CN112409479B

CN112409479B - Humanized anti-neocoronavirus neutralizing antibody nCoV-121 and application thereof

Info

Publication number: CN112409479B
Application number: CN202011324505.5A
Authority: CN
Inventors: 梁米芳; 孙丽娜; 曲圆圆; 殷强玲; 李德新; 王世文; 李川; 芜为; 李建东; 李阿茜; 黄晓霞
Original assignee: National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention
Current assignee: National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2022-04-26
Anticipated expiration: 2040-11-23
Also published as: CN112409479A

Abstract

The invention discloses a humanized anti-new coronavirus neutralizing antibody nCoV-121 and application thereof. The invention successfully obtains a humanized neutralizing antibody nCoV-121 with specificity aiming at the surface antigen of the novel coronavirus by utilizing a phage antibody library technology, has the neutralizing function of preventing the novel coronavirus from infecting sensitive cells in vitro, has high affinity to the antigen, and is expected to be prepared into a specific antibody medicament for clinically preventing and treating the novel coronavirus pneumonia.

Description

Humanized anti-neocoronavirus neutralizing antibody nCoV-121 and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a humanized anti-new coronavirus neutralizing antibody nCoV-121 and application thereof.

Background

The novel coronavirus SARS-CoV-2 is a new virus discovered in 2019, and can cause human viral pneumonia or lung infection. The coronavirus genome encodes spinous process protein, envelope protein, membrane protein and nucleocapsid protein in sequence. Among them, Spike protein (Spike) is the most important surface membrane protein of coronavirus, and contains two subunits, S1 and S2, of which S1 mainly comprises a Receptor Binding Domain (RBD) responsible for recognizing the receptor of the cell. The new coronavirus spike protein interacts with human angiotensin converting enzyme 2(ACE2) to infect human respiratory epithelial cells.

Although various drugs like Reidesivir, hydroxychloroquine, lopinavir and interferon beta 1a have been shown to be effective against neocorona in vitro, recent studies by WHO have shown that none of these drugs significantly reduces the mortality of neocorona virus and does not improve the course of treatment for patients. Therefore, the development of monoclonal antibodies with neutralizing activity and the combination therapy method of various monoclonal antibodies is particularly important. Antibody cocktail therapy refers to a therapeutic regimen combining two neutralizing monoclonal antibodies that bind to key receptors of the spike protein of new coronavirus in a non-competitive manner, which can attenuate escape of the variant virus and thereby more effectively block infection by the new coronavirus. There is therefore a great need to further develop neutralizing antibodies against different binding sites of the neo-corona antigen.

Disclosure of Invention

The invention aims to provide a human-derived anti-new coronavirus neutralizing antibody nCoV-121 and application thereof.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a human anti-neocoronaviruse neutralizing antibody nCoV-121, whose light and heavy chain hypervariable regions CDR1, CDR2 and CDR3 have the following amino acid sequences:

the amino acid sequences of the variable regions of the light chain and the heavy chain of antibody nCoV-121 are shown as

SEQ ID NO

1 and 2, respectively.

In a second aspect, the invention provides nucleic acid molecules encoding the antibody nCoV-121. Wherein the nucleotide sequences encoding the light chain variable region and the heavy chain variable region are shown in

SEQ ID NO

3 and 4, respectively.

In a third aspect, the present invention provides biological materials containing the above-described nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

In a fourth aspect, the invention provides a single chain antibody or a full antibody engineered from the antibody nCoV-121.

In a fifth aspect, the invention provides a medicament for preventing or treating infection by a novel coronavirus and related diseases caused by the infection, wherein the effective component of the medicament is antibody nCoV-121 or a single-chain antibody or full antibody obtained by modifying the antibody nCoV-121.

In one embodiment of the present invention, the amino acid sequences of the light chain and heavy chain of the whole antibody IgG are shown in SEQ ID NOS: 5-6, respectively, and the nucleotide sequences encoding the light chain and heavy chain are shown in SEQ ID NOS: 7-8, respectively.

