Monoclonal antibody for resisting monkey pox virus A35R protein or antigen binding fragment and application thereof

Abstract

The invention discloses a monoclonal antibody for resisting monkey pox virus A35R protein or an antigen binding fragment of the monoclonal antibody and application of the monoclonal antibody. The monkey pox virus antibody A35R-10G7 with a high protection effect is obtained by screening memory B cells specifically combined with a fully human antibody humanized mouse anti-A35R, and the monkey pox virus antibody A35R-10G7 has strong combining capacity with monkey pox virus, strong affinity and in-vivo protection effect.

Description

Technical Field

[0001] The present application belongs to the field of antibody engineering, and specifically relates to a monoclonal antibody or an antigen-binding fragment thereof against monkeypox virus A35R protein and its application. Background Art

[0002] Monkeypox is a zoonotic infectious disease caused by Monkeypox virus (MPXV), with typical clinical manifestations including fever, headache, swollen lymph nodes and muscle pain, accompanied by rash after fever, etc. MPXV is a double-stranded DNA virus with two infectious virus particle forms: intracellular mature virus particles (IMV) and extracellular enveloped virus particles (EEV), and can be transmitted among people through direct contact (sexual contact or skin contact), respiratory droplets, etc.

[0003] Monkeypox virus belongs to the Poxviridae family, the Chordopoxvirinae subfamily, and the Orthopoxvirus genus (OPXV). Its genus also includes smallpox virus (VARV), vaccinia (smallpox vaccine) virus (VACV), rinderpest virus (CPXV), and camelpox virus (CMLV). According to a comparison between VACV and MPXV, the surface proteins between poxviruses show approximately 93-98% sequence similarity, suggesting that they play similar roles in the process of entering the body.

[0004] Since the eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination, young people now have low immunity to orthopoxviruses, which may be one of the reasons for the current MPOX outbreak. With the spread of multiple strains of monkeypox virus in non-endemic countries, not only are the transmission routes diverse and hidden, and the symptoms of infection atypical, but the monkeypox virus also accelerates mutation during human-to-human transmission, and its mutation rate is far beyond expectations, posing an increasing threat to global public health security. Although there are not many cases of monkeypox in China now, with the flow of populations within and outside the country, how to prevent and control monkeypox infection has become a key issue that needs to be solved urgently. And currently there is no specific antiviral therapy approved for the prevention or treatment of human MPXV infection.

[0005] It is very necessary to develop new monkeypox virus monoclonal antibodies that have strong binding affinity to antigen proteins and have in vivo protective effects. Summary of the invention

[0006] Based on this, an embodiment of the present application provides a monoclonal antibody against the A35R protein of monkeypox virus, which has a high affinity for the A35R protein of monkeypox virus and can provide protective effects in mice.

[0007] The technical solutions include:

[0008] An embodiment of the present application provides a monoclonal antibody against the A35R protein of monkeypox virus or an antigen-binding fragment thereof, the heavy chain variable region of which comprises HCDR1, HCDR2 and HCDR3 as shown below: HCDR1 is as shown in SEQ ID NO:5, HCDR2 is as shown in SEQ ID NO:6, and HCDR3 is as shown in SEQ ID NO:7; and, the light chain variable region of which comprises LCDR1, LCDR2 and LCDR3 as shown below: LCDR1 is as shown in SEQ ID NO:8, LCDR2 is as shown in SEQ ID NO:9, and LCDR3 is as shown in SEQ ID NO:10.

[0009] In some embodiments, the sequences of the heavy chain framework region and the light chain framework region independently comprise one of the antibody framework region sequences derived from humans and the antibody framework region sequences derived from mice. Optionally, the sequences of the heavy chain framework region and the light chain framework region are all derived from human antibody framework region sequences.

[0010] In some embodiments, the heavy chain variable region and the light chain variable region are as shown in SEQ ID NO:1 and SEQ ID NO:2 respectively.

[0011] In some embodiments, the monoclonal antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region.

[0012] In some embodiments, the species of the heavy chain constant region and the light chain constant region are derived from humans.

