CN116217714B - Full porcine PEDV monoclonal antibody and epitope thereof - Google Patents
Full porcine PEDV monoclonal antibody and epitope thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody of a whole pig-source porcine epidemic diarrhea virus, which comprises a heavy chain, a light chain variable region and a heavy chain and light chain constant region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:3, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 5. The invention also discloses an epitope peptide, the amino acid sequence of which is shown in SEQ ID NO:14, the epitope peptide is capable of specifically binding to the monoclonal antibody. The monoclonal antibody provided by the invention is obtained from a pig body by applying a single B cell antibody technology, can react with various strains of PEDV, has the advantages of strong specificity, high sensitivity and the like, and the epitope obtained by using the antibody is a real epitope of the antibody generated by PEDV in the pig body, and is relatively conservative. The invention lays a foundation for the development of the PEDV diagnosis kit.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a full porcine PEDV monoclonal antibody and an epitope thereof.
Background
Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an acute, highly contagious infectious disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), the main symptoms being vomiting, watery diarrhea and dehydration. The morbidity of PED is high, and the death rate of piglets under 7 days old is as high as 80-100%, which causes serious economic loss to pig industry in China. The rapid and simple diagnosis kit plays an important role in the prevention and control of PED, and a conserved epitope and a highly reactive antibody play an irreplaceable role in the development of the diagnosis kit.
The single B cell antibody technology is the most important method for screening functional diversity antibodies in high flux at present, and the core of the method is to separate antigen specific B cells from immune animal tissues or peripheral blood, then amplify heavy chain and light chain variable region genes of antibodies from single antibody secretion B cells through a single cell antibody gene amplification technology, and finally express the monoclonal antibodies with biological activity in mammalian cells. The method can ensure the natural pairing of the light chain and the heavy chain variable region, can screen antibody genes aiming at different antigenic sites, has good diversity and high preparation efficiency, and has a structure similar to that of an antibody generated in a natural animal body. Because the antibody is obtained from the natural animal body, the identified antigen epitope is the real epitope of the antibody generated by the pathogen in the natural animal body, and the antibody and the antigen epitope obtained by the method can lay a foundation for the diagnosis of the pathogen.
The PEDV has not found the epitope identified by the whole swine antibody, and the conserved epitope is identified by the whole swine antibody and matched with the specific antibody, thereby being beneficial to the diagnosis of pathogens.
Disclosure of Invention
The invention aims to provide a novel conserved PEDV epitope, a specific antibody of the epitope and a preparation method thereof.
The invention is realized in the following way:
one of the purposes of the invention is to provide a monoclonal antibody of porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus) of whole pig source, which comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:3, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 5.
Specifically, the heavy chain constant region is of the IgG1 type and the light chain constant region is of the Lambda type.
The second object of the present invention is to provide a DNA fragment encoding the monoclonal antibody of porcine epidemic diarrhea virus of whole pig origin.
Specifically, the DNA fragment encoding the heavy chain variable region is shown in SEQ ID NO:2, the DNA fragment encoding the light chain variable region is shown in SEQ ID NO: 4.
The third object of the present invention is to provide a recombinant expression vector comprising the DNA fragment.
It is a fourth object of the present invention to provide a host cell comprising said recombinant expression vector.
The invention aims at providing an epitope peptide, the amino acid sequence of which is shown in SEQ ID NO:14, the epitope peptide is capable of specifically binding to the monoclonal antibody.
The invention also provides the application of the monoclonal antibody or the epitope peptide in preparing a porcine epidemic diarrhea virus detection reagent or a kit. For example, a direct ELISA test is performed on porcine epidemic diarrhea virus by using monoclonal antibodies, or an indirect ELISA test is performed on antibodies produced in animals by using epitope peptides.
