Anti-porcine epidemic diarrhea virus broad-spectrum monoclonal antibody and application thereof

Abstract

The invention relates to an anti-porcine epidemic diarrhea virus broad-spectrum monoclonal antibody and application thereof in the field of medical preparations. The antibody developed by the invention is a monoclonal antibody or an antigen binding fragment thereof, and the monoclonal antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region; amino acid sequences of HCDR1, HCDR2 and HCDR3 in a heavy chain variable region of the antibody are sequentially shown as the 26th site to the 33rd site, the 51st site to the 57th site and the 96th site to the 103rd site of SEQ ID No: 1; the HCDR1, the HCDR2 and the HCDR3 are complementary determining regions; amino acid sequences of LCDR1, LCDR2 and LCDR3 in a light chain variable region of the antibody are sequentially shown as the 27th site to the 37th site, the 55th site to the 57th site and the 94th site to the 102nd site of SEQ ID No: 2, and the LCDR1, the LCDR2 and the LCDR3 are complementary determining regions.

Description

TECHNICAL FIELD

[0001] The present application relates to a broad-spectrum monoclonal antibody against porcine epidemic diarrhea virus and its application in the field of medical formulations. BACKGROUND

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

[0003] PEDV is a member of the alpha coronavirus genus of the Coronaviridae family, a single-stranded positive-sense RNA virus with an envelope. The genome consists of two non-coding regions at both ends and seven open reading frames, with a size of about 28 kb. The viral genome encodes four structural proteins: Spike (S), Membrane (M), Envelope (E), and Nucleocapsid (N), as well as non-structural proteins pp1a, pp1b, and accessory protein ORF3. The S protein is the main antigenic protein of PEDV virus, which can stimulate the body to produce neutralizing antibodies. According to the different functions, the protein can be divided into S1 and S2 two regions, in which S1 is mainly involved in the binding of virus and cell receptors. This region contains multiple antigen epitopes and is the main target protein for PEDV detection and vaccine development.

[0004] PEDV was first discovered in the 1970s. At that time, the PEDV epidemic was mostly sporadic infection, with low morbidity and mortality, and did not attract widespread attention. In 2010, the emergence of a highly pathogenic mutant strain was first reported in China. This variant strain has the characteristics of fast spread and high mortality. Subsequently, the prevalence of the variant strain has been reported in many countries such as the United States, Germany, and Mexico. Genetic evolution analysis results show that PEDV strains can be divided into two genotypes, GI and GII. The GI genotype mainly includes the classic strains and cell-attenuated strains prevalent before 2010, while the GII type mainly includes the current world epidemic variant strains. Whole genome analysis results show that the main difference between different subtypes of strains is located in the S gene.

[0005] The continuous variation of the strain has brought great challenges to the detection and vaccine prevention of PEDV. In order to cope with the rapid evolution of the virus, the development of PEDV broad-spectrum recognition antibodies has important significance and potential application value for clinical diagnosis and prevention of porcine epidemic diarrhea. SUMMARY

[0006] The main problem to be solved by the present application is how to obtain an antibody capable of recognizing a porcine epidemic diarrhea virus (PEDV) in a broad spectrum.

[0007] To solve the above problems, the present application provides a monoclonal antibody capable of recognizing a PEDV in a broad spectrum.

[0008] The antibody provided by the present application is a monoclonal antibody or an antigen-binding fragment thereof, which comprises 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 shown in SEQ ID No: 1, 26-33, 51-57 and 96-103, respectively; the 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 shown in SEQ ID No: 2, 27-37, 55-57 and 94-102, respectively, and the LCDR1, LCDR2 and LCDR3 are complementarity determining regions.

[0009] The "antibody" in the present application can include polyclonal antibodies and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof. The term encompasses immunoglobulins that are genetically engineered and / or otherwise modified, such as intrabodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific antibodies such as bispecific antibodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term "antibody" is not limited by any particular method of producing the antibody. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and subtypes thereof (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgM, IgE, IgA and IgD.

[0010] The term "monoclonal antibody" or "Antibody," in the absence of specific label, generally refers to an immunoglobulin molecule that is generally composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. In general, the heavy chain can be understood as the polypeptide chain of larger molecular weight in an antibody, and the light chain is the polypeptide chain of smaller molecular weight in an antibody. The light chain can be classified as kappa and lambda light chain. The heavy chain can be generally classified as mu, delta, gamma, alpha, or epsilon, and defines the isotype of an antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light chain and the heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy chain / light chain pair form the antibody binding site. Assignment of amino acids to each region or domain follows the definition set forth 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 further comprise more than 3 CDRs, e.g., 6, 9, or 12. For example, in a bifunctional antibody, the heavy chain can be the C-terminal of the heavy chain of an IgG antibody linked to a ScFv of another antibody, in which case the heavy chain contains 9 CDRs.

[0011] In the present invention, the light chain variable region (VL) or the heavy chain variable region (VH) of the antibody is composed of "framework" regions separated by three "complementarity determining regions" or "CDRs". The framework regions serve to align the CDRs that specifically bind to the epitope of the antigen. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. The VL domain and the VH domain each comprise, from amino-terminus to carboxy-terminus, the following framework regions (FRs) and CDRs: 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 above CDRs are defined sequences according to the IMGT numbering system. Within the scope of the present invention, the antibody can be in various forms such as a full-length antibody, a Fab fragment, a F(ab')2 fragment, or a single-chain Fv fragment.

