Broad-spectrum monoclonal antibodies against porcine epidemic diarrhea virus and their applications

Abstract

This invention relates to a broad-spectrum monoclonal antibody against porcine epidemic diarrhea virus (PEDV) and its applications in the field of medical preparations. The antibody developed in this invention is a monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are as shown in positions 26-33, 51-57, and 96-103 of SEQ ID No:1, respectively; HCDR1, HCDR2, and HCDR3 are complementarity-determining regions; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody are as shown in positions 27-37, 55-57, and 94-102 of SEQ ID No:2, respectively; LCDR1, LCDR2, and LCDR3 are complementarity-determining regions.

Description

Technical Field

[0001] This invention relates to a broad-spectrum monoclonal antibody against porcine epidemic diarrhea virus and its application in the field of medical preparations. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a highly contagious enterovirus that causes infection in pigs, with vomiting, diarrhea, and dehydration as the main clinical symptoms. Pigs of all ages are susceptible, but young piglets are most severely affected. Infected piglets often die from severe dehydration caused by diarrhea, with a mortality rate that can reach 100%.

[0003] PEDV, a member of the genus Alphacoronavirus in the family Coronaviridae, is an enveloped, single-stranded, positive-sense RNA virus. Its genome consists of non-coding regions at both ends and seven open reading frames, approximately 28 kb in size. The viral genome encodes four structural proteins: the spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N), as well as non-structural proteins pp1a, pp1b, and the accessory protein ORF3. The S protein is the main antigenic protein of PEDV, capable of stimulating the body to produce neutralizing antibodies. Based on its function, this protein can be divided into two regions, S1 and S2, with S1 primarily involved in viral binding to cell receptors. This region contains multiple antigenic epitopes and is a major target protein for PEDV detection and vaccine development.

[0004] PEDV was first discovered in the 1870s. At that time, PEDV outbreaks were mostly sporadic infections with low morbidity and mortality rates, and did not attract widespread attention. It wasn't until 2010 that a highly pathogenic mutant strain was first reported in my country. This mutant strain was characterized by rapid transmission and a high mortality rate. Subsequently, the United States, Germany, Mexico, and other countries reported the spread of the mutant strain. Genetic evolutionary analysis showed that PEDV strains can be divided into two genotypes: GI and GII. The GI genotype mainly includes classic strains and cellular attenuated strains that circulated before 2010, while the GII genotype mainly includes the currently prevalent mutant strains worldwide. Whole-genome analysis of the virus showed that the main difference between different subtypes lies in the S gene.

[0005] The continuous mutation of PEDV strains poses a significant challenge to the detection and vaccination of PEDV. To address the rapid evolution of the virus, developing broad-spectrum PEDV recognition antibodies is of great importance and has potential application value for the clinical diagnosis and prevention of porcine epidemic viral diarrhea. Summary of the Invention

[0006] The main problem to be solved by this invention is how to obtain antibodies that can broadly recognize porcine epidemic diarrhea virus.

[0007] To address the above problems, this invention provides a monoclonal antibody with broad-spectrum PEDV recognition.

[0008] The antibody provided by this invention is a monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are as shown in positions 26-33, 51-57 and 96-103 of SEQ ID No:1, respectively; HCDR1, HCDR2 and HCDR3 are complementarity-determining regions; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody are as shown in positions 27-37, 55-57 and 94-102 of SEQ ID No:2, respectively; LCDR1, LCDR2 and LCDR3 are complementarity-determining regions.

[0009] The term "antibody" as used in this invention may include polyclonal and monoclonal antibodies, including intact antibodies and their functional (antigen-binding) antibody fragments. The term covers genetically engineered and / or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, and multispecific antibodies, such as bispecific antibodies, tri- and tetra-antibodies, tandem biscFvs, and tandem triscFvs. Unless otherwise stated, the term "antibody" should be understood to encompass its functional antibody fragment. The term "antibody" is not limited to any particular method of antibody production. The term also covers intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subtypes (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgM, IgE, IgA, and IgD.

[0010] The term "monoclonal antibody," or simply "antibody," generally refers to an immunoglobulin molecule, typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). In general terms, the heavy chain can be understood as the larger polypeptide chain in the antibody, while the light chain refers to the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The constant region of the light chain consists of a single CL domain. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. In particular, the heavy chain can also contain more than three CDRs, such as 6, 9, or 12. For example, in bifunctional antibodies, the heavy chain can be the C-terminus of the heavy chain of an IgG antibody linked to the ScFv of another antibody; in this case, the heavy chain contains 9 CDRs.

[0011] In this invention, the light chain variable region (VL) or heavy chain variable region (VH) of the antibody is composed of a "framework" region separated by three "complementarity-determining regions" or "CDRs". The framework regions are used to align the CDRs that specifically bind to the antigenic epitopes. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; and the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0012] The aforementioned CDR is a sequence defined according to the IMGT numbering system. Within the scope of protection of this invention, the antibody may be in various forms such as a full-length antibody, a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment.

[0013] In this invention, the term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. This antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target.

