Monoclonal antibodies for distinguishing between lapinized attenuated swine fever vaccine strains and epidemic strains and their application

Abstract

The present invention discloses a monoclonal antibody for distinguishing a lapinized attenuated vaccine strain of classical swine fever from an epidemic strain and its application, belonging to the field of immunoglobulins. Specifically disclosed is an E2 protein monoclonal antibody or an antigen-binding fragment thereof, including a heavy chain variable region (SEQ ID No.1) and a light chain variable region (SEQ ID No.3). The present invention also discloses an ELISA method for distinguishing a lapinized attenuated vaccine strain of classical swine fever from an epidemic strain using the E2 protein monoclonal antibody. The monoclonal antibody of the present invention specifically reacts with an epidemic strain of classical swine fever virus, but does not react with a lapinized attenuated vaccine strain of classical swine fever. The ELISA competition method established using the monoclonal antibody can accurately distinguish a lapinized attenuated vaccine strain of classical swine fever from an epidemic strain. The monoclonal antibody of the present invention can be used in the fields of serological diagnosis of CSFV immunization and wild virus, immune effect evaluation, and related experiments, and provides antibody resources for effective detection and purification of CSFV.

Description

Technical Field

[0001] The present invention relates to the field of immunoglobulins, and in particular to monoclonal antibodies for distinguishing between lapinized attenuated swine fever vaccine strains and epidemic strains and applications thereof. Background Art

[0002] Classical swine fever is a disease that seriously threatens the global pig industry. The World Organization for Animal Health (WOAH) lists it as a notifiable animal disease and it is a Category II animal disease in my country. Domestic pigs and wild boars are the main susceptible animals to classical swine fever. Its main clinical manifestations are widespread systemic bleeding, reproductive disorders, dyspnea and prolonged fever. Acute classical swine fever has a short incubation period and a high mortality rate. Affected animals may die only 10 to 20 days after infection. Subacute and chronic classical swine fever can cause intermittent fever, slow growth and development, and ultimately death in affected animals. Persistently infected animals have no obvious clinical symptoms, but pregnant sows are prone to miscarriage and stillbirth, and are also a more harmful source of infection.

[0003] Classical Swine Fever Virus (CSFV) is the pathogen that causes swine fever. It is a single-stranded positive-strand RNA virus with an envelope. The virus particles are spherical with a diameter of about 40 to 60 nm. The genome size is 11.3 to 13.0 kb, containing a large open reading frame, encoding a polyprotein composed of 3898 amino acid residues, which is further cleaved by protease to produce four structural proteins (Core, E rns , E1 and E2) and 8 nonstructural proteins (N pro , P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B), among which the structural protein E rns Together with E2 and nonstructural protein NS3, they can stimulate the body to produce antibodies. E2 protein is the main protective antigen of CSFV, and its size is about 51-58 kDa. The amino terminus of E2 protein has four antigenic domains, B, C, D, and A, which can be further divided into two antigenic structural units, B / C and D / A, where most of the antigenic epitopes are located.

[0004] With the widespread use of HCLV (C-strain), swine fever has been widely controlled in my country, and the current epidemic is characterized by multi-point sporadic. There are 3 genotypes of CSFV (genotypes 1 to 3), which can be further divided into 11 genotypes (1.1 to 1.4; 2.1 to 2.3; 3.1 to 3.4). The current prevalent strain is subtype 2.1, and the 2.1 subtype epidemic strain can be further divided into 10 subtypes (2.1a to 2.1j). The existing CSFV serological detection technology cannot distinguish between C-strain immunity and epidemic strain infection, which greatly restricts the purification of CSFV. Therefore, it is urgent to develop a detection method that can distinguish between CSFV vaccine strains and epidemic strains. Summary of the invention

[0005] One of the technical problems to be solved by the present invention is how to identify and distinguish between the lapinized attenuated vaccine strain of swine fever and the epidemic strain. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] To solve the above technical problems, the present invention first provides an E2 protein monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal 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 three complementary determining regions whose amino acid sequences are respectively positions 26-33 of SEQ ID No.1, positions 51-58 of SEQ ID No.1 and positions 97-111 of SEQ ID No.1; and the light chain variable region comprises three complementary determining regions whose amino acid sequences are respectively positions 27-32 of SEQ ID No.3, positions 50-52 of SEQ ID No.3 and positions 89-97 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 heavy chain constant region may be a heavy chain constant region of mouse IgG2a.

