CN113527472A - Specific antibody of SARS-CoV-2S protein or its fragment and its application - Google Patents

Abstract

The invention separates coronavirus spike protein RBD specific memory B cell from recovered person PBMC after new coronavirus infection, amplifies to obtain antibody light and heavy chain variable region sequence, connects and introduces carrier, recombines and expresses to obtain recombinant humanized monoclonal antibody, selects monoclonal antibody with high affinity with SARS-CoV-2 spike protein RBD, strong blocking power to SARS-CoV-2 spike protein RBD and host receptor ACEII, and strong neutralizing activity to virus infected cell, and then screens ADE effect of the monoclonal antibody, finally obtains anti-SARS-CoV-2S protein specific antibody MW07 without ADE phenomenon.

Description

Specific antibody of SARS-CoV-2S protein or its fragment and its application

Technical Field

The invention belongs to the field of antibody engineering, in particular relates to a monoclonal antibody aiming at coronavirus and application thereof, and particularly relates to a specific antibody of SARS-CoV-2S protein or a fragment thereof, a preparation method and application thereof.

Background

Although the plasma therapy of the rehabilitation patients achieves certain clinical effect, the plasma source of the antigen patients is limited, the potential safety hazard of purified antibodies is high, and the titer of specific antibodies is unstable. The monoclonal antibody with high titer, stable performance and good safety has good application prospect for controlling the epidemic situation of the new coronavirus. The prior literature currently discloses or teaches reports of protective neutralizing mabs against novel coronavirus RBDs. Protective Neutralizing antibodies against the new coronavirus are generated using the new coronavirus Spike protein RBD (e.g., bioRxiv, "SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding company and Potential improvements on neural antibodies and Vaccine Development", 20200220). The SARS spike protein RBD and the new coronavirus spike protein RBD are present as cross-neutralizing epitope peptides, and the anti-SARS monoclonal antibody CR3022 is capable of binding to the new coronavirus spike protein RBD (Emerging Microbes & Infections, 9 (1): 382. sup. 385, 20200217). A homologous modeling method is adopted to define hot spots and key residues of a protein-protein interaction interface of a new coronavirus CTD 1/human ACE2 compound, screen candidate inhibitors targeting the combination surface of a CTD1 region and ACE2, and block recognition and combination of the virus and human ACE2 protein.

With the continued advance of anti-SARS-CoV-2 vaccines and therapeutic antibody research, there have been recent reports that show that ADE phenomenon can occur when a portion of therapeutic antibodies are used in SARS-CoV-2 treatment. The term ADE refers to an antibody-dependent enhancement, and some viruses have a significantly enhanced ability to replicate or infect with the aid of specific antibodies, which can cause more serious pathological damage during infection. The popular explanation is that the antibody cannot neutralize the virus, but acts as a "trojan horse" to make the virus more powerful in infecting immune cells, producing more progeny virus, causing severe symptoms.

So far, the monoclonal antibody for blocking the binding of the new coronavirus to host ACE2 still stays at the theoretical research stage, and no prior art report of the monoclonal antibody with clear sequence structure and definite binding function for blocking the new coronavirus exists. In addition, the research on anti-SARS-CoV-2S protein antibody in the prior art finds that there is an antibody-dependent enhancing effect (ADE) in the process of SARS-CoV-2 infecting host cells, which brings difficulty to the research of SARS-CoV-2 vaccine and therapeutic antibody.

Disclosure of Invention

In order to solve the problems, the invention separates the coronary virus spike protein RBD specific memory B cell from the recovered person PBMC after the infection of the new coronary virus, amplifies to obtain the light and heavy chain variable region sequence of the antibody, connects with a guide vector, recombines and expresses to obtain the recombinant humanized monoclonal antibody, selects the monoclonal antibody which has high affinity with SARS-CoV-2 spike protein RBD, strong blocking power to SARS-CoV-2 spike protein RBD and host receptor ACEII and strong neutralizing activity to virus infected cells from the recombinant humanized monoclonal antibody, and then screens the ADE effect of the monoclonal antibody to finally obtain the anti-SARS-CoV-2S protein specific antibody MW07 which does not generate ADE phenomenon. Specifically speaking:

in a first aspect, the present invention provides an antibody or fragment thereof specific for SARS-CoV-2S protein, characterized in that the epitope to which the antibody or fragment thereof specifically binds is an epitope present on SARS-CoV-2S 1 protein and is not present on SARS-CoV S1 protein, MERS-CoV S1 protein.

Further, the SARS-CoV-2S protein specific antibody or its fragment of the present invention is characterized in that the epitope specifically bound by the antibody or its fragment is located in the receptor binding domain RBD of SARS-CoV-2.

Further, the SARS-CoV-2S protein-specific antibody or fragment thereof of the present invention is characterized in that the antibody or fragment thereof inhibits infection of a host cell by SARS-CoV-2 in a dose-dependent manner.

Further, the antibody or fragment thereof specific for SARS-CoV-2S protein of the invention is characterized in that the antibody or fragment thereof does not cause significant antibody-dependent potentiation effect (ADE) when used for treating SARS-CoV-2 infection.

Further, the antibody or fragment thereof specific for SARS-CoV-2S protein according to the invention is characterized in that the antibody or fragment thereof is capable of exerting a neutralizing effect by blocking the binding of SARS-CoV-2 to the host cell receptor ACEII, wherein the IC50 blocking the binding of SARS-CoV-2 to the host cell receptor ACEII is less than 500ng/mL, such as less than 200ng/mL, preferably less than 100ng/mL, more preferably less than 80 ng/mL.

Furthermore, the SARS-CoV-2S protein specific antibody or its fragment of the invention is characterized in that the heavy chain variable region CDR1-3 of the antibody or its fragment is respectively identical to the heavy chain variable region CDR1-3 of SEQ ID NO: 10 has 100% sequence homology to CDR1-3 of the heavy chain variable region shown in seq id no; the light chain variable region CDR1-3 is identical to SEQ ID NO: the CDR1-3 of the light chain variable region shown in FIG. 9 has 100% sequence homology.

Furthermore, the SARS-CoV-2S protein specific antibody or its fragment of the invention is characterized in that the amino acid sequences of the heavy chain variable region CDR1-3 of the antibody or its fragment are respectively SEQ ID NO: 14-16, the amino acid sequences of the light chain variable region CDR1-3 are respectively SEQ ID NO: 11-13.

Further, the antibody or fragment specific to SARS-CoV-2S protein of the present invention is characterized in that the heavy chain variable region of the antibody or fragment thereof is identical to the heavy chain variable region of SEQ ID NO: 10 has a sequence homology of 70% or more; the light chain variable region is identical to SEQ ID NO: 9 has a sequence homology of 70% or more.

In a second aspect, the present invention provides an antibody or fragment thereof specific for SARS-CoV-2S protein, characterized in that: the monoclonal antibody or fragment thereof has a competitive relationship with the antibody or fragment thereof of the first aspect of the invention as detected by a Fortebio epitope competitive binding assay.

Further, the antibody or a fragment thereof of the present invention includes a murine antibody, a rabbit antibody, a goat antibody, a sheep antibody, a camel antibody, an alpaca antibody, a chimeric antibody, a human antibody, a humanized antibody, Fab, F (ab')2, Fv, scFv, Fd, a heavy chain antibody, a nanobody, and the like.

In a third aspect, the present invention provides a composition comprising one or more antibodies or fragments thereof specific for SARS-CoV-2S protein, said antibodies or fragments thereof being selected from the group consisting of antibodies or fragments thereof specific for SARS-CoV-2S protein as described in the first aspect of the invention.

