Porcine epidemic diarrhea virus g2c type mutant strain monoclonal antibody, preparation, antigen epitope identification and application thereof

Abstract

This invention relates to the field of biology, specifically providing a hybridoma cell line targeting the S1 gene of the G2c variant of porcine epidemic diarrhea virus (PEDV) and the specific antigenic epitope recognized by the antibody secreted by this cell line. 680 SGQLLAFKNVTSGAVYSV 697 This invention also discloses the heavy and light chain variable region sequences of the antibody. The hybridoma cell line exhibits high specificity and stability, and the antibody it secretes can specifically recognize the S1 protein of the PEDV G2c variant. This provides key biomaterials and technical support for the rapid diagnosis, vaccine development, and pathogenic mechanism research of the PEDV G2c variant, and has significant application value in the field of livestock disease prevention and control.

Description

Technical Field

[0001] This invention relates to the field of biology, and in particular to a method for preparing a monoclonal antibody against the spike protein S1 of a G2c variant of porcine epidemic diarrhea virus, its antigenic epitope, and its identification. Background Technology

[0002] Porcine epidemic diarrhea (PED) is an acute infectious disease of the digestive tract caused by porcine epidemic diarrhea virus (PEDV), and is classified as a Class II animal disease in my country. First discovered in Europe in 1978, the virus belongs to the alphacoronavirus family and is an enveloped, single-stranded, positive-sense RNA virus (with a genome length of approximately 28 kb). It encodes four structural proteins: spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). The S protein (especially the S1 subunit) is a key protein mediating viral invasion of host cells, a major protective antigen inducing the production of neutralizing antibodies, and a core target for vaccine development and the preparation of specific diagnostic reagents. Typical clinical symptoms of PED include watery diarrhea, vomiting, and dehydration. It has a mortality rate of up to 100% in newborn piglets, causing enormous economic losses to my country's pig farming industry. Precise prevention and control of PED has become a critical issue that urgently needs to be addressed by the industry.

[0003] The PEDV genome has a discontinuous transcriptional mechanism, making it prone to mutation and recombination, leading to continuous variations in viral antigenicity. Since 2010, highly pathogenic PEDV G2 genotype variants have emerged and become widespread in my country. Their antigenicity differs significantly from classic strains (such as CV777), resulting in a substantial decrease in the cross-protective efficacy of traditional vaccines based on classic strains, and frequent PEDV outbreaks still occur in immunized pig herds. In recent years, G2c variants have evolved into the absolutely dominant circulating PEDV strain in my country (such as isolate TJbc2023). Characteristic variations in the key antigenic site of its S protein further weaken the specificity of existing control technologies, leading to bottlenecks in the accurate typing and detection of G2c variants, vaccine evaluation, and research on pathogenic mechanisms. Furthermore, the specific antigenic epitopes of G2c variants have not yet been accurately identified, and there is a lack of monoclonal antibody tools that can specifically identify this type of strain, hindering the development of targeted control technologies.

[0004] Currently, there are significant technological gaps in antibody and epitope research targeting PEDV, making it difficult to meet the control requirements of G2c variants. Specific shortcomings include: (1) Existing antibodies are mostly targeted at classic G1 type or early G2 subtype (such as G2a / G2b), lacking monoclonal antibodies that can specifically identify the dominant circulating strain of G2c type, and some antibodies have the risk of cross-reaction, making it impossible to achieve accurate typing detection of G2c type. (2) The immunogenic linear epitope of the S1 protein of the G2c variant has not been precisely located, and there is a lack of reports on the clear amino acid sequence and corresponding gene sequence, which leads to a lack of core basis for the design of targeted antigens and the development of detection technology. Summary of the Invention To address the aforementioned technological gaps, this study used the PEDV G2c variant (TJbc2023), currently the dominant strain in my country, as the research object. Based on its S1 protein reference sequence, the S1 gene was cloned into the pCold-TF prokaryotic expression vector. After induction of expression, the recombinant S1 protein was purified using imidazole elution at different concentrations. BALB / c mice were immunized with this recombinant protein, and spleen cells were fused with SP2 / 0 myeloma cells. Positive hybridoma cells were obtained through indirect ELISA screening and subcloning purification, ultimately successfully preparing a high-titer, high-specificity monoclonal antibody (1E10). IFA and Western blot experiments verified that the antibody specifically recognizes the S1 protein, and subtype identification showed that its heavy chain is IgG2a and its light chain is Kappa. Furthermore, by constructing a series of truncated S1 protein expressions, the antigenic epitope recognized by the antibody was precisely identified as amino acids 680-697 of the S1 protein, corresponding to the encoded amino acid sequence SGQLLAFKNVTSGAVYSV (SEQ ID NO.28).

