Antibody for resisting monkey pox virus A35R protein as well as preparation method and application thereof

Abstract

The invention discloses an anti-monkey pox virus antibody as well as a preparation method and application thereof. The invention specifically discloses eight kinds of antibodies (A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6, A35R-18E2 and A35R-18H2) which have very good affinity to monkey pox virus A35R protein, and the eight kinds of antibodies have very good neutralizing activity and a certain in-vivo protection effect.

Description

Technical Field

[0001] This application relates to the field of antibody engineering, and specifically relates to antibodies against the A35R protein of monkeypox virus, their preparation methods and applications. Background Art

[0002] Monkeypox virus (MPXV) belongs to the family Poxviridae, subfamily Chordopoxvirinae, genus Orthopoxvirus (OPXV). The genus Orthopoxvirus has more than 10 members, including variola virus (VARV), vaccinia (smallpox vaccine) virus (VACV), cowpox virus (CPXV), camelpox virus (CMLV), and several new species isolated from infected humans or primates since 2010. Based on the comparison between VACV and MPXV, the surface proteins among poxviruses show approximately 93 - 98% sequence similarity, suggesting that they play similar roles during the process of entering the body.

[0003] In 1958, monkeypox virus was first discovered in a group of monkeys used for research and was named "Monkeypoxvirus (MPXV)". In 1970, a 9 - month - old baby boy was diagnosed, which was the first human case, indicating that monkeypox virus can infect humans.

[0004] Monkeypox is a zoonosis, and its natural host has not been clearly identified. It mainly spreads among rodents and non - human primates. It can be transmitted through various forms such as contact with damaged skin tissues. In addition, recently, the spread of monkeypox virus in non - endemic countries has shown a phenomenon related to sexual contact. Monkeypox virus has been found in body fluids such as semen, but whether it is transmitted through sexual contact is not clear. After infection, symptoms such as swollen lymph nodes, muscle soreness, and permanent scars left after pustules rupture will appear.

[0005] Since the eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination in humans, the current young population has a relatively low immune ability to orthopoxviruses, which may be one of the reasons for the current MPOX outbreak. With the spread of multiple monkeypox virus strains in non - endemic countries, not only are the transmission routes diverse and hidden, and the infection symptoms are atypical, but also the monkeypox virus accelerates mutation during human - to - human transmission, and its mutation rate far exceeds expectations, posing an increasingly greater threat to global public health security. Although there are not many monkeypox cases in China at present, with the flow of people inside and outside the country, how to prevent and control monkeypox infection has become a key problem to be solved urgently. And currently, no specific antiviral therapy has been approved for the treatment of human MPXV infection. We urgently need more research on this virus to better prevent and treat monkeypox.

[0006] To date, neutralizing antibodies have proven to be an effective method for treating viral diseases, including human immunodeficiency virus (HIV), influenza virus, and other flaviviruses, etc. Monkeypox virus (MPXV) exists in two forms: extracellular enveloped virus (EEV) and intracellular mature virus (IMV) particles.

[0007] It is very important and necessary to develop new monkeypox virus antibodies with high antigen affinity and good in vitro neutralization. Summary of the Invention

[0008] Based on this, an embodiment of the present application provides an antibody against monkeypox virus A35R protein, which has good affinity with monkeypox virus A35R protein and good neutralizing activity.

[0009] The technical solution includes:

[0010] An embodiment of the present application provides an antibody against monkeypox virus, and the antibody includes a heavy chain variable region and a light chain variable region;

[0011] The CDR sequences of the heavy chain variable region and the CDR sequences of the light chain variable region are selected from any one of items a1) to a8);

[0012] a1) HCDR1 with the sequence shown in SEQ ID NO:5, HCDR2 with the sequence shown in SEQ ID NO:6, HCDR3 with the sequence shown in SEQ ID NO:7; and, LCDR1 with the sequence shown in SEQ ID NO:8, LCDR2 with the sequence shown in SEQ ID NO:9, LCDR3 with the sequence shown in SEQ ID NO:10;

[0013] a2) HCDR1 with the sequence shown in SEQ ID NO:21, HCDR2 with the sequence shown in SEQ ID NO:22, HCDR3 with the sequence shown in SEQ ID NO:23; and, LCDR1 with the sequence shown in SEQ ID NO:24, LCDR2 with the sequence shown in SEQ ID NO:25, LCDR3 with the sequence shown in SEQ ID NO:26;

[0014] a3) HCDR1 with the sequence shown in SEQ ID NO:37, HCDR2 with the sequence shown in SEQ ID NO:38, HCDR3 with the sequence shown in SEQ ID NO:39; and, LCDR1 with the sequence shown in SEQ ID NO:40, LCDR2 with the sequence shown in SEQ ID NO:41, LCDR3 with the sequence shown in SEQ ID NO:42;

[0015] a4) An HCDR1 having a sequence as set forth in SEQ ID NO:53, an HCDR2 having a sequence as set forth in SEQ ID NO:54, and an HCDR3 having a sequence as set forth in SEQ ID NO:55; and, an LCDR1 having a sequence as set forth in SEQ ID NO:56, an LCDR2 having a sequence as set forth in SEQ ID NO:57, and an LCDR3 having a sequence as set forth in SEQ ID NO:58;

