Broadly neutralizing monoclonal antibodies that recognize seven pestiviruses

Abstract

The present invention discloses a broad-spectrum neutralizing monoclonal antibody that recognizes 7 kinds of pestiviruses, belonging to the field of immunoglobulins. Specifically disclosed are anti-plague virus monoclonal antibodies or antigen-binding fragments thereof, including a heavy chain variable region (SEQ ID No.1) and a light chain variable region (SEQ ID No.3). The monoclonal antibody of the present invention reacts with BVDV1, BVDV2, CSFV, BDV, HoBiPeV, AydinPeV and OVPV, and is broader in spectrum than other pestivirus monoclonal antibodies, and can recognize all pestivirus members currently known to infect cattle and sheep, and can also recognize pestivirus member CSFV, and has good neutralizing ability, and can be made into diagnostic reagents and products for pestivirus in clinical use. The antibody of the present invention can be used for serological diagnosis of pestiviruses, evaluation of immune effects, and detection of pestivirus pathogens in experimental serum products, etc., and has broad application prospects in clinical diagnosis and other fields.

Description

Technical Field

[0001] The invention belongs to the field of immunoglobulins, and in particular relates to broad-spectrum neutralizing monoclonal antibodies capable of recognizing seven pestiviruses. Background Art

[0002] Pestivirus is a type of virus that seriously harms livestock and wild animals. Its member virus particles are spherical, with a diameter of about 40-60 nm, with an envelope, and are single-stranded positive-strand RNA viruses. The genome size is 11.3-13.0 kb, containing a large open reading frame and non-coding regions at both ends of the genome, encoding 4 structural proteins and 8 non-structural proteins, of which the structural protein E rns (gp44 / 48) and E2 (gp55), non-structural protein NS3 can stimulate the body to produce antibodies, and E2 protein is an important protective antigen of pestivirus. The amino terminus of E2 protein of Classical swine fever virus (CSFV) has four independent antigenic domains, namely B, C, D, and A domains, which can be further divided into B / C antigenic region and D / A antigenic region. Most of the antigenic epitopes are located on these two antigenic regions.

[0003] According to the latest report of the International Committee on Taxonomy of Viruses (ICTV), pestiviruses include 11 known members ( Pestivirus A - Pestivirus K), in addition, some viruses with large genetic differences from known members such as Batpestivirus, Pangolin pestivirus, Bamboo rat pestivirus, Linda pestivirus, Ovinepestivirus (OVPV) have also been discovered. Some members are widely distributed and pose a serious threat to the global aquaculture industry. Pestivirus A (BVDV1) and Pestivirus B (BVDV2) can infect livestock such as cattle, sheep, and pigs, as well as wild animals such as deer and antelopes, and can cause diarrhea in calves, mucosal shedding, abortion, stillbirth, and congenital malformations in fetuses; Pestivirus C (CSFV) is one of the pathogens that seriously harm the pig farming industry. The main symptoms of sick pigs are high fever, breathing difficulties, extensive bleeding throughout the body and reproductive disorders. Pestivirus The main hosts of D(BDV) are sheep and goats. After infection with BDV, clinical manifestations include abortion, stillbirth, lamb tremors, immunosuppression and hirsutism. Pestivirus H (HoBiPeV) mainly infects cattle and other ruminants and is also known as BVDV3. In addition, pestiviruses that are genetically distant from BDV PestivirusI (AydinPeV) and OVPV were also detected in sheep flocks. These two new pestiviruses are more closely related to CSFV strains than BDV. In addition to endangering animal health, pestiviruses and their antibodies are also important contaminants of bovine serum products, causing great damage to the healthy development of life science research and vaccine production.

[0004] At present, only a few monoclonal antibodies that can recognize multiple pestivirus members have been identified. Monoclonal antibody m912 can react with BDV and some CSFV, monoclonal antibody 15C5 can react with most BVDV1, BVDV2, BVDV3, BDV and Bungowannah virus, and NS3 protein monoclonal antibody C16 is also a broad-spectrum monoclonal antibody for pestiviruses, which can react with pestiviruses such as CSFV, BVDV1, BVDV2, BVDV3 and BDV, but these two broad-spectrum monoclonal antibodies do not have neutralizing ability. The monoclonal antibodies for pestiviruses identified so far have problems such as few types of recognized members and no neutralizing ability, which greatly limits the application of these monoclonal antibodies. Therefore, it is urgent to research and develop broad-spectrum neutralizing antibodies that can specifically bind to more pestiviruses and have neutralizing effects. Summary of the invention

[0005] One of the purposes of the present invention is to provide a broad-spectrum neutralizing monoclonal antibody that recognizes seven pestiviruses and its application. The technical problems to be solved are not limited to the described technical themes, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0006] To achieve the above objectives, the present invention first provides an anti-plague virus monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 with an amino acid sequence of positions 26-33 of SEQ ID No.1, HCDR2 with an amino acid sequence of positions 51-58 of SEQ ID No.1, and HCDR3 with an amino acid sequence of positions 97-108 of SEQ ID No.1; the light chain variable region comprises LCDR1 with an amino acid sequence of positions 27-37 of SEQ ID No.3, LCDR2 with an amino acid sequence of positions 55-57 of SEQ ID No.3, and LCDR3 with an amino acid sequence of positions 94-102 of SEQ ID No.3.

[0007] The antigen-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2, antibody variable region (Fv), disulfide-stabilized Fv (dsFv), single-chain antibody (ScFv), single-domain antibody (sdAb, i.e., nanoantibody), minibody and minimum recognition unit (MRU).

[0008] The sequences of the complementarity determining regions (CDRs) can be defined according to the Kabat numbering system.

[0009] The heavy chain variable region and the light chain variable region both contain a framework region (FR, the region outside the CDR in the variable region). The framework region may be derived from humans or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, camels, etc.).

[0010] Furthermore, the amino acid sequence of the heavy chain variable region may be as shown in SEQ ID No.1; and the amino acid sequence of the light chain variable region may be as shown in SEQ ID No.3.

[0011] Furthermore, the monoclonal antibody or antigen-binding fragment thereof may also include a heavy chain constant region (CH) and a light chain constant region (CL). The heavy chain constant region may be selected from the heavy chain constant regions of IgG, IgA, IgM, IgD and IgE. The heavy chain constant region may also be selected from CH1, Fc and CH3 domains. The light chain constant region may be selected from Kappa (κ) and lambda (λ) type light chain constant regions. The heavy chain constant region and the light chain constant region may be derived from humans or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, camels, etc.).