[0014] The term "Fab fragment" refers to a heterodimer composed of a heavy chain (Fd) and a complete light chain linked by disulfide bonds, containing only one antigen-binding site. The aforementioned heavy chain (Fd) refers to approximately half of the H chain portion of the Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0015] The term "Fv fragment" refers to a vector containing VH and VL genes that can be constructed separately, co-transfected into cells to express them separately, and then assembled into a functional Fv antibody; alternatively, a stop codon can be set between VH and VL in the vector to express two small protein fragments, which can then be bound together by non-covalent bonds to form an Fv antibody (Fv fragment).

[0016] The term "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab' fragments to form the F(ab')2 molecule.

[0017] The term "F(ab')2 segment" contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

[0018] The term "single-chain antibody (ScFv)" refers to a polypeptide formed by linking a light chain variable region and a heavy chain variable region. This polypeptide can spontaneously fold into its native conformation, maintaining the specificity and affinity of Fv.

[0019] The monoclonal antibody is TEH346.

[0020] Further, the amino acid sequence of the light chain variable region in the antibody is SEQ ID No:2, or has at least 90% identity with SEQ ID No:2; and / or the amino acid sequence of the heavy chain variable region is SEQ ID No:1, or has at least 90% identity with SEQ ID No:1.

[0021] Furthermore, the heavy chain type of the antibody is IgG; and / or the light chain type of the antibody is kappa.

[0022] The present invention also provides the following antibody, obtained by immunizing animals with the S1 protein of porcine epidemic diarrhea virus as an immunogen, wherein the amino acid sequence of the S1 protein is SEQ ID No:6.

[0023] This invention also provides any of the following biomaterials:

[0024] (A1) The active fragment of the antibody is derived from any of the antibodies described above or any of the following: single-chain antibody, antibody Fab region and antigen-binding fragment;

[0025] (A2) A nucleic acid molecule that encodes the antibody described above or the active fragment of the antibody described above or (A1);

[0026] (A3) An expression cassette, recombinant vector, recombinant cell or recombinant bacterium containing the nucleic acid molecule described in (A2);

[0027] (A4) A kit containing the antibody described above, or the active fragment of the antibody described in (A1), or the nucleic acid molecule described in (A2), or the expression cassette, recombinant vector, recombinant cell, or recombinant bacterium described in (A3).

[0028] In the above-mentioned biological material, in the nucleic acid molecule described in (A2), the nucleotide sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region encoding the antibody are shown as positions 76-99, 151-171 and 286-309 of SEQ ID No:3, respectively;

[0029] and / or

[0030] In the nucleic acid molecule described in (A2), the nucleotide sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region encoding the antibody are shown in positions 79-111, 163-171 and 280-306 of SEQ ID No:4, respectively.

[0031] In the aforementioned biological materials, the expression cassette described in (A3) refers to DNA capable of expressing the antibody or the antigen-binding fragment in a host cell. The expression cassette may also include all regulatory sequences necessary to initiate the expression of a nucleic acid molecule encoding the antibody or the antigen-binding fragment. These regulatory sequences, under compatible conditions, guide the coding sequence to express the antibody or the antigen-binding fragment in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a adapter-equipped regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively linked to the 3' end of the nucleic acid sequence encoding the antibody or the antigen-binding fragment. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleic acid sequence encoding the protein (i.e., the antibody or the antigen-binding fragment). Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of the protein (i.e., the antibody or the antigen-binding fragment) that guides the protein (i.e., the antibody or the antigen-binding fragment) into the cellular secretory pathway. Signal peptide coding regions that can guide the expressed protein (i.e., the antibody or the antigen-binding fragment) into the secretory pathway of the host cell used can be used in this invention. Adding a regulatory sequence that can regulate protein (i.e., the antibody or the antigen-binding fragment) expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that enable gene amplification. In these examples, the nucleic acid sequence encoding the protein (i.e., the antibody or the antigen-binding fragment) should be operatively linked to the regulatory sequence.

[0032] In the aforementioned biological materials, the recombinant vector described in (A3) can be a cloning vector or an expression vector. As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site.

[0033] In preparing an expression vector, a nucleic acid molecule encoding a protein (i.e., the antibody or the antigen-binding fragment) is placed within the vector so that it can be operatively linked to an appropriate expression regulatory sequence. The recombinant expression vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of vector typically depends on its compatibility with the host cell into which it will be introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector (i.e., a complete structure existing outside the chromosome that can replicate independently of the chromosome), such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, integrates into the chromosome and replicates along with the integrated chromosome. The vector contains one or more selection markers that facilitate the selection of transformed cells. A selection marker is a gene whose product confers resistance to viruses, resistance to heavy metals, or confers a protrophic type for auxotrophs, etc. Examples of bacterial selection markers include resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. The vector contains elements that enable stable integration of the vector into the host cell genome or ensure autonomous replication of the vector independently of the cell genome. In the case of autonomous replication, the vector may also contain an origin of replication, enabling autonomous replication within the target host cell. The origin of replication may carry a mutation that makes it temperature-sensitive in the host cell (see, for example, f. Ehrlich, 1978, Proceedings of the National Academy of Sciences 75:1433). The yield of the gene product can be increased by inserting one or more copies of a nucleic acid molecule encoding the aforementioned protein (i.e., the antibody or the antigen-binding fragment) into the host cell. This copy number increase can be achieved by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or by inserting an amplifiable selection marker along with the nucleic acid molecule, and by culturing cells in the presence of a suitable selection reagent to select cells containing the amplified copy of the selection marker gene, thereby containing the additional copy of the nucleic acid molecule. The operations used to connect the above-mentioned elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0034] In the aforementioned biological materials, the recombinant cells described in (A3) can be animal cells, and the animal cell line can be non-reproductive material. The animal cells can be isolated mammalian cells. The mammals include humans or mice. The mammalian cells may not include animal germ cells, animal fertilized eggs, and animal embryonic stem cells, and may be somatic cells or cell lines. The animal cells may be cell lines or somatic cells derived from mice.

