A monoclonal antibody with broad-spectrum neutralizing activity against novel coronavirus and its application

Abstract

The present invention discloses a monoclonal antibody with broad-spectrum neutralizing activity against a novel coronavirus and its application, belonging to the field of immunology and biotechnology. The present invention prepares and screens a monoclonal antibody with broad-spectrum neutralizing activity, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.2; the amino acid sequence of its light chain variable region is shown in SEQ ID NO.4. The present invention also discloses the application of the monoclonal antibody with broad-spectrum neutralizing activity in identifying highly conserved neutralizing antigen epitopes and preparing genetic engineering therapeutic antibodies.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and immunology, and specifically relates to a monoclonal antibody with broad-spectrum neutralizing activity against the novel coronavirus and its application. Background Art

[0002] The novel coronavirus (also known as COVID-19 in the early days) is an infectious disease caused by the novel coronavirus (SARS-CoV-2), which can cause pneumonia, fever, cough and other symptoms, and in severe cases, death. The virus has caused huge losses and impacts on human health and economic activities worldwide. Since the outbreak, researchers around the world have conducted large-scale research on the prevention and control of the disease and have achieved many important results. For example, a variety of therapeutic drugs and preventive vaccines have been approved for use, and these drugs and vaccines have made important contributions to the prevention and control of the epidemic. The novel coronavirus (SARS-CoV-2) is an RNA virus that is prone to mutation. From the initial epidemic strain to the present, hundreds of variant strains have appeared, including dozens of variant strains identified as VOC (variant of concern) by the World Health Organization. The occurrence of these mutations has changed the transmission and infection characteristics of the virus strains, especially some important antigenic epitopes have mutated, resulting in the existing vaccine immune effect being affected, especially some epitope-specific therapeutic antibodies The therapeutic effect has also changed. This immune escape mutation of the virus poses a huge challenge to the research of vaccines and therapeutic antibodies.

[0003] Immune escape mutations in existing technologies result in antibodies being unable to achieve broad spectrum. Summary of the invention

[0004] The purpose of the present invention is to provide a broad-spectrum neutralizing monoclonal antibody against the new coronavirus.

[0005] The present invention provides a monoclonal antibody for a novel coronavirus, wherein the amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody is the amino acid sequence at positions 48 to 54 as shown in SEQ ID NO.2, the amino acid sequence of CDR-H2 is the amino acid sequence at positions 69 to 84 as shown in SEQ ID NO.2; the amino acid sequence of CDR-H3 is the amino acid sequence at positions 117 to 130 as shown in SEQ ID NO.2;

[0006] The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody is the amino acid sequence at positions 44-60 shown in SEQ ID NO.4, the amino acid sequence of CDR-H2 is the amino acid sequence at positions 76-82 shown in SEQ ID NO.4; and the amino acid sequence of CDR-H3 is the amino acid sequence at positions 115-122 shown in SEQ ID NO.4.

[0007] The present invention provides a gene sequence encoding the above-mentioned monoclonal antibody, including a gene sequence encoding the heavy chain variable region CDR-H1, a gene sequence encoding CDR-H2, and a gene sequence encoding CDR-H3 of the monoclonal antibody;

[0008] The monoclonal antibody comprises a light chain variable region CDR-L1 coding gene sequence, a CDR-L2 coding gene sequence, and a CDR-L3 coding gene sequence.

[0009] It is further defined that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.2; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.4.

[0010] The present invention provides a gene sequence encoding the above monoclonal antibody, wherein the gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1; and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.3.

[0011] The present invention provides a recombinant vector for expressing the above monoclonal antibody, wherein the recombinant vector contains the above gene sequence.

[0012] It is further defined that the starting vector of the recombinant vector is a pCAGneo vector.

[0013] The present invention provides a recombinant microbial cell expressing the above monoclonal antibody, wherein the recombinant microbial cell expresses the above recombinant vector.

[0014] It is further defined that the recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

[0015] The present invention provides the use of the above-mentioned monoclonal antibody, the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the preparation of a kit for detecting the new coronavirus.

[0016] The present invention provides the use of the above-mentioned monoclonal antibody, the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the preparation of a kit for treating or diagnosing the new coronavirus.

