Porcine epidemic diarrhea virus broad-spectrum neutralizing antibody and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a porcine epidemic diarrhea virus broad-spectrum neutralizing antibody and application thereof. A porcine epidemic diarrhea virus broad-spectrum neutralizing antibody 6G2 is screened, the virus neutralizing activity of the antibody is evaluated through a cell model, and it is found that the antibody can effectively block infection of G1a, G1b, G2a, G2b, G2c and S-INDEL (porcine epidemic diarrhea virus) gene subtype strains with high epidemic rate on cells. Further, animal infection experiments of pigs prove that the neutralizing antibody provided by the invention has good prevention and treatment effects on the porcine epidemic diarrhea virus, and has important significance on improving the capability of preventing and controlling porcine epidemic diarrhea in a pig farm.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a broad-spectrum neutralizing antibody against porcine epidemic diarrhea virus and an application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease caused primarily by the porcine epidemic diarrhea virus (PEDV). Clinical symptoms primarily include diarrhea, vomiting, and general dehydration. PEDV belongs to the Coronaviridae family, genus Coronavirus, and is a linear, positive-strand RNA virus with a genome size of approximately 28 kb. It is primarily transmitted through fecal-oral and fecal-nasal routes. PED affects pigs of all ages, with suckling piglets being particularly affected, with a mortality rate as high as 90%. This causes significant economic losses to the pig industry, severely impacting its development and making it a global swine disease.

[0003] The primary means of preventing and treating PEDV is vaccination. However, with the emergence of highly pathogenic strains and the mutation of the virus, vaccination has become less effective, and PEDV infections continue to occur. Therefore, vaccination is no longer sufficient for current situations. Currently, there is no specific treatment for PEDV. Therefore, the development of new drugs that can effectively prevent and treat PEDV is urgently needed.

[0004] Neutralizing antibodies, produced by B cells, bind to surface antigens on pathogens, thereby preventing them from binding to target cell receptors. This is a key step in the body's pathogen clearance. Piglets acquire PEDV-neutralizing antibodies from sows' milk, providing protection in their digestive tract and playing a crucial role in preventing and controlling PEDV infection. Therefore, screening for broad-spectrum neutralizing antibodies against PEDV could support effective prevention and treatment of PEDV. Summary of the Invention

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

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

[0007] In a first aspect of the present invention, a broadly neutralizing monoclonal antibody against porcine epidemic diarrhea virus is provided. 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 the heavy chain variable region: 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: AFRSGSVTATNYPS;

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

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

[0014] The amino acid sequence of the heavy chain variable region and the amino acid sequence of 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, a 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 a biological material comprising a nucleic acid encoding the monoclonal antibody, wherein the biological material is an expression cassette, a transposon, a plasmid vector, a viral vector, or a host cell. By using the biological material to express the nucleic acid encoding the monoclonal antibody, a broadly neutralizing monoclonal antibody against porcine epidemic diarrhea virus can be obtained through genetic engineering.

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

[0019] (1) Neutralization function that inhibits 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 biomaterial in any of the following aspects:

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

[0023] (2) Used for preparing porcine epidemic diarrhea virus detection reagents or kits.

[0024] The examples 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 further provides a porcine epidemic diarrhea virus detection reagent or kit, wherein the reagent or kit contains the monoclonal antibody, the nucleic acid or the biological material.

[0026] The beneficial effects of the present invention are:

[0027] The present invention screened a broad-spectrum neutralizing antibody that can effectively prevent and treat G1a, G1b, G2a, G2b, G2c, and S-INDEL types of PEDV, and constructed a CHO cell line that can stably express the antibody. The broad-spectrum neutralizing antibody can effectively treat and prevent PEDV infection. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figure 2 Relative affinity curves for different genotypes of the virus. The horizontal axis represents the antibody concentration, and the vertical axis represents the OD value at 450 nm. DETAILED DESCRIPTION

[0030] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

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

[0032] 1.1 Cloning of Antibody-Encoding Genes

[0033] Using a commercially available porcine epidemic diarrhea live vaccine (Kefij, Wuhan) as the immunogen, single B cell antibody preparation technology was used to screen and obtain 37 monoclonal B cells positive for antibodies to the PEDV-S1 protein (amino acid sequence shown in SEQ ID NO: 1). Total RNA from the monoclonal B cells was extracted and reverse transcribed into cDNA. The cDNA was then used as a template for PCR amplification of the heavy and light chain variable regions of the naturally paired monoclonal antibodies. The PCR amplification procedure was as follows: initial denaturation at 95°C for 30 seconds, followed by 40 cycles of amplification at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, with a final extension at 72°C for 5 minutes. Electrophoresis of the amplified fragments showed that the fragment sizes were consistent with the expected values. After sequencing verification, the constant region sequences were obtained from the IMGT online database (www.imgt.org), and the antibody gene sequences for the complete light and heavy chains were obtained.