Furthermore, the composition is characterized by further comprising a pharmaceutically acceptable carrier, and is used as a pharmaceutical composition, preferably the pharmaceutical composition is a water agent, an injection and a powder injection.

In a fourth aspect, the present invention provides the use of an antibody or fragment thereof in the manufacture of a medicament for the treatment of a coronavirus infection, wherein: the antibody or fragment thereof comprises one or more antibodies selected from the group consisting of antibodies specific for the SARS-CoV-2S protein or fragments thereof according to the first aspect of the invention.

Further, the use according to the invention, characterized in that the coronavirus infection comprises a SARS-CoV-2 infection.

In a fifth aspect, the present invention provides a polynucleotide encoding an antibody specific for the SARS-CoV-2S protein according to the first aspect of the invention or a fragment thereof.

In a sixth aspect, the present invention provides a vector comprising a polynucleotide according to the fifth aspect of the invention.

In a seventh aspect, the present invention provides a host cell comprising a polynucleotide according to the fifth aspect of the present invention or a vector according to the sixth aspect of the present invention.

In an eighth aspect, the present invention provides a method for preparing an antibody or fragment thereof specific for SARS-CoV-2S protein, comprising the steps of:

(1) culturing a host cell according to the seventh aspect of the invention under conditions suitable for expression of an antibody or fragment thereof specific for the recombinant SARS-CoV-2S protein;

(2) separating and purifying SARS-CoV-2S protein specific antibody or its fragment from cell culture. .

For a better understanding of the present invention, certain terms are first defined. Other definitions are set forth throughout the detailed description.

The term "Coronavirus" refers to a member of the order Filovirales (Nidovirales), the family Coronaviridae (Coronaviridae), the genus Coronaviridae (Coronaviridus). The coronavirus of the invention mainly relates to human-infecting coronavirus, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2(2019-nCov), and the coronavirus of the invention particularly relates to SARS-CoV, MERS-CoV and SARS-CoV-2 (2019-nCov).

The term "specificity" refers to the determination of the presence or absence of a protein in a heterogeneous population of proteins and/or other organisms, e.g., the binding reaction of a monoclonal antibody of the invention to a SARS-CoV-2 RBD protein. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can also be divided into hypervariable regions, termed Complementarity Determining Regions (CDRs), which are separated by more conserved Framework (FR) regions. Each VH and VL is composed of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "antigen-binding fragment" of an antibody (or simply antibody portion), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH 1; (ii) a F (ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bridge; (iii) an Fd fragment consisting of VH and CH 1; (iv) an Fv fragment consisting of VL and VH antibody single arms; (v) dAb fragments consisting of VH (Ward et al, (1989) Nature 341: 544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined by recombinant methods via a synthetic linker that makes the two single protein chains, in which the VL and VH regions pair to form monovalent molecules (known as single chain Fc (scFv); see, e.g., Bird et al., (1988) Science 242: 423-. These single chain antibodies are also intended to be included within the term meaning. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

Antigen-binding fragments of the invention include those capable of specifically binding coronavirus RBD. Examples of antibody binding fragments include, for example, but are not limited to, Fab ', F (ab')₂Fv fragments, single chain Fv (scFv) fragments and single domain fragments.

The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab 'fragments are generated by cleavage of the disulfide bond at the hinge cysteine of the F (ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F (ab')2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than intact antibodies (see, e.g., Wahl et al, 1983, J.Nucl. Med.24: 316).

As is generally understood in the art, an "Fc" region is a fragment crystallizable constant region of an antibody that does not comprise an antigen-specific binding region. In IgG, IgA and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of an antibody (CH2 and CH3 domains, respectively). The IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

The "Fv" fragment is the smallest fragment of an antibody that contains the entire target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain (VH-VL dimer) in tight non-covalent association. In this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer target binding specificity on an antibody. However, in some cases, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) may have the ability to recognize and bind to a target, although at a lower affinity than the entire binding site.

A "single chain Fv" or "scFv" antibody binding fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a structure that facilitates target binding.

A "single domain fragment" consists of a single VH or VL domain that exhibits sufficient affinity for a coronavirus RBD. In a particular embodiment, the single domain fragments are camelized (see, e.g., Riechmann, 1999, Journal of immunological Methods 231: 25-38).

The anti-coronavirus RBD antibodies of the invention include derivatized antibodies. For example, derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins. Any of a number of chemical modifications can be made by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more unnatural amino acid, e.g., using ambrx technology (see, e.g., Wolfson, 2006, chem. biol. 13 (10): 1011-2).

"human antibodies" include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or an animal that is transgenic for one or more human immunoglobulins and does not express endogenous immunoglobulins. Human antibodies can be made by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. nos. 4,444,887 and 4,716,111; and PCT publication WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741. Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins, but can express human immunoglobulin genes. See, for example, PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. patent nos. 5,413,923; 5,625,126, respectively; 5,633,425, respectively; 5,569,825; 5,661,016, respectively; 5,545,806; 5,814, 318; 5,885,793, respectively; 5,916,771, respectively; and 5,939,598. Alternatively, using techniques similar to those described above, companies such as LakePharma, inc. (Belmont, CA) or Creative BioLabs (Shirley, NY) may be engaged in providing human antibodies to selected antigens. Fully human antibodies that recognize selected epitopes can be generated using a technique known as "guided selection". In this method, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope (see Jespers et al, 1988, Biotechnology 12: 899-903).

The terms "antibody recognizing an antigen" and "antibody specific for an antigen" are used herein interchangeably with the term "antibody specifically binding to an antigen".

The term "high affinity" for an IgG antibody means a KD for the antigen of 1.0x 10^-6M or less, preferably 5.0x 10^-8M or less, more preferably 1.0x 10^-8M below, 5.0x 10^-9M or less, more preferably 1.0x 10^-9M is less than or equal to M. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of an IgM subtype means a KD of 10^-6M is less, preferably 10^-7M is less, more preferably 10^-8M is less than or equal to M.

The term "Kassoc" or "Ka" refers to the association rate of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, derived from the KD to Ka ratio (KD/Ka), and expressed in molar concentration (M). The KD value of an antibody can be determined by methods known in the art. A preferred way of determining the KD of an antibody is by measurement using a Surface Plasmon Resonance (SPR), preferably a biosensing system such as the Biacore (TM) system.

The term "EC 50," also called half maximal effect concentration, refers to the concentration of antibody that causes 50% of the maximal effect.

Compared with the prior art, the technical scheme of the invention has the following advantages:

first, the SARS-CoV-2S protein specific antibody or its fragment provided by the invention has definite amino acid sequence structure, high affinity to coronavirus RBD, and definite effect of inhibiting or blocking coronavirus RBD from combining with host cell, and has lower heterogeneity and higher clinical application potential when used as a medicament.

Second, the SARS-CoV-2S protein specific antibody or its fragment provided by the invention can specifically bind to SARS-CoV-2, but not to SARS-CoV and MERS-CoV-2. In particular, it does not produce detectable antibody-dependent enhancement effect (ADE) when used for treating SARS-CoV-2 infection, and has safety superior to other anti-SARS-CoV-2S protein specific antibodies.

Thirdly, the SARS-CoV-2S protein specific antibody or its fragment provided by the invention is separated from a large amount of candidate human antibodies, and can compete with host cell ACEII to bind SARS-CoV-2, thereby neutralizing the infection of SARS-CoV-2 to host cells. According to the molecular mechanism, the blocking experiment and the neutralizing experiment, the SARS-CoV-2 neutralizing monoclonal antibody or the fragment thereof provided by the invention can be expected to play a role in treating COVID-19 by reducing the virus load of SARS-CoV-2.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1: the fusion expression of SARS-CoV-2S 1 protein RBD and His.