[0016] a5) An HCDR1 having a sequence as set forth in SEQ ID NO:69, an HCDR2 having a sequence as set forth in SEQ ID NO:70, and an HCDR3 having a sequence as set forth in SEQ ID NO:71; and, an LCDR1 having a sequence as set forth in SEQ ID NO:72, an LCDR2 having a sequence as set forth in SEQ ID NO:73, and an LCDR3 having a sequence as set forth in SEQ ID NO:74;

[0017] a6) An HCDR1 having a sequence as set forth in SEQ ID NO:85, an HCDR2 having a sequence as set forth in SEQ ID NO:86, and an HCDR3 having a sequence as set forth in SEQ ID NO:87; and, an LCDR1 having a sequence as set forth in SEQ ID NO:88, an LCDR2 having a sequence as set forth in SEQ ID NO:89, and an LCDR3 having a sequence as set forth in SEQ ID NO:90;

[0018] a7) An HCDR1 having a sequence as set forth in SEQ ID NO:101, an HCDR2 having a sequence as set forth in SEQ ID NO:102, and an HCDR3 having a sequence as set forth in SEQ ID NO:103; and, an LCDR1 having a sequence as set forth in SEQ ID NO:104, an LCDR2 having a sequence as set forth in SEQ IDNO:105, and an LCDR3 having a sequence as set forth in SEQ ID NO:106;

[0019] a8) An HCDR1 having a sequence as set forth in SEQ ID NO:117, an HCDR2 having a sequence as set forth in SEQ ID NO:118, and an HCDR3 having a sequence as set forth in SEQ ID NO:119; and, an LCDR1 having a sequence as set forth in SEQ ID NO:120, an LCDR2 having a sequence as set forth in SEQ IDNO:121, and an LCDR3 having a sequence as set forth in SEQ ID NO:122.

[0020] In one specific embodiment, the amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, and 113; the amino acid sequence of the light chain variable region is shown as any one of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, and 114.

[0021] Optionally, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 2.

[0022] Optionally, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 18.

[0023] Optionally, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 33, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 34.

[0024] Optionally, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 49, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 50.

[0025] Optionally, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 65, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 66.

[0026] Optionally, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 81, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 82.

[0027] Optionally, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 97, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 98.

[0028] Optionally, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 113, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 114.

[0029] In one specific embodiment, it further includes a heavy chain constant region and a light chain constant region.

[0030] In one specific embodiment, the species of the heavy chain constant region and the light chain constant region is derived from humans.

[0031] In one specific embodiment, the sequence of the light chain constant region is selected from the sequences of the kappa-type light chain constant region or the lambda-type light chain constant region.

[0032] In one specific embodiment, the sequence of the heavy chain constant region is selected from the sequences of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0033] One embodiment of the present application provides a nucleic acid molecule encoding the antibody.

[0034] One embodiment of the present application provides an expression vector comprising the nucleic acid molecule.

[0035] In one specific embodiment, the expression vector includes, but is not limited to, a plasmid.

[0036] In one specific embodiment, the plasmid includes, but is not limited to, pCAGGS.

[0037] One embodiment of the present application provides a host cell comprising the nucleic acid molecule or the expression vector.

[0038] In one specific embodiment, the host cell includes eukaryotic cells.

[0039] In one specific embodiment, the eukaryotic cells include yeast cells, insect cells, and mammalian cells.

[0040] In one specific embodiment, the mammalian cells include 293T cells, 293F cells, CHO cells, or HEK293 cells, etc.

[0041] One embodiment of the present application provides a method for preparing the antibody, the preparation method comprising the following steps: culturing the host cell and isolating the antibody from the obtained culture.

[0042] One embodiment of the present application provides a detection reagent or a drug comprising the antibody.

[0043] One embodiment of the present application provides the use of the antibody in detecting the A35R protein of monkeypox virus for non-diagnostic purposes.

[0044] One embodiment of the present application provides the use of the antibody in preparing a detection product, a preventive drug, or a therapeutic drug for orthopoxvirus.

[0045] In one specific embodiment, the orthopoxvirus includes monkeypox virus and / or vaccinia virus.

[0046] Compared with the traditional technology, the present application has the following beneficial effects:

[0047] The monoclonal antibody of the present application has a strong binding ability to monkeypox virus, good in vitro neutralization, high affinity, and has an in vivo protective effect. It is completely different from the reported monkeypox virus antibody sequences, providing products for the detection and neutralization of monkeypox virus, and making it possible to provide monkeypox virus products with in vivo protective effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following described drawings are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0049] Figure 1 It is the SDS-PAGE result of the purification molecular sieve of monkeypox virus A35R protein;

[0050] Figure 2 It is the kinetic curve result of A35R antibody and A35R protein;

[0051] Figure 3 It is the neutralization curve result of A35R antibody against live virus VACV-WR;

[0052] Figure 4 It is the experimental schematic diagram and the curves of the survival and weight changes of mice after virus infection. Among them, Figure A is the schematic diagram of the experimental implementation, and Figure B is the curves of the survival and weight changes of mice after virus infection. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] To facilitate the understanding of the present application, the following will give a more comprehensive description of the present application. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0055] The term "and / or" used herein includes any one of two or more related listed items, as well as any and all combinations of the related listed items. The any and all combinations include any two related listed items, any more related listed items, or the combination of all related listed items.