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

[0014] The term "Fab fragment" is a heterodimer formed by the heavy chain Fd and the complete light chain through a disulfide bond, containing only one antigen binding site. The heavy chain Fd refers to about 1 / 2 of the H chain portion in Fab (about 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0015] The term "Fv fragment" refers to a construct that can be separately constructed with a VH and VL gene, co-transfected into cells to be separately expressed, and then assembled to form a functional Fv antibody; or a construct that can be separately expressed as two small protein fragments by setting a stop codon between VH and VL in the vector, and then combined to form an Fv antibody (Fv fragment) through non-covalent bonding.

[0016] The term "Fab' fragment" contains one light chain and a portion of one heavy chain that includes the VH domain and the CH1 domain and the region between CH1 and CH2, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

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

[0018] The term "single chain antibody (ScFv)" refers to a polypeptide obtained by connecting a light chain variable region and a heavy chain variable region. The polypeptide can spontaneously fold into a natural conformation, maintaining the specificity and affinity of Fv.

[0019] The monoclonal antibody is monoclonal antibody 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] Further, the heavy chain type of the antibody is IgG; and / or the light chain type of the antibody is kappa.

[0022] The present application also provides an antibody obtained after immunizing an animal with the S1 protein of porcine epidemic diarrhea virus as an immunogen, the amino acid sequence of the S1 protein being SEQ ID No: 6.

[0023] The present application also provides any of the following biological materials: (A1) an active fragment of the antibody, which is derived from any of the antibodies described in the foregoing or any of the antibodies described in the foregoing: single chain antibody, antibody Fab region and antigen binding fragment; (A2) a nucleic acid molecule, which encodes the antibody described in the foregoing or the antibody described in the foregoing or the active fragment of the antibody of (A1); (A3) an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacteria containing the nucleic acid molecule described in (A2); (A4) a kit containing the antibody described in the foregoing or the active fragment of the antibody of (A1) or the nucleic acid molecule of (A2) or the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacteria of (A3).

[0024] The above-mentioned biological material, in the nucleic acid molecule of (A2), the nucleotide sequences encoding HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are sequentially shown as SEQ ID No: 3 at positions 76-99, 151-171 and 286-309; and / or In the nucleic acid molecule of (A2), the nucleotide sequence encoding LCDR1, LCDR2 and LCDR3 in the variable region of the light chain of the antibody is shown in SEQ ID No: 4 as positions 79-111, 163-171 and 280-306, respectively.

[0025] Among the above-mentioned biomaterials, the expression cassette described in (A3) refers to a DNA capable of expressing the antibody or the antigen-binding fragment in a host cell. The expression cassette can further include all regulatory sequences necessary for initiating expression of the nucleic acid molecule encoding the antibody or the antigen-binding fragment. The regulatory sequences can direct the expression of the coding sequence in a suitable host cell to express the antibody or the antigen-binding fragment under compatible conditions. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences include a promoter and termination signals for transcription and translation. The regulatory sequences can be provided with linkers for the introduction of restriction sites facilitating the joining of the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein. The regulatory sequences can be a suitable promoter sequence, i.e., a nucleic acid sequence that is recognized by a host cell to initiate the expression of a nucleic acid sequence. The promoter sequence contains transcriptional control sequences that mediate the transcription of the protein. The promoter can be any nucleic acid sequence that shows transcriptional activity in the host cell of choice including mutated, truncated, and hybrid promoters, and can be derived from genes encoding proteins either homologous or heterologous to the host cell. The regulatory sequences can also be a suitable transcription terminator sequence, i.e., a sequence that is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the antibody or the antigen-binding fragment. Any terminator that is functional in the host cell of choice can be used in the present application. The regulatory sequences can also be a suitable leader sequence, i.e., an mRNA untranslated region that is vital for translation of the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protein, i.e., the antibody or the antigen-binding fragment. Any leader sequence that is functional in the host cell of choice can be used in the present application. The regulatory sequences can also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of the protein, i.e., the antibody or the antigen-binding fragment, that directs the expressed protein, i.e., the antibody or the antigen-binding fragment, to the secretory pathway of the host cell. Any signal peptide coding region that directs the expressed protein, i.e., the antibody or the antigen-binding fragment, to the secretory pathway of the host cell of choice can be used in the present application. It can also be desirable to add regulatory sequences that allow the regulation of the expression of the protein, i.e., the antibody or the antigen-binding fragment, relative to the growth of the host cell. Examples of regulatory sequences are those that allow expression of the nucleic acid sequence encoding the protein, i.e., the antibody or the antigen-binding fragment, in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other examples of regulatory sequences are those that allow the amplification of the gene. In these cases, the nucleic acid sequence encoding the protein, i.e., the antibody or the antigen-binding fragment, is operably linked with the regulatory sequence.

[0026] Among the above-mentioned biomaterials, the recombinant vector in (A3) can be a cloning vector or an expression vector. As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of mediating the expression of an inserted polynucleotide- encoded protein, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda 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 viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain various elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can contain a replication initiation site.

