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPYSFGQGTKLEIKR SEQ ID No:32; or alternatively
2)6J19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：33
light chain variable region:
QSVLTQPPSASGTPGERVTISCSGSSSNIGRSTVSWYQQLPGTAPKLLMYSSYQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG SEQ ID No:34; or alternatively
3)7N13
Heavy chain variable region:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTMYYGDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVGATGELFDYWGQGTLVTASS SEQ ID No：35
light chain variable region:
AIQMTQSPSSLSASVGDRVTITCRATQGIGNELGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIKR SEQ ID No:36; or alternatively
4)8L19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSNYSMNWVRQAPGKGLEWVSSISTTGTYTHYAGSVKGRFTISRDNAKNSLFLRMNSLRAEDTAVYYCARPYYYGSGSPDYWGQGTLVTVSS SEQ ID No：37
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQSISTFLNWYQQKPGKAPNLLIYAASSLQRGVPSRFTGSGSGTDFTLTISSLQPEDFATYYCHQTYSKPWTFGRGTKVEIER SEQ ID No:38, a step of carrying out the process; or alternatively
5)6M9
Heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFRNYDINWVRQAPGKGLEWVSSISGSGIDTYYGDSVEGRFTVSRDNAESSVLLEMNSLRADDTAVFYCVRGLAGAFDYWGQGTLVTVSS SEQ ID No：39
light chain variable region:
EIVLTQSPGTLSLSPGERATLSCRASQSVTSGYLAWYQQKPGQAPRLLIHGTSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHRSSPMYSFGQGSKLEIKR SEQ ID No：40。
in the embodiments of the present invention, the antibody or antigen-binding fragment thereof is a humanized antibody, more preferably a fully humanized antibody.
In the technical scheme of the invention, the antibody is a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.
In the technical scheme of the invention, the antibody or the antigen binding fragment thereof specifically binds to the SAR-COV-2 surface S protein.
In a further aspect the invention provides a nucleotide sequence encoding an antibody or antigen binding fragment thereof as hereinbefore described.
In a further aspect, the invention provides a vector comprising a nucleotide sequence as hereinbefore described.
In a further aspect the invention provides a host cell comprising the aforementioned vector or vector set, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells or other cells suitable for the preparation of antibodies or antigen-binding fragments thereof.
In a further aspect the invention provides a kit comprising an antibody or antigen binding fragment thereof as hereinbefore described.
In a further aspect the invention provides a detection reagent comprising an antibody or antigen-binding fragment thereof as hereinbefore described.
In a further aspect the present invention provides the use of an antibody or antigen binding fragment thereof as described above as a detection reagent for: enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western Blot), flow cytometry (FACS), immunohistochemical (IHC) assay, or immuno-PCR.
In the immunological detection described above, the antibody or antigen-binding fragment thereof may be coupled alone or with a conjugate such as a conjugate of horseradish peroxidase (HRP), alkaline Phosphatase (AP), biotin (Biotin), fluorescein Isothiocyanate (FITC), cy3, cy5, magnetic beads, agarose, etc., by electrostatic adsorption or hydrophilic-hydrophobic adsorption.
In the present embodiment, the detection reagent can be used for detection for non-diagnostic or therapeutic purposes.
In a further aspect the invention provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof as hereinbefore described and a pharmaceutically acceptable adjuvant.
In a further aspect of the invention, wherein the antibody or antigen binding fragment thereof blocks or reduces binding of the S protein of SAR-COV-2 to a cell surface receptor of the subject, preferably a cell angiotensin converting enzyme related carboxypeptidase (ACE 2).
In a further aspect, the invention provides the use of an antibody or antigen binding fragment thereof against SAR-COV-2 in the manufacture of a medicament for preventing, treating or alleviating at least one symptom or indication of SAR-COV-2 infection.
In the technical scheme of the invention, the medicine is an oral or injection preparation.
In a further aspect, the invention provides a method of preventing, treating or alleviating at least one symptom or indication of a SAR-COV-2 infection, the method comprising administering to a subject an antibody or antigen-binding fragment thereof of any of the preceding or a pharmaceutical composition of the preceding.
In a further aspect of the invention, wherein the at least one symptom or indication is selected from the group consisting of: pulmonary inflammation, alveolar injury, fever, cough, dyspnea, hypoxia, acute respiratory distress syndrome, septic shock, coagulation dysfunction, metabolic acidosis, nasal obstruction, runny nose, sore throat, diarrhea, organ failure, septic shock and death.
In a further embodiment of the invention, the pharmaceutical composition or the antibody or antigen binding fragment thereof is administered in combination with a second therapeutic agent. Wherein the second therapeutic agent is selected from the group consisting of: anti-inflammatory drugs (e.g., corticosteroids and non-steroidal anti-inflammatory drugs), antiviral drugs, different antibodies to the S protein of SAR-COV-2, vaccines for SAR-COV-2, antibiotics, dietary supplements such as antioxidants, and any other palliative therapy for treating SAR-COV-2 infection, drugs that alleviate the symptoms or indications described above.
In a technical aspect of the invention, wherein the pharmaceutical composition or the antibody or antigen binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially.
Advantageous effects
(1) The anti-SAR-COV-2 antibody disclosed by the invention can be used for targeting and combining the S protein of the SAR-COV-2 virus, has high specificity, and can effectively block the combination of the S protein on the surface of the SAR-COV-2 virus and a receptor on the surface of a cell of a subject.
(2) Compared with the murine antibody, the gene of the fully human antibody is fully derived from human genes, has no other species components, does not generate toxic or side effects such as anti-mouse anti-antibody and the like in human body, has better biocompatibility, and is more suitable and potential to become a macromolecular medicament for treating influenza virus.
(3) Compared with the method for preparing the SAR-COV-2 virus-resistant humanized monoclonal antibody by using the phage display technology provided by the prior art, the method for developing the SAR-COV-2 virus-resistant antibody by using the single B cell has the advantages of simplicity and rapidness in operation, high affinity and specificity of the produced humanized antibody and the like.
Drawings
FIG. 1 is a graph showing the results of a flow assay for CD40L expression of NTH-3T3 of example 1.
FIG. 2 is a graph showing the results of sorting memory B cells using the flow cytometer of example 1.
FIG. 3 is a graph showing the results of ELISA experiments in example 1.
FIG. 4 is a graph showing the result of agarose gel electrophoresis in example 2.
Detailed Description
In order to more clearly understand the technical features, objects and advantages of the present application, the following detailed description will now be made in connection with specific embodiments, and it should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In the examples, each of the starting reagent materials is commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.
As used herein, the term "antibody" refers to a molecule comprising at least one antigen binding site that immunospecifically binds to a particular antigen target of interest. Thus, the term "antibody" includes, but is not limited to, full length antibodies and/or variants thereof, fragments, 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 mimics of the structure and/or function of an antibody or designated fragments or portions thereof, including single chain antibodies and fragments thereof. Binding of an antibody to a target may cause a variety of effects, such as, but not limited to, modulation, reduction, increase, antagonism, agonism, alleviation, slowing, blocking, inhibition, elimination and/or interference 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 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 (sdAb), variable fragments (Fv), single chain variable fragments (scFv), or disulfide-linked Fv (sdFv); a bifunctional antibody or a bivalent bifunctional antibody; a linear antibody; a single chain antibody molecule; multispecific antibodies formed from antibody fragments. Antibodies may be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) or subclass.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of small amounts of mutations that may occur naturally. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibodies have the advantage that they can be synthesized without contaminating other antibodies. The modifier "monoclonal" is not to be construed as requiring antibody production by any particular method.
