A monoclonal antibody against monkeypox virus protein and its application

Abstract

Some embodiments of the present specification provide a monoclonal antibody to a monkeypox virus protein and its application. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: the amino acid sequence of CDRH1 as shown in SEQ ID NO: 1, the amino acid sequence of CDRH2 as shown in SEQ ID NO: 2, and the amino acid sequence of CDRH3 as shown in SEQ ID NO: 3; and the light chain variable region comprises: the amino acid sequence of CDRL1 as shown in SEQ ID NO: 4, the amino acid sequence YAS of CDRL2, and the amino acid sequence of CDRL3 as shown in SEQ ID NO: 6.

Description

Technical Field

[0001] This specification relates to the field of bioengineering technology, and in particular to a monoclonal antibody against monkeypox virus protein or an antigen-binding fragment thereof and related applications. Background Art

[0002] Monkeypox virus, classified in the genus Orthopoxvirus within the family Poxviridae, is a highly contagious, linear, double-stranded DNA virus. Monkeypox virus poses a significant threat to human health and is currently spreading worldwide. Therefore, rapid response to potential monkeypox virus outbreaks is crucial, often relying on appropriate analytical methods to rapidly and accurately detect monkeypox virus before it becomes widespread.

[0003] Therefore, it is necessary to provide an antibody that can specifically bind to monkeypox virus protein and a method for detecting whether monkeypox virus exists in a sample using the antibody. Summary of the Invention

[0004] Some embodiments of the present specification provide an antibody or an antigen-binding fragment thereof that can bind to monkeypox virus protein, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises: the amino acid sequence of CDRH1 as shown in SEQ ID NO: 1, the amino acid sequence of CDRH2 as shown in SEQ ID NO: 2, and the amino acid sequence of CDRH3 as shown in SEQ ID NO: 3; the light chain variable region comprises: the amino acid sequence of CDRL1 as shown in SEQ ID NO: 4, the amino acid sequence YAS of CDRL2, and the amino acid sequence of CDRL3 as shown in SEQ ID NO: 6.

[0005] Some embodiments of this specification also provide a nucleic acid molecule. In some embodiments, the nucleic acid molecule encodes the aforementioned antibody or antigen-binding fragment thereof.

[0006] Some embodiments of this specification also provide an expression vector, characterized in that the expression vector comprises the aforementioned nucleic acid molecule.

[0007] Some embodiments of the present specification also provide a host cell, characterized in that the host cell comprises the aforementioned expression vector.

[0008] Some embodiments of this specification also provide a kit for detecting monkeypox virus, characterized in that the kit comprises the aforementioned antibody or antigen-binding fragment thereof.

[0009] Some embodiments of this specification also provide the use of the aforementioned antibodies or antigen-binding fragments thereof in preparing a kit for detecting monkeypox virus.

[0010] Some embodiments of this specification also provide a method for detecting monkeypox virus, characterized in that it includes using the aforementioned kit to detect whether monkeypox virus is present in a sample. DETAILED DESCRIPTION

[0011] As used in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not refer to the singular but also include the plural. Generally speaking, the terms "comprises" and "include" only indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list. A method or apparatus may also include other steps or elements.

[0012] Unless defined otherwise, 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 disclosure belongs.

[0013] The terms "about" and "around" may describe a range of values ​​within a certain value, such as plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the value, etc. For example, the term "about 10 mL" may include 9 mL to 11 mL.

[0014] As used herein, the term "antibody" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. For example, the light chains of an antibody can be divided into kappa and lambda light chains. Heavy chains can be divided into μ, δ, γ, α, and ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)). Each VH and VL is generally composed of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form an antigen binding site, respectively. Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0015] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to an antigen to which the full-length antibody specifically binds, and / or competes with the full-length antibody for binding to the same antigen. In some embodiments, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., a monoclonal antibody to the monkeypox virus A35R antigen provided in this application) by conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and can be screened for specificity in the same manner as intact antibodies.

[0016] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared.

[0017] The following is a detailed description of the methods involved in the examples of this specification. It should be noted that the following examples are only used to explain this specification and do not constitute a limitation of this specification.