[0056] In this text, "multiple", unless otherwise specified, means greater than 2 or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0057] In this text, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0058] In this text, "optionally", "optional", "option", mean optional, that is, it means either selected from one of the two parallel options of "yes" or "no". If "optional" appears multiple times in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction relationship, each "optional" is independent.

[0059] In this application, by screening memory B cells that specifically bind to A35R of a fully humanized mouse against human antibodies, human-derived high-neutralizing activity MPXV antibodies A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6, A35R-18E2, and A35R-18H2 are obtained, and finally a new humanized monoclonal antibody against monkeypox virus that is specific, has strong binding ability to monkeypox virus, good in vitro neutralization, and high affinity is screened out.

[0060] Among them, the specific solutions are as follows:

[0061] I. Preparation and purification of monkeypox virus A35R monoclonal antibody

[0062] Through the expression and purification of monkeypox virus A35R protein, the sorting of specific memory B cells that bind to A35R protein, and the expression and purification of monkeypox virus A35R antibody, 8 monkeypox virus A35R monoclonal antibodies are obtained, namely A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6, A35R-18E2, and A35R-18H2. The amino acid sequences of the light and heavy chains, CDR1-CDR3 of the light chain variable region, and CDR1-CDR3 of the heavy chain variable region of these 8 monkeypox virus monoclonal antibodies are as follows. The light chain variable region and the heavy chain variable region are different from the existing sequences and are specific.

[0063] II. Performance verification of monoclonal antibody

[0064] (I) Detection of antibody-antigen affinity by surface plasmon resonance technology

[0065] Eight antibodies, namely A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6, A35R-18E2 and A35R-18H2, have good affinity with the antigen.

[0066] (II) ELISA for detecting the binding activity between A35R antigen and antibody

[0067] Antibodies A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6, A35R-18E2 and A35R-18H2 can all bind to the antigen with strong binding ability.

[0068] (III) Antigen-antibody neutralization test

[0069] The neutralization curve results of A35R antibody against VACV-WR were detected by plaque assay. The PRNT50 values of A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6, A35R-18E2 and A35R-18H2 were 0.022 μg / mL, 0.176 μg / mL, 0.118 μg / mL, 0.080 μg / mL, 0.283 μg / mL, 1.177 μg / mL, 0.044 μg / mL and 5.185 μg / mL, respectively.

[0070] In this article, "PRNT50" is the 50% Plaque Reduction Neutralization Test (PRNT).

[0071] (IV) Animal protection test

[0072] Under the condition of low-dose virus infection, administration of antibodies A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6 and A35R-18E2 can all provide protection for mice infected with monkeypox virus.

[0073] The following is a detailed description in combination with specific embodiments. Unless otherwise specified, the following embodiments do not include other components except inevitable impurities. The reagents and instruments used in the embodiments are all conventional selections in the art unless otherwise specified. The experimental methods without specific conditions in the embodiments are carried out under conventional conditions, such as the conditions described in the literature, books or the methods recommended by the manufacturers.

[0074] The main experimental materials involved in the following embodiments include but are not limited to:

[0075] The key drugs and reagents are shown in Table 1:

[0076] Table 1

[0077] Name Model Manufacturer Humanized mouse with fully human antibodies RenMab Beijing Biocytogen Co., Ltd. 14K Chip 750-00021 Berkeley lights

[0078] The main instruments are shown in Table 2:

[0079] Table 2

[0080] Instrument Name Model Manufacturer Beacon Cell Sorter Beacon Berkeley Lights

[0081] Example 1 Preparation and purification of monkeypox virus monoclonal antibody

[0082] 1. Expression and purification of monkeypox virus A35R protein

[0083] The gene of A35R was constructed into the eukaryotic expression vector pCAGGS and transfected into 293F cells. The A35R protein was secreted and expressed by the cells. The cell supernatant was collected and purified by affinity chromatography and molecular sieve, and identified by SDS-PAGE.

[0084] Among them, the S90-T181 segment (SEQ ID NO: 129) of the A35R protein was intercepted, and after gene sequence optimization, it was constructed into the vector pCAGGS:

[0085] STTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT.

[0086] The results of SDS-PAGE of the molecular sieve purification of monkeypox virus A35R protein are as Figure 1 shown. The protein flowed through Superdex200GL, and an absorption peak at UV280 nM could be detected at the ~16 mL position. The molecular weight of the protein in SDS-PAGE was about ~15 kDa, and the obtained protein had a high purity.

