Anti-coronavirus monoclonal antibody and application thereof

Abstract

The invention relates to the field of medicine, in particular to an anti-coronavirus monoclonal antibody and application thereof in detection of coronavirus. The anti-coronavirus monoclonal antibody at least has one of the following technical characteristics: (1) the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the amino acid sequence of the heavy chain CDR1 is GFAFSSYA; the amino acid sequence of the heavy chain CDR2 is ISSYEGRNT; the amino acid sequence of the heavy chain CDR3 is AKERDDILTGFQDYFDY, and the amino acid sequence of the heavy chain CDR3 is The light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3, and the amino acid sequence of the light chain CDR1 is QDIAGW; the amino acid sequence of the light chain CDR2 is DAS; and the amino acid sequence of the light chain CDR3 is HQSNSFPYT. The antibody provided by the invention can effectively recognize various coronaviruses and mutants.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an anti-pan-coronavirus monoclonal antibody and an application thereof in detecting pan-coronavirus. Background Art

[0002] The well-known high-mortality coronaviruses (SARS, 9.5%; MERS, 34.4%; COVID-19, ~1-2%) are all transmitted from animals across species, causing severe respiratory infections in humans, posing a major threat to public health and safety and a serious social and economic burden. These three coronaviruses all use bats as natural hosts, and then infect intermediate host animals (the intermediate hosts of SARS-CoV and MERS-CoV are civets and camels, respectively, while SARS-CoV-2 may be bats and pangolins), obtain virus recombination products, and then spread across species. However, SARS-CoV-2 is unlikely to be the last coronavirus spillover infection event. Based on differences in protein sequences, coronaviruses can be divided into four genera - Alph coronavirus, Beta coronavirus, Gamma virus, and Delta coronavirus, with bats being the main host of coronaviruses. Serological evidence of human exposure to bat coronaviruses in rural China shows that there is a frequent bat coronavirus spillover effect, but it has not yet caused a pandemic. In addition to wild animals, domesticated livestock or pets also have the ability to spread the virus. For example, ferrets and cats are susceptible to SARS-CoV-2, especially cats can spread it between cats through respiratory droplets, indicating that coronavirus can spread among animals and can quickly adapt to new hosts. Different coronavirus strains can produce new variants through natural recombination, which can shuttle between animals and humans, leading to a potential new round of viral infection outbreaks.

[0003] In addition, another issue worth noting is that there is a time lag between new human coronavirus infections and the development of new treatments or preventive vaccines. Therefore, it is necessary to develop methods that can be used to detect future new coronavirus infections, establish a prediction and early warning mechanism, and conduct regular monitoring to achieve prediction and early warning of possible new coronavirus infections.

[0004] Prior art CN 114409774 A discloses a broad-spectrum humanized monoclonal neutralizing antibody against SARS-COV-2 virus. The neutralizing antibody is obtained by screening by single B cell flow sorting-antibody gene amplification paired expression technology, has a unique CDR region, can specifically bind to SARS-COV-2 and can effectively neutralize the "variants of concern" (SARS-CoV-2WT, alpha, Beta, Gamma, Delta, Omicron) currently defined by the World Health Organization, especially the IC50 of neutralizing Omicron variant strains is less than 0.1 μg / mL. The present invention also relates to the preparation method and use of this group of neutralizing antibodies. The 4 antibodies of this invention can be used for emergency prevention and or treatment of the global epidemic of COVID-19 variant strains. However, it is a modified body that connects the antibody to ACE2, and its preparation process is complicated. Prior art CN 114716541 A discloses a full-human broad-spectrum neutralizing antibody against coronavirus and its application. Specifically, this invention discloses a fully human monoclonal antibody 76E1 targeting the S2 region of the coronavirus S protein, a nucleic acid sequence encoding the antibody and antibody fragments, and a method for preparing the same. In vitro and in vivo experiments have confirmed that the 76E1 antibody can effectively and broadly bind to and neutralize a variety of coronaviruses, prevent and control coronavirus infections, has low immunogenicity to the human body, and can be used clinically to prevent and treat coronavirus infection-related diseases such as SARS-CoV-2, as well as its potential application as a vaccine design. However, the antibody only has binding ability to seven human coronaviruses, and the EC 50 Between 40-300ng / mL, binding performance needs to be optimized. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-pan-coronavirus monoclonal antibody and its application in detecting pan-coronavirus. The anti-pan-coronavirus monoclonal antibody can effectively identify coronaviruses of various animal and human origins and has a strong and broad-spectrum binding ability.

[0006] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0007] An anti-pan-coronavirus monoclonal antibody, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region, and the anti-pan-coronavirus monoclonal antibody has at least one of the following technical features:

[0008] (1) the heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the amino acid sequence of heavy chain CDR1 is GFAFSSYA; the amino acid sequence of heavy chain CDR2 is ISYEGRNT; the amino acid sequence of heavy chain CDR3 is AKERDDILTGFQDYFDY; the light chain variable region includes light chain CDR1, light chain CDR2 and light chain CDR3, the amino acid sequence of light chain CDR1 is QDIAGW; the amino acid sequence of light chain CDR2 is DAS; the amino acid sequence of light chain CDR3 is HQSNSFPYT;

[0009] (2) the heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the amino acid sequence of the heavy chain CDR1 is GFTFRDAW, the amino acid sequence of the heavy chain CDR2 is IKQDGSEK, and the amino acid sequence of the heavy chain CDR3 is ARDLILSP; the light chain variable region includes a light chain CDR1, a light chain CDR2 and a light chain CDR3, the amino acid sequence of the light chain CDR1 is SANIGRNY, the amino acid sequence of the light chain CDR2 is WNN, and the amino acid sequence of the light chain CDR3 is AAWDNRLSAYV;

[0010] (3) the heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the amino acid sequence of the heavy chain CDR1 is GFTFSDYP, the amino acid sequence of the heavy chain CDR2 is ISYDAANE, and the amino acid sequence of the heavy chain CDR3 is ARASYDSKWSVGAALDM; the light chain variable region includes a light chain CDR1, a light chain CDR2 and a light chain CDR3, the amino acid sequence of the light chain CDR1 is QSVLLSSNNKNY, the amino acid sequence of the light chain CDR2 is WAS, and the amino acid sequence of the light chain CDR3 is QQYYSIPQT;

[0011] (4) the heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the amino acid sequence of the heavy chain CDR1 is GFTFSRYA, the amino acid sequence of the heavy chain CDR2 is VSFDGRNV, and the amino acid sequence of the heavy chain CDR3 is VRDSYIVASEDGAFDI; the light chain variable region includes a light chain CDR1, a light chain CDR2 and a light chain CDR3, the amino acid sequence of the light chain CDR1 is QRLTSR, the amino acid sequence of the light chain CDR2 is GAS, and the amino acid sequence of the light chain CDR3 is QQYDDWPLIS;

[0012] (5) The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the amino acid sequence of heavy chain CDR1 is GFNFDDYA, the amino acid sequence of heavy chain CDR2 is ISSSGKGT, and the amino acid sequence of heavy chain CDR3 is VKGGRSDSSGNPYFYFMEV; the light chain variable region includes light chain CDR1, light chain CDR2 and light chain CDR3, the amino acid sequence of light chain CDR1 is QSISPH, the amino acid sequence of light chain CDR2 is GAS, and the amino acid sequence of light chain CDR3 is QEYNDWPLFLT.

[0013] In one preferred embodiment, the heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is QVQLVESGGGVVQPGRSLRLSCAAS, the amino acid sequence of heavy chain FR2 is IHWVRQAPGKGLEWVAV, the amino acid sequence of heavy chain FR3 is YYADSVKGRCTISRDNSRNAVYLQMDSLRPEDTAVYYC, and the amino acid sequence of heavy chain FR4 is WGQGTLVTVSS.

[0014] In one preferred embodiment, the light chain further includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4, the amino acid sequence of light chain FR1 is DIQMTQSPSSVSVSVGDRVTITCRAS, the amino acid sequence of light chain FR2 is IAWYQQKPGTAPKLLIY, the amino acid sequence of light chain FR3 is TLQSGVPSRFSGSGSGTHFTLTINNLQPEDFATYYC, and the amino acid sequence of light chain FR4 is FGQGTRLQIK.

[0015] In one preferred embodiment, the heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is EVQLVESGGGLVQPGGSLRLSCAAS, the amino acid sequence of heavy chain FR2 is MNWVRQAPGKGLEWVAN, the amino acid sequence of heavy chain FR3 is YYLDSVRGRFTISRDNAKSSVYLQMNSLRVEDTALYYC, and the amino acid sequence of heavy chain FR4 is FPQGVLVTVSS.

[0016] In one preferred embodiment, the light chain further includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4; the amino acid sequence of light chain FR1 is QSVLTQPPAVSGTPGQRVIISCSGS, the amino acid sequence of light chain FR2 is VYWFQILPGTAPKLLIY, the amino acid sequence of light chain FR3 is QRPSGVPDRFSGSKSGTSGSLAISGLRSEDEADYYC, and the amino acid sequence of light chain FR4 is FATGTKVTVL.

[0017] In one preferred embodiment, the heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is QVQLVESGGGVVQPGRSLRLSCAAS, the amino acid sequence of heavy chain FR2 is MHWVRLSPGKGLQWVAV, the amino acid sequence of heavy chain FR3 is FYADSVKGRFTISRDTLKSTLYLQMDTLSAEDTAVYYC, and the amino acid sequence of heavy chain FR4 is WGLGTRVTVSS.