[0013] In some embodiments, the antibody independently has the sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

[0014] In some embodiments, the sequence of the light chain constant region is selected from the sequence of the κ-type light chain constant region or the λ-type light chain constant region.

[0015] An embodiment of the present application further provides a detection reagent or a detection kit, comprising the monoclonal antibody or its antigen-binding fragment.

[0016] An embodiment of the present application further provides a drug, comprising the monoclonal antibody or its antigen-binding fragment.

[0017] One embodiment of the present application also provides a nucleic acid encoding the monoclonal antibody or its antigen-binding fragment.

[0018] One embodiment of the present application also provides a recombinant vector comprising the nucleic acid.

[0019] One embodiment of the present application also provides a host cell comprising the nucleic acid or the recombinant vector.

[0020] In some embodiments, the host cell includes eukaryotic cells. Optionally, the eukaryotic cells include yeast cells, insect cells, and mammalian cells. Optionally, the mammalian cells include 293T cells, 293F cells, CHO cells, or HEK293 cells, etc.

[0021] One embodiment of the present application also provides a method for preparing the monoclonal antibody, the preparation method comprising the following steps: using the host cell to prepare the monoclonal antibody or its antigen-binding fragment.

[0022] One embodiment of the present application also provides the use of the monoclonal antibody or its antigen-binding fragment in detecting the A35R protein of monkeypox virus for non-diagnostic purposes.

[0023] One embodiment of the present application also provides the use of the monoclonal antibody or its antigen-binding fragment in preparing detection products, prophylactic drugs, or therapeutic drugs for monkeypox virus and / or vaccinia virus.

[0024] Compared with the traditional technology, the present application has the following beneficial effects:

[0025] The monoclonal antibody or its antigen-binding fragment of the present application has a strong affinity for the A35R protein of monkeypox virus and has an in vivo protective effect. It is completely different from the reported monkeypox virus antibody sequences, providing products for the detection of monkeypox virus and the possibility for developing monkeypox virus products with in vivo protective effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for description in the embodiments. Obviously, the following drawings are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1 Results of SDS-PAGE of molecular sieve purification of monkeypox virus A35R protein;

[0028] Figure 2Survival of antibody A35R-10G7 Fab and antigen A35R on a molecular sieve;

[0029] Figure 3 Kinetic curve results of antibody A35R-10G7 and A35R;

[0030] Figure 4 Neutralization curve results of antibody A35R-10G7 against VACV-WR;

[0031] Figure 5 Schematic diagram of antibody protection experiment, curves of survival and body weight changes of mice after infection. Among them, the upper figure is a schematic diagram of the experiment implementation, and the lower figure is the curves of survival and body weight changes of mice after low-dose infection. Detailed implementation mode

[0032] To facilitate the understanding of this application, the following will provide a more comprehensive description of this application. This 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 this application more thorough and comprehensive.

[0033] 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 herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0034] The term "and / or" used herein includes any one of two or more related listed items, and also includes 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 a combination of all related listed items.

[0035] In this article, "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.

[0036] In this article, 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.

[0037] In this article, "optionally", "optional", "option", mean optional, that is, it means any one of the two parallel options of "yes" or "no". If "optional" appears multiple times in a technical solution, without special explanation and without contradiction or mutual restriction relationship, each "optional" is independent of each other.

[0038] As used herein, a "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous population of antibodies or antigen-binding fragments that participate in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" or antigen-binding fragment thereof encompasses intact and full-length monoclonal antibodies as well as antibody fragments such as Fab, Fab', (Fab')2, Fv, scFv mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. In addition, a "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and their antigen-binding fragments prepared in any number of ways, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0039] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region generally refers to a portion of an antibody, typically a portion of the light or heavy chain, usually about 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which varies in sequence between antibodies and is responsible for the binding and specificity of a particular antibody to its particular antigen. The variability in sequence is concentrated in those regions called CDRs, while the more conserved regions in the variable domain are called FR regions (Framework regions). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region is a rodent or murine variable region.

[0040] As used herein, a "Fab fragment" (Antigen-binding fragment) is also called an antigen-binding fragment and is the region of an antibody structure that can bind to an antigen. It consists of the entire light chain (variable and constant regions) and a portion of the heavy chain structure (variable region and one constant region fragment CH1), and the light chain and heavy chain are linked by a disulfide bond.