[0027] In preparing the expression vector, nucleic acid molecules encoding the protein (i.e., the antibody or the antigen binding fragment) can be placed into the vector so as to be operatively linked to the appropriate expression control sequences. The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can conveniently be subjected to recombinant DNA procedures and allows expression of the nucleic acid sequence in a host cell. The choice of vector depends primarily on the host cell it is designed to transform. The vector can be linear or closed circular. The vector can be an autonomously replicating vector, i.e., a vector that exists as an episome in a host cell, allowing it to replicate independently of the chromosomal DNA of the host cells into which it is introduced (e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector can comprise any mechanism for replication in the host cell, e.g., an origin of replication. Alternatively, the vector can be one that, when introduced into a host cell, is integrated into the cell chromosome and replicated with the cell chromosome. The vector system can further comprise one or more selectable marker genes to facilitate selection of host cells into which the vector is introduced. A selectable marker gene is a gene whose product confers antibiotic resistance, resistance to heavy metals, or the like, on a host cell into which the gene is introduced. Examples of bacterial selectable marker genes are those that code for antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. The vector can further comprise a multiple cloning region to allow insertion of a nucleic acid molecule. The vector can also comprise a selectable marker gene to allow selection of host cells into which the vector is introduced. The vector can further comprise a replication origin to allow autonomous replication of the vector in a host cell. The replication origin can be mutated to render it temperature sensitive (see, e.g., fEhrlich, 1978, Proc. Natl. Acad. Sci. USA 75:1433). More than one copy of a nucleic acid molecule encoding the protein (i.e., the antibody or the antigen binding fragment) can be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid molecule can be brought about by including an appropriate origin of replication in the vector. The copy number of the vector can be increased by introducing a selectable marker gene in the vector, which can amplify the vector and thus the inserted nucleic acid molecule along with it. The procedures used to ligate the elements of the recombinant expression vectors of the application will be apparent to the skilled worker (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0028] Among the above-mentioned biological materials, the recombinant cell described in (A3) can be an animal cell, and the animal cell can be a non-reproductive material. The animal cell can be an isolated mammalian cell. The mammal can be a human or a mouse. The mammalian cell can not include an animal reproductive cell, an animal zygote, and an animal embryonic stem cell, and can be a somatic cell or a cell line. The animal cell can be a cell line or a somatic cell derived from a mouse.

[0029] Further, the cell can be a 293T cell.

[0030] The present application also provides a method for preparing the antibody as described above, comprising the following steps: introducing a nucleic acid molecule encoding the antibody as described above into a recipient cell to obtain a transgenic cell expressing the antibody, and culturing the transgenic cell to obtain the antibody.

[0031] The present application also provides the use of the antibody as described above, or the biomaterial as described above, in any one of the following: (B1) recognizing or assisting in recognizing porcine epidemic diarrhea virus; (B2) binding or assisting in binding porcine epidemic diarrhea virus; (B3) detecting or assisting in detecting whether a sample to be tested contains porcine epidemic diarrhea virus; (B4) preparing a product for detecting or assisting in detecting whether a sample to be tested contains porcine epidemic diarrhea virus; (B5) detecting or assisting in detecting the content of porcine epidemic diarrhea virus in a sample to be tested; (B6) preparing a product for detecting or assisting in detecting the content of porcine epidemic diarrhea virus in a sample to be tested.

[0032] The present application also provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition is the antibody as described above.

[0033] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent or carrier.

[0034] In the pharmaceutical composition, the term "pharmaceutically acceptable excipient" refers to an additive substance other than the active ingredient in a pharmaceutical preparation, which is used to improve the physical properties, stability or bioavailability of the drug. According to the function, it can be divided into various types. Disintegrants can promote the rapid disintegration of drugs in the body, releasing active ingredients, such as starch, sodium carboxymethyl starch, etc.; binding agents are used to bind drug powders into granules or tablets, and common ones are povidone, hydroxypropyl methyl cellulose; lubricants can reduce the friction between drug particles and the surface of the equipment, ensuring the smooth molding and release of the drug, magnesium stearate, talc is a commonly used lubricant. These excipients need to meet the conditions of non-toxic, non-irritating, not chemically reacting with active ingredients, etc., to ensure the safety and effectiveness of the drug.

[0035] In the pharmaceutical composition, the term "diluent" is mainly used to adjust the concentration of the drug to achieve the appropriate dosage specification. When the content of the active ingredient is low, a diluent is added to increase the volume during the preparation of solid preparations such as tablets and capsules. Lactose, sucrose, mannitol, etc. are commonly used diluents, which are chemically stable and do not affect the activity of the antibody or antigen-binding fragment, and have good fluidity and compressibility, facilitating the formation of the preparation. In the injection, the diluent is used to dissolve or dilute the active ingredient, such as water, physiological saline, glucose solution, etc., to ensure that the concentration of the drug meets the requirements of clinical use, while maintaining the osmotic pressure balance of the solution to avoid damage to the pig tissue.