As used herein, the term HCDR is synonymous with heavy chain complementarity determining region and LCDR is synonymous with light chain complementarity determining region.
As used herein, monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, and fragments of such antibodies exhibit the desired biological activity.
As used herein, the term "SAR-COV-2" also referred to as "novel coronavirus" refers to a newly occurring virus that causes novel coronavirus pneumonia (COVID-19).
As used herein, S protein refers to Spike protein (Spike protein) on coronaviruses, and SARS-CoV-2 recognizes ACE2 on the surface of cells in humans through Spike protein on the surface of the virus and infects host cells. The S protein on the surface of coronavirus SARS-CoV-2 can inhibit the adhesion of virus to target cell receptor effectively to prevent virus from invading cell.
The term "humanized antibody" as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The humanized antibodies of the invention may comprise amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or in vitro site-specific mutagenesis or by in vivo somatic mutation).
The term "antigen-binding fragment" or the like as used herein includes any naturally occurring, enzymatically available, 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 has the ability to bind to one or more fragments of the S protein of SAR-COV-2.
In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof directed against SAR-COV-2, which specifically binds to SAR-COV-2 surface S protein; it has three heavy chain complementarity determining regions (HCDR) and three light chain complementarity determining regions (LCDR) of any of the following groups:
1)9g8
HCDR1：GGSITTSSDY SEQ ID No：1；
HCDR2：IYYSGRT SEQ ID No：2；
HCDR3：ARRLTYYYDSSGYANWYFDL SEQ ID No：3；
LCDR1：QRFSTF SEQ ID No：4；
LCDR:2：AAS SEQ ID No：5；
LCDR3: QQSYSIPYS SEQ ID No:6, preparing a base material; or alternatively
2)6J19
HCDR1：GFTFSSYS SEQ ID No：7；
HCDR2：ISSSGTFI SEQ ID No：8；
HCDR3：ARERFVGVLDI SEQ ID No：9；
LCDR1：SSNIGRST SEQ ID No：10；
LCDR:2：SSY SEQ ID No：11；
LCDR3: AAWDDSLNGPV SEQ ID No:12; or alternatively
3)7N13
HCDR1：GFTFSSYS SEQ ID No：13；
HCDR2：ISSSSSTM SEQ ID No：14；
HCDR3：ARGVGATGELFDY SEQ ID No：15；
LCDR1：QGIGNE SEQ ID No：16；
LCDR:2：AAS SEQ ID No：17；
LCDR3: LQDYNYPRT SEQ ID No:18; or alternatively
4)8L19
HCDR1：GFTFSNYS SEQ ID No：19；
HCDR2：ISTTGTYT SEQ ID No：20；
HCDR3：ARPYYYGSGSPDY SEQ ID No：21；
LCDR1：QSISTF SEQ ID No：22；
LCDR:2：AAS SEQ ID No：23；
LCDR3: HQTYSKPWT SEQ ID No:24, a step of detecting the position of the base; or alternatively
5)6M9
HCDR1：GFTFRNYD SEQ ID No：25；
HCDR2：ISGSGIDT SEQ ID No：26；
HCDR3：VRGLAGAFDY SEQ ID No：27；
LCDR1：QSVTSGY SEQ ID No：28；
LCDR:2：GTS SEQ ID No：29；
LCDR3：QQHRSSPMYS SEQ ID No：30。
In another aspect, an isolated antibody or antigen binding fragment thereof against SAR-COV-2 is provided having a heavy chain variable region and a light chain variable region as shown below;
1)9g8
heavy chain variable region:
QLQLQESGPGLVKPSETLSLTCTVSGGSITTSSDYWGWIRQPPGKGLEWIGSIYYSGRTYYNPSLKSRVTISVDTSKNDFSLKLSSVTAADTAVYYCARRLTYYYDSSGYANWYFDLWGRGTLVTVSS SEQ ID No：31
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQRFSTFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPYSFGQGTKLEIKR SEQ ID No:32; or alternatively
2)6J19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：33
light chain variable region:
QSVLTQPPSASGTPGERVTISCSGSSSNIGRSTVSWYQQLPGTAPKLLMYSSYQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG SEQ ID No:34; or alternatively
3)7N13
Heavy chain variable region:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTMYYGDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVGATGELFDYWGQGTLVTASS SEQ ID No：35
light chain variable region:
AIQMTQSPSSLSASVGDRVTITCRATQGIGNELGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIKR SEQ ID No:36; or alternatively
4)8L19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSNYSMNWVRQAPGKGLEWVSSISTTGTYTHYAGSVKGRFTISRDNAKNSLFLRMNSLRAEDTAVYYCARPYYYGSGSPDYWGQGTLVTVSS SEQ ID No：37
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQSISTFLNWYQQKPGKAPNLLIYAASSLQRGVPSRFTGSGSGTDFTLTISSLQPEDFATYYCHQTYSKPWTFGRGTKVEIER SEQ ID No:38, a step of carrying out the process; or alternatively
5)6M9
Heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFRNYDINWVRQAPGKGLEWVSSISGSGIDTYYGDSVEGRFTVSRDNAESSVLLEMNSLRADDTAVFYCVRGLAGAFDYWGQGTLVTVSS SEQ ID No：39
light chain variable region:
EIVLTQSPGTLSLSPGERATLSCRASQSVTSGYLAWYQQKPGQAPRLLIHGTSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHRSSPMYSFGQGSKLEIKR SEQ ID No：40。
in some embodiments, the antibody is a humanized antibody, and in some specific examples, a fully humanized antibody is preferred.
In some embodiments, the antibody is a monoclonal or polyclonal antibody, preferably a monoclonal antibody.
In some embodiments, the antibody or antigen binding fragment thereof specifically binds to a SAR-COV-2 surface S protein.
In some embodiments, the amino acid sequence of the heavy chain variable region or the light chain variable region of the antibody may be an amino acid sequence with equivalent function formed by substitution, deletion, or addition of one or several amino acids.
In some embodiments, ELISA experiments prove that the anti-SAR-COV-2 antibody or antigen binding fragment can target and bind to the S protein of SAR-COV-2 virus. The antibody is a fully humanized monoclonal antibody, and compared with a murine antibody, the gene of the fully humanized antibody is completely derived from a human gene, has no other species components, does not generate toxic or side effects such as an anti-mouse antibody and the like in a human body, has better biocompatibility, and is more suitable and potential to become a macromolecular medicament for treating influenza virus.
In another aspect, the present application provides genes encoding the anti-SAR-COV-2 fully human monoclonal antibodies described herein. In some embodiments, the gene comprises a nucleotide sequence encoding an amino acid having the above-described amino acids.