[0005] Monoclonal antibody 1E10, characterized in that the amino acid sequence of the heavy chain variable region of monoclonal antibody 1E10 is as follows: QVQLQQSGAELARPGASVKLSCKAS GYTFTAYW VQWVRQRPGQGLEWIGA IYPGNDDI NYSPKFWGRATVTVDKSSSTAYMQLSSLATEDSAVYFC ARGGY WGQGTSVTVSS (SEQ ID NO.21), HCDR1-3 are respectively: GYTFTAYW (SEQ ID NO.22); IYPGNDDI (SEQ ID NO.23) and ARGGY (SEQ ID NO.24); The amino acid sequence of the light chain variable region is as follows: DVLMTQTPLSLPVSLGDQASISCRSS QSIIHSNGNTY LEWYLQKPGQSPKLLIY KGA NRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYC FQASHVPWTFGGGTKLEIK (SEQ ID NO.25), LCDR1-3 are respectively: QSIIHSNGNTY (SEQ ID NO.26); KGA and FQASHVPWT (SEQ ID NO.27).

[0006] This study is the first to obtain the G2c variant-specific monoclonal antibody 1E10 and its antigenic epitope, overcoming the technical limitations of existing long-fragment epitopes. This epitope has the advantages of small molecular weight, easy recombination expression, and strong binding specificity. It lays the foundation for the development of G2c-specific diagnostic reagents (such as IFA and Western blot detection kits), vaccine evaluation, and research on the pathogenic mechanism of PEDV G2c variants. It also has important application value for improving the precise prevention and control capabilities of dominant PED strains in my country. Attached Figure Description

[0007] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0008] Figure 1 This is an electrophoresis diagram of the PCR amplification of the gene sequence fragment encoding PEDV S1 in Example 1 of this invention. Lane 1 represents the DNA molecular weight standard DL2000, and lanes 2-3 represent the PEDV S1 gene fragment, which is 2187 bp in size, consistent with the expected result, indicating that the correct size gene fragment was successfully amplified.

[0009] Figure 2-3 This is a Coomassie brilliant blue staining identification image of S1-induced expression in Example 2. Among them, Figure 2 Lane M represents the relative molecular mass standard of the protein; lane 1 is the pCold-TF empty vector induction control; lane 2 is the whole bacteria after pCold-TF-S1 induction; lane 3 is the supernatant after pCold-TF-S1 induction; and lane 4 is the precipitate after pCold-TF-S1 induction. This indicates that the expression level of recombinant S1 in the supernatant is high, which can be used to purify the antigen protein. After elution with different gradient imidazoles, Figure 3 Lanes 3-5 contain recombinant proteins eluted with 200mM imidazole. Figure 3 Lanes 6-10 contain recombinant protein eluted with 400 mM imidazole. This indicates that the recombinant S1 antigen protein was purified effectively, with a single band approximately 126 kDa, consistent with expectations. This suggests that the purified protein is correct and suitable for subsequent immunization.

[0010] Figure 4 The image shown in Example 5 is an IFA diagram of Vero-E6 cells infected with PEDV TJbc2023 strain using the 1E10 monoclonal antibody, demonstrating that the antibody can specifically recognize the PEDV S1 protein.

[0011] Figure 5 This is a Western blotting image of Vero-E6 cells infected with PEDV TJbc2023 strain using the 1E10 monoclonal antibody in Example 5, demonstrating that the antibody can specifically recognize the PEDV S1 protein.

[0012] Figure 6 The results of the 1E10 monoclonal antibody subtype detection in Example 5 indicate that the heavy chain of this antibody is IgG2a and the light chain is Kappa.

[0013] Figure 7 This is a Western blotting image of the antigen epitopes recognized by the 1E10 monoclonal antibody in the preliminary segmentation screening in Example 6, indicating that the truncated S1 fragments recognized by this antibody all contain peptides. 680 SGQLLAFKNVTSGAVYSV 697 .

[0014] Figure 8 These are the sequencing results of the mAb-1E10 heavy and light chain variable regions from Example 7 of this invention.