[0014] Furthermore, the light chain constant region may be a mouse Kappa (κ) type light chain constant region.

[0015] Furthermore, the nucleotide sequence of the heavy chain constant region may be as shown in SEQ ID No.13.

[0016] Furthermore, the nucleotide sequence of the light chain constant region may be as shown in SEQ ID No.14.

[0017] The present invention also provides a biomaterial, which may be any of the following:

[0018] 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;

[0019] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0020] A3) a recombinant vector containing the nucleic acid molecule described in A1);

[0021] A4) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0022] A5) A recombinant host cell containing the nucleic acid molecule described in A1).

[0023] The biological materials can all express the monoclonal antibodies or antigen-binding fragments thereof described herein.

[0024] In the above biological material, the nucleic acid molecule may be any of the following:

[0025] B1) a DNA molecule whose coding sequence is SEQ ID No. 2;

[0026] B2) The coding sequence is a DNA molecule of SEQ ID No.4.

[0027] 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.

[0028] 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.

[0029] The present invention also provides the use of the monoclonal antibody or antigen-binding fragment thereof, or the biomaterial in any of the following:

[0030] C1) Application in distinguishing between lapinized attenuated swine fever vaccine strains and epidemic strains;

[0031] C2) Use in the preparation of products for identifying and distinguishing between lapinized attenuated swine fever vaccine strains and epidemic strains;

[0032] C3) Use in the preparation of products for diagnosing or assisting in the diagnosis of classical swine fever virus infection or diseases caused by classical swine fever virus infection;

[0033] C4) Use in the preparation of products for screening or assisting in screening for classical swine fever virus infection or diseases caused by classical swine fever virus infection;

[0034] C5) Use in the preparation of products for detecting epidemic strains of classical swine fever virus or E2 protein of epidemic strains of classical swine fever virus;

[0035] C6) Use in the preparation of a product for binding to a classical swine fever virus epidemic strain or the E2 protein of a classical swine fever virus epidemic strain.

[0036] The diagnosis or auxiliary diagnosis of classical swine fever virus infection or a disease caused by classical swine fever virus 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 classical swine fever virus E2 protein or the content of classical swine fever virus E2 protein in the sample, and diagnosing or auxiliary diagnosis of classical swine fever virus infection or a disease caused by classical swine fever virus infection is performed based on the test results.

[0037] The screening or auxiliary screening of classical swine fever virus infection or diseases caused by classical swine fever virus infection can be performed by isolating samples from subjects, and then using the monoclonal antibody or its antigen-binding fragment to detect whether classical swine fever virus E2 protein or the content of classical swine fever virus E2 protein in the samples, and screening for classical swine fever virus infection or diseases caused by classical swine fever virus infection is performed based on the test results.

[0038] The detection of the epidemic strain of classical swine fever virus or the E2 protein of the epidemic strain of classical swine fever virus includes any in vivo or in vitro detection of the E2 protein based on the principle of antigen-antibody specific reaction. The detection of the E2 protein can be to detect whether the sample to be tested contains the E2 protein and / or to detect the content of the E2 protein in the sample to be tested.

[0039] The product that binds to the epidemic strain of classical swine fever virus or the E2 protein of the epidemic strain of classical swine fever virus can be an E2 protein inhibitor, or a product for separating or purifying the 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 the environmental conditions such as the pH value.

[0040] Furthermore, the classical swine fever virus infection may be an infection caused by an epidemic strain of classical swine fever virus.

[0041] The products described herein may include reagents, kits, chips, test strips, test cards, and immunosensors.