FIG. 2: and (3) performing fusion expression of SARS-CoV-2S 1 protein RBD and mFc.

FIG. 3: the fusion expression of SARS-CoV-2S 1 protein and mFc.

FIG. 4: fusion expression of human ACE2 with human Fc.

FIG. 5: MW07 affinity assay with recombinant protein SARS-CoV2-S1 RBD-his.

FIG. 6: analysis of the cross-reactivity of MW07 with coronavirus S protein.

FIG. 7: graph of the blocking effect of MW07 on S1RBD-mFc binding to ACEII-hFc.

FIG. 8: MW07 blockade of S1RBD-His binding to ACEII-His.

FIG. 9: MW07 neutralizing activity in vitro against pseudovirus infected Huh7 cells.

FIG. 10: observation of ADE effect of wtlgG antibody mediated pseudovirus infection of Raji cells.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Example 1: recombinant expression of SARS-CoV-2 antigen and host receptor

Cloning a fully synthesized gene S1RBD (Access: QHD43416.1, 319-541aa) into a eukaryotic transient expression vector with a His label or a mFc label at the C end by an enzyme digestion method, transferring the obtained expression plasmid into escherichia coli for amplification, separating to obtain an S1RBD-His expression plasmid and an S1RBD-mFc expression plasmid, and transferring the plasmids into HEK293 cells for recombinant expression according to the operational instruction of a transfection reagent 293 fectamine (Cat: 12347019, Gibco). And 5-6 days after cell transfection, taking culture supernatant, and purifying the expression supernatant by using a ProA affinity chromatography column through S1RBD-mFc to obtain S1RBD-mFc protein. And purifying the expression supernatant by using a HisTrap HP column affinity chromatography column to obtain the S1RBD-his protein. And the purity of the obtained recombinant protein was checked by SDS-PAGE (FIGS. 1-2). The coding nucleic acid sequence of S1RBD-his is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2 is shown in the specification; the coding nucleic acid sequence of the S1RBD-mFc is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4, respectively.

Cloning S1 gene (Accession: QHD43416.1, 1-685aa) from purchased full-length SARS-CoV-2 expression vector (Cat: VG40589-UT, Beijing Yiqian Shenzhou) by a PCR method, respectively cloning the gene into eukaryotic transient expression vector with mFc label at C end by an enzyme cutting method, transferring the obtained expression plasmid into escherichia coli for amplification, separating to obtain S1-mFc expression plasmid, and transferring the plasmid into HEK293 cells for recombinant expression according to the operation instruction of transfection reagent 293fectin (Cat: 12347019, Gibco). 5-6 days after cell transfection, culture supernatant is taken, and S1-mFc is purified by utilizing a ProA affinity chromatography column to obtain S1-mFc protein. And the purity of the obtained recombinant protein was checked by SDS-PAGE (FIG. 3). The coding nucleic acid sequence of S1-mFc is shown in SEQ ID NO: 5, and the amino acid sequence is shown as SEQ ID NO: and 6.

ACEII extracellular region gene ACEII (1-615) (Accession: NP-068576.1, 1-615aa) is cloned from a purchased full-length human ACEII expression vector (Cat: HG10108-ACR, Beijing Yiqiaoshengzhou) by a PCR method, and is respectively cloned into a eukaryotic transient expression vector with a hFc label at the C end by an enzyme digestion method, the obtained expression plasmid is transferred into escherichia coli for amplification, separated to obtain the ACEII (1-615) -hFc expression plasmid, and the plasmid is transferred into HEK293 cells for recombinant expression according to the operation instruction of a transfection reagent 293fectin (Cat: 12347019, Gibco). 5-6 days after cell transfection, culture supernatant was taken, and expression supernatant was purified by using a ProA affinity column to obtain ACEII (1-615) -hFc protein, and the purity of the obtained recombinant protein was checked by SDS-PAGE (FIG. 4). The coding nucleic acid sequence of ACEII (1-615) -hFc is shown as SEQ ID NO: 7, and the amino acid sequence is shown as SEQ ID NO: shown in fig. 8.

Example 2: b cell screening to obtain specific antibody for resisting new coronavirus S1RBD

And (3) sorting the specific memory B cells of the novel coronavirus infection rehabilitation patients by using FITC-S1RBD-hFc as an antigen. Single cell PCR technology (methods and primers reference New Biotechnology, 2010 Volume 27, Number 2, P110-117 and Human Monoclonal Antibodies on pages 114 to 117) amplify the antibody light and heavy chain genes and perform sequencing analysis. And (3) synthesizing the light and heavy chain variable region gene of the antibody with correct sequencing, cloning the gene into a transient expression vector pKN019 containing a light chain constant region and a transient expression vector pKN041 containing a heavy chain constant region IgG1 gene, and performing transient expression and purification by using an HEK293 system to obtain a recombinant antibody MW 07.

The method for single cell PCR amplification of antibody light and heavy chain gene is as follows:

first, RNA magnetic beads (nunzozan) are used to extract RNA from single B cells, which is reverse-transcribed into cDNA.

1. Mu.l of Catch Buffer B (TCL + 1% β -ME) was dispensed into each well and individual memory B cells were sorted.

2. Pasting the membrane, and centrifuging at 2000rpm for 1 min.

3. Add 10. mu. l H2O and 33. mu.l Beads to each well, mix them by blowing and sucking, and react at room temperature for 10 min.

4. The magnetic frame was placed at room temperature for 5min, and the supernatant was discarded.

5. The beads were rinsed with 200. mu.l of freshly prepared 80% ethanol in nuclease-free water at room temperature for 30s, and the supernatant was discarded.

6. Rinsing is repeated once, the supernatant is discarded, and the mixture is air-dried for 3 min.

7. Moving down the magnetic frame, adding 12 μ l of Mix 1 into each hole, blowing and sucking for 5 times, and acting at room temperature for 5 min.

8. Place on magnetic stand, room temperature for 2min, transfer 10. mu.l to new plate, centrifuge at 300g for 30s, run procedure 1.

9. Add 10. mu.l Mix 2 to each well, Mix, centrifuge, run procedure 2.

10. The synthesized cDNA was subjected to PCR as soon as possible.

Mix 1：310μl H2O+50μl dNTP+20μl Random 6+20μl Oligo_dT

Mix 2：170μl H2O+160μl Buffer+40μl DTT+20μl RNase I+10μl RTase IV (Cat：EN0601 and 18090010，ThermoFisher)

Procedure 1: 5min at 65 ℃ → 4 ℃infinity

Procedure 2: 10min at 23 ℃ → 30min at 50 ℃ → 10min at 80 ℃ → infinity at 4 ℃ ∞

However, two-step PCR was used to amplify the antibody heavy and light chain (Kappa) variable region genes. The primer sequences were derived from Human Monoclonal Antibodies at pages 114 to 117.

First round PCR (Ig-VH1, Ig-VK1), reaction (20. mu.l):

and (3) running a program:

94℃ 5min→(94℃ 30s→51℃ 30s→72℃ 55s)×15Cycles

→(94℃ 30s→56℃ 30s→72℃ 55s)×30Cycles

→72℃ 8min

→4℃ ∞

second round PCR (Ig-VH2, Ig-VK2), reaction (20. mu.l):

and (3) running a program:

94℃ 5min→(94℃ 30s→57℃ 30s→72℃ 45s)×50Cycles

→72℃ 10min

→4℃ ∞

and separating and purifying the PCR product by agarose gel electrophoresis and sequencing the variable regions of the light chain and the heavy chain of the antibody.