[0087] 2. Sorting of specific memory B cells that bind to the A35R protein

[0088] The gene of A35R was constructed into an mRNA vector. After in vitro transcription and capping, the concentration was measured and the quality was identified. The fully humanized antibody humanized mouse (RenMab) was immunized 3 times at a dose of 2.5 μg per mouse. Serum was collected before immunization and 12 days after each immunization to detect the antibody titer. When the antibody titer reached an appropriate level, after a booster immunization, the lymph nodes of the humanized mice were taken, ground into single cells, incubated with antigen molecules, and then specific single B cells were sorted using a Beacon sorter. After reverse transcription and PCR amplification, the variable region gene sequences of the antibodies were obtained.

[0089] Eight monoclonal antibodies against monkeypox virus A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6, A35R-18E2, and A35R-18H2 were obtained. The amino acid sequences of the light and heavy chains, CDR1-CDR3 of the light chain variable region, and CDR1-CDR3 of the heavy chain variable region of these eight monkeypox virus monoclonal antibodies are as follows. The light chain variable region and the heavy chain variable region are different from the existing sequences and are specific.

[0090] Monoclonal antibody A35R-9E4:

[0091] Amino acid sequence of the heavy chain variable region, SEQ ID NO:1:

[0092] QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISFDGSNEYYADSVK GRFTISRDNSRSTLSLQVNSLRTEDTAVYYCAKRGHSNYFYYGMDVWGQGTTVTVSS

[0093] Amino acid sequence of the light chain variable region, SEQ ID NO:2:

[0094] EIVMTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATDIPGRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPFTFGQGTKLEIK

[0095] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:3:

[0096] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTTATTTCATTTGATGGAAGTAATGAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAGGAGCACGCTGTCTCTGCAAGTGAACAGCCTGAGAACTGAGGACACGGCTGTGTATTACTGTGCGAAAAGGGGTCACTCAAACTACTTCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0097] Nucleic acid sequence of the light chain variable region, SEQ ID NO:4:

[0098] GAAATTGTGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGACATCCCAGGCAGGTTCAGTGGCAGTGGGTCTGGGACTGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA

[0099] Among them, the amino acids of the CDR region of the A35R-9E4 antibody are shown in Table 3; the nucleotide sequence of the CDR region of the A35R-9E4 antibody is shown in Table 4.

[0100] Table 3

[0101] Amino Acid Sequence Serial Number H-CDR1 GFTFSSYG SEQ ID NO:5 H-CDR2 ISFDGSNE SEQ ID NO:6 H-CDR3 AKRGHSNYFYYGMDV SEQ ID NO:7 L-CDR1 QSVSSTY SEQ ID NO:8 L-CDR2 GAS SEQ ID NO:9 L-CDR3 QQYGSSPPFT SEQ ID NO:10

[0102] Table 4

[0103]

[0104] A35R-9F8 monoclonal antibody:

[0105] Amino acid sequence of the heavy chain variable region, SEQ ID NO:17:

[0106] QVQLVQSGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWVAVISFDGGNKYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDSAVYYCAKRGYSDNDFPHFFYYGLDVWGQGTTVTVSS

[0107] Amino acid sequence of the light chain variable region, SEQ ID NO:18:

[0108] EIVMTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGFGTDFTFTITRLEPEDFAVYYCQQYGSSPPFTFGPGTKVEIK

[0109] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:19:

[0110] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGAAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCTGTTATATCATTTGATGGAGGTAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACTCGGCTGTGTATTACTGTGCGAAACGTGGATACAGTGACAACGATTTCCCTCACTTCTTCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0111] Nucleic acid sequence of the light chain variable region, SEQ ID NO:20:

[0112] GAAATTGTGATGACCCAGTCTCCAGGCACTCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTTTGGTACAGACTTCACTTTCACCATCACCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTACGGTAGTTCACCTCCATTCACTTTCGGCCCAGGGACCAAAGTGGAAATCAAA

[0113] Among them, the amino acids of the CDR region of the A35R-9F8 antibody are shown in Table 5; the nucleotide sequence of the CDR region of the A35R-9F8 antibody is shown in Table 6.

[0114] Table 5

[0115]

[0116]

[0117] Table 6

[0118]

[0119] A35R-10C4 monoclonal antibody:

[0120] Amino acid sequence of the heavy chain variable region, SEQ ID NO:33:

[0121] QVQLVQSGGGVVQPGRSLRLSCVAFGFTFSSYGMHWVRQAPGKGLEWVAVISYDGNNKYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRGYSYVFYYGMDVWGQGTTVTVSS

[0122] Amino acid sequence of the light chain variable region, SEQ ID NO:34:

[0123] EIVMTQSPGTLSLSPGERATLSCRASQSISSSYLAWYQQKPGQAPRLLIYGAFSRVTGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYGSSPPWTFGQGTKVEIK

[0124] Heavy chain variable region nucleic acid sequence, SEQ ID NO:35:

[0125] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGTAGCCTTTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAGCGTGGATACAGCTATGTTTTCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0126] Light chain variable region nucleic acid sequence, SEQ ID NO:36:

[0127] GAAATTGTGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCAGCTACTTAGCCTGGTACCAGCAAAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATTCAGCAGGGTCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTCTGCAGTGTATTATTGTCAGCAGTATGGTAGTTCACCTCCGTGGACGTTCGGCCAAGGGACCAAGGTTGAAATCAAA

[0128] Among them, the amino acids of the CDR region of the A35R-10C4 antibody are shown in Table 7; the nucleotide sequence of the CDR region of the A35R-10C4 antibody is shown in Table 8.