[0017] Beneficial effects: The present invention uses the RBD protein expressed in the cell line as an antigen to immunize mice, and a monoclonal antibody with broad-spectrum neutralizing activity against the S protein (RBD domain) is obtained after screening. The broad-spectrum monoclonal antibody was sequenced to obtain the amino acid sequences of the variable regions of the antibody heavy chain and light chain. The monoclonal antibody was titered by ELISA. The monoclonal antibody titer in the cultured cell supernatant was 1:1280, and the monoclonal antibody titer in the mouse ascites was 1:204800. The affinity constant of the monoclonal antibody binding to O-RBD is kD=1.17×10 -7 Mol. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 To express and purify the RBD protein of the new coronavirus (SARS-COV-2) for electrophoresis analysis;

[0019] Figure 2 The western blot results of monoclonal antibodies and three variants of the new coronavirus RBD protein. E2 is the swine fever E2 protein, which serves as a negative control;

[0020] Figure 3 This is the result of indirect immunofluorescence reaction between monoclonal antibody 22.9-1 and RBD protein in cells;

[0021] Figure 4 These are the results of the virus neutralization test of monoclonal antibody 22.9-1. DETAILED DESCRIPTION

[0022] Example 1: Expression and purification of novel coronavirus RBD protein

[0023] With reference to the S gene sequence of the SARS-COV-2Omicron variant (GISAID database gene sequence number: EPI_ISL_7160037, GISAID database website: https: / / gisaid.org / ), the gene sequence encoding the RBD region was codon-optimized, a signal peptide gene sequence (atgaagttctcctgggtgatgttcttcctgatggccgtggtgaccggcgtgaactcc) was added to the 5' end, and two 6×His tag coding sequences were added to the 3' end to form an expression cassette. The optimized expression cassette gene sequence is shown in SEQ ID NO.3, and the codon-optimized gene sequence is named opti-O-RBD. The opti-O-RBD gene was synthesized by a biological company and cloned into the eukaryotic expression vector pCAGneo ((Hua et al., 2014. Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate. DOI: 10.1186 / 1472-6750-14-62)) between the SacI and BglII restriction sites to construct the recombinant expression plasmid pCAG-opti-O-RBD. The recombinant plasmid pCAG-opti-O-RBD was transfected into BHK-21 cells, and the transfected cells were selected and cultured in a medium containing G418 to obtain a stable expression cell line B-ORBD. After the stable expression cell line was expanded and cultured, the cell culture supernatant was harvested, and the cell expression supernatant was purified by affinity chromatography using Ni-NTA resin. The purified O-RBD protein was analyzed by SDS-PAGE electrophoresis, indicating that the purified S protein ( Figure 1 ).

[0024] Example 2: Preparation and identification of monoclonal antibodies

[0025] The purified recombinant protein O-RBD obtained in Example 1 was used as an immunogen to immunize 6-week-old BALB / c mice, and conventional spleen lymphocyte hybridoma technology was used for fusion. After cloning by limiting dilution method, a hybridoma cell that secretes specific SARS-COV-2RBD protein was obtained and named 22.9-1. The monoclonal antibody was identified as IgG1 / κ type by a subtype identification kit. The monoclonal antibody was titered by ELISA method. The monoclonal antibody titer in the culture cell supernatant was 1:1280, and the monoclonal antibody titer in the mouse ascites was 1:204800. The affinity constant of the monoclonal antibody binding to O-RBD is kD=1.17×10 -7 Mol. The ELISA test of the monoclonal antibody showed that the monoclonal antibody could react with wild-type RBD protein (WT-RBD), Delta variant RBD protein (D-RBD), and Omicron variant (O-RBD) at the same time, and the results are shown in Table 1.

[0026] Both WT-RBD and D-RBD were prepared and provided by the Zoonotic Disease Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The expression and purification methods of WT-RBD (GenBank accession No. NC_045512) and D-RBD (GISAID, EPI_ISL_2863933) proteins were the same as those in Example 1. Western blot detection showed that both could specifically react with RBD proteins of different variants of SARS-COV-2. The test results are shown in Figure 2 Indirect immunofluorescence assay also showed that the monoclonal antibodies were able to recognize SARS-COV-2RBD protein expressed by cells. Figure 3 .

[0027] Identification of monoclonal antibody virus neutralization activity: Antibody neutralization test was performed using a chimeric virus expressing the S protein of the new coronavirus, VSVΔG-GFP. The four chimeric viruses expressing the S protein of the wild-type (WT), Delta (D), BA.1 and BA.2 variants were prepared, identified and provided by the zoonosis team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The preparation method was carried out according to the literature method (Shan, D. et al. Immunogenicity of a recombinant VSV-Vectored SARS-CoV vaccine induced robust immunity in rhesus monkeys after single-dose immunization. Virol. Sin. 37, 248-255 (2022).).

