CN114437205A - Anti-coronavirus antibody and application thereof - Google Patents

Anti-coronavirus antibody and application thereof Download PDF

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CN114437205A
CN114437205A CN202011225733.7A CN202011225733A CN114437205A CN 114437205 A CN114437205 A CN 114437205A CN 202011225733 A CN202011225733 A CN 202011225733A CN 114437205 A CN114437205 A CN 114437205A
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郭亚娟
杨柳
沈月雷
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Doma Pharmaceutical Technology Suzhou Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/125—Adult respiratory distress syndrome
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Abstract

The invention provides an anti-coronavirus antibody or an antigen-binding fragment thereof, and a preparation method and application thereof. The anti-coronavirus antibody or the antigen-binding fragment thereof has high binding affinity with coronavirus S protein, and can be used for detection of coronavirus.

Description

Anti-coronavirus antibody and application thereof
Technical Field
The invention relates to the technical field of anti-coronavirus antibodies, in particular to an anti-coronavirus antibody or an antigen binding fragment thereof, and a preparation method and application thereof.
Background
The coronavirus belongs to genus coronavirus of family Coronaviridae of order Togaviridae, and is in corona coronaria or crown shape under microscope. It is a group of related RNA viruses that cause disease in mammals and birds. The two ends of the coronavirus genome respectively comprise an untranslated region (5 'UTR and 3' UTR), wherein about 3/4 at the 5 'end comprises 2 large overlapped open reading frames ORF1a and ORF1b which mainly encode nonstructural proteins such as enzymes related to virus replication and transcription, and about 1/4 at the 3' end mainly encodes major structural proteins such as surface spike protein (S protein), membrane protein (M protein), small membrane protein (E protein) and nucleocapsid protein (N protein).
At present, for the detection of coronavirus, the research progress of the nucleic acid detection method of human coronavirus in the non-patent literature (nipehua et al, journal of international virology, 2016) discloses the methods of nucleic acid detection, serological detection, antigen detection, virus separation, electron microscopy detection and the like mainly for the detection of human coronavirus, and the nucleic acid detection is mainly explained.
Another non-patent document, the research progress of serological detection method of coronavirus of respiratory syndrome in the middle east (Marek's vaccine, etc., J. Virol. China, 2018) discloses that technical reference is provided for controlling the prevalence of MERS-CoV, the serological detection method of MERS-CoV at home and abroad is summarized, and specifically evaluates the serological method for detecting specific antibody or antigen, which is simple and convenient to operate, relatively low in cost, not easy to pollute and easy to popularize and apply compared with nucleic acid detection and virus separation, moreover, for mild or asymptomatic MERS-CoV infection cases, the animal origin and transmission characteristics of the virus can be determined, serological detection of susceptible animals in epidemic areas can provide an early warning and prevention and control strategy for epidemic outbreak, and more importantly, the serological detection method can also assist in researching the immune response process of organisms to virus infection and provide scientific reference for research of pathogenic mechanisms and vaccines.
However, the number of antibodies of coronavirus is still small, the detection method is single and needs several hours, and further research and development of each scientific research unit are urgently needed. Thus, the present application provides a novel anti-coronavirus antibody that binds coronavirus S protein with high affinity and specificity, and is useful for relevant diagnostic assays and treatments against coronaviruses.