[0042] The subject may be a human or a non-human animal (eg, pig, cow, sheep, rabbit, cat, horse, deer, monkey, chicken, dog, etc.).

[0043] 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.

[0044] 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).

[0045] Furthermore, the conjugated moiety may be a detectable label.

[0046] 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.

[0047] The present invention also provides a kit, which contains the monoclonal antibody or antigen-binding fragment thereof described herein.

[0048] The kit may have at least one of the following uses:

[0049] (1) Identify and differentiate between the lapinized vaccine strain and the epidemic strain of swine fever;

[0050] (2) Diagnosis or auxiliary diagnosis of classical swine fever virus infection or diseases caused by classical swine fever virus infection;

[0051] (3) Screening or assisting in screening for classical swine fever virus infection or diseases caused by classical swine fever virus infection;

[0052] (4) Detection of classical swine fever virus epidemic strains or E2 protein of classical swine fever virus epidemic strains;

[0053] (5) Combining with the epidemic strain of classical swine fever virus or the E2 protein of the epidemic strain of classical swine fever virus.

[0054] 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.

[0055] 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.).

[0056] 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.

[0057] 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.

[0058] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0059] The components of the kit may be provided in the form of a solution, such as an aqueous solution. In the case of being in the form of an aqueous solution, the concentration or content of these components can be easily determined by those skilled in the art according to different needs. For example, for storage purposes, the concentration of the components may be present in a higher form, and when in working state or use, the concentration may be reduced to the working concentration by diluting the above-mentioned higher concentration solution.

[0060] The kit may also include, for example, a buffer, a preservative, or a protein stabilizer. The kit may also include components necessary for detecting the detectable label, such as an enzyme or a substrate. The kit may also contain a control sample or a series of control samples that can be assayed and compared to the test sample. The kit may include a written product on or in the kit container. The written product describes how to use the reagents included in the kit.

[0061] The present invention also provides a method for preparing the monoclonal antibody or its antigen-binding fragment, which comprises expressing the monoclonal antibody or its antigen-binding fragment described herein in a host cell, and recovering or isolating the monoclonal antibody or its antigen-binding fragment.

[0062] 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.

[0063] 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).

[0064] 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).

[0065] 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.

[0066] The present invention also provides a method for identifying and distinguishing a lapinized attenuated classical swine fever vaccine strain from an epidemic strain, the method comprising using the monoclonal antibody or its antigen-binding fragment, the antibody conjugate or the kit described herein to identify and distinguish a lapinized attenuated classical swine fever vaccine strain from an epidemic strain.

[0067] Furthermore, the method for identifying and distinguishing the classical swine fever lapinized attenuated vaccine strain and the epidemic strain can be an ELISA competitive method.

[0068] Furthermore, the ELISA competition method can be performed by labeling the monoclonal antibody or its antigen-binding fragment described in this article with a detectable label, and then contacting it with a test sample and a standard antigen (E2 protein) of known concentration, so that the test antigen in the test sample and the known antigen compete with each other, and distinguishing the classical swine fever lapinized attenuated vaccine strain and the epidemic strain by detecting the competitive relationship (such as blocking rate) between the test sample and the known antigen.

[0069] The ELISA competition method generally refers to an ELISA method that uses the competitive relationship between antigen and antibody for detection. The principle of the competition method is based on the binding reaction between antigen and antibody. When the antigen in the sample to be tested and the standard antigen of known concentration react with the antibody at the same time, a competitive relationship will be formed between them. In the present invention, if the sample to be tested contains the epidemic strain of classical swine fever virus, then since the E2 protein of the epidemic strain of classical swine fever virus can specifically bind to the monoclonal antibody TCH065 of the present invention, it will occupy more antibodies, thereby reducing the binding opportunity of the known antigen and the antibody TCH065, and when the sample to be tested contains the attenuated swine fever rabbit vaccine strain, then since the attenuated swine fever rabbit vaccine strain does not specifically bind to the monoclonal antibody TCH065 of the present invention, the known antigen will occupy more antibodies at this time, therefore, the results of the competitive reaction can be detected to identify and distinguish the attenuated swine fever rabbit vaccine strain and the epidemic strain.

