Porcine epidemic diarrhea virus broad spectrum neutralizing antibodies and uses thereof

Abstract

This invention belongs to the field of biotechnology, specifically relating to a broad-spectrum neutralizing antibody against porcine epidemic diarrhea virus (PEDV) and its applications. This invention screened a broad-spectrum neutralizing antibody against PEDV, 6G2. Cell model evaluation revealed that the antibody effectively blocked infection of cells by several highly prevalent PEDV genotypes: G1a, G1b, G2a, G2b, G2c, and S-INDEL. Further animal infection experiments in pigs demonstrated that the neutralizing antibody provided by this invention has good preventive and therapeutic effects against PEDV, which is of great significance for improving the ability of pig farms to control PEDV.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a broad-spectrum neutralizing antibody against porcine epidemic diarrhea virus and its application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease mainly caused by porcine epidemic diarrhea virus (PEDV). Clinical symptoms primarily include diarrhea, vomiting, and systemic dehydration. PEDV belongs to the family Coronaviridae, genus Coronavirus, and is a linear positive-sense RNA virus with a genome of approximately 28 kb. It is mainly transmitted through fecal-oral and fecal-nasal transmission. Pigs of all ages can be infected with PED, but suckling piglets are most severely affected, with mortality rates reaching up to 90%. It causes enormous economic losses to the pig industry, seriously impacting its development and becoming one of the most prevalent pig diseases worldwide.

[0003] The primary means of preventing PED is vaccination. However, with the mutation of virus strains and the emergence of highly pathogenic strains, the protective effect of vaccination is not ideal, and PEDV infection occurs frequently. Therefore, vaccination is no longer sufficient to meet the actual needs. Currently, there is no specific drug for PEDV. Therefore, there is an urgent need to develop new drugs that can effectively prevent and treat PED.

[0004] Neutralizing antibodies, produced by B cells, bind to the surface antigens of pathogenic microorganisms, thereby preventing the pathogen from binding to target cell receptors. This is a crucial step in the body's clearance of pathogens. Piglets acquire PEDV neutralizing antibodies from sow's milk, forming a protective barrier in their digestive tract, which is the most important factor in controlling PEDV infection. Therefore, screening for broad-spectrum neutralizing antibodies against PEDV can support effective prevention and treatment of PED. Summary of the Invention

[0005] This invention screened a broad-spectrum neutralizing antibody that can effectively prevent and treat G1a, G1b, G2a, G2b, G2c, and S-INDEL PEDV, and constructed a CHO cell line that can stably express the antibody. This broad-spectrum neutralizing antibody can effectively treat and prevent PEDV infection, providing a new technical means for controlling the spread of PED.

[0006] The present invention specifically achieves the following technical effects:

[0007] In a first aspect, the present invention provides a broad-spectrum neutralizing monoclonal antibody against porcine epidemic diarrhea virus, wherein the amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody are as follows:

[0008] CDR1 of heavy chain variable regions: DTAFS;

[0009] CDR2 of the heavy chain variable region: SIATGGGGTYYAGSVSG;

[0010] CDR3 of the heavy chain variable region: RTEADAIGPVMDL;

[0011] CDR1 of the light chain variable region: AFRGSVTATNYPS;

[0012] CDR2 of the light chain variable region: NTDNRPT;

[0013] CDR3 of the light chain variable region: ALYQSGNVI.

[0014] The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO:3 and SEQ ID NO:5, respectively.

[0015] The monoclonal antibody may also be a single-chain antibody or an antigen-binding fragment, wherein the antigen-binding fragment includes a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment.

[0016] In a second aspect, the present invention provides a nucleic acid encoding the monoclonal antibody, wherein the nucleic acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO:7 and SEQ ID NO:9, respectively.

[0017] In a third aspect, the present invention provides biological materials comprising nucleic acids encoding the monoclonal antibody, wherein the biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell. By expressing the nucleic acid encoding the monoclonal antibody using the biological material, a broad-spectrum neutralizing monoclonal antibody against porcine epidemic diarrhea virus can be obtained through genetic engineering.