[0015] Figure 9 It is a homology analysis of antigenic epitopes in different strains. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0017] Example 1: Construction of the recombinant prokaryotic expression vector pCold-TF-S1 The inventors first constructed the pCold-TF-S1 prokaryotic expression plasmid, as follows: Specific primers targeting the S1 gene were designed (primer information is shown in Table 1, SEQ NO: 1-2). Using PEDV TJbc2023 strain cDNA as a template, the S1 gene was amplified by PCR using these specific primers. The amplification product was then ligated into the pCold-TF prokaryotic expression vector. Sequencing alignment results showed that the inserted gene sequence was completely identical to the S1 gene sequence of the PEDV TJbc2023 strain. This recombinant plasmid was named pCold-TF-S1.

[0018] Table 1. Specific primers for the S1 gene

[0019] The specific PCR reaction conditions were set as follows: pre-denaturation at 98℃ for 3 min; followed by 35 cycles, each cycle consisting of 98℃ denaturation for 10 s, 60℃ annealing for 30 s, and 72℃ extension for 2 min 30 s; and a final extension at 72℃ for 10 min. After electrophoresis, the PCR products were observed and photographed under UV light. The recombinant plasmid was identified by PCR using the primers designed above. Observation under UV light after electrophoresis showed that a specific band of approximately 2187 bp was amplified. Figure 1 The size of the recombinant plasmid is consistent with the expected fragment size, indicating that the recombinant plasmid was successfully constructed.

[0020] Example 2: Induction and purification of recombinant prokaryotic expression vector pCold-TF-S1 The pCold-TF-S1 plasmid and the pCold-TF empty vector plasmid were transformed into BL21 competent cells and evenly spread on LB solid agar medium containing ampicillin. The cells were then incubated upside down at 37°C. Single colonies were picked and inoculated into LB liquid medium containing ampicillin, and incubated overnight at 37°C with shaking. The next day, 1 mL of each colony was added to fresh LB liquid medium containing ampicillin and incubated at 37°C with shaking. When the OD of the bacterial culture was approximately 0.6–0.8, IPTG was added, and the culture was induced at 16°C for 16 h. The bacterial culture was collected by centrifugation and sonicated, and then subjected to SDS-PAGE electrophoresis for identification. The results were used to analyze the induced expression of plasmid pCold-TF-S1. After successful identification, the remaining bacterial culture from the induction group was centrifuged at high speed, and the bacterial cells were collected and sonicated. SDS-PAGE electrophoresis was then performed to analyze whether the recombinant S1 protein was expressed in the supernatant or in inclusion bodies. Figure 2 As shown, the recombinant S1 protein was mainly expressed in the supernatant. The recombinant protein was purified using Ni-NTA agarose from Beyotime Biotechnology. The general procedure was as follows: a large amount of His-tagged S1 bacterial culture was collected, washed twice with sterile PBS, and the cells were disrupted using an ultrasonic homogenizer. The cells were then centrifuged at low temperature and high speed, and the supernatant was collected. The supernatant was bound to pre-treated Ni-NTA agarose at 4°C for 2 hours, followed by washing with a wash buffer containing 200 mM and 400 mM imidazole. The eluent containing 200 mM and 400 mM imidazole was collected and subjected to SDS-PAGE gel electrophoresis to observe the purification effect of the recombinant protein. The purified protein was then subjected to SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue.

[0021] Example 3 Animal Immunization Eight 6-week-old female BALB / c mice were randomly divided into an immunization group and a negative control group, with four mice in each group. Before immunization, the concentration of recombinant S1 protein derived from the PEDV G2c variant TJbc 2023 was quantified using the BCA method to ensure accurate immunization dosage. Immunization was performed via subcutaneous injection in the back, with immunization intervals of 14 days. Each mouse received 100 μg of recombinant S1 protein per immunization. For the first immunization, the recombinant S1 protein was thoroughly emulsified with an equal volume of Freund's complete adjuvant; for the second and third immunizations, an equal volume of Freund's incomplete adjuvant was used to emulsify the protein, thereby enhancing immunogenicity and reducing local irritation. Seven days after the last immunization, blood was collected from all mice via the orbital venous plexus, and serum was separated by centrifugation after standing. The titer of anti-S1 protein-specific antibodies in the serum of the immunized group was measured using an indirect ELISA method, while the serum of the negative control group was also measured to exclude interference from non-specific reactions.