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

[0035] The complete antibody light and heavy chain genes were ligated into the downstream signal peptide of the expression vector pcDNA3.1 via homologous recombination. Sequencing verified the successful construction of the recombinant plasmids. These plasmids were designated as pPED1-VH, pPED1-VL, pPED2-VH, pPED2-VL, pPED3-VH, pPED3-VL, and finally pPED37-VH and pPED37-VL, totaling 37 pairs of expression plasmids.

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

[0037] Plasmids expressing antibody light and heavy chains from the same B cell line were co-transfected into 293F cells. The specific steps are 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 5 The cell suspension was added to a 24-well cell culture plate at 1 mL / well and cultured in a 37°C, 5% carbon dioxide incubator for 20 hours.

[0040] (2) Co-transfection of 293F cells with plasmids expressing antibody light and heavy chains

[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 plasmid 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 highly reactive strains with monoclonal antibodies

[0043] PEDV-S1 protein was prepared at a concentration of 0.5 μg / mL in carbonate buffer (pH 9.6) and added to an ELISA plate at 100 μL / well. The plate was coated 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 blocked at 37°C for 1 hour. After washing with PBST, the cell supernatant collected in 1.3 was diluted 5-fold in blocking buffer and added to the ELISA plate at 100 μL / well. The plate was incubated at 37°C for 1 hour. After washing with PBST, HRP-conjugated goat anti-swine IgG antibody was added and incubated at 37°C for 1 hour. After washing with PBST, 100 μL / well of 0.3 g / L TMB colorimetric solution was added and incubated at 25°C in the dark for 15 minutes. The reaction was terminated by adding 100 μL / well of 2 M H₂SO₄ solution. The absorbance of each well was measured at 450 nm. The results are shown in Table 1, which indicate that 16 of the antibodies had good reactivity with PEDV-S1 protein (the result of S / P>1 was judged as high reactivity; N was the serum of PEDV double-negative SPF pigs, and P was the mixed positive serum sample of pigs routinely immunized with PEDV vaccine).

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

[0045] Antibody No. <![CDATA[OD 450 ]]> Antibody No. <![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: Virus neutralization activity detection of 16 highly reactive antibodies

[0047] 2.1 Virus neutralization activity detection of antibody strains

[0048] The transient expression levels of 16 highly reactive antibody strains in 293F cells were expanded and purified. The purified recombinant porcine monoclonal antibodies were used to detect the virus neutralization activity of each antibody strain against six genotypes of PEDV, namely G1a, G1b, G2a, G2b, G2c, and S-INDEL.

[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) Serially dilute the antibody in MEM basal medium.

[0052] (3) Each dilution of antibody was mixed with 200 TCID 50 / 50μL of G1a, G1b, G2a, G2b, G2c, S-INDEL six genotypes of PEDV were mixed in equal volumes. At the same time, a virus control group was set up: 200TCID 50 Mix 50 μL of G1a, G1b, G2a, G2b, G2c, and S-INDEL PEDV strains with equal volumes of MEM culture medium and incubate at 37°C for 1 hour.

[0053] (4) Each mixed solution was inoculated into the prepared Vero cell culture plate at 100 μL / well, with 4 wells inoculated for each group. At the same time, a 4-well normal cell control group was set up.

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

[0055] (6) The 96-well cell culture plate was placed in a 37°C, 5% carbon dioxide incubator for 48 hours, and the cell pathological changes were observed.

[0056] (7) The neutralizing activity of antibodies was calculated using the Reed-Muench method, and the lowest concentration of antibodies that could protect 50% of cells from pathological changes (IC 50 , μg / mL) was used as an indicator of the neutralizing activity of the antibody.