[0018] The monoclonal antibody, the nucleic acid, and the biomaterial described above have at least one of the following functions:

[0019] (1) Neutralizing function to inhibit the activity of porcine epidemic diarrhea virus, specifically inhibiting the activity of six subtypes of porcine epidemic diarrhea virus: G1a, G1b, G2a, G2b, G2c, and S-INDEL.

[0020] (2) Detection of porcine epidemic diarrhea virus.

[0021] In a fourth aspect, the present invention provides the use of the monoclonal antibody, the nucleic acid, and the biological material in any of the following aspects:

[0022] (1) A product for the preparation of a vaccine or drug for the prevention or treatment of porcine epidemic diarrhea virus infection. The porcine epidemic diarrhea virus includes G1a, G1b, G2a, G2b, G2c and / or S-INDEL type porcine epidemic diarrhea virus.

[0023] (2) Used to prepare reagents or kits for detecting porcine epidemic diarrhea virus.

[0024] The embodiments of the present invention have verified through experiments that the monoclonal antibody 6G2 of the present invention has a good effect in preventing and treating porcine epidemic diarrhea. Therefore, the present invention also provides a product for preventing or treating porcine epidemic diarrhea virus infection, wherein the product contains the monoclonal antibody, the nucleic acid or the biological material, and the product is a vaccine or a drug.

[0025] The present invention also provides a reagent or kit for detecting porcine epidemic diarrhea virus, wherein the reagent or kit contains the monoclonal antibody, the nucleic acid or the biological material described above.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention screened a broad-spectrum neutralizing antibody that can effectively prevent and treat G1a, G1b, G2a, G2b, G2c, and S-INDEL PEDV, and constructed a CHO cell line that can stably express the antibody. This broad-spectrum neutralizing antibody can effectively treat and prevent PEDV infection. Attached Figure Description

[0028] Figure 1 Electrophoresis diagram of the specific amplified fragment of the .6G2 neutralizing antibody gene. M represents the DL2000 DNA Marker, VH represents the amplified fragment of the heavy chain gene, and VL represents the amplified fragment of the light chain gene.

[0029] Figure 2 Relative affinity curves of different genotype strains. The horizontal axis represents antibody concentration, and the vertical axis represents the OD value at 450 nm. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] Example 1: Screening of high-affinity, whole-porcine-derived broad-spectrum neutralizing antibodies against porcine epidemic diarrhea.

[0032] 1.1 Cloning of antibody-encoding genes

[0033] Using commercially available porcine epidemic diarrhea (PED) live vaccine (Wuhan Keqian, Kefujing) as the immunogen, 37 PEDV-S1 protein antibody-positive monoclonal B cells were screened using single-cell B-cell antibody preparation technology. Total RNA was extracted from the monoclonal B cells and reverse transcribed into cDNA. Subsequently, the heavy chain and light chain variable regions of naturally paired monoclonal antibodies were amplified by PCR using the cDNA as a template. The PCR amplification program was: 95℃ pre-denaturation for 30 s, followed by 40 cycles of 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 1 min, with a final extension at 72℃ for 5 min. Electrophoresis of the amplified fragments showed that the fragment size was consistent with expectations. After sequencing verification, the constant region sequence was obtained from the IMGT online database (www.imgt.org), yielding the complete light chain and heavy chain antibody gene sequences.

[0034] 1.2 Construction of antibody light and heavy chain expression plasmids

[0035] The complete antibody light and heavy chain genes were ligated downstream of the signal peptide of the expression vector pcDNA3.1 via homologous recombination. Sequencing confirmed the successful construction of the recombinant plasmids. These plasmids were named pPED1-VH, pPED1-VL, pPED2-VH, pPED2-VL, pPED3-VH, pPED3-VL, and so on, up to pPED37-VH and pPED37-VL, for a total of 37 pairs.

[0036] 1.3 Transfection of 293F cells to express antibodies

[0037] Plasmids expressing the light and heavy chains of antibodies derived from the same B cell line were co-transfected into 293F cells. The specific procedure is as follows:

[0038] (1) 293F cell plating

[0039] 293F cells in good growth condition were digested with trypsin, the cell suspension was counted, and cell growth medium was added to adjust the cell density to 2 × 10⁻⁶ cells / cells. 5 Add 1 mL of cell suspension to each well of a 24-well cell culture plate and incubate at 37°C with 5% CO2 for 20 hours.