In a sixth aspect, the invention provides a novel reagent for detecting coronaviruses, which comprises an antibody nCoV-121 or a single-chain antibody or a full antibody obtained by modifying the antibody nCoV-121.

In a seventh aspect, the invention provides any of the following uses of antibody nCoV-121 or a single chain antibody or a full antibody engineered from antibody nCoV-121:

1) for the preparation of a medicament for the prevention or treatment of infections with new coronaviruses and associated diseases caused by infections therewith;

2) used for preparing a new coronavirus detection reagent or a kit;

3) for the prevention or treatment of infections by new coronaviruses and related diseases caused by infections therewith;

4) used for detecting new coronavirus.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention successfully obtains a humanized neutralizing antibody nCoV-121 specifically aiming at the surface antigen of the novel coronavirus by using a phage antibody library technology, has a neutralizing function of preventing the novel coronavirus from infecting sensitive cells in vitro, and has high affinity to the antigen. The humanized neutralizing anti-new type coronavirus surface antigen gene engineering antibody variable region gene, Fab antibody gene and whole antibody gene under the characteristics of each antibody gene obtained by the method can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombination system or any other gene containing the antibody gene after reconstruction based on the expression and production of the antibody, so that an antibody product with the effect of neutralizing new type coronavirus infection can be obtained, and a specific antibody medicament for clinically preventing and treating new type coronavirus pneumonia can be prepared.

Drawings

FIG. 1 shows the Fab antibody titer in lysates of 12 monoclonal bacteria in a preferred embodiment of the present invention.

FIG. 2 is an SDS-PAGE purified gel of the neutralizing antibody nCoV-121 of human origin in a preferred embodiment of the invention.

FIG. 3 shows the results of the analysis of the neutralization of the novel coronavirus (live virus) by the antibody nCoV-121 in the preferred embodiment of the present invention.

FIG. 4 shows the results of ELISA detection of the binding activity of antibody nCoV-121 and the surface antigen of the novel coronavirus in the preferred embodiment of the present invention.

FIG. 5 shows the result of IFA detection of the binding activity of antibody nCoV-121 to the surface antigen of a novel coronavirus in a preferred embodiment of the present invention.

FIG. 6 shows the results of flow cytometry on the binding activity of antibody nCoV-121 to the novel coronavirus S protein in the preferred embodiment of the invention.

FIG. 7 is a graph showing the affinity assay of the antibody nCoV-121 in the preferred embodiment of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 preparation and function of a neutralizing antibody nCoV-121 against a human New coronavirus

Materials (I) and (II)

1. Sample, carrier: new coronavirus antibody positive sera and lymphocytes: is provided by Wuhan disease control and Shandong disease control. Strain XLI-Blue was purchased from Stratagene, USA, and antibody library construction vector pComb3H (40kb) was complimentary to Scripps research, USA.

2. Antigen: the novel coronavirus RBD protein and S1 protein are provided by Hualan bioengineering GmbH, novel coronavirus S protein trimer is provided by Jiangsu Dongzhi anti-biological medicine science and technology GmbH, RBD protein expression plasmid, the full-length S protein expression plasmid (see Chi X, Yan R, Zhang J, Zhang G, Zhang Y, Hao M, Zhang Z, Fan P, Dong Y, Yang Y, Chen Z, Guo Y, Zhang J, Li Y, Song X, Chen Y, Xia L, Fu L, Hou L, Xu J, Yu C, Li J, Zhou Q, Chen W.A neutral human antibody binding to the N-terminal domain of the Spike protein of SARS-CoV-2.science 2020 Aug7; 6504, 650-655.doi, 10.1126/science. abc6952.Epub Jun 22.PMID: 32571838; ID: PMC 7373) was given by Qinghua university.

3. Novel coronaviruses: the new coronavirus 19nCoV-CDC-Tan-Strain (HB02) used in the experiment was isolated and preserved from the virus disease of the Chinese center for disease prevention and control.