[0057] Through the above experiments, four monoclonal antibodies with neutralizing activity were screened (7C1, 6G2, 2F5, and 1H12), of which one (6G2) could broadly neutralize the 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 heavy chain variable region is shown in SEQ ID NO: 3. The heavy chain variable region includes a CDR1 with the sequence of DTAFS, a CDR2 with the sequence of SIATGGGGTYYAGSVSG, and a CDR3 with the sequence of 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 light chain variable region is shown in SEQ ID NO:5. The light chain variable region includes a CDR1 with a sequence of AFRSGSVTATNYPS, a CDR2 with a sequence of NTDNRPT, and a CDR3 with a sequence of 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 a CHO cell line stably expressing the neutralizing antibody 6G2 from whole pigs against porcine epidemic diarrhea virus 3.1 Transfection of CHO cells

[0064] (1) CHO cell plating

[0065] The CHO cells in good growth state were digested with trypsin, the cell suspension was counted, and DMEM culture medium containing 10% fetal bovine serum was added to adjust the cell density to 2×10 5 The cell suspension was added to a 24-well cell culture plate at 1 mL / well and cultured in a 37°C, 5% carbon dioxide incubator for 20 hours.

[0066] (2) Linearization of expression plasmid

[0067] In this example, the neutralizing antibody 6G2, which exhibits high neutralizing activity, was selected to construct a stable cell line. Large quantities of the recombinant antibody expression plasmids, pPED-6G2-VH and pPED-6G2-VL, were extracted (i.e., pPED13-VH and pPED13-VL in 1.2). To improve the efficiency of stable cell line screening, the plasmids were digested with NheI into linear fragments, purified, and recovered for transfection.

[0068] (3) Transfection

[0069] Dilute 4 μL of Lipofectamine 2000 reagent with 50 μL of Opti-MEM medium. Then, dilute 1 μg of the linearized heavy chain plasmid and 2 μg of the linearized light chain 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] 3.2G418 antibiotic pressure screening

[0071] 24 hours after transfection, pressure selection with G418 antibiotics was initiated in DMEM supplemented with 800 μg / mL G418 and 10% fetal bovine serum. The medium was changed every 3-5 days. After 10 days, the cells grew into clusters.

[0072] 3.3 Monoclonal cell line screening

[0073] The cells were trypsinized, an appropriate amount of screening medium was added, and the cells were counted and diluted to a density of 1 cell / 200 μL with additional screening medium. The cells were seeded into a 96-well cell culture plate at a density of 200 μL / well and cultured in a 37°C, 5% CO2 incubator. Cell growth was observed, and wells containing only single clones were marked. When the cells grew to 70%-80%, the supernatant was collected and the antibody titer was determined using an established indirect ELISA method (see Section 1.4 of Example 1 for the experimental procedure). Five highly expressing positive clones were screened, as shown in Table 3. The five positive clones were transferred to cell culture flasks for expansion and cryopreservation. A portion of the culture was subjected to limiting dilution and seeded into a 96-well cell culture plate. Positive clones were further screened using the indirect ELISA method. The clone with the highest average signal value among the five positive clones after dilution was selected. After two more limiting dilutions, ELISA screening was performed. All wells containing cells were positive clones, thus obtaining the stable transfected cell line PED-6G2-CHO. Cells from 10 wells were selected for expansion and cryopreservation.

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

[0075] Cell well number 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] The RNA of the stably transfected cell line PED-6G2-CHO was extracted, and the neutralizing antibody gene was amplified after reverse transcription and sequenced. The electrophoresis results of the amplified product were as follows: Figure 1 As shown, the neutralizing antibody gene was successfully amplified. The sequencing results were completely consistent with expectations.

[0078] Example 4: Analysis of relative affinity of broadly neutralizing antibodies to PEDV genotypes

[0079] The purified G1a, G1b, G2a, G2b, G2c, and S-INDEL six genotypes of PEDV were prepared into 10 μg / mL with carbonate buffer (pH 9.6), added to the ELISA plate at 100 μL / well, and placed at 4°C overnight. After washing, 200 μL / well of blocking solution was added, and the plate was blocked at 37°C for 2 hours. After washing, the broad-spectrum neutralizing antibody 6G2 was prepared into 100 ng / mL with blocking solution. After continuous 2-fold dilution, each dilution of the antibody was added to the ELISA plate at 100 μL / well and incubated at 37°C for 1 hour. After washing, enzyme-labeled secondary antibody was added and incubated at 37°C for 1 hour. After washing, TMB colorimetric solution was added at 100 μL / well and incubated at 25°C for 15 minutes. The reaction was terminated by adding the stop solution. The absorbance of each well was detected at 450 nm. The curve was drawn with the antibody concentration as the horizontal axis and OD450nm as the vertical axis (see Figure 2 ) and calculated the antibody concentration corresponding to 50% binding between the antigen and antibody using the curve fitting formula. Lower antibody concentrations indicate a higher relative affinity between the antigen and antibody 6G2. Based on these results, the relative affinity of the broadly neutralizing antibody 6G2 for the six genotypes of the virus, from highest to lowest, is: G2b, G2a, G2c, S-INDEL, and G1a / G1b. See Table 4 for specific data.