[0018] Monkeypox virus is quite similar to smallpox virus. In biological classification, monkeypox virus belongs to the genus Orthopoxvirus in the family Poxviridae, a group of single linear, double-stranded DNA viruses. Orthopoxvirus, a genus within the subfamily Chorpodopoxvirinae of the Poxviridae family, includes vaccinia virus, variola virus, rabbitpox virus, ectromelia (mice) virus, buffalopoxvirus, camelpox virus, and monkeypox virus, with vaccinia virus being the representative species of the genus.

[0019] Orthopoxviruses (vaccinia, variola / smallpox, monkeypox) have two virion forms: intracellular mature virion (IMV) and extracellular enveloped virion (EEV), each with distinct structural and biological properties. Importantly, these two virion forms do not share any surface proteins, making them immunologically distinct and not recognized by a single specific antibody. IMV possesses a hardened coat protein that facilitates host-to-host transmission, while the more fragile EEV is adapted for intercellular spread in blood, making it a target designed to limit host immune clearance. The EEV membrane specifically encodes seven proteins: A33, A34, A36, A56, B5, F12, and F13.

[0020] Recent research, using proteomic analysis, has shown that early-stage human IgM antibodies in the blood of monkeypox virus-infected individuals primarily recognize the EEV proteins F13L, A35R, and B6R, as well as an unknown protein, A44R. Meanwhile, late-stage human IgG antibodies in the blood of monkeypox virus-infected individuals primarily recognize the EEV proteins F13L, A35R, and B6R, as well as the IMV proteins D8L, A29L, and H3L, and the core protein A4L.

[0021] The present invention immunizes mice with prokaryotically expressed monkeypox virus A35R protein, then fuses mouse spleen cells with myeloma cells. Highly specific hybridoma cells are obtained through specific high-throughput screening. Large amounts of mouse ascites are obtained through culture and re-immunization. High-purity, high-sensitivity, and high-specificity monoclonal antibodies against the monkeypox virus A35R antigen are then obtained through multi-step separation and purification. This provides the necessary raw materials for the development of kits for detecting monkeypox virus. The monoclonal antibodies against the monkeypox virus A35R antigen provided by the present invention have been shown to have good specific binding ability and can be used in immunological assays such as immunoblotting and immunofluorescence.

[0022] Some embodiments of the present disclosure provide an antibody or antigen-binding fragment thereof that can bind to a monkeypox virus protein, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3; and the light chain variable region comprises CDRL1, CDRL2, and CDRL3. CDRH refers to the complementarity determining region in the heavy chain variable region; CDRL refers to the complementarity determining region in the light chain variable region.

[0023] In some embodiments, the amino acid sequence of CDRH1 is: GYTFTSYW (SEQ ID NO: 1), the amino acid sequence of CDRH2 is: INPSNGRF (SEQ ID NO: 2), and the amino acid sequence of CDRH3 is: ARNNPTGNFAF (SEQ ID NO: 3).

[0024] In some embodiments, the amino acid sequence of CDRL1 is QSISNY (SEQ ID NO: 4), the amino acid sequence of CDRL2 is YAS, and the amino acid sequence of CDRL3 is QQNNSWPQLT (SEQ ID NO: 6).

[0025] In some embodiments, the heavy chain variable region comprises the following sequence:

[0026] VKLQQSGAELVKPGASVRLSCKASGYTFTSYWMHWVRQRPGQGLEWIGEIN

[0027] PSNGRFNCNEKFRTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARNNPTGN FAFWGQGTTLTVSS (SEQ ID NO: 7).

[0028] In some embodiments, the light chain variable region comprises the following sequence:

[0029] DIVLIQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQS ISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQNNSWPQLTFGAGTK (SEQ ID NO: 8).

[0030] In some embodiments, the above-mentioned heavy chain variable region or light chain variable region may contain no more than 2 amino acids or no more than 1 amino acid conservative substitutions. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted by another amino acid residue with a similar side chain, such as physical or functionally similar residues (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent bonds or hydrogen bonds, etc.) to corresponding amino acid residues. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family.