Second, method

1. Construction and screening of humanized anti-new coronavirus surface antigen antibody library

1.1 construction of phage antibody libraries

Lymphocytes were isolated from convalescent peripheral blood of patients with the novel coronavirus using lymphocyte separation medium (Sigma in USA), total cellular RNA was extracted using RNeasy Mini Kit (QIAGEN, Germany), the extracted RNA was reverse-transcribed into cDNA using Oligo-dT primer using First chain Synthesis Kit (SuperScriptTM III First-Strand Synthesis System for RT-PCR. Cat No.18080-051) from Invitrogen, and human light and heavy chain Fab genes were PCR-amplified using a set of primers for amplifying human IgG1 heavy chain Fd and light chain Kappa and Lambda. The PCR amplification conditions were: 1min at 94 ℃, 1min at 52 ℃, 1min at 72 ℃ and 35 cycles. The library construction method is essentially performed according to the literature (Barbas, C.F III., Kang, A.S., and girder, R.A.Assembly of combinatorial antibody libraries on phase surface: the gene III site, Proc.Natl.Acad.Sci.USA 1991; 88(18): 7978-.

1.2 enrichment and screening of phage antibody library and inducible expression of Fab fragment antibody

The screening antigen is purified novel coronavirus RBD protein and S1 protein. With 0.1M NaHCO₃(pH8.6) diluting the antigen with a solution, and coating the immune wells of a 96-well plate with 150. mu.l of the antigen per well; blocking with 2% skimmed milk-PBST at 37 deg.C for 2h, adding the phage antibody library, incubating at 37 deg.C for 2h with 150 μ l per well, and repeatedly washing with 5% Tween-20-PBST for 10 times; finally, eluting each well with 150 μ l of glycine-hydrochloric acid eluent with pH2.2, and neutralizing with Tris solution with pH 9.6; the eluted phage was further infected with 20ml of fresh OD₆₀₀About 0.5 XL1-Blu was infected with the helper phage VCSM13 (Stratagene, usa) for the next round of selection. This was repeated 3 times. Specific enrichment screening methods and the inducible expression of Fab fragments were performed essentially as described in the literature (Barbas, C.F III., Kang, A.S., and finer, R.A.Assembly of combinatorial antibody libraries on phase surface: the gene III site.Proc.Natl.Acad.Sci.USA 1991; 88(18): 7978-.

2. Detection of new coronavirus surface antigen genetic engineering Fab antibody

2.1 detection of Fab antibody expression

With 0.1m/L NaHCO₃(pH9.6) solution coated anti-human Fab antibody (Sigma, 1:2000 for dilution) in ELISA plate, 4 degrees C overnight; sealing with 5% skimmed milk, adding expressed Fab antibody at 37 deg.C for 1 hr, and sealing at 37 deg.C for 1 hr; adding enzyme-labeled anti-human Fab secondary antibody (Sigma in USA, 1:2000 for dilution) at 37 ℃ for 1 h; color developing solution for color development, 2M H₂SO₄The reaction is stopped, and the absorbance A value is detected by a microplate reader.

2.2 Indirect ELISA for detecting the binding Activity of Fab antibodies to the surface antigens of novel Coronaviridae

The purified novel coronavirus RBD protein and S1 protein are used as coating antigens, and the subsequent steps are the same as the above.

3. Nucleic acid sequence analysis of human Fab antibody variable region genes:

plasmid DNA was prepared with the Qiagen Miniprep Kit (QIAGEN, Germany) for nucleic acid sequence analysis. The sequencing primers of the light chain and the heavy chain are respectively 5 '-ATTGAATTCAGGAGGAA-3' and 5 '-TGAAATACCTATTGCCTA-3'. The sequencing results were compared to the IgG gene sequence in the Internet V-Base gene bank (http:// www.vbase2.org /).

