Monoclonal antibodies against monkeypox virus A35R protein or their antigen-binding fragments and their applications

Abstract

This application discloses a monoclonal antibody against monkeypox virus A35R protein or its antigen-binding fragment and its application. A monkeypox virus antibody A35R-10G7 with high protective efficacy was obtained by screening humanized mouse memory B cells that specifically bind to A35R using fully human antibodies. This antibody exhibits strong binding ability to monkeypox virus, high affinity, and in vivo protective activity.

Description

Technical Field

[0001] This application belongs to the field of antibody engineering, specifically relating to monoclonal antibodies against monkeypox virus A35R protein or their antigen-binding fragments and their applications. Background Technology

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

[0003] Monkeypox virus belongs to the family Poxviridae, subfamily Chordopoxvirinae, and genus Orthopoxvirus (OPXV). Other viruses in this genus include smallpox virus (VARV), cowpox vaccine virus (VACV), rinderpest virus (CPXV), and camelpox virus (CMLV). Comparisons between VACV and MPXV show that the surface proteins of these poxviruses exhibit approximately 93-98% sequence similarity, suggesting they play similar roles in the process of entering the body.

[0004] The eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination have resulted in lower immunity to ornithovirus among younger populations, which may be one of the reasons for the current MPXV outbreak. With the spread of various monkeypox virus strains in non-endemic countries, not only are transmission routes diverse and insidious, and infection symptoms atypical, but monkeypox virus also mutates rapidly during human-to-human transmission, with a mutation rate far exceeding expectations, posing an increasing threat to global public health security. Although monkeypox cases are currently low in China, with the increasing movement of people both domestically and internationally, how to prevent and control monkeypox infection has become a critical issue that urgently needs to be addressed. Furthermore, there are currently no specific antiviral therapies approved for the prevention or treatment of human MPXV infection.

[0005] It is essential to develop novel monkeypox virus monoclonal antibodies that have strong affinity for binding antigen proteins and provide in vivo protection. Summary of the Invention

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

[0007] The technical solution includes:

[0008] One embodiment of this application provides a monoclonal antibody against monkeypox virus A35R protein or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown below: HCDR1 is shown as SEQ ID NO:5, HCDR2 is shown as SEQ ID NO:6 and HCDR3 is shown as SEQ ID NO:7; and its light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown below: LCDR1 is shown as SEQ ID NO:8, LCDR2 is shown as SEQ ID NO:17 and LCDR3 is shown as SEQ ID NO:9.

[0009] In some embodiments, the sequences of the heavy chain backbone region and the light chain backbone region independently include one of an antibody backbone region sequence derived from humans and an antibody backbone region sequence derived from mice. Optionally, the sequences of the heavy chain backbone region and the light chain backbone region are all antibody backbone region sequences derived from humans.

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

[0011] In some embodiments, the monoclonal antibody or its antigen-binding fragment further includes 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 a 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] One embodiment of this application also provides a detection reagent or detection kit, including the monoclonal antibody or its antigen-binding fragment.

[0016] One embodiment of this application also provides a drug comprising the monoclonal antibody or an antigen-binding fragment thereof.

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

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

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

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

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

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

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

[0024] Compared with traditional technologies, this application has the following advantages:

[0025] The monoclonal antibody or its antigen-binding fragment of this application has a strong affinity for the monkeypox virus protein A35R and has in vivo protective effects. It is completely different from the previously reported monkeypox virus antibody sequence, providing a product for the detection of monkeypox virus and making it possible to develop monkeypox virus products with in vivo protective effects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0028] Figure 2To assess the survival of antibody A35R-10G7 Fab and antigen A35R on molecular sieves;

[0029] Figure 3 The results show the kinetic curves of antibodies A35R-10G7 and A35R.

[0030] Figure 4 The results show the neutralization curve of antibody A35R-10G7 against VACV-WR.

[0031] Figure 5 The diagram below illustrates the survival and weight changes of mice after infection in an antibody protection experiment. The top diagram shows the experimental procedure, while the bottom diagram shows the survival and weight changes of mice after low-dose infection. Detailed Implementation

[0032] To facilitate understanding of this application, a more complete description will be provided below. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of this application will be more thorough and complete.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0034] The term “and / or” as used herein includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including 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 refers to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0036] In this article, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions that include the listed features.