[0028] The experimental process is: the serially diluted monoclonal antibody is mixed with 100 TCID 50 The various chimeric viruses were incubated at 37°C for 1 hour. The virus-antibody complex was then added to the Vero E6 cells that had grown into a monolayer in a 96-well plate. Negative and positive controls were set up. The cells were cultured in a 5% CO2 incubator at 37°C. After 24 hours, the green fluorescence signal was observed and recorded. The results are shown in Figure 2. Figure 4 As shown, monoclonal antibody 22.9-1 has a good neutralizing effect on the four variants of the new coronavirus, and the neutralization titer is more than 1,000 times.

[0029] Table 1 ELISA reaction results of monoclonal antibodies and three novel coronavirus RBD proteins

[0030]

[0031] Note: The coated antigens are all RBD proteins of different mutant strains expressed in cell lines, and the coating amount is 2μg / ml. The remaining operation steps are the same as the conventional ELISA method. The test is set up with 3 replicate wells.

[0032] Example 3: Determination of monoclonal antibody variable region gene sequence

[0033] The monoclonal antibody obtained in Experimental Example 2 has a broad-spectrum virus neutralizing effect on a variety of new coronaviruses. The site of antibody-antigen interaction is mainly present in the Fab structure formed by the heavy chain variable region and the light chain variable region. In order to determine the gene sequence of the light and heavy chain variable regions of the monoclonal antibody, the hybridoma cells 22.9-1 in the logarithmic growth phase were first washed once with PBS, the cell pellets were collected after centrifugation, and then the cell pellets were lysed with 1ml Trizol reagent. The hybridoma cell lysate was sent to a biotechnology company for total RNA extraction and sequencing of the light and heavy chain variable regions of mouse IgG1 / Kappa antibodies. The monoclonal antibody heavy chain variable region gene sequence is shown in SEQ ID NO 1.

[0034] SEQ ID NO.1:

[0035] ATGAACTTCGGGTTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGA

[0036] AGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACT

[0037] TTCCTGTTCAGCCTCTGGATTCACTTTAAATAGTTATGCCATGTCTTGGGTTCGCCAGAC

[0038] TCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTACTACTGGTGGTTACACCTACTATC

[0039] TTGACAGTGTGAAGGGCCGATTTACCATCTCCAGAGAGAATGCCAGGAACATCCTGTA

[0040] CCTGCAAATGAGCAGTCTGCGATCTGAGGACACGGCCATGTATTACTGTGCAAGAGGC

[0041] CGGGGCAGCTATGATTACGAAACTGGGACATTTGCTTATTGGGGCCAAGGGACTCTGGT

[0042] CACTGTCTCTGCA

[0043] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2.

[0044] SEQ ID NO.2:

[0045] MNFGFSLIFLVLVLKGVQCEVKLVESGGGLVKPGGSLKLSCSASGFTLNSYAMSWVRQTPE

[0046] KRLEWVASITTGGYTYYLDSVKGRFTISRENARNILYLQMSSLRSEDTAMYYCARGRGSY

[0047] DYETGTFAYWGQGTLVTVSA

[0048] The specific domain division of the heavy chain variable region is shown in Table 2:

[0049] Table 2 Monoclonal antibody heavy chain variable region domain

[0050] Domain Signal peptide FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Amino acid sequence 1-19 20-47 48-54 55-68 69-84 85-116 117-130 131-141

[0051] The monoclonal antibody light chain variable region gene sequence is shown in SEQ ID NO 3.

[0052] SEQ ID NO.3:

[0053] ATGGATTCACAGGCCCAGGTTCTTATATTGCTGCTGCTATGGGTATCTGGTACCTGTGGG

[0054] GACATTTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCA

[0055] GTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAATTACTT

[0056] GGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCC

[0057] ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCAC

[0058] TCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTT

[0059] ATAATCTTCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0060] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0061] SEQ ID NO.4:

[0062] MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVSMSCKSSQSLLNSRTRKNYLA

[0063] WYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLP

[0064] TFGGGTKLEIK

[0065] The specific domains of the light chain variable region are divided as shown in Table 3:

[0066] Table 3 Monoclonal antibody light chain variable region domain

[0067] Domain Signal peptide FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Amino acid sequence 1-20 21-43 44-60 61-75 76-82 83-114 115-122 123-132