The amino acid sequence of the variable domain of MW07 is shown below.

MW07-VL (SEQ ID NO: 9, LCDR1-3 are SEQ ID NO: 11-13, respectively)

DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPKLLLYAASTLESGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQFYSTPRTFGQGTKVEIK

MW07-VH (SEQ ID NO: 10, HCDR1-3 are SEQ ID NO: 14-16, respectively)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDASEKYYLDSLKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGILWFGDYPWGQGTLVTVSS

Example 3 affinity of MW07 for S1RBD

The binding affinity of the antibody and the S1-RBD protein antigen is measured by using a Fortebio protein interaction system. The method mainly comprises the following steps:

binding and dissociation changes between the test antibody and monovalent S1RBD were captured using Octet QKe system from Fortebio using capture Antibody (AHC) bioprobes against the Fc fragment of the human antibody at different concentrations. 4 different concentrations of MW07(45nM, 30nM, 15nM, 7.5nM) were flowed over the AHC probe (Cat: 18-5060, PALL) for 120s as determined. S1RBD-His diluted at different concentrations was used as the mobile phase. The binding time was 300s and the dissociation time was 300 s. After the experiment, blank control response values are deducted, and 1: 1Langmuir binding pattern fitting is carried out by software, and the kinetic constant of antigen-antibody binding is calculated. The results are shown in FIG. 5. The affinity constants of MW07 and SARS-CoV-2S 1RBD were fitted, with a KD of 0.424nM, which is higher than the affinity of MW01-MW06 (the alternative application).

Example 4 specificity and Cross-reactivity of MW07

SARS-CoV-2S 1RBD-His recombinant protein (expressed by Kono sincerity technologies, Inc., lot: 20200213A), SARS-CoV-2S 1-His recombinant protein (expressed by Kono sincerity technologies, Inc., lot: 20200221H), recombinant protein SARS S1-His: (Beijing Yi Qiao Shenzhou, Cat: 40150-V08B1), recombinant protein MERS-CoV S1-His (Beijing Yi Qiao Shenzhou, Cat: 40069-V08H) and negative control NC-His with the concentration of 1 mug/mL; the cells were coated overnight at 4 ℃ and blocked with 5% BSA in a 37 ℃ incubator for 60 min. The antibody to be tested (initial concentration of 10. mu.g/mL, 3-fold serial dilution, 12 gradients) was added and reacted for 60min in a constant temperature incubator at 37 ℃. PBST plate washing 4 times; then HRP-anti-human Fc (Cat: 109. sup. + 035. sup. + 098. sup. Jackson Immuno Research) diluted 1: 5000 was added for reaction for 45min, TMB (Cat: ME 142. sup.; Beijing Taitianhe Biol.) substrate was added for color development for 15min, and the plate was read after 2M HCl termination. The absorbance values OD450nm-630nm were read and recorded for a well plate at a wavelength of 450nm, using 630nm as the reference wavelength.

The results are shown in FIG. 6. MW07 specifically binds only to the S protein of the novel coronavirus SARS-CoV-2 (S1RBD and S1), but not to SARS S1-His, MERS-CoV S1-His, and the negative control NC-His.

Example 3 blocking Activity of MW07 on S1RBD/ACEII

3.1ELSIA analysis of the blocking Effect of antibodies on the binding of ACEII-hFc to S1RBD-mFc

The human ACEII-hFc (1-615aa) recombinant protein, concentration 0.75. mu.g/mL, 100. mu.l/well, was coated overnight at 4 ℃ and after blocking with 5% BSA in a 37 ℃ incubator for 120min, the plates were washed 4 times with PBST. Respectively taking 100uL of antibodies MW01 and MW07 (the initial concentration is 40 mu g/mL, 1.5 times of serial dilution and 12 gradients) to be detected and S1-RBD-mFc 70ng/mL, uniformly mixing in equal volume, placing for 50min at 37 ℃, and taking two 100 mu l of each sample and adding the two 100 mu l of each sample into an ACE2-hFc coated hole in parallel; after reacting for 60min in a constant temperature incubator at 37 ℃, washing the plate for 4 times by PBST; then adding HRP-anti-mouse IgG (Cat: 115-; TMB (Cat: ME142, Beijing Taitianhe organism) substrate was added for color development for 15min, and the plate was read after 2M HCl stop. The wavelength 450nm was read and recorded.

As a result, as shown in FIG. 7, antibody MW07 was able to effectively block the binding of S1RBD to ACEII, and the blocking activity was higher than that of antibody MW 01.

3.2Fortebio competitive binding assay analysis of blocking Effect of antibody on binding of ACEII-his and S1RBD-his

Capturing antibodies MW01, MW07 and CR3022 (a monoclonal antibody of anti-SARS-Cov RBD disclosed in US2010172917A1, which has cross-binding with new crown S1 RBD) to be analyzed by using an AHC biological probe), balancing PBS, taking 100nM S1RBD-his as fluidity, fully binding 240S, further taking ACEII-his as a second binding phase, fully binding 300S, and transferring into PBS for dissociation.

The results are shown in FIG. 8, and antibody MW07 is effective in blocking the binding of S1RBD to ACEII.

Example 4 neutralizing Activity of MW07 against New coronavirus pseudovirus infected Huh7 cells

The neutralizing activity of Huh7 cells infected by new corona pseudovirus in the hospital is experimentally observed. After neutralization reaction of antibodies to be evaluated and 750TCID 50/well pseudovirus particles (transfected luciferase reporter gene) at 37 ℃, 2x 104/well huh7 cells are respectively inoculated and cultured in a CO2 incubator at 37 ℃ for 20-28 h. And (3) taking out the cell plate after 20-28h, adding 100 mu l of luciferase detection reagent into each hole, reacting for 2min in a dark place, reading by using a fluorescence detector, calculating the neutralization inhibition rate, and calculating IC50 by using a Reed-Muench method according to the result of the neutralization inhibition rate.

The results are shown in FIG. 9, MW07 is capable of dose-dependently inhibiting entry of pseudovirions into host cells, and IC50 values were 62ng/mL, far higher than the neutralizing activity of MW01(IC50, 281ng/mL) as calculated by Reed-Muench method.

Example 5 Effect of MW07 on ADE of SARS-CoV-2 infected Raji cells

ADE detection of antibodies was performed by infecting Raji cells with new corona pseudovirus particles from the hospital. Different concentrations of the novel crown antibody to be evaluated were combined with 750TCID₅₀Perwell pseudovirions (transfected luciferase reporter genes) were neutralized at 37 ℃ and then inoculated with 1X10⁵Raji cells in well, CO at 37 ℃₂Culturing in an incubator for 20-28 h. And taking out the cell plate after 20-28h, adding 100 mul of luciferase detection reagent into each hole, avoiding light reaction for 2min, reading by a fluorescence detector, and evaluating the strength of ADE according to the intensity of a fluorescence signal.

As a result, as shown in FIG. 10, ADE was not observed in both MW07 in the form of wtlgG1 at a concentration range of 10-10000ng/mL, whereas both MW05 and MW01(8B4) in the form of wtlgG1 produced a stronger ADE at a concentration range (100-1500 ng/mL).

Note: the antibody 8B4(MW01) is described in the patent application No. CN202010178099. X. The antibody MW05 is described in patent application CN 202010298015.6.