[0035] Furthermore, the cells may be 293T cells.

[0036] The present invention also provides a method for preparing the antibody described above, comprising the following steps: introducing a nucleic acid molecule encoding the antibody described above into a recipient cell to obtain a transgenic cell expressing the antibody, culturing the transgenic cell, and obtaining the antibody.

[0037] The present invention also provides the use of the antibodies or biomaterials described above in any of the following:

[0038] (B1) Identification or assistance in the identification of porcine epidemic diarrhea virus;

[0039] (B2) Binds to or assists in binding to porcine epidemic diarrhea virus;

[0040] (B3) Detection or auxiliary detection of whether the sample to be tested contains porcine epidemic diarrhea virus;

[0041] (B4) Prepare products for detecting or assisting in the detection of whether a sample contains porcine epidemic diarrhea virus;

[0042] (B5) Detection or auxiliary detection of porcine epidemic diarrhea virus content in the sample to be tested;

[0043] (B6) Prepare products for detecting or assisting in the detection of porcine epidemic diarrhea virus content in the sample to be tested.

[0044] The present invention also provides a pharmaceutical composition wherein the active ingredient of the pharmaceutical composition is the antibody described above.

[0045] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients, diluents, or carriers.

[0046] In the pharmaceutical composition described above, the term "pharmaceuticalally acceptable excipient" refers to an additive substance in the pharmaceutical formulation other than the active ingredient, used to improve the physical properties, stability, or bioavailability of the drug. Excipients can be categorized into several types based on their function. Disintegrants promote rapid disintegration of the drug in vivo, releasing the active ingredient; examples include starch and sodium carboxymethyl starch. Binders are used to bind drug powders into granules or tablets; common examples include povidone and hydroxypropyl methylcellulose. Lubricants reduce friction between drug particles and between particles and equipment surfaces, ensuring smooth drug formation and release; magnesium stearate and talc are commonly used lubricants. These excipients must meet conditions such as being non-toxic, non-irritating, and not chemically reacting with the active ingredient to ensure the safety and efficacy of the drug.

[0047] In the pharmaceutical composition described above, the term "diluent" is primarily used to adjust the drug concentration to achieve the appropriate dosage. When preparing solid dosage forms (such as tablets and capsules), diluents are added to increase volume when the content of the active ingredient is low. Lactose, sucrose, and mannitol are commonly used diluents; they are chemically stable, do not affect the activity of antibody or antigen-binding fragments, and have good flowability and compressibility, facilitating formulation. In injectable preparations, diluents are used to dissolve or dilute the active ingredient, such as water, physiological saline, and glucose solution, ensuring that the drug concentration meets clinical requirements while maintaining osmotic pressure balance to avoid damage to porcine tissues.

[0048] In the pharmaceutical composition described above, the term "carrier" not only functions as an excipient and diluent, but also plays a special role in drug delivery, especially in targeted drug delivery systems. Liposomes, a common carrier composed of a phospholipid bilayer, can encapsulate antibody or antigen-binding fragments. This not only protects the active ingredient from degradation by enzymes and other substances in the body, but also allows for surface modification to make them more targeted, such as by attaching targeting ligands, making them easier for diseased cells to take up. Nanoparticle carriers (such as polymer nanoparticles and solid lipid nanoparticles) also have similar functions; their nanoscale size facilitates penetration of biological barriers, enabling efficient drug delivery.

[0049] The present invention also provides an ELISA detection kit targeting porcine epidemic diarrhea virus, the kit comprising the antibodies or the biological materials described above.

[0050] In this invention, the kit includes an immunohistochemistry kit, an immunofluorescence kit, a flow cytometry detection kit, and an immunoblotting kit.

[0051] This invention also provides a method for preparing the antibody described above, comprising the following steps:

[0052] 1) Immunize animals with porcine epidemic diarrhea virus S1 protein as an immunogen;

[0053] 2) Isolate spleen lymphocytes from immunized animals;

[0054] 3) Screening of antibody-secreting cells in the spleen lymphocytes described in step 2) yields single porcine epidemic diarrhea virus-specific antibody-secreting cells;

[0055] 4) Sequencing and expression purification were performed to obtain a monoclonal antibody against porcine epidemic diarrhea virus.