[0040] (2) Plasmids expressing antibody light and heavy chains were co-transfected into 293F cells.

[0041] Dilute 4 μL of Lipofectamine 2000 reagent with 50 μL of Opti-MEM medium. Dilute 1 μg of heavy chain linearized plasmid and 2 μg of light chain linearized plasmid with 50 μL of Opti-MEM medium. Add the diluted plasmids to the diluted Lipofectamine 2000 transfection reagent and incubate at room temperature for 5 minutes. Add the resulting DNA-liposome complex to the 293F cells prepared in (1). Four days after transfection, collect the cell supernatant for detection.

[0042] 1.4 Indirect ELISA screening of high-reactive strains of monoclonal antibodies

[0043] PEDV-S1 protein was prepared into a 0.5 μg / mL solution using carbonate buffer (pH 9.6) and added to an ELISA plate at 100 μL / well, incubated overnight at 4°C. After washing with PBST, 200 μL / well of blocking buffer (0.2 M phosphate buffer containing 0.5% casein, pH 7.4 ± 0.1) was added, and the plate was blocked at 37°C for 1 hour. After washing with PBST, the cell supernatant collected in step 1.3 was diluted 5-fold with blocking buffer and added to an ELISA plate at 100 μL / well, incubated at 37°C for 1 hour. After washing with PBST, HRP-labeled goat anti-pig IgG antibody was added, and the plate was incubated at 37°C for 1 hour. After washing with PBST, 100 μL / well of 0.3 g / L LTMB chromogenic solution was added, and the plate was incubated at 25°C in the dark for 15 minutes. The reaction was terminated by adding 2 M H₂SO₄ solution at 100 μL / well. The absorbance of each well was measured at 450 nm. The results are shown in Table 1. These results indicate that 16 of the antibodies showed good reactivity with PEDV-S1 protein (S / P>1 was considered as high reactivity; where N is PEDV double-negative SPF swine serum and P is a mixed positive serum sample from pigs routinely immunized with PEDV vaccine).

[0044] Table 1. Reactivity of positive antibody strains as determined by ELISA

[0045] Antibody number <![CDATA[OD 450 ]]> Antibody number <![CDATA[OD 450 ]]> N (negative control) 0.069 9B11 1.752 N (negative control) 0.076 9G3 1.676 P (positive control) 1.838 12F9 1.636 P (positive control) 1.882 11C2 1.579 10B7 1.942 11B4 1.537 5A9 1.996 7C2 1.398 9F1 2.004 5G5 1.385 7D8 2.058 5G4 1.324 8F5 2.153 5G10 1.317 12F7 2.292 8C1 1.244 11F8 2.483 8E6 1.236 7H3 2.571 8C2 1.15 2C6 2.738 5A10 1.147 1A3 2.741 3G4 1.033 7C1 2.767 3F1 1.017 4F2 2.775 1B12 0.982 6G2 2.807 4C7 0.931 2F5 2.836 1G12 0.814 5C8 2.849 4H6 0.793 1H12 2.926 3D5 0.728 - - 1F4 0.683

[0046] Example 2: Detection of virus neutralizing activity of 16 high-reactive antibodies

[0047] 2.1 Detection of virus neutralizing activity of antibody strains

[0048] The transient transexpression levels of 16 highly reactive antibody strains in 293F cells were increased and purified. The virus neutralizing activity of each antibody strain against six PEDV genotypes (G1a, G1b, G2a, G2b, G2c, and S-INDEL) was detected using the purified recombinant whole-pig monoclonal antibody.

[0049] The specific steps are as follows:

[0050] (1) Dilute each genotype of PEDV to 200 TCID using MEM basal culture medium. 50 / 50μL.

[0051] (2) Continuously dilute the antibody with MEM basal medium.

[0052] (3) Mix each dilution of antibody with 200 TCID200. 50 Equal volumes of 50 μL of PEDV containing the six genotypes G1a, G1b, G2a, G2b, G2c, and S-INDEL were mixed. A viral control group of 200 TCID50 was also included. 50 Mix 50 μL of PEDV strains of type G1a, G1b, G2a, G2b, G2c, and S-INDEL with an equal volume of MEM culture medium. Incubate at 37°C for 1 hour.