[0070] Furthermore, the method for identifying and distinguishing the attenuated swine fever lapinized vaccine strain and the epidemic strain may include the following steps:

[0071] (1) Coat the ELISA plate with a known concentration of antigen E2 protein;

[0072] (2) Add the sample to be tested, the positive control (such as swine fever antibody positive serum) and the negative control (such as swine fever antibody negative serum) to the ELISA plate coated with the antigen and incubate them together;

[0073] (3) after washing the plate, adding the monoclonal antibody or antigen-binding fragment thereof of the present invention that has been labeled with a detectable marker and incubating the mixture;

[0074] (4) Detection by detecting a detectable label.

[0075] Furthermore, the antigen E2 protein in step (1) can be directly purchased commercially, or can be obtained by a recombinant expression method (such as the preparation method of CSFV-JL23 E2 protein described in Example 1 of the present invention).

[0076] The method for identifying and distinguishing the attenuated classical swine fever lapinized vaccine strain and the epidemic strain can be for the purpose of disease diagnosis, disease prognosis and / or disease treatment, or can be for non-disease diagnosis, non-disease prognosis and non-disease treatment purposes.

[0077] The non-disease diagnosis purpose, non-disease prognosis purpose and non-disease treatment purpose can be used to evaluate the effect of the vaccine. Accurately distinguishing the attenuated lapinized vaccine strain of classical swine fever from the epidemic strain is crucial to evaluating the effect of the vaccine. The protective effect of the vaccine can be more accurately evaluated, thereby guiding the development and use of the vaccine. The non-disease diagnosis purpose, non-disease prognosis purpose and non-disease treatment purpose can also be used for epidemiological research. For example, by distinguishing the lapinized lapinized vaccine strain of classical swine fever from the epidemic strain, the transmission law, epidemic trend and genetic relationship between different strains of classical swine fever virus can be better understood.

[0078] The present invention also provides a method for detecting the epidemic strain of classical swine fever virus or the E2 protein of the epidemic strain of classical swine fever virus, which comprises using the monoclonal antibody or its antigen-binding fragment, the antibody conjugate or the kit described herein to detect the epidemic strain of classical swine fever virus or the E2 protein of the epidemic strain of classical swine fever virus.

[0079] 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.

[0080] Furthermore, the method for detecting the epidemic strain of classical swine fever virus or the E2 protein of the epidemic strain of classical swine fever virus comprises contacting the monoclonal antibody or its antigen-binding fragment, the antibody conjugate or the kit described in this article with a sample to be tested, and detecting whether the classical swine fever virus E2 protein is present in the sample to be tested through an antigen-antibody reaction.

[0081] Furthermore, the method for detecting the epidemic strain of classical swine fever virus or the E2 protein of the epidemic strain of classical swine fever virus comprises first labeling the monoclonal antibody or its antigen-binding fragment described in this article with a detectable marker; then contacting it with a sample to be tested, and detecting whether the E2 protein exists in the sample to be tested, or the content of the E2 protein in the sample to be tested, by detecting the detectable marker.

[0082] 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, etc.

[0083] 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.

[0084] The non-disease diagnosis purpose, non-disease prognosis purpose and non-disease treatment purpose may be detecting whether there is classical swine fever virus contamination in environmental samples.

[0085] Furthermore, the method for detecting E2 protein may be an ELISA detection method.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] The membrane carrier may be a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane but is not limited thereto.

[0090] The monoclonal 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.