[0012] Further, the heavy chain constant region can be selected from the heavy chain constant region of human IgG subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant region can also be selected from the heavy chain constant region of mouse IgG subclasses such as IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgG5 and IgG6.

[0013] Furthermore, the nucleotide sequence of the heavy chain constant region may be as shown in SEQ ID No.14.

[0014] Furthermore, the nucleotide sequence of the light chain constant region may be as shown in SEQ ID No.15.

[0015] The present invention also provides a biomaterial, which may be any of the following:

[0016] A1) a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof;

[0017] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0018] A3) a recombinant vector containing the nucleic acid molecule described in A1);

[0019] A4) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0020] A5) A recombinant host cell containing the nucleic acid molecule described in A1).

[0021] The biological materials can all express the monoclonal antibodies or antigen-binding fragments thereof described herein.

[0022] In the above biological material, the nucleic acid molecule may be any of the following:

[0023] B1) a DNA molecule whose coding sequence is SEQ ID No. 2;

[0024] B2) The coding sequence is a DNA molecule of SEQ ID No.4.

[0025] The amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 2 is the heavy chain variable region of SEQ ID No. 1. The amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 4 is the light chain variable region of SEQ ID No. 3.

[0026] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 4. Considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for expression of specific species as needed.

[0027] The present invention also provides the use of the monoclonal antibody or antigen-binding fragment thereof, or the biomaterial described herein in any of the following:

[0028] C1) Use in the preparation of products for inhibiting or neutralizing pestivirus activity;

[0029] C2) Use in the preparation of products for diagnosing or assisting in the diagnosis of pestivirus infection or diseases caused by pestivirus infection;

[0030] C3) Use in the preparation of products for screening or assisting in screening of pestivirus infection or diseases caused by pestivirus infection;

[0031] C4) Use in the preparation of products for detecting pestivirus or pestivirus E2 protein;

[0032] C5) Use in the preparation of a product for binding pestivirus or pestivirus E2 protein.

[0033] The inhibition or neutralization of pestivirus activity includes binding to pestivirus E2 protein, thereby causing the pestivirus to lose the ability to bind to receptors and then lose the ability to infect host cells, thereby achieving the purpose of inhibiting or neutralizing pestivirus activity.

[0034] The diagnosis or auxiliary diagnosis of pestivirus infection or a disease caused by pestivirus infection can be performed by isolating a sample from a subject, and then using the monoclonal antibody or its antigen-binding fragment to detect whether the pestivirus E2 protein or the content of the pestivirus E2 protein in the sample, and diagnosing or auxiliary diagnosis of pestivirus infection or a disease caused by pestivirus infection based on the test results.

[0035] The screening or auxiliary screening for pestivirus infection or diseases caused by pestivirus infection can be performed by isolating a sample from a subject, and then using the monoclonal antibody or its antigen-binding fragment to detect whether the pestivirus E2 protein or the content of the pestivirus E2 protein in the sample, and screening for pestivirus infection or diseases caused by pestivirus infection is performed based on the test results.

[0036] The detection of pestivirus or pestivirus E2 protein includes any in vivo or in vitro detection of E2 protein based on the principle of antigen-antibody specific reaction. The detection of E2 protein can be to detect whether the sample to be tested contains E2 protein and / or to detect the content of E2 protein in the sample to be tested.

[0037] The product that binds to pestivirus or pestivirus E2 protein can be an E2 protein inhibitor, or a product for separating or purifying E2 protein, but is not limited thereto. For example, the monoclonal antibody or antigen-binding fragment thereof of the present invention can be prepared into an immunoaffinity chromatography column, and the E2 protein can be screened and separated based on the principle that the antigen can be captured by the antibody when passing through the chromatography column, and the antigen can be dissociated from the antigen by changing environmental conditions such as pH value.

[0038] The products described herein may include reagents, kits, chips, test strips, test cards, and immunosensors.

[0039] The subject may be a human or a non-human animal (eg, pig, cow, sheep, rabbit, cat, horse, deer, monkey, chicken, dog, etc.).

[0040] In the above application, the pestivirus may be bovine viral diarrhea virus type 1, bovine viral diarrhea virus type 2, classical swine fever virus, border disease virus, HoBi-like Pestivirus, Aydin-like Pestivirus and / or Ovine Pestivirus.

[0041] The present invention also provides a kit, which contains the monoclonal antibody or antigen-binding fragment thereof described herein.

[0042] The kit may have at least one of the following uses: (1) diagnosing or assisting in the diagnosis of pestivirus infection or a disease caused by pestivirus infection; (2) screening or assisting in the screening of pestivirus infection or a disease caused by pestivirus infection; (3) detecting pestivirus or pestivirus E2 protein; (4) binding to pestivirus or pestivirus E2 protein.

[0043] The test samples of the kit can be environmental samples, blood samples (such as whole blood, plasma, serum), sputum samples, tissue samples, cell samples, feces samples, etc., but are not limited thereto.

[0044] Furthermore, the environmental samples may include feed factory environmental samples (such as silos, conveyor belts, workbenches, floors, etc.), breeding environmental samples (such as sludge, breeding feed, soil, feeding troughs, rags for wiping the environment, swabs, breeding water, air, etc.) and surrounding environmental samples (such as transport vehicles, surrounding farmland, etc.).

[0045] The kit may be a chemiluminescent immunoassay kit, an enzyme-linked immunosorbent assay kit, an immunoprecipitation assay kit, an immunoblotting assay kit, an immunochromatography assay kit, a flow cytometry assay kit, an immunohistochemistry assay kit, a colloidal gold immunoassay kit or a fluorescent immunoassay kit, but is not limited thereto.

[0046] Furthermore, the kit may also include reagents required for immunoassay, such as labeled antibodies or antigens, magnetic particles, blocking solution, diluent, washing solution, color developing solution, stop solution, etc., but not limited thereto.

[0047] The present invention also provides an antibody conjugate, comprising an antibody portion and a conjugated portion, wherein the antibody portion comprises the monoclonal antibody or antigen-binding fragment thereof described herein.

[0048] Furthermore, the antibody portion and the coupling portion can be directly linked, or covalently linked via a linker (eg, a hydrazone bond, a disulfide bond, a thioether bond, a peptide bond).

[0049] Furthermore, the conjugated moiety may be a detectable label.