[0053] (4) Take each mixture and inoculate it into the prepared Vero cell culture plate, 100 μL / well, 4 wells per group, and set up a 4-well normal cell control group.

[0054] (5) Add 100 μL of MEM culture medium containing 20 μg / mL trypsin.

[0055] (6) Place the 96-well cell culture plate in a 37°C, 5% carbon dioxide incubator for 48 hours and observe the cell pathogenesis.

[0056] (7) The antibody neutralizing activity was calculated using the Reed-Muench method to determine the minimum antibody concentration (IC50) that protects 50% of cells from cytopathic effects. 50 (μg / mL) is used as an indicator of antibody neutralizing activity.

[0057] Through the above experiments, four monoclonal antibodies with neutralizing activity were screened (7C1, 6G2, 2F5, 1H12), of which one (6G2) could broadly neutralize infection of six subtypes of the virus. The results are shown in Table 2.

[0058] Table 2. Neutralization test results of recombinant antibodies against porcine epidemic diarrhea virus

[0059]

[0060] The full-length amino acid sequence of the heavy chain of the above-mentioned 6G2 monoclonal antibody is shown in SEQ ID NO:2, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:3. The variable region of the heavy chain includes CDR1 with the sequence DTAFS, CDR2 with the sequence SIATGGGGTYYAGSVSG, and CDR3 with the sequence RTEADAIGPVMDL.

[0061] The full-length amino acid sequence of the light chain of the 6G2 monoclonal antibody is shown in SEQ ID NO:4, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:5. The variable region of the light chain includes CDR1 with the sequence AFRGSVTATNYPS, CDR2 with the sequence NTDNRPT, and CDR3 with the sequence ALYQSGNVI.

[0062] The DNA sequence encoding the full-length heavy chain of the 6G2 monoclonal antibody is shown in SEQ ID NO:6, the DNA sequence encoding the variable region of the heavy chain of the 6G2 monoclonal antibody is shown in SEQ ID NO:7, the DNA sequence encoding the full-length light chain of the 6G2 monoclonal antibody is shown in SEQ ID NO:8, and the DNA sequence encoding the variable region of the light chain of the 6G2 monoclonal antibody is shown in SEQ ID NO:9.

[0063] Example 3: Construction and identification of CHO cell lines stably expressing whole-pig neutralizing antibody 6G2 against porcine epidemic diarrhea virus 3.1 Transfection of CHO cells

[0064] (1) CHO cell plating

[0065] CHO cells in good growth condition were digested with trypsin, and the cell suspension was counted. DMEM culture medium containing 10% fetal bovine serum was added to adjust the cell density to 2 × 10⁶ cells / year. 5 Add 1 mL of cell suspension to each well of a 24-well cell culture plate and incubate at 37°C with 5% CO2 for 20 hours.

[0066] (2) Linearization of expression plasmids

[0067] In this embodiment, the neutralizing antibody 6G2, which has high neutralizing activity, was selected to construct a stable cell line. The expression plasmids pPED-6G2-VH and pPED-6G2-VL of this recombinant antibody were extracted in large quantities, namely pPED13-VH and pPED13-VL in section 1.2. To improve the screening efficiency of the stable cell line, the plasmids were digested with NheI to obtain linear copies, purified, and recovered for transfection.

[0068] (3) Transfection

[0069] Dilute 4 μL of Lipofectamine 2000 reagent with 50 μL of Opti-MEM medium. Dilute 1 μg of heavy chain linearized plasmid and 2 μg of light chain linearized plasmid with 50 μL of Opti-MEM medium. Add the diluted plasmids to the diluted Lipofectamine 2000 transfection reagent and incubate at room temperature for 5 minutes. Add the resulting DNA-liposome complex to the prepared CHO cells. Change the medium after 6 hours.

[0070] 3.2 G418 antibiotic stress screening

[0071] Twenty-four hours after transfection, selection was initiated using G418 antibiotic under pressure in DMEM medium containing 800 μg / mL G418 and 10% fetal bovine serum. The medium was changed every 3–5 days. After 10 days, cells began to grow in clusters.