[0041] As used herein, "PRNT50" is a commonly used assay in virology research, which stands for "50% Plaque Reduction Neutralization Test", that is, the 50% plaque reduction neutralization test. Calculate PRNT50: Based on the plaque count results, calculate the serum or antibody dilution that can reduce the number of plaques by 50%, and this dilution is the PRNT50 value.

[0042] In this application, memory B cells that specifically bind to the humanized mouse anti-A35R against the whole human antibody were screened to obtain the human-derived MPXV antibody A35R-10G7. A35R-10G7 is a novel humanized monoclonal antibody against the monkeypox virus with strong specific binding ability and high affinity to the monkeypox virus, and can provide high protection in vivo.

[0043] Among them, the specific scheme includes:

[0044] I. Preparation and purification of monoclonal antibody

[0045] By expressing and purifying the A35R protein of the monkeypox virus, sorting specific memory B cells that bind to the A35R protein, and expressing and purifying the A35R antibody of the monkeypox virus, the monkeypox virus A35R monoclonal antibody A35R-10G7 was obtained. The amino acid sequences of the light chain and heavy chain of this monkeypox virus monoclonal antibody, CDR1-CDR3 of the light chain variable region, and CDR1-CDR3 of the heavy chain variable region are as follows. The light chain variable region and the heavy chain variable region are different from the existing sequences and are specific.

[0046] II. Performance verification of monoclonal antibody

[0047] (I) Detection of the affinity between the antibody and antigen A35R by surface plasmon resonance technology

[0048] The A35R-10G7 antibody has a good affinity for the antigen A35R.

[0049] (II) Survival of antibody A35R-10G7 Fab and antigen A35R on the molecular sieve

[0050] Antibody A35R-10G7 Fab and antigen A35R can bind and survive on the molecular sieve.

[0051] (III) Antibody neutralization test

[0052] The neutralizing activity of the antibody was detected by the plaque method. The PRNT50 value of the neutralization curve result of the A35R-10G7 antibody against VACV-WR > 100 μg / mL, and its in vitro neutralizing activity is poor.

[0053] (IV) Animal protection test

[0054] Under the condition of low-dose virus infection, administration of the A35R-10G7 antibody can provide protection for mice infected with the monkeypox virus.

[0055] The following is a detailed description in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, they 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. For the experimental methods without specific conditions noted in the embodiments, they are carried out under conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the manufacturers.

[0056] The main key drugs and reagents involved in the following embodiments are shown in Table 1:

[0057] Table 1

[0058] Name Model Manufacturer Humanized mouse with fully human antibodies RenMab Beijing Biocytogen Co., Ltd. 14K Chip 750-00021 Berkeley lights

[0059] The main instruments involved in the following embodiments are shown in Table 2:

[0060] Table 2

[0061]

[0062]

[0063] Example 1 Preparation and Purification of Monoclonal Antibodies Against Monkeypox Virus

[0064] 1. Expression and Purification of Monkeypox Virus A35R Protein

[0065] 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.

[0066] Among them, S90-T181 (SEQ ID NO: 17) of A35R was intercepted, and after gene sequence optimization, it was constructed into the vector pCAGGS:

[0067] STTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSD VLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT

[0068] As Figure 1 shown, the results of SDS-PAGE of the molecular sieve purification of monkeypox virus A35R protein showed that the protein flowed through Superdex200GL, and an absorption peak at UV280 nM could be detected at the position of ~17 mL. The molecular weight of the protein in SDS-PAGE was about ~15 kDa, and the obtained protein had a high purity.

[0069] 2. Sorting of Specific Memory B Cells Binding to A35R Protein

[0070] 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. Humanized mice with fully human antibodies (RenMab) were immunized 3 times at a dose of 2.5 μg / mouse / time. 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 cell sorter. The variable region gene sequences of the antibodies were obtained after reverse transcription and PCR amplification.

[0071] The monoclonal antibody of monkeypox virus A35R-10G7 was obtained. The amino acid sequences of the light chain and heavy chain of this monkeypox virus monoclonal antibody, CDR1-CDR3 of the light chain variable region, and CDR1-CDR3 of the heavy chain variable region are as follows. The light chain variable region and the heavy chain variable region are different from the existing sequences and are specific.