[0018] In one preferred embodiment, the light chain further includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4; the amino acid sequence of light chain FR1 is DIQMTQSPDSLAVSLGERATLNCKSS, the amino acid sequence of light chain FR2 is LAWYQQKPGQPPKLLIH, the amino acid sequence of light chain FR3 is TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC, and the amino acid sequence of light chain FR4 is FGQGTKLEIK.

[0019] In one preferred embodiment, the heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is QVQLVESGGGVVQPGRSLRLSCAAS, the amino acid sequence of heavy chain FR2 is IHWVRQAPGRGLEWVSI, the amino acid sequence of heavy chain FR3 is NYAGAVRGRFTISRDSSKKTVYLQMNRLRSEDTAVYYC, and the amino acid sequence of heavy chain FR4 is WGQGTMVTVSS.

[0020] In one preferred embodiment, the light chain also includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4; the amino acid sequence of light chain FR1 is EIVMTQSPATLSVSPGESATLACRAS, the amino acid sequence of light chain FR2 is LAWYQQKPGQAPRLLIY, the amino acid sequence of light chain FR3 is TRAPGIPARFSGSGSGTEFSLTISSLQSEDSALYYC, and the amino acid sequence of light chain FR4 is FGQGTRLEIK.

[0021] In one preferred embodiment, the heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is EVQLVESGGDLVQPGGSLRLSCSAF, the amino acid sequence of heavy chain FR2 is MHWVRQAPGKGLNCVSV, the amino acid sequence of heavy chain FR3 is TYAEAVKGRFSVSRDNVKNTVSLQMNSLRAEDTGIYYC, and the amino acid sequence of heavy chain FR4 is WGKGTMVTVSS.

[0022] In one preferred embodiment, the light chain further includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4; the amino acid sequence of light chain FR1 is EIVMTQSPATLSVSPGERATLSCRAS, the amino acid sequence of light chain FR2 is LAWYQQKPGQSPRLLIY, the amino acid sequence of light chain FR3 is IRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC, and the amino acid sequence of light chain FR4 is FGGGTKVEIK.

[0023] In one preferred embodiment, the anti-pan-coronavirus monoclonal antibody has at least one of the following technical features:

[0024] (1) The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 2;

[0025] (2) the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 3; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 4;

[0026] (3) the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 5; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 6;

[0027] (4) the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 8;

[0028] (5) The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 9; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 10.

[0029] SEQ ID NO: 1:

[0030] QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYAIHWVRQAPGKGLEWVAVISYEGRNTYYAD SVKGRCTISRDNSRNAVYLQMDSLRPEDTAVYYCAKERDDILTGFQDYFDYWGQGTLVTVSS.

[0031] SEQ ID NO: 2:

[0032] DIQMTQSPSSVSVSVGDRVTITCRASQDIAGWIAWYQQKPGTAPKLLIYDASTLQSGVPSRFSGSGSGTHFTLTINNLQPEDFATYYCHQSNSFPYTFGQGTRLQIK.

[0033] SEQ ID NO: 3:

[0034] EVQLVESGGGLVQPGGSLRLSCAASGFTFRDAWMNWVRQAPGKGLEWVANIKQDGSEKYYL DSVRGRFTISRDNAKSSVYLQMNSLRVEDTALYYCARDLILSPFPQGVLVTVSS.

[0035] SEQ ID NO: 4:

[0036] QSVLTQPPAVSGTPGQRVIISCSGSSANIGRNYVYWFQILPGTAPKLLIYWNNQRPSGVPDRFSG SKSGTSGSLAISGLRSEDEADYYCAAWDNRLSAYVFATGTKVTVL.

[0037] SEQ ID NO: 5:

[0038] QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYPMHWVRLSPGKGLQWVAVISYDAANEFYA DSVKGRFTISRDTLKSTLYLQMDTLSAEDTAVYYCARASYDSKWSVGAALDMWGLGTRVTVSS。

[0039] SEQ ID NO:6:

[0040] DIQMTQSPDSLAVSLGERATLNCKSSQSVLLSSNNKNYLAWYQQKPGQPPKLLIHWASTRESG VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPQTFGQGTKLEIK。

[0041] SEQ ID NO:7:

[0042] QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYAIHWVRQAPGRGLEWVSIVSFDGRNVNYAG AVRGRFTISRDSSKKTVYLQMNRLRSEDTAVYYCVRDSYIVASEDGAFDIWGQGTMVTVSS。

[0043] SEQ ID NO:8:

[0044] EIVMTQSPATLSVSPGESATLACRASQRLTSRLAWYQQKPGQAPRLLIYGASTRAPGIPARFSGS GSGTEFSLTISSLQSEDSALYYCQQYDDWPLISFGQGTRLEIK。

[0045] SEQ ID NO:9:

[0046] EVQLVESGGDLVQPGGSLRLSCSAFGFNFDDYAMHWVRQAPGKGLNCVSVISSSGKGTTYAE AVKGRFSVSRDNVKNTVSLQMNSLRAEDTGIYYCVKGGRSDSSGNPYFYFMEVWGKGTMVTVSS。

[0047] SEQ ID NO:10:

[0048] EIVMTQSPATLSVSPGERATLSCRASQSISPHLAWYQQKPGQSPRLLIYGASIRATGIPARFSGSG SGTEFTLTISSLQSEDFAVYYCQEYNDWPLFLTFGGGTKVEIK.

[0049] The present invention also claims to protect the nucleotide encoding the above-mentioned anti-pan-coronavirus monoclonal antibody.

[0050] In a preferred embodiment, the nucleotide comprises the nucleotide sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12.

[0051] In a preferred embodiment, the nucleotide comprises the nucleotide sequence shown in SEQ ID NO: 13 and SEQ ID NO: 14.

[0052] In a preferred embodiment, the nucleotide comprises the nucleotide sequence shown in SEQ ID NO: 15 and SEQ ID NO: 16.

[0053] In a preferred embodiment, the nucleotide comprises the nucleotide sequence shown in SEQ ID NO: 17 and SEQ ID NO: 18.

[0054] In a preferred embodiment, the nucleotide comprises the nucleotide sequence shown in SEQ ID NO: 19 and SEQ ID NO: 20.

[0055] SEQ ID NO: 11:

[0056] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCGCCTTCAGTAGCTATGCCATTCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGAGGGAAGGAATACATACTATGCAGACTCCGTGAAGGGCCGATGCACCATCTCCAGAGACAATTCCAGGAATGCGGTGTATCTGCAAATGGACAGCCTGAGACCTGAGGACACGGCTGTCTATTACTGTGCGAAAGAACGGGACGATATATTGACTGGTTTTCAGGACTACTTTGACTACTGGGGCCAGGGAACCCTCGTCACCGTCTCCTCAGC。

[0057] SEQ ID NO:12:

[0058] GACATCCAGATGACCCAGTCTCCCTCTTCCGTGTCTGTTTCTGTAGGGGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTGCCGGCTGGATAGCCTGGTATCAGCAGAAACCAGGGACAGCCCCTAAGCTCCTGATTTATGATGCCTCCACTTTGCAAAGTGGGGTCCCATCTAGGTTCAGCGGCAGTGGATCTGGGACACATTTCACTCTCACCATCAACAACCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCACCAGTCTAACTCTTTCCCGTACACTTTTGGCCAGGGGACCAGGCTTCAGATCAAA。

[0059] SEQ ID NO:12:

[0060] GAAGTGCAGCTGGTGGAATCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACATTTAGAGACGCTTGGATGAACTGGGTTCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAATATAAAACAAGATGGAAGTGAGAAATACTATCTGGACTCTGTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGTTCAGTCTATCTGCAAATGAACAGCCTGAGAGTCGAAGACACGGCTCTCTATTACTGTGCGAGAGACTTAATTTTATCACCGTTCCCCCAGGGAGTCCTGGTCACCGTCTCCTCAGC。

[0061] SEQ ID NO:14:

[0062] CAGTCTGTGCTGACTCAGCCACCCGCAGTGTCTGGGACCCCCGGGCAGAGGGTCATCATCTCTTGTTCTGGAAGCAGCGCCAACATCGGAAGGAACTATGTATATTGGTTCCAGATCCTCCCAGGAACGGCCCCCAAACTCCTCATCTATTGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGGTTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGACTATTACTGTGCAGCATGGGATAACAGACTGAGTGCTTATGTCTTCGCAACTGGGACCAAGGTCACCGTCCTA。

[0063] SEQ ID NO:15:

[0064] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCTTCAGTGACTATCCTATGCACTGGGTCCGCCTCTCTCCGGGCAAGGGACTGCAGTGGGTGGCAGTTATATCATATGATGCAGCCAATGAGTTCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACACTTTGAAGAGCACGCTGTATCTGCAAATGGACACCCTGAGCGCTGAGGACACGGCTGTGTATTATTGTGCGCGAGCCTCTTATGACAGCAAGTGGTCAGTAGGGGCTGCTCTTGATATGTGGGGCCTGGGGACAAGGGTCACCGTCTCTTCAGC。

[0065] SEQ ID NO:16:

[0066] GACATCCAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCCTCAACTGCAAGTCCAGTCAGAGTGTCTTACTCAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAACTGCTCATTCACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTCCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTATTCCTCAGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA。

[0067] SEQ ID NO:17:

[0068] CAGGTGCAGTTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTCAGTCGCTATGCTATCCACTGGGTCCGCCAGGCTCCAGGCAGGGGTCTGGAGTGGGTGTCAATTGTTTCATTTGATGGAAGGAATGTTAACTATGCAGGCGCCGTGAGGGGCCGATTCACCATCTCCAGAGACAGTTCGAAGAAAACGGTGTATCTGCAAATGAACAGGCTGAGATCTGAGGACACGGCTGTCTATTATTGTGTGAGAGATTCTTATATAGTGGCCTCTGAAGATGGTGCTTTTGACATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGC。

[0069] SEQ ID NO:18:

[0070] GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTCTCTCCTGGGGAAAGCGCCACTCTCGCCTGCAGGGCCAGTCAGCGCCTTACCAGCAGGTTAGCCTGGTACCAGCAGAAGCCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCCCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAATTCTCTCTCACCATCAGCAGCCTGCAGTCTGAGGATTCTGCACTTTATTACTGTCAGCAGTATGATGACTGGCCTTTGATCAGCTTCGGCCAAGGGACACGACTGGAGATTAAA。

[0071] SEQ ID NO:19:

[0072] GAAGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTTCTGCCTTTGGGTTCAACTTCGATGACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGGAAAGGGCTGAATTGTGTTTCAGTCATCAGTTCCAGCGGAAAAGGAACAACCTATGCAGAGGCAG TGAAGGGCAGATTCAGTGTCTCCAGAGACAATGTCAAGAACACAGTGTCTCTTCAAATGAACAGTCTGAGAGCTGAGGACACGGGTATTTATTATTGTGTGAAAGGAGGCCGTAGTGATTCCAGCGGCAACCCCTATTTTTATTTCATGGAAGTCTGGGGCAAGGGGACCATGGTCACCGTCTCCTCAGC.