Disclosure of Invention
The present invention provides an anti-coronavirus antibody or an antigen-binding fragment thereof, which has high binding affinity to coronavirus S protein and can be used for detection of coronavirus (for example, uniport numbers Q3LZX1, Q3I5J5, Q0Q475, SARS of A0A0K1Z074, SARS-CoV-2, and the like). The specific scheme is as follows:
in a first aspect of the invention, there is provided an anti-coronavirus antibody or antigen-binding fragment thereof comprising a VHCDR1, a VHCDR2 and a VHCDR3 of the heavy chain variable region and a VLCDR1, a VLCDR2 and a VLCDR3 of the light chain variable region, wherein,
the amino acid sequence of VHCDR1 comprises SEQ ID NO:1, the amino acid sequence of VHCDR2 comprises SEQ ID NO: 2, the amino acid sequence of VHCDR3 comprises SEQ ID NO: 3, the amino acid sequence of VLCDR1 comprises SEQ ID NO:4, the amino acid sequence of VLCDR2 comprises SEQ ID NO: 5, the amino acid sequence of VLCDR3 comprises SEQ ID NO: 6(Kabat coding mode);
alternatively, the amino acid sequence of VHCDR1 comprises SEQ ID NO:7, the amino acid sequence of VHCDR2 comprises SEQ ID NO: 8, the amino acid sequence of VHCDR3 comprises SEQ ID NO: 9, the amino acid sequence of VLCDR1 comprises SEQ ID NO: 10, the amino acid sequence of VLCDR2 comprises SEQ ID NO: 11, the amino acid sequence of VLCDR3 comprises SEQ ID NO: 12(Chothia coding scheme).
An antibody may comprise two identical copies of the light chain and two identical copies of the heavy chain. Heavy chains, each comprising one variable domain (or variable region, VH) and multiple constant domains (or constant regions), are bound to each other by disulfide bonds within their constant domains to form the "handle" of the antibody. Light chains, each comprising a variable domain (or variable region, VL) and a constant domain (or constant region), are each bound to a heavy chain by disulfide bonding. The variable region of each light chain is paired with the variable region of the heavy chain to which it is bound. The variable regions of both light and heavy chains contain three hypervariable regions located between more conserved Framework Regions (FRs).
These hypervariable regions, termed Complementarity Determining Regions (CDRs), form loops that comprise the primary antigen-binding surface of an antibody. The four framework regions adopt predominantly a β -sheet conformation, and the CDRs form loops that connect and in some cases form part of the β -sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen binding region. Methods for identifying CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and many definitions of CDRs are commonly used. The Kabat definition is based on sequence variability and Chothia definition is based on the position of the structural loop regions. These methods and definitions are described in the following: for example, Martin, "Protein sequence and structure analysis of Antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; abhinandan, et al, "Analysis and improvements to Kabat and structural core number of antibody variable domains," Molecular immunology 45.14(2008): 3832-; wu, T.T.and Kabat, E.A. (1970) J.exp.Med.132: 211-250; martin et al, Methods enzymol.203:121-53 (1991); morea et al, Biophys chem.68(1-3):9-16 (Oct.1997); morea et al, J Mol biol.275(2):269-94 (Jan.1998); chothia et al, Nature 342(6252) 877-83(Dec. 1989); ponomarenko and Bourne, BMC Structural Biology 7:64 (2007).
Preferably, the amino acid sequence of the heavy chain variable region comprises SEQ ID NO 13 or has more than 80% homology with SEQ ID NO 13 and retains the ability to bind to coronavirus S protein.
Preferably, the amino acid sequence of the light chain variable region comprises SEQ ID NO:14 or has more than 80% homology with SEQ ID NO:14 and retains the ability to bind to coronavirus S protein.
Preferably, the coronavirus is SARS or SARS-CoV-2 with the numbers Q3LZX1, Q3I5J5, Q0Q475 and A0A0K1Z 074.
Preferably, the anti-coronavirus antibody or antigen-binding fragment thereof is a monoclonal antibody or a polyclonal antibody.
Preferably, the anti-coronavirus antibody or antigen-binding fragment thereof is a single chain antibody, Fv antibody, Fd, dAb, bispecific antibody, bispecific single chain antibody, linear antibody, single chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising an antibody binding domain or homologous antibody binding domain. The antibody binding domain may comprise, among other things, an intact heavy and/or light chain CDR, an intact heavy and/or light chain variable region of an antibody, an intact full-length heavy and/or light chain, or a single, two, three, four, five or six CDR from the antibody. Single chain antibodies comprise a heavy chain variable region and a light chain variable region.