[0129] Table 7

[0130]

[0131]

[0132] Table 8

[0133]

[0134] A35R-10D5 monoclonal antibody:

[0135] Amino acid sequence of the heavy chain variable region, SEQ ID NO:49:

[0136] QVQLVQSGGGVVQPGRSLRLACAASGFTFSSYGMHWVRQSPGKGLEWVAVISYDGDKKYYADSVK GRFTISRDNSKNTLYLQMNSLRSEDTAVYYCAKRGLNHYFYYGLDVWGQGTTVTVSS

[0137] Amino acid sequence of the light chain variable region, SEQ ID NO:50:

[0138] EIVMTQSPGTLSLSPGERATLSCRASQSVSTNFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCHQYGSSPPFTFGQGTKLEIK

[0139] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:51:

[0140] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCGCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGTCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTTATATCATATGATGGAGATAAAAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGATCTGAGGACACGGCTGTGTATTACTGTGCGAAAAGGGGCTTGAACCACTACTTCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0141] Nucleic acid sequence of the light chain variable region, SEQ ID NO:52:

[0142] GAAATTGTGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCACCAACTTCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGGAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAATATGGGAGCTCACCTCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA

[0143] Among them, the amino acids of the CDR region of the A35R-10D5 antibody are shown in Table 9; the nucleotide sequence of the CDR region of the A35R-10D5 antibody is shown in Table 10.

[0144] Table 9

[0145]

[0146]

[0147] Table 10

[0148]

[0149] A35R-10E7 monoclonal antibody:

[0150] Amino acid sequence of the heavy chain variable region, SEQ ID NO:65:

[0151] QVQLVQSGGGVVQPGRSLRLSCAASGFTFNSYGMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAKRGNSGWFYYGMDVWGQGSTVTVSS

[0152] Amino acid sequence of the light chain variable region, SEQ ID NO:66:

[0153] EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGSSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFVVYYCHQYGSSPPFTFGPGTKVEIK

[0154] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:67:

[0155] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTATCTATTACTGTGCGAAAAGGGGGAACAGTGGCTGGTTCTACTACGGTATGGACGTCTGGGGCCAAGGGTCCACGGTCACCGTCTCCTCA

[0156] Nucleic acid sequence of the light chain variable region, SEQ ID NO:68:

[0157] GAAATTGTGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTTCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCCGAAGATTTTGTAGTTTATTATTGTCATCAATATGGTAGTTCACCTCCTTTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAA

[0158] Among them, the amino acids in the CDR region of the A35R-10E7 antibody are shown in Table 11; the nucleotide sequence of the CDR region of the A35R-10E7 antibody is shown in Table 12.

[0159] Table 11

[0160] Amino Acid Sequence Serial Number H-CDR1 GFTFNSYG SEQ ID NO:69 H-CDR2 ISYDGNNK SEQ ID NO:70 H-CDR3 AKRGNSGWFYYGMDV SEQ ID NO:71 L-CDR1 QSVSSSY SEQ ID NO:72 L-CDR2 GSS SEQ ID NO:73 L-CDR3 HQYGSSPPFT SEQ ID NO:74

[0161] Table 12

[0162]

[0163] A35R-10H6 monoclonal antibody:

[0164] Amino acid sequence of the heavy chain variable region, SEQ ID NO:81:

[0165] QVQLVQSGGGVVQPGRSLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVGVISYDGSNKYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDSAVYYCAKRSNYGGIFYYGMDVWGQGTTVTVSS

[0166] Amino acid sequence of the light chain variable region, SEQ ID NO:82:

[0167] EIVMTQSPGTLSLSPGERVTLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATAIPDRFSGSGSGTDFTLTISRLEPEDCAVYYCQQYGNSPPFTFGPGTKVEIK

[0168] Heavy chain variable region nucleic acid sequence, SEQ ID NO:83:

[0169] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGGAGTTATATCATATGATGGAAGTAATAAATACTATGCAGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACTCGGCTGTGTATTACTGTGCGAAACGGAGTAACTACGGGGGGATCTTTTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0170] Light chain variable region nucleic acid sequence, SEQ ID NO:84:

[0171] GAAATTGTGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGTCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTGTGCAGTGTATTACTGTCAACAGTATGGTAACTCACCTCCATTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAA

[0172] Among them, the amino acids of the CDR region of the A35R-10H6 antibody are shown in Table 13; the nucleotide sequence of the CDR region of the A35R-10H6 antibody is shown in Table 14.