[0056] This invention screened and identified a hybridoma cell line targeting the S1 protein of porcine epidemic diarrhea virus (PEDV) using hybridoma technology. The monoclonal antibody (TEH346) secreted by this cell line has broad-spectrum binding activity and can effectively recognize a variety of circulating PEDV strains, including the classic strain (PEDV-CV777) and variant strains. It can be used as a raw material for the establishment of various detection methods, including but not limited to enzyme-linked immunosorbent assay (ELISA), immunochromatographic test strips (colloidal gold / fluorescence), immunohistochemistry (IHC), and Western blot, providing key materials for the development of a new generation of universal PEDV detection reagents. Attached Figure Description

[0057] Figure 1 The results are from indirect immunofluorescence experiments of monoclonal antibody TEH346 and different genotype strains of PEDV.

[0058] Figure 2 The results of Western blot experiments on the expression of S1 protein baculovirus in 19 different genotypes of PEDV were presented.

[0059] Figure 3 The results of Western blot experiments on the S1 protein of monoclonal antibody TEH346 and 19 PEDV strains with different genotypes are presented.

[0060] Figure 4 The results were used to verify the expression activity of monoclonal antibody TEH346 via indirect immunofluorescence.

[0061] Figure 5 The results validate the purification effect of monoclonal antibody TEH346. Lane 1 is for samples treated with non-reducing buffer, and lane 2 is for samples treated with reducing buffer. Detailed Implementation

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0064] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0065] The insect baculovirus expression vector pFastBac 1 used in the following examples has been described in: Mi Shijiang. Identification of monoclonal antibodies and broad-spectrum monoclonal antibodies for differentiating between prevalent and vaccine strains of classical swine fever virus and their antigenic epitope analysis [D]. Jilin University, 2022. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0066] The Sf9 cells used in the following examples are described in: Mishijiang. Identification of Monoclonal Antibodies and Broad-Spectrum Monoclonal Antibodies for Differentiating Between Epidemic and Vaccine Strains of Classical Swine Fever Virus and Their Antigenic Epitope Analysis [D]. Jilin University, 2022. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0067] The SP2 / 0 cells used in the following examples are described in: Mishijiang. Identification of Monoclonal Antibodies and Broad-Spectrum Monoclonal Antibodies for Differentiating Between Epidemic and Vaccine Strains of Classical Swine Fever Virus and Their Antigenic Epitope Analysis [D]. Jilin University, 2022. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0068] The information about the vaccine virus involved in this embodiment is as follows:

[0069] PEDV-AJ1102: Wuhan Keqian Biotechnology Co., Ltd., Approval Number: Veterinary Drug Production Permit No. 170041133.

[0070] PEDV-SCSZ-1: Chongqing Aolong Biological Products Co., Ltd., Approval Number: Veterinary Drug Production Permit No. 230241134.

[0071] PEDV-LW / L: Luoyang Huizhong Biotechnology Co., Ltd., Approval Number: Veterinary Drug Production Permit No. 163001140.

[0072] PEDV-ZJ08: Ruipu (Baoding) Biopharmaceutical Co., Ltd., Approval Number: Veterinary Drug Production Permit No. 030381109.

[0073] PEDV-CV777: Harbin Weike Biotechnology Co., Ltd., Approval Number: Veterinary Drug Production Permit No. 080011097.

[0074] Example 1: Preparation and identification of monoclonal antibodies against porcine epidemic diarrhea virus S1 protein

[0075] 1. Preparation of immunoantigens

[0076] The immunogenic antigen used in the experiment was the S1 protein of the prevalent porcine epidemic diarrhea virus (PEDV) strain CH / GDZQ / 2014 (PEDV-CH / GDZQ / 2014). The gene encoding the S1 protein of PEDV-CH / GDZQ / 2014 (sequence 20634-23009 of the sequence shown in GenBank accession number: KM242131, with a His tag added to the 3' end of the sequence, the His tag nucleotide sequence being 5'-CATCATCACCATCACCAT-3', SEQ ID No:7) was cloned into the insect baculovirus expression vector pFastBac1 to obtain the recombinant expression vector pFastBac1-S1.

[0077] The structure of the pFastBac 1-S1 carrier is described as follows: It is based on the starting carrier pFastBac 1... BamH I and EcoR The recombinant vector is obtained by inserting a DNA fragment with the sequence SEQ ID No:5 between the two restriction enzyme sites, while keeping the other sequences of the vector pFastBac 1 unchanged. The pFastBac 1-S1 vector can express the PEDV-CH / GDZQ / 2014 S1 protein, whose amino acid sequence is SEQ ID No:6.

[0078] The recombinant expression vector pFastBac 1-S1 was transformed into DH10Bac competent cells to obtain recombinant rod particles containing the target gene fragment. The rod particles were transfected into Sf9 cells for protein expression. The collected PEDV-CH / GDZQ / 2014 S1 protein was purified using fixed metal ion affinity chromatography. The purified protein can be used as an immunogenic antigen.

[0079] 2. Mouse immunization

[0080] 50 μg of purified PEDV-CH / GDZQ / 2014 S1 protein was mixed with an equal volume of 206 adjuvant (purchased from Seppic, catalog number ISA206VG) and emulsified. SPF-grade 6-8 week old female BALB / c mice (purchased from Jilin Jintai Meidi Biotechnology Co., Ltd., catalog number 1010003) were immunized. Mice were selected for subcutaneous injection at multiple sites, immunized every two weeks for a total of 3 immunizations. One week after the third immunization, 100 μg of unadjuvanted purified PEDV-CH / GDZQ / 2014 S1 protein was injected intraperitoneally for shock immunization. The mice were then fed for another 3 days.