[0072] 3.3 Screening of monoclonal cell lines

[0073] Cells were digested with trypsin, and an appropriate amount of selection medium was added. Cells were counted, and selection medium was added to dilute the cells to a density of 1 cell / 200 μL. The cells were seeded into 96-well cell culture plates at 200 μL / well and incubated at 37°C in a 5% CO2 incubator. Cell growth was observed, and wells with only a single clone were marked. When the cells reached 70%–80% confluence, the supernatant was collected, and antibody titers were detected using the established indirect ELISA method (experimental procedure described in Section 1.4 of Example 1). Five high-expressing positive clones were screened, as shown in Table 3. The five positive clones were transferred to cell culture flasks for expansion culture and cryopreservation. A portion of the culture was used for limiting dilution and seeded into 96-well cell culture plates. Positive clones were screened again using the indirect ELISA method. The clone with the highest average signal value among the diluted positive wells of the original five positive clones was selected and subjected to two more limiting dilution cultures before ELISA screening. All wells containing cells were considered positive clones, thus obtaining the stable cell line PED-6G2-CHO. Ten wells of cells were selected for expansion culture and cryopreservation.

[0074] Table 3. Results of ELISA screening of stable cell lines with high expression of PED-6G2-CHO.

[0075] Cell pore numbering 2N8 3D22 1P19 1P21 3E21 OD450 1.597 1.576 1.619 1.585 1.664

[0076] 3.4 Identification of stable cell lines

[0077] RNA was extracted from the stable cell line PED-6G2-CHO, and the neutralizing antibody gene was amplified after reverse transcription and sequenced. The electrophoresis results of the amplification product are shown below. Figure 1 As shown, this indicates that the neutralizing antibody gene was successfully amplified. The sequencing results are completely consistent with expectations.

[0078] Example 4: Analysis of the relative affinity of broad-spectrum neutralizing antibodies to different PEDV genotypes

[0079] Purified PEDV for six genotypes (G1a, G1b, G2a, G2b, G2c, and S-INDEL) was prepared at 10 μg / mL using carbonate buffer (pH 9.6), and 100 μL / well was added to each well of an ELISA plate and incubated overnight at 4°C. After washing, 200 μL / well of blocking buffer was added, and the plates were blocked at 37°C for 2 hours. After washing, the broad-spectrum neutralizing antibody 6G2 was prepared at 100 ng / mL using blocking buffer, and serially diluted 2-fold. Each dilution of antibody was added to each well at 100 μL / well and incubated at 37°C for 1 hour. After washing, enzyme-labeled secondary antibody was added, and the plates were incubated at 37°C for 1 hour. After washing, TMB chromogenic solution was added at 100 μL / well, and the plates were incubated at 25°C for 15 minutes. The reaction was terminated by adding stop solution. The absorbance of each well was measured at 450 nm. A curve was plotted with antibody concentration on the x-axis and OD450nm on the y-axis (see [link to relevant documentation]). Figure 2 The antibody concentration corresponding to 50% binding between the antigen and antibody was calculated using the fitted curve formula. A lower antibody concentration indicates a higher relative affinity between the antigen and antibody 6G2. Based on the above results, the relative affinity of the broad-spectrum neutralizing antibody 6G2 for the six genotype strains, from highest to lowest, is: G2b, G2a, G2c, S-INDEL, and G1a / G1b. Specific data are shown in Table 4.

[0080] Table 4. Relative affinity of broad-spectrum neutralizing antibodies for different PEDV genotypes

[0081]

[0082] Example 5: Evaluation of the efficacy in preventing PEDV infection in piglets