[0173] Table 13

[0174] Amino Acid Sequence Serial Number H-CDR1 GFTFSSYG SEQ ID NO:85 H-CDR2 ISYDGSNK SEQ ID NO:86 H-CDR3 AKRSNYGGIFYYGMDV SEQ ID NO:87 L-CDR1 QSVSSSF SEQ ID NO:88 L-CDR2 GAS SEQ ID NO:89 L-CDR3 QQYGNSPPFT SEQ ID NO:90

[0175] Table 14

[0176]

[0177] A35R-18E2 monoclonal antibody:

[0178] Amino acid sequence of the heavy chain variable region, SEQ ID NO:97:

[0179] QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVK GRFTMSRDNSKNTLFLQMNSLRAEDTAVYYCAKRGYSYGRFYYGMDVWGQGTTVTVSS

[0180] Amino acid sequence of the light chain variable region, SEQ ID NO:98:

[0181] EIVMTQSPGTLSLSPGERATLSCRTSQSISSSYLAWYQQKPGQAPRLLIYGASSGATGIPDRFSGSGSGT DFSLTISRLEPEDFAVYYCHQYGSSPPFTFGPGTKVEIK

[0182] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:99:

[0183] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATGTCCAGAGACAATTCCAAGAACACGCTATTTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAACGTGGATATAGCTATGGCCGCTTCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0184] Nucleic acid sequence of the light chain variable region, SEQ ID NO:100:

[0185] GAAATTGTGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGACCAGTCAGAGTATTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCGGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCTCTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTATGGTAGCTCACCTCCATTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAA

[0186] Among them, the amino acids in the CDR region of the A35R-18E2 antibody are shown in Table 15; the nucleotide sequence of the CDR region of the A35R-18E2 antibody is shown in Table 16.

[0187] Table 15

[0188] Amino Acid Sequence Serial Number H-CDR1 GFTFSSYG SEQ ID NO:101 H-CDR2 ISYDGSNK SEQ ID NO:102 H-CDR3 AKRGYSYGRFYYGMDV SEQ ID NO:103 L-CDR1 QSISSSY SEQ ID NO:104 L-CDR2 GAS SEQ ID NO:105 L-CDR3 HQYGSSPPFT SEQ ID NO:106

[0189] Table 16

[0190]

[0191] A35R-18H2 monoclonal antibody:

[0192] Amino acid sequence of the heavy chain variable region, SEQ ID NO:113:

[0193] QVQLVQSGGGVVQPGRSLRLSCAASGFTFNSYGMHWVRQAPGKGLEWVAVIYYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRIRGNYYFHYGLDVWGQGTTVTVSS

[0194] Amino acid sequence of the light chain variable region, SEQ ID NO:114:

[0195] EIVMTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNSPPYTFGQGTKVEIK

[0196] Heavy chain variable region nucleic acid sequence, SEQ ID NO: 115:

[0197] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAATAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATATTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAAGGATCCGTGGAAACTACTATTTCCACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0198] Light chain variable region nucleic acid sequence, SEQ ID NO: 116:

[0199] GAAATTGTGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAACTCACCTCCGTACACTTTTGGCCAGGGGACCAAGGTGGAAATCAAA

[0200] Among them, the amino acids of the CDR region of the A35R-18H2 antibody are shown in Table 17; the nucleotide sequence of the CDR region of the A35R-18H2 antibody is shown in Table 18.

[0201] Table 17

[0202] Amino Acid Sequence Serial Number H-CDR1 GFTFNSYG SEQ ID NO:117 H-CDR2 IYYDGSNK SEQ ID NO:118 H-CDR3 AKRIRGNYYFHYGLDV SEQ ID NO:119 L-CDR1 QSVSSNY SEQ ID NO:120 L-CDR2 GAS SEQ ID NO:121 L-CDR3 QQYGNSPPYT SEQ ID NO:122

[0203] Table 18

[0204]

[0205] 3. Expression and purification of antibodies

[0206] (1) Antibody clone construction:

[0207] The variable heavy chain gene (VH) of the antibody was fused with the IgG1 constant region gene (CH) and constructed into the pCAGGS vector (named pCAGGS-Heavy chain-full length). The variable light chain gene (VL) of the antibody was fused with the constant region gene (CL) and constructed into the pCAGGS vector (named pCAGGS-Light chain-full length).

[0208] (2) Antibody expression and purification:

[0209] When co-transfecting 100 mL of 293F cells with the light chain and heavy chain plasmids, 36 μg of the heavy chain plasmid and 75 μg of the light chain plasmid were taken, dissolved in 1 mL of HBS, and left standing for 5 min. 300 μg of PEI was dissolved in 1 mL of HBS and also left standing for 5 min. The above plasmid solution was mixed with the above PEI solution, left standing for 20 min, and then added to the 293F cells. The cells were cultured in a constant temperature shaker at 37°C. After 5 days, the cells were centrifuged at 8000 r / min for 90 min, and the supernatant was harvested. The supernatant was passed through an affinity column Protein A, and the change in the UV280 absorbance value was detected by an AKTA Purifier machine. After elution with 0.1 M Glycine, pH 3.0, the antibody was collected, concentrated and exchanged into PBS. After detection by SD-PAGE, the antibody had high purity. The size of the heavy chain after antibody denaturation was approximately 50 KD, and the size of the light chain was approximately 25 KD.