4. Construction and expression purification of whole antibody recombinant expression plasmid

4.1 construction of recombinant expression plasmid for Whole antibody

By reference to antibody sequences in the V-base database, primers were designed to amplify Fab antibody light and heavy chain variable region gene fragments, Age1/BsiW1 cleavage sites at the 5 ' and 3 ' ends of the kappa chain, Age1/Xho1 cleavage sites at the 5 ' and 3 ' ends of the lambda chain, and Age1/Sal1 cleavage sites at the 5 ' and 3 ' ends of the heavy chain, respectively, and the light and heavy chains were cloned into the full antibody expression vector Michell series vector (this vector is given by professor of Qinghua university Schwary, see Chi, Yan R, Zhang J, Zhang G, Zhang Y, Hao M, Zhang Z, Fan P, Dong Y, Yang Y, Cheng Z, Guo Y, Zhang J, Li Y, Song X, Chen Y, Xia L, Fu L, Hou L, Xu J, Yu C, Li, Chen J, Zhang Q W.A, Zhang J, K J W.A, K5-T7, 9, 5, 3 ', 9, 5, 3, 2, 3, 9, 2, 9, 2, 3, 2, 9, 3, 2, 3, 9, 2, 9, 2, 9, 2, 9, 2 -655.doi:10.1126/science. abc6952.epub 2020Jun 22.PMID: 32571838; PMCID: PMC7319273.) to construct a whole antibody expression vector.

4.2 Total antibody expression and purification

Mixing the constructed expression plasmid with 1mg/mL Polyethyleneimine (PEI) solution according to a ratio of 1:5(w/v), and then carrying out instantaneous transfection with the density of 2 × 10⁶The cells of Expi293F were incubated at 37 ℃ for 4 days with shaking at 120rpm, and then the supernatants were purified and collected by using Protein-G affinity chromatography column of GE corporation, respectively. Antibody purity was analyzed by SDS-polyacrylamide gel (SDS-PAGE) and antibody concentration was determined using the BCATM protein assay kit (Thermo, USA).

5. Function detection of new coronavirus surface antigen gene engineering antibody

5.1 measurement of antibody affinity by BIAcore method

Surface Plasmon Resonance (SPR) is an optical physical phenomenon. When a beam of polarized light is incident on the end face of the prism within a certain angle range, surface plasma waves are generated at the interface between the prism and the metal film. When the propagation constant of the incident light wave matches the propagation constant of the surface plasmon wave, free electrons in the metal film are caused to resonate, i.e., surface plasmon resonance. By using the SPR technology, the intermolecular interaction can be obtained in real time without marking. When the conjugate is analyzed, the conjugate is firstly coupled to a chip, then the analyte flows through the surface of the chip, if the conjugate and the analyte have binding activity, the refractive index of the surface of the metal film is changed, and finally, the SPR angle is changed, and the change is represented by a response signal value RU of Biacore. By detecting the change of SPR angle, information such as kinetic constants (an association rate constant ka, an dissociation rate constant KD and an affinity constant KD) and specificity is obtained.

Data results processing raw data was output using Biacore instrumentation Evaluation program software and subjected to kinetic fitting analysis in a 1:1Binding mode. From the fitting results, the association rate constant (ka) and dissociation rate constant (KD) and affinity constant (KD) were obtained.

5.2 neutralizing Activity of genetically engineered antibody against surface antigen of novel human coronavirus

Vero cells were supplied by China Biotechnology Ltd, and 19nCoV-CDC-Tan-Strain (HB02, P5) was used as a virus. 2 times of gradient dilution is carried out on the purified monoclonal antibody, 50ul of diluted antibody and equal volume of 2000TCID are taken₅₀Mixing the viruses per ml, and incubating for 2 hours at 37 ℃; vero cells were infected with the above mixture at 37 ℃ with 5% CO₂Incubators were incubated for 96h to observe cytopathic effect (CPE), and the highest dilution of the sample with complete protection of the cells was recorded.

5.3ELISA detection of anti-human novel coronavirus surface antigen gene engineering antibody and antigen binding Activity

The binding activity of the purified monoclonal antibody and the antigen is respectively detected by using the purified novel coronavirus RBD protein, the S1 protein and the S protein as coating antigens, and the subsequent steps are the same as 2.1.