[0031] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 85%, 90%, or 95% identical in the framework regions to the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the heavy chain variable region comprises an amino acid sequence that has one or more amino acid additions, deletions, and / or substitutions in the framework regions compared to the amino acid sequence shown in SEQ ID NO: 7.

[0032] In some embodiments, the light chain variable region comprises an amino acid sequence that is at least 85%, 90%, or 95% identical in the framework region to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the light chain variable region comprises an amino acid sequence that has one or more amino acid additions, deletions, and / or substitutions in the framework region compared to the amino acid sequence set forth in SEQ ID NO: 8.

[0033] In some embodiments, the monkeypox virus protein is the monkeypox virus A35R protein. The monkeypox virus A35R protein (MPXV-A35R) is a type II membrane glycoprotein specific for extracellular enveloped viruses (EEVs) that plays a key role in the efficient formation of EEVs and helps viral particles spread efficiently between cells.

[0034] Some embodiments of this specification also provide a nucleic acid molecule that encodes the aforementioned antibody or antigen-binding fragment thereof, and is a potential target for developing monkeypox virus detection antibodies and therapeutic antibodies.

[0035] In some embodiments, the nucleic acid molecule may include a nucleotide sequence encoding CDRH1 in the heavy chain variable region: GGCTACACCTTCACCAGCTACTGG (SEQ ID NO: 5). The nucleic acid molecule may include a nucleotide sequence encoding CDRH2 in the heavy chain variable region: ATTAATCCTAGCAACGGTCGTTTT (SEQ ID NO: 11). The nucleic acid molecule may include a nucleotide sequence encoding CDRH3 in the heavy chain variable region: GCAAGAAATAACCCAACTGGGAACTTTGCCTTC (SEQ ID NO: 12).

[0036] In some embodiments, the nucleic acid molecule may include a nucleotide sequence encoding CDRL1 in the light chain variable region: CAAAGTATTAGCAACTAC (SEQ ID NO: 13). The nucleic acid molecule may include a nucleotide sequence encoding CDRL2 in the light chain variable region: TATGCTTCC. The nucleic acid molecule may include a nucleotide sequence encoding CDRL3 in the light chain variable region: CAACAGAATAACAGCTGGCCTCAGCTCACG (SEQ ID NO: 14).

[0037] In some embodiments, the nucleic acid molecule comprises the following nucleotide sequence encoding the heavy chain variable region:

[0038] GTCAAGCTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGT

[0039] GAGGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGC

[0040] ACTGGGTGAGGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGAT

[0041] TAATCCTAGCAACGGTCGTTTTAACTGCAATGAGAAGTTCAGGACCAAGG

[0042] CCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGC

[0043] AGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAATAACCC

[0044] AACTGGGAACTTTGCCTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 9).

[0045] In some embodiments, the nucleic acid molecule comprises the following nucleotide sequence encoding the light chain variable region:

[0046] GATATTGTGCTAATTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGAT

[0047] AGAGTCAGTCTTTCCTGCAGGGCCAGTCAAAGTATTAGCAACTACCTACA

[0048] CTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATG

[0049] CTTCCCAGTCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCA

[0050] GGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGG

[0051] AATGTATTTCTGTCAACAGAATAACAGCTGGCCTCAGCTCACGTTCGGTGCTGGGACCAAG (SEQ ID NO: 10).

[0052] Some embodiments of this specification also provide an expression vector, which includes the aforementioned nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10. In some embodiments, the aforementioned nucleic acid molecule can be inserted into one or more expression vectors using, for example, recombinant DNA technology and gene transfection methods well known in the art, so that the gene is operably linked to the transcription and translation regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is connected to the vector so that the transcription and translation control sequences within the vector play their intended functions of regulating the transcription and translation of the antibody gene, thereby expressing the monkeypox virus protein antibody provided by the present invention.