The amino acid and nucleotide sequences of the antigen molecules involved in the invention are as follows:

＞S1 RBD-His

nucleotide sequence SEQ ID NO: 1 (the marking part is a label part)

ATGCCTCTGCTGCTGCTGCTGCCTCTGCTGTGGGCTGGAGCCCTGGCTCGGGTGCAG CCCACCGAGTCCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGT TCAACGCCACCCGGTTCGCCTCCGTGTACGCCTGGAACCGGAAGCGGATCTCCAACTGCGT GGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAGTGCTACGGCGTGT CTCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAG AGGCGACGAGGTGCGGCAGATCGCTCCTGGCCAGACCGGCAAGATCGCCGACTACAACTA CAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAACCTGGACTC CAAGGTGGGAGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCC CTTCGAGCGGGACATCTCCACCGAGATCTACCAGGCTGGCTCCACACCCTGCAACGGCGT GGAGGGCTTCAACTGCTACTTCCCTCTGCAGTCCTACGGCTTCCAGCCCACCAACGGCGTG GGCTACCAGCCCTACCGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCTCCTGCCACCG TGTGCGGACCCAAGAAGTCCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCGCTAGCG GATCCCATCACCATCACCATCAC

＞S1 RBD-His

Amino acid sequence SEQ ID NO: 2 (the marking part is a label part)

MPLLLLLPLLWAGALARVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCV ADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPD DFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFASGSHHHHHH

＞S1 RBD-mFc

Nucleotide sequence SEQ ID NO: 3 (the marking part is a label part)

ATGCCTCTGCTGCTGCTGCTGCCTCTGCTGTGGGCTGGAGCCCTGGCTCGGGTGCAG CCCACCGAGTCCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGT TCAACGCCACCCGGTTCGCCTCCGTGTACGCCTGGAACCGGAAGCGGATCTCCAACTGCGT GGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAGTGCTACGGCGTGT CTCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAG AGGCGACGAGGTGCGGCAGATCGCTCCTGGCCAGACCGGCAAGATCGCCGACTACAACTA CAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAACCTGGACTC CAAGGTGGGAGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCC CTTCGAGCGGGACATCTCCACCGAGATCTACCAGGCTGGCTCCACACCCTGCAACGGCGT GGAGGGCTTCAACTGCTACTTCCCTCTGCAGTCCTACGGCTTCCAGCCCACCAACGGCGTG GGCTACCAGCCCTACCGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCTCCTGCCACCG TGTGCGGACCCAAGAAGTCCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCGCTAGCG TGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTC ATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGT TGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTG GAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTC AGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAG GGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAG ACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAA AGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAG TGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGC TCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTT TCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCA CTCTCCTGGTAAA

＞S1 RBD-mFc

Amino acid sequence SEQ ID NO: 4 (the marking part is a label part)

MPLLLLLPLLWAGALARVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCV ADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPD DFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFASVPRDCGCKPC ICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQ FNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQM AKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWE AGNTFTCSVLHEGLHNHHTEKSLSHSPGK

＞S1-mFc

Nucleotide sequence SEQ ID NO: 5 (the marking part is a label part)

ATGTTTGTGTTCCTGGTGCTGCTGCCACTGGTGTCCAGCCAGTGTGTGAACCTGACCA CCAGGACCCAACTTCCTCCTGCCTACACCAACTCCTTCACCAGGGGAGTCTACTACCCTGA CAAGGTGTTCAGGTCCTCTGTGCTGCACAGCACCCAGGACCTGTTCCTGCCATTCTTCAGC AATGTGACCTGGTTCCATGCCATCCATGTGTCTGGCACCAATGGCACCAAGAGGTTTGACA ACCCTGTGCTGCCATTCAATGATGGAGTCTACTTTGCCAGCACAGAGAAGAGCAACATCAT CAGGGGCTGGATTTTTGGCACCACCCTGGACAGCAAGACCCAGTCCCTGCTGATTGTGAA CAATGCCACCAATGTGGTGATTAAGGTGTGTGAGTTCCAGTTCTGTAATGACCCATTCCTGG GAGTCTACTACCACAAGAACAACAAGTCCTGGATGGAGTCTGAGTTCAGGGTCTACTCCTC TGCCAACAACTGTACCTTTGAATATGTGAGCCAACCATTCCTGATGGACTTGGAGGGCAAG CAGGGCAACTTCAAGAACCTGAGGGAGTTTGTGTTCAAGAACATTGATGGCTACTTCAAG ATTTACAGCAAACACACACCAATCAACCTGGTGAGGGACCTGCCACAGGGCTTCTCTGCCT TGGAACCACTGGTGGACCTGCCAATTGGCATCAACATCACCAGGTTCCAGACCCTGCTGGC TCTGCACAGGTCCTACCTGACACCTGGAGACTCCTCCTCTGGCTGGACAGCAGGAGCAGC AGCCTACTATGTGGGCTACCTCCAACCAAGGACCTTCCTGCTGAAATACAATGAGAATGGC ACCATCACAGATGCTGTGGACTGTGCCCTGGACCCACTGTCTGAGACCAAGTGTACCCTGA AATCCTTCACAGTGGAGAAGGGCATCTACCAGACCAGCAACTTCAGGGTCCAACCAACAG AGAGCATTGTGAGGTTTCCAAACATCACCAACCTGTGTCCATTTGGAGAGGTGTTCAATGC CACCAGGTTTGCCTCTGTCTATGCCTGGAACAGGAAGAGGATTAGCAACTGTGTGGCTGAC TACTCTGTGCTCTACAACTCTGCCTCCTTCAGCACCTTCAAGTGTTATGGAGTGAGCCCAAC CAAACTGAATGACCTGTGTTTCACCAATGTCTATGCTGACTCCTTTGTGATTAGGGGAGATG AGGTGAGACAGATTGCCCCTGGACAAACAGGCAAGATTGCTGACTACAACTACAAACTGC CTGATGACTTCACAGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACAGCAAGGTGG GAGGCAACTACAACTACCTCTACAGACTGTTCAGGAAGAGCAACCTGAAACCATTTGAGA GGGACATCAGCACAGAGATTTACCAGGCTGGCAGCACACCATGTAATGGAGTGGAGGGC TTCAACTGTTACTTTCCACTCCAATCCTATGGCTTCCAACCAACCAATGGAGTGGGCTACCA ACCATACAGGGTGGTGGTGCTGTCCTTTGAACTGCTCCATGCCCCTGCCACAGTGTGTGGA CCAAAGAAGAGCACCAACCTGGTGAAGAACAAGTGTGTGAACTTCAACTTCAATGGACTG ACAGGCACAGGAGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAACAGTTTGG CAGGGACATTGCTGACACCACAGATGCTGTGAGGGACCCACAGACCTTGGAGATTCTGGA CATCACACCATGTTCCTTTGGAGGAGTGTCTGTGATTACACCTGGCACCAACACCAGCAAC CAGGTGGCTGTGCTCTACCAGGATGTGAACTGTACTGAGGTGCCTGTGGCTATCCATGCTG ACCAACTTACACCAACCTGGAGGGTCTACAGCACAGGCAGCAATGTGTTCCAGACCAGGG CTGGCTGTCTGATTGGAGCAGAGCATGTGAACAACTCCTATGAGTGTGACATCCCAATTGG AGCAGGCATCTGTGCCTCCTACCAGACCCAGACCAACAGCCCAAGGAGGGCAAGGGCTA GCGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTC TTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTG TGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGAT GTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCG CTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGC AGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGC AGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGAT AAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGC AGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGAT GGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAAT ACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCT CCCACTCTCCTGGTAAA

＞S1-mFc

Amino acid sequence SEQ ID NO: 6 (the marking part is a label part)

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSN VTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNAT NVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN FKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPG DSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTS NFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSK VGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQP YRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIAD TTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWR VYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARASVPRDCGCKPCI CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQ FNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQM AKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWE AGNTFTCSVLHEGLHNHHTEKSLSHSPGK