The seventh object of the present invention is to provide a method for preparing the monoclonal antibody of porcine epidemic diarrhea virus of whole pig origin, which comprises the following steps: immunizing a pig with a PEDV vaccine, taking pig peripheral blood, separating PBMCs from the pig peripheral blood, further obtaining specific B cells capable of recognizing PEDV-S1 protein through flow separation, designing nested PCR primers, amplifying antibody heavy chain variable region and light chain variable region sequences from the B cells, inserting the heavy chain variable region sequences into a carrier plasmid containing a pig IgG1 heavy chain constant region, inserting the light chain variable region sequences into a carrier plasmid containing a Lambda type light chain constant region, constructing heavy chain and light chain complete plasmids, and finally co-transfecting CHO cells with the heavy chain plasmid and the light chain plasmid to express monoclonal antibodies.
The invention has the following beneficial effects:
the monoclonal antibody provided by the invention is obtained from pig body for the first time by using single B cell antibody technology, can react with various strains of PEDV, has the advantages of strong specificity and high sensitivity, and the epitope obtained by using the antibody is a real epitope of the antibody generated by PEDV in pig body, and is relatively conservative. The antigen epitope is matched with a specific antibody thereof, and lays a foundation for the development of a PEDV diagnosis kit.
See the specific examples for more details.
Drawings
Fig. 1: heavy chain plasmid map of the constructed monoclonal antibody.
Fig. 2: constructed monoclonal antibody light chain plasmid map.
Fig. 3: monoclonal antibody protein electrophoresis pattern.
Fig. 4: and (5) detecting the reactivity of the monoclonal antibody.
Fig. 5: schematic representation of truncated fragments of PEDV-S1 protein.
Fig. 6: reactivity of truncated fragments of PEDV-S1 protein with monoclonal antibodies.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Key material description:
SF9 cells: used as host cells in baculovirus expression systems for expressing foreign proteins, stored in the laboratory;
anti-porcine IgM-PE-Cy7 antibody: PE-Cy7 is a compound fluorescein, and the two fluorescein types PE and Cy7 are combined together through covalent bonding, and the anti-pig IgM antibody is marked by PE-Cy7, so that the anti-pig IgM-PE-Cy7 antibody is obtained. Anti-porcine IgM antibodies can specifically bind to porcine IgM molecules and are used to label B cells expressing porcine IgM molecules. The anti-porcine IgM antibodies used in the examples were purchased from BioRad, and PE-Cy7 fluorochromes were purchased from AAT Bioquest.
Anti-porcine IgG-FITC: anti-porcine IgG-FITC antibodies can specifically bind to porcine IgG molecules and are used to label B cells expressing porcine IgG antibodies, available from mybio source corporation.
anti-porcine-CD 3-PE antibody: CD3 is a characteristic molecule on the surface of T cells, and anti-porcine-CD 3-PE can specifically bind to the CD3 molecule, and is used for marking the T cells expressing the CD3 molecule, and is purchased from Bio-Rad company.
An anti-biotin-APC antibody: the anti-biotin-APC antibody can specifically bind to biotin and can be used to bind to biotin-labeled antigen, available from MiLtenyi Biotec.
CH-pcDNA3.4 vector: the CH-pcDNA3.4 vector contained a porcine IgG1 heavy chain constant region, optimized for synthesis by Jin Weizhi biological Co., ltd, and was stored in this laboratory.
CL-pcdna3.4 vector: the CL-pcDNA3.4 vector contains the porcine lambda light chain constant region, synthesized optimally by Jin Weizhi BioLimited, and stored in this laboratory.
CHO-K1-Hi cells: supplied by the pearl Kair company.
EXAMPLE 1 preparation of PEDV-specific monoclonal
1. Preparation of screening antigen
According to the known PEDV-S1 protein (JN 980698.1) nucleotide sequence, designing a primer according to a conventional method, amplifying an S1 protein sequence, recovering a PCR product, performing enzyme digestion, connecting the PCR product to a pFastBacI vector to obtain a pFastBacI-S1 plasmid, then converting the pFastBacI-S1 plasmid into DH10Bac competent cells, obtaining positive pole grains through blue and white spot screening, and transfecting the pole grains into SF9 cells for S1 protein expression.
The primer sequences are shown below:
upstream of S1: ATGAGGTCTTTAATTTACTTCTGG;
downstream of S1: CCTAATACTCATACTAAAGTTGGTG;
the nucleotide sequence of the encoded PEDV-S1 protein is shown as SEQ ID NO: 1.