[0036] In the pharmaceutical composition, the term "carrier" has a special function in drug delivery in addition to the functions of excipients and diluents, especially in targeted drug delivery systems. Liposomes, as a common carrier, are composed of phospholipid bilayers, which can encapsulate antibodies or antigen-binding fragments, not only protecting the active ingredients from degradation by enzymes and other substances in the body, but also making them more easily taken up by diseased cells by modifying the surface of the liposomes to have targeting properties, such as connecting targeting ligands. Nanoparticle carriers (such as polymer nanoparticles, solid lipid nanoparticles) also have similar functions, and their nanoscale size helps them penetrate biological barriers to achieve efficient drug delivery.

[0037] The present application also provides an ELISA detection kit for porcine epidemic diarrhea virus, which comprises the antibody or the biological material described above.

[0038] In the present application, the kit includes an immunohistochemical kit, an immunofluorescence kit, a flow cytometry detection kit, and an immunoblotting kit.

[0039] The present application also provides a method for preparing the antibody described above, comprising the following steps: 1) Immunizing animals with porcine epidemic diarrhea virus S1 protein as an immunogen; 2) Isolating the spleen lymphocytes of the immunized animals; 3) Screening the antibody-secreting cells in the spleen lymphocytes of step 2) to obtain individual porcine epidemic diarrhea virus-specific antibody-secreting cells; 4) Sequencing and expressing and purifying to obtain monoclonal antibodies against porcine epidemic diarrhea virus.

[0040] The application screens and identifies a hybridoma cell strain against the S1 protein of porcine epidemic diarrhea virus through the hybridoma technology. The monoclonal antibody (TEH346) secreted by the cell strain has broad-spectrum binding activity and can effectively recognize various PEDV epidemic strains including the classic strain (PEDV-CV777) and the mutant strain. The monoclonal antibody can be used as a raw material for the establishment of various detection methods including but not limited to enzyme-linked immunosorbent assay (ELISA), immunochromatography test strip (colloidal gold / fluorescence), immunohistochemistry (IHC), Western blot and the like. The monoclonal antibody provides a key material for the development of a new generation of PEDV universal detection reagent. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Figure 4 is an indirect immunofluorescence experiment result of the monoclonal antibody TEH346 and different genotypes of PEDV strains.

[0042] Figure 2 Figure 6 is a Western blot experiment result of the S1 protein of 19 PEDV strains of different genotypes.

[0043] Figure 3 Figure 7 is a Western blot experiment result of the monoclonal antibody TEH346 and the S1 protein of 19 PEDV strains of different genotypes.

[0044] Figure 4 Figure 8 is an indirect immunofluorescence experiment result of the monoclonal antibody TEH346.

[0045] Figure 5 Figure 9 is a result of the verification of the purification effect of the monoclonal antibody TEH346. Lane 1 is a non-reducing buffer treatment sample, and lane 2 is a reducing buffer treatment sample. DETAILED DESCRIPTION

[0046] The application will be further described in detail below in conjunction with the specific embodiments. The embodiments provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.

[0047] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0048] In the quantitative test in the following examples, three repeated experiments were set, and the average value was taken, unless otherwise specified.

[0049] The insect baculovirus expression vector pFastBac 1 in the following examples has been described in: Misjiang. Identification of Differentiating Monoclonal Antibodies and Broad-Spectrum Monoclonal Antibodies for Epidemic Strains and Vaccine Strains of Porcine Pestivirus and Epitope Analysis of Antigens[D]. Jilin University, 2022. The biological material can be obtained from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0050] The Sf9 cells in the following examples have been described in: Misjiang. Identification of Differentiating Monoclonal Antibodies and Broad-Spectrum Monoclonal Antibodies for Epidemic Strains and Vaccine Strains of Porcine Pestivirus and Epitope Analysis of Antigens[D]. Jilin University, 2022. The biological material can be obtained from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0051] The SP2 / 0 cells in the following examples have been described in: Misjiang. Identification of Differentiating Monoclonal Antibodies and Broad-Spectrum Monoclonal Antibodies for Epidemic Strains and Vaccine Strains of Porcine Pestivirus and Epitope Analysis of Antigens[D]. Jilin University, 2022. The biological material can be obtained from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0052] The information of the vaccine virus involved in the present example is as follows: PEDV-AJ1102: Wuhan Keqian Biological Co., Ltd., approval number: veterinary drug Sheng Zi 170041133.

[0053] PEDV-SCSZ-1: Chongqing Aolong Biological Products Co., Ltd., approval number: veterinary drug Sheng Zi 230241134.

[0054] PEDV-LW / L: Luoyang Huizhong Biological Technology Co., Ltd., approval number: veterinary drug Sheng Zi 163001140.

[0055] PEDV-ZJ08: Ruipu (Baoding) Biological Pharmaceutical Co., Ltd., approval number: veterinary drug Sheng Zi 030381109.

[0056] PEDV-CV777: Harbin Weike Biological Technology Co., Ltd., approval number: veterinary drug Sheng Zi 080011097.

[0057] Example 1, Preparation and identification of monoclonal antibody of porcine epidemic diarrhea virus S1 protein 1. Preparation of immunogen The immunogen used in the experiment is the S1 protein of the epidemic strain PEDV-CH / GDZQ / 2014 (PEDV-CH / GDZQ / 2014), and the S1 protein coding gene of PEDV-CH / GDZQ / 2014 (the sequence of 20634-23009 of GenBank accession number: KM242131, the His tag is added at the 3' end of the sequence, and the His tag nucleotide sequence is 5'-CATCATCACCATCACCAT-3', SEQ ID No: 7) is cloned into the insect baculovirus expression vector pFastBac 1 to obtain the recombinant expression vector pFastBac 1-S1.