Preferably, the anti-coronavirus antibody or antigen-binding fragment thereof specifically binds to coronavirus S protein. Preferably, the anti-coronavirus antibody or antigen-binding fragment thereof specifically binds to coronavirus S protein, S1 protein, S2 protein or RBD protein.
Further preferably, the anti-coronavirus antibody or antigen-binding fragment thereof specifically binds to RBD, S2 or S protein of SARS-CoV-2, or S protein of SARS.
In a specific embodiment of the invention, the amino acid sequence of the S protein of SARS-CoV-2 is shown in SEQ ID NO 16.
In a specific embodiment of the invention, the amino acid sequence of the S2 protein of SARS-CoV-2 is shown in SEQ ID NO. 15.
In a specific embodiment of the invention, the amino acid sequence of the RBD protein of SARS-CoV-2 is shown in SEQ ID NO: 17.
In one embodiment of the present invention, the amino acid sequence of SARS protein S is shown in SEQ ID NO 18.
In a second aspect of the invention, there is provided a nucleic acid encoding an anti-coronavirus antibody or antigen-binding fragment thereof as described above.
In a third aspect of the invention, there is provided a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:
(1) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region comprising VHCDRs 1, 2 and 3 of SEQ ID NOs 1, 2 and 3, and wherein the heavy chain variable region binds to a coronavirus S protein when paired with a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 14; and/or the presence of a gas in the gas,
(2) an immunoglobulin light chain or fragment thereof comprising a light chain variable region comprising the VLCDR1, 2, and 3 of SEQ ID NOs 4, 5, and 6, and wherein the light chain variable region binds to coronavirus S protein when paired with a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 13.
In a fourth aspect of the invention, there is provided a vector comprising the nucleic acid described above.
Preferably, the vector is capable of expression in vivo or in vitro or ex vivo. Further preferably, the expression vector is expressed at a high level in vivo in cells. Preferably, the expression vector is a prokaryotic expression vector or a lentiviral expression vector. Further preferably, the prokaryotic expression vector is an escherichia coli series.
In a fifth aspect of the invention, there is provided a cell comprising a nucleic acid as described above or a vector as described above.
Preferably, the cell may be eukaryotic or prokaryotic. More preferably, the cell may be a yeast cell, 293 cell, CHO cell, escherichia coli, or the like.
In a sixth aspect of the invention, there is provided a hybridoma cell producing the anti-coronavirus antibody or antigen-binding fragment thereof described above.
In a seventh aspect of the present invention, there is provided a method for producing a hybridoma, said method comprising immunizing a non-human animal with a coronavirus S protein or a coding sequence thereof to obtain a hybridoma, collecting splenocytes from the immunized non-human animal, and fusing the collected splenocytes with SP2/0 cells to obtain the hybridoma.
In an eighth aspect of the present invention, there is provided a method for producing an anti-coronavirus antibody or an antigen-binding fragment thereof, wherein a cell containing the above-mentioned nucleic acid or vector is cultured to obtain the anti-coronavirus antibody or the antigen-binding fragment thereof.
In a ninth aspect of the invention, there is provided a method of producing an anti-coronavirus antibody or antigen-binding fragment thereof, said method comprising a protein immunization method or a DNA immunization method.
The non-human animal of the invention is a non-human mammal. More preferably, the animal is a rodent.
In one embodiment of the invention, the non-human animal is a rat or a mouse.
In an eleventh aspect of the present invention, there is provided a test kit comprising the above anti-coronavirus antibody or antigen-binding fragment thereof, the above nucleic acid, the above vector, the above cell, the above hybridoma cell.
Preferably, the detection kit further comprises a detection reagent. Specifically, the detection reagent includes, but is not limited to, a detection chip, a reagent required for enzyme-linked immunosorbent assay (ELISA), a reagent required for Immunofluorescence (IFA) detection, a reagent required for immunoblot (WB) detection, a reagent required for protein microarray detection, a reagent required for Immunochromatography (ICA) detection, and the like.