[0072] Monoclonal antibody A35R-10G7:

[0073] Amino acid sequence of the heavy chain variable region, SEQ ID NO:1:

[0074] QVQLVQSGGGVVQPGRSLRLSCAASGFIFRSYVMHWVRQAPGKGLEWVAVISYDGTNKDYADSVKGRFTFSRDNSKNTLYLQMNSLRTEDTAVYYCVKGGYRYGPFDYWGQGTLVTVSS

[0075] Amino acid sequence of the light chain variable region, SEQ ID NO:2:

[0076] EIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFRGSGSGTDFTLKISRVEAEDVGVYYCMQPLQTPFTFGPGTKVEIK

[0077] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO:3:

[0078] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCATCTTCAGAAGTTATGTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAACTAATAAAGACTACGCAGACTCCGTGAAGGGCCGATTCACCTTCTCCAGAGACAATTCCAAGAACACGCTGTATTTGCAAATGAACAGCCTGAGAACTGAGGACACGGCTGTGTATTACTGTGTGAAAGGTGGATATAGATATGGTCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0079] Nucleic acid sequence of the light chain variable region, SEQ ID NO:4:

[0080] GAAATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGAGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAACCTCTACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAA

[0081] Among them, the amino acids of the CDR region of the A35R-10G7 antibody are shown in Table 3; the nucleotide sequence of the CDR region of the A35R-10G7 antibody is shown in Table 4.

[0082] Table 3

[0083]

[0084] Table 4

[0085]

[0086] 3. Expression, purification and digestion of antibodies

[0087] 3.1 Antibody cloning and construction: 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), and 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-fulllength).

[0088] 3.2 Antibody expression and purification: 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 and 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 plasmid and PEI were mixed, left standing for 20 min, and then added to the cells. Incubate in a constant temperature shaker at 37°C. After 5 days, the supernatant was harvested by centrifugation at 8000 r for 90 min. The supernatant was passed through an affinity column Protein A, and the antibody was obtained after elution with 0.1 M Glycine, pH 3.0 by an AKTA Purifier machine, and concentrated and exchanged into PBS.

[0089] 3.3 Obtaining Fab fragments by digesting the antibody: Use a commercial reagent (Pierce TM Fab Preparation Kit, CatNo: 44985, Thermo Scientific) to digest the antibody, and the Fab fragments were separated by a Protein A affinity column and identified by SDS-PAGE. As Figure 2 shown, the molecular weight of Fab is ~25 kDa.

[0090] Example 2 Detection of the binding of the antibody Fab fragment to the antigen on the column

[0091] The antigen protein A35R, the antibody Fab fragment, and their mixture were passed through the chromatography column Superdex 200GL in sequence, and UV280 nM absorption peaks could be detected at positions ~17 mL, ~18 mL, and ~13 mL respectively. Among them, the UV280 absorption peak position of the mixture of antigen A35R and antibody Fab fragment was less than that of the individual protein, indicating that antigen A35R binds to antibody 10G7-Fab.

[0092] Example 3 Detection of the affinity between the antibody and the antigen by surface plasmon resonance technology

[0093] Surface plasmon resonance (SPR) experiments can be used to detect the interaction between proteins in vitro. By detecting the change in the response value of the mobile phase flowing over the chip surface, the kinetic parameters of the binding of two proteins can be known. It is carried out on the instrument BIACORE 8K. In surface plasmon resonance experiments, usually one of the two proteins to be analyzed is immobilized on the surface of the metal chip, and the other is used as the mobile phase to flow over the chip surface, and the change in the response value caused by it is detected. Before the experiment, PBST should be filtered through a 0.22 μM filter membrane and then autoclaved. The antibody concentration is 10 μg / mL (can be adjusted appropriately), and the antigen concentration titers are 400 nM, 200 nM, 100 nM, 50 nM, 25 nM (can be adjusted appropriately). The affinity and binding characteristics of the A35R antigen-antibody are as Figure 3 shown.