[0050] Furthermore, the detectable markers include enzymes (such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, etc.), chemiluminescent reagents (such as acridinium ester compounds, acridinium sulfonamide compounds, luminol and its derivatives, ruthenium derivatives, etc.), fluorescent dyes (such as AMCA, FITC, CFSE, GFP, DAPI, 7-AAD, Hoechst 33342, Pacific Blue, PE, PE-TR, PE-Cy7, PE-Cy5, PI, PerCP-Cy5.5, APC, APC-CY7, APC-H7, V500, Alexa 700, BV605, BV480, BV785, BV510, BV711, BV421, etc.), near-infrared dyes (such as cyanine dyes, BODIPY dyes, rhodamine dyes, squaric acid dyes, porphyrin dyes, etc.), radionuclides (such as 125I, 18F, 11C, 99mTc, 123I, etc.), biotin, nanoparticles for magnetic resonance imaging, quantum dots for magnetic resonance imaging, magnetic substances (such as magnetic beads, nanoparticles containing gadolinium complexes, superparamagnetic iron oxide nanoparticles) and colloidal gold, but not limited to these.

[0051] The present invention also provides a method for detecting pestivirus or pestivirus E2 protein, which comprises using the monoclonal antibody or antigen-binding fragment thereof, the kit or the antibody conjugate described herein to detect pestivirus or pestivirus E2 protein.

[0052] The detection may include detecting whether the E2 protein exists in the sample, or the expression level or content of the E2 protein in the sample.

[0053] Furthermore, the method for detecting pestivirus or pestivirus E2 protein comprises contacting the monoclonal antibody or antigen-binding fragment thereof, the antibody conjugate or the kit described herein with a sample to be tested, and detecting whether pestivirus E2 protein is present in the sample to be tested through an antigen-antibody reaction.

[0054] Furthermore, the method for detecting pestivirus or pestivirus E2 protein comprises first labeling the monoclonal antibody or antigen-binding fragment thereof described herein with a detectable marker; then contacting it with a sample to be tested, and detecting whether E2 protein is present in the sample to be tested, or the content of E2 protein in the sample to be tested, by detecting the detectable marker.

[0055] The method for detecting E2 protein can be an immunological detection, such as precipitation reaction, agglutination test, Western Blot, enzyme immunoassay (such as ELISA), double antibody sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), chemiluminescent immunoassay, fluorescent immunoassay, radioimmunoassay (RIA), colloidal gold immunoassay (GIC), colloidal gold immunochromatography (GICA), immunohistochemistry (IHC), complement fixation reaction, multiplex immunoassay and fluorescent immunochromatography.

[0056] The method for detecting E2 protein may be for the purpose of disease diagnosis, disease prognosis and / or disease treatment, or may be for non-disease diagnosis, non-disease prognosis and non-disease treatment purposes.

[0057] The non-disease diagnosis purpose, non-disease prognosis purpose and non-disease treatment purpose may be detecting whether there is pestivirus contamination in environmental samples.

[0058] Furthermore, the method for detecting E2 protein may be an ELISA detection method.

[0059] Furthermore, the method comprises the step of coating the monoclonal antibody or antigen-binding fragment thereof described herein on a solid phase carrier to obtain a solid phase carrier coated with the antibody. The coating means being bound to the solid phase carrier by physical adsorption.

[0060] Furthermore, the solid phase carrier may be an ELISA plate, a membrane carrier, a microsphere, a biochip or a magnetic bead but is not limited thereto.

[0061] The material of the solid phase carrier may be polystyrene, cellulose, cross-linked dextran, polyacrylamide, polyethylene, polypropylene, polyvinyl chloride, cross-linked dextran, glass, silicone rubber or agarose gel, but is not limited thereto.

[0062] The membrane carrier may be a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane but is not limited thereto.

[0063] The present invention also provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof described herein, which comprises expressing the monoclonal antibody or antigen-binding fragment thereof described herein in a host cell, and recovering or isolating the monoclonal antibody or antigen-binding fragment thereof.

[0064] Furthermore, the preparation method may include the following steps: constructing a recombinant expression vector containing a nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment (such as a DNA molecule shown in SEQ ID No. 2 and SEQ ID No. 4); introducing the recombinant expression vector into a host cell to obtain a recombinant host cell expressing the monoclonal antibody or its antigen-binding fragment; culturing the recombinant cell, and recovering or isolating the monoclonal antibody or its antigen-binding fragment from the culture of the recombinant host cell.

[0065] Furthermore, the recovery or separation can be separated from the culture (including all substances in the culture container) by precipitation method (such as salting out method, organic solvent precipitation method, caprylic acid-saturated ammonium sulfate precipitation method, isoelectric point precipitation method) or chromatography technology (such as ion exchange chromatography, gel filtration chromatography, affinity chromatography).

[0066] Furthermore, the expression vector can be selected from prokaryotic expression vectors (including Escherichia coli (such as BL21 series expression cells, M15 expression cells, Top10 expression cells and Origamai series expression cells) expression vectors but not limited thereto) and eukaryotic expression vectors (including yeast (such as X33 cells, GS115 cells and SMD1168 cells) expression vectors, insect cell (such as Sf21 cells, Sf-9 cells and Hi-5 cells) expression vectors, mammalian cell (such as HET293 cells and CHO cells) expression vectors but not limited thereto).

[0067] The introduction method may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria by chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation or metal cation-mediated transformation) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria by phage transduction. (3) directly transferring the target gene into plant receptor cells by physical or chemical methods, such as chemical stimulation, electric shock, liposome-mediated method, microinjection, gene gun method, laser microbeam method, pollen tube channel method, ultrasound method, air gun method and vortex method. (4) transferring the target gene into plant receptor cells by vector, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method. (5) Introducing the target gene into in vitro animal cells (transfection) through calcium phosphate co-precipitation, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation (i.e. electrotransfection method), microinjection, gene gun method or virus-mediated method (such as adenovirus infection method, lentivirus infection method), etc.

[0068] The anti-plague virus monoclonal antibodies described herein may be mouse monoclonal antibodies, humanized monoclonal antibodies (including chimeric antibodies, CDR-grafted antibodies and SDR-grafted antibodies) and fully human monoclonal antibodies.