[0073] SEQ ID NO: 20:

[0074] GAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCGGGGGAAAGAGCCACCCTCTCGTGCAGGGCCAGTCAGAGTATTAGCCCCCACTTAGCCTGGTACCAGCAAAAGCCTGGCCAGTCACCCAGGCTCCTCATCTACGGTGCATCAATCAGGGC CACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTTACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTACTATTGTCAAGAGTATAATGACTGGCCTCTTTTCCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA.

[0075] A recombinant vector comprising the anti-pan-coronavirus monoclonal antibody as described above or a nucleotide encoding the anti-pan-coronavirus monoclonal antibody.

[0076] Use of the anti-pan-coronavirus monoclonal antibody, the nucleotide encoding the anti-pan-coronavirus monoclonal antibody or the recombinant vector as described above in the preparation of a reagent for diagnosing coronavirus or a kit for diagnosing coronavirus.

[0077] The diagnostic reagent can be a protein detection immunoassay strip. In some embodiments, the anti-pan-coronavirus monoclonal antibody can be used for immunological tests such as ELISA, immunochromatography, immunoblotting, and immunofluorescence, providing the required raw materials for immunological detection reagents.

[0078] The detection kit developed with the anti-pan-coronavirus monoclonal antibody, the nucleotide encoding the anti-pan-coronavirus monoclonal antibody or the recombinant vector as raw materials has good application value, high sensitivity and specificity, and can detect coronavirus at an early stage. The vector can be an animal or a human.

[0079] In one preferred embodiment, the diagnostic reagent or diagnostic kit has the function of identifying or binding to a coronavirus; preferably, the coronavirus includes one or more of the novel coronavirus SARS-CoV-2 strain, SARS-CoV strain, MERS-CoV strain, OC43 strain, HKU1 strain, NL63 strain, 229E strain, bat SARS-like coronavirus RaTG13 and WIV1, civet SARS-like coronavirus SZ3 and Civet007, pangolin SARS-like coronavirus GX-P5L and GD18, bovine coronavirus BCoV, red-necked duck coronavirus HKU20, chicken coronavirus IBV, novel coronavirus Alpha mutant, novel coronavirus Beta mutant, novel coronavirus Gamma mutant, novel coronavirus Delta mutant, and novel coronavirus Omicron mutant.

[0080] A recombinant bacterium, wherein the recombinant bacterium contains the anti-pan-coronavirus monoclonal antibody as described above or a nucleotide encoding the anti-pan-coronavirus monoclonal antibody.

[0081] A recombinant cell, the recombinant cell contains the anti-pan-coronavirus monoclonal antibody as described above or a nucleotide encoding the anti-pan-coronavirus monoclonal antibody as described above.

[0082] The present invention also claims a method for preparing the anti-pan-coronavirus monoclonal antibody, comprising the following steps:

[0083] S1. Memory B cells specific for spike proteins were isolated from the peripheral blood of patients who recovered from coronavirus infection.

[0084] S2, amplifying the Ig variable sequence of the memory B cell to obtain the amplified product;

[0085] S3. Construct expression plasmid, perform in vitro transfection, expression, and purification to obtain anti-pan-coronavirus monoclonal antibodies.

[0086] The present invention uses flow cytometry single-cell analysis and sorting technology and single B cell PCR amplification antibody preparation technology to sort single memory B cells specific to the spike protein from PBMCs of recovered patients, directly clone the antibody heavy chain and light chain variable region sequences, construct expression plasmids, and express and purify to obtain pan-coronavirus monoclonal antibodies (SVM7-28, SVM7-75, SVM6-5, SVM6-39 and SSM14-3).

[0087] The pan-coronavirus monoclonal antibody of the present invention is a monoclonal antibody that specifically targets S2, can specifically bind to a variety of human and animal coronaviruses, and has strong binding force. The specific monoclonal antibody can widely bind to a variety of coronavirus spike proteins, including Alpha (NL63 and 229E), Beta (SARS-CoV, MERS-CoV, SARS-CoV-2 and SARS-like coronaviruses, etc.), Gamma (chicken coronavirus IBV) and Delta (ruddy duck coronavirus HKU20) coronaviruses, and is an effective pan-coronavirus monoclonal antibody.

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] 1) The anti-pan-coronavirus monoclonal antibody of the present invention recognizes non-linear conserved epitopes, which are conserved epitopes present in both non-mutated viruses and mutant viruses. Therefore, the anti-pan-coronavirus monoclonal antibody of the present invention can effectively recognize a variety of coronaviruses and popular new coronavirus mutant strains (Alpha, Beta, Gamma, Delta and Omicron), and has a strong and broad-spectrum binding ability.

[0090] 2) The anti-pan-coronavirus monoclonal antibody of the present invention recognizes cross-regional conformational targets, which have the advantages of higher specificity, higher functionality, and wider application in development and detection than linear targets.

[0091] 3) The broad-spectrum pan-coronavirus monoclonal antibody of the present invention can potently and broadly bind to SARS-like coronaviruses from a variety of animal sources and even coronaviruses of the Gamma and Delta genera. These viruses have the ability to spread across species and can be detected at the source for early prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 It is a flow chart of the flow cytometry sorting of SARS-CoV-2S protein-specific memory B cells in the present invention.

[0093] Figure 2 It is a sequence diagram of the heavy chain variable region of the anti-pan-coronavirus antibody SVM7-28 of the present invention.

[0094] Figure 3 It is a sequence diagram of the light chain variable region of the anti-pan-coronavirus antibody SVM7-28 of the present invention.

[0095] Figure 4 It is a sequence diagram of the heavy chain variable region of the anti-pan-coronavirus antibody SSM14-3 of the present invention.

[0096] Figure 5 It is a sequence diagram of the light chain variable region of the anti-pan-coronavirus antibody SSM14-3 of the present invention.

[0097] Figure 6 It is a sequence diagram of the heavy chain variable region of the anti-pan-coronavirus antibody SVM6-5 of the present invention.

[0098] Figure 7 It is a sequence diagram of the light chain variable region of the anti-pan-coronavirus antibody SVM6-5 of the present invention.

[0099] Figure 8 It is a sequence diagram of the heavy chain variable region of the anti-pan-coronavirus antibody SVM6-39 of the present invention.

[0100] Fig. 9 It is a sequence diagram of the light chain variable region of the anti-pan-coronavirus antibody SVM6-39 of the present invention.

[0101] Fig.10 It is a sequence diagram of the heavy chain variable region of the anti-pan-coronavirus antibody SVM7-75 of the present invention.

[0102] Fig.11 It is a sequence diagram of the light chain variable region of the anti-pan-coronavirus antibody SVM7-75 of the present invention.

[0103] Fig.12 This is the binding status of the anti-pan-coronavirus antibodies SVM7-28, SSM14-3, SVM6-5, SVM6-39, and SVM7-75 of the present invention with different subunits of the SARS-CoV-2S protein.

[0104] Fig.13 The present invention relates to the ability of the anti-pan-coronavirus antibodies SVM7-28, SSM14-3, SVM6-5, SVM6-39, and SVM7-75 to bind to denatured coronavirus spike protein.

[0105] Fig.14 This is a graph of the binding of SVM7-28 to seven human coronaviruses, where the horizontal axis is the antibody dilution concentration after log conversion and the vertical axis is the absorbance at a wavelength of 450nm.

[0106] Fig.15This is a graph of the binding of SVM7-28 to the original strain of the new coronavirus, Alpha, Beta, Gamma, Delta, and Omicron mutant strains, where the horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0107] Fig.16 This is a graph showing the binding of SVM7-28 to animal-derived SARS-like coronavirus, bovine coronavirus BCoV, red-necked duck coronavirus HKU20, and chicken coronavirus IBV, where the horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0108] Fig.17 This is a graph of the binding of SSM14-3 to seven human coronaviruses, where the horizontal axis is the antibody dilution concentration after log conversion and the vertical axis is the absorbance at a wavelength of 450nm.