5.4 Indirect Immunofluorescence (IFA) detection of the binding Activity of the genetically engineered antibody against the surface antigen of the novel human coronavirus with the antigen

5.4.1 binding Activity with New coronavirus

Culturing vero cells infected by 19nCoV-CDC-Tan-Strain (HB02) of new coronavirus for 4 days to prepare antigen tablets, and fixing for 30 minutes by 95% alcohol; adding antibody expression supernatant, incubating at 37 deg.C for 30min, cleaning, and blow-drying; adding anti-human Fc fluorescent antibody (Sigma) diluted with Evans blue staining solution diluted at a ratio of 1:30000 according to a ratio of 1:40, incubating at 37 deg.C for 30min, washing, and drying. And (5) detecting and photographing by a fluorescence microscope.

5.4.2 binding Activity to New coronavirus RBD protein

Mixing RBD protein expression plasmid with 1mg/mL Polyethyleneimine (PEI) solution at a ratio of 1:5(w/v), transiently transfecting cells with Expi293F with confluence of 80%, and placing the cells in CO at 37 DEG C₂Incubating in incubator for 48 hr to obtain antigen tablet, and the subsequent steps are 5.4.1.

5.4.3 binding Activity to the New coronavirus S protein

Mixing the S protein full-length expression plasmid with 1mg/mL Polyethyleneimine (PEI) solution according to a ratio of 1:5(w/v), transiently transfecting Expi293F cells with confluence of 80%, and placing the cells in a CO (carbon monoxide) solution at 37 DEG C₂Incubating in incubator for 48 hr to obtain antigen tablet, and the subsequent steps are 5.4.1.

5.5 flow cytometry detection of the binding Activity of anti-human novel coronavirus surface antigen genetically engineered antibody and novel coronavirus S protein

Mixing the S protein full-length expression plasmid with 1mg/mL Polyethyleneimine (PEI) solution according to a ratio of 1:5(w/v), transiently transfecting Expi293F cells with confluence of 80%, and placing the cells in a CO (carbon monoxide) solution at 37 DEG C₂And (5) incubating for 36 h. The cells were digested and washed 3 times with PBS and the test antibody was added to 5.5X 10 cells at a concentration of 20ug/ml⁵In each cell, incubation was performed at room temperature for 30 min. Washed 3 times with PBS, anti-human Fc-FITC antibody (Sigma) was added to the cells at a concentration of 1:40 and incubated for 30min at room temperature. After washing 3 times with PBS, the cells were resuspended in 200ul PBS and then tested on the machine (BD FACSAria)^TMⅡ)。

Three, result in

1.1 construction of humanized anti-New coronavirus surface antigen antibody library

Successfully constructing 4 Kappa libraries and 1 Lambda library with the library capacity of 1 × 10⁷Above, the insertion rate of the light and heavy chains is above 90%. Respectively packaging Kappa and Lambda seed libraries, mixing at a ratio of 1:1, eluting with RBD, S1 and S2 proteins of new purified coronavirus, and gradually increasing library volume600 clones, ELISA-detected and sequenced to find 12 antibody clones with different sequences, as shown in Table 1.

TABLE 1 antibody library packaging and enrichment screening throughput

1.2 specific binding of humanized anti-Neocoronavirus Fab antibodies to Neocrown surface antigen

ELISA detection results of 12 strain clone Fab induced lysate anti-human Fab and new crown RBD antigen, S1 antigen and S2 antigen are shown (figure 1), wherein the titers of nCoV-121 antibody anti-human Fab, new crown RBD antigen and S1 antigen are all higher, so that the strain is selected to construct IgG whole antibody.

2. Sequence analysis of human anti-neocoronavirus surface antigen antibody

Analyzing by DNAstar Lasergene software, comparing with IgG sequence in Internet V-Base gene bank, and screening out 12 Fab antibodies with different sequences. The total number of 6 heavy chains and 12 light chains. One of the heavy chain variable regions was classified under VH3, and the light chain variable region was classified under VL6 and named nCoV-121. The amino acid sequences of the light chain variable region and the heavy chain variable region are respectively shown as

SEQ ID NO

1 and 2, and the nucleotide sequences of the coding light chain variable region and the heavy chain variable region are respectively shown as

SEQ ID NO

3 and 4.