In a twelfth aspect of the present invention, there is provided the use of the above-mentioned anti-coronavirus antibody or antigen-binding fragment thereof, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cell or the above-mentioned hybridoma cell for detecting coronavirus S protein.
Preferably, the S protein may be S, S1, S2 or RBD protein. Further preferably, the RBD, S2 or S protein of SARS-CoV-2 or the S protein of SARS is used.
Preferably, the coronavirus is SARS or SARS-CoV-2 with the numbers Q3LZX1, Q3I5J5, Q0Q475 and A0A0K1Z 074.
In a thirteenth aspect of the invention, there is provided the use of the above-mentioned anti-coronavirus antibody or antigen-binding fragment thereof, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cell or the above-mentioned hybridoma cell for the preparation of a product for the diagnosis of a disease associated with coronavirus infection.
Preferably, the related diseases caused by coronavirus infection are selected from the group consisting of cold, middle east respiratory syndrome, severe acute respiratory syndrome, tumor, pneumonia, renal failure, and the like.
In a fourteenth aspect of the present invention, there is provided a method for detecting coronavirus or coronavirus S protein, said method comprising contacting a sample to be detected with an anti-coronavirus antibody or an antigen-binding fragment thereof according to the present invention, and detecting a complex formed between coronavirus S protein and the anti-coronavirus antibody or the antigen-binding fragment thereof.
Preferably, the detection method is to detect the presence or amount of coronavirus or coronavirus S protein. Wherein the presence or absence is indicated, and the content may be an expression amount, a protein concentration, or the like.
In a fifteenth aspect of the present invention, there is provided a method for diagnosing a disease associated with coronavirus infection, said method comprising sampling, contacting the sample with an anti-coronavirus antibody or antigen-binding fragment thereof according to the present invention, and detecting a complex formed between coronavirus S protein and the anti-coronavirus antibody or antigen-binding fragment thereof.
An "antigen-binding fragment" as described herein is a portion of an antibody that retains the specific binding activity of an intact antibody, i.e., any portion of the antibody is capable of specifically binding to an epitope on a target molecule of an intact antibody. It includes, for example, Fab ', F (ab')2 and variants of these fragments. For example, a heavy and/or light chain CDR of a whole antibody, a heavy and/or light chain variable region of a whole antibody, a full length heavy or light chain of a whole antibody, or a single CDR from a heavy or light chain of a whole antibody.
"diagnosis" as used herein refers to the determination of whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or the determination of the progression or likely progression of a disease in the future, or the assessment of a patient's response to a therapy.
The term "and/or" as used herein includes a list of items in the alternative as well as any number of combinations of items.
The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that specify the presence of the stated elements or steps, as well as any other elements or steps that do not materially affect the technical effects specified. When used herein to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein.
Kabat numbering is used by default in this disclosure unless specifically noted otherwise in this disclosure.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: and (3) flow result graph of the binding of each selected antibody with S protein and RBD protein. The flow results for two antibodies are provided exemplarily in the figure, wherein CT150, CT183 are the numbers of the two antibodies, NC is the negative control, and PC is the positive control.
FIG. 2: flow results of binding of each antibody selected to the S2 protein. The flow results for two antibodies are provided exemplarily in the figure, wherein CT150, CT183 are the numbers of the two antibodies, and NC is a negative control.
FIG. 3: the absorbance of the binding of antibody CT150 to S2 protein changed, specifically, the OD450 value change curve with increasing CT150 concentration.