[0091] The present invention screened and identified a monoclonal antibody hybridoma cell line that can distinguish between the attenuated lapinized vaccine strain of classical swine fever and the epidemic strain through hybridoma technology. The monoclonal antibody (TCH065) secreted by the cell line specifically reacts with the epidemic strain of classical swine fever virus, but does not react with the lapinized attenuated vaccine strain of classical swine fever. Based on this, the present invention prepared the monoclonal antibody TCH065 through genetic engineering, and established an ELISA competition method that can identify and distinguish the lapinized attenuated vaccine strain of classical swine fever and the epidemic strain using the antibody TCH065. The monoclonal antibody TCH065 of the present invention can be used in the fields of serological diagnosis of CSFV immunity and wild virus, immune effect evaluation, and related experiments, providing antibody resources for the effective detection and purification of CSFV. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1These are the results of indirect immunofluorescence experiments between monoclonal antibody TCH065 and different genotype strains.

[0093] Figure 2 These are the results of Western blot experiments between monoclonal antibody TCH065 and different genotype strains.

[0094] Figure 3 The results of IFA were used to verify the expression activity of monoclonal antibody TCH065.

[0095] Figure 4 This is the result of verifying the purification effect of monoclonal antibody TCH065. DETAILED DESCRIPTION

[0096] 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.

[0097] 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.

[0098] The insect baculovirus expression vector pFastBac 1 in the following examples is recorded in the following literature: 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.

[0099] The CSFV epidemic strains of various genotypes isolated and preserved in this laboratory in the following embodiments (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. The public can obtain the biological material from the applicant (Jilin University) in accordance with the relevant provisions of national biosafety. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0100] The swine fever epidemic strains AH1 and GD23 in the following embodiments are recorded in the following literature: Gong W, Li J, Wang Z, et al. Commercial E2 subunit vaccine provides full protection to pigs against lethal challenge with 4 strains of classical swine fever virus genotype 2. Vet Microbiol. 2019;237:108403 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.

[0101] The protein tag antibody anti-His Tag mAb in the following examples was purchased from Solebol, catalog number K200060M.

[0102] Example 1. Preparation and identification of monoclonal antibodies against classical swine fever virus E2 protein

[0103] The immune antigen in this embodiment is the E2 protein of the 2.1b subtype epidemic strain JL23 of classical swine fever virus (CSFV) (CSFV-JL23) (named as CSFV-JL23 E2 protein). The CSFV-JL23 E2 protein is prepared using an insect baculovirus expression system, and the steps are as follows: the E2 protein encoding gene containing a signal peptide of CSFV-JL23 (nucleotide sequence is SEQ ID No.5) is added to the 3' end with a His tag (His tag nucleotide sequence is 5'-CATCATCACCATCACCAT-3' (SEQ ID No.6)) and then cloned into the insect baculovirus expression vector pFastBac 1 to obtain a recombinant expression vector, the recombinant expression vector is transformed into DH10Bac competent cells to obtain a recombinant bacmid containing the target gene fragment, the bacmid is transfected into Sf9 cells for protein expression, and the CSFV-JL23 E2 purified protein is collected by fixed metal ion affinity chromatography, and the immune antigen is obtained after purification.

[0104] (1) Mouse immunization

[0105] SPF-grade female BALB / c mice aged 6-8 weeks were immunized. The purified CSFV-JL23 E2 protein was added to an equal volume of 206 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-JL23 E2 protein without adjuvant was injected intraperitoneally for shock immunization and the mice were kept for 3 days.

[0106] (2) Cell fusion

[0107] 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. After activation for 10 h with Advanced RPMI 1640 medium containing 5% FBS (purchased from Thermo Fisher, catalog number 12633012), the fused cells were transferred to semi-solid culture medium (purchased from Bio-Long, catalog number BTYA0607) and cultured for 10 days.

[0108] (3) Hybridoma cell screening

[0109] The monoclonal cell clusters in the semi-solid culture medium in step (2) were picked up and transferred to 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 (named hybridoma cell TCH065) was identified, and the monoclonal antibody secreted by it was named monoclonal antibody TCH065.

[0110] (4) Identification of monoclonal antibody type

[0111] The indirect ELISA method was used to identify the monoclonal antibody Ig class and light chain type. 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 monoclonal antibody TCH065 was of IgG2b, κ light chain.