[0109] Fig.18 This is a graph of the binding of SSM14-3 to the original strain of the new coronavirus, Alpha, Beta, Gamma, Delta, and Omicron mutant strains, where the horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0110] Fig.19 This is a graph showing the binding of SSM14-3 to animal-derived SARS-like coronavirus, bovine coronavirus BCoV, red-necked duck coronavirus HKU20, and chicken coronavirus IBV, where the horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0111] Fig. 20 This is a graph of the binding of SVM6-5 to seven human coronaviruses, where the horizontal axis is the log-converted antibody dilution concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0112] Fig.21 This is a graph showing the binding of SVM6-5 to the original strain, Alpha, Beta, Gamma, Delta, and Omicron mutant strains of the new coronavirus. The horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0113] Fig. 22 This is a graph showing the binding of SVM6-5 to animal-derived SARS-like coronavirus, bovine coronavirus BCoV, red-necked duck coronavirus HKU20, and chicken coronavirus IBV, where the horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0114] Fig.23This is a graph of the binding of SVM6-39 to seven human coronaviruses, where the horizontal axis is the log-converted antibody dilution concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0115] Fig.24 This is a graph of the binding of SVM6-39 to the original strain of the new coronavirus, Alpha, Beta, Gamma, Delta, and Omicron mutant strains, where the horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0116] Fig.25 This is a graph showing the binding of SVM6-39 to animal-derived SARS-like coronavirus, bovine coronavirus BCoV, red-necked duck coronavirus HKU20, and chicken coronavirus IBV, where the horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0117] Fig.26 This is a graph of the binding of SVM7-75 to seven human coronaviruses, where the horizontal axis is the log-converted antibody dilution concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0118] Fig. 27 This is a graph of the binding of SVM7-75 to the original strain, Alpha, Beta, Gamma, Delta, and Omicron mutant strains of the new coronavirus. The horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0119] Fig.28 This is a graph showing the binding of SVM7-75 to animal-derived SARS-like coronavirus, bovine coronavirus BCoV, red-necked duck coronavirus HKU20, and chicken coronavirus IBV, where the horizontal axis is the antibody concentration and the vertical axis is the absorbance at a wavelength of 450nm.

[0120] Fig.29 This is a graph of the binding of non-broad-spectrum binding antibodies obtained from the same batch to seven human coronaviruses, where the horizontal axis is the antibody dilution concentration after log conversion and the vertical axis is the absorbance at a wavelength of 450nm.

[0121] Fig.30 This is a graph showing the binding of inactive antibodies obtained from the same batch to seven human coronaviruses, where the horizontal axis is the log-converted antibody dilution concentration and the vertical axis is the absorbance at a wavelength of 450 nm. DETAILED DESCRIPTION

[0122] The present invention will be described in detail below in conjunction with the embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0123] Example 1

[0124] See also Figure 1 The specific method for obtaining pan-coronavirus monoclonal antibodies (SVM7-28, SSM14-3, SVM6-5, SVM6-39, SVM7-75) is as follows:

[0125] (1) Flow cytometry single-cell sorting of SARS-CoV-2-specific memory B cells

[0126] a. Collect peripheral blood from several patients who have recovered from SARS-CoV-2 infection, separate PBMCs, and freeze them in liquid nitrogen for later use;

[0127] b. Resuscitate the collected peripheral blood PBMCs from patients who have recovered from the novel coronavirus SARS-CoV-2 infection from liquid nitrogen;

[0128] c. Add complete medium and incubate at 37°C overnight, wherein the complete medium is 1640 (Gibco, Catalog No.: C11875500CP) medium containing 10% fetal bovine serum (Gibco, Catalog No.: 10270-106);

[0129] d. Then, cells were stained with Live / dead (ThermoFisher, Catalog No.: L34962), CD3 (BD ​​Biosciences, Catalog No.: 612752), CD19 (Biolegend, Catalog No.: 302230), IgD (Biolegend, Catalog No.: 348240), CD27 (Biolegend, Catalog No.: 356412), IgG (BD Biosciences, Catalog No.: 555787), and SARS-CoV-2S probe, which is Alexa Fluor TM SARS-CoV-2S ECD protein (Sino Biological, Catalog No.: 40589-V08B1) labeled with 488 Protein Labeling Kit (ThermoFisher, Catalog No.: A20181);

[0130] e. Stream sorting Live / dead - CD3 - CD19 + IgD - CD27 + IgG + As well as B cells positive for SARS-CoV-2S probe, SARS-CoV-2S protein-specific Memory B cells were obtained.

[0131] (2) Amplification of SARS-CoV-2-specific memory B cell Ig variable region sequences (reference: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.)

[0132] a. Add the prepared cell lysate into a 96-well PCR plate. The system is as follows;

[0133]

[0134]

[0135] b. Add the sorted SARS-CoV-2S protein-specific Memory B cells to a 96-well PCR plate containing the above-mentioned cell lysate;

[0136] c. Place the 96-well PCR plate on dry ice and quickly freeze the lysed cells;

[0137] d. Incubate at 68°C for 1 minute and place on ice;

[0138] e. Configure the cDNA synthesis system as shown in the following table:

[0139]

[0140] f. PCR reaction conditions are: 42℃5min, 25℃10min, 50℃60min, 94℃5min; antibody Ig variable region cDNA is obtained by PCR amplification.

[0141] g. The first round of PCR amplification of Ig variable region, the reaction system is as follows:

[0142]

[0143] The above 5'First PCR primer mix and 3'First PCR primer mix refer to the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.

[0144] The first round of PCR amplification obtained the first round of PCR amplification products, wherein the PCR reaction conditions were: 94°C for 15 min pre-denaturation; 94°C for 30 s, 58°C for 30 s (IgH and Igκ) or 60°C for 30 s (Igλ), 72°C for 55 s amplification for 50 cycles; 72°C for 10 min;

[0145] h. The second round of amplification of Ig variable region, the reaction system is as follows:

[0146]

[0147]

[0148] Among them, the above-mentioned 5'Second PCR primer mix and 3'Second PCR primer mix are both referenced from the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.

[0149] The second round of PCR amplification obtained the second round of PCR amplification products, wherein the PCR reaction conditions were: 94°C for 15 min pre-denaturation; 94°C for 30 s, 58°C for 30 s (IgH and Igκ) or 60°C for 30 s (Igλ), 72°C for 45 s amplification for 50 cycles; 72°C for 10 min;

[0150] i. Recover the second round of PCR amplification products using an agarose gel DNA recovery kit (Tian Gen, Cat. No.: DP209-03);

[0151] j. Specific amplification of Ig variable region, obtaining 5 pairs of specific PCR products, the reaction system is as follows:

[0152]

[0153] Among them, the above-mentioned 5'Specific PCR primer mix and 3'Specific PCR primer mix refer to the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.

[0154] PCR reaction conditions for specific amplification: 94℃15min pre-denaturation; 94℃30s, 58℃30s (IgH and Igκ) or 60℃30s (Igλ), 72℃45s amplification for 50 cycles; 72℃10min; H is the heavy chain vector, κ is the light chain vector, and λ is another light chain vector. Antibodies have heavy and light chains, and light chains are divided into K light chains and λ light chains. Four of the five antibodies are K light chains, and one is a λ light chain (SSM14-3)

[0155] k. Agarose gel electrophoresis was used to detect the specific PCR products of the five pairs of amplifications. The results were as follows: Figure 2-Figure 11 As shown;

[0156] l. Recover 5 pairs of specific PCR products using an agarose gel DNA recovery kit (Tian Gen, catalog number: DP209-03).

[0157] (3) Construction of expression plasmids, in vitro transfection, expression, and purification of monoclonal antibodies

[0158] 3.1 Construction of expression plasmid

[0159] a. Take 30.4 μL of each of the above specific PCR products (IgH, Igκ and Igλ), add 3.4 μL of CutSmart buffer (NEB, catalog number: B7204S) and mix well to obtain the corresponding mixture;

[0160] b. Configure the enzyme digestion system as follows:

[0161]

[0162] Among them, the above-mentioned AgeI-HF, SalI-HF, BsiWI-HF and XhoI are NEB restriction endonucleases, and the product numbers are R3552L (AgeI-HF), R3138L (SalI-HF), R3553L (BsiWI-HF) and R0146L (XhoI), respectively.

[0163] c. Add the corresponding configured mixture of step b to each mixture in step a;

[0164] d. Enzyme digestion at 37°C for 2h;

[0165] e. Use agarose gel DNA recovery kit (Tian Gen, Cat. No.: DP209-03) to recover the digested product;

[0166] f. Connect the specific PCR product after enzyme digestion with the corresponding vector. The connection system is as follows:

[0167]

[0168] Among them, the above-mentioned IgH vector is AbVec2.0-IGHG1 (AddGene, catalog number: 80795), the Igκ vector is AbVec1.1-IGKC (AddGene, catalog number: 80796), and the Igλ vector is AbVec1.1-IGLC2-XhoI (AddGene, catalog number: 99575).

[0169] g. Connect at 16°C overnight;

[0170] h. Add the overnight ligation product to a centrifuge tube containing 100 μL DH5α competent cells (Tian Gen, Catalog No.: CB101-02) and place on ice for 30 minutes;

[0171] i. Place the mixture of the ligation product and competent cells in step h in a 42°C water bath and heat shock for 90 seconds;

[0172] j. Take out and place on ice for 3-5 minutes, then add 900 μL TB medium (ThermoFisher, catalog number: 22711022);

[0173] k. After shaking culture for 45-60 minutes, centrifuge at 6000 rpm for 1 minute;

[0174] l. Aspirate 800 μL of supernatant and use the remaining liquid to mix the bacteria (derived from the competent cells described above);

[0175] m. The bacterial suspension in step l is evenly spread on an agar plate with ampicillin;

[0176] n. Place the agar plate upside down in a 37°C bacterial incubator and culture for 16 hours;

[0177] o. Pick a single full colony on the agar plate and send it to the company for sequencing together with the second round of PCR products;

[0178] p. Select the colonies that match the second round PCR product sequence 100%, use the endotoxin-free plasmid mini-extraction kit (Tian Gen, catalog number: DP118-02) to extract the plasmids, and obtain 5 groups of heavy chain plasmids and light chain plasmids respectively.