[0210] Example 2 Detection of the affinity between antibody and antigen by surface plasmon resonance technology

[0211] Surface plasmon resonance (SPR) experiments can be used to detect in vitro protein interactions. The kinetic parameters of the binding of two proteins can be known by detecting the change in response value of the mobile phase flowing through the chip surface. It is performed on the instrument BIACORE 8K. In the surface plasmon resonance experiment, one of the two proteins to be analyzed is usually fixed on the surface of a metal chip, and the other is used as the mobile phase to flow through the chip surface to detect the change in response value caused by it. Before the experiment, PBST should be filtered using a 0.22μM filter membrane and then sterilized with high-pressure steam. The antibody concentration is 10μg / mL (can be adjusted appropriately), and the antigen concentration titer is 400nM, 200nM, 100nM, 50nM, 25nM (can be adjusted appropriately). The characteristics of the A35R antigen-antibody affinity test are shown in Tables 19 and Figure 2 .

[0212] Table 19 Characteristics of A35R antigen-antibody affinity assay

[0213]

[0214]

[0215] Example 3 Antibody Neutralization Test

[0216] The neutralizing activity of the antibody was detected by the plaque assay. The operation steps are briefly described as follows: the starting concentration of the antibody is 200 μg / mL, and the dilution is 4-fold, with 10 gradients. Take 400 μL of each gradient and mix it with an equal volume of vaccinia virus VACV-WR dilution (containing 10% complement and 100 μg / mL of targeted IMV antibody), and incubate at 37°C for 1 hour. Take out the 12-well plate inoculated with Vero cells in advance. When the cell confluence is ~100%, it can be used for neutralization experiments. After rinsing the cells twice, add the mixture of antibodies and viruses, 400 μL / well, set two replicate wells for each antibody gradient, set a control well with only virus but no antibody, and incubate at 37°C for 1h. Discard the antibody and virus mixture, rinse the cells once, and add 1 mL / well of fixative, which is composed of methylcellulose and high-glucose culture medium (2×) in a volume ratio of 1:1. Place the cell plate in a cell culture incubator for culture. After 48 hours, the cells were fixed with 8% tissue cell fixative, stained with crystal violet, counted, and the PRNT50 value was calculated.

[0217] like Figure 3As shown, the neutralization curve results of A35R antibody against VACV-WR, the PRNT50 values of A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6, A35R-18E2 and A35R-18H2 are 0.022 μg / mL, 0.176 μg / mL, 0.118 μg / mL, 0.080 μg / mL, 0.283 μg / mL, 1.177 μg / mL, 0.044 μg / mL and 5.185 μg / mL respectively.

[0218] Example 4 Animal Protection Test

[0219] To further evaluate the antibody applicability, antibodies with higher neutralizing activities were selected: A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6 and A35R-18E2, and their protective effects were evaluated by administration at the mouse level. Since the animal experiment of monkeypox virus infection needs to be carried out in a high-level biosafety level 3 laboratory, the vaccinia virus (belonging to the same family of poxviruses as monkeypox virus) Western Reserve (VACV-WR) strain was initially used for evaluation. The mice at 7-8 weeks old were infected with VACV-WR virus by intranasal drip at 5LD 50 and 10LD 50 respectively. Antibodies (200 μg / mouse) were intraperitoneally injected 4 h before and 4 h after infection.

[0220] As shown in Table 20 and Figure 4 shown, after infection with 5LD 50 virus, the body weights of mice in the irrelevant antibody group all decreased by more than 20% on the 6th day, while the body weights of mice injected with A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6 and A35R-18E2 antibody groups showed no obvious changes. After infection with 10LD 50 virus, the body weights of mice in the irrelevant antibody group all decreased by more than 20% on the 5th day, while the body weights of mice injected with A35R-9E4, A35R-9F8, A35R-10D5, A35R-10E7 and A35R-10H6 antibody groups showed no obvious changes. One mouse in the A35R-10H6 group had a body weight decrease of more than 20% on the 6th day.

[0221] Table 20 Survival rate of mice after injection of A35R antibody

[0222]

[0223] In summary, in this application, memory B cells that specifically bind to humanized mouse anti-A35R of fully human antibodies were screened, and human-derived high-neutralizing activity MPXV antibodies A35R-9E4, A35R-9F8, A35R-10C4, A35R-10D5, A35R-10E7, A35R-10H6, A35R-18E2, and A35R-18H2 were obtained. These antibodies are completely different from the reported monkeypox virus antibody sequences, are newly discovered antibodies, have a strong ability to bind to antigens, and at the same time, have strong neutralizing VACV activity. Moreover, they can protect mice from the attack of lethal dose of VACV-WR. The results suggest that these 8 human-derived antibodies have application value in the clinical treatment and prevention of monkeypox virus.