[0069] The anti-pestivirus monoclonal antibodies described in this article can bind to the E2 proteins of multiple pestiviruses (such as bovine viral diarrhea virus type 1 (BVDV1), bovine viral diarrhea virus type 2 (BVDV2), swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhea virus type 3 (HoBiPeV), Aydin-like pestivirus (AydinPeV) and / or ovine plague virus (OVPV)), have the effect of neutralizing pestiviruses, and are also called broad-spectrum neutralizing monoclonal antibodies that recognize 7 pestiviruses.

[0070] The antibodies of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors, and then transfected into host cells, and the transfected host cells are cultured under specific conditions to express the antibodies of the present invention. Those skilled in the art can select conventional host cells, expression vectors, methods for introducing expression vectors into host cells, and methods for separating and purifying antibodies as needed.

[0071] The present invention screened and identified anti-plague virus broad-spectrum neutralizing monoclonal antibodies (broad-spectrum neutralizing monoclonal antibodies that recognize 7 kinds of pestiviruses) through hybridoma technology. The antibody can be expressed and produced in prokaryotic cells, eukaryotic cells and any recombinant system, and can be made into diagnostic reagents and products for pestivirus in clinical practice. The antibody of the present invention reacts with BVDV1, BVDV2, CSFV, BDV, HoBiPeV, AydinPeV and OVPV. Compared with other pestivirus monoclonal antibodies, it is more broad-spectrum, can recognize all pestivirus members currently known to infect cattle and sheep, and has good neutralizing ability. The monoclonal antibodies provided by the present invention and the diagnostic reagents and products developed based on the antibodies can be used for serological diagnosis of pestivirus, evaluation of immune effects, and detection of pestivirus pathogens in experimental bovine serum products, etc., and have broad application prospects in the fields of clinical diagnosis and so on. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 These are the results of indirect immunofluorescence experiments between monoclonal antibody TCH052 and different genotype strains.

[0073] Figure 2 These are the results of Western blot experiments between monoclonal antibody TCH052 and different genotype strains.

[0074] Figure 3These are the results of Western blot experiments between monoclonal antibody TCH052 and different pestivirus species.

[0075] Figure 4 The results of the SDS-PAGE experiment to identify the purification effect of monoclonal antibody TCH052.

[0076] Figure 5 This is the result of identifying the neutralizing ability of monoclonal antibody TCH052.

[0077] Figure 6 This is the result of verifying the purification effect of monoclonal antibody TCH052.

[0078] Figure 7 The results of Western blot were used to verify the expression activity of monoclonal antibody TCH052.

[0079] Figure 8 This is the functional verification result of monoclonal antibody TCH052. DETAILED DESCRIPTION

[0080] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0081] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0082] The CSFV epidemic strains of various genotypes isolated and preserved in this laboratory in the following examples (SM, CN021, CN069, GD53, CN049, CN057, CN063, CN068, CN002, HuB2) and the swine fever rabbit attenuated vaccine C strain (HCLV) are recorded in the following literature: Shijiang Mi, Lihua Wang, Hongwei Li, et al. Characterization of monoclonal antibodies that specifically differentiate field isolates from vaccine strains of classical swine fever virus. Frontiers in Immunology, 2022, 13, 930631. BVDV1 strain (BVDV-1) and BVDV2 strain (BVDV-2) were kindly donated by Zhu Guoqiang, a professor at the School of Veterinary Medicine of Yangzhou University, and are recorded in the following documents: Jiang Ying, Lin Yanqing, Tao Jie, et al. Preparation of monoclonal antibodies against bovine viral diarrhea virus and establishment of double antibody sandwich ELISA detection method [J]. Chinese Journal of Preventive Veterinary Medicine, 2012, 34(12):4. The public may obtain the biological material from the applicant (Jilin University) in accordance with the relevant national biosafety regulations. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0083] The genotype 2.1b subtype epidemic strain JL23 in the following embodiment is recorded in the following document: Gong W, Li J, Wang Z, et al. Virulence evaluation of classical swine fever virus subgenotype 2.1 and 2.2 isolates circulating in China[J]. Veterinary Microbiology, 2019, 232:114-120. The public may obtain the biological material from the applicant (Jilin University) in accordance with relevant national biosafety regulations. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0084] The insect baculovirus expression vector pFastBac 1 in the following example is recorded in the following document: Mi Shijiang. Identification of monoclonal antibodies for distinguishing epidemic strains and vaccine strains of classical swine fever virus and identification of broad-spectrum monoclonal antibodies and their antigenic epitope analysis [D]. Jilin University, 2022.

[0085] The protein tag antibody anti-His Tag mAb in the following examples was purchased from Solebol, catalog number K200060M.

[0086] The semi-solid culture medium in the following examples was purchased from Bio-Long Company, with the product number BTYA0607.

[0087] The HAT additives in the following examples were purchased from Thermo Fisher, catalog number 21060017.

[0088] The nucleotide sequence of the gene encoding the CSFV-LPC E2 protein in the following examples is GenBank Accession No. AY526732 (Update Date 26-JUL-2016), positions 1171-2259.

[0089] The English abbreviations and their corresponding English full names and Chinese annotations in this article are shown in Table 1.

[0090]

[0091] Example 1. Preparation and identification of monoclonal antibodies against pestivirus E2 protein

[0092] The immune antigen is the E2 protein of the classical swine fever virus (CSFV) vaccine strain LPC, named CSFV-LPC E2 protein. In this embodiment, the insect baculovirus expression system is used to prepare the E2 protein of CSFV-LPC, and the steps are as follows: the E2 protein encoding gene of CSFV-LPC (GenBank accession number: AY526732, position 1171-2259 of the sequence shown) is cloned into the insect baculovirus expression vector pFastBac 1 after adding a His tag (the nucleotide sequence of the His tag is 5'-CATCATCACCATCACCAT-3', SEQ ID No. 5) at the 3' end to obtain a recombinant expression vector, and the recombinant expression vector is transformed into DH10Bac competent cells to obtain a recombinant bacmid containing the target gene fragment, and the bacmid is transfected into Sf9 cells for protein expression, and the CSFV-LPC E2 protein is collected by fixed metal ion affinity chromatography, and the immune antigen (CSFV-LPC E2 purified protein) is obtained after purification.

[0093] (1) Mouse immunization

[0094] SPF-grade female BALB / c mice aged 6-8 weeks were immunized. The purified CSFV-LPC E2 protein was added to an equal volume of 201 adjuvant (Seppic) for emulsification and injected subcutaneously at multiple points to immunize the mice. Each mouse was immunized with 50 μg of emulsified protein. The immunization was performed once every two weeks for a total of 3 times. One week after the three immunizations, 100 μg of purified CSFV-LPC E2 protein without adjuvant was injected intraperitoneally for shock immunization and the mice were kept for 3 days.