[0081] 3. Cell fusion

[0082] Three days after the initial immunization, mice were euthanized by cervical dislocation and immersed in 75% alcohol for 5 minutes. The spleen was aseptically removed and placed in a 200-mesh nylon sieve. The cells were gently ground using a syringe plunger, and the sieve was rinsed with serum-free Advanced RPMI-1640 (Thermo Fisher Scientific, catalog number 12633012). The spleen cell suspension was collected in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes, discarding the supernatant. This washing process was repeated once, and the spleen cells were counted. SP2 / 0 cells in the logarithmic growth phase were mixed with spleen cells at a ratio of 1:5 to 1:10. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded. The bottom of the centrifuge tube was gently tapped to loosen the cell pellet. Within 1 minute, 1 mL of preheated PEG 1500 (Sigma-Aldrich, catalog number 25322-68-3) at 37°C was added dropwise while gently rotating the centrifuge tube. Over the next 10 minutes, add 10 mL of preheated (37°C) Advanced RPMI-1640 medium dropwise in the same manner. After this, centrifuge the cells at 800 rpm for 5 minutes, discard the supernatant, resuspend the cell pellet in 100 mL of HY semi-solid medium (STEMCELL Technologies, catalog number 03804), and evenly spread it into six-well plates at 2.5-3 mL / well. Incubate the plates at 37°C with 5% CO2 for 7-10 days.

[0083] 4. Hybridoma cell screening

[0084] After 7-10 days, monoclonal cell clusters from the semi-solid culture medium in step 2 were observed under a microscope and transferred to Advanced RPMI-1640 medium containing 1% HAT (Thermo Fisher Scientific, catalog number 21060017), 10% FBS (Biological Industries, catalog number 04-001-1ACS), and 1% penicillin antibody (Cytiva HyClone, catalog number SV30010). After 3 days of culture, antibody-positive cell wells were screened using an indirect immunofluorescence assay. After subcloning, a hybridoma cell line that secreted a monoclonal antibody specifically binding to the S1 protein was identified and named TEH346. The monoclonal antibody secreted by the hybridoma cell line TEH346 is called monoclonal antibody TEH346.

[0085] 5. Identification of monoclonal antibody types

[0086] The Ig class and light chain type of the monoclonal antibody were identified using an indirect ELISA method. The secondary antibodies used for different antibody types were all from the Enzyme-Label Secondary Antibody Kit for Ig Class Identification of Mouse Monoclonal Antibody (purchased from Suzhou Biotron Immunotherapy Co., Ltd., catalog number BF16002X). The results showed that the monoclonal antibody type of TEH346 was IgG1, κ light chain.

[0087] 6. Identification of Monoclonal Antibody-Viral Response Types - IFA

[0088] The monoclonal antibody TEH346 was subjected to indirect immunofluorescence assay (IFA) with vaccine strains PEDV-AJ1102, PEDV-SCSZ-1, PEDV-LW / L, PEDV-ZJ08, and PEDV-CV777, as follows:

[0089] a) Cell plating: Vero cells were passaged at a certain ratio and seeded into 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator.

[0090] b) Cell inoculation: Dilute the virus with DMEM medium at 100 TCID50. 50 PEDV virus was inoculated into each well. The virus dilution was added to the 96-well plate and incubated at 37°C in a 5% CO2 incubator for 72 h.

[0091] c) Cell fixation: Discard the cell culture supernatant, add 200 μL PBS to each well of a 96-well plate and wash 3 times, add 50 μL of 80% cold acetone stored at -20℃, and fix in a -20℃ freezer for 1 h.

[0092] d) Primary antibody incubation: Discard the cold acetone fixative, add 200 μL of PBS to each well and wash 3 times, add 100 μL of hybridoma cell culture supernatant to each well, and incubate at 37°C for 1 h.

[0093] e) Secondary antibody incubation: Discard the primary antibody incubation solution, add 200 μL of PBS to each well and wash 3 times. Dilute Alexa Fluor 488 fluorescent secondary antibody (purchased from Thermo Fisher Scientific, catalog number A-21202) 1:500 with PBS, and add 0.01% Evans Blue (purchased from Beijing Solarbio Biotechnology Co., Ltd., catalog number G1810). Mix thoroughly and add 100 μL to each well of the cell plate. Incubate at 37°C for 1 h.

[0094] f) Fluorescence observation: Discard the secondary antibody incubation solution, add 200 μL of PBS to each well and wash 3 times, then observe the reaction between the antibody and infected cells under a fluorescence microscope.

[0095] The results are as follows Figure 1 As shown, the monoclonal antibody TEH346 exhibited a specific reaction with all strains and showed significant fluorescence.