FIG. 4: the absorbance of binding of antibody CT150 to SARS S protein changes, specifically, the OD450 value change curve with increasing CT150 concentration.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: generation of anti-SARS-CoV-2 antibodies
To generate antibodies against SARS-CoV-2-S2, 6 human 6-8 weeks old RenMab was immunized with DNA encoding the SARS-CoV-2S protein (SEQ ID NO:16)TMMouse (Beijing Baiosai Tou Geneva Biotechnology Co., Ltd.). 3 times after immunization, the mice were boosted with CHO-S- (SARS-CoV-2-S) (i.e., SARS-CoV-2-S protein was expressed by transfer into CHO cells), 4 days later, and the mouse spleen cells were collected and enriched in IgG type B cells by magnetic bead negative selection using Miltenyi Pan B kit (from Miltenyi Biotec, Cat. No. 130104443) and IgM kit (from Miltenyi Biotec, Cat. No. 130047301)7Flow sorting of antigen-specific IgG type B cells 5600, then 1310 pairs of antibody sequences are finally obtained by using a high-throughput sequencing technology.
After data generation, incomplete sequences are removed, the frequency of sequence occurrence is further analyzed, sequences with the frequency of occurrence larger than 1 are selected for antibody in-vitro synthesis, and finally 110 antibodies are obtained.
1. The binding of these antibodies to the S protein, RBD protein and S2 protein of SARS-CoV-2 was detected by flow assay, and the affinity of positive antibodies was detected by Biacore. Specifically, SARS-CoV-2-S2 protein (SEQ ID NO:
15) SARS-CoV-2-RBD protein (SEQ ID NO: 17) and SARS-CoV-2-S protein (SEQ ID NO:16) the coding sequence is transferred into CHO cells or CHO-GFP cells for protein expression, and is used for detecting the binding reaction of the antibody and the protein. The flow detection results show that the binding capacity of the 110 antibodies to different proteins is different. Specifically, there were 84 antibodies binding to SARS-CoV-2-S protein, 22 antibodies binding to SARS-CoV-2-RBD protein, and 59 antibodies binding to SARS-CoV-2-S2 protein. Exemplary streaming assay results are shown in fig. 1 and 2.
The specific experimental operations were as follows: taking 96-well cell culture plate, adding the CHO cells (25. mu.l, 2X 10) expressing different SARS-CoV-2-S protein, SARS-CoV-2-S2 protein or SARS-CoV-2-RBD protein into each well4Cells/well), 50ul of the antibody to be detected was added thereto, respectively, and the mixture was allowed to stand at 4 ℃ for 30 minutes. The 96-well cell culture plates were centrifuged at 1200rpm for 5 minutes and washed twice with PBS. Subsequently, 50 μ l of 1: 500 diluted anti-human Fc antibody Alexa
Figure BDA0002763594510000071
647anti-human IgG Fc Antibody (purchased from Biolegend, cat # 410713), and left to stand at 4 ℃ for 30 minutes. Subsequently, the 96-well cell culture plate was centrifuged at 1200rpm for 5 minutes, washed once with PBS, and then 200 μ l PBS was added per well and subjected to flow cytometry.
2. The method of capturing antibodies using the Protein A sensor chip of Biacore T200(Biacore) detects the binding affinity of these antibodies to SARS-CoV-2-S1 ECD-his (from Acro, cat # S1N-C52H4), SARS-CoV-2-S2 ECD-his (from Acro, cat # S2N-C52H5) and SARS S (R667A) -his (from Acro, cat # SPN-S52H 5).
The specific experimental operation is as follows: each antibody was injected separately into the sensor chip (10. mu.l/min, 50s) and the protein was captured as 45-65 RU. Then, the 2019-nCoV-S1 ECD-his, 2019-nCoV-S2 ECD-his or SARS S (R667A) -his protein is injected into the sensor chip (30 μ l/min, 100-. The chip was regenerated with glycine (30. mu.l/min, 10-20s) at pH2.0 and the assay results were read. Kinetic association rates (kon) and Kinetic dissociation rates (koff) were measured by the Bioacore T200 evaluation software for 1: the 1 langmuir binding model was fit to ((Karlsson, r.roos, h.fagerstam, l.petersson, b.,1994.Methods Enzymology 6.99-110). affinity rate constant KD ═ koff/kon.