3. Construction and expression purification of whole antibody recombinant expression plasmid

Respectively cloning the variable region genes of the light chain and the heavy chain of the nCoV-121 antibody into a whole antibody expression vector, then transiently transfecting an Expi293F cell, and realizing the secretory expression of whole antibody IgG by using the mammalian cell system. The expression supernatant was directly purified by Protein-G affinity column chromatography of GE company, and the expression and purification of whole IgG antibody were checked by SDS-PAGE, which confirmed that a relatively pure Protein was obtained, and that the light and heavy chains of the antibody after melting were located at about 28kD and 55kD, respectively (FIG. 2). The amino acid sequences of the light chain and the heavy chain of the whole antibody IgG are respectively shown in SEQ ID NO. 5-6, and the nucleotide sequences of the coding light chain and the coding heavy chain are respectively shown in SEQ ID NO. 7-8.

4. Function detection of novel coronavirus surface antigen genetic engineering IgG antibody

4.1 affinity assay

The biosensor BIAcore (TM) 8000 is used for measuring the affinity of the human monoclonal antibody, the working principle and the basic process of the biosensor are that the surface plasma resonance (surface plasmon resonance) phenomenon is used for monitoring the change of a resonance angle caused by the change of the refractive index of a surface medium of the sensing piece, the change is in direct proportion to the quantity of biomacromolecules on a surface junction of the sensing piece, and free molecules in a solution do not influence the size of the resonance angle, so the biosensor is very specific and sensitive. The detection of any biomacromolecule interaction by a biosensor is carried out by the following steps: association, dissociation and regeneration. The invention uses purified new crown S1 protein as antigen to couple to a biosensing chip to determine the affinity of the humanized monoclonal antibody, and the affinity constant is calculated by Biacore Insight Evaluation as shown in Table 2. nCoV-121 has an affinity for the S protein of the novel coronavirus of < 2.83X 10^-12(FIG. 7).

TABLE 2 measurement of antibody affinity by surface plasmon resonance

4.2 neutralization assay

The result of the virus neutralization experiment shows that the nCoV-121 antibody has neutralization activity, IC₅₀The value was 0.48. mu.g/ml, and the neutralizing activity was good. Meanwhile, after nCoV-61 and nCoV-121 are used together, IC can be obviously reduced₅₀Value (0.12. mu.g/ml) (FIG. 3).

4.3ELISA detection of the binding Activity of antibodies to the surface antigen of the New coronavirus

The nCoV-121 antibody can be combined with novel coronavirus RBD protein, S1 protein and S protein, and the result of ELISA detection performed on the nCoV-121 antibody and the proteins is still positive after the nCoV-121 antibody is diluted by 64000 times by taking 0.2mg/ml as an initial dilution concentration, which indicates that the nCoV-121 antibody and the proteins have good binding activity (figure 4).

4.4 immunofluorescence detection of binding Activity of genetically engineered antibodies with novel coronavirus, novel coronavirus RBD protein and S protein

The indirect immunofluorescence assay of nCoV-121 antibody was positive for both the novel coronavirus, the novel coronavirus RBD protein expressed in the cells, and the S protein, indicating that nCoV-121 antibody binds to the antigen (FIG. 5).

4.5 flow cytometry detection of the binding Activity of anti-human novel coronavirus surface antigen genetically engineered antibody and novel coronavirus S protein

The results of flow cytometry for the binding activity of antibody nCoV-121 to the S protein of the novel coronavirus are shown in FIG. 6. The abscissa of the graph shows the FITC fluorescence intensity, and when the antibody binds to the cell surface, a FITC fluorescence signal can be detected. Compared with a control group which is not transfected with the S protein, the experimental group which is transfected with the S protein can see obvious cell grouping, and isotype control antibodies can not see grouping. This indicates that nCoV-121 antibody can bind to a novel coronavirus S protein expressed on the cell surface.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 7