[0083] Fifteen healthy, unvaccinated piglets aged 50 days (purchased from Rugao Peiqi Agricultural and Animal Husbandry Technology Co., Ltd.) were selected. Antibodies against porcine epidemic diarrhea (PED), classical swine fever (CSF), porcine reproductive and respiratory syndrome (PRRS), pseudorabies, foot-and-mouth disease (FMD), and porcine pneumonia were all negative using a commercially available ELISA kit. The piglets were randomly divided into three groups of five each. Group 1 received oral milk (10 mL / pig), Group 2 received oral milk containing 10 mg of antibody 6G2 (10 mL / pig), and Group 3 received oral milk containing 10 mg of nonspecific porcine IgG (10 mL / pig). Twenty-four hours after administration, the piglets were challenged with viruses. Group 1 received oral DMEM culture medium as a control, while Groups 2 and 3 received oral DMEM culture medium. 5 TCID 50Piglets were challenged with 5 mL of G2a PEDV. Twenty-four hours later, they were given a second dose. The first group received oral milk (10 mL / pig), the second group received oral milk containing 10 mg of antibody 6G2 (10 mL / pig), and the third group received oral milk containing 10 mg of nonspecific porcine IgG (10 mL / pig). The piglets' mental state and clinical symptoms were observed daily after challenge, and the results are shown in Table 5. Anal swabs were collected from piglets before challenge, on day 0 after challenge, and on days 1, 5, and 10 after challenge. Nucleic acid was extracted, and the PEDV viral load was detected using quantitative real-time PCR. The results are shown in Table 6.

[0084] Table 5. Evaluation results of the efficacy of antibody 6G2 in preventing PEDV infection in piglets.

[0085]

[0086]

[0087] Table 5 shows the following diarrhea assessment (stool consistency score): 0: solid; 1: pasty; 2: semi-liquid; 3: liquid. The following mental status scores are also provided: 0: normal; 1: lethargic, slow to move; 2: prefers to lie down, depressed; 3: lying down, barely alive.

[0088] Table 6. Results of quantitative real-time PCR from anal swabs of piglets

[0089]

[0090] The results showed that the negative control group had no diarrhea symptoms and remained in good spirits. Two pigs in the second group (antibody 6G2) experienced mild diarrhea but recovered on day 6 post-challenge. All five pigs in the third group (non-specific porcine IgG) experienced severe diarrhea, and four died on day 9 post-challenge. Quantitative real-time PCR results showed that in the second group (antibody 6G2), two pigs were positive on day 5 post-challenge, while the other three were negative. All PCR results were negative on day 10 post-challenge. The third group (non-specific porcine IgG) continued to shed the virus after challenge, and all PCR results were positive. These results indicate that the monoclonal antibody 6G2 of this invention has a good preventive effect against porcine epidemic diarrhea (PED).

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A broadly neutralizing monoclonal antibody to porcine epidemic diarrhea virus, characterized in that, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain and light chain variable region of the monoclonal antibody are as follows: CDR1 of the heavy chain variable region: DTAFS; CDR2 of the heavy chain variable region: SIATGGGGTYYAGSVSG; CDR3 of the heavy chain variable region: RTEADAIGPVMDL; CDR1 of the light chain variable region: AFRSGSVTATNYPS; CDR2 of the light chain variable region: NTDNRPT; CDR3 of the light chain variable region: ALYQSGNVI.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is an antigen-binding fragment, which includes a Fab fragment, a F(ab')2 fragment or a single-chain antibody.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 3 and SEQ ID NO: 5, respectively.

4. A nucleic acid encoding the monoclonal antibody of claim 1.

5. The nucleic acid of claim 4, wherein The nucleic acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 7 and SEQ ID NO: 9, respectively.

6. A biological material comprising the nucleic acid of claim 4 or 5, which is an expression cassette, a transposon, a plasmid vector, a viral vector or a host cell.

7. Use of the monoclonal antibody of any one of claims 1 to 3, the nucleic acid of claim 4 or 5 or the biological material of claim 6 in any of the following aspects: (1) for preparing a product for preventing or treating porcine epidemic diarrhea virus infection; the product is a medicament; (2) for preparing a porcine epidemic diarrhea virus detection reagent or kit.

8. Use according to claim 7, characterized in that, The porcine epidemic diarrhea virus in (1) is a G1a, G1b, G2a, G2b, G2c and / or S-INDEL type porcine epidemic diarrhea virus.

9. A product for the prevention or treatment of porcine epidemic diarrhea virus infection, characterized in that, The product contains the monoclonal antibody of any one of claims 1 to 3, the nucleic acid of claim 4 or 5 or the biological material of claim 6, and the product is a medicament.

10. A porcine epidemic diarrhea virus detection reagent or kit, characterized by, The reagent and kit contain the monoclonal antibody of any one of claims 1 to 3, the nucleic acid of claim 4 or 5 or the biological material of claim 6.

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