[0022] Example 4: Preparation of PEDV S1 monoclonal antibody (1) Resuscitation and culture of SP2 / 0 cells Resuscitate SP2 / 0 cells and adjust cell density approximately 5 days before fusion. The procedure is as follows: Remove SP2 / 0 frozen cells from liquid nitrogen, place them in a sealed bag, and quickly place them in a 37°C water bath to thaw. After complete thawing, centrifuge horizontally at 1000 rpm for 5 min, aspirate the supernatant in a biosafety cabinet, resuspend the cell clumps in 1 mL of complete culture medium, and then seed them into cell culture dishes for culture.

[0023] (2) Preparation of feeder cells One day prior to fusion, blood was collected from the eyes of mice in the negative control group (serum was used for subsequent negative controls). The mice were then euthanized and disinfected by immersion in 75% alcohol. In a biosafety cabinet, the mice were placed supine on sterile foam boards. The abdominal skin was lifted with forceps, and a small hole was cut at the corresponding location in the abdominal fat layer. The skin was then torn open along the hole to expose the peritoneal cavity. 5 ml of LDM-MEM culture medium was injected into the peritoneal cavity. The peritoneum was gently rubbed with alcohol swabs to suspend the peritoneal macrophages. The culture medium was aspirated after flicking a centrifuge tube, and this procedure was repeated. The collected culture medium was added at 100 μL / well to a 96-well plate and incubated at 37°C in a 5% CO2 incubator. Cell growth was observed the following day; cells in good condition were ready for subsequent fusion.

[0024] (3) Spleen cell preparation Blood was collected from the eyes of immunized mice (serum was used as a subsequent positive control), and the mice were then sacrificed and soaked in 75% alcohol for 7 min. After fixing the mice, the abdominal skin was cut open with sterile scissors, and the spleen was aseptically removed and placed in a culture dish. A 40 μm cell sieve was placed on a 50 mL centrifuge tube, and the spleen was ground with a disposable grinder. DMEM culture medium was added to rinse the spleen, and the homogenate was collected and centrifuged at 1000 rpm for 5 min.

[0025] (4) Hybridoma cell fusion SP2 / 0 myeloma cell suspension and immune spleen cell suspension were mixed at a ratio of 1:3 in a 50 mL centrifuge tube. DMEM culture medium was added to a final volume of 30 mL, and the mixture was thoroughly mixed. The tube was centrifuged at 1000 rpm for 10 min. The supernatant was discarded, and the bottom of the tube was gently tapped to form a cell pellet. The centrifuge tube was placed in a 37°C warm water foam box, and 1 mL of preheated PEG at 37°C was slowly added dropwise over 1 min while rotating the tube. The mixture was then allowed to stand for 2 min. Subsequently, DMEM basal culture medium was slowly added dropwise to dilute the PEG and terminate the fusion. The tube was centrifuged again at 1000 rpm for 10 min. The cells were resuspended in 20% serum HAT culture medium preheated at 37°C and seeded at 100 μL / well into a 96-well plate containing feeder cells. The plates were then incubated at 37°C in a 5% CO2 incubator. The fusion status was observed 5 days after fusion, and HAT culture medium was added as needed. On day 10 after fusion, 50 μL of cell supernatant was aspirated from each well using a multipipe, and positive hybridoma cells secreting the target antibody were screened using an indirect ELISA.

[0026] (5) Screening of positive hybridoma cells Screening was performed using an indirect ELISA method. The supernatant of fusion cells was used as the primary antibody, SP2 / 0 cell supernatant as the negative control, serum from pre-fusion immunized mice as the positive control, and HRP-labeled goat anti-mouse IgG as the secondary antibody. The specific procedure was as follows: Purified S1 protein was used to coat ELISA plates, 4 μg per well (100 μL volume), and coated overnight at 4°C. The next day, 100 μL of 5% skim milk was added to each well, and the plates were blocked at 37°C for 2 hours. After washing three times with PBST, 100 μL of cell supernatant was added, and the plates were incubated at 37°C for 1 hour. The plates were then washed three times with PBST containing 5% Tween-20 (5 min each time), and 100 μL of a 1:5000 diluted secondary antibody was added, and the plates were incubated at 37°C for 45 minutes. After washing three times with PBST, 100 μL of TMB chromogenic solution was added to each well, and the plates were incubated at room temperature in the dark for 15 minutes. Finally, 50 μL of 2M H2SO4 was added to stop the reaction, and the OD was detected using a microplate reader. 450 The positive cell wells with the highest OD values ​​were selected for expansion culture, followed by three subcloning operations. After each subcloning, the above-mentioned indirect ELISA method was used for screening. Finally, the hybridoma cell line 1E10, which can stably secrete S1 antibody, was obtained and expanded cultured.