2. Flow-sorting antigen-specific B cells
Biotin labeling of PEDV-S1 antigen: reference to EZ-Link from Thermo Inc TM Sulfo-NHS-LC-Biotin reagent instructions: a piece of biotin was equilibrated at room temperature, centrifuged transiently, and 180. Mu.L of DNase/RNase free water was added and vortexed. The biotin solution was added to 1mL of antigen and incubated on ice for 3h. And adding the incubated antigen biotin mixed solution into a 30kDa ultrafiltration tube, replacing the PBS buffer solution for 3 times, and removing unlabeled biotin molecules in the solution.
Anti-porcine IgM-PE-Cy7 antibody preparation: reference buckutite TM Rapid PE-Cy7 tandem Antibody labeling Kit labeling: 1.25. Mu.L of Component C was added to 25. Mu.L of anti-porcine IgM antibody; 10 μL of LDMSO was added to Component B and 50 μL of DNase/RNase free water was added to Component A; mu.L of DMSO-Component B mixture was added to IgM-Component C (vortexed), incubated at room temperature for 1h, and 50. Mu.L of Component A was added to the incubated antibody, incubated at room temperature for 1h.
PEDV-S1 positive B cell sorting: peripheral blood of PEDV vaccine immunized pig was collected, density gradient centrifuged, and peripheral blood PBMCs were isolated, counted and diluted to 1X 10 with PBS (1% BSA) 7 mu.L of anti-pig IgG-FITC antibody, 1. Mu.L of anti-pig IgM-PE-Cy7 antibody, 2. Mu.L of PEDV-S1-biotin antigen, 10. Mu.L of anti-pig-CD 3-PE antibody are added to 200. Mu.L of cell suspension per mL, and a control (without antigen PEDV-S1-biotin group) is set, mixed uniformly, incubated at 4℃in the absence of light for 30min,400g/min centrifuged for 5min, PBS (containing 1% BSA) is used to wash the cells 3 times, 200. Mu.L of PBS (containing 1% BSA) is used to resuspend the cells, 2. Mu.L of anti-biotin-APC antibody is added,incubation was performed at 4℃for 30min in the absence of light, centrifuged at 400g/min for 5min and the cells were washed 3 times with PBS (1% BSA).
The separation was performed using a BD FACS Aria II flow cytometer, a 70 μm nozzle was selected during the separation, and after the separation was stabilized, a liquid flow device was turned on, and a droplet delay experiment was performed. After the instrument is debugged, single B cell sorting is carried out, lymphocyte populations are circled firstly, then adhesion cells are eliminated according to FSC-A and FSC-H, and diagonal single lymphocytes are circled. To increase the probability of screening antigen-specific B cells, we first selected a population of anti-porcine IgM-PE-Cy7, anti-porcine-CD 3-PE double negative lymphocytes according to the fluorescent marker score, removed B cells expressing IgM molecules and T cells expressing CD3 molecules from the lymphocyte population, and then further screened antigen-specific B cells double positive for anti-porcine IgG-FITC, PEDV-S1-APC into 96-well PCR plates containing 10. Mu.L lysate.
3. Amplification of antibody variable region genes
After cell sorting was completed, 1. Mu.L of stop solution was added to each well to terminate the lysis process. Then according to SuperScript TM VILO TM The instructions for MasterMix reagent prepared a reverse transcription system cocktail, and 10 μl of reverse transcription cocktail was added to each well for reverse transcription to prepare cDNA molecules.
The amplification of the antibody variable region adopts nested PCR, firstly, the first round uses reverse transcribed cDNA as a template, and H/L-Out-F, H/L-Out-R is used for respectively amplifying; the second round of PCR takes the product of the first round of PCR as a template, and uses nested primers H/L-In-F, H/L-In-R for amplification, and the variable region genes of the heavy chain and the light chain of the antibody can be obtained after the amplification is completed.