[0095] (2) Cell fusion

[0096] Three days after the shock immunization, the mice were killed by dislocation and the spleen was removed. The spleen was gently crushed in a nylon sieve, washed with sterile PBS and collected. The rinsing fluid was used to remove red blood cells and count the spleen cells. SP2 / 0 cells and spleen cells were mixed at a ratio of 1:5 and centrifuged at 1200 r / min for 3 min to discard the supernatant. The precipitate was washed twice with 10 mL of electrofusion solution (BTX), resuspended in 9 mL of electrofusion solution, and the cells were transferred to the electrofusion instrument for fusion. The fusion parameters were set to 800 V, 40 μs, and 1 time. The fused cells were activated for 10 h using Advanced RPMI-1640 complete medium containing 5% FBS (purchased from Thermo Fisher, catalog number 12633012), and then transferred to semi-solid medium (purchased from Bio-Long, catalog number BTYA0607) for culture for 10 days.

[0097] (3) Hybridoma cell screening

[0098] The monoclonal cell clusters in the semi-solid culture medium in step (2) were picked up and placed in Advanced RPMI 1640 culture medium containing HAT (purchased from Thermo Fisher, catalog number 21060017). After culturing for 3 days, the antibody-positive cell wells were screened using an indirect immunofluorescence test. After subcloning, a hybridoma cell line that can secrete a monoclonal antibody that specifically binds to the E2 protein was identified and named TCH052. The monoclonal antibody secreted by the hybridoma cell line TCH052 is called monoclonal antibody TCH052.

[0099] (4) Antibody type identification

[0100] The indirect ELISA method was used to identify the Ig class and light chain type of the monoclonal antibody. The secondary antibodies used in the experiment for different types of antibodies were all from the enzyme-labeled secondary antibody kit for mouse monoclonal antibody Ig subclass identification (purchased from Biolon, product number BF16002X). The results showed that the type of monoclonal antibody TCH052 was IgG1, κ light chain.

[0101] (5) Identification of the type of monoclonal antibody-virus reaction

[0102] The monoclonal antibody TCH052 was used for indirect immunofluorescence assay (IFA) with various genotypes of CSFV epidemic strains (SM, CN021, CN069, GD53, CN049, CN057, CN063, CN068, CN002, HuB2) isolated and stored in our laboratory, as well as hog cholera rabbit attenuated vaccine C strain (HCLV), and one BVDV1 strain (BVDV-1) and one BVDV2 strain (BVDV-2) kindly donated by Professor Zhu Guoqiang of the School of Veterinary Medicine of Yangzhou University. The steps are as follows:

[0103] ① Inoculation of cells: Press 100 TCID 50 CSFV cytotoxicity was inoculated into each well, and the cells were added to a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 72 h.

[0104] ② Cell fixation: discard the cell culture supernatant, add 200 μL PBS to each well of the 96-well plate and wash three times, add 80% cold acetone stored at -20℃ (50 μL / well), and fix in a -20℃ refrigerator for 1 h.

[0105] ③ Primary antibody incubation: discard the cold acetone fixative, add 200 μL of PBS to each well and wash three times, add 100 μL of hybridoma cell culture supernatant to each well, and incubate at 37°C for 1 h.

[0106] ④ Secondary antibody incubation: discard the primary antibody incubation solution, add 200 μL PBS to each well and wash three times, dilute the Alexa Fluor 488 fluorescent secondary antibody 1:500 with PBS, add 0.01% Evans blue and 5% FBS at the same time, mix thoroughly, add 100 μL to each well of the cell plate, and incubate at 37°C for 1 h.

[0107] ⑤Fluorescence observation: discard the secondary antibody incubation solution, add 200 μL of PBS to each well and wash three times, and observe the reaction between serum antibodies and infected cells under a fluorescence microscope.

[0108] The results are as follows Figure 1 The results showed that the monoclonal antibody reacted with all strains and exhibited obvious fluorescence.

[0109] The E2 proteins of different genotypes of CSFV and BVDV were expressed by the insect baculovirus expression system, including the E2 proteins of the following 17 CSFV strains: HCLV, SM, Brescia (1.2), CSF0650 (1.3), CSF1056 (1.4), CN021 (2.1a), CN069 (2.1b), GD53 (2.1c), CN049 (2.1g), CN057 (2.1h), CN063 (2.1i), CN068 (2.1j), CN002 (2.2), HuB2 (2.3), CSF0410 (3.1), JJ9811 (3.2), 94.4 / IL / 94 / TWN (3.4); and the E2 proteins of two BVDVs: BVDV1 and BVDV2. The preparation method of the E2 protein of the above strains refers to the preparation method of the CSFV-LPCE2 protein in Example 1. The coding genes of each E2 protein cloned into the insect baculovirus expression vector pFastBac 1 are as follows:

[0110] The nucleotide sequence of the gene encoding the HCLV E2 protein is at positions 2345-3433 of GenBank Accession No. Z46258.1 (Update Date 25-NOV-2005).

[0111] The nucleotide sequence of the gene encoding the SM E2 protein: GenBank Accession No. AY775178.2 (Update Date 27-NOV-2007), positions 2345-3433.

[0112] The nucleotide sequence of the gene encoding Brescia (1.2) E2 protein is at positions 2333-3421 of GenBank Accession No.AY578687.1 (Update Date 31-MAR-2005).

[0113] The nucleotide sequence of the gene encoding the E2 protein of CSF0650 (1.3) is 2147-3235 of GenBank Accession No. JX028200.1 (Update Date 19-AUG-2012).

[0114] The nucleotide sequence of the gene encoding the CSF1056 (1.4) E2 protein is: GenBank Accession No. JX028202.1 (Update Date 19-AUG-2012), positions 2147-3235.

[0115] The nucleotide sequence of the gene encoding CN021 (2.1a) E2 protein is shown in SEQ ID No.6.

[0116] The nucleotide sequence of the gene encoding CN069 (2.1b) E2 protein is shown in SEQ ID No.7.

[0117] The nucleotide sequence of the gene encoding the GD53 (2.1c) E2 protein is at positions 2344-3432 of GenBank Accession No. KP343640.1 (Update Date 01-MAY-2016).