[0027] Example 5: Specificity detection and subtype identification of monoclonal antibodies To verify the specificity of the monoclonal antibody, Vero-E6 cells were infected with PEDV TJbc2023 strain, and IFA and Western blotting analyses were performed. IFA results showed that the 1E10 monoclonal antibody specifically recognized PEDV-infected cells, exhibiting specific green fluorescence, while uninfected control cells showed no obvious fluorescence. Figure 4 Western blotting results showed that the 1E10 monoclonal antibody could recognize specific natural S protein bands with a molecular weight of approximately 180 kDa or higher, with clear bands and no obvious non-specific contaminants. Figure 5 ).

[0028] Further identification of antibody isotypes using a Proteintech monoclonal antibody isotype identification kit revealed that only the OD450 nm values ​​corresponding to the IgG2a heavy chain and Kappa light chain were significantly elevated, while the absorbance signals corresponding to the other isotypes remained at low levels without significant increases. This confirmed that the heavy chain isotype of this monoclonal antibody was IgG2a, and the light chain isotype was Kappa. Figure 6 ).

[0029] Example 6: Identification of the recognition epitope of the S1 monoclonal antibody To clarify the epitope region recognized by the 1E10 monoclonal antibody, PEDV S1 (1-729 aa) was first divided into three fragments (S1-1: 1~486 aa; S1-2: 244~486 aa; S1-3: 486~729 aa). Expression plasmids were constructed using primers from SEQ NO:3-8 in Table 1. A schematic diagram of the fragment division is shown below. Figure 7 A. PCR reaction conditions were set as follows: pre-denaturation at 98℃ for 3 min; followed by 35 cycles of denaturation (98℃ for 10 s), annealing (60℃ for 30 s), and extension (72℃ for 1 min); finally, a final extension at 72℃ for 10 min. Each fragment was ligated into the pCold-TF vector, transformed into BL21 competent cells, and positive clones were screened. Single colonies were picked to verify their validity before induction of expression. Bacteria were cultured at 37℃ in a shaker until the OD value reached 0.6–0.8, then 1 mM IPTG was added, and the cells were induced at 16℃ for 16 h. The cells were then collected and sonicated. Results showed that the epitope recognized by the 1E10 monoclonal antibody was located in the S3 region (244–729 aa). Figure 7 B).

[0030] The S1-3 fragment was further truncated into three sub-fragments (S1-3-1: 487~579 aa, S1-3-2: 562~654 aa, and S1-3-3: 637~729 aa). Using the primers listed in Table 1 (SEQ NO: 9-14), the same PCR amplification, induced expression, and identification methods were employed. The results showed that the epitope recognized by this monoclonal antibody was confined to the S1-3-3 region (654~729 aa). Figure 7 C).

[0031] To determine the minimal epitope, the S1-3-3 fragment was further truncated into three fragments (S1-3-3-1: 654~680 aa; S1-3-3-2: 669~719 aa; S1-3-3-3: 697~729 aa). Using the primers of SEQ NO:15-20 in Table 1, amplification, expression, and identification were completed using the aforementioned experimental method. The results confirmed that the minimal epitope of PEDV S1 recognized by the 1E10 monoclonal antibody is amino acids 680~697, corresponding to the following sequence: 680 SGQLLAFKNVTSGAVYSV 697 ( Figure 7 D).

[0032] Example 7: mAb-1E10 variable region sequence determination After complete lysis of the 1E10 monoclonal cell line with an appropriate amount of Trizol Reagent, total RNA was extracted and reverse transcribed into cDNA. The cDNA was then transferred to Suzhou Genewise Biotech Co., Ltd. for sequencing. The sequencing results of the antibody heavy chain (VH) and light chain (VL) were compared with antibody databases (IMGT, etc.) to obtain relevant information such as CDR1 / 2 / 3, and to determine the gene sequence of the variable region. The variable region sequences of mAb-1E10 heavy chain (VH) and light chain (VL) are shown below. Figure 8 As shown.