[0112] (5) Identification of the type of monoclonal antibody-virus reaction

[0113] The monoclonal antibody TCH065 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 the C strain of hog cholera rabbit attenuated vaccine (HCLV). The steps are as follows:

[0114] ① Inoculation of cells: Press 100 TCID 50 CSFV cytotoxicity was inoculated into each well, and PK-15 cells were added into 96-well plates and cultured in a 37°C, 5% CO2 incubator for 72 h.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] The results are as follows Figure 1 The results showed that monoclonal antibody TCH065 reacted with all strains except the vaccine strain HCLV and exhibited obvious fluorescence.

[0120] The E2 proteins of different genotypes of CSFV were expressed by an insect baculovirus expression system, including the E2 proteins of the following 18 CSFV strains: LPC, 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). The preparation method of the E2 proteins of the above vaccine strains and strains refers to the preparation method of the CSFV-JL23 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:

[0121] The nucleotide sequence of the gene encoding the LPC E2 protein of the classical swine fever virus (CSFV) vaccine strain: GenBank Accession No. AY526732 (Update Date 26-JUL-2016) positions 1171-2259.

[0122] The nucleotide sequence of the gene encoding the E2 protein of classical swine fever rabbit attenuated vaccine C strain (HCLV): positions 2345-3433 of GenBank Accession No. Z46258.1 (Update Date 25-NOV-2005).

[0123] The nucleotide sequence of the gene encoding the SM E2 protein: GenBank Accession No. AY775178.2 (Update Date 27-NOV-2007), positions 2345-3433.

[0124] 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).

[0125] 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).

[0126] 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.

[0127] The nucleotide sequence of the gene encoding CN021 (2.1a) E2 protein is shown in SEQ ID No.7.

[0128] The nucleotide sequence of the gene encoding CN069 (2.1b) E2 protein is shown in SEQ ID No.8.

[0129] 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).

[0130] 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).

[0131] The nucleotide sequence of the gene encoding CN057 (2.1h) E2 protein is shown in SEQ ID No.9.

[0132] Nucleotide sequence of the gene encoding CN063 (2.1i) E2 protein: GenBank Accession No.KY132096.1 (Update Date 01-FEB-2017), positions 2345-3433.

[0133] The nucleotide sequence of the gene encoding CN068 (2.1j) E2 protein is shown in SEQ ID No.10.

[0134] The nucleotide sequence of the gene encoding CN002 (2.2) E2 protein is shown in SEQ ID No.11.

[0135] The nucleotide sequence of the gene encoding HuB2 (2.3) E2 protein is shown in SEQ ID No.12.

[0136] 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).

[0137] 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).

[0138] 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).

[0139] Western blot experiments were performed with monoclonal antibody TCH065 and different genotypes of CSFV E2 proteins expressed by insect baculovirus expression system, and protein tag antibody anti-His Tag mAb was used as a control. The steps are as follows:

[0140] ① Protein treatment: Add E2 protein into 4× loading buffer according to the proportion and boil for 10 min.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] The results are as follows Figure 2 As shown, monoclonal antibody TCH065 reacts with E2 proteins of all strains except vaccine strains HCLV and LPC.

[0148] (6) Antibody sequence determination

[0149] The hybridoma cell TCH065 was sent to Nanjing Detai Biotechnology Co., Ltd. for sequencing, and the sequence was as follows:

[0150] The amino acid sequence of the heavy chain variable region of monoclonal antibody TCH065 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 TCH065 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:

[0151] The amino acid sequence of the heavy chain variable region CDR1 of the monoclonal antibody TCH065 is shown at positions 26-33 of SEQ ID No. 1;

[0152] The amino acid sequence of the heavy chain variable region CDR2 of the monoclonal antibody TCH065 is shown in positions 51-58 of SEQ ID No. 1;

[0153] The amino acid sequence of the heavy chain variable region CDR3 of the monoclonal antibody TCH065 is shown at positions 97-111 of SEQ ID No. 1;

[0154] The amino acid sequence of the light chain variable region CDR1 of the monoclonal antibody TCH065 is shown at positions 27-32 of SEQ ID No. 3;

[0155] The amino acid sequence of the light chain variable region CDR2 of the monoclonal antibody TCH065 is shown at positions 50-52 of SEQ ID No. 3;

[0156] The amino acid sequence of the light chain variable region CDR3 of monoclonal antibody TCH065 is shown at positions 89-97 of SEQ ID No.3.