[0094] Example 4 Antibody Neutralization Test

[0095] The neutralizing activity of the antibody was detected by the plaque method, which is briefly described as follows: The initial concentration of the antibody is 200 μg / mL, and it is serially diluted 4-fold for 10 gradients. Take 400 μL of each gradient and mix it with an equal volume of the dilution of vaccinia virus VACV-WR (containing 10% complement and 100 μg / mL of the targeting IMV antibody), and incubate at 37 °C for 1 h. Vero cells were seeded into 12-well plates in advance and can be used for neutralization experiments when the cell confluence is ~100%. After the cells are rinsed twice, add the mixture of antibody and virus, 400 μL / well. Set two replicate wells for each antibody gradient, and set a control well with only virus without antibody. Incubate at 37 °C for 1 h. Discard the mixture of antibody and virus, rinse the cells once, and add 1 mL / well of the fixative. The fixative is prepared by mixing methyl cellulose and high-glucose culture medium (2×) in a volume ratio of 1:1. Place the cell plate in the cell culture incubator. After 48 hours, fix with 8% tissue cell fixative, then add crystal violet for staining and counting, and calculate the neutralizing activity value PRNT50 of the antibody according to the degree of plaque reduction.

[0096] As Figure 4 shown, for the neutralization curve result of the A35R antibody against VACV-WR, the PRNT50 value of A35R-10G7 > 100 μg / mL, and no neutralizing activity was demonstrated in the neutralization test.

[0097] Example 5 Animal Protection Test

[0098] To further evaluate the applicability of the antibody, the protective effect was evaluated at the mouse level. Since animal experiments on monkeypox virus infection need to be carried out in a high-level biosafety level 3 laboratory, initially, the Western Reserve (VACV-WR) strain of vaccinia virus (which belongs to the same family of poxviruses as monkeypox virus) was used for evaluation. 7- to 8-week-old mice were infected with 5 LD50 of VACV-WR by intranasal instillation. 4 hours before infection and 4 hours after infection, the antibody A35R-10G7 was intraperitoneally injected at a dose of 200 μg per mouse.

[0099] As Figure 5 shown, after infection with 5 LD50 of the virus, the body weights of mice in the group with irrelevant antibodies all decreased by more than 20% on the 5th day, while the body weights of mice in the group injected with the A35R-10G7 antibody showed no obvious change.

[0100] In summary, in this application, memory B cells specifically binding to humanized mouse anti-A35R of fully human antibodies were screened to obtain the highly protective human MPXV antibody A35R-10G7, which is completely different from the reported monkeypox virus antibody sequences. It is a newly discovered antibody with a strong ability to bind to antigens. Moreover, it can protect mice from attack by a lethal dose of VACV-WR. The results suggest that this human antibody has application value in the clinical treatment and prevention of monkeypox virus.

[0101] 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 recorded in this specification.

[0102] The above-described embodiments merely represent several implementation manners of this application. The description is relatively specific and detailed, but it should not be construed as a limitation on 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 the patent of this application should be subject to the appended claims.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against monkeypox virus A35R protein, characterized in that: Its heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown below: HCDR1 is shown in SEQ ID NO:5, HCDR2 is shown in SEQ ID NO:6, HCDR3 is shown in SEQ ID NO:7; And, its light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown below: LCDR1 is shown in SEQ ID NO:8, LCDR2 is shown in SEQ ID NO:9, LCDR3 is shown in SEQ ID NO:

10.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region and light chain variable region are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

3. A detection reagent or a detection kit, characterized in that: The invention comprises the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A drug, characterized in that The invention comprises the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

5. A nucleic acid, characterized in that It encodes the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

6. A recombinant vector, characterized in that: Comprising the nucleic acid of claim 5.

7. A host cell, characterized in that Comprising the nucleic acid according to claim 5 or the recombinant vector according to claim 6.

8. The method for preparing a monoclonal antibody according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: using the host cell according to claim 7 to prepare a monoclonal antibody or an antigen-binding fragment thereof.

9. Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in detecting monkeypox virus A35R protein for non-diagnostic purposes.

10. Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a detection product, preventive drug or therapeutic drug for monkeypox virus and / or vaccinia virus.

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