[0037] In this article, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "options" in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, then each "option" is independent.

[0038] In this document, "monoclonal" antibody or its antigen-binding fragment refers to a homogeneous group of antibodies or antigen-binding fragments that participate in the highly specific recognition and binding of a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically consist of different antibodies targeting different antigenic determinants. The term "monoclonal antibody" or its antigen-binding fragment encompasses full-length and complete monoclonal antibodies, as well as antibody fragments such as Fab, Fab', (Fab')2, Fv, scFv mutants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" or its antigen-binding fragment refers to any number of such antibodies and their antigen-binding fragments prepared in any manner, including but not limited to hybridoma, 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. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differs in sequence between antibodies and is responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called CDRs, while more conserved regions within a 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 antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region is a rodent or mouse variable region.

[0040] In this article, the "Fab fragment" (Antigen-binding fragment) is also called the antigen-binding fragment, which is the region in the antibody structure that can bind to the antigen. It consists of a complete light chain (variable region and constant region) and a partial heavy chain structure (variable region and a constant region fragment CH1), with the light chain and heavy chain linked by a disulfide bond.

[0041] In this article, "PRNT50" is a commonly used detection method in virology research, short for "50% Plaque Reduction Neutralization Test." PRNT50 is calculated by determining the serum or antibody dilution that reduces the number of plaques by 50% based on the plaque count results; this dilution is the PRNT50 value.

[0042] This application describes the development of a human MPXV antibody, A35R-10G7, by screening humanized mouse memory B cells that specifically bind to A35R using fully human antibodies. A35R-10G7 is a novel humanized monoclonal antibody against monkeypox virus that exhibits strong specific binding ability and affinity, providing high in vivo protection.

[0043] The specific plans include:

[0044] I. Preparation and Purification of Monoclonal Antibodies

[0045] 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, a monkeypox virus A35R monoclonal antibody A35R-10G7 was obtained. The amino acid sequences of the light and heavy chains, the variable regions CDR1-CDR3 of the light chain, and the variable regions CDR1-CDR3 of the heavy chain of this monkeypox virus monoclonal antibody are as follows. The variable regions of the light chain and the variable regions of the heavy chain are different from existing sequences, and have specificity.

[0046] II. Performance Validation of Monoclonal Antibodies

[0047] (I) Detection of antibody-antigen A35R affinity using 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 molecular sieves

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

[0051] (III) Antibody Neutralization Test

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

[0053] (iv) Animal protection experiments

[0054] In low-dose viral infection, administration of A35R-10G7 antibody can provide protection against monkeypox virus infection in mice.

[0055] The following detailed description is provided with reference to specific embodiments. Unless otherwise specified, the embodiments do not include components other than unavoidable impurities. Unless otherwise specified, the reagents and instruments used in the embodiments are conventionally selected in the art. Experimental methods not specifying specific conditions in the embodiments are implemented according to conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

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

[0057] Table 1

[0058] name model Manufacturers Fully humanized antibody mice RenMab Biocytok Pharmaceutical Technology Co., Ltd. 14K chip 750-00021 Berkeleylights

[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 monkeypox virus monoclonal antibody

[0064] 1. Expression and purification of monkeypox virus A35R protein

[0065] The A35R gene was constructed and transfected into the eukaryotic expression vector pCAGGS into 293F cells. The A35R protein was expressed by cell secretion. The cell supernatant was collected and purified using affinity chromatography and molecular sieves, and then identified by SDS-PAGE.

[0066] Specifically, the S90-T181 (SEQ ID NO:15) segment of A35R was extracted, and after gene sequence optimization, it was constructed into the vector pCAGGS.

[0067] STTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSD VLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT

[0068] like Figure 1 As shown, the SDS-PAGE results of monkeypox virus A35R protein purification molecular sieve showed that when the protein flowed through a Superdex 200GL, a UV 280 nM absorption peak could be detected at a position of ~17 mL. The molecular weight of the protein in the SDS-PAGE was approximately ~15 kDa, and the obtained protein had high purity.