In some embodiments, the nucleotide sequence is as follows (the following sequences are merely exemplary, and one skilled in the art can design other nucleotide sequences that can be translated into the desired amino acid sequence depending on the particular amino acid sequence):
1)9g8
the nucleotide sequence encoding the heavy chain variable region is:
gtctctggtggctctatcaccactagtagtgactactggggctggatccgccagcccccagggaaggggctggagtggattgggagtatctattatagtgggagaacctactacaacccgtccctcaagagtcgagtcaccatatccgtagacacgtccaagaacgacttctctctgaagctgagctctgtgaccgccgcagacacggctgtgtattactgtgcgagacgccttacgtattactatgatagtagtggttatgcgaactggtacttcgatctctggggccgtggcaccctggtcactgtctcctca；SEQ ID No：41
the nucleotide sequence encoding the light chain variable region is:
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagaggttcagcacctttttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcacgatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtatcccgtactcttttggccaggggaccaagctggagatcaaacga SEQ ID No：42
2)6J19
the sequence encoding the heavy chain variable region is:
gaggtgcagctggtggagtctgggggaggccTggtcaAgcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagttatagtatgaagtgggtccgccaggctccagggaaggggctggagtgggtctcaaccatcagtagtagtggtactttcataaagtatgcagactcactgcagggccgattcaccatcaccagagacaacgccaagaccgcagtgtatctgcaaatgaacagcctgagagtcgaggacacggctgtttattactgtgcgagagaacgattcgttggtgttttggatatctggggccaagggacaatggtcaccgtctcttca；SEQ ID No：43
the nucleotide sequence encoding the light chain variable region is:
Cagtctgtgctgactcagccaccctcagcgtctgggacccccggggagagggtcaccatctcttgttctggaagcagctccaacatcggaaggagtactgtaagctggtaccagcagctcccaggaacggcccccaaactcctcatgtatagtagttatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgaatggtccggtgttcggcggagggaccaagctgaccgtcctaggt SEQ ID No：44
3)7N13
the nucleotide sequence encoding the heavy chain variable region is:
GaggtgcagctggtggagtctgggggaggcttgGtacagcCTGgggggTCcctgagactctcctgtgcagcctctggattcaccttcagtagttatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtttcatatattagtagtagtagtagtaccatgtactacggagactctgtgaagggccgattcaccatctccagagacaatgccaagaactcactgtatctgcaaatgaacagcctgagagacgaggacacggctgtgtattactgtgcgagaggagtgggagccacgggggaactctttgactactggggccagggaaccctggtcaccgcctcctca：SEQ ID No：45
the nucleotide sequence encoding the light chain variable region is:
gccaTccagatgacccaGTCtccatCctccctgtctgcatCtgtaggagacagagtcaccatcacttgccgggcaactcagggaattggaaatgaattagggtggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttacaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatTttgcaacttattactgtctacaagattacaattaccctcgtacgttcggccaagggaccaaggtggaaatcaaacga SEQ ID No：46
4)8L19
the nucleotide sequence encoding the heavy chain variable region is:
gaggtgcagctggtggagtctgggggaggccTggtcaAgcctggggggtccctgagactctcctgtgcagtctctggattcaccttcagtaactatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtactactggtacttacacacactacgccggctcagtgaagggccgattcaccatctccagagacaatgccaagaactcgctgtttttgcgaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggccctattactatggttcggggagtcctgactactggggccagggaaccctggtcaccgtctcctca SEQ IDNo：47
the nucleotide sequence encoding the light chain variable region is:
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcacctttttgaattggtatcagcagaagcccgggaaagcccctaatctcctgatctatgctgcatccagtttgcaacgtggggtcccatcaaggttcactggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaccagacttacagtaagccctggacgttcggccgagggaccaaggtggaaatcgaacga SEQ ID No：48
5)6M9
the nucleotide sequence encoding the heavy chain variable region is:
Gaggtgcagctgttggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcaggaactatgacatcaactgggtccgccaggctccagggaaggggctggagtgggtctcatcgattagtggtagtggtattgacacatactacggagactcagtggagggccgattcaccgtctccagagacaacgccgagagctcagtattactggagatgaacagcctgagagccgacgatacggctgtattttactgtgtgaggggtctggctggcgcctttgactactggggccagggaaccctggtcaccgtctcctca SEQ ID No：49
the nucleotide sequence encoding the light chain variable region is:
GaaattGtGttgacGcagTCtccagGcAccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttaccagcggctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatccatggtacatccaggagggccactggcatcccagacaggttcagtggcagtgggtctgggacagatttcactctcaccatcagcagactggagcctgaagattttgcagtctattactgtcagcagcatcgtagctcacccatgtacagttttggccaggggagcaagctggagatcaaacga SEQ ID No：50
in another aspect, the invention provides a vector comprising a nucleotide sequence as described above.
In yet another aspect, the present application provides a cell comprising a nucleotide sequence as described above or a vector as described above.
In yet another aspect, the present application provides a method for producing the anti-SAR-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody that is capable of specifically binding SAR-COV-2, the method comprising culturing genetically engineered cells containing the above gene encoding the heavy and light chain of the anti-SAR-COV-2 fully human monoclonal antibody or the above vector or directly culturing the above cells, collecting, and purifying the anti-SAR-COV-2 fully human monoclonal antibody.
In the prior art, a method for preparing the anti-SAR-COV-2 virus humanized monoclonal antibody by adopting a phage display technology has the advantages of low production cost, no complicated work such as immunization and cell fusion, and the like, but has obvious disadvantages that the antibodies obtained from a non-immune antibody library often have insufficient affinity, are limited by the conversion rate of exogenous genes, have insufficient library capacity of the antibody library to cover the antibody diversity of animals, and the like. The present invention thus provides a method for screening antibodies by isolating B cells secreting functional antibodies from the blood of a patient, then extracting RNA and synthesizing cDNA, cloning genes secreting antibodies of interest therefrom, and finally recombining and expressing fully human monoclonal antibodies. The technology is simple and quick to operate, the produced humanized antibody has high affinity and specificity, and in addition, the monoclonal antibody technology with the virus neutralization function or tumor killing function can be further separated from memory B cells by adopting the improved technology, so that complicated operation and cost are greatly reduced.
In another aspect, the present application provides a pharmaceutical composition comprising an anti-SAR-COV-2 fully human monoclonal antibody as described herein or a biologically active fragment derived from the monoclonal antibody that is capable of specifically binding SAR-COV-2.
In another aspect, the present application provides the use of said anti-SAR-COV-2 fully human monoclonal antibody or a biologically active fragment derived from said monoclonal antibody capable of specifically binding SAR-COV-2 or said pharmaceutical composition for the manufacture of a medicament for the treatment of a disease caused by SAR-COV-2 virus.
In another aspect, the present application provides a kit for detecting SAR-COV-2 virus levels comprising an anti-SAR-COV-2 fully human monoclonal antibody described herein or a biologically active fragment derived from the monoclonal antibody that is capable of specifically binding SAR-COV-2; in some embodiments, the kit further comprises a second antibody and an enzyme or fluorescent or radiolabel for detection, and a buffer; the second antibody is, for example, an anti-antibody against a monoclonal antibody described herein.
Example 1
(1) Construction of NTH-3T3 cell line stably expressing CD40L (3T 3-CD 40L)
3T3-CD40L feeder cells were established using lentiviruses. The lentiviral expression vector pLVX-CD40L was constructed, 293T cells were transfected, and viral supernatants were collected on day four of transfection. NIH-3T3 cells were activated, cultured for 3 passages, infected with lentivirus, and cultured and passaged 3 additional times. Sorting cells with FITC fluorescence intensity near MFI by flow cytometry, re-adding to culture flask at 37deg.C, 5% CO 2 The culture and detection in the incubator, the detection results are shown in FIG. 1, wherein 3T3 cells expressing CD40L and 3T3 cells transfected with empty vector pLVX (with ZxGreen) were stained with anti-CD 40L with APC, respectively, and then analyzed by an up-flow cytometer. As a result, it was found that all 3T3-CD40L feeder cells expressed CD40L. When the cells grow to 80% -90%, the cells are digested and collected, and the concentration is 1×10 per milliliter 7 And (3) cells. Placing into a radiometer for 5000rads radiation, and re-suspending cells in frozen solution with concentration of 3.5X10 per ml 7 The cells were aliquoted in 1ml frozen vials and stored frozen in liquid nitrogen (2 years of storage).