[0080] Table 4. Relative affinity of broadly neutralizing antibodies to PEDV genotypes

[0081]

[0082] Example 5: Evaluation of the effectiveness of preventing piglets from being infected with PEDV

[0083] Fifteen healthy piglets (purchased from Rugao Peiqi Agriculture and Animal Husbandry Technology Co., Ltd.) aged 50 days without immunization were selected and tested for antibodies to porcine epidemic diarrhea, classical swine fever, porcine reproductive and respiratory syndrome, pseudorabies, foot-and-mouth disease, and porcine pneumonia using a commercial Elisa test kit. The antibodies were all negative. They were randomly divided into three groups, with 5 pigs in each group. The first group was orally administered with milk, 10 mL / head; the second group was orally administered with milk containing 10 mg of antibody 6G2, 10 mL / head; and the third group was orally administered with milk containing 10 mg of nonspecific porcine IgG, 10 mL / head. 24 hours after administration, the virus was challenged. The first group was orally administered with DMEM culture medium as a control, and the second and third groups were orally administered with 10 5 TCID 5010 mL / pig of G2a PEDV. 24 hours after challenge, another dose was administered: the first group received 10 mL / pig of milk; the second group received 10 mL / pig of milk containing 10 mg of antibody 6G2; and the third group received 10 mL / pig of milk containing 10 mg of nonspecific porcine IgG. The mental state and clinical symptoms of the piglets were observed daily after challenge, and the results are shown in Table 5. Anal swabs were collected from the piglets before challenge, on day 0, and on days 1, 5, and 10 after challenge, for nucleic acid extraction. PEDV viral load was detected using fluorescent quantitative PCR, and the results are shown in Table 6.

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

[0085]

[0086]

[0087] In Table 5, diarrhea assessment (stool consistency score) is as follows: 0: solid; 1: pasty; 2: semi-fluid; 3: liquid. Mental status is scored as follows: 0: normal; 1: listless and slow to move; 2: prone to depression; 3: prone to the ground and dying.

[0088] Table 6. Results of Fluorescence Quantitative PCR of Piglet Anal Swabs

[0089]

[0090] The above results show that the negative control group did not have diarrhea symptoms and was in good mental state. Two pigs in the second group (antibody 6G2) had mild diarrhea and recovered on the 6th day after the infection. All five pigs in the third group (non-specific pig IgG) had severe diarrhea symptoms, and four pigs died on the 9th day after the infection. The results of fluorescence quantitative PCR showed that the second group (antibody 6G2) had two pigs with positive fluorescence quantitative PCR results on the 5th day after the infection, and the remaining three were negative. The PCR results were all negative on the 10th day after the infection. The third group (non-specific pig IgG) has been excreting toxins after the infection, and the fluorescence quantitative PCR results are all positive. The above results show that the monoclonal antibody 6G2 of the present invention has a good effect in preventing porcine epidemic diarrhea.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A broadly neutralizing monoclonal antibody against porcine epidemic diarrhea virus, characterized in that: The amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2, and CDR3 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 a single-chain antibody or an antigen-binding fragment, and the antigen-binding fragment includes a Fab fragment, a F(ab')2 fragment or a single-chain Fv fragment.

3. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region and the amino acid sequence of 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 according to claim 1.

5. The nucleic acid according to claim 4, characterized in that 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 according to claim 4 or 5, wherein the biological material is an expression cassette, a transposon, a plasmid vector, a viral vector or a host cell.

7. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid according to claim 4 or 5, or the biomaterial according to claim 6 in any of the following aspects: (1) Products for the prevention or treatment of porcine epidemic diarrhea virus infection; the products are vaccines or drugs; (2) Used for the preparation of porcine epidemic diarrhea virus detection reagents or kits.

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

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

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

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