[0069] 2. Sorting of specific memory B cells that bind to A35R protein

[0070] The A35R gene was constructed into an mRNA vector, transcribed in vitro, and capped. Concentration and quality were then determined. Humanized mice (RenMab) received three immunizations at a dose of 2.5 μg / mouse / immunization. Serum was collected before immunization and 12 days after each immunization to detect antibody titers. Once the antibody titer reached an appropriate level, a booster immunization was administered. Lymph nodes from the humanized mice were then collected, ground into single cells, incubated with antigen molecules, and specifically sorted using a Beacon sorter. The variable region gene sequence of the antibody was obtained after reverse transcription and PCR amplification.

[0071] A monoclonal antibody against monkeypox virus A35R-10G7 was obtained. The amino acid sequences of the light and heavy chains, the variable regions CDR1-CDR3 of the light chain, and the variable regions CDR1-CDR3 of the heavy chain are as follows. The variable regions of the light chain and the variable regions of the heavy chain are different from existing sequences, and have specificity.

[0072] A35R-10G7 monoclonal antibody:

[0073] Heavy chain variable region amino acid sequence, SEQ ID NO:1:

[0074] QVQLVQSGGGVVQPGRSLRLSCAASGFIFRSYVMHWVRQAPGKGLEWVAVISYDGTNKDYADSVKGRFTFSRDNSKNTLYLQMNSLRTEDTAVYYCVKGGYRYGPFDYWGQGTLVTVSS

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

[0076] EIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFRGSGSGTDFTLKISRVEAEDVGVYYCMQPLQTPFTFPGGTKVEIK

[0077] Heavy chain variable region nucleic acid sequence, SEQ ID NO:3:

[0078] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCATCTTCAGAAGTTATGTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAACTAATAAAGACTACGCAGACTCCGTGAAGGGCCGATTCACCTTCTCCAGAGACAATTCCAAGAACACGCTGTATTTGCAAATGAACAGCCTGAGAACTGAGGACACGGCTGTGTATTACTGTGTGAAAGGTGGATATAGATATGGTCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

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

[0080] GAAATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGAGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAACCTCTACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAA

[0081] 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. Antibody expression, purification, and enzyme digestion

[0087] 3.1 Antibody Cloning and Construction: The antibody heavy chain variable region gene (VH) and the IgG1 constant region gene (CH) were fused into the pCAGGS vector (named pCAGGS-Heavy chain-full length), and the antibody light chain variable region gene (VL) and the constant region gene (CL) were fused into the pCAGGS vector (named pCAGGS-Light chain-full length).

[0088] 3.2 Antibody Expression and Purification: When co-transfecting 100 mL of 293F cells with light and heavy chain plasmids, 36 μg of heavy chain plasmid and 75 μg of light chain plasmid were dissolved in 1 mL of HBS and allowed to stand for 5 min. 300 μg of PEI was dissolved in 1 mL of HBS and allowed to stand for 5 min. The plasmid and PEI were mixed, allowed to stand for 20 min, and then added to the cells. The cells were cultured at 37°C in a shaker. After 5 days, the supernatant was harvested after centrifugation at 8000 rpm for 90 min. The supernatant was passed through a Protein A affinity column and eluted with 0.1 M Glycine at pH 3.0 using an AKTA Purifier to obtain the antibody. The antibody was then concentrated and the solution was changed to PBS.

[0089] 3.3 Obtaining Fab fragments by enzyme digestion of antibodies: Using commercially available reagents (Pierce) TM The antibody was digested with enzymes using the Fab Preparation Kit (CatNo: 44985, Thermo Scientific), and the Fab fragment was obtained by separation using a Protein A affinity column and identified by SDS-PAGE. Figure 2 As shown, the molecular weight of Fab is ~25kDa.

[0090] Example 2: On-column survival detection of antibody Fab fragment binding to antigen

[0091] The antigen protein A35R, the antibody Fab fragment, and their mixture were sequentially passed through a Superdex 200GL chromatography column. UV 280 nM absorption peaks were detected at ~17 mL, ~18 mL, and ~13 mL, respectively. The UV 280 absorption peak of the mixture of antigen A35R and antibody Fab fragment was lower than that of the individual protein, indicating that antigen A35R binds to antibody 10G7-Fab.