＞ACE2 1-615-hFc

Nucleotide sequence SEQ ID NO: 7 (the marking part is a label part)

ATGTCAAGCTCTTCCTGGCTCCTTCTCAGCCTTGTTGCTGTAACTGCTGCTCAGTCCAC CATTGAGGAACAGGCCAAGACATTTTTGGACAAGTTTAACCACGAAGCCGAAGACCTGTT CTATCAAAGTTCACTTGCTTCTTGGAATTATAACACCAATATTACTGAAGAGAATGTCCAAAA CATGAATAATGCTGGGGACAAATGGTCTGCCTTTTTAAAGGAACAGTCCACACTTGCCCAA ATGTATCCACTACAAGAAATTCAGAATCTCACAGTCAAGCTTCAGCTGCAGGCTCTTCAGCA AAATGGGTCTTCAGTGCTCTCAGAAGACAAGAGCAAACGGTTGAACACAATTCTAAATACA ATGAGCACCATCTACAGTACTGGAAAAGTTTGTAACCCAGATAATCCACAAGAATGCTTATT ACTTGAACCAGGTTTGAATGAAATAATGGCAAACAGTTTAGACTACAATGAGAGGCTCTGG GCTTGGGAAAGCTGGAGATCTGAGGTCGGCAAGCAGCTGAGGCCATTATATGAAGAGTAT GTGGTCTTGAAAAATGAGATGGCAAGAGCAAATCATTATGAGGACTATGGGGATTATTGGA GAGGAGACTATGAAGTAAATGGGGTAGATGGCTATGACTACAGCCGCGGCCAGTTGATTG AAGATGTGGAACATACCTTTGAAGAGATTAAACCATTATATGAACATCTTCATGCCTATGTGA GGGCAAAGTTGATGAATGCCTATCCTTCCTATATCAGTCCAATTGGATGCCTCCCTGCTCATT TGCTTGGTGATATGTGGGGTAGATTTTGGACAAATCTGTACTCTTTGACAGTTCCCTTTGGA CAGAAACCAAACATAGATGTTACTGATGCAATGGTGGACCAGGCCTGGGATGCACAGAGA ATATTCAAGGAGGCCGAGAAGTTCTTTGTATCTGTTGGTCTTCCTAATATGACTCAAGGATT CTGGGAAAATTCCATGCTAACGGACCCAGGAAATGTTCAGAAAGCAGTCTGCCATCCCACA GCTTGGGACCTGGGGAAGGGCGACTTCAGGATCCTTATGTGCACAAAGGTGACAATGGAC GACTTCCTGACAGCTCATCATGAGATGGGGCATATCCAGTATGATATGGCATATGCTGCACA ACCTTTTCTGCTAAGAAATGGAGCTAATGAAGGATTCCATGAAGCTGTTGGGGAAATCATG TCACTTTCTGCAGCCACACCTAAGCATTTAAAATCCATTGGTCTTCTGTCACCCGATTTTCAA GAAGACAATGAAACAGAAATAAACTTCCTGCTCAAACAAGCACTCACGATTGTTGGGACTC TGCCATTTACTTACATGTTAGAGAAGTGGAGGTGGATGGTCTTTAAAGGGGAAATTCCCAA AGACCAGTGGATGAAAAAGTGGTGGGAGATGAAGCGAGAGATAGTTGGGGTGGTGGAA CCTGTGCCCCATGATGAAACATACTGTGACCCCGCATCTCTGTTCCATGTTTCTAATGATTAC TCATTCATTCGATATTACACAAGGACCCTTTACCAATTCCAGTTTCAAGAAGCACTTTGTCAA GCAGCTAAACATGAAGGCCCTCTGCACAAATGTGACATCTCAAACTCTACAGAAGCTGGAC AGAAACTGTTCAATATGCTGAGGCTTGGAAAATCAGAACCCTGGACCCTAGCATTGGAAAA TGTTGTAGGAGCAAAGAACATGAATGTAAGGCCACTGCTCAACTACTTTGAGCCCTTATTTA CCTGGCTGAAAGACCAGAACAAGAACTCTTTTGTGGGATGGAGTACCGACTGGAGTCCAT ATGCAGACGCTAGCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCTCCATCTCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACC GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

The amino acid sequence of ACE 21-615-hFc is SEQ ID NO: 8 (the marking part is a label part)

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILN TMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEY VVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYV RAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDA QRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKV TMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPASEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