The affinity detection result shows that 59 positive antibodies are combined with SARS-CoV-2-S1 protein and SARS S protein, and the affinity is not obviously different, and 21 antibodies are combined with SARS-CoV-2-S2 protein (see tables 1-3).
TABLE 1 affinity results for the S1 protein (SARS-CoV-2-S1 ECD-his)
Figure BDA0002763594510000081
Figure BDA0002763594510000091
TABLE 2 affinity results for the S2 protein (SARS-CoV-2-S2 ECD-his)
Figure BDA0002763594510000092
Figure BDA0002763594510000101
Figure BDA0002763594510000111
Figure BDA0002763594510000121
TABLE 3 results of affinity for SARS S protein (SARS S (R667A) -his)
Figure BDA0002763594510000122
Figure BDA0002763594510000131
Figure BDA0002763594510000141
Figure BDA0002763594510000151
And combining the flow detection result and the affinity detection result, and selecting the antibody CT150 with high affinity of S2 for further detection. The heavy chain CDR1, CDR2 and CDR3 of CT150 and the light chain CDR1, CDR2 and CDR3 amino acid sequences are shown in SEQ ID NOS: 1-6(Kabat numbering see Table 4) or SEQ ID NOS: 7-12(Chothia numbering see Table 5). The amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the CT150 antibody are shown in SEQ ID NO: 13-14.
Table 4: kabat CDR
Figure BDA0002763594510000152
Table 5: chothia CDR
Figure BDA0002763594510000153
The antibody was detected for EC50 using an ELISA method. The results showed that the antibody CT150 had an EC50 of 8.176ng/ml for SARS-CoV-2-S2 protein (see FIG. 3) and an EC50 of 129.3ng/ml for SARS S protein (see FIG. 4). Comparing the SARS-CoV-2-S2 protein to the SARS S protein sequence to find a 90% protein identity, further searching for the SARS-CoV-2-S2 protein sequence using the BLAST tool on Uniport, found that the sequence identity of various coronaviruses (e.g., the coronaviruses numbered Q3LZX1, Q3I5J5, Q0Q475, A0A0K1Z 074) is over 85%, and it is expected that antibody CT150 can bind to these coronaviruses.
The above data demonstrate that CT150 antibody can bind with high affinity to S2 protein of SARS-CoV-2 and can cross-recognize S protein (SEQ ID NO: 18) or MERS B chain of SARS virus (7C02_ B is shown as SEQ ID NO: 19). Because the sequence of coronavirus S2 is relatively conserved, the CT150 antibody can be used for developing a detection reagent for rapidly detecting various coronaviruses including SARS-CoV-2 virus and SARS virus.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are all within the protection scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Sequence listing
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<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gln Gln Leu Asn Ser Tyr Pro Ile Phe Thr
1 5 10
<210> 7
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Phe Thr Phe Ser Ser Tyr Ala Met His
1 5 10
<210> 8
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Ser Tyr Asp Gly Asp Asn
1 5
<210> 9
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Asp Arg Gly Ser Tyr Phe Tyr Gly Val Asp Val
1 5 10
<210> 10
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ala
1 5 10
<210> 11
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Ala Ala Ser Thr Leu Gln Ser
1 5
<210> 12
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gln Gln Leu Asn Ser Tyr Pro Ile Phe Thr
1 5 10
<210> 13
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gln Glu Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val
35 40 45
Ala Leu Ile Ser Tyr Asp Gly Asp Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Thr Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Arg Gly Ser Tyr Phe Tyr Gly Val Asp Val Trp Gly Gln
100 105 110
Gly Ala Thr Val Thr Val Ser Ser
115 120
<210> 14
<211> 108
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Ile
85 90 95
Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
<210> 15
<211> 588
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala
1 5 10 15
Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn
20 25 30
Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys
35 40 45
Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys
50 55 60
Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg
65 70 75 80
Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val
85 90 95
Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe
100 105 110
Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser
115 120 125
Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala
130 135 140
Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala
145 150 155 160
Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
165 170 175
Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu
180 185 190
Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala
195 200 205
Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
210 215 220
Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn
225 230 235 240
Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr
245 250 255
Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln
260 265 270
Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile
275 280 285
Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala
290 295 300
Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
305 310 315 320
Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser
325 330 335
Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser
340 345 350
Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
355 360 365
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
370 375 380
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly
385 390 395 400
Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His
405 410 415
Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr
420 425 430
Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val
435 440 445
Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys
450 455 460
Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp
465 470 475 480
Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys
485 490 495
Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
500 505 510
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro
515 520 525
Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met
530 535 540
Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
545 550 555 560
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser
565 570 575
Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
580 585
<210> 16
<211> 1261
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln
1010 1015 1020
Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe
1025 1030 1035 1040
Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1045 1050 1055
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1060 1065 1070