[0157] (7) Preparation of antibodies using genetic engineering methods

[0158] 1) Construction of recombinant expression plasmid

[0159] In order to express the monoclonal antibody TCH065, 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 TCH065 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. 13). The nucleotide sequence of the light chain gene of the monoclonal antibody TCH065 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. 14).

[0160] The heavy chain gene and light chain gene of monoclonal antibody TCH065 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.

[0161] 2) Antibody expression:

[0162] a) Inoculate freshly digested 293T cells onto 175 cm 2 In 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%.

[0163] 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.

[0164] c) combining the two solutions in step b) above and mixing them to obtain a composite.

[0165] d) Add the above complex directly into the cell culture medium in step a) and mix well by pipetting with a pipette.

[0166] e) Move the cell plate to a 37ºC / 5% CO2 incubator for culture. After 3 days of culture, collect the culture supernatant for verification.

[0167] 3) Verification of antibody expression:

[0168] An IFA experiment was used to verify whether the antibody was expressed. The expression supernatant was used as the primary antibody, and the CSFV broad-spectrum antibody WH303 was used as the control. Indirect immunofluorescence assay (IFA) was performed with the CSFV epidemic strain CN069 and the swine fever rabbit attenuated vaccine C strain (HCLV). The specific steps are the same as those in Example 1, step (5) identification of the type of reaction between the monoclonal antibody and the virus.

[0169] The results are as follows Figure 3 As shown, the expression supernatant reacted with cells infected with the epidemic strain CN069 and the control antibody to show obvious green fluorescence, indicating that the infection was successful. The expression supernatant reacted with cells infected with the epidemic strain CN069 to show obvious green fluorescence, and did not react with the vaccine strain HCLV, indicating that the antibody was successfully expressed.

[0170] 4) Antibody purification:

[0171] a) Buffer preparation: Add Na2HPO4·12H2O to sterile ddH2O to a final concentration of 0.2 M and vortex thoroughly to mix.

[0172] b) Preparation of pre-elution buffer: Add 0.1 M citric acid to the buffer to make the volume percentage of citric acid 20%.

[0173] c) Preparation of eluent: Add 0.1 M citric acid to the buffer solution so that the volume proportion of citric acid is 60%.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 5) Verification of antibody expression:

[0181] 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 4 As shown in Figure 1, lane 1 has a distinct single band at greater than 180 kDa. After the antibody is treated with reducing buffer, as shown in lane 2, there are two distinct bands of light chain and heavy chain at approximately 25 kDa and 50 kDa, indicating that the antibody is well purified.

[0182] Example 2: Preparation of conjugated antibodies

[0183] HRP was conjugated to monoclonal antibody TCH065 using an HRP conjugation kit (Sangong, D601047) to prepare HRP-TCH065 conjugated antibody. The specific steps are as follows:

[0184] (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.

[0185] (2) Add 200 μL HRP coupling buffer and let stand at room temperature for 30 min.

[0186] (3) Add 1 mL of purified monoclonal antibody TCH065 and transfer to a dialysis bag. Dialyze in 2 L of dialysis solution at room temperature for 2 h.

[0187] (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.

[0188] Example 3: Application of monoclonal antibody TCH065 in distinguishing between lapinized attenuated swine fever vaccine strains and epidemic strains

[0189] This example exemplifies an ELISA method using monoclonal antibody TCH065 to identify and distinguish between classical swine fever lapinized attenuated vaccine strains and epidemic strains. The specific operation method is as follows:

[0190] (1) The coating concentration in the ELISA plate is 0.1 μg / mL CSFV epidemic strain E2 protein (i.e., CSFV-JL23 E2 protein in Example 1). After coating, the coating solution is discarded and the plate is vacuum-sealed for storage.