[0179] 3.2 In vitro transfection and expression

[0180] a. Use FreeStyle one day before transfection TM 293 expression medium (Gibco, catalog number: 12338018) was used to adjust the 293F cell density to 1×10 6 Pieces / mL;

[0181] b. Count the cells on the day of transfection and use fresh FreeStyle TM 293 expression medium (Gibco, catalog number: 12338018) was used to adjust the 293F cell density to 2×10 6 Pieces / mL;

[0182] c. Prepare PEI mixture: Add Polyethylenimine (PEI) (Polysciences, Catalog No.: 23996-2) to OptiPRO TM SMF medium (ThermoFisher, catalog number: 12309019) was used to make the concentration at the time of transfection 4 μg / mL;

[0183] d. Prepare plasmid mixture: Add the extracted paired heavy chain plasmid and light chain plasmid into OptiPRO TMMix well in SFM medium (ThermoFisher, catalog number: 12309019) to make the total plasmid concentration at the time of transfection 1 μg / mL;

[0184] e. Finally, add the PEI mixture to the plasmid mixture and mix gently to form a transfection mixture system, and let it stand at room temperature for 20 minutes;

[0185] f. Add the transfection mixture in step e to the 293F cells in step b and shake gently to mix;

[0186] g. The 293F cells from step f were placed in a 37°C suspension incubator containing 8% CO2 and cultured at 125 rpm for 7 days.

[0187] 3.3 Purification of monoclonal antibodies

[0188] a. The transfected suspension culture medium was centrifuged at 4000 rpm for 15 min to collect the expression supernatant;

[0189] b. The supernatant was filtered using a 0.22 μm filter (JET, catalog number: FPE204030);

[0190] c. Open Protein purification instrument, use PBS to balance the Protein A column, flow rate 3mL / min;

[0191] d. The filtered expression supernatant was then loaded onto the Protein A column at a flow rate of 3 mL / min;

[0192] e. Use PBS to wash away non-specific binding proteins on the Protein A column at a flow rate of 3 mL / min;

[0193] f. Use glycine buffer at pH = 3.0 to elute the antibody bound to the Protein A column at a flow rate of 1 mL / min;

[0194] g. Collect the eluate to obtain 5 pairs of purified antibodies.

[0195] (4) ELISA screening of antibodies that bind to the S protein of the original strain of the new coronavirus SARS-CoV-2

[0196] a. Coat the ELISA plate with SARS-CoV-2Wuhan-Hu-1 S protein (Sino Biological, Catalog No.: 40589-V08B1) at a concentration of 2 μg / mL (100 μL / well);

[0197] b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

[0198] c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

[0199] d. After washing away the blocking solution with 1×PBST, add the purified antibody to the ELISA plate at a concentration of 1 μg / mL and incubate at 37°C for 1 hour;

[0200] e. After washing away the unbound antibody with 1×PBST, add RHP-labeled anti-human IgG antibody (Jacksonimmunoresearch, catalog number: 109-035-003) and incubate at 37°C for 1 h;

[0201] f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

[0202] g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

[0203] h. The OD value was measured using a Varioskan Flash full wavelength scanning multifunctional reader (Thermo Fisher Scientific), and the OD value was used to reflect the binding strength of the antibody.

[0204] Anti-pan-coronavirus antibodies (SVM7-28, SVM7-75, SVM6-5, SVM6-39, and SSM14-3) were selected based on their binding strength to the SARS-CoV-2 S protein.

[0205] After sequencing, the amino acid sequence of the heavy chain variable region of the anti-coronavirus antibody SVM7-28 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2. The gene sequence encoding the heavy chain variable region of the anti-pan-coronavirus antibody SVM7-28 is shown in SEQ ID NO: 11; the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 12. The amino acid sequence of the full-length heavy chain of the monoclonal antibody is shown in SEQ ID NO: 21; the amino acid sequence of the full-length light chain of the monoclonal antibody is shown in SEQ ID NO: 22.

[0206] SEQ ID NO: 21:

[0207] MGWSCIILFLVATATGVHSQVQLVESGGGVVQPGRSLRLSCAASGFAFSSYAIHWVRQAPGKGLEWVAVISYEGRNTYYADSVKGRCTISRDNSRNAVYLQMDSLRPEDTAVYYCAKERDDILTGFQDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0208] SEQ ID NO:22:

[0209] MGWSCIILFLVATAVHSDIQMTQSPSSVSVSVGDRVTITCRASQDIAGWIAWYQQKPGTAPKLLIYDASTLQSGVPSRFSGSGSGTHFTLTINNLQPEDFATYYCHQSNSFPYTFGQGTRLQIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0210] The amino acid sequence of the heavy chain variable region of the anti-pan-coronavirus antibody SSM14-3 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4. The gene sequence encoding the heavy chain variable region of the anti-pan-coronavirus antibody SVM7-28 is shown in SEQ ID NO: 13; the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 14. The amino acid sequence of the full-length heavy chain of the monoclonal antibody is shown in SEQ ID NO: 23; the amino acid sequence of the full-length light chain of the monoclonal antibody is shown in SEQ ID NO: 24.

[0211] SEQ ID NO: 23:

[0212] MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFRDAWMNWVRQAPGKGLEWVANIKQDGSEKYYLDSVRGRFTISRDNAKSSVYLQMNSLRVEDTALYYCA RDLILSPFPQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0213] SEQ ID NO: 24:

[0214] MGWSCIILFLVATATGSWAQSVLTQPPAVSGTPGQRVIISCSGSSANIGRNYVYWFQILPGTAPKLLIYWNNQRPSGVPDRFSGSKSGTSGSLAISGLRSEDEADYYCAAWDNRLSAY VFATGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0215] The amino acid sequence of the heavy chain variable region of the anti-pan-coronavirus antibody SVM6-5 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6. The gene sequence encoding the heavy chain variable region of the anti-pan-coronavirus antibody SVM7-28 is shown in SEQ ID NO: 15; the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 16. The amino acid sequence of the full-length heavy chain of the monoclonal antibody is shown in SEQ ID NO: 25; the amino acid sequence of the full-length light chain of the monoclonal antibody is shown in SEQ ID NO: 26.

[0216] SEQ ID NO: 25:

[0217] MGWSCIILFLVATATGVHSQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYPMHWVRLSPGKGLQWVAVISYDAANEFYADSVKGRFTISRDTLKSTLYLQMDTLSAEDTAVYYCARASYDSKWSVGAALDMWGLGTRVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0218] SEQ ID NO:26:

[0219] MGWSCIILFLVATAVHSDIQMTQSPDSLAVSLGERATLNCKSSQSVLLSSNNKNYLAWYQQKPGQPPKLLIHWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPQTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0220] The amino acid sequence of the heavy chain variable region of the anti-pan-coronavirus antibody SVM6-39 is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. The gene sequence encoding the heavy chain variable region of the anti-pan-coronavirus antibody SVM7-28 is shown in SEQ ID NO: 17; the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 18. The amino acid sequence of the full-length heavy chain of the monoclonal antibody is shown in SEQ ID NO: 27; the amino acid sequence of the full-length light chain of the monoclonal antibody is shown in SEQ ID NO: 28.

[0221] SEQ ID NO: 27:

[0222] MGWSCIILFLVATATGVHSQVQLVESGGGVVQPGRSLRLSCAASGFTFSRYAIHWVRQAPGRGLEWVSIVSFDGRNVNYAGAVRGRFTISRDSSKKTVYLQMNRLRSEDTAVYYCVRD SYIVASEDGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0223] SEQ ID NO: 28:

[0224] MGWSCIILFLVATAVHSEIVMTQSPATLSVSPGESATLACRASQRLTSRLAWYQQKPGQAPRLLIYGASTRAPGIPARFSGSGSGTEFSLTISSLQSEDSALYYCQQYDDWPLISF GQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0225] The amino acid sequence of the heavy chain variable region of the anti-pan-coronavirus antibody SVM7-75 is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10. The gene sequence encoding the heavy chain variable region of the anti-pan-coronavirus antibody SVM7-28 is shown in SEQ ID NO: 19; the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 20. The amino acid sequence of the full-length heavy chain of the monoclonal antibody is shown in SEQ ID NO: 29; the amino acid sequence of the full-length light chain of the monoclonal antibody is shown in SEQ ID NO: 30.

[0226] SEQ ID NO: 29:

[0227] MGWSCIILFLVATATGVHSEVQLVESGGDLVQPGGSLRLSCSAFGFNFDDYAMHWVRQAPGKGLNCVSVISSSGKGTTYAEAVKGRFSVSRDNVKNTVSLQMNSLRAEDTGIYYCVKGGRSDSSGNPYFYFMEVWGKGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0228] SEQ ID NO: 30:

[0229] MGWSCIILFLVATAVHSEIVMTQSPATLSVSPGERATLSCRASQSISPHLAWYQQKPGQSPRLLIYGASIRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQEYNDWPLFLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0230] Example 2: Binding experiment

[0231] (1) ELISA was used to determine the binding target of the broad-spectrum anti-pantropic coronavirus monoclonal antibody to the SARS-CoV-2 S protein

[0232] a. Coat the ELISA plate with SARS-CoV-2 S1 protein (Sino Biological, Catalog No.: 40591-V08H) or SARS-CoV-2 S2 protein (Sino Biological, Catalog No.: 40590-V08B) at a concentration of 2 μg / mL (100 μL / well);

[0233] b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

[0234] c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

[0235] d. After washing away the blocking solution with 1×PBST, the purified antibody and control antibody (HNC-5, an anti-HCV antibody, patent number: ZL 2019 1 0640392.0) were added to the ELISA plate at a concentration of 1 μg / mL and incubated at 37°C for 1 h;

[0236] e. After washing away the unbound antibody with 1×PBST, add RHP-labeled anti-human IgG antibody (Jacksonimmunoresearch, catalog number: 109-035-003) and incubate at 37°C for 1 h;

[0237] f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

[0238] g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

[0239] h. The OD value was measured using a Varioskan Flash full wavelength scanning multifunctional reader (Thermo Fisher Scientific), and the OD value was used to reflect the binding strength of the antibody.