Primers for amplifying heavy chain variable regions:
H-Out-F：GTTTCGGCTGAACTGGGTGGTC；
H-Out-R：GGTCACTGRCTCGGGGAAGTAGC；
H-In-F：GGTGGAGTSTGGRGGAGGCCTG；
H-In-R：CAGGGGGCCAGAGGGTAGACC；
primers for amplifying the light chain variable region:
L-Out-F：ATGGCCTGGACGGTGCTTCTGATC
L-Out-R：CCTCCAGGTCACSGTCACG
L-In-F：TCTCAGACTGTGATCCAGGAG
L-In-R：GTCACTTATTAGACACACCAGGGTG
first round amplification system:
first round amplification procedure: 95 ℃ for 5min;95℃30s,56℃50s,72℃1min,34 cycles; and at 72℃for 10min.
Second round amplification system:
second round amplification procedure: 95 ℃ for 5min;95℃30s,60℃50s,72℃1min,35 cycles; and at 72℃for 10min.
The variable region (VH) gene sequence of the antibody obtained by PCR amplification is shown as SEQ ID NO. 2, and the amino acid sequence is shown as SEQ ID NO. 3; the light chain variable region (VL) gene sequence is shown as SEQ ID NO. 4, and the amino acid sequence is shown as SEQ ID NO. 5.
4. Vector construction
4.1 construction of vector expressing the heavy chain constant region of porcine IgG1
Vectors containing the heavy chain constant region of porcine IgG1 were optimized and synthesized by Jin Weizhi biosystems. The gene sequence of the heavy chain constant region (CH) of the pig IgG1 is shown as SEQ ID NO. 6, and the amino acid sequence is shown as SEQ ID NO. 7.
4.2 vector construction for expressing the porcine Lambda light chain constant region
Vectors containing the porcine Lambda light chain constant region were optimized and synthesized by Jin Weizhi biosystems. The gene sequence of the porcine Lambda light chain constant region (CL) is shown as SEQ ID NO. 8, and the amino acid sequence is shown as SEQ ID NO. 9.
4.3 construction of heavy and light chain complete plasmids
The amplified heavy chain variable region (VH) gene codon is optimized and then inserted into a CH-pcDNA3.4 vector containing a pig IgG1 heavy chain constant region through NotI and BbvCI enzyme cutting sites, so as to construct a vector plasmid for expressing the complete heavy chain of the antibody; after codon optimization of the light chain variable region (VL) gene, the vector plasmid for expressing the complete light chain of the antibody was constructed by inserting NotI and AleI into a CL-pcDNA3.4 vector containing a porcine lambda light chain constant region. The constructed heavy chain plasmid map is shown in figure 1, and the constructed light chain plasmid map is shown in figure 2.
The nucleotide sequence of the complete heavy chain of the antibody is shown as SEQ ID NO. 10, and the amino acid sequence is shown as SEQ ID NO. 11; the nucleotide sequence of the complete light chain of the antibody is shown as SEQ ID NO. 12, and the amino acid sequence is shown as SEQ ID NO. 13.
The recombinant expression plasmid converts DH5 alpha competent amplified plasmid, and uses the endotoxin removal plasmid extraction kit to extract plasmid, the operation steps are carried out according to the kit instruction.
5. Antibody expression
The plasmids were co-transfected into CHO cells at a ratio of 1:1 to express antibodies according to the instructions of the pearl Kaiser company.
The specific operation is as follows:
after counting CHO cells, centrifugation was carried out and the density was adjusted to 2X 10 with fresh medium 7 10mL of the cell suspension was transferred to a 125mL shake flask and returned to the incubator, and after shaking culture for 1h, the cell suspension was removed from the incubator for transfection.
85 mu g of the light chain plasmid and 85 mu g of the heavy chain plasmid are added into CHO cells and are fully shaken, then 350 mu L of transfection reagent (TA-CHO) is added and mixed, and after being fully mixed, the cells are put back into an incubator for culture for 6 hours; after 6h of cultivation in the incubator, 20mL of fresh culture solution was added to dilute the transfected cells, and the cells were returned to the incubator for continuous cultivation. After 24h of transfection, 360. Mu.L KE-CHO (CHO cell protein expression enhancer) and 600. Mu.L KT-Feed (instant recombinant protein expression phytone nutrient supplement) were added and transferred to a 32℃incubator for low-temperature expression, 300. Mu.L glucose was supplemented the second day, 600. Mu.L glucose and 600. Mu.L KT-Feed were supplemented the third day, 600. Mu.L glucose was supplemented four and five days, respectively, and the cell supernatant was harvested at 12000rpm/30min on the sixth day.