[0096] 7. Identification of the type of reaction between monoclonal antibodies and viruses - Western blot

[0097] The S1 protein of different PEDV genotypes was expressed using an insect baculovirus expression system, including the S1 proteins of the following 19 PEDV strains: PEDV-SM98, PEDV-LZC, PEDV-CV777, PEDV-HLJDQ5, PEDV-AHHY6, PEDV-HLJJW1, PEDV-SCPM47, PEDV-AHLY1, PEDV-AHWH8, PEDV-FJZZ2, PEDV-GSYT1, PEDV-XJJY6, PEDV-YNQW1, PEDV-SD, PEDV-GDZQ, PEDV-AJ1102, PEDV-NMBB1, PEDV-HuNYF3, and PEDV-GDYL13. The preparation method of the S1 protein of the above strains is the same as the preparation method of the PEDV-CH / GDZQ / 2014 S1 protein in Example 1. The encoding genes for each S1 protein cloned into the insect baculovirus expression vector pFastBac1 are as follows:

[0098] The nucleotide sequence of the gene encoding the PEDV-HLJDQ5 S1 protein: GenBank Accession No. OR085241, positions 20620-22983, updated on August 20, 2025.

[0099] The nucleotide sequence of the gene encoding the PEDV-NMBB1 S1 protein: GenBank Accession No. OR085300, positions 20709-23084, updated on August 20, 2025.

[0100] The nucleotide sequence of the gene encoding the PEDV-SCPM47 S1 protein: GenBank Accession No. OR085252, positions 20634-23009, updated on August 20, 2025.

[0101] The nucleotide sequence of the gene encoding the PEDV-LZC S1 protein: GenBank Accession No. EF185992, positions 20638-23004, updated on January 16, 2007.

[0102] The nucleotide sequence of the gene encoding the PEDV-XJJY6 S1 protein: GenBank Accession No. OR085287, positions 20629-23004, updated on August 20, 2025.

[0103] The nucleotide sequence of the gene encoding the PEDV-AHWH8 S1 protein: GenBank Accession No. OR085281, positions 20758-23133, updated on August 20, 2025.

[0104] The nucleotide sequence of the gene encoding the PEDV-GSYT1 S1 protein: GenBank Accession No. PV536086, positions 20622-22997, updated on August 20, 2025.

[0105] The nucleotide sequence of the gene encoding the PEDV-SD S1 protein: GenBank Accession No. PP958821, positions 20622-22997, updated on July 2, 2025.

[0106] The nucleotide sequence of the gene encoding the PEDV-AHLY1 S1 protein: GenBank Accession No. OR085262, positions 20653-23028, updated on August 20, 2025.

[0107] The nucleotide sequence of the gene encoding the PEDV-YNQW1 S1 protein: GenBank Accession No. PV536117, positions 20646-23021, updated on August 20, 2025.

[0108] The nucleotide sequence of the gene encoding the PEDV-FJZZ2 S1 protein: GenBank Accession No. OR085266, positions 20616-22991, updated on August 20, 2025.

[0109] The nucleotide sequence of the gene encoding the PEDV-CH / GDZQ S1 protein: GenBank Accession No. KM242131, positions 20634-23009, updated on February 3, 2015.

[0110] The nucleotide sequence of the gene encoding the PEDV-HLJJW1 S1 protein: GenBank Accession No. PV536091, positions 20649-23015, updated on August 20, 2025.

[0111] The nucleotide sequence of the gene encoding the PEDV-CV777 S1 protein: GenBank Accession No. LT905450, positions 20633-22999, updated on June 15, 2018.

[0112] The nucleotide sequence of the gene encoding the PEDV-AJ1102 S1 protein: GenBank Accession No. OQ589489, positions 20634-23009, updated on April 11, 2025.

[0113] The nucleotide sequence of the gene encoding the PEDV-HuNYF3 S1 protein: GenBank Accession No. OR085310, positions 20612-22987, updated on August 20, 2025.

[0114] The nucleotide sequence of the gene encoding the PEDV-GDYL13 S1 protein: GenBank Accession No. OR085305, positions 20633-23008, updated on August 20, 2025.

[0115] The nucleotide sequence of the gene encoding the PEDV-AHHY6 S1 protein: GenBank Accession No. PV536074, positions 20631-22994, updated on August 20, 2025.

[0116] The nucleotide sequence of the gene encoding the PEDV-SM98 S1 protein: GenBank Accession No. GU937797, positions 20627-23005, updated on March 27, 2011.

[0117] The expressed proteins were validated using a commercially available His antibody (purchased from Beijing Solarbio Biotechnology Co., Ltd., catalog number K200060M). Results showed that all 19 proteins were successfully expressed. Figure 2 ).

[0118] To verify the reactivity of TEH346 monoclonal antibody, TEH346 hybridoma cells were diluted 1:50 as the primary antibody, and Western blot reactions were performed on the proteins expressed by 19 strains. The results showed that TEH346 monoclonal antibody reacted with the S1 protein of all 19 PEDV strains. Figure 3 This indicates that the antibody is a broad-spectrum PEDV antibody.

[0119] 8. Antibody sequencing

[0120] All primers used in this part of the experiment were synthesized by Jilin Kumei Biotechnology Co., Ltd. Reverse transcription: After digesting and resuspending hybridoma cells in good growth condition, 0.5 μL of the cell suspension was placed in a clean 200 μL PCR tube. 4 μL of cell lysis buffer (prepared by 1 μL Recombinant RNase Inhibitor (Takara, catalog number 2313A) + 19 μL 0.2% Triton™ X-100 (Sigma, T9284)), 1 μL Loligo dt(30) VN primer, and 1 μL dNTP (Takara, catalog number 4030) were added to the cell suspension and mixed with a fingertip. The cells were incubated at 72℃ for 3 min using a PCR instrument to fully lyse them, followed by brief centrifugation and placement on ice. Biotin-labeled tag primers (TSO) and other reagents were added to the cell suspension as shown in Table 1 below to prepare the reverse transcription system.