[0118] The nucleotide sequence of the gene encoding the CN049 (2.1 g) E2 protein is at positions 2347-3435 of GenBank Accession No. KU504339.1 (Update Date 28-JUN-2016).

[0119] The nucleotide sequence of the gene encoding CN057 (2.1h) E2 protein is shown in SEQ ID No.8.

[0120] Nucleotide sequence of the gene encoding CN063 (2.1i) E2 protein: GenBank Accession No.KY132096.1 (Update Date 01-FEB-2017), positions 2345-3433.

[0121] The nucleotide sequence of the gene encoding CN068 (2.1j) E2 protein is shown in SEQ ID No.9.

[0122] The nucleotide sequence of the gene encoding CN002 (2.2) E2 protein is shown in SEQ ID No.10.

[0123] The nucleotide sequence of the gene encoding HuB2 (2.3) E2 protein is shown in SEQ ID No.11.

[0124] The nucleotide sequence of the gene encoding the E2 protein of CSF0410 (3.1) is 2146-3234 of GenBank Accession No. JQ411575.1 (Update Date 09-MAR-2016).

[0125] The nucleotide sequence of the gene encoding the E2 protein of JJ9811 (3.2) is at positions 2346-3434 of GenBank Accession No. KF669877.1 (Update Date 20-DEC-2012).

[0126] The nucleotide sequence of the gene encoding the 94.4 / IL / 94 / TWN(3.4) E2 protein is at positions 2344-3432 of GenBank Accession No. AY646427.1 (Update Date 29-OCT-2007).

[0127] The nucleotide sequence of the gene encoding the BVDV1 E2 protein is shown in SEQ ID No.12.

[0128] The nucleotide sequence of the gene encoding the BVDV2 E2 protein is shown in SEQ ID No.13.

[0129] Western blot experiments were performed with monoclonal antibody TCH052 and different genotypes of CSFV and BVDV E2 proteins expressed by insect baculovirus expression system (i.e., E2 proteins of the above 19 strains and CSFV-LPC), and CSFV antibody protein tag antibody anti-His Tag mAb was used as a control. The steps are as follows:

[0130] ① Protein treatment: Add E2 protein into 4× loading buffer according to the proportion and boil for 10 min.

[0131] ② Protein electrophoresis: Load the treated protein (10 μL per lane) into a 10% density SDS-PAGE gel and perform protein electrophoresis at 55 V for 50 min and 110 V for 80 min.

[0132] ③ Transfer: Use the semi-dry transfer method to transfer the protein after electrophoresis to the NC membrane, the program is 23 V-25 min.

[0133] ④ Blocking: Add 5 g skim milk powder to every 100 mL PBS and shake thoroughly to mix as blocking solution. Add 5 mL of blocking solution to a ziplock bag and place it on a shaker to block the NC membrane at room temperature for 1 h.

[0134] ⑤ Incubate with primary antibody: dilute the hybridoma cell culture supernatant with blocking solution at 1:100 and dilute the Anti-His Tag antibody at 1:3000 as a positive control, and incubate overnight at 4°C on a shaker.

[0135] ⑥ Incubate with secondary antibody: Wash the membrane three times with PBS, add fluorescent secondary antibody labeled with Alexa Fluor 680 diluted 1:5000 in PBS, and incubate on a shaker at room temperature and away from light for 1 h.

[0136] ⑦ Scanning NC membrane: Wash the membrane three times with PBS, place it in a two-color infrared laser imaging system for scanning, and save the image.

[0137] The results are as follows Figure 2 As shown, monoclonal antibody TCH052 reacts with E2 proteins of all strains.

[0138] The E2 proteins of the following pestiviruses were expressed by the insect baculovirus expression system: BDV, PAPeV, PPeV, GPeV, HoBiPeV, AydinPeV, RPeV, APPV and OVPV. The preparation methods of the E2 proteins of the above pestiviruses refer to the preparation method of the CSFV-LPC E2 protein in Example 1. Among them, the coding genes of each E2 protein cloned into the insect baculovirus expression vector pFastBac 1 are as follows:

[0139] The nucleotide sequence of the gene encoding the BDV E2 protein: GenBank Accession No. NC_003679 (Update Date 13-AUG-2018), positions 2347-3435.

[0140] The nucleotide sequence of the gene encoding the PAPeV E2 protein: GenBank Accession No. NC_024018 (Update Date 04-JUN-2019), positions 2335-3417.

[0141] The nucleotide sequence of the gene encoding the PPeV E2 protein: GenBank Accession No. NC_023176 (Update Date 13-AUG-2018), positions 2386-3486.

[0142] The nucleotide sequence of the gene encoding the GPeV E2 protein: GenBank Accession No. NC_003678 (Update Date 13-AUG-2018), positions 2351-3442.

[0143] The nucleotide sequence of the gene encoding the HoBiPeV E2 protein: GenBank Accession No. NC_012812.1 (Update Date 13-AUG-2018), positions 2361-3449.

[0144] The nucleotide sequence of the gene encoding AydinPeV E2 protein: GenBank Accession No. NC_018713 (Update Date 13-AUG-2018), positions 2352-3440.

[0145] The nucleotide sequence of the gene encoding the RPeV E2 protein: GenBank Accession No. KJ950914 (Update Date 02-JUL-2018), positions 2809-3915.

[0146] The nucleotide sequence of the gene encoding the APPV E2 protein: GenBank Accession No. NC_038964 (Update Date 24-AUG-2018), positions 2122-2949.

[0147] The nucleotide sequence of the gene encoding the OVPV E2 protein: positions 2323-3411 of GenBank Accession No. MG770617 (Update Date 26-AUG-2021).

[0148] Western blot experiments were performed using monoclonal antibody TCH052 and E2 proteins of 12 pestivirus members (CSFV-LPC, BVDV1, BVDV2, BDV, PAPeV, PPeV, GPeV, HoBiPeV, AydinPeV, RPeV, APPV and OVPV), and protein tag antibody anti-His Tag mAb was used as a control. Western blot experiments were performed using hybridoma cell culture supernatant as primary antibody and Alexa Fluor 680-labeled fluorescent antibody as secondary antibody.

[0149] The results are as follows Figure 3 As shown, monoclonal antibody TCH052 can react with the E2 proteins of seven pestivirus species, including BVDV1, BVDV2, CSFV, BDV, HoBiPeV, AydinPeV and OVPV, which are widely prevalent and highly pathogenic. These pestiviruses mainly come from livestock such as pigs, cattle and sheep.