6. Antibody purification and validation
After filtration of the expressed supernatant with a 0.22 μm filter, the antibody was purified using a pre-packed column of 1mL Protein A from the Ind, as follows: the Protein A column was assembled, 5 column volumes were rinsed with ultrapure water at a flow rate of 1mL/min, 20 column volumes were equilibrated with PBS, the filtered cell supernatant was then loaded at 0.2mL/min, 10 column volumes of PBS were loaded at a flow rate of 1mL/min after loading, and finally 10 column volumes were eluted with elution buffer. The column was washed with ultrapure water for 5 column volumes and kept with 20% ethanol. After purification, the antibody was replaced with PBS using ultrafiltration tube and stored at-20 ℃. 40. Mu.L of the antibody was added to 10. Mu.L of 5 Xprotein reducing loading, the mixture was boiled at 100℃for 10min, and the expression was identified by SDS-PAGE protein electrophoresis, and the results are shown in FIG. 3.
After protein electrophoresis, the antibody (B5) shows two bands, one is about 55KD, the other is about 25KD, and the other is a light chain.
EXAMPLE 2 reactivity of Whole porcine monoclonal antibody
1. ELISA for detecting antibody reactivity
The expressed and purified PEDV-S1 protein is coated in ELISA coating plates at the concentration of 2 mug/mL overnight at 4 ℃, after PBST is washed 5 times, 5% skim milk powder is added for sealing at 37 ℃ for 2h, after PBST is washed 5 times, 2 times of diluted whole pig source antibody is added, incubation is carried out at 37 ℃ for 1.5h, PBST is washed 5 times, 1:5000 diluted anti-pig IgG-HRP antibody is added, incubation is carried out at 37 ℃ for 0.5h, after PBST is washed 5 times, chromogenic solution is added, after incubation is carried out at 37 ℃ for 10min, stop solution is added, and the value of OD630nm is read by an enzyme-labeled instrument.
2. Indirect immunofluorescence detection of antibody reactivity
Different PEDV strains (YN, WHLL, SX, HNAY, HNXX strains) were inoculated in 24-well plates, after inoculation, when typical syncytia of cells appeared, the cells were washed 3 times with PBS, 3 times with 4% paraformaldehyde, 10min with PBS, 3 times with 0.2% Triton-100 permeabilization, 3 times with PBS, 10 μg/mL of antibody, 1.5h incubation at 37 ℃,3 times with PBS, 1:100 dilution of anti-pig IgG-FITC antibody, 0.5h incubation at 37 ℃,3 times with PBS, 1:5000 dilution of DAPI, 3 times with PBS, 1mL of PBS, observation with an inverted fluorescence microscope, and photographing.
The results are shown in FIG. 4.
In ELISA experiments, the PEDV-S1 protein is used for detecting the antibody, the antibody has good reactivity with the PEDV-S1 protein (figure 4A), the reactivity of the antibody with different PEDV strains is further detected through indirect immunofluorescence, the antibody can be specifically reacted with all 5 PEDV strains in our laboratory (figure 4B), and the specific fluorescence appears through indirect immunofluorescence experiment detection, so that the antibody has reactivity with the PEDV strains.
Example 3 identification of antibody-recognizing epitopes
To identify the epitope recognized by the antibody, we constructed a vector of truncated PEDV-S1 gene and pET-32a, expressed the truncated protein (see fig. 5), subjected to SDS-PAGE protein electrophoresis, and transferred the protein to NC membrane by transfer. Blocking for 3h with TBST containing 1% BSA on a shaking table at room temperature, adding 2 μg/mL of antibody diluted with TBST, incubating for 3h on a shaking table at room temperature, incubating anti-pig IgG-HRP antibody diluted 1:5000 after 5 times of TBST washing, incubating for 1h on a shaking table at room temperature, washing for 5 times with TBST, developing with a developing solution, and photographing.