[0121]

[0122] Add the reverse transcription mixture to the cell sample to prepare a 10 μL reaction system. Gently tap to mix, being careful not to form air bubbles. After a brief low-speed centrifugation, proceed with the reverse transcription reaction according to the programmed procedure. The reaction program is 42℃ for 90 min, followed by 10 cycles of (50℃ for 2 min, 42℃ for 2 min) × 72℃ for 15 min.

[0123] a) Antibody library construction

[0124] Antibody libraries were constructed by PCR using specific primers and tag primers targeting the constant regions of antibodies and the reverse transcription products. The reaction system is shown in Table 2.

[0125]

[0126] The reaction program was 98℃ for 3 min, (98℃ for 20 s, 67℃ for 15 s, 72℃ for 6 min) × 20 cycles, 72℃ for 5 min.

[0127] b) Antibody light / heavy chain specific amplification

[0128] Using the tag primer as the upstream primer, the antibody's light / heavy chains were amplified by combining specific primers targeting the heavy / light chains. The reaction system is shown in Table 3.

[0129]

[0130] The reaction program was 98℃ for 30 s, (98℃ for 30 s, 60℃ for 10 s, 72℃ for 30 s) × 32 cycles, and 72℃ for 5 min.

[0131] c) Antibody light / heavy chain sequencing

[0132] After successful identification of the amplified product by 1% agarose gel electrophoresis and recovery of DNA, the DNA was ligated into the vector pCE2-TA / blunt-zero (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number C601-01), transformed into Escherichia coli DH5α (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number C502-03), and the bacteria were picked and sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing.

[0133] Predictive analysis showed that the amino acid sequence of the heavy chain variable region of monoclonal antibody TEH346 is shown in SEQ ID No:1, and the nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID No:3; the amino acid sequence of the light chain variable region of monoclonal antibody TEH346 is shown in SEQ ID No:2, and the nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID No:4. Among them:

[0134] The amino acid sequence of the CDR1 heavy chain variable region of monoclonal antibody TEH346 is shown as positions 26-33 of SEQ ID No:1;

[0135] The amino acid sequence of the CDR2 heavy chain variable region of monoclonal antibody TEH346 is shown in positions 51-57 of SEQ ID No:1;

[0136] The amino acid sequence of the CDR3 heavy chain variable region of monoclonal antibody TEH346 is shown in positions 96-103 of SEQ ID No:1;

[0137] The amino acid sequence of the CDR1 variable region of the light chain of monoclonal antibody TEH346 is shown as positions 27-37 of SEQ ID No:2;

[0138] The amino acid sequence of the CDR2 variable region of the light chain of monoclonal antibody TEH346 is shown in positions 55-57 of SEQ ID No:2;

[0139] The amino acid sequence of the CDR3 variable region of the light chain of monoclonal antibody TEH346 is shown in positions 94-102 of SEQ ID No:2.

[0140] Example 2: Preparation of recombinant antibody TEH346 using genetic engineering methods

[0141] 1. Construction of expression antibody plasmids

[0142] To express the monoclonal antibody TEH346, the nucleotide sequences of its light chain variable region and heavy chain variable region were linked to mouse-kappa and mouse-IgG2a template sequences, respectively, to obtain the TEH346 light chain gene and heavy chain gene. The TEH346 light chain gene and heavy chain gene were then cloned into the vector pcDNA3.4 (purchased from Hunan Fenghui Biotechnology Co., Ltd., catalog number ZT179), respectively, to obtain the light chain expression vector pcDNA3.4-TEH346-L and the heavy chain expression vector pcDNA3.4-TEH346-H.

[0143] 2. Antibody expression

[0144] 293T cell culture: Prepare 293T cells at a cell density of 90% T175 specification and culture them in DMEM medium with 8% FBS (purchased from Corning, catalog number 10-013-CVRC).

[0145] Transfection of recombinant expression plasmids: 200 μg of recombinant expression plasmids (100 μg each of TEH346 recombinant light chain expression plasmid pcDNA3.4-TEH346-L and recombinant heavy chain expression plasmid pcDNA3.4-TEH346-H) and 200 μL of QuickShuttle-293 cell transfection reagent (purchased from Suzhou Botron Immunotherapy Co., Ltd., catalog number KX0110044) were diluted with 1 mL of physiological saline. After dilution, the two were mixed well and added directly to the cell culture flask for transfection.

[0146] Cell culture: Transfected cells were cultured in a 37ºC 5% CO2 incubator. After 3 days of culture, the cell supernatant was collected for verification.

[0147] 3. Validation of antibody expression

[0148] The expressed antibodies were identified using an indirect immunofluorescence assay (IFA). The supernatant of cells transfected with the antibody expression plasmid was used as the primary antibody and subjected to IFA with the PEDV vaccine strain. The specific steps were the same as those in step 6 of Example 1 for the identification of the monoclonal antibody-virus reaction type.