[0058] The structure of the pFastBac 1-S1 vector is described as follows: the DNA fragment of SEQ ID No: 5 is inserted between the two enzyme cutting sites of the starting vector pFastBac 1, and the other sequences of the vector pFastBac 1 remain unchanged to obtain the recombinant vector. The pFastBac 1-S1 vector can express the PEDV-CH / GDZQ / 2014 S1 protein, and the amino acid sequence is SEQ ID No: 6. BamH I and EcoR The DNA fragment of SEQ ID No: 5 is inserted between the two enzyme cutting sites of the starting vector pFastBac 1, and the other sequences of the vector pFastBac 1 remain unchanged to obtain the recombinant vector. The pFastBac 1-S1 vector can express the PEDV-CH / GDZQ / 2014 S1 protein, and the amino acid sequence is SEQ ID No: 6.

[0059] The recombinant expression vector pFastBac 1-S1 is transformed into DH10Bac competent cells to obtain a recombinant bacmid containing the target gene fragment, and the bacmid is transfected into Sf9 cells for protein expression. The collected PEDV-CH / GDZQ / 2014 S1 protein is purified by fixed metal ion affinity chromatography, and the purified protein can be used as an immunogen.

[0060] 2, Mouse immunization Take 50 μg PEDV-CH / GDZQ / 2014 S1 purified protein and mix with an equal volume of 206 adjuvant (purchased from Seppic Company, item number ISA206VG) and emulsify, and immunize SPF level 6-8 week old female BALB / c mice (purchased from Jilin Province Jintai Medici Biotechnology Co., Ltd., item number 1010003), and select subcutaneous multiple point injection of immunized mice, immunize every two weeks, a total of 3 times, one week after the third immunization, intraperitoneally inject 100 μg of PEDV-CH / GDZQ / 2014 S1 purified protein without adjuvant for impact immunization, and then continue to feed for 3 days.

[0061] 3, Cell fusion Three days after the impact immunization, the mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 5 min. The spleen was removed aseptically and placed in a 200-mesh nylon screen, gently ground using a syringe core, and the nylon screen was rinsed with serum-free Advanced RPMI-1640 (purchased from Thermo Fisher Scientific, Cat. No. 12633012), and the spleen cell suspension was collected in a centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Repeat the washing once, and count the spleen cells. Mix the SP2 / 0 cells in the logarithmic growth phase with the spleen cells at a cell ratio of 1:5-1:10, centrifuge at 1000 rpm for 5 min, discard the supernatant, tap the bottom of the centrifuge tube to loosen the cell sediment, and add 1 mL of 37°C preheated PEG 1500 (purchased from Sigma-Aldrich, Cat. No. 25322-68-3) to the sedimented cells within 1 min, while gently rotating the centrifuge tube and adding PEG 1500. In the next 10 min, continue to add 10 mL of 37°C preheated Advanced RPMI-1640 medium in the same way. After the end, centrifuge the cells at 800 rpm for 5 min, discard the supernatant, resuspend the cell sediment with 100 mL of HY semi-solid medium (purchased from STEMCELL Technologies, Cat. No. 03804), and evenly spread in a six-well plate, 2.5-3 mL / well. The spread six-well plate was placed in a 37°C, 5% CO2 incubator for 7-10 days.

[0062] 4. Screening of hybridoma cells After 7-10 days, monoclonal cell clusters in the semi-solid medium of step 2 were picked up under a microscope to Advanced RPMI-1640 medium containing 1% HAT (purchased from Thermo Fisher Scientific, Cat. No. 21060017), 10% FBS (purchased from Biological Industries, Cat. No. 04-001-1ACS), and 1% double-antibiotic (purchased from Cytiva HyClone, Cat. No. SV30010). After 3 days of culture, antibody-positive cell wells were screened using an indirect immunofluorescence test, and a hybridoma cell strain that could secrete a monoclonal antibody specifically binding to S1 protein was determined after subcloning, named TEH346. The monoclonal antibody secreted by the hybridoma cell strain TEH346 is called mAb TEH346.

[0063] 5. Identification of antibody type of mAb The Ig class and light chain type of the monoclonal antibody were identified using an indirect ELISA method. The secondary antibodies used in the experiment were from mouse monoclonal Ig class subunit identification enzyme-labeled secondary antibody kit (purchased from Suzhou Boterlong Immunotechnology Co., Ltd., item number BF16002X). The results showed that the monoclonal antibody TEH346 was IgG1 and κ light chain.

[0064] 6. Identification of the reaction type of the monoclonal antibody with the virus - IFA 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. The steps were as follows: a) Cell plating: Vero cells were passaged at a certain ratio and inoculated in a 96-well plate, and cultured overnight at 37°C in a 5% CO2 incubator.

[0065] b) Cell infection: The virus was diluted with DMEM medium, and PEDV virus was inoculated at 100 TCID 50 / well. The virus dilution was added to the 96-well plate, and cultured at 37°C in a 5% CO2 incubator for 72 h.