[0053] Some embodiments of this specification also provide a host cell comprising the aforementioned expression vector. Such host cells include, but are not limited to, microbial cells and animal cells. For example, when an expression vector containing a nucleotide sequence encoding a monkeypox virus protein antibody or antigen-binding fragment thereof provided herein is introduced into a mammalian host cell, the monkeypox virus protein antibody or antigen-binding fragment thereof can be obtained by culturing the host cell. Similar methods can be used to mass-produce the monkeypox virus protein antibody or antigen-binding fragment thereof provided herein.

[0054] Some embodiments of this specification also provide a kit for detecting monkeypox virus protein, wherein the kit comprises the aforementioned antibody or antigen-binding fragment thereof.

[0055] In some embodiments, the kit may be a colloidal gold detection kit, an immunochromatographic detection kit, an enzyme immunoassay kit, a chemiluminescence kit, an immunoturbidimetric detection kit, etc. The present invention is not limited thereto.

[0056] Some embodiments of this specification also provide the use of the aforementioned antibodies or antigen-binding fragments thereof in preparing a kit for detecting monkeypox virus.

[0057] Some embodiments of this specification also provide a method for detecting monkeypox virus, comprising using the aforementioned kit to detect whether monkeypox virus is present in a sample.

[0058] In some embodiments, the method can also be used for non-diagnostic purposes. For example, the method for detecting monkeypox virus can be used to detect whether an environment or certain objects are contaminated with monkeypox virus. As an example, the sample can be a sample taken from the environment, such as drinking water. As another example, the sample can be a sample obtained by smearing or other means from the surface of an object, such as a food packaging bag. In some embodiments, the method for detecting monkeypox virus can also be used for scientific research and other purposes, which are not limited by the present invention.

[0059] Example

[0060] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are all repeated three times unless otherwise specified, and the results are averaged. Example 1 Preparation of anti-A35R antigen monoclonal antibodies

[0061] 1. Construction of recombinant plasmid

[0062] The monkeypox virus A35R protein sequence used in this embodiment was obtained from Genebank (accession number: 928958). The restriction enzyme site GGATCC of BamH I was added upstream of the synthesized monkeypox virus A35R protein sequence, and the restriction enzyme site GAATTC of EcoR I was added downstream. Utilize BamH I and EcoR I to double-digest a 546bp A35R fragment, which was then ligated into the pGEX-6p-1 vector that was also double-digested by BamH I and EcoR I after recovery to obtain the bacterial expression plasmid pGEX-6p-1-A35R. The pGEX-6p-1-A35R plasmid with correct sequencing was transformed into a BL21 bacterial strain and induced to express.

[0063] 2. Preparation of Monkeypox Virus A35R Antigen

[0064] The BL21 strain containing pGEX-6p-1-A35R was cultured overnight at 37°C (200 rpm) in 10 mL of LB+0.5% glucose medium containing 50 μg / mL ampicillin. The next day, the strain was amplified 1:100 and transferred to 500 mL of culture medium. IPTG was added at 37°C and 200 rpm to an OD600 of approximately 0.6. After induction at 16°C for 16 hours, the cells were harvested (in a 500 mL collection flask, at 4000 rpm for 10 minutes at 4°C), and the supernatant discarded. 30 mL of phosphate-buffered saline buffer (PBS) was added, the cells were suspended by vortexing, and the suspension was placed on ice (all subsequent steps were performed on ice). The suspension was sonicated at 4°C (70 W, 90 cycles of 10 seconds each, with 10 seconds rest intervals) until clear. The precipitate was then removed by centrifugation, and the target protein was collected by column chromatography according to the GE Glutathione Sepharose 4B instructions.