[0240] The results are as follows Fig.12 As shown, SVM7-28, SSM14-3, SVM6-5, SVM6-39, and SVM7-75 showed strong binding ability to SARS-CoV-2S2 protein, while SSM14-3, SVM6-5, SVM6-39, and SVM7-75 did not bind to SARS-CoV-2S1, and SVM7-28 had weak binding to SARS-CoV-2S1 at high concentrations, indicating that these antibodies are antibodies with S2 as the main target.

[0241] (2) Expression and purification of pan-coronavirus S2 protein

[0242] a. Use FreeStyle one day before transfection TM 293 expression medium (Gibco, catalog number: 12338018) was used to adjust the 293F cell density to 1 × 106 cells / mL;

[0243] b. Count the cells on the day of transfection and use fresh FreeStyle TM 293 expression medium (Gibco, catalog number: 12338018) was used to adjust the 293F cell density to 2 × 106 cells / mL;

[0244] c. The gene sequences encoding the pan-coronavirus RaTG13, WIV1, GX-P5L, GD18, SZ3, Civet007, BCoV, HKU20, IBV and the new coronavirus mutant strain Omicron BA.3S2 proteins were synthesized by Nanjing GenScript and inserted into the pcDNA3.1 vector with a Strep-Tactin tag (SA-WSHPQFEK-(GGGS)2-GGSAWSHPQFEK). Among them, the gene numbers of the above-mentioned pan-coronavirus are: RaTG13 (Genebank: MN996532.2), WIV1 (Gendbank: KC881007.1), GX-P5L (Genebrank: MT040335.1), GD18 (Genebbank: MT799524.1), SZ3 (Geneban k: AY304486.1), Civet007 (Genebank: AY572034.1), BCoV (Genebbank: MN982198.1), HKU20 (Genebbank: NC_016995.1), IBV (Genebbank: JN192154.1) and Omicron BA.3 (Genebank: OW901148.1).

[0245] d. Prepare transfection mixture: Add the plasmid synthesized in step c to Opti-MEM TM (ThermoFisher, Catalog No.: 31985062) medium and mix well to make the transfection plasmid concentration 1 μg / mL, then add Polyethylenimine (PEI) (Polysciences, Catalog No.: 23996-2) and mix well to make the PEI transfection concentration 4 μg / mL;

[0246] e. Add the transfection mixture in step d to the 293F cells in step a and shake gently to mix;

[0247] f. The 293F cells of step e were placed in a 37°C suspension incubator containing 8% CO2 and cultured in suspension at 125rpm for 7 days;

[0248] g. The transfected suspension culture was centrifuged at 4000 rpm for 15 min to collect the expression supernatant;

[0249] h. The supernatant was filtered using a 0.22 μm filter (JET, catalog number: FPE204030);

[0250] i. Open Protein purification instrument, using Buffer W (IBA Lifesciences, catalog number: 2-1003-100) to separate Strep- The column was balanced with a high capacity cartridge (IBA Lifesciences, catalog number: 2-1237-001) at a flow rate of 1 mL / min.

[0251] j. Then the filtered expression supernatant was loaded with Strep- High capacity cartridge column, flow rate 1mL / min;

[0252] k. Use Buffer W to wash away non-specific binding proteins on the column at a flow rate of 1 mL / min;

[0253] l. Use Buffer BXT (IBA Lifesciences, Catalog No.: 2-1042-025) to elute the protein bound to the column at a flow rate of 1 mL / min;

[0254] m. Collect the eluate to obtain the purified pan-coronavirus S2 protein.

[0255] (3) ELISA detection of constructed antibody binding epitopes

[0256] a. Coat the ELISA plate with SARS-CoV-2S ECD protein (Sino Biological, Cat. No. 40591-V08H) at a concentration of 2 μg / mL (100 μL / well);

[0257] b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

[0258] c. Add 200 mM DTT (Thermo Scientific TM , Catalog No.: R0861) and 4% SDS (Solebol, Catalog No.: S8010) denaturation buffer, 37°C for 1 h;

[0259] d. Wash away unbound proteins using 1×PBST;

[0260] e. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

[0261] f. After washing away the blocking solution with 1×PBST, the antibodies purified in Example 1 (SVM7-28, SVM7-75, SVM6-5, SVM6-39, SSM14-3) were diluted (using a blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933)) to obtain antibody solutions with concentrations of 10, 3.33, 1.11, 0.37, 0.123, 0.041, 0.0137, 0.0046, 0.0015, 0.0005, 0.0017 and 0 μg / mL, respectively. The antibody solutions were added to the ELISA plate and incubated at 37°C for 1 h;

[0262] g. Unbound antibodies were washed away with 1× PBST, and then RHP-labeled anti-human IgG antibody (Jackson ImmunoResearch, Catalog No.: 109-035-003) was added and incubated at 37°C for 1 h;

[0263] h. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

[0264] i. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

[0265] j. OD values ​​were measured using Varioskan Flash full wavelength scanning multifunctional reader (ThermoFisher);

[0266] k. The data were calculated using Prism 8.0 software (GraphPad) to obtain the half-effective concentration (EC50) of the antibody against human and animal coronavirus spike protein.

[0267] The results are as follows Fig.13 As shown, the antibody (SVM7-28) has a weak binding ability to the denatured coronavirus spike protein. The antibody partially targets linear epitopes and partially targets non-linear epitopes. However, the antibodies (SVM7-75, SVM6-5, SVM6-39, SSM14-3) have no binding ability to the denatured coronavirus spike protein, indicating that the antibody targets non-linear epitopes.

[0268] (4) ELISA detection of the half effective concentration (EC50) of the constructed antibodies and the spike proteins of human and animal coronaviruses

[0269] a. The purified coronavirus S2 protein or SARS-CoV-2 S ECD protein (Sino Biological, Catalog No.: 40591-V08H) or SARS-CoV S ECD protein (Sino Biological, Catalog No.: 40150-V08B1) or MERS-CoV S ECD protein (Sino Biological, Catalog No.: 40069-V08B) or MERS-CoV S ECD protein (Sino Biological, Catalog No.: ) or MERS-CoV S ECD protein (Sino Biological, Catalog No.: ) or OC43 S ECD protein (Sino Biological, Catalog No.: 40607-V08B) or HKU1 S ECD protein (Sino Biological, Catalog No.: 40606-V08B) or 229E S ECD protein (Sino Biological, Catalog No.: 40605-V08B) or NL63 S SARS-CoV-2 ECD protein (Sino Biological, Catalog No.: 40604-V08B) or SARS-CoV-2 Alpha mutant S ECD protein (Sino Biological, Catalog No.: 40589-V08B6) or SARS-CoV-2 Beta mutant S ECD protein (Sino Biological, Catalog No.: 40589-V08B11) or SARS-CoV-2 Gamma mutant S ECD protein (Sino Biological, Catalog No.: 40589-V08B10) or SARS-CoV-2 Delta mutant S ECD protein (Sino Biological, Catalog No.: 40589-V08B16) or SARS-CoV-2 Omicron BA.1 mutant S ECD protein (Sino Biological, Catalog No.: 40589-V08B33) or SARS-CoV-2 Omicron BA.2 S ECD protein (Sino Biological, Catalog No.: 40589-V08B45) The ELISA plate was coated with 2 μg / mL (100 μL / well) of SARS-CoV-2OmicronBA.4 / 5S ECD protein (Sino Biological, Catalog No. 40589-V08H32) or SARS-CoV-2OmicronBA.2.75S ECD (Sino Biological, Catalog No. 40589-V08H36) protein;

[0270] b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

[0271] c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

[0272] d. After washing away the blocking solution with 1×PBST, the purified antibody in Example 1 was diluted (using a blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933)) to obtain antibody solutions with concentrations of 10, 3.33, 1.11, 0.37, 0.123, 0.041, 0.0137, 0.0046, 0.0015, 0.0005, 0.0017 and 0 μg / mL, respectively. The antibody solutions were added to the ELISA plate and incubated at 37°C for 1 h.

[0273] e. Use 1×PBST to wash away unbound antibodies, then add RHP-labeled anti-human IgG antibody (Jackson ImmunoResearch, catalog number: 109-035-003) and incubate at 37°C for 1 h;

[0274] f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

[0275] g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

[0276] h. OD values ​​were measured using Varioskan Flash full wavelength scanning multifunctional reader (ThermoFisher);

[0277] i. The data were calculated using Prism 8.0 software (GraphPad) to obtain the half effective concentration (EC50) of the antibody against human and animal coronavirus spike protein.