[0092] Example 3: Detection of antibody-antigen affinity using surface plasmon resonance technology

[0093] Surface plasmon response (SPR) assays can be used to detect interactions between proteins in vitro. The kinetic parameters of protein binding are determined by detecting changes in the response value as the mobile phase flows over the chip surface. This is performed on a BIACORE 8K instrument. In SPR assays, one of the two proteins to be analyzed is typically immobilized on a metal chip surface, while the other is used as the mobile phase flowing over the chip surface, and the resulting change in response value is detected. Before the experiment, PBST should be filtered through a 0.22 μM filter and then autoclaved. The antibody concentration is 10 μg / mL (adjustable as needed), and the antigen titers are 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM (adjustable as needed). The affinity and binding characteristics of the A35R antigen antibody are as follows: Figure 3 As shown.

[0094] Example 4 Antibody Neutralization Test

[0095] The neutralizing activity of the antibody was detected using the plaque assay, as briefly described below: The initial antibody concentration was 200 μg / mL, serially diluted 4-fold to create 10 gradients. 400 μL of each gradient was mixed with an equal volume of vaccinia virus VACV-WR dilution buffer (containing 10% complement and 100 μg / mL targeting IMV antibody) and incubated at 37°C for 1 h. Vero cells were pre-seeded into 12-well plates; cells with ~100% confluence were suitable for the neutralization experiment. After washing the cells twice, 400 μL of the antibody-virus mixture was added to each well, with two replicate wells for each antibody gradient and a control well containing only virus and no antibody. The cells were incubated at 37°C for 1 h. The antibody-virus mixture was discarded, and the cells were washed once. 1 mL of fixative was added to each well, prepared by mixing methylcellulose and high-glucose medium (2×) at a 1:1 volume ratio. The cell culture plates were then placed in a cell culture incubator. After 48 hours, the cells were fixed with 8% tissue cell fixative, then stained with crystal violet, counted, and the neutralizing activity value PRNT50 of the antibody was calculated based on the degree of reduction in plaques.

[0096] like Figure 4 As shown in the figure, the neutralization curve results of A35R antibody against VACV-WR show that the PRNT50 value of A35R-10G7 is >100μg / mL, and no neutralizing activity was observed in the neutralization test.

[0097] Example 5 Animal Protection Experiment

[0098] To further evaluate the applicability of the antibody, its protective efficacy was assessed in mice. Since animal experiments involving monkeypox virus infection require a high-level biosafety laboratory (B3), the Western Reserve (VACV-WR) strain of vaccinia virus (belonging to the same family as monkeypoxvirus) was initially used for evaluation. Seven- to eight-week-old mice were infected intranasally with 5LD50 VAV-WR. Four hours before and four hours after infection, 200 μg of antibody A35R-10G7 was injected intraperitoneally per mouse.

[0099] like Figure 5 As shown, after infection with the 5LD50 virus, the body weight of all mice in the unrelated antibody group decreased by more than 20% on day 5, while the body weight of mice in the A35R-10G7 antibody group did not change significantly.

[0100] In summary, this application obtained a newly discovered, highly protective human MPXV antibody, A35R-10G7, by screening humanized mouse memory B cells that specifically bind to A35R using fully human antibodies. This antibody has a completely different sequence from previously reported monkeypox virus antibodies and exhibits strong antigen-binding ability. Furthermore, it can protect mice from lethal doses of VCV-WR. These results suggest that this human antibody has potential applications in the clinical treatment and prevention of monkeypox virus.

[0101] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0102] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against the A35R protein of monkeypox virus, characterized in that, The heavy chain variable region thereof 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, HCDR3 is as shown in SEQ ID NO: 7; and the light chain variable region thereof 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: 17, LCDR3 is as shown in SEQ ID NO:

9.

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

3. A detection reagent or a detection kit, characterized by, The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2.

4. A medicament, characterized by comprising: The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2.

5. A nucleic acid, characterized in that, The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2.

6. A recombinant vector, characterized in that, The nucleic acid of claim 5.

7. A host cell, characterized in that, The nucleic acid of claim 5 or the recombinant vector of claim 6.

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

9. Use of the monoclonal antibody or antigen-binding fragment thereof of 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 of claim 1 or 2 in preparing a detection product, a prophylactic drug or a therapeutic drug for monkeypox virus and / or vaccinia virus.

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