[0150] (6) Preparation and purification of monoclonal antibody in ascites

[0151] Female Balb / C mice aged 6-8 weeks were intraperitoneally injected with 300 μL of mouse ascites adjuvant. Twelve days later, 1×10 6The mouse abdominal circumference was significantly enlarged, and the ascites was extracted with a syringe. The supernatant was collected and stored after centrifugation at 12000 r / min for 10 min. The antibody in the ascites was purified using a Protein A / G pre-packed chromatographic column, and the antibody purification effect was verified by SDS-PAGE experiment. The results are as follows: Figure 4 As shown, there is an obvious and single band greater than 180 kDa, indicating that the experiment obtained a monoclonal antibody of higher purity.

[0152] (7) Identification of neutralizing ability of monoclonal antibodies

[0153] The ability of monoclonal antibody TCH052 to neutralize CSFV vaccine strain LPC, genotype 2.1b subtype epidemic strain JL23, BVDV1 and BVDV2 strains was identified by indirect immunofluorescence assay. The identification method was as follows: CSFV strains and BVDV strains of different genotypes were diluted to 2000 TCID using MEM and DMEM, respectively. 50 / mL, 50 μL of virus dilution was added to a 96-well plate. The purified antibody TCH052 was diluted 2-fold using MEM or DMEM, and 50 μL of each concentration gradient was added to the corresponding virus solution and incubated at 37°C for 1 h. 100 μL of 1:4 passaged PK-15 cells or MDBK cells were added to the virus / antibody neutralization solution, respectively, and cultured at 37°C and 5% CO2 for 3 days. IFA was used to identify whether the broad-spectrum monoclonal antibody against pestivirus can neutralize CSFV and BVDV.

[0154] The results are as follows Figure 5 As shown in the figure, when a higher concentration of purified monoclonal antibody was added, cells infected with CSFV or BVDV strains had no obvious green fluorescence. As the antibody dilution multiple continued to increase, cells infected with different strains all showed green fluorescence, indicating that monoclonal antibody TCH052 has good neutralizing ability.

[0155] (8) Determination of monoclonal antibody sequence

[0156] The monoclonal antibody purified by Nanjing Detai Bioengineering Co., Ltd. was commissioned to be sequenced. Based on the nucleic acid sequence obtained by Sanger sequencing, the corresponding amino acid sequence was obtained using the rule that a triplet codon encodes an amino acid. It was finally determined that the amino acid sequence of the heavy chain variable region of monoclonal antibody TCH052 is shown in SEQ ID No.1, and the nucleotide sequence of the heavy chain variable region encoding gene is shown in SEQ ID No.2; the amino acid sequence of the light chain variable region of monoclonal antibody TCH052 is shown in SEQ ID No.3, and the nucleotide sequence of the light chain variable region encoding gene is shown in SEQ ID No.4. Among them:

[0157] The amino acid sequence of the heavy chain variable region CDR1 of the monoclonal antibody TCH052 is shown at positions 26-33 of SEQ ID No. 1;

[0158] The amino acid sequence of the heavy chain variable region CDR2 of the monoclonal antibody TCH052 is shown in positions 51-58 of SEQ ID No. 1;

[0159] The amino acid sequence of the heavy chain variable region CDR3 of the monoclonal antibody TCH052 is shown in positions 97-108 of SEQ ID No. 1;

[0160] The amino acid sequence of the light chain variable region CDR1 of the monoclonal antibody TCH052 is shown at positions 27-32 of SEQ ID No. 3;

[0161] The amino acid sequence of the light chain variable region CDR2 of the monoclonal antibody TCH052 is shown at positions 50-52 of SEQ ID No. 3;

[0162] The amino acid sequence of the light chain variable region CDR3 of monoclonal antibody TCH052 is shown at positions 94-102 of SEQ ID No.3.

[0163] Example 2: Genetic engineering preparation of antibodies and functional verification

[0164] 1. Genetic engineering method to prepare antibodies

[0165] 1) Construction of recombinant expression plasmid

[0166] In order to express the monoclonal antibody TCH052, a heavy chain expression vector and a light chain expression vector were prepared respectively: the nucleotide sequence of the heavy chain gene of the monoclonal antibody TCH052 was obtained by directly connecting the nucleotide sequence of the heavy chain variable region encoding gene (SEQ ID No. 2) and the mouse-IgG2a template sequence (heavy chain constant region sequence, SEQ ID No. 14). The nucleotide sequence of the light chain gene of the monoclonal antibody TCH052 was obtained by directly connecting the nucleotide sequence of the light chain variable region encoding gene (SEQ ID No. 4) and the mouse-kappa template sequence (light chain constant region sequence, SEQ ID No. 15).

[0167] The heavy chain gene and light chain gene of monoclonal antibody TCH052 were cloned into the vector pcDNA3.4, respectively, to obtain the heavy chain expression vector and the light chain expression vector. The recombinant vectors were synthesized by Nanjing Detai Bioengineering Co., Ltd.

[0168] 2) Antibody expression:

[0169] a) Inoculate freshly digested 293T cells onto 175 cm 2In the culture flask, 35 mL of DMEM medium containing 8% FBS (purchased from Corning, catalog number 10-013-CVRC) was added to culture the cells to a density of 90%.

[0170] b) Dilute 200 μg of the plasmid containing the light chain and heavy chain (100 μg each) and 200 μL of QuickShuttle-293 cell-specific transfection reagent (Biolon, KX0110044) into 1 mL of normal saline.

[0171] c) combining the two solutions in step b) above and mixing them to obtain a composite.

[0172] d) Add the above complex directly into the cell culture medium in step a) and mix well by pipetting with a pipette.

[0173] e) Move the cell plate to a 37ºC / 5% CO2 incubator for culture. After 3 days of culture, collect the culture supernatant for verification.

[0174] 3) Verification of antibody expression:

[0175] Take the cell expression supernatant, add non-reducing buffer without DTT (lane 1) and reducing buffer with DTT in proportion, and perform Western blot experiment. The results are as follows: Figure 6 As shown in Figure 1, lane 1 has a single obvious band at greater than 180 kDa. After the antibody was treated with reducing buffer, as shown in lane 2, there were two obvious bands of light chain and heavy chain at about 25 kDa and 50 kDa, indicating that the antibody was well purified. The expression supernatant was subjected to Western blot experiment with E2 protein of CSFV-LPC and BVDV1. The results are shown in Figure 1. Figure 7 As shown, monoclonal antibody TCH052 reacted with E2 proteins of all strains, indicating that the expressed antibody was active. The above experiments showed that the antibody was successfully expressed.