[0066] c) Cell fixation: The cell culture supernatant was discarded, and each well of the 96-well plate was washed with 200 μL of PBS for 3 times. 80% cold acetone (50 μL / well) stored at -20°C was added, and fixed in a -20°C refrigerator for 1 h.

[0067] d) Primary antibody incubation: The cold acetone fixative was discarded, each well was washed with 200 μL of PBS for 3 times, 100 μL of hybridoma cell culture supernatant was added to each well, and incubated at 37°C for 1 h.

[0068] e) Secondary antibody incubation: The primary antibody incubation solution was discarded, each well was washed with 200 μL of PBS for 3 times, Alexa Fluor 488 fluorescent secondary antibody (purchased from Thermo Fisher Scientific Company, item number A-21202) was diluted 1:500 with PBS, 0.01% Evans blue (purchased from Beijing Solaybao Biotechnology Co., Ltd., item number G1810) was added, and 100 μL of the mixture was added to each well of the cell plate after thorough mixing, and incubated at 37°C for 1 h.

[0069] f) Fluorescence observation: The secondary antibody incubation solution was discarded, each well was washed with 200 μL of PBS for 3 times, and the reaction of the antibody with the infected cells was observed under a fluorescence microscope.

[0070] The results are shown in Table 1.Figure 1 As shown, the monoclonal antibody TEH346 specifically reacted with all strains and showed obvious fluorescence.

[0071] 7. Identification of the reaction type of the monoclonal antibody with the virus - Western blot The S1 proteins of different genotypes of PEDV were expressed by an insect baculovirus expression system, including the S1 proteins of the following 19 strains of PEDV: 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 proteins of the above strains refers to the preparation method of PEDV-CH / GDZQ / 2014 S1 protein in Example 1. The coding genes of each S1 protein cloned into the insect baculovirus expression vector pFastBac 1 are as follows: The nucleotide sequence of the coding gene of PEDV-HLJDQ5 S1 protein: positions 20620-22983 of GenBank Accession No. OR085241, updated on August 20, 2025.

[0072] The nucleotide sequence of the coding gene of PEDV-NMBB1 S1 protein: positions 20709-23084 of GenBank Accession No. OR085300, updated on August 20, 2025.

[0073] The nucleotide sequence of the coding gene of PEDV-SCPM47 S1 protein: positions 20634-23009 of GenBank Accession No. OR085252, updated on August 20, 2025.

[0074] The nucleotide sequence of the coding gene of PEDV-LZC S1 protein: positions 20638-23004 of GenBank Accession No. EF185992, updated on January 16, 2007.

[0075] Nucleotide sequence of the coding gene of PEDV-XJJY6 S1 protein: positions 20629-23004 of GenBank Accession No. OR085287, updated on August 20, 2025.

[0076] Nucleotide sequence of the coding gene of PEDV-AHWH8 S1 protein: positions 20758-23133 of GenBank Accession No. OR085281, updated on August 20, 2025.

[0077] Nucleotide sequence of the coding gene of PEDV-GSYT1 S1 protein: positions 20622-22997 of GenBank Accession No. PV536086, updated on August 20, 2025.

[0078] Nucleotide sequence of the coding gene of PEDV-SD S1 protein: positions 20622-22997 of GenBank Accession No. PP958821, updated on July 2, 2025.

[0079] Nucleotide sequence of the coding gene of PEDV-AHLY1 S1 protein: positions 20653-23028 of GenBank Accession No. OR085262, updated on August 20, 2025.

[0080] Nucleotide sequence of the coding gene of PEDV-YNQW1 S1 protein: positions 20646-23021 of GenBank Accession No. PV536117, updated on August 20, 2025.

[0081] Nucleotide sequence of the coding gene of PEDV-FJZZ2 S1 protein: positions 20616-22991 of GenBank Accession No. OR085266, updated on August 20, 2025.

[0082] Nucleotide sequence of the coding gene of PEDV-CH / GDZQ S1 protein: positions 20634-23009 of GenBank Accession No. KM242131, updated on February 3, 2015.

[0083] Nucleotide sequence of the coding gene of PEDV-HLJJW1 S1 protein: positions 20649-23015 of GenBank Accession No. PV536091, updated on August 20, 2025.

[0084] Nucleotide sequence of the coding gene of PEDV-CV777 S1 protein: 20633-22999 of GenBank Accession No. LT905450, updated on June 15, 2018.

[0085] Nucleotide sequence of the coding gene of PEDV-AJ1102 S1 protein: 20634-23009 of GenBank Accession No. OQ589489, updated on April 11, 2025.

[0086] Nucleotide sequence of the coding gene of PEDV-HuNYF3 S1 protein: 20612-22987 of GenBank Accession No. OR085310, updated on August 20, 2025.

[0087] Nucleotide sequence of the coding gene of PEDV-GDYL13 S1 protein: 20633-23008 of GenBank Accession No. OR085305, updated on August 20, 2025.

[0088] Nucleotide sequence of the coding gene of PEDV-AHHY6 S1 protein: 20631-22994 of GenBank Accession No. PV536074, updated on August 20, 2025.

[0089] Nucleotide sequence of the coding gene of PEDV-SM98 S1 protein: 20627-23005 of GenBank Accession No. GU937797, updated on March 27, 2011.