[0068] Example 4: Preparation of genetically engineered humanized antibodies

[0069] Construction of humanized monoclonal antibody 22.9-1 expression plasmid: The monoclonal antibody heavy chain variable region amino acid sequence obtained in Example 3 was combined with the human IgG1 heavy chain constant region sequence (reference sequence number: QDA39867) to obtain the monoclonal antibody heavy chain amino acid sequence shown in SEQ ID NO.5; the coding codon of SEQ ID NO.5 sequence was optimized to obtain the monoclonal antibody heavy chain gene sequence shown in SEQ ID NO.6. The SEQ ID NO.6 gene sequence was artificially synthesized and cloned into the SacI and XhoI cloning sites of the eukaryotic expression vector pCAGneo (Hua et al., 2014. Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate. DOI: 10.1186 / 1472-6750-14-62), and the monoclonal antibody heavy chain expression plasmid was verified by sequencing. The amino acid sequence of the light chain variable region of the monoclonal antibody obtained in Example 3 and the human IgG Kappal light chain constant region (reference sequence number: CAA75031) were connected to obtain the complete light chain amino acid sequence SEQ ID NO.7. The gene sequence encoding the protein of SEQ ID NO.7 was codon optimized to obtain the gene coding sequence shown in SEQ ID NO.8. The gene sequence of SEQ ID NO.8 was artificially synthesized and cloned between the SacI and XhoI sites of the eukaryotic expression plasmid pCAGneo to construct an antibody light chain expression plasmid, and the plasmid was sequenced for verification.

[0070] Transfect cells to express antibodies: Transform the correctly sequenced plasmid into DH5α bacterial cells, pick a single colony for expansion culture, and use a plasmid extraction kit to extract the plasmid. The extracted plasmid is frozen and stored for later use after the concentration is determined. HEK293 cells are expanded and subcultured, and HEK293 cells are cultured in a 37°C incubator containing 5% CO2. The culture is DMEM containing 10% fetal bovine serum. Transfection is performed when HEK293 cells grow to 80% to 90% full. During transfection, the heavy chain and light chain expression plasmids are mixed in a ratio of 1:1, and the cells are transfected with PEI transfection reagent. 12 hours after transfection, the cell culture medium is replaced with serum-free cell culture medium, and the expression is continued. When the cell activity decreases significantly and nearly 40% of the cells fall off and die, the cell culture medium is collected. The recombinant full-length antibody in the cell supernatant is purified by protein A affinity chromatography, and the purified antibody is measured and then aliquoted and frozen for later use.

[0071] Expression antibody activity detection: The recombinant RBD protein (WT-RBD, D-RBD, O-RBD) was coated on the ELISA plate with carbonate buffer at a concentration of 2 μg / ml, and blocked overnight at 4°C. The coated plate was washed three times with PBST and blocked with 5% skim milk, and blocked overnight at 4°C. The blocked coated plate was washed three times with PBST, and the purified humanized monoclonal antibody diluted in multiples was added, and the reaction was performed at 37°C for 1 hour, and washed three times with PBST; 10000 times diluted HRP-labeled goat anti-human IgG secondary antibody was added, and the reaction was performed at 37°C for 30 minutes, and PBST was washed three times, and 100 μl TMB substrate solution was added to each well for color development, and the color was developed at room temperature for 8 minutes, and 100 μl of 2M sulfuric acid stop solution was added to each well. The OD450 value was measured within 10 minutes. The results are shown in Table 4. The recombinant humanized monoclonal antibody maintained reactivity with the three RBD proteins.

[0072] Table 4 ELISA reactivity of recombinant humanized monoclonal antibodies with RBD protein

[0073]

[0074]

[0075] SEQ ID NO.5 (humanized monoclonal antibody heavy chain amino acid sequence)

[0076] MNFGFSLIFLVLVLKGVQCEVKLVESGGGLVKPGGSLKLSCSASGFTLNSYAMSWVRQTPE

[0077] KRLEWVASITTGGYTYYLDSVKGRFTISRENARNILYLQMSSLRSEDTAMYYCARGRGSY

[0078] DYETGTFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW

[0079] NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0080] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG

[0081] VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0082] QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

[0083] SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO.6 (Codon-optimized humanized monoclonal antibody heavy chain gene sequence)

[0084] atgaacttcggcttctccctgatcttcctggtgctggtgctgaagggcgtgcagtgcgaggtgaagctggtggagtccggcggcggcctggtg

[0085] aagcccggcggctccctgaagctgtcctgctccgcctccggcttcaccctgaactcctacgccatgtcctgggtgcgccagacccccgagaa

[0086] gcgcctggagtgggtggcctccatcaccaccggcggctacacctactacctggactccgtgaagggccgcttcaccatctcccgcgagaac