(2) Sorting and activation of memory B cells
Isolation and cryopreservation of Peripheral Blood Mononuclear Cells (PBMC) from patients once infected with SAR-COV-2 virus by using lymph separation solution, 10-50×10 per tube 6 Cells were frozen in a liquid nitrogen tank. PBMC flow-type staining solution was prepared, and the composition thereof is shown in Table 1 below
TABLE 1 PBMC flow staining solution
Antibodies to Volume (mu L)
CD19-PE-Cy7 0.5
IgM-PE 1.0
IgA-APC 2.5
IgD-FITC 2.5
PBS-1％(wt/vol)BSA 43.5
Thawing PBMC, adding the PBMC flow staining solution, and sorting on flow cytometer to obtain CD19 as shown in FIG. 2 + IgM – IgA – IgD – The purity of the memory B cells is more than 90%, if the purity is less than 90%, the sorting process is repeated. A mixed medium for activating B cells was prepared as shown in table 2 below:
TABLE 2
Component (A) Volume of
Complete IMDM medium 336mL
IL-2(10,000U mL -1 ) 3.5mL
IL-21(100μg mL -1 ) 175μL
3T3-CD40L obtained in step (1) 10mL
Adding memory B cells into mixed culture medium, mixing, limiting dilution in 384 well plate, 1 cell per well, 50 μl volume, placing at 37deg.C, 5% CO 2 And (5) standing and culturing in an incubator. After 13 days, the supernatant was taken to obtain human monoclonal antibodies 9g8, 6J19, 7N13, 8L19 and 6M9.
(3) Surface antigen S protein experiment of human monoclonal antibody combined with SAR-COV-2 virus
The surface antigen S protein of the SAR-COV-2 virus is purchased from Yinqiao Shenzhou company, has immunogenicity, and can be used for detecting and treating the SAR-COV-2 coronavirus. ELISA experiments were performed on the 5 human monoclonal antibodies 6J19, 7N13, 8L19 and 6M9 obtained above, specifically:
(1) 100ng/100 μl of HA protein of SAR-COV-2 virus was coated in 96-well ELISA plates, 100 μl per well;
(2) Placing in a refrigerator at 4 ℃ overnight;
(3) Washing with PBST solution three times, adding 200 μl of 5% skimmed milk powder solution into each well, and incubating at 37deg.C for 1 hr;
(4) Three times with PBST solution, 100. Mu.l of normal human serum (negative control) or supernatant without virus infection was added, each three replicates;
(5) After incubation for 1 hour at 37 ℃ the cells were washed three times with PBST solution;
(6) HRP-loaded anti-human IgG antibody (abcam) was diluted 1:5000 and added to the microplate at 100 μl per well;
(7) After incubation for 1 hour at 37 ℃ the cells were washed three times with PBST solution;
(8) Mu.l of TMB substrate solution (Thermo Scientific) was added to each well, at 37℃for 5 minutes;
(9) 100 μl of 2M sulfuric acid was added to each well, and the absorbance was immediately measured at 450nm in a microplate reader. The results are shown in figure 3, and ELISA experiments show that the human monoclonal antibodies 6J19, 7N13, 8L19 and 6M9 obtained by the invention can all target and bind the S protein of SAR-COV-2 virus.
EXAMPLE 2 cloning of humanized monoclonal antibody Gene
The B cells obtained in example 1 capable of secreting an antibody binding to SAR-COV-2 virus were lysed, and the lysate was subjected to reverse transcription of RNA to obtain PCR template cDNA of the humanized antibody gene. Primers for cloning antibody genes were designed and synthesized, and the heavy and light chain genes of the antibodies were cloned using cDNA as a template, and sequenced by Jinwei corporation. Specifically:
(1) The lysed B cell fluid was transferred to a 96-well plate (Eppendorf, 030133366).
(2) Reverse transcription system: 150ng of random primer (Invitrogen, 481190-011), 0.5. Mu.l of 10mM dNTP (Invitrogen, 18497-088), 1. Mu.l of 0.1M DTT (Invitrogen, 18080-044), 0.5% v/v Igepal CA-630 (Sigma, I3021-50 ML), 4U RNAsin (Promega), 6U Prime RNAse Inhibitor (Eppendorf) and 50U
Figure BDA0002529166390000151
III reverse transcriptase (Invitrogen, 18080-044) was supplemented with DEPC water to 14. Mu.l/well.
(3) Reverse transcription reaction procedure: 42 ℃ for 10min;25 ℃ for 10min;50 ℃ for 60min;94℃for 5min.
(4) The cDNA was stored at-20 ℃.
(5) Primer design and synthesis:
forward Primer 5'-3' sequence (Forward Primer 5'-3' sequence)
Heavy chain variable region PCR primers:
5′VH1 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGGTGCAG(SEQ ID NO:51)
5′VH1/5 CTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGCAG(SEQ ID NO:52)
5′VH3 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGGTGGAG(SEQ ID NO:53)
5′VH3-23 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGTTGGAG(SEQ ID NO:54)
5′VH4 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGCAGGAG(SEQ ID NO:55)
5′VH 4-34CTGCAACCGGTGTACATTCCCAGGTGCAGCTACAGCAGTG(SEQ ID NO:56)
5′VH 1-18 CTGCAACCGGTGTACATTCCCAGGTTCAGCTGGTGCAG(SEQ ID NO:57)
5′VH 1-24 CTGCAACCGGTGTACATTCCCAGGTCCAGCTGGTACAG(SEQ ID NO:58)
5′VH3-33 CTGCAACCGGTGTACATTCTCAGGTGCAGCTGGTGGAG(SEQ ID NO:59)
5′VH 3-9 CTGCAACCGGTGTACATTCTGAAGTGCAGCTGGTGGAG(SEQ ID NO:60)
5′VH4-39 CTGCAACCGGTGTACATTCCCAGCTGCAGCTGCAGGAG(SEQ ID NO:61)
5′VH 6-1 CTGCAACCGGTGTACATTCCCAGGTACAGCTGCAGCAG(SEQ ID NO:62)
3′JH 1/2/4/5 TGCGAAGTCGACGCTGAGGAGACGGTGACCAG(SEQ ID NO:63)
3′JH 3 TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG(SEQ ID NO:64)
3′JH 6 TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG(SEQ ID NO:65)
kappa light chain variable region PCR primers
5′Vκ 1-5 CTGCAACCGGTGTACATTCTGACATCCAGATGACCCAGTC(SEQ ID NO:66)
5′Vκ 1-9 TTGTGCTGCAACCGGTGTACATTCAGACATCCAGTTGACCC AGTCT(SEQ ID NO:67)
5′Vκ 1D-43 CTGCAACCGGTGTACATTGTGCCATCCGGATGACCCAGTC(SEQ ID NO:68)
5′Vκ 2-24 CTGCAACCGGTGTACATGGGGATATTGTGATGACCCAGAC(SEQ ID NO:69)
5′Vκ 2-28 CTGCAACCGGTGTACATGGGGATATTGTGATGACTCAGTC(SEQ ID NO:70)
5′Vκ 2-30 CTGCAACCGGTGTACATGGGGATGTTGTGATGACTCAGTC(SEQ ID NO:71)
5′Vκ 3-11 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACAC AGTC(SEQ ID NO:72)
5′Vκ 3-15 CTGCAACCGGTGTACATTCAGAAATAGTGATGACGCAGTC(SEQ ID NO:73)
5′Vκ 3-20 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACG CAGTCT(SEQ ID NO:74)
5′Vκ 4-1 CTGCAACCGGTGTACATTCGGACATCGTGATGACCCAGTC(SEQ ID NO:75)
3′Jκ 1/4 GCCACCGTACGTTTGATYTCCACCTTGGTC(SEQ ID NO:76)
3′Jκ 2 GCCACCGTACGTTTGATCTCCAGCTTGGTC(SEQ ID NO:77)
3′Jκ 3 GCCACCGTACGTTTGATATCCACTTTGGTC(SEQ ID NO:78)
3′Jκ 5 GCCACCGTACGTTTAATCTCCAGTCGTGTC(SEQ ID NO:79)
(6) Heavy and light chains of antibody genes were amplified by PCR using KOD-Plus-Neo (TOYOBO, KOD 401) kit, 40. Mu.L system: 3.5. Mu.L cDNA,20nM mixed primer, 4. Mu.L buffer, 4. Mu.L 2mM dNTPs, 2.4. Mu.L MgSO 4 ，1μL KOD。
(7) The reaction procedure: 94 ℃ for 2min;45 cycles: 98 ℃ for 10s;58 ℃ for 30s;68℃for 28s.