[0176] 4) Antibody purification:

[0177] a) Buffer preparation: Add Na2HPO4·12H2O to sterile ddH2O to a final concentration of 0.2 M and vortex thoroughly to mix.

[0178] b) Preparation of pre-elution buffer: Add 0.1 M citric acid to the buffer to make the volume percentage of citric acid 20%.

[0179] c) Preparation of eluent: Add 0.1 M citric acid to the buffer solution so that the volume proportion of citric acid is 60%.

[0180] d) Sample processing: Take 30 mL of the cell expression supernatant verified in step 3), add the buffer prepared in step a) at a volume ratio of 1:1, and filter through a pore size of 0.22 μm before loading onto the column.

[0181] e) Equilibrate the column: Use a constant flow pump to slowly pass 10 mL of buffer through a Protein A / G4FF prepacked chromatographic column (Sanggong, C600983) at a flow rate of 1 mL / min.

[0182] f) Sample loading: Use a constant flow pump to slowly pass the solution in step d) through the Protein A / G column at a flow rate of 1 mL / min.

[0183] g) Washing: Use a constant flow pump to slowly pass 10 mL of wash buffer through the Protein A / G column at a flow rate of 1 mL / min.

[0184] h) Pre-elution: Use a constant flow pump to slowly pass 10 mL of pre-elution buffer through the Protein A / G column at a flow rate of 1 mL / min.

[0185] i) Elution: Use a constant flow pump to slowly pass 15 mL of eluent through the Protein A / G column at a flow rate of 1 mL / min, and dispense the eluted product into 1.5 mL centrifuge tubes.

[0186] 2. Preparation of conjugated antibodies

[0187] HRP was conjugated to monoclonal antibody TCH052 using an HRP conjugation kit (Sangong, D601047) to prepare HRP-TCH052 conjugated antibody. The specific steps are as follows:

[0188] (1) Mix 500 μL of HRP solution and 200 μL of HRP activation buffer, place on a shaker and shake slowly, and react at room temperature for 30 min.

[0189] (2) Add 200 μL HRP coupling buffer and let stand at room temperature for 30 min.

[0190] (3) Add 1 mL of purified monoclonal antibody TCH052 and transfer to a dialysis bag. Dialyze in 2 L of dialysis solution at room temperature for 2 h.

[0191] (4) Add 100 μL of reducing agent to the dialyzed product, let it stand at room temperature for 2 h, stir it every 30 min, and aliquot and store the coupled product.

[0192] 3. Functional verification of monoclonal antibody TCH052

[0193] Western blot experiments were performed with the labeled monoclonal antibody HRP-TCH052 prepared in the previous step and the E2 proteins of the seven pestivirus members (BVDV1, BVDV2, CSFV-LPC, BDV, HoBiPeV, AydinPeV and OVPV) that can be recognized by monoclonal antibody TCH052. The protein tag antibody anti-His Tag mAb primary antibody and goat anti-mouse IgG H&L (HRP) pre-adsorbed secondary antibody (purchased from abcam, catalog number ab97040) were used as controls. The labeled monoclonal antibody HRP-TCH052 was diluted 1:1000 with PBS for Western blot experiments. The color was developed using the Omni-ECL™ Ultrasensitive Chemiluminescence Detection Kit (purchased from Yamase, catalog number SQ201). The results are shown in Figure 2. Figure 8 As shown, the labeled monoclonal antibody HRP-TCH052 can react with the E2 proteins of 7 pestivirus species.

[0194] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An anti-plague virus monoclonal antibody or an antigen-binding fragment thereof, 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 HCDR1 with an amino acid sequence of positions 26 to 33 of SEQ ID No.1, HCDR2 with an amino acid sequence of positions 51 to 58 of SEQ ID No.1, and HCDR3 with an amino acid sequence of positions 97 to 108 of SEQ ID No.1; the light chain variable region comprises LCDR1 with an amino acid sequence of positions 27 to 37 of SEQ ID No.3, LCDR2 with an amino acid sequence of positions 55 to 57 of SEQ ID No.3, and LCDR3 with an amino acid sequence of positions 94 to 102 of SEQ ID No.3; The pestivirus is bovine viral diarrhea virus type 1, bovine viral diarrhea virus type 2, classical swinefever virus, border disease virus, HoBi-like Pestivirus, Aydin-like Pestivirus and / or Ovine Pestivirus.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.1; the amino acid sequence of the light chain variable region is shown in SEQ ID No.

3.

3. Biomaterial, characterized in that The biological material is any of the following: A1) a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1); A4) a recombinant microorganism containing the nucleic acid molecule described in A1); A5) A recombinant host cell containing the nucleic acid molecule described in A1).

4. The biomaterial according to claim 3, characterized in that The nucleic acid molecules are B1) and B2): B1) a DNA molecule whose coding sequence is SEQ ID No. 2; B2) The coding sequence is a DNA molecule of SEQ ID No.

4.

5. Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, or the biomaterial according to claim 3 or 4 in any of the following: C1) Use in the preparation of products for diagnosing or assisting in the diagnosis of pestivirus infection or diseases caused by pestivirus infection; C2) Use in the preparation of products for detecting pestivirus or pestivirus E2 protein; The pestivirus is bovine viral diarrhea virus type 1, bovine viral diarrhea virus type 2, classical swinefever virus, border disease virus, HoBi-like Pestivirus, Aydin-like Pestivirus and / or Ovine Pestivirus.

6. A kit, characterized in that The kit contains the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. An antibody conjugate, comprising an antibody portion and a conjugate portion, characterized in that: The antibody portion comprises the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

8. A method for detecting pestivirus or pestivirus E2 protein for non-disease diagnosis and treatment purposes, characterized in that: The method comprises detecting pestivirus or pestivirus E2 protein using the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, the kit according to claim 6, or the antibody conjugate according to claim 7; The pestivirus is bovine viral diarrhea virus type 1, bovine viral diarrhea virus type 2, classical swinefever virus, border disease virus, HoBi-like Pestivirus, Aydin-like Pestivirus and / or Ovine Pestivirus.

9. The method for preparing the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The preparation method comprises expressing the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in a host cell, and recovering or isolating the monoclonal antibody or antigen-binding fragment thereof.

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