<110> Applicant's name

<120> SARS-CoV-2S protein specific antibody or its fragment and its application

<130> None

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 747

<212> DNA

<213> artificial sequence

<400> 1

atgcctctgc tgctgctgct gcctctgctg tgggctggag ccctggctcg ggtgcagccc 60

accgagtcca tcgtgcggtt ccccaacatc accaacctgt gccccttcgg cgaggtgttc 120

aacgccaccc ggttcgcctc cgtgtacgcc tggaaccgga agcggatctc caactgcgtg 180

gccgactact ccgtgctgta caactccgcc tccttctcca ccttcaagtg ctacggcgtg 240

tctcccacca agctgaacga cctgtgcttc accaacgtgt acgccgactc cttcgtgatc 300

agaggcgacg aggtgcggca gatcgctcct ggccagaccg gcaagatcgc cgactacaac 360

tacaagctgc ccgacgactt caccggctgc gtgatcgcct ggaactccaa caacctggac 420

tccaaggtgg gaggcaacta caactacctg taccggctgt tccggaagtc caacctgaag 480

cccttcgagc gggacatctc caccgagatc taccaggctg gctccacacc ctgcaacggc 540

gtggagggct tcaactgcta cttccctctg cagtcctacg gcttccagcc caccaacggc 600

gtgggctacc agccctaccg ggtggtggtg ctgtccttcg agctgctgca cgctcctgcc 660

accgtgtgcg gacccaagaa gtccaccaac ctggtgaaga acaagtgcgt gaacttcgct 720

agcggatccc atcaccatca ccatcac 747

<210> 2

<211> 249

<212> PRT

<213> artificial sequence

<400> 2

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

20 25 30

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

35 40 45

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

50 55 60

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

65 70 75 80

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

85 90 95

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

100 105 110

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

115 120 125

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

130 135 140

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

145 150 155 160

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

165 170 175

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

180 185 190

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

195 200 205

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

210 215 220

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala

225 230 235 240

Ser Gly Ser His His His His His His

245

<210> 3

<211> 1404

<212> DNA

<213> Artificial sequence

<400> 3

atgcctctgc tgctgctgct gcctctgctg tgggctggag ccctggctcg ggtgcagccc 60

accgagtcca tcgtgcggtt ccccaacatc accaacctgt gccccttcgg cgaggtgttc 120

aacgccaccc ggttcgcctc cgtgtacgcc tggaaccgga agcggatctc caactgcgtg 180

gccgactact ccgtgctgta caactccgcc tccttctcca ccttcaagtg ctacggcgtg 240

tctcccacca agctgaacga cctgtgcttc accaacgtgt acgccgactc cttcgtgatc 300

agaggcgacg aggtgcggca gatcgctcct ggccagaccg gcaagatcgc cgactacaac 360

tacaagctgc ccgacgactt caccggctgc gtgatcgcct ggaactccaa caacctggac 420

tccaaggtgg gaggcaacta caactacctg taccggctgt tccggaagtc caacctgaag 480

cccttcgagc gggacatctc caccgagatc taccaggctg gctccacacc ctgcaacggc 540

gtggagggct tcaactgcta cttccctctg cagtcctacg gcttccagcc caccaacggc 600

gtgggctacc agccctaccg ggtggtggtg ctgtccttcg agctgctgca cgctcctgcc 660

accgtgtgcg gacccaagaa gtccaccaac ctggtgaaga acaagtgcgt gaacttcgct 720

agcgtgccca gggattgtgg ttgtaagcct tgcatatgta cagtcccaga agtatcatct 780

gtcttcatct tccccccaaa gcccaaggat gtgctcacca ttactctgac tcctaaggtc 840

acgtgtgttg tggtagacat cagcaaggat gatcccgagg tccagttcag ctggtttgta 900

gatgatgtgg aggtgcacac agctcagacg caaccccggg aggagcagtt caacagcact 960

ttccgctcag tcagtgaact tcccatcatg caccaggact ggctcaatgg caaggagttc 1020

aaatgcaggg tcaacagtgc agctttccct gcccccatcg agaaaaccat ctccaaaacc 1080

aaaggcagac cgaaggctcc acaggtgtac accattccac ctcccaagga gcagatggcc 1140

aaggataaag tcagtctgac ctgcatgata acagacttct tccctgaaga cattactgtg 1200

gagtggcagt ggaatgggca gccagcggag aactacaaga acactcagcc catcatggac 1260

acagatggct cttacttcgt ctacagcaag ctcaatgtgc agaagagcaa ctgggaggca 1320

ggaaatactt tcacctgctc tgtgttacat gagggcctgc acaaccacca tactgagaag 1380

agcctctccc actctcctgg taaa 1404

<210> 4

<211> 468

<212> PRT

<213> Artificial sequence

<400> 4

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

20 25 30

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

35 40 45

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

50 55 60

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

65 70 75 80

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

85 90 95

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

100 105 110

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

115 120 125

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

130 135 140

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

145 150 155 160

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

165 170 175

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

180 185 190

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

195 200 205

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

210 215 220

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala

225 230 235 240

Ser Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

245 250 255

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

260 265 270

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

275 280 285

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

290 295 300

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

305 310 315 320

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

355 360 365

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

370 375 380

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

385 390 395 400

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

405 410 415

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

420 425 430

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

435 440 445

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

450 455 460

Ser Pro Gly Lys

465

<210> 5

<211> 2742

<212> DNA

<213> Artificial sequence

<400> 5

atgtttgtgt tcctggtgct gctgccactg gtgtccagcc agtgtgtgaa cctgaccacc 60

aggacccaac ttcctcctgc ctacaccaac tccttcacca ggggagtcta ctaccctgac 120

aaggtgttca ggtcctctgt gctgcacagc acccaggacc tgttcctgcc attcttcagc 180

aatgtgacct ggttccatgc catccatgtg tctggcacca atggcaccaa gaggtttgac 240

aaccctgtgc tgccattcaa tgatggagtc tactttgcca gcacagagaa gagcaacatc 300

atcaggggct ggatttttgg caccaccctg gacagcaaga cccagtccct gctgattgtg 360

aacaatgcca ccaatgtggt gattaaggtg tgtgagttcc agttctgtaa tgacccattc 420

ctgggagtct actaccacaa gaacaacaag tcctggatgg agtctgagtt cagggtctac 480

tcctctgcca acaactgtac ctttgaatat gtgagccaac cattcctgat ggacttggag 540

ggcaagcagg gcaacttcaa gaacctgagg gagtttgtgt tcaagaacat tgatggctac 600

ttcaagattt acagcaaaca cacaccaatc aacctggtga gggacctgcc acagggcttc 660

tctgccttgg aaccactggt ggacctgcca attggcatca acatcaccag gttccagacc 720

ctgctggctc tgcacaggtc ctacctgaca cctggagact cctcctctgg ctggacagca 780

ggagcagcag cctactatgt gggctacctc caaccaagga ccttcctgct gaaatacaat 840

gagaatggca ccatcacaga tgctgtggac tgtgccctgg acccactgtc tgagaccaag 900

tgtaccctga aatccttcac agtggagaag ggcatctacc agaccagcaa cttcagggtc 960

caaccaacag agagcattgt gaggtttcca aacatcacca acctgtgtcc atttggagag 1020

gtgttcaatg ccaccaggtt tgcctctgtc tatgcctgga acaggaagag gattagcaac 1080

tgtgtggctg actactctgt gctctacaac tctgcctcct tcagcacctt caagtgttat 1140

ggagtgagcc caaccaaact gaatgacctg tgtttcacca atgtctatgc tgactccttt 1200

gtgattaggg gagatgaggt gagacagatt gcccctggac aaacaggcaa gattgctgac 1260

tacaactaca aactgcctga tgacttcaca ggctgtgtga ttgcctggaa cagcaacaac 1320

ctggacagca aggtgggagg caactacaac tacctctaca gactgttcag gaagagcaac 1380

ctgaaaccat ttgagaggga catcagcaca gagatttacc aggctggcag cacaccatgt 1440

aatggagtgg agggcttcaa ctgttacttt ccactccaat cctatggctt ccaaccaacc 1500

aatggagtgg gctaccaacc atacagggtg gtggtgctgt cctttgaact gctccatgcc 1560

cctgccacag tgtgtggacc aaagaagagc accaacctgg tgaagaacaa gtgtgtgaac 1620

ttcaacttca atggactgac aggcacagga gtgctgacag agagcaacaa gaagttcctg 1680

ccattccaac agtttggcag ggacattgct gacaccacag atgctgtgag ggacccacag 1740

accttggaga ttctggacat cacaccatgt tcctttggag gagtgtctgt gattacacct 1800

ggcaccaaca ccagcaacca ggtggctgtg ctctaccagg atgtgaactg tactgaggtg 1860

cctgtggcta tccatgctga ccaacttaca ccaacctgga gggtctacag cacaggcagc 1920

aatgtgttcc agaccagggc tggctgtctg attggagcag agcatgtgaa caactcctat 1980

gagtgtgaca tcccaattgg agcaggcatc tgtgcctcct accagaccca gaccaacagc 2040

ccaaggaggg caagggctag cgtgcccagg gattgtggtt gtaagccttg catatgtaca 2100

gtcccagaag tatcatctgt cttcatcttc cccccaaagc ccaaggatgt gctcaccatt 2160

actctgactc ctaaggtcac gtgtgttgtg gtagacatca gcaaggatga tcccgaggtc 2220

cagttcagct ggtttgtaga tgatgtggag gtgcacacag ctcagacgca accccgggag 2280

gagcagttca acagcacttt ccgctcagtc agtgaacttc ccatcatgca ccaggactgg 2340

ctcaatggca aggagttcaa atgcagggtc aacagtgcag ctttccctgc ccccatcgag 2400

aaaaccatct ccaaaaccaa aggcagaccg aaggctccac aggtgtacac cattccacct 2460

cccaaggagc agatggccaa ggataaagtc agtctgacct gcatgataac agacttcttc 2520

cctgaagaca ttactgtgga gtggcagtgg aatgggcagc cagcggagaa ctacaagaac 2580

actcagccca tcatggacac agatggctct tacttcgtct acagcaagct caatgtgcag 2640

aagagcaact gggaggcagg aaatactttc acctgctctg tgttacatga gggcctgcac 2700

aaccaccata ctgagaagag cctctcccac tctcctggta aa 2742

<210> 6

<211> 914

<212> PRT

<213> Artificial sequence

<400> 6

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ala Ser Val

675 680 685

Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val

690 695 700

Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile

705 710 715 720

Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp

725 730 735

Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His

740 745 750

Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

755 760 765

Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys

770 775 780

Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu

785 790 795 800

Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr

805 810 815

Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu

820 825 830

Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp

835 840 845

Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile

850 855 860

Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln

865 870 875 880

Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His

885 890 895

Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro

900 905 910

Gly Lys

<210> 7

<211> 2547

<212> DNA

<213> Artificial sequence

<400> 7

atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60

attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120

tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180

aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240

caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300

cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360

aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420

tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480

aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540

gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600

gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660

cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720

catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780

tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840

ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900

gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960

cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020

aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080

tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140

tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200

catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260

attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320

aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380

tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440

aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500

gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560

taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620

aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680

ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740

gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800

aactcttttg tgggatggag taccgactgg agtccatatg cagacgctag cgagcccaaa 1860

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 1920

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 1980

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 2040

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 2100

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 2160

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 2220

gccaaagggc agccccgaga accacaggtg tacaccctgc ctccatctcg ggatgagctg 2280

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 2340

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 2400

gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 2460

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 2520

aagagcctct ccctgtctcc gggtaaa 2547

<210> 8

<211> 649

<212> PRT

<213> Artificial sequence

<400> 8

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe

20 25 30

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

65 70 75 80

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Ala

405 410 415

Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

420 425 430

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

435 440 445

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

450 455 460

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

465 470 475 480

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

485 490 495

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

500 505 510

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

515 520 525

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

530 535 540

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

545 550 555 560

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

565 570 575

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

580 585 590

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

595 600 605

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

610 615 620

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

625 630 635 640

Lys Ser Leu Ser Leu Ser Pro Gly Lys

645

<210> 9

<211> 107

<212> PRT

<213> MW07-VL

<400> 9

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 Gly Ile Ser Asn Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

35 40 45

Tyr Ala Ala Ser Thr Leu Glu 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 Phe Tyr Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 10

<211> 120

<212> PRT

<213> MW07-HL

<400> 10

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

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Ala Ser Glu Lys Tyr Tyr Leu Asp Ser Leu

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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Gly Ile Leu Trp Phe Gly Asp Tyr Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 11

<212> PRT

<213> MW07-LCDR1

<400> 11

Arg Ala Ser Gln Gly Ile Ser Asn Ser Leu Ala

1 5 10

<210> 12

<211> 7

<212> PRT

<213> MW07-LCDR2

<400> 12

Ala Ala Ser Thr Leu Glu Ser

1 5

<210> 13

<211> 9

<212> PRT

<213> MW07-LCDR3

<400> 13

Gln Gln Phe Tyr Ser Thr Pro Arg Thr

1 5

<210> 14

<211> 5

<212> PRT

<213> MW07-HCDR1

<400> 14

Ser Tyr Trp Met Ser

1 5

<210> 15

<211> 17

<212> PRT

<213> MW07-HCDR2

<400> 15

Asn Ile Lys Gln Asp Ala Ser Glu Lys Tyr Tyr Leu Asp Ser Leu Lys

1 5 10 15

Gly

<210> 16

<211> 11

<212> PRT

<213> MW07-HCDR3

<400> 16

Asp Leu Gly Ile Leu Trp Phe Gly Asp Tyr Pro

1 5 10

Claims (18)

1. An antibody or fragment thereof specific for a SARS-CoV-2S protein, characterized in that the epitope to which the antibody or fragment thereof specifically binds is an epitope present on the SARS-CoV-2S 1 protein and is not present on the SARS-CoV S1 protein, MERS-CoV S1 protein.

2. The antibody or fragment thereof specific for SARS-CoV-2S protein according to claim 1, wherein the epitope to which the antibody or fragment thereof specifically binds is located within the receptor binding domain RBD of SARS-CoV-2.

3. The antibody or fragment thereof specific for SARS-CoV-2S protein of claim 1, wherein the antibody or fragment thereof inhibits infection of host cells by SARS-CoV-2 in a dose dependent manner.

4. The antibody or fragment thereof specific for SARS-CoV-2S protein of claim 1, wherein the antibody or fragment thereof does not cause a significant antibody-dependent enhancing effect (ADE) when used to treat SARS-CoV-2 infection.

5. Antibody or fragment thereof specific for the SARS-CoV-2S protein according to claim 1, wherein the antibody or fragment thereof is capable of exerting a neutralizing effect by blocking the binding of SARS-CoV-2 to the host cell receptor ACEII, wherein the IC50 blocking the binding of SARS-CoV-2 to the host cell receptor ACEII is less than 500ng/mL, such as less than 200ng/mL, preferably less than 100ng/mL, more preferably less than 80 ng/mL.

6. The antibody or fragment thereof specific for SARS-CoV-2S protein as claimed in any of claims 1 to 5, wherein the heavy chain variable region CDR1-3 of the antibody or fragment thereof is identical to the amino acid sequence as set forth in SEQ ID NO: 10 has 100% sequence homology to CDR1-3 of the heavy chain variable region shown in seq id no; the light chain variable region CDR1-3 is identical to SEQ ID NO: the CDR1-3 of the light chain variable region shown in FIG. 9 has 100% sequence homology.

7. The antibody or fragment thereof specific for SARS-CoV-2S protein according to claim 6, wherein the amino acid sequences of CDR1-3 in the heavy chain variable region of the antibody or fragment thereof are respectively SEQ ID NO: 14-16, the amino acid sequences of the light chain variable region CDR1-3 are respectively SEQ ID NO: 11-13.

8. Antibody or fragment thereof specific for the SARS-CoV-2S protein according to claim 6 or 7, wherein the heavy chain variable region of the antibody or fragment thereof is identical to the heavy chain variable region of SEQ ID NO: 10 has a sequence homology of 70% or more; the light chain variable region is identical to SEQ ID NO: 9 has a sequence homology of 70% or more.

9. A SARS-CoV-2S protein specific antibody or fragment thereof, characterized in that: the monoclonal antibody or fragment thereof has a competitive relationship with the antibody or fragment thereof of any one of claims 6-8 as detected by a Fortebio epitope competitive binding assay.

10. The antibody or fragment thereof of any one of claims 1 to 9, comprising a murine antibody, a rabbit antibody, a goat antibody, a sheep antibody, a camel antibody, an alpaca antibody, a chimeric antibody, a human antibody, a humanized antibody, a Fab, F (ab')₂Fv, scFv, Fd, heavy chain antibody, nanobody, etc.

11. A composition comprising one or more SARS-CoV-2S protein-specific antibodies or fragments thereof selected from the group consisting of SARS-CoV-2S protein-specific antibodies or fragments thereof according to any one of claims 1 to 10.

12. The composition of claim 11, further comprising a pharmaceutically acceptable carrier, and being used as a pharmaceutical composition, preferably the pharmaceutical composition is a liquid, an injection, or a powder injection.

13. The application of the antibody or the fragment thereof in preparing the medicine for treating coronavirus infection is characterized in that: the antibody or fragment thereof comprises one or more antibodies or fragments thereof selected from the group consisting of antibodies specific for the SARS-CoV-2S protein of any one of claims 1 to 10.

14. Use according to claim 13, wherein the coronavirus infection comprises a SARS-CoV-2 infection.

15. A polynucleotide encoding an antibody or fragment thereof specific for SARS-CoV-2S protein according to any one of claims 1 to 10.

16. A vector comprising the polynucleotide of claim 15.

17. A host cell comprising the polynucleotide of claim 15 or the vector of claim 16.

18. A method of making a SARS-CoV-2S protein specific antibody or fragment thereof, comprising the steps of:

(1) culturing the host cell of claim 17 under conditions suitable for expression of recombinant SARS-CoV-2S protein-specific antibodies or fragments thereof;

(2) separating and purifying SARS-CoV-2S protein specific antibody or its fragment from cell culture.

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