(8) Agarose gel was performed and as shown in FIG. 4, the light chain sizes of the five antibodies were about 300bp and the heavy chain sizes were about 400bp, respectively.
(9) The PCR amplified products were sent to Jin Weizhi Biotechnology Co.Ltd for sequencing
(10) The 5 antibody variable region sequences are as follows:
1)9g8
heavy chain variable region:
QLQLQESGPGLVKPSETLSLTCTVSGGSITTSSDYWGWIRQPPGKGLEWIGSIYYSGRTYYNPSLKSRVTISVDTSKNDFSLKLSSVTAADTAVYYCARRLTYYYDSSGYANWYFDLWGRGTLVTVSS SEQ ID No：31
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQRFSTFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPYSFGQGTKLEIKR SEQ ID No:32; or alternatively
2)6J19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：33
light chain variable region:
QSVLTQPPSASGTPGERVTISCSGSSSNIGRSTVSWYQQLPGTAPKLLMYSSYQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG SEQ ID No:34; or alternatively
3)7N13
Heavy chain variable region:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTMYYGDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVGATGELFDYWGQGTLVTASS SEQ ID No：35
light chain variable region:
AIQMTQSPSSLSASVGDRVTITCRATQGIGNELGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIKR SEQ ID No:36; or alternatively
4)8L19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSNYSMNWVRQAPGKGLEWVSSISTTGTYTHYAGSVKGRFTISRDNAKNSLFLRMNSLRAEDTAVYYCARPYYYGSGSPDYWGQGTLVTVSS SEQ ID No：37
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQSISTFLNWYQQKPGKAPNLLIYAASSLQRGVPSRFTGSGSGTDFTLTISSLQPEDFATYYCHQTYSKPWTFGRGTKVEIER SEQ ID No:38, a step of carrying out the process; or alternatively
5)6M9
Heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFRNYDINWVRQAPGKGLEWVSSISGSGIDTYYGDSVEGRFTVSRDNAESSVLLEMNSLRADDTAVFYCVRGLAGAFDYWGQGTLVTVSS SEQ ID No：39
light chain variable region:
EIVLTQSPGTLSLSPGERATLSCRASQSVTSGYLAWYQQKPGQAPRLLIHGTSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHRSSPMYSFGQGSKLEIKR SEQ ID No：40。
correspondingly, the CDR region sequences are as follows:
1)9g8
HCDR1：GGSITTSSDY SEQ ID No：1；
HCDR2：IYYSGRT SEQ ID No：2；
HCDR3：ARRLTYYYDSSGYANWYFDL SEQ ID No：3；
LCDR1：QRFSTF SEQ ID No：4；
LCDR:2：AAS SEQ ID No：5；
LCDR3: QQSYSIPYS SEQ ID No:6, preparing a base material; or alternatively
2)6J19
HCDR1：GFTFSSYS SEQ ID No：7；
HCDR2：ISSSGTFI SEQ ID No：8；
HCDR3：ARERFVGVLDI SEQ ID No：9；
LCDR1：SSNIGRST SEQ ID No：10；
LCDR:2：SSY SEQ ID No：11；
LCDR3: AAWDDSLNGPV SEQ ID No:12; or alternatively
3)7N13
HCDR1：GFTFSSYS SEQ ID No：13；
HCDR2：ISSSSSTM SEQ ID No：14；
HCDR3：ARGVGATGELFDY SEQ ID No：15；
LCDR1：QGIGNE SEQ ID No：16；
LCDR:2：AAS SEQ ID No：17；
LCDR3: LQDYNYPRT SEQ ID No:18; or alternatively
4)8L19
HCDR1：GFTFSNYS SEQ ID No：19；
HCDR2：ISTTGTYT SEQ ID No：20；
HCDR3：ARPYYYGSGSPDY SEQ ID No：21；
LCDR1：QSISTF SEQ ID No：22；
LCDR:2：AAS SEQ ID No：23；
LCDR3: HQTYSKPWT SEQ ID No:24, a step of detecting the position of the base; or alternatively
5)6M9
HCDR1：GFTFRNYD SEQ ID No：25；
HCDR2：ISGSGIDT SEQ ID No：26；
HCDR3：VRGLAGAFDY SEQ ID No：27；
LCDR1：QSVTSGY SEQ ID No：28；
LCDR:2：GTS SEQ ID No：29；
LCDR3：QQHRSSPMYS SEQ ID No：30。
The last explanation is: the above embodiments are only for illustrating the implementation procedure and features of the present application, and not for limiting the technical solution of the present application, and although the present application is described in detail with reference to the above embodiments, it should be understood by those of ordinary skill in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the present application, and any modifications or partial substitutions are intended to be included within the scope of the present application.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute of China academy of sciences
<120> an anti-SAR-COV-2 antibody or antigen-binding fragment thereof and use thereof
<130> CP120010319C
<160> 79
<170> PatentIn version 3.3
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<210> 4
<211> 6
<212> PRT
<213> artificial sequence
<400> 4
Gln Arg Phe Ser Thr Phe
1 5
<210> 5
<211> 3
<212> PRT
<213> artificial sequence
<400> 5
Ala Ala Ser
1
<210> 6
<211> 9
<212> PRT
<213> artificial sequence
<400> 6
Gln Gln Ser Tyr Ser Ile Pro Tyr Ser
1 5
<210> 7
<211> 8
<212> PRT
<213> artificial sequence
<400> 7
Gly Phe Thr Phe Ser Ser Tyr Ser
1 5
<210> 8
<211> 8
<212> PRT
<213> artificial sequence
<400> 8
Ile Ser Ser Ser Gly Thr Phe Ile
1 5
<210> 9
<211> 11
<212> PRT
<213> artificial sequence
<400> 9
Ala Arg Glu Arg Phe Val Gly Val Leu Asp Ile
1 5 10
<210> 10
<211> 8
<212> PRT
<213> artificial sequence
<400> 10
Ser Ser Asn Ile Gly Arg Ser Thr
1 5
<210> 11
<211> 3
<212> PRT
<213> artificial sequence
<400> 11
Ser Ser Tyr
1
<210> 12
<211> 11
<212> PRT
<213> artificial sequence
<400> 12
Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val
1 5 10
<210> 13
<211> 8
<212> PRT
<213> artificial sequence
<400> 13
Gly Phe Thr Phe Ser Ser Tyr Ser
1 5
<210> 14
<211> 8
<212> PRT
<213> artificial sequence
<400> 14
Ile Ser Ser Ser Ser Ser Thr Met
1 5
<210> 15
<211> 13
<212> PRT
<213> artificial sequence
<400> 15
Ala Arg Gly Val Gly Ala Thr Gly Glu Leu Phe Asp Tyr
1 5 10
<210> 16
<211> 6
<212> PRT
<213> artificial sequence
<400> 16
Gln Gly Ile Gly Asn Glu
1 5
<210> 17
<211> 3
<212> PRT
<213> artificial sequence
<400> 17
Ala Ala Ser
1
<210> 18
<211> 9
<212> PRT
<213> artificial sequence
<400> 18
Leu Gln Asp Tyr Asn Tyr Pro Arg Thr
1 5
<210> 19
<211> 8
<212> PRT
<213> artificial sequence
<400> 19
Gly Phe Thr Phe Ser Asn Tyr Ser
1 5
<210> 20
<211> 8
<212> PRT
<213> artificial sequence
<400> 20
Ile Ser Thr Thr Gly Thr Tyr Thr
1 5
<210> 21
<211> 13
<212> PRT
<213> artificial sequence
<400> 21
Ala Arg Pro Tyr Tyr Tyr Gly Ser Gly Ser Pro Asp Tyr
1 5 10
<210> 22
<211> 6
<212> PRT
<213> artificial sequence
<400> 22
Gln Ser Ile Ser Thr Phe
1 5
<210> 23
<211> 3
<212> PRT
<213> artificial sequence
<400> 23
Ala Ala Ser
1
<210> 24
<211> 9
<212> PRT
<213> artificial sequence
<400> 24
His Gln Thr Tyr Ser Lys Pro Trp Thr
1 5
<210> 25