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr
1075 1080 1085
His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr
1090 1095 1100
Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile
1105 1110 1115 1120
Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe
1125 1130 1135
Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val
1140 1145 1150
Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln
1155 1160 1165
Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser
1170 1175 1180
Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp
1185 1190 1195 1200
Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val
1205 1210 1215
Met Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu
1220 1225 1230
Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp
1235 1240 1245
Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
1250 1255 1260
<210> 17
<211> 194
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
1 5 10 15
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
20 25 30
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
35 40 45
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
50 55 60
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
65 70 75 80
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
85 90 95
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
100 105 110
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
115 120 125
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
130 135 140
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
145 150 155 160
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
165 170 175
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
180 185 190
Thr Val
<210> 18
<211> 588
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Ser Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala
1 5 10 15
Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn
20 25 30
Phe Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys
35 40 45
Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys
50 55 60
Ala Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg
65 70 75 80
Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val
85 90 95
Phe Ala Gln Val Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe
100 105 110
Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr
115 120 125
Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala
130 135 140
Asp Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn
145 150 155 160
Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
165 170 175
Pro Pro Leu Leu Thr Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu
180 185 190
Val Ser Gly Thr Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala
195 200 205
Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
210 215 220
Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn
225 230 235 240
Gln Phe Asn Lys Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr
245 250 255
Ser Thr Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln
260 265 270
Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile
275 280 285
Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala
290 295 300
Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
305 310 315 320
Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser
325 330 335
Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser
340 345 350
Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
355 360 365
Gln Ala Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
370 375 380
Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly
385 390 395 400
Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser
405 410 415
Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr
420 425 430
Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile
435 440 445
Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys
450 455 460
Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp
465 470 475 480
Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys
485 490 495
Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
500 505 510
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro
515 520 525
Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met
530 535 540
Val Thr Ile Leu Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
545 550 555 560
Gly Ala Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser
565 570 575
Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
580 585
<210> 19
<211> 246
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Glu Ala Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu
1 5 10 15
Cys Asp Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn
20 25 30
Phe Lys Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu
35 40 45
Leu Ser Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro
50 55 60
Ala Ala Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe
65 70 75 80
Ser Tyr Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly
85 90 95
Pro Ile Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys
100 105 110
Leu Ile Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro
115 120 125
Leu Lys Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp
130 135 140
Arg Thr Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys
145 150 155 160
Val Ser Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg
165 170 175
Lys Gln Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly
180 185 190
Ser Thr Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr
195 200 205
Val Gln Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu Phe
210 215 220
Ala Asn Asp Thr Lys Ile Ala Ser Gln Leu Gly Asn Cys Val Glu Tyr
225 230 235 240
His His His His His His
245

Claims (10)

1. An anti-coronavirus antibody or antigen-binding fragment thereof, wherein said anti-coronavirus antibody or antigen-binding fragment thereof comprises a VHCDR1, VHCDR2 and VHCDR3 of the heavy chain variable region and a VLCDR1, VLCDR2 and VLCDR3 of the light chain variable region, wherein,
the amino acid sequence of VHCDR1 comprises SEQ ID NO:1, the amino acid sequence of VHCDR2 comprises SEQ ID NO: 2, the amino acid sequence of VHCDR3 comprises SEQ ID NO: 3, the amino acid sequence of VLCDR1 comprises SEQ ID NO:4, the amino acid sequence of VLCDR2 comprises SEQ ID NO: 5, the amino acid sequence of VLCDR3 comprises SEQ ID NO: 6 is shown in the specification;
alternatively, the amino acid sequence of VHCDR1 comprises SEQ ID NO:7, the amino acid sequence of VHCDR2 comprises SEQ ID NO: 8, the amino acid sequence of VHCDR3 comprises SEQ ID NO: 9, the amino acid sequence of VLCDR1 comprises SEQ ID NO: 10, the amino acid sequence of VLCDR2 comprises SEQ ID NO: 11, the amino acid sequence of VLCDR3 comprises SEQ ID NO: shown at 12.