[0090] The expressed proteins were verified using a commercial His antibody (purchased from Beijing Solabio Biotechnology Co., Ltd., item number K200060M). The results showed that 19 proteins were successfully expressed ( Figure 2 ).

[0091] To verify the reaction characteristics of TEH346 monoclonal antibody, TEH346 hybridoma cells were used as primary antibodies at a dilution of 1:50 to perform Western blot reaction on 19 expressed proteins. The results showed that TEH346 monoclonal antibody reacted with S1 proteins of 19 PEDV strains ( Figure 3 ), indicating that the antibody is a broad-spectrum PEDV antibody.

[0092] 8. Antibody sequence determination The primers involved in this part of the experiment are synthesized by Jilin Province Kumai Biotechnology Co., Ltd. Reverse transcription: well-growing hybridoma cells were resuspended after digestion, and 0.5 μL of cell suspension was taken into a clean 200 μL PCR tube. Add 4 μL of cell lysate (lysis solution is prepared by 1 μL Recombinant RNase Inhibitor (Takara, item number 2313A) + 19 μL 0.2% Triton™ X-100 (Sigma, T9284)), 1 μL oligo dt (30) VN primer and 1 μL dNTP (Takara, item number 4030) to the cell suspension, and mix well. Use PCR instrument 72℃ incubation 3min, make the cell fully lysis, then centrifuge briefly, and place on ice. Add biotin-labeled tag primer (TSO) and other reagents to the cell suspension according to Table 1 below to prepare the reverse transcription system.

[0093]

[0094] Add the reverse transcription mixture to the cell sample to configure a 10 μL reaction system, mix well with a pipette, and pay attention not to form bubbles. After low-speed short centrifugation, perform the reverse transcription reaction according to the program. The reaction program is 42℃ for 90 min, (50℃ for 2 min, 42℃ for 2 min) x 10 cycles, 72℃ for 15 min.

[0095] a) Construction of antibody library PCR was performed on the reverse transcription product using specific primers for the constant region of the antibody and tag primers to construct the antibody library. The reaction system is shown in Table 2.

[0096]

[0097] The reaction program is 98℃ for 3 min, (98℃ for 20 s, 67℃ for 15 s, 72℃ for 6 min) x 20 cycles, 72℃ for 5 min.

[0098] b) Specific amplification of antibody light / heavy chains The light / heavy chains of the antibody were amplified using the tag primer as the upstream primer and the specific primer for the heavy / light chain. The reaction system is shown in Table 3.

[0099]

[0100] The reaction program is 98℃ for 30 s, (98℃ for 30 s, 60℃ for 10 s, 72℃ for 30 s) x 32 cycles, 72℃ for 5 min.

[0101] c) Sequencing of antibody light / heavy chains After the amplification product was identified successfully by 1% agarose gel electrophoresis, the DNA was recovered, ligated to the vector pCE2-TA / blunt-zero (purchased from Nanjing Novizen Biotech Co., Ltd., Catalog No. C601-01), transformed into E. coli DH5a (purchased from Nanjing Novizen Biotech Co., Ltd., Catalog No. C502-03) and picked bacteria, and sent to Jilin Kumai Biotechnology Co., Ltd. for sequencing.

[0102] The prediction result analysis showed that the amino acid sequence of the heavy chain variable region of the monoclonal antibody TEH346 was as shown in SEQ ID No: 1, the nucleotide sequence of the heavy chain variable region encoding gene was as shown in SEQ ID No: 3; the amino acid sequence of the light chain variable region of the monoclonal antibody TEH346 was as shown in SEQ ID No: 2, and the nucleotide sequence of the light chain variable region encoding gene was as shown in SEQ ID No: 4. Among them: The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody TEH346 was as shown in SEQ ID No: 1 at positions 26-33; The amino acid sequence of CDR2 of the heavy chain variable region of the monoclonal antibody TEH346 was as shown in SEQ ID No: 1 at positions 51-57; The amino acid sequence of CDR3 of the heavy chain variable region of the monoclonal antibody TEH346 was as shown in SEQ ID No: 1 at positions 96-103; The amino acid sequence of CDR1 of the light chain variable region of the monoclonal antibody TEH346 was as shown in SEQ ID No: 2 at positions 27-37; The amino acid sequence of CDR2 of the light chain variable region of the monoclonal antibody TEH346 was as shown in SEQ ID No: 2 at positions 55-57; The amino acid sequence of CDR3 of the light chain variable region of the monoclonal antibody TEH346 was as shown in SEQ ID No: 2 at positions 94-102.

[0103] Example 2, preparation of recombinant antibody TEH346 by genetic engineering method 1. Construction of antibody plasmid expression In order to express the monoclonal antibody TEH346, the nucleotide sequences of the light chain variable region and the heavy chain variable region thereof were connected with the mouse-kappa and mouse-IgG2a template sequences respectively to obtain the light chain gene and the heavy chain gene of TEH346. The light chain gene and the heavy chain gene of TEH346 were cloned into the vector pcDNA3.4 (purchased from Hunan Fenghui Biotechnology Co., Ltd., Catalog No. ZT179) respectively to obtain the light chain expression vector pcDNA3.4-TEH346-L and the heavy chain expression vector pcDNA3.4-TEH346-H.