[0033] To further verify the broad-spectrum detection performance of the 1E10 monoclonal antibody obtained in this invention against porcine epidemic diarrhea virus (PEDV), this study used the 1E10 monoclonal antibody to conduct specific detection experiments on wild-type PEDV strains carrying different mutation sites. Systematic detection and verification results showed that the 1E10 monoclonal antibody could produce a specific positive immune response with all tested wild-type PEDV strains, with no negative or false-negative results. This fully demonstrates that the monoclonal antibody has the ability to broadly recognize wild-type PEDV strains and can be stably and efficiently applied to PEDV detection scenarios, possessing excellent application potential and practical value as a dedicated PEDV detection reagent.

[0034] Based on this, the present invention further performs bioinformatics alignment and sequence conservation analysis on the core region of the antigenic epitope of the above-mentioned wild-type epidemic strain of porcine epidemic diarrhea virus. The corresponding sequence conservation analysis results are detailed in [link to relevant documentation]. Figure 9 Sequence analysis results showed that the antigenic epitope region targeted by the 1E10 monoclonal antibody was highly conserved and stable in all tested wild-type circulating strains of porcine epidemic diarrhea virus (PEDV), without any key amino acid mutations or epitope deletions. The above experimental and sequence analysis results corroborate each other, further confirming that the 1E10 monoclonal antibody screened in this invention can broadly, accurately, and stably recognize different variants of wild-type PEDV strains, effectively achieving efficient detection of PEDV in clinical settings and samples, and providing a reliable core detection raw material for the clinical diagnosis, epidemiological monitoring, and epidemic prevention and control of PEDV.

[0035] In summary, this invention provides a method for preparing a monoclonal antibody targeting the antigenic epitope of PEDV S1 and its application. A prokaryotic expression vector capable of expressing PEDV S1 was constructed by cloning the PEDV S1 coding sequence into the pCold-TF vector, and the PEDV S1 protein was obtained through His tag purification. The purified protein was used as an immunogen to immunize BALB / c mice, and a monoclonal antibody against PEDV S1 was obtained through cell fusion and subclonal selection. Identification results showed that the prepared monoclonal antibody had an IgG2a heavy chain and a Kappa light chain. IFA and Western blotting analyses showed that the obtained monoclonal antibody specifically recognizes PEDV S1, and its recognized epitope sequence is [epitope sequence missing]. 680 SGQLLAFKNVTSGAVYSV 697 .

[0036] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A monoclonal antibody 1E10 targeting the G2c type PEDV S1 protein, characterized in that... The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E10 is as follows: QVQLQQSGAELARPGASVKLSCKAS GYTFTAYW VQWVRQRPGQGLEWIGA IYPGNDDI NYSPKFWGRATVTVDKSSSTAYMQLSSLATEDSAVYFC ARGGY WGQGTSVTVSS The amino acid sequence of the light chain variable region is as follows: DVLMTQTPLSLPVSLGDQASISCRSS QSIIHSNGNTY LEWYLQKPGQSPKLLIY KGA NRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYC FQASHVPWT FGGGTKLEIK, where the underlined part is CDR1-3.

2. An antigenic epitope of the S1 protein of G2c porcine epidemic diarrhea virus (PEDV), characterized in that, The antigenic epitope is the peptide segment at positions 680-697 of the PEDV S1 protein, and its amino acid sequence is as follows: 680 SGQLLAFKNVTSGAVYSV 697 The antigenic epitope can be specifically recognized and bound by the monoclonal antibody 1E10 according to claim 1, and can be used for the specific recognition of G2c type PEDV S1 protein in indirect immunoassay and Western blotting experiments.

3. An antibody reagent, characterized in that, The antibody reagent comprises an antibody or a functional fragment thereof that specifically binds to the G2c type PEDV S1 protein antigenic epitope of claim 2; the antibody is the monoclonal antibody 1E10 of claim 1.

4. The use of the monoclonal antibody of claim 1, the antigenic epitope of claim 2, and the antibody reagent of claim 3 in the preparation of reagents for detecting PEDV.

5. A method for detecting porcine epidemic diarrhea virus (PEDV) for non-disease diagnostic purposes, characterized in that, The monoclonal antibody of claim 1 or the antibody reagent of claim 3 was used to detect PEDV in the test sample.

6. The detection method according to claim 5, characterized in that, The detection method is applicable to rapid detection of environmental or water samples, disease screening in aquaculture farms, and epidemiological strain monitoring.

7. The use of the monoclonal antibody according to claim 1 in the preparation of a targeted drug for the prevention or treatment of porcine epidemic diarrhea virus infection.

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