[0065] The column operation is as follows:

[0066] 1. Sample clarification and filtration: Use a 50 mL syringe and a 0.22 μm filter membrane to clarify the prepared bacterial supernatant;

[0067] 2. Use protein chromatography columns to capture and purify on an AKTA chromatography system;

[0068] 3. Flush the system, then flush the A1 pump of the AKTA chromatography system with the equilibration solution and flush the B1 pump with the eluent;

[0069] 4. Set the system flow rate to 0.1 mL / min, select the corresponding column position 1 to connect the protein chromatography column, and balance the AKTA chromatography system and column with PBS. After the balance is completed, adjust the UV to zero;

[0070] 5. Start loading the sample and transfer the A1 pump to the sample loading centrifuge tube;

[0071] 6. After loading, transfer the A1 pump to PBS and rinse until the detection wavelength is stable. Then transfer the A1 pump to the eluent (1mM PBS + 15mM reduced glutathione, pH 8.0-9.0) for eluation and collect the eluate; then rinse with equilibrium solution A (PBS), and finally rinse the column with 20% ethanol.

[0072] 7. Elute the recombinant GST (glutathione sulfhydryltransferase)-tagged A35R antigen from the column using elution buffer (1 mM PBS + 15 mM reduced glutathione, pH 8.0-9.0). Analyze the purified antigen using SDS-PAGE and observe using Coomassie Brilliant Blue staining. Digest the GST-A35R protein overnight with recombinant HRV 3C protease to remove the GST tag. Pass the protein through a GE gel filtration prepacked column (Superdex 200 Increase 10 / 300 GL) and, through molecular sieving, isolate and obtain highly pure monkeypox virus A35R antigen. III. Preparation of Monoclonal Antibodies to the A35R Antigen

[0073] Healthy female Balb / c mice aged 6-8 weeks were used for animal immunization experiments according to a pre-specified immunization protocol. Splenic lymphocytes from these successfully immunized Balb / c mice were extracted and fused with mouse myeloma SP2 / 0 cells via cell fusion. After two rounds of subcloning, a hybridoma cell line stably secreting a monoclonal antibody against the monkeypox virus A35R antigen was obtained, thereby obtaining a monoclonal antibody against the monkeypox virus A35R antigen.

[0074] The specific steps of animal immunization experiments include:

[0075] 1. Balb / c mice of similar weight and age were randomly divided into two groups: group A with aluminum adjuvant (aluminum hydroxide adjuvant) and group B without aluminum adjuvant. The dose of immune antigen in group A was 100 μg / mouse, and the dose of immune antigen in group B was 150 μg / mouse. Each group of mice had a control group.

[0076] 2. Before the start of the experiment, the pre-immune serum of each mouse was collected (pre-immune serum was collected on the fifth day, blood was collected through the eyeball, and an appropriate amount of blood was collected to ensure the normal condition of the mice), and the collected serum was stored at -80°C.

[0077] 3. Preparation of the aluminum adjuvant (aluminum hydroxide adjuvant) group: Before immunization, each antigen was diluted to the corresponding dose (100 μg / mouse or 150 μg / mouse) in 75 μL PBS and mixed with alum adjuvant (1 mg / mouse) at a volume ratio of antigen:adjuvant = 3:1 (i.e., 25 μL of adjuvant was added to 75 μL of immunogen diluent). The adjuvant was shaken well before use, and the injection adjuvant (25 μL) was slowly added dropwise to the immunogen solution. After the adjuvant and immunogen diluent were thoroughly mixed, the two were thoroughly mixed for 30 minutes to allow the adjuvant to effectively adsorb the antigen. Subsequent procedures were carried out according to the animal immunization experimental procedures.

[0078] 4. Preparation of the group without aluminum adjuvant: The antigen was diluted in 100 μL PBS to the corresponding dose in Table 1 (100 μg / mouse or 150 μg / mouse), and 100 μL of immunogen was added. Subsequent procedures were carried out according to the animal immunization experiment.

[0079] 5. Subcutaneous injection at intervals of 2 weeks: The experimental design is a three-time immunization method, but blood is collected from the eyeball 7 days after each immunization injection, and part of the mouse supernatant is obtained by centrifugation. The serum titer is first tested. 7 days after the last immunization, blood is collected from the heart to obtain the maximum amount of blood, centrifuged to obtain the supernatant, and stored at -80℃.

[0080] 6. Detect serum titer.

[0081] (1) Five mice were immunized, numbered A0, A1, A2, B0, and B1. After three immunizations, serum titers were measured. The test data are shown in Table 1.