[0087] gcccgcaacatcctgtacctgcagatgtcctccctgcgctccgaggacaccgccatgtactactgcgcccgcggccgcggctcctacgacta

[0088] cgagaccggcaccttcgcctactggggccagggcaccctggtgaccgtgtcctccgcctccaccaagggcccctccgtgttccccctggccc

[0089] cctcctccaagtccacctccggcggcaccgccgccctgggctgcctggtgaaggactacttccccgagcccgtgaccgtgtcctggaactcc

[0090] ggcgccctgacctccggcgtgcacaccttccccgccgtgctgcagtcctccggcctgtactccctgtcctccgtggtgaccgtgccctcctcct

[0091] ccctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagaaggtggagcccaagtcctgcgaca

[0092] agacccacacctgccccccctgccccgcccccgagctgctgggcggcccctccgtgttcctgttcccccccaagcccaaggacaccctgat

[0093] gatctcccgcacccccgaggtgacctgcgtggtggtggacgtgtcccacgaggaccccgaggtgaagttcaactggtacgtggacggcgtg

[0094] gaggtgcacaacgccaagaccaagccccgcgaggagcagtacaactccacctaccgcgtggtgtccgtgctgaccgtgctgcaccaggac

[0095] tggctgaacggcaaggagtacaagtgcaaggtgtccaacaaggccctgcccgcccccatcgagaagaccatctccaaggccaagggccag

[0096] ccccgcgagccccaggtgtacaccctgcccccctcccgcgacgagctgaccaagaaccaggtgtccctgacctgcctggtgaagggcttct

[0097] acccctccgacatcgccgtggagtgggagtccaacggccagcccgagaacaactacaagaccaccccccccgtgctggactccgacggct

[0098] ccttcttcctgtactccaagctgaccgtggacaagtcccgctggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaa

[0099] ccactacacccagaagtccctgtccctgtcccccggcaagtaa

[0100] SEQ ID NO.7 (Amino acid sequence of humanized monoclonal antibody light chain)

[0101] MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVSMSCKSSQSLLNSRTRKNYLA

[0102] WYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLP

[0103] TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0104] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0105] SEQ ID NO.8 (Codon-optimized humanized monoclonal antibody light chain gene sequence)

[0106] Atggactcccaggcccaggtgctgatcctgctgctgctgtgggtgtccggcacctgcggcgacatcgtgatgtcccagtccccctcctccctg

[0107] gccgtgtccgccggcgagaaggtgtccatgtcctgcaagtcctcccagtccctgctgaactcccgcacccgcaagaactacctggcctggta

[0108] ccagcagaagcccggccagtcccccaagctgctgatctactgggcctccacccgcgagtccggcgtgcccgaccgcttcaccggctccgg

[0109] ctccggcaccgacttcaccctgaccatctcctccgtgcaggccgaggacctggccgtgtactactgcaagcagtcctacaacctgcccacctt

[0110] cggcggcggcaccaagctggagatcaagcgcaccgtggccgccccctccgtgttcatcttccccccctccgacgagcagctgaagtccggc

[0111] accgcctccgtggtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaact

[0112] cccaggagtccgtgaccgagcaggactccaaggactccacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagca

[0113] caaggtgtacgcctgcgaggtgacccaccagggcctgtcctcccccgtgaccaagtccttcaaccgcggcgagtgc。

Claims

1. A monoclonal antibody with broad-spectrum neutralizing activity against the novel coronavirus, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

4.

2. A nucleic acid molecule encoding the monoclonal antibody according to claim 1.

3. A nucleic acid molecule encoding the monoclonal antibody according to claim 1, characterized in that: The nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1; the nucleic acid molecule encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

3.

4. A recombinant vector expressing the monoclonal antibody according to claim 1, characterized in that: The recombinant vector contains the nucleic acid molecule according to claim 3.

5. A recombinant microbial cell expressing the monoclonal antibody according to claim 1, characterized in that: The recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

6. A mammalian cell line expressing the monoclonal antibody according to claim 1, characterized in that: The mammalian cell line is HEK293 cells.

7. Use of the monoclonal antibody according to claim 1, the recombinant vector according to claim 4, or the recombinant microbial cell according to claim 5 in the preparation of a reagent for detecting a new coronavirus.

8. Use of the monoclonal antibody according to claim 1, the recombinant vector according to claim 4, or the recombinant microbial cell according to claim 5 in the preparation of an agent for treating the new coronavirus.

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