<211> 8
<212> PRT
<213> artificial sequence
<400> 25
Gly Phe Thr Phe Arg Asn Tyr Asp
1 5
<210> 26
<211> 8
<212> PRT
<213> artificial sequence
<400> 26
Ile Ser Gly Ser Gly Ile Asp Thr
1 5
<210> 27
<211> 10
<212> PRT
<213> artificial sequence
<400> 27
Val Arg Gly Leu Ala Gly Ala Phe Asp Tyr
1 5 10
<210> 28
<211> 7
<212> PRT
<213> artificial sequence
<400> 28
Gln Ser Val Thr Ser Gly Tyr
1 5
<210> 29
<211> 3
<212> PRT
<213> artificial sequence
<400> 29
Gly Thr Ser
1
<210> 30
<211> 10
<212> PRT
<213> artificial sequence
<400> 30
Gln Gln His Arg Ser Ser Pro Met Tyr Ser
1 5 10
<210> 31
<211> 128
<212> PRT
<213> artificial sequence
<400> 31
Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Thr Ser
20 25 30
Ser Asp Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Arg Thr Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Asp Phe
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg Arg Leu Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Ala Asn
100 105 110
Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 32
<211> 108
<212> PRT
<213> artificial sequence
<400> 32
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg Phe Ser Thr Phe
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Tyr
85 90 95
Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 33
<211> 118
<212> PRT
<213> artificial sequence
<400> 33
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Ser Ser Ser Gly Thr Phe Ile Lys Tyr Ala Asp Ser Leu
50 55 60
Gln Gly Arg Phe Thr Ile Thr Arg Asp Asn Ala Lys Thr Ala Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Arg Phe Val Gly Val Leu Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 34
<211> 111
<212> PRT
<213> artificial sequence
<400> 34
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Glu
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Arg Ser
20 25 30
Thr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Met Tyr Ser Ser Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95
Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
<210> 35
<211> 120
<212> PRT
<213> artificial sequence
<400> 35
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Ser Ser Thr Met Tyr Tyr Gly Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Val Gly Ala Thr Gly Glu Leu Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Ala Ser Ser
115 120
<210> 36
<211> 108
<212> PRT
<213> artificial sequence
<400> 36
Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Thr Gln Gly Ile Gly Asn Glu
20 25 30
Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Asn Tyr Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 37
<211> 120
<212> PRT
<213> artificial sequence
<400> 37
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Thr Thr Gly Thr Tyr Thr His Tyr Ala Gly Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe
65 70 75 80
Leu Arg Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Pro Tyr Tyr Tyr Gly Ser Gly Ser Pro Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 38
<211> 108
<212> PRT
<213> artificial sequence
<400> 38
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Phe
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Thr Tyr Ser Lys Pro Trp
85 90 95
Thr Phe Gly Arg Gly Thr Lys Val Glu Ile Glu Arg
100 105
<210> 39
<211> 117
<212> PRT
<213> artificial sequence
<400> 39
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr
20 25 30
Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Gly Ser Gly Ile Asp Thr Tyr Tyr Gly Asp Ser Val
50 55 60
Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Glu Ser Ser Val Leu
65 70 75 80
Leu Glu Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Phe Tyr Cys
85 90 95
Val Arg Gly Leu Ala Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 40
<211> 110
<212> PRT
<213> artificial sequence
<400> 40
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Gly
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile His Gly Thr Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Arg Ser Ser Pro
85 90 95
Met Tyr Ser Phe Gly Gln Gly Ser Lys Leu Glu Ile Lys Arg
100 105 110
<210> 41
<211> 315
<212> DNA
<213> artificial sequence
<400> 41
gtctctggtg gctctatcac cactagtagt gactactggg gctggatccg ccagccccca 60
gggaaggggc tggagtggat tgggagtatc tattatagtg ggagaaccta ctacaacccg 120
tccctcaaga gtcgagtcac catatccgta gacacgtcca agaacgactt ctctctgaag 180
ctgagctctg tgaccgccgc agacacggct gtgtattact gtgcgagacg ccttacgtat 240
tactatgata gtagtggtta tgcgaactgg tacttcgatc tctggggccg tggcaccctg 300
gtcactgtct cctca 315
<210> 42
<211> 324
<212> DNA
<213> artificial sequence
<400> 42
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gaggttcagc acctttttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca cgatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta tcccgtactc ttttggccag 300
gggaccaagc tggagatcaa acga 324
<210> 43
<211> 354
<212> DNA
<213> artificial sequence
<400> 43
gaggtgcagc tggtggagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt agttatagta tgaagtgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaacc atcagtagta gtggtacttt cataaagtat 180
gcagactcac tgcagggccg attcaccatc accagagaca acgccaagac cgcagtgtat 240
ctgcaaatga acagcctgag agtcgaggac acggctgttt attactgtgc gagagaacga 300
ttcgttggtg ttttggatat ctggggccaa gggacaatgg tcaccgtctc ttca 354
<210> 44
<211> 333
<212> DNA
<213> artificial sequence
<400> 44
cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccggggagag ggtcaccatc 60
tcttgttctg gaagcagctc caacatcgga aggagtactg taagctggta ccagcagctc 120
ccaggaacgg cccccaaact cctcatgtat agtagttatc agcggccctc aggggtccct 180
gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240
tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggtccggtg 300
ttcggcggag ggaccaagct gaccgtccta ggt 333
<210> 45
<211> 359
<212> DNA
<213> artificial sequence
<400> 45
gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt agttatagca tgaactgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtttcatat attagtagta gtagtagtac catgtactac 180
ggagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agacgaggac acggctgtgt attactgtgc gagaggagtg 300
ggagccacgg gggaactctt tgactactgg ggccagggaa ccctggtcac cgcctcctc 359
<210> 46
<211> 324
<212> DNA
<213> artificial sequence
<400> 46
gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaactca gggaattgga aatgaattag ggtggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tacaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtctacaa gattacaatt accctcgtac gttcggccaa 300
gggaccaagg tggaaatcaa acga 324
<210> 47
<211> 360
<212> DNA
<213> artificial sequence
<400> 47
gaggtgcagc tggtggagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60
tcctgtgcag tctctggatt caccttcagt aactatagca tgaactgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcatcc attagtacta ctggtactta cacacactac 180
gccggctcag tgaagggccg attcaccatc tccagagaca atgccaagaa ctcgctgttt 240
ttgcgaatga acagcctgag agccgaggac acggctgtgt attactgtgc gaggccctat 300
tactatggtt cggggagtcc tgactactgg ggccagggaa ccctggtcac cgtctcctca 360
<210> 48
<211> 324
<212> DNA
<213> artificial sequence
<400> 48
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc acctttttga attggtatca gcagaagccc 120
gggaaagccc ctaatctcct gatctatgct gcatccagtt tgcaacgtgg ggtcccatca 180
aggttcactg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaccag acttacagta agccctggac gttcggccga 300
gggaccaagg tggaaatcga acga 324
<210> 49
<211> 351
<212> DNA
<213> artificial sequence
<400> 49
gaggtgcagc tgttggagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagg aactatgaca tcaactgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcatcg attagtggta gtggtattga cacatactac 180
ggagactcag tggagggccg attcaccgtc tccagagaca acgccgagag ctcagtatta 240
ctggagatga acagcctgag agccgacgat acggctgtat tttactgtgt gaggggtctg 300
gctggcgcct ttgactactg gggccaggga accctggtca ccgtctcctc a 351
<210> 50
<211> 330
<212> DNA
<213> artificial sequence
<400> 50
gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttacc agcggctact tagcctggta ccagcagaaa 120
cctggccagg ctcccaggct cctcatccat ggtacatcca ggagggccac tggcatccca 180
gacaggttca gtggcagtgg gtctgggaca gatttcactc tcaccatcag cagactggag 240
cctgaagatt ttgcagtcta ttactgtcag cagcatcgta gctcacccat gtacagtttt 300
ggccagggga gcaagctgga gatcaaacga 330
<210> 51
<211> 38
<212> DNA
<213> artificial sequence
<400> 51
ctgcaaccgg tgtacattcc caggtgcagc tggtgcag 38
<210> 52
<211> 38
<212> DNA
<213> 52
<400> 52
ctgcaaccgg tgtacattcc gaggtgcagc tggtgcag 38
<210> 53
<211> 38
<212> DNA
<213> artificial sequence
<400> 53
ctgcaaccgg tgtacattct gaggtgcagc tggtggag 38
<210> 54
<211> 38
<212> DNA
<213> artificial sequence
<400> 54
ctgcaaccgg tgtacattct gaggtgcagc tgttggag 38
<210> 55
<211> 38
<212> DNA
<213> artificial sequence
<400> 55
ctgcaaccgg tgtacattcc caggtgcagc tgcaggag 38
<210> 56
<211> 40
<212> DNA
<213> artificial sequence
<400> 56
ctgcaaccgg tgtacattcc caggtgcagc tacagcagtg 40
<210> 57
<211> 38
<212> DNA
<213> artificial sequence
<400> 57
ctgcaaccgg tgtacattcc caggttcagc tggtgcag 38
<210> 58
<211> 38
<212> DNA
<213> artificial sequence
<400> 58
ctgcaaccgg tgtacattcc caggtccagc tggtacag 38
<210> 59
<211> 38
<212> DNA
<213> artificial sequence
<400> 59
ctgcaaccgg tgtacattct caggtgcagc tggtggag 38
<210> 60
<211> 38
<212> DNA
<213> artificial sequence
<400> 60
ctgcaaccgg tgtacattct gaagtgcagc tggtggag 38
<210> 61
<211> 38
<212> DNA
<213> artificial sequence
<400> 61
ctgcaaccgg tgtacattcc cagctgcagc tgcaggag 38
<210> 62
<211> 38
<212> DNA
<213> artificial sequence
<400> 62
ctgcaaccgg tgtacattcc caggtacagc tgcagcag 38
<210> 63
<211> 32
<212> DNA
<213> artificial sequence
<400> 63
tgcgaagtcg acgctgagga gacggtgacc ag 32
<210> 64
<211> 34
<212> DNA
<213> artificial sequence
<400> 64
tgcgaagtcg acgctgaaga gacggtgacc attg 34
<210> 65
<211> 33
<212> DNA
<213> artificial sequence
<400> 65
tgcgaagtcg acgctgagga gacggtgacc gtg 33
<210> 66
<211> 40
<212> DNA
<213> artificial sequence
<400> 66
ctgcaaccgg tgtacattct gacatccaga tgacccagtc 40
<210> 67
<211> 46
<212> DNA
<213> artificial sequence
<400> 67
ttgtgctgca accggtgtac attcagacat ccagttgacc cagtct 46
<210> 68
<211> 40
<212> DNA
<213> artificial sequence
<400> 68
ctgcaaccgg tgtacattgt gccatccgga tgacccagtc 40
<210> 69
<211> 40
<212> DNA
<213> artificial sequence
<400> 69
ctgcaaccgg tgtacatggg gatattgtga tgacccagac 40
<210> 70
<211> 40
<212> DNA
<213> artificial sequence
<400> 70
ctgcaaccgg tgtacatggg gatattgtga tgactcagtc 40
<210> 71
<211> 40
<212> DNA
<213> artificial sequence
<400> 71
ctgcaaccgg tgtacatggg gatgttgtga tgactcagtc 40
<210> 72
<211> 45
<212> DNA
<213> artificial sequence
<400> 72
ttgtgctgca accggtgtac attcagaaat tgtgttgaca cagtc 45
<210> 73
<211> 40
<212> DNA
<213> artificial sequence
<400> 73
ctgcaaccgg tgtacattca gaaatagtga tgacgcagtc 40
<210> 74
<211> 46
<212> DNA
<213> artificial sequence
<400> 74
ttgtgctgca accggtgtac attcagaaat tgtgttgacg cagtct 46
<210> 75
<211> 40
<212> DNA
<213> artificial sequence
<400> 75
ctgcaaccgg tgtacattcg gacatcgtga tgacccagtc 40
<210> 76
<211> 30
<212> PRT
<213> artificial sequence
<400> 76
Gly Cys Cys Ala Cys Cys Gly Thr Ala Cys Gly Thr Thr Thr Gly Ala
1 5 10 15
Thr Tyr Thr Cys Cys Ala Cys Cys Thr Thr Gly Gly Thr Cys
20 25 30
<210> 77
<211> 30
<212> DNA
<213> artificial sequence
<400> 77
gccaccgtac gtttgatctc cagcttggtc 30
<210> 78
<211> 30
<212> DNA
<213> artificial sequence
<400> 78
gccaccgtac gtttgatatc cactttggtc 30
<210> 79
<211> 30
<212> DNA
<213> artificial sequence
<400> 79
gccaccgtac gtttaatctc cagtcgtgtc 30

Claims (13)

1. An isolated antibody against SAR-COV-2 or an antigen-binding fragment thereof that specifically binds to SAR-COV-2 surface S protein; it has three heavy chain complementarity determining regions (HCDR) and three light chain complementarity determining regions (LCDR) of any of the following groups:
1)9g8
HCDR1：GGSITTSSDY SEQ ID No：1；
HCDR2：IYYSGRT SEQ ID No：2；
HCDR3：ARRLTYYYDSSGYANWYFDL SEQ ID No：3；
LCDR1：QRFSTF SEQ ID No：4；
LCDR2: AAS SEQ ID No:5, a step of; and
LCDR3: QQSYSIPYS SEQ ID No:6, preparing a base material; or alternatively