[0082] Table 1: Serum titer test data

[0083]

[0084] Immune spleen cells from mice A0, B0, and B1 were fused with the myeloma cell line SP2 / 0. Fusion cells were selected using HAT selection medium (containing hypoxanthine, aminopterin, and thymidin). The fused cells were then screened for positive results using an enzyme-linked immunosorbent assay (ELISA) and subcloned. Ascites fluid from selected positive clones was collected, and the ascites antibodies produced by the hybridoma cells were purified using a Protein A / G antibody purification column. The purity of the purified ascites antibodies was >90%.

[0085] Example 2 Screening of anti-A35R antigen monoclonal antibodies

[0086] (1) Cell fusion and clone screening

[0087] Three mice numbered A0, B0, and B1 in the embodiment were selected to complete three rounds of fusion.

[0088] Fusion screening of mice A0, B0, and B1 yielded 25 positive wells for subcloning. Fusion screening identified 80 positive clones with OD450 values ​​>2.2, which were titered by serial dilution and then subjected to a second subclone screening. Nine hybridoma cell lines were obtained, designated A0-1, A0-2, A0-3, B0-1, B0-2, B0-3, B1-1, B1-2, and B1-3.

[0089] (2) Ascites preparation and testing data

[0090] Each hybridoma cell line was injected into the peritoneal cavity of F1 mice, and a total of 9 ascites samples were collected. The titer data of all ascites tests are shown in Table 2 below:

[0091] Table 2: Ascites test titer

[0092]

[0093] (3) Antibody purification conditions and test data

[0094] The above ascites were purified by 3.3% n-octanoic acid-thiamine precipitation method, and antibodies corresponding to 9 hybridoma cell lines were obtained. The titer test data of all antibodies are shown in the table below:

[0095] Table 3: Antibody titer detection

[0096] Dilution concentration A0-1 A0-2 A0-3 B0-1 B0-2 B0-3 B1-1 B1-2 B1-3 20 μg / mL 3.3986 3.2062 3.0380 3.0501 3.0699 3.1823 3.1736 3.2625 3.3681 4ug / mL 3.6199 3.3263 3.1059 3.1774 3.1081 3.5701 3.2811 3.1167 3.3728 800ug / mL 3.3729 3.1843 3.0528 3.0719 3.0222 3.2752 3.2167 3.0001 3.3659 160 ng / mL 3.5558 3.1849 2.9123 3.1115 2.9032 3.1238 3.1836 2.8315 3.4020 32 ng / mL 3.0831 2.0089 1.4791 3.01 1.5582 1.8511 2.9987 1.5366 2.8988 6.4 ng / mL 1.4789 0.6346 0.5663 2.092 0.4544 0.6648 1.7001 0.6162 1.2542 1.28 ng / mL 0.5495 0.3317 0.2434 0.8273 0.3590 0.2543 0.6144 0.2648 0.3162 PBS 0.2301 0.1528 0.1165 0.1832 0.1647 0.1692 0.1961 0.1149 0.1705

[0097] The above data show that the monoclonal antibodies of the 9 hybridoma cell lines have good specific binding ability to A35R antigen.

[0098] Example 3 ELISA detection of the binding activity of monoclonal anti-A35R antigen monoclonal antibodies

[0099] The binding activity of anti-A35R antigen monoclonal antibodies was detected by IgG antibody titer detection. The specific steps are as follows:

[0100] (1) Bottom plate coating: Dilute the antigen to be used to 3 μg / mL with coating diluent, add 100 μL of the prepared coating solution to each well, and place in a 4°C refrigerator for 24 hours.

[0101] (2) After 24 hours, take the wells out of the refrigerator and place them at 37°C for 30 minutes. Then discard the liquid in the wells and wash the wells three times with washing solution, each time for 3 minutes.

[0102] (3) Blocking the enzyme-labeled reaction wells: Add 200 μL of 5% calf serum to each well and block at 37°C for 90 min. After blocking, wash the wells three times with washing solution, each time for 3 min.