2. The anti-coronavirus antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region comprises SEQ ID No. 13 and the amino acid sequence of the light chain variable region comprises SEQ ID No. 14.
3. The anti-coronavirus antibody or antigen-binding fragment thereof of claim 1, wherein the anti-coronavirus antibody or antigen-binding fragment thereof is a single chain antibody, Fv antibody, Fd, dAb, bispecific antibody, bispecific single chain antibody, linear antibody, or multispecific antibody.
4. The anti-coronavirus antibody or antigen-binding fragment thereof of claim 1, wherein said anti-coronavirus antibody or antigen-binding fragment thereof specifically binds to coronavirus S protein.
5. A nucleic acid encoding the anti-coronavirus antibody or antigen-binding fragment thereof of any one of claims 1-4.
6. A vector comprising the nucleic acid of claim 5.
7. A cell comprising the nucleic acid of claim 5 or the vector of claim 6.
8. A method for producing an anti-coronavirus antibody or an antigen-binding fragment thereof, comprising culturing the cell of claim 7 to obtain the anti-coronavirus antibody or the antigen-binding fragment thereof.
9. A test kit comprising the anti-coronavirus antibody or antigen-binding fragment thereof of any one of claims 1-4.
10. Use of the anti-coronavirus antibody or antigen-binding fragment thereof according to any one of claims 1-4 for the detection of coronavirus S protein or for the preparation of a product for the diagnosis of a disease associated with coronavirus infection.
CN202011225733.7A 2020-11-05 2020-11-05 Anti-coronavirus antibody and application thereof Pending CN114437205A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108778328A (en) * 2016-03-16 2018-11-09 加利福尼亚大学董事会 Albumin A combination polypeptide, anti-EPHA 2 antibody and its application method
WO2020043670A1 (en) * 2018-08-27 2020-03-05 Affimed Gmbh Cryopreserved nk cells preloaded with an antibody construct
CN111197058A (en) * 2018-11-20 2020-05-26 北京百奥赛图基因生物技术有限公司 Preparation method and application of humanized CD73 gene animal model
US20200231661A1 (en) * 2017-02-17 2020-07-23 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108778328A (en) * 2016-03-16 2018-11-09 加利福尼亚大学董事会 Albumin A combination polypeptide, anti-EPHA 2 antibody and its application method
US20200231661A1 (en) * 2017-02-17 2020-07-23 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
WO2020043670A1 (en) * 2018-08-27 2020-03-05 Affimed Gmbh Cryopreserved nk cells preloaded with an antibody construct
CN111197058A (en) * 2018-11-20 2020-05-26 北京百奥赛图基因生物技术有限公司 Preparation method and application of humanized CD73 gene animal model

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI LIU ET AL.,: "Anti–spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection", JCI.INSIGHT., vol. 4, no. 4, 21 February 2019 (2019-02-21), pages 1 - 19 *
高原等: "2019新型冠状病毒的抗原抗体检测", 计量学报, vol. 41, no. 5, 31 May 2020 (2020-05-31), pages 513 - 517 *

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