[0224] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0225] The above-described embodiments only represent several implementation manners of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of this application, several modifications and improvements can still be made, and these all belong to the protection scope of this application. Therefore, the protection scope of this application patent should be subject to the appended claims.

Claims

1. An antibody against monkeypox virus A35R protein, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; The CDR sequence of the heavy chain variable region and the CDR sequence of the light chain variable region are selected from any one of items a1) to a8); a1) HCDR1 with a sequence as shown in SEQ ID NO:5, HCDR2 with a sequence as shown in SEQ ID NO:6, and HCDR3 with a sequence as shown in SEQ ID NO:7; and LCDR1 with a sequence as shown in SEQ ID NO:8, LCDR2 with a sequence as shown in SEQ ID NO:9, and LCDR3 with a sequence as shown in SEQ ID NO:10; a2) HCDR1 with a sequence as shown in SEQ ID NO:21, HCDR2 with a sequence as shown in SEQ ID NO:22, and HCDR3 with a sequence as shown in SEQ ID NO:23; and LCDR1 with a sequence as shown in SEQ ID NO:24, LCDR2 with a sequence as shown in SEQ ID NO:25, and LCDR3 with a sequence as shown in SEQ ID NO:26; a3) HCDR1 with a sequence as shown in SEQ ID NO:37, HCDR2 with a sequence as shown in SEQ ID NO:38, and HCDR3 with a sequence as shown in SEQ ID NO:39; and LCDR1 with a sequence as shown in SEQ ID NO:40, LCDR2 with a sequence as shown in SEQ ID NO:41, and LCDR3 with a sequence as shown in SEQ ID NO:42; a4) HCDR1 with a sequence as shown in SEQ ID NO:53, HCDR2 with a sequence as shown in SEQ ID NO:54, and HCDR3 with a sequence as shown in SEQ ID NO:55; and LCDR1 with a sequence as shown in SEQ ID NO:56, LCDR2 with a sequence as shown in SEQ ID NO:57, and LCDR3 with a sequence as shown in SEQ ID NO:58; a5) HCDR1 with a sequence as shown in SEQ ID NO:69, HCDR2 with a sequence as shown in SEQ ID NO:70, and HCDR3 with a sequence as shown in SEQ ID NO:71; and LCDR1 with a sequence as shown in SEQ ID NO:72, LCDR2 with a sequence as shown in SEQ ID NO:73, and LCDR3 with a sequence as shown in SEQ ID NO:74; a6) HCDR1 with a sequence as shown in SEQ ID NO:85, HCDR2 with a sequence as shown in SEQ ID NO:86, and HCDR3 with a sequence as shown in SEQ ID NO:87; and LCDR1 with a sequence as shown in SEQ ID NO:88, LCDR2 with a sequence as shown in SEQ ID NO:89, and LCDR3 with a sequence as shown in SEQ ID NO:90; a7) HCDR1 with a sequence as shown in SEQ ID NO: 101, HCDR2 with a sequence as shown in SEQ ID NO: 102, and HCDR3 with a sequence as shown in SEQ ID NO: 103; and LCDR1 with a sequence as shown in SEQ ID NO: 104, LCDR2 with a sequence as shown in SEQ ID NO: 105, and LCDR3 with a sequence as shown in SEQ ID NO: 106; a8) HCDR1 with a sequence as shown in SEQ ID NO:117, HCDR2 with a sequence as shown in SEQ ID NO:118, and HCDR3 with a sequence as shown in SEQ ID NO:119; and LCDR1 with a sequence as shown in SEQ ID NO:120, LCDR2 with a sequence as shown in SEQ ID NO:121, and LCDR3 with a sequence as shown in SEQ ID NO:

122.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID Nos: 1, 17, 33, 49, 65, 81, 97 and 113; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID Nos: 2, 18, 34, 50, 66, 82, 98 and 114; Optionally, the amino acid sequence of the heavy chain variable region 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; Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 18; Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 34; Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 49, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 50; Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 65, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 66; Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 81, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 82; Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 97, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 98; Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:113, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:

114.

3. The antibody according to claim 1 or 2, characterized in that Also included are the heavy chain constant region and the light chain constant region; Optionally, the species of the heavy chain constant region and the light chain constant region are derived from human; Optionally, the sequence of the light chain constant region is selected from the sequence of a κ-type light chain constant region or a λ-type light chain constant region; Optionally, the sequence of the heavy chain constant region is selected from the sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

4. A nucleic acid molecule, characterized in that It encodes the antibody according to any one of claims 1 to 3.

5. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 4.

6. A host cell, characterized in that Comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. The method for preparing an antibody according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: Cultivating the host cell according to claim 6, and isolating the antibody from the obtained culture.

8. A detection reagent or drug, characterized in that: The invention comprises the antibody according to any one of claims 1 to 3.

9. Use of the antibody according to any one of claims 1 to 3 in detecting monkeypox virus A35R protein for non-diagnostic purposes.

10. Use of the antibody according to any one of claims 1 to 3 in the preparation of a detection product, a preventive drug or a therapeutic drug for orthopoxvirus; Optionally, the orthopoxvirus comprises monkeypox virus and / or vaccinia virus.

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