[0191] (2) Add 50 μL of sample diluent to each well of the coated plate, and then add 50 μL of classical swine fever virus-infected sera (numbered 234, 260, 262, 264, 272, 232, 265, 268, 281, 215, 233, 238, 244, 253, 275, 288, 468, 473 and 475), sera obtained from immunization with classical swine fever vaccine strain C-strain (numbered 2305), classical swine fever antibody negative control (classic swine fever antibody negative serum, numbered 13), and classical swine fever antibody positive control (serum obtained from infection with classical swine fever epidemic strain GD23, numbered 285) to the corresponding wells. Set up duplicate wells for negative and positive controls and incubate at 37°C for 1 h. The above serum and preparation method are recorded in the following literature: Gong W, Li J, Wang Z, et al. Commercial E2 subunit vaccine provides full protection to pigs against lethal challenge with 4 strains of classical swine fever virus genotype 2. Vet Microbiol. 2019;237:108403.

[0192] (3) Discard the liquid in each well and wash the wells with 300 μL of washing solution for a total of 5 times. After the last wash, pat the washing solution in the wells dry.

[0193] (4) Add 100 μL of 1:5000 diluted HRP-TCH065 conjugated antibody to each well and incubate at 37°C for 1 h.

[0194] (5) Repeat step (3).

[0195] (6) Add 50 μL of TMB colorimetric solution to each well and incubate at room temperature in the dark for 10 min.

[0196] (7) Add 50 μL of stop solution to each well to terminate the reaction and measure the OD with an ELISA reader. 450 The blocking rate was calculated using the formula (negative control average OD 450 - Sample OD 450 ) / mean OD of negative control 450 ×100%.

[0197] The results are shown in Table 1. For the serum infected with the epidemic strain, the blocking rate was above 70%, and for the vaccine immune serum, the blocking rate was 3.25%, indicating that the blocking ELISA method established based on monoclonal antibody TCH065 can clearly distinguish between swine fever vaccine strain immunity and epidemic strain infection, and monoclonal antibody TCH065 can be used for the research and development and preparation of swine fever blocking kits.

[0198]

[0199] 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. A monoclonal antibody against the E2 protein of classical swine fever virus or an antigen-binding fragment thereof, characterized in that: The monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementary determining regions whose amino acid sequences are respectively positions 26-33 of SEQ ID No.1, positions 51-58 of SEQ ID No.1 and positions 97-111 of SEQ ID No.1; and the light chain variable region comprises three complementary determining regions whose amino acid sequences are respectively positions 27-32 of SEQ ID No.3, positions 50-52 of SEQ ID No.3 and positions 89-97 of SEQ ID No.

3.

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) Application in the preparation of products for identifying and distinguishing lapinized attenuated swine fever vaccine strains from epidemic strains; C2) Use in the preparation of products for diagnosing or assisting in the diagnosis of classical swine fever virus infection or diseases caused by classical swine fever virus infection; C3) Use in the preparation of products for screening or assisting in screening for classical swine fever virus infection or diseases caused by classical swine fever virus infection; C4) Use in the preparation of products for detecting the epidemic strain of classical swine fever virus or the E2 protein of the epidemic strain of classical swine fever virus.

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

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

8. 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.

9. A method for non-disease diagnosis and treatment of classical swine fever lapinized attenuated vaccine strains and epidemic strains, characterized in that: The method comprises using the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, the antibody conjugate according to claim 6 or the kit according to claim 7 to identify and differentiate between the lapinized attenuated vaccine strain of classical swine fever and the epidemic strain.

10. A method for non-disease diagnosis and treatment for detecting a swine fever virus epidemic strain or an E2 protein of a swine fever virus epidemic strain, characterized in that: The method comprises using the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, the antibody conjugate according to claim 6 or the kit according to claim 7 to detect the epidemic strain of classical swine fever virus or the E2 protein of the epidemic strain of classical swine fever virus.

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