WB found that the antibody could specifically bind to PEDV-S1, we further truncated PEDV-S1 protein to SA, SB, SC, SD, SE, found that the antibody specifically bound to the truncated SE (fig. 6A), then we truncated SE1, SE2, SE3, SE4, found that the antibody specifically bound to SE1 (fig. 6B), further truncated SE1 to SE5, SE6, found that the antibody specifically bound to SE5 (fig. 6C), truncated SE5 again to SE7, SE8, SE9, SE10, found that the antibody significantly reduced reactivity with SE10, but consistent with SE7, SE8, SE9 specific binding capacity (fig. 6D), further truncated SE9 to SE11, SE12, SE13, SE14, SE15, found that the antibody specifically reacted with SE11, SE12, SE13, none of SE14, SE15 (fig. 6E), further truncated SE13 to SE16, SE17, SE18 found that the antibody specifically reacted only with SE16, none of SE17, SE18 (fig. 6F). Epitope SE16 is the major reactive epitope, 16 amino acids FLAGVYYTSDSGQLLA, has not been previously reported, and this epitope is identified and recognized by the antibodies that are actually present in pigs, the true epitope for PEDV to produce antibodies in pigs.
The method comprises the following steps: description of sequence Listing
SEQ ID NO:1: a nucleotide sequence which is PEDV-S1 protein;
SEQ ID NO:2: nucleotide sequence of monoclonal antibody heavy chain variable region (VH);
SEQ ID NO:3: amino acid sequence of monoclonal antibody heavy chain variable region (VH);
SEQ ID NO:4: nucleotide sequence of monoclonal antibody light chain variable region (VL);
SEQ ID NO:5: amino acid sequence of monoclonal antibody light chain variable region (VL);
SEQ ID NO:6: nucleotide sequence of heavy chain constant region (CH) of monoclonal antibody;
SEQ ID NO:7: amino acid sequence of heavy chain constant region (CH) of monoclonal antibody;
SEQ ID NO:8: nucleotide sequence of monoclonal antibody light chain constant region (CL);
SEQ ID NO:9: amino acid sequence of monoclonal antibody light chain constant region (CL);
SEQ ID NO:10: the nucleotide sequence of the complete heavy chain of the monoclonal antibody;
SEQ ID NO:11: the amino acid sequence of the complete heavy chain of the monoclonal antibody;
SEQ ID NO:12: the nucleotide sequence of the complete light chain of the monoclonal antibody;
SEQ ID NO:13: the monoclonal antibody completes the amino acid sequence of the light chain;
SEQ ID NO:14: amino acid sequence of epitope peptide.
Claims (8)
1. A monoclonal antibody to porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus) of whole swine origin, characterized in that: the monoclonal antibody comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 5.
2. The whole pig-derived porcine epidemic diarrhea virus monoclonal antibody of claim 1, wherein: the heavy chain constant region is of the IgG1 type and the light chain constant region is of the lambda type.
3. A DNA fragment encoding the whole porcine epidemic diarrhea virus monoclonal antibody of claim 1.
4. A DNA fragment encoding a monoclonal antibody to porcine epidemic diarrhea virus of whole swine origin as claimed in claim 3, wherein: the DNA fragment encoding the heavy chain variable region is shown as SEQ ID NO:2, the DNA fragment encoding the light chain variable region is shown in SEQ ID NO: 4.
5. A recombinant expression vector comprising the DNA fragment of claim 3 or 4.
6. A host cell comprising the recombinant expression vector of claim 5.
7. An epitope peptide, wherein the amino acid sequence of the epitope peptide is shown in SEQ ID NO:14, which is capable of specifically binding to the monoclonal antibody of claim 1 or 2.
8. Use of the monoclonal antibody of claim 1 or the epitope peptide of claim 7 in the preparation of a porcine epidemic diarrhea virus detection reagent or kit.
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