2)6J19
HCDR1：GFTFSSYS SEQ ID No：7；
HCDR2：ISSSGTFI SEQ ID No：8；
HCDR3：ARERFVGVLDI SEQ ID No：9；
LCDR1：SSNIGRST SEQ ID No：10；
LCDR2: SSY SEQ ID No:11; and
LCDR3: AAWDDSLNGPV SEQ ID No:12; or alternatively
3)7N13
HCDR1：GFTFSSYS SEQ ID No：13；
HCDR2：ISSSSSTM SEQ ID No：14；
HCDR3：ARGVGATGELFDY SEQ ID No：15；
LCDR1：QGIGNE SEQ ID No：16；
LCDR2: AAS SEQ ID No:17; and
LCDR3: LQDYNYPRT SEQ ID No:18; or alternatively
4)8L19
HCDR1：GFTFSNYS SEQ ID No：19；
HCDR2：ISTTGTYT SEQ ID No：20；
HCDR3：ARPYYYGSGSPDY SEQ ID No：21；
LCDR1：QSISTF SEQ ID No：22；
LCDR2: AAS SEQ ID No:23; and
LCDR3: HQTYSKPWT SEQ ID No:24, a step of detecting the position of the base; or alternatively
5)6M9
HCDR1：GFTFRNYD SEQ ID No：25；
HCDR2：ISGSGIDT SEQ ID No：26；
HCDR3：VRGLAGAFDY SEQ ID No：27；
LCDR1：QSVTSGY SEQ ID No：28；
LCDR2: GTS SEQ ID No:29; and
LCDR3：QQHRSSPMYS SEQ ID No：30。
2. an isolated antibody against SAR-COV-2, or an antigen-binding fragment thereof, having a heavy chain variable region and a light chain variable region as shown below;
1)9g8
heavy chain variable region:
QLQLQESGPGLVKPSETLSLTCTVSGGSITTSSDYWGWIRQPPGKGLEWIGSIYYSGRTYYN PSLKSRVTISVDTSKNDFSLKLSSVTAADTAVYYCARRLTYYYDSSGYANWYFDLWGRGTL VTVSS SEQ ID No：31
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQRFSTFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQSYSIPYSFGQGTKLEIKR SEQ ID No:32; or alternatively
2)6J19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYA DSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：33
light chain variable region:
QSVLTQPPSASGTPGERVTISCSGSSSNIGRSTVSWYQQLPGTAPKLLMYSSYQRPSGVPDRF SGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG SEQ ID No:34; or alternatively
3)7N13
Heavy chain variable region:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTMYYG DSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVGATGELFDYWGQGTLVTASS SEQ ID No：35
light chain variable region:
AIQMTQSPSSLSASVGDRVTITCRATQGIGNELGWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIKR SEQ ID No:36; or alternatively
4)8L19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSNYSMNWVRQAPGKGLEWVSSISTTGTYTHYA GSVKGRFTISRDNAKNSLFLRMNSLRAEDTAVYYCARPYYYGSGSPDYWGQGTLVTVSS SEQ ID No：37
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQSISTFLNWYQQKPGKAPNLLIYAASSLQRGVPSRFT GSGSGTDFTLTISSLQPEDFATYYCHQTYSKPWTFGRGTKVEIER SEQ ID No:38, a step of carrying out the process; or alternatively
5)6M9
Heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFRNYDINWVRQAPGKGLEWVSSISGSGIDTYYG DSVEGRFTVSRDNAESSVLLEMNSLRADDTAVFYCVRGLAGAFDYWGQGTLVTVSS SEQ ID No：39
light chain variable region:
EIVLTQSPGTLSLSPGERATLSCRASQSVTSGYLAWYQQKPGQAPRLLIHGTSRRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHRSSPMYSFGQGSKLEIKR SEQ ID No：40。
3. the antibody or antigen-binding fragment thereof of any one of claim 1 or claim 2, which is a humanized antibody or antigen-binding fragment thereof.
4. A nucleotide, characterized in that: encoding the antibody or antigen binding fragment thereof of any one of claims 1-3.
5. A carrier, characterized in that: comprising the nucleotide sequence of claim 4.
6. A host cell, characterized in that: comprising the vector of claim 5.
7. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.
8. A detection reagent comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.
9. Use of the antibody or antigen binding fragment thereof of any one of claims 1-3 in the preparation of a detection reagent for use in: enzyme-linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemical assay, or immuno-PCR.
10. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1-3 and a pharmaceutically acceptable adjuvant.
11. Use of an antibody or antigen-binding fragment thereof against SAR-COV-2 according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 10 for the manufacture of a medicament for preventing, treating or alleviating at least one symptom or indication of SAR-COV-2 infection.
12. The use of claim 11, wherein the at least one symptom or indication is selected from the group consisting of: novel coronavirus pneumonia, alveolar injury, fever, cough, dyspnea, hypoxia, acute respiratory distress syndrome, sepsis shock, coagulation dysfunction, metabolic acidosis, nasal obstruction, runny nose, pharyngalgia, diarrhea, organ failure, septic shock.
13. The use of claim 11, wherein the symptom or indication is pulmonary inflammation.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB202003632D0 (en) * 2020-03-12 2020-04-29 Harbour Antibodies Bv SARS-Cov-2 (SARS2, COVID-19) antibodies
CN111153991A (en) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 Human SARS-CoV-2 monoclonal antibody and its preparation method and use
CN111233985A (en) * 2020-02-09 2020-06-05 北京丹大生物技术有限公司 Preparation method of novel coronavirus IgA antibody rapid detection test strip
CN111239392A (en) * 2020-02-26 2020-06-05 浙江诺迦生物科技有限公司 Novel coronavirus pneumonia (COVID-19) serological diagnosis kit

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7396914B2 (en) * 2003-08-04 2008-07-08 University Of Massachusetts SARS nucleic acids, proteins, antibodies, and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111233985A (en) * 2020-02-09 2020-06-05 北京丹大生物技术有限公司 Preparation method of novel coronavirus IgA antibody rapid detection test strip
CN111153991A (en) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 Human SARS-CoV-2 monoclonal antibody and its preparation method and use
CN111239392A (en) * 2020-02-26 2020-06-05 浙江诺迦生物科技有限公司 Novel coronavirus pneumonia (COVID-19) serological diagnosis kit
GB202003632D0 (en) * 2020-03-12 2020-04-29 Harbour Antibodies Bv SARS-Cov-2 (SARS2, COVID-19) antibodies

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Baoqing Sun等.Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 patients.《Emerg Microbes Infect 》.2020,第9卷(第1期),第940-948页. *
张茜等.新型冠状病毒肺炎的抗体应对策略.南京医科大学学报(自然科学版).2020,(02),第11-15页. *

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