[0103] (4) Add the sample to be tested: dilute the sample according to the required ratio, add the diluted sample to the enzyme-labeled reaction well, 100 μL per well, incubate at 37°C for 90 min; wash the well three times with washing solution, each time for 3 min.

[0104] (5) Add enzyme-labeled antibody: Add secondary antibody at an appropriate concentration according to the instructions; incubate at 37°C for 90 min, add 100 μL per well and wash as before.

[0105] (6) Add substrate solution: Add 100 μL of substrate to each well and incubate at 37°C in the dark for 15 to 30 minutes.

[0106] (7) Termination reaction: Add 50 μL of stop solution to each well to terminate the reaction and measure the experimental results within 20 minutes.

[0107] The purified antibodies obtained in Example 1 were used to perform ELISA titer detection according to the above steps. The results showed that the ELISA titers were all >1:128,000, indicating that the anti-A35R antigen monoclonal antibodies had good binding activity.

[0108] Example 4 Application of anti-A35R antigen monoclonal antibody in the kit

[0109] This example uses the immunocolloidal gold detection method as an example, and verifies the anti-A35R antigen monoclonal antibody using the immunocolloidal gold platform.

[0110] Specifically, we purchased A35R antigens from other companies, including A35R antigen from Suzhou Jinan Protein Technology Co., Ltd., Catalog No. DRA209; A35R antigen from Nanjing Oukai Biotechnology Co., Ltd., Catalog No. C1620; and A35R antigen from Beijing Fubo Biotechnology Co., Ltd., Catalog No. FB0606. The binding activity of the nine monoclonal antibodies generated in Example 2 against the purchased A35R antigens was tested. The antigens were diluted to 3 μg / mL in coating diluent, and 100 μL of the prepared coating diluent was added to each well. The ELISA test data are shown in the table below:

[0111] Table 4: Preliminary evaluation results of 9 monoclonal antibodies in immunocolloidal gold assay

[0112]

[0113]

[0114] As can be seen from Table 4, monoclonal antibody B1-2 has good binding activity to the A35R antigen purchased from the other three companies. It is named Anti-A35R-1 antibody. Monoclonal antibody Anti-A35R-1 can be used for immunological detection such as immunoblotting and immunofluorescence.

[0115] Example 5 Sequence Analysis of the Heavy Chain Variable Region (VH) and Light Chain Variable Region (VL) of Monoclonal Antibody Anti-A35R-1

[0116] The primers for amplifying the heavy chain V region (VH) and light chain V region (VL) genes are as follows:

[0117] Heavy chain variable region forward primer (VH-FOR): GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG

[0118] (SEQ ID NO: 15);

[0119] Heavy chain variable region reverse primer (VH-BACK): GGAAGGTGTGCACACCGCTGGAC (SEQ ID NO: 16);

[0120] Light chain variable region forward primer (VL-FOR): ATGGAATCACAGRCYCWGGT (SEQ ID NO: 17);

[0121] Light chain variable region reverse primer (VL-BACK): GATGGTGGGAAGATGGATACAGTT (SEQ ID NO: 18).

[0122] Take the hybridoma cell line Anti-A35R-1 in the logarithmic growth phase (about 10 7 cells), and total RNA of the cells was extracted according to the instructions of the Trizol RNA extraction kit. The total RNA was used as a template for reverse transcription to synthesize the first chain cDNA, and the above amplified product was used as a template for PCR amplification of the antibody VH / VL gene.

[0123] The heavy chain VH (about 360 bp) and light chain VL (about 300 bp) fragments of Anti-A35R-1 were recovered and sequenced.

[0124] Then analyze the VH / VL gene sequences:

[0125] The resulting sequence is as follows:

[0126] Heavy chain variable region sequence:

[0127] Anti-A35R-1-VH:351bp

[0128] GTCAAGCTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCT TCAGTGAGGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAACGGTCGTTTTAACTGCAATGAGAAGTTCAGGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAATAACCCAACTGGGAACTTTGCCTTCTGGGGCCAAGGCACCACTCTCACAGT CTCCTCA(SEQ ID NO:9).