[0104] 2. Expression of antibody 293T cell culture: Prepare 293T cells in T175 scale culture with cell density of 90%, and use DMEM medium with 8% FBS (purchased from Corning, item number 10-013-CVRC) for culture.

[0105] Transfection of recombinant expression plasmid: Dilute 200 μg of recombinant expression plasmid (100 μg of each of recombinant light chain expression plasmid pcDNA3.4-TEH346-L and recombinant heavy chain expression plasmid pcDNA3.4-TEH346-H) and 200 μL QuickShuttle-293 cell-specific transfection reagent (purchased from Suzhou Boterlong Immunotechnology Co., Ltd., item number KX0110044) with 1 mL of normal saline, mix them after dilution, and directly add them to the cell culture bottle for transfection.

[0106] Cell culture: After transfection, the cells were cultured in a 37ºC 5% CO2 incubator, and the cell supernatant was collected after 3 days of culture for verification.

[0107] 3. Verification of antibody expression Use indirect immunofluorescence assay (IFA) to identify the expressed antibody. The cell supernatant expressing the antibody expression plasmid was used as the primary antibody for IFA experiment with PEDV vaccine strain. The specific steps are the same as those in Example 1, step 6, identification of monoclonal antibody reaction type with virus.

[0108] The results are shown in Figure 4 The expression supernatant reacted with PEDV strain infected cells, showing obvious green fluorescence, and did not react with normal cells, indicating that the antibody was successfully expressed.

[0109] 4. Antibody purification Use Protein A / G 4FF pre-packed chromatography column (purchased from Shengong Bioengineering (Shanghai) Co., Ltd., item number C600983-0501) to purify the expressed antibody.

[0110] Buffer preparation: Use sterile ddH2O to prepare 0.2 M Na2HPO4·12H2O.

[0111] Pre-elution buffer preparation: Use buffer to prepare 0.1 M citric acid, so that the volume fraction of citric acid is 20%.

[0112] Elution buffer preparation: Use buffer to prepare 0.1 M citric acid, so that the volume fraction of citric acid is 60%.

[0113] All reagents should be filtered with 0.22 μm filter before use.

[0114] Sample treatment: Take 30 mL of the cell expression supernatant described above, add the prepared buffer at a volume ratio of 1:1, filter with a 0.22 μm filter, and then prepare for column loading.

[0115] Equilibrium column: slowly pass 10 mL of buffer through the Protein A / G 4FF pre-packed chromatography column at a flow rate of 1 mL / min using a constant flow pump; Loading: slowly pass the treated cell supernatant solution through the Protein A / G 4FF pre-packed chromatography column at a flow rate of 1 mL / min using a constant flow pump.

[0116] Washing: slowly pass 10 mL of washing buffer through the Protein A / G 4FF pre-packed chromatography column at a flow rate of 1 mL / min using a constant flow pump.

[0117] Elution: slowly pass 15 mL of eluent through the Protein A / G 4FF pre-packed chromatography column at a flow rate of 1 mL / min using a constant flow pump, and fractionate the eluted product into 1.5 mL centrifuge tubes.

[0118] 5. Verification of purified antibody Take the purified antibody, prepare the Western blot loading sample using non-reducing Buffer without DTT and reducing Buffer with DTT, and verify the purified antibody.

[0119] The results are shown in the figure: lane 1 is the sample treated with non-reducing Buffer, and there is a single band at greater than 180 kDa, and the results of the antibody treated with reducing Buffer are shown in lane 2, and there are two obvious bands at about 25 kDa and 50 kDa, which are light chain and heavy chain, respectively, indicating that the antibody is well purified, and the recombinant antibody TEH346 is successfully obtained.

[0120] The above describes the present application in detail. For those skilled in the art, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application, and without unnecessary experiments. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

Claims

1. An antibody characterized in that: The antibody is a monoclonal antibody or an antigen-binding fragment thereof, which 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 shown in SEQ ID No: 1, 26-33, 51-57, 96-103, respectively; the 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 shown in SEQ ID No: 2, 27-37, 55-57, 94-102, respectively, and the LCDR1, LCDR2 and LCDR3 are complementarity determining regions.

2. The antibody of claim 1, wherein: 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.

3. The antibody of 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) an active fragment of an antibody, which 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 encoding the antibody of claim 1 or 2 or the active fragment of the antibody of (A1); (A3) an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacteria containing the nucleic acid molecule of (A2); (A4) a kit containing the antibody of claim 1 or 2 or the active fragment of the antibody of (A1) or the nucleic acid molecule of (A2) or the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacteria of (A3).

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

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

7. Use of the antibody of any of claim 1 or 2, or the biological material of claim 4 or 5, in any of the following: (B1) preparing a product for detecting or assisting in detecting whether a sample to be tested contains porcine epidemic diarrhea virus; (B2) preparing a product for detecting or assisting in detecting the content of porcine epidemic diarrhea virus in a sample to be tested. ​ ​ 8. A pharmaceutical composition, characterized by, The active ingredient of the pharmaceutical composition is the antibody according to claim 1 or 2.

9. An ELISA detection kit targeting porcine epidemic diarrhea virus, characterized in that, The kit comprises the antibody according to claim 1 or 2, the biomaterial according to claim 4 or 5.

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