[0278] The results are as follows Fig.14 As shown in Figure 2, the SVM7-28 antibody exhibited strong binding ability to seven human coronavirus spike proteins, with EC50 ranging from 8.2-29 ng / mL. Fig.15 As shown in the results, SVM7-28 showed strong binding ability to 6 of the 9 new coronavirus mutants, with EC50 between 4.4-10.9 ng / mL, while it had weak binding activity to the other three mutants (Omicron BA.2, BA.4 / 5 and BA.2.75). Fig.16As shown, SVM7-28 exhibited potent binding ability to nine animal coronaviruses, with EC50 ranging from 1.6-19.7 ng / mL.

[0279] The results are as follows Fig.17 As shown in Figure 2, the SSM14-3 antibody exhibited strong binding ability to seven human coronavirus spike proteins, with EC50 ranging from 6.6 to 200.5 ng / mL. Fig.18 As shown in the results, SSM14-3 exhibited strong binding ability to the new coronavirus mutants Alpha, Delta, Omicron BA.1 and BA.3, with EC50 between 10.8-422.89 ng / mL, while it had weak binding activity to the new coronavirus mutants Beta, Gamma, and Omicron BA.2. Fig.19 As shown, SSM14-3 exhibited potent binding ability to nine animal coronaviruses, with EC50 ranging from 1.5-7.4 ng / mL.

[0280] The results are as follows Fig. 20 As shown in the figure, the SVM6-5 antibody exhibited strong binding ability to the spike proteins of four human coronaviruses (SARS-CoV, SARS-CoV-2, MERS-CoV and OC43), with EC50 between 19-61.3 ng / mL, and had weak binding activity to HKU1, 229E and NL63. Fig.21 As shown in the results, SVM6-5 showed strong binding ability to 7 of the 9 new coronavirus mutants, with EC50 ranging from 2-104.9 ng / mL, while it had weak binding activity to the other two mutants (Omicron BA.1 and BA.3). Fig. 22 As shown, SVM6-5 exhibited strong binding ability to 7 of the 9 animal coronavirus species, with EC50 ranging from 5.7-261.1 ng / mL, and had no binding activity to bat coronavirus WIV1 and chicken coronavirus IBV.

[0281] The results are as follows Fig.23 As shown in the figure, the SVM6-39 antibody exhibited strong binding ability to the spike proteins of three human coronaviruses (SARS-CoV, SARS-CoV-2 and OC43), with EC50 ranging from 54.3 to 167.9 ng / mL, and had weak binding activity to the other four human coronaviruses. Fig.24 As shown in the results, SVM6-39 exhibited strong binding ability to 5 of the 9 novel coronavirus mutants, with EC50 ranging from 19.9 to 403.6 ng / mL, while it had weak binding activity to the other 4 mutants (Omicron BA.1, BA.2, BA.4 / 5, and BA.2.75). Fig.25As shown, SVM6-39 exhibited strong binding ability to 6 animal SARS-like coronaviruses and bovine coronavirus BCoV, with EC50 between 2.4-33.1 ng / mL, while it had weak binding activity to red-necked duck coronavirus HKU20 and chicken coronavirus IBV, with EC50 of 506.3 ng / mL and 987.1 ng / mL, respectively.

[0282] The results are as follows Fig.26 As shown in the figure, the SVM7-75 antibody showed strong binding ability to human coronavirus SARS-CoV, MERS-CoV, SARS-CoV-2, NL63 and OC43 spike proteins, with EC50 between 21.3-65.8ng / mL. It also had binding ability to human coronavirus HKU1 and 229E, with EC50 of 982.9ng / mL and 3849ng / mL, respectively. Fig. 27 As shown in the results, SVM7-75 exhibited strong binding ability to 6 of the 9 novel coronavirus mutants, with EC50 ranging from 17.3-208.6 ng / mL, while it had weak binding activity to the other three mutants (Omicron BA.2, BA.4 / 5, and BA.2.75). Fig.28 As shown, SVM7-75 exhibited potent binding ability to nine animal coronaviruses, with EC50 ranging from 4.3-139.3 ng / mL.

[0283] However, through testing, the fully human monoclonal antibody 76E1 disclosed in the prior art CN 114716541 A only has binding ability to seven human coronaviruses, and the EC50 is approximately between 40-300 ng / mL, which is much higher than the EC50 of the present invention (6.6-29 ng / mL); and the EC50 of 76E1 to the spike protein peptide of animal coronavirus IBV and HKU11 is 40-100 ng / mL, which is much higher than the present invention. The minimum EC50 of the present invention is 1.5 ng / mL.

[0284] (5) ELISA detection of the half effective concentration (EC50) of the constructed non-broad-spectrum antibody and the human coronavirus spike protein

[0285] In fact, according to the method of Example 1, the present invention amplified more than 300 antibodies, of which a small number were antibodies without binding activity, most were non-broad-spectrum antibodies, and only 5 (as described above) were antibodies with good activity, broad spectrum, and targeting non-linear site targets. The following is a specific process for evaluating the performance of one of the non-broad-spectrum antibodies (SCM14-52) and one of the antibodies without binding activity (SCM9-7):

[0286] SCM14-52

[0287] Heavy chain variable region DNA sequence:

[0288] SEQ ID NO: 31:

[0289] CAGGTCCACCTGGTGCAATCTGGGGCTGAGGTGAAGAGGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGGACCTTTTCTATCAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAAGGATCAGCCCTATTCGGACTATAGCAGACCACGCACTGAACTTCCAGGGCAGACTCACGATTACCGCGGACAAATCCACGACCACAGTCTACATGGAGCTGAGCAGCCTGAGTTCTGAGGACACGGCCATTTACTACTGTACGAGAGACGGTTCTGGTCTGGGCCTCTTTGACTTCTGGGCCCAGGGAACTCAGGTCACCGTCTCCTCAGC

[0290] Heavy chain variable region amino acid sequence:

[0291] SEQ ID NO: 32:

[0292] QVHLVQSGAEVKRPGSSVKVSCKASGGTFRTFSINWVRQAPGQGLEWLGRISPIRTIADHALN FQGRLTITADKSTTTVYMELSSLSSEDTAIYYCTRDGSGLGLFDFWAQGTQVTVSS

[0293] Light chain variable region DNA sequence:

[0294] SEQ ID NO: 33:

[0295] TCCTATGAGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAACTAAAAGTGTGCAGTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATGTATTATGATGCCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAACAGGGTCGAAGCCGGGGATGAGGCCGACTATCACTGTCAGGCGTGGGCTACTAGTAGTGATCATCCGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGC

[0296] Amino acid sequence of the light chain variable region:

[0297] SEQ ID NO: 34:

[0298] SYELTQPPSVSVAPGKTARITCGGNNIGTKSVQWYQQKPGQAPVLVMYYDADRPSGIPERFSG SNSGNTATLTINRVEAGDEADYHCQAWATSSDHPVFGGGTKLTVLGQ

[0299] SCM9-7

[0300] DNA sequence of the heavy chain variable region:

[0301] SEQ ID NO: 35:

[0302] CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAGCCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCACTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAACAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTCACAATGACCAACATGGACCCTGTGGACACAGCCACATACTACTGTGCACACAGAGGGTGGCTGACTCCTAACTCGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGC

[0303] Amino acid sequence of the heavy chain variable region:

[0304] SEQ ID NO: 36:

[0305] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIYWDDDKRYSPS LNSRLTITKDTSKNQVVLTMTNMDPVDTATYYCAHRGWLTPNSFDPWGQGTLVTVSS

[0306] DNA sequence of the light chain variable region:

[0307] SEQ ID NO: 37:

[0308] TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACCTGCTCTGGAGATAAATTGGGGGATTATTATGCTTCCTGGTATCAACAGAAGCCAGGCCAGTCCCCTGTGCTGGTCATGTATCAGGATAATAAGCG GCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCGGCGGGACCCAGGCTATGGATGAGGGTGACTATTACTGTCAGGCGTGGGACAGCAGCCCGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAG

[0309] Light chain variable region amino acid sequence:

[0310] SEQ ID NO: 37:

[0311] SYELTQPPSVSVSPGQTASITCSGDKLGDYYASWYQQKPGQSPVLVMYQDNKRPSGIPERFSGSNSGNTATLTIGGTQAMDEGDYYCQAWDSSPVFGGGTKLTVL

[0312] The specific process is as follows: a. SARS-CoV-2 S ECD protein (Sino Biological, Catalog No.: 40591-V08H) or SARS-CoV S ECD protein (Sino Biological, Catalog No.: 40150-V08B1) or MERS-CoV S ECD protein (Sino Biological, Catalog No.: 40069-V08B) or OC43 S ECD protein (Sino Biological, Catalog No.: 40607-V08B) or HKU1 S ECD protein (Sino Biological, Catalog No.: 40606-V08B) or 229E S ECD protein (Sino Biological, Catalog No.: 40605-V08B) or NL63 S ECD protein (Sino Biological, Catalog No.: 40604-V08B) was coated on the ELISA plate at a concentration of 2 μg / mL (100 μL / well);

[0313] b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

[0314] c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

[0315] d. After washing away the blocking solution with 1×PBST, the non-broad-spectrum antibody (SCM14-52) or the antibody without binding activity (SCM9-7) purified in Example 1 was diluted (using a blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933)) to obtain antibody solutions with concentrations of 10, 3.33, 1.11, 0.37, 0.123, 0.041, 0.0137, 0.0046, 0.0015, 0.0005, 0.0017 and 0 μg / mL, respectively. The antibody solutions were added to the ELISA plate and incubated at 37°C for 1 h;

[0316] e. Use 1×PBST to wash away unbound antibodies, then add RHP-labeled anti-human IgG antibody (Jackson ImmunoResearch, catalog number: 109-035-003) and incubate at 37°C for 1 h;

[0317] f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

[0318] g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

[0319] h. OD values ​​were measured using Varioskan Flash full wavelength scanning multifunctional reader (ThermoFisher);

[0320] i. The data were calculated using Prism 8.0 software (GraphPad) to obtain the half effective concentration (EC50) of the antibody against human and animal coronavirus spike protein.