[0129] Anti-A35R-1-VH protein: 117aa

[0130] VKLQQSGAELVKPGASVRLSCKASGYTFTSYWMHWVRQRPGQGLE WIGEINPSNGRFNCNEKFRTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR NNPTGNFAFWGQGTTLTVSS(SEQ ID NO:7)

[0131] Variable region sequence of the light chain:

[0132] Anti-A35R-1LVκ: 312bp

[0133] GATATTGTGCTAATTCAGTCTCCAGCCACCCTGTCTGTGACTCCAG GAGATAGAGTCAGTCTTTCCTGCAGGGCCAGTCAAAGTATTAGCAACTACCTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCCA GGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAATAACAGCTGGCCTCAGCTCACGTTCGGTGCTGGGACCAAG(SEQ ID NO:10).

[0134] Anti-A35R-1LVκprotein:104aa

[0135] DIVLIQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKY ASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQNNSWPQLTFGAGTK (SEQ ID NO: 8).

[0136] The monoclonal antibody or antigen-binding fragment thereof for a monkeypox virus protein antigen disclosed in the present invention and related applications thereof have beneficial effects including but not limited to: (1) the novel monoclonal antibody for a monkeypox virus protein antigen provided by the present invention has high titer and high specificity; (2) the monoclonal antibody for a monkeypox virus protein provided by the present invention can be used in immunological assays such as immunoblotting and immunofluorescence, for example, to detect the presence of monkeypox virus in a sample using a colloidal gold detection platform. It should be noted that different embodiments may produce different beneficial effects. In different embodiments, the beneficial effects that may be produced may be any one or a combination of the above, or any other possible beneficial effects.

[0137] Those skilled in the art will appreciate that the above embodiments are intended only to illustrate the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and variations made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against a monkeypox virus protein, characterized in that: The monkeypox virus protein is the monkeypox virus A35R protein, and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes: the amino acid sequence of CDRH1 as shown in SEQ ID NO: 1, the amino acid sequence of CDRH2 as shown in SEQ ID NO: 2, and the amino acid sequence of CDRH3 as shown in SEQ ID NO: 3; The light chain variable region includes: the amino acid sequence of CDRL1 as shown in SEQ ID NO: 4, the amino acid sequence of CDRL2 as YAS, and the amino acid sequence of CDRL3 as shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region consists of any of the following: The amino acid sequence shown in SEQ ID NO: 7; an amino acid sequence that is at least 85%, 90%, or 95% identical in the framework regions to the amino acid sequence shown in SEQ ID NO: 7; and An amino acid sequence having one or more additions, deletions and / or substitutions of amino acids in the framework regions compared to the amino acid sequence shown in SEQ ID NO:

7.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein The light chain variable region is composed of any of the following composition: The amino acid sequence shown in SEQ ID NO: 8; an amino acid sequence that is at least 85%, 90%, or 95% identical in the framework regions to the amino acid sequence shown in SEQ ID NO: 8; and An amino acid sequence having one or more additions, deletions and / or substitutions of amino acids in the framework regions compared to the amino acid sequence shown in SEQ ID NO:

8.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein The ELISA titer of the antibody or antigen-binding fragment thereof binding to the monkeypox virus protein is greater than 1:128,000.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, wherein The nucleic acid molecule includes a nucleotide sequence as shown in SEQ ID NO: 9 encoding the heavy chain variable region, and a nucleotide sequence as shown in SEQ ID NO: 10 encoding the light chain variable region.

7. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 5.

8. A host cell, characterized in that The host cell comprises the expression vector according to claim 7.

9. A kit for detecting monkeypox virus, characterized in that: The kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

10. The kit according to claim 9, wherein The kit is a colloidal gold detection kit, an immunochromatographic detection kit, an enzyme immunoassay kit, a chemiluminescence kit or an immunoturbidimetric detection kit.

11. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in preparing a kit for detecting monkeypox virus.

12. A method for detecting monkeypox virus for non-diagnostic purposes, characterized in that The method comprises using the kit as claimed in claim 9 to detect whether monkeypox virus is present in a sample.

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