<211> 744

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggttc ttgggccaat 60

tttatgctga ctcagcccca ctctgtgtcg gagtctccgg ggaagacggt aaccatctcc 120

tgcacccgca gcagtggcag cattgccagc aactatgtgc agtggtacca gcagcgcccg 180

ggcagttccc ccaccactgt gatctatgag gataaccaaa gaccctctgg ggtccctgat 240

cggttctctg gctccatcga cagctcctcc aactctgcct ccctcaccat ctctggactg 300

aagactgagg acgaggctga ctactactgt cagtcttatg atagcagcaa tcagtgggtg 360

ttcggcggag ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcggtcact 420

ctgggtcagc ccaaggctgc cccctcggtc actctgttcc cgccctcgag tgaggagctt 480

caagccaaca aggccacact ggtgtgtctc ataagtgact tctacccggg agccgtgaca 540

gtggcctgga aggcagatag cagccccgtc aaggcgggag tggagaccac cacaccctcc 600

aaacaaagca acaacaagta cgcggccagc agctacctga gcctgacgcc tgagcagtgg 660

aagtcccaca gaagctacag ctgccaggtc acgcatgaag ggagcaccgt ggagaagaca 720

gtggccccta cagaatgttc atag 744

<210> 8

<211> 1419

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattccgaa 60

gtgcagctgg tgcagtctgg gggaggcttg gtacagcctg gccggtccct gacactctcc 120

tgtggagcct ctggattcac ctttgaggat tatgccatgc actgggtccg gcaagctcca 180

gggaagggcc tggagtgggt ctcaggtatt gattggaaca gtggtgttat aggctatgcg 240

gactctgtga agggccgatt catcatctcc agagacaacg ccaagaactc cctgtatctg 300

cacatgagaa gtctgacagc agaggacacg gccttgtatt actgtgcaaa agatgtatat 360

agcgaatcgg gttcggggag ttattacgac tattggggcc agggaaccct ggtcaccgtc 420

tcctcagcgt cgaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 480

tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga acctgtgacg 540

gtctcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 600

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 660

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt 720

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 780

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1140

gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1200

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260

cccgtgctgg actccgacgg ctccttcttc ctctatagca agctcaccgt ggacaagagc 1320

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380

tacacgcaga agagcctctc cctgtccccg ggtaaatga 1419

Claims

1. The human anti-SARS-CoV-2 neutralizing antibody nCoV-121 is characterized in that the amino acid sequences of the light chain and heavy chain hypervariable regions CDR1, CDR2 and CDR3 are as follows:

the amino acid sequences of the light chain CDRs 1, CDR2, and CDR3 are SGSIASNY, EDN, and QSYDSSNQWV, respectively;

the amino acid sequences of heavy chain CDR1, CDR2, and CDR3 are GFTFEDYA, IDWNSGVI, and AKDVYSESGSGSYYDY, respectively.

2. The neutralizing antibody of claim 1, wherein the amino acid sequences of the light and heavy chain variable regions are set forth in SEQ ID NO 1 and 2, respectively.

3. A nucleic acid molecule encoding the neutralizing antibody of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequences encoding the light chain variable region and the heavy chain variable region are set forth in SEQ ID NOs 3 and 4, respectively.

5. Biological material comprising a nucleic acid molecule according to claim 3 or 4, said biological material being an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

6. A single chain antibody comprising the hypervariable regions of the light and heavy chains of the neutralizing antibody of claim 1 or 2.

7. A medicament for preventing or treating SARS-CoV-2 infection and related diseases caused by the infection, which comprises the neutralizing antibody according to claim 1 or 2 or the single chain antibody according to claim 6 as an active ingredient.

SARS-CoV-2 detection reagent, characterized in that it comprises the neutralizing antibody according to claim 1 or 2 or the single chain antibody according to claim 6.

9. Use of the neutralizing antibody of claim 1 or 2 or the single chain antibody of claim 6 for any one of the following:

1) for preparing a medicament for preventing or treating SARS-CoV-2 infection and related diseases caused by the infection;

2) is used for preparing a SARS-CoV-2 detection reagent or a reagent kit.