[0149] The results are as follows Figure 4 As shown: the expression supernatant reacted with PEDV strain-infected cells, exhibiting obvious green fluorescence, and did not react with normal cells, indicating that antibody expression was successful.

[0150] 4. Antibody purification

[0151] The expressed antibody was purified using a Protein A / G 4FF pre-packed column (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number C600983-0501).

[0152] Buffer preparation: Prepare a final concentration of 0.2 M Na2HPO4·12H2O using sterile ddH2O.

[0153] Pre-elution buffer preparation: Prepare 0.1 M citric acid using buffer solution to make the citric acid volume ratio 20%.

[0154] Elution buffer preparation: Prepare 0.1 M citric acid using buffer solution to make the citric acid volume ratio 60%.

[0155] All reagents must be filtered through a 0.22 μm filter before use.

[0156] Sample preparation: Take 30 mL of the cell expression supernatant and add the prepared buffer at a volume ratio of 1:1. Filter the solution using a 0.22 μm filter before loading onto the column.

[0157] Equilibrate the column: Use a constant flow pump to slowly pass 10 mL of buffer solution through a pre-packed Protein A / G4FF column at a flow rate of 1 mL / min;

[0158] Sample loading: The prepared cell supernatant solution was slowly passed through a pre-packed Protein A / G 4FF column at a flow rate of 1 mL / min using a constant flow pump.

[0159] Washing: Use a constant flow pump to slowly pass 10 mL of washing buffer through a pre-packed Protein A / G4FF column at a flow rate of 1 mL / min.

[0160] Elution: Using a constant flow pump, 15 mL of eluent was slowly passed through a pre-packed Protein A / G 4FF column at a flow rate of 1 mL / min. The elution product was then aliquoted into 1.5 mL centrifuge tubes.

[0161] 5. Validation of purified antibodies

[0162] Take the purified antibody and prepare Western blot samples using a non-reducing buffer without DTT and a reducing buffer containing DTT to validate the purified antibody.

[0163] The results are shown in the figure: Lane 1 is the sample treated with non-reducing buffer, with a clear single band at greater than 180 kDa. The results after the antibody was treated with reducing buffer are shown in lane 2, with two clear bands for the light chain and heavy chain at approximately 25 kDa and 50 kDa, respectively, indicating that the antibody was well purified and the recombinant antibody TEH346 was successfully obtained.

[0164] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. An antibody against porcine epidemic diarrhea virus, characterized in that: The antibody is a monoclonal antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are as shown in positions 26-33, 51-57, and 96-103 of SEQ ID No:1, respectively; HCDR1, HCDR2, and HCDR3 are complementarity-determining regions; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody are as shown in positions 27-37, 55-57, and 94-102 of SEQ ID No:2, respectively; LCDR1, LCDR2, and LCDR3 are complementarity-determining regions. CDR is a sequence defined according to the IMGT numbering system.

2. The antibody according to claim 1, characterized in that: The amino acid sequence of the light chain variable region in the antibody has at least 90% identity with SEQ ID No:2; and / or the amino acid sequence of the heavy chain variable region has at least 90% identity with SEQ ID No:

1.

3. The antibody according to claim 1 or 2, characterized in that: The heavy chain type of the antibody is IgG1; and / or the light chain type of the antibody is kappa.

4. Any of the following biological materials: (A1) The active fragment of the antibody is any of the following derived from the antibody of claim 1 or 2: a single-chain antibody, an antibody Fab region, and an antigen-binding fragment; (A2) A nucleic acid molecule that encodes the antibody of claim 1 or 2 or the active fragment of the antibody of claim (A1); (A3) An expression cassette, recombinant vector, recombinant cell or recombinant bacterium containing the nucleic acid molecule described in (A2); (A4) A kit containing the antibody of claim 1 or 2, or the active fragment of the antibody of claim (A1), or the nucleic acid molecule of claim (A2), or the expression cassette, recombinant vector, recombinant cell, or recombinant bacterium of claim (A3).

5. The biomaterial according to claim 4, characterized in that: In the nucleic acid molecule described in (A2), the nucleotide sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region encoding the antibody are shown in positions 76-99, 151-171 and 286-309 of SEQ ID No:3, respectively. and / or In the nucleic acid molecule described in (A2), the nucleotide sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region encoding the antibody are shown in positions 79-111, 163-171 and 280-306 of SEQ ID No:4, respectively.

6. A method for preparing the antibody according to claim 1 or 2, comprising the following steps: introducing a nucleic acid molecule encoding the antibody according to claim 1 or 2 into a recipient cell to obtain a transgenic cell expressing the antibody, culturing the transgenic cell to obtain the antibody.

7. The use of the antibody of claim 1 or 2 or the biomaterial of claim 4 or 5 in any of the following: (B1) Prepare products for detecting or assisting in the detection of whether a sample contains porcine epidemic diarrhea virus; (B2) Prepare products for detecting or assisting in the detection of porcine epidemic diarrhea virus content in the sample to be tested.

8. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition is the antibody as described in claim 1 or 2.

9. An ELISA detection kit targeting porcine epidemic diarrhea virus, characterized in that, The kit contains the antibody as described in claim 1 or 2 or the biological material as described in claim 4 or 5.

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