[0321] The results are as follows Fig.29 As shown in the figure, the non-broad-spectrum antibody (SCM14-52) has a strong binding ability to the spike protein of the new coronavirus, with an EC50 of 33.1 ng / mL, and does not bind to the other six human coronaviruses. Fig.30 As shown, the non-binding active antibody (SCM9-7) does not bind to any of the seven human coronaviruses.

Claims

1. An anti-pan-coronavirus monoclonal antibody, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region, characterized in that the anti-pan-coronavirus monoclonal antibody has at least one of the following technical features: (1) the heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the amino acid sequence of heavy chain CDR1 is GFAFSSYA; the amino acid sequence of heavy chain CDR2 is ISYEGRNT; the amino acid sequence of heavy chain CDR3 is AKERDDILTGFQDYFDY; the light chain variable region includes light chain CDR1, light chain CDR2 and light chain CDR3, the amino acid sequence of light chain CDR1 is QDIAGW; the amino acid sequence of light chain CDR2 is DAS; the amino acid sequence of light chain CDR3 is HQSNSFPYT; (2) the heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the amino acid sequence of the heavy chain CDR1 is GFTFRDAW, the amino acid sequence of the heavy chain CDR2 is IKQDGSEK, and the amino acid sequence of the heavy chain CDR3 is ARDLILSP; the light chain variable region includes a light chain CDR1, a light chain CDR2 and a light chain CDR3, the amino acid sequence of the light chain CDR1 is SANIGRNY, the amino acid sequence of the light chain CDR2 is WNN, and the amino acid sequence of the light chain CDR3 is AAWDNRLSAYV; (3) the heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the amino acid sequence of the heavy chain CDR1 is GFTFSDYP, the amino acid sequence of the heavy chain CDR2 is ISYDAANE, and the amino acid sequence of the heavy chain CDR3 is ARASYDSKWSVGAALDM; the light chain variable region includes a light chain CDR1, a light chain CDR2 and a light chain CDR3, the amino acid sequence of the light chain CDR1 is QSVLLSSNNKNY, the amino acid sequence of the light chain CDR2 is WAS, and the amino acid sequence of the light chain CDR3 is QQYYSIPQT; (4) the heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the amino acid sequence of the heavy chain CDR1 is GFTFSRYA, the amino acid sequence of the heavy chain CDR2 is VSFDGRNV, and the amino acid sequence of the heavy chain CDR3 is VRDSYIVASEDGAFDI; the light chain variable region includes a light chain CDR1, a light chain CDR2 and a light chain CDR3, the amino acid sequence of the light chain CDR1 is QRLTSR, the amino acid sequence of the light chain CDR2 is GAS, and the amino acid sequence of the light chain CDR3 is QQYDDWPLIS; (5) The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the amino acid sequence of heavy chain CDR1 is GFNFDDYA, the amino acid sequence of heavy chain CDR2 is ISSSGKGT, and the amino acid sequence of heavy chain CDR3 is VKGGRSDSSGNPYFYFMEV; the light chain variable region includes light chain CDR1, light chain CDR2 and light chain CDR3, the amino acid sequence of light chain CDR1 is QSISPH, the amino acid sequence of light chain CDR2 is GAS, and the amino acid sequence of light chain CDR3 is QEYNDWPLFLT.

2. The anti-pan-coronavirus monoclonal antibody according to claim 1, characterized in that The heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is QVQLVESGGGVVQPGRSLRLSCAAS, the amino acid sequence of heavy chain FR2 is IHWVRQAPGKGLEWVAV, the amino acid sequence of heavy chain FR3 is YYADSVKGRCTISRDNSRNAVYLQMDSLRPEDTAVYYC, and the amino acid sequence of heavy chain FR4 is WGQGTLVTVSS; preferably, the light chain also includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4, the amino acid sequence of light chain FR1 is DIQMTQSPSSVSVSVGDRVTITCRAS, the amino acid sequence of light chain FR2 is IAWYQQKPGTAPKLLIY, the amino acid sequence of light chain FR3 is TLQSGVPSRFSGSGSGTHFTLTINNLQPEDFATYYC, and the amino acid sequence of light chain FR4 is FGQGTRLQIK.

3. The anti-pan-coronavirus monoclonal antibody according to claim 1, characterized in that The heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is EVQLVESGGGLVQPGGSLRLSCAAS, the amino acid sequence of heavy chain FR2 is MNWVRQAPGKGLEWVAN, the amino acid sequence of heavy chain FR3 is YYLDSVRGRFTISRDNAKSSVYLQMNSLRVEDTALYYC, and the amino acid sequence of heavy chain FR4 is FPQGVLVTVSS; preferably, the light chain also includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4; the amino acid sequence of light chain FR1 is QSVLTQPPAVSGTPGQRVIISCSGS, the amino acid sequence of light chain FR2 is VYWFQILPGTAPKLLIY, the amino acid sequence of light chain FR3 is QRPSGVPDRFSGSKSGTSGSLAISGLRSEDEADYYC, and the amino acid sequence of light chain FR4 is FATGTKVTVL.

4. The anti-pan-coronavirus monoclonal antibody according to claim 1, characterized in that The heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is QVQLVESGGGVVQPGRSLRLSCAAS, the amino acid sequence of heavy chain FR2 is MHWVRLSPGKGLQWVAV, the amino acid sequence of heavy chain FR3 is FYADSVKGRFTISRDTLKSTLYLQMDTLSAEDTAVYYC, and the amino acid sequence of heavy chain FR4 is WGLGTRVTVSS; preferably, the light chain also includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4; the amino acid sequence of light chain FR1 is DIQMTQSPDSLAVSLGERATLNCKSS, the amino acid sequence of light chain FR2 is LAWYQQKPGQPPKLLIH, the amino acid sequence of light chain FR3 is TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC, and the amino acid sequence of light chain FR4 is FGQGTKLEIK.

5. The anti-pan-coronavirus monoclonal antibody according to claim 1, characterized in that The heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is QVQLVESGGGVVQPGRSLRLSCAAS, the amino acid sequence of heavy chain FR2 is IHWVRQAPGRGLEWVSI, the amino acid sequence of heavy chain FR3 is NYAGAVRGRFTISRDSSKKTVYLQMNRLRSEDTAVYYC, and the amino acid sequence of heavy chain FR4 is WGQGTMVTVSS; preferably, the light chain also includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4; the amino acid sequence of light chain FR1 is EIVMTQSPATLSVSPGESATLACRAS, the amino acid sequence of light chain FR2 is LAWYQQKPGQAPRLLIY, the amino acid sequence of light chain FR3 is TRAPGIPARFSGSGSGTEFSLTISSLQSEDSALYYC, and the amino acid sequence of light chain FR4 is FGQGTRLEIK.

6. The anti-pan-coronavirus monoclonal antibody according to claim 1, characterized in that The heavy chain also includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4; the amino acid sequence of heavy chain FR1 is EVQLVESGGDLVQPGGSLRLSCSAF, the amino acid sequence of heavy chain FR2 is MHWVRQAPGKGLNCVSV, the amino acid sequence of heavy chain FR3 is TYAEAVKGRFSVSRDNVKNTVSLQMNSLRAEDTGIYYC, and the amino acid sequence of heavy chain FR4 is WGKGTMVTVSS; preferably, the light chain also includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4; the amino acid sequence of light chain FR1 is EIVMTQSPATLSVSPGERATLSCRAS, the amino acid sequence of light chain FR2 is LAWYQQKPGQSPRLLIY, the amino acid sequence of light chain FR3 is IRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC, and the amino acid sequence of light chain FR4 is FGGGTKVEIK.

7. The anti-pan-coronavirus monoclonal antibody according to claim 1, characterized in that The anti-coronavirus monoclonal antibody has at least one of the following technical features: (1) The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 2; (2) the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 3; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 4; (3) the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 5; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 6; (4) the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 8; (5) The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 9; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

10.

8. Nucleotides encoding the anti-pan-coronavirus monoclonal antibody according to any one of claims 1 to 7; preferably, the nucleotides include the nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, the nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, the nucleotides include the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, the nucleotides include the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18, or the nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO:

20.

9. A recombinant vector, characterized in that: The recombinant vector contains the anti-pan-coronavirus monoclonal antibody according to any one of claims 1 to 7 or the nucleotide encoding the anti-pan-coronavirus monoclonal antibody according to claim 8.

10. Use of the anti-pan-coronavirus monoclonal antibody according to any one of claims 1 to 7, the nucleotide encoding the anti-pan-coronavirus monoclonal antibody according to claim 8, or the recombinant vector according to claim 9 in the preparation of a reagent for diagnosing coronavirus or a kit for diagnosing coronavirus.

11. The use according to claim 10, characterized in that: The coronavirus includes one or more of the novel coronavirus SARS-CoV-2 strain, SARS-CoV strain, MERS-CoV strain, OC43 strain, HKU1 strain, NL63 strain, 229E strain, bat SARS-like coronavirus RaTG13, WIV1, civet SARS-like coronavirus SZ3, Civet007, pangolin SARS-like coronavirus GX-P5L, GD18, bovine coronavirus BCoV, red-necked duck coronavirus HKU20, chicken coronavirus IBV, novel coronavirus Alpha mutant strain, novel coronavirus Beta mutant strain, novel coronavirus Gamma mutant strain, novel coronavirus Delta mutant strain, and novel coronavirus Omicron mutant strain.

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