A monoclonal antibody against porcine epidemic diarrhea virus n protein and an antigen epitope peptide recognized by the monoclonal antibody and application thereof

Abstract

The application discloses a kind of anti-swine epidemic diarrhea virus N protein monoclonal antibody and the antigen epitope peptide and application identified by it, belong to monoclonal antibody technical field.The heavy chain variable region of the monoclonal antibody 8G2 includes CDR1 with the amino acid sequence of NHGMN, CDR2 with the amino acid sequence of WINTNSGEPTYAEEFKG and CDR3 with the amino acid sequence of EGYYP;Light chain variable region includes CDR1 with the amino acid sequence of KSSQSLLDSDGQTYLN, CDR2 with the amino acid sequence of LVSKLDS and CDR3 with the amino acid sequence of WQGTHFPL.The amino acid sequence of the antigen epitope identified is DLKDIPEWR.The 8G2 monoclonal antibody prepared in the application is widely applied, and can be used in laboratory and clinical application, and lays an important foundation for the clinical detection of PEDV and its pathogenesis research.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, and in particular to a monoclonal antibody against the N protein of porcine epidemic diarrhea virus, its recognized antigenic epitope peptide, and its applications. Background Technology

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious infectious disease caused by porcine epidemic diarrhea virus (PEDV). The main symptoms are vomiting, watery diarrhea, and dehydration. PED has a high morbidity rate and is one of the most serious diseases affecting the livestock industry, causing huge economic losses to the global pig farming industry every year. PEDV can infect pigs of all ages; adult pigs mainly show symptoms such as weight loss and malnutrition, but the mortality rate in suckling piglets under one week old is as high as 80-100%.

[0003] PEDV belongs to the genus Coronavirus of the family Coronaviridae. It is an enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 28 kb, including a 5' cap and a 3' polyadenylated end. It contains seven open reading frames (ORFs): ORF1a, ORF1b, nucleocapsid protein (N), membrane protein (M), spike protein (S), envelope protein (E), and accessory protein (ORF3). The PEDV N protein is highly conserved and possesses high immunogenicity; early viral infection can induce a large number of antibodies against the N protein. Therefore, the N protein can serve as a target for early detection of PEDV infection and has significant application value in clinical research.

[0004] During their research, the inventors unexpectedly obtained a monoclonal antibody against PEDV N protein based on PEDV N protein. This monoclonal antibody can specifically recognize and bind to N protein, providing a reliable tool for exploring the function of PEDV N protein and also providing raw materials for the development of PEDV diagnostic reagents. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody against the N protein of porcine epidemic diarrhea virus (PEDV), its recognized antigenic epitope peptide, and its applications, in order to solve the problems existing in the prior art. The 8G2 monoclonal antibody prepared by this invention has a wide range of applications and can be used in laboratory and clinical applications, laying an important foundation for the clinical detection of PEDV and its N protein antigen.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a monoclonal antibody 8G2 against the N protein of porcine epidemic diarrhea virus. The heavy chain variable region of the monoclonal antibody 8G2 includes CDR1 with the amino acid sequence NHGMN, CDR2 with the amino acid sequence WINTNSGEPTYAEEFKG, and CDR3 with the amino acid sequence EGYYP; the light chain variable region of the monoclonal antibody 8G2 includes CDR1 with the amino acid sequence KSSQSLLDSDGQTYLN, CDR2 with the amino acid sequence LVSKLDS, and CDR3 with the amino acid sequence WQGTHFPL.

[0008] Optionally, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 8G2 is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of the monoclonal antibody 8G2 is shown in SEQ ID NO.2.

[0009] The present invention also provides a gene encoding the monoclonal antibody 8G2, wherein the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8G2 is shown in SEQ ID No. 3, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8G2 is shown in SEQ ID No. 4.

[0010] The present invention also provides an expression cassette or recombinant vector containing the gene.

[0011] The present invention also provides a host bacterium containing the recombinant vector.

[0012] Optionally, the monoclonal antibody 8G2 can induce a specific immune response with porcine epidemic diarrhea virus or its N protein antigen.

[0013] Optionally, the amino acid sequence of the antigenic epitope peptide recognized by the monoclonal antibody 8G2 is DLKDIPEWR.

[0014] Optionally, the heavy chain subtype of the monoclonal antibody 8G2 is IgG1, and the light chain subtype is Kappa.

[0015] The present invention also provides the use of the monoclonal antibody 8G2, the gene, the expression cassette, the recombinant vector, or the host bacterium in the preparation of a detection product for porcine epidemic diarrhea virus or its N protein.

[0016] The present invention also provides a product for detecting porcine epidemic diarrhea virus or its N protein antigen, the product comprising the monoclonal antibody 8G2 or the gene or the expression cassette or recombinant vector or the host bacterium.

[0017] The present invention discloses the following technical effects:

[0018] This invention provides a monoclonal antibody, 8G2, against the nucleocapsid protein of porcine epidemic diarrhea virus (PEDV). This monoclonal antibody specifically recognizes PEDV and can be used in ELISA, Western blotting, in vitro anatomical analysis (IFA), intracellular immunoassay (IHC), and flow cytometry. The heavy chain isotype was identified as IgG1, and the light chain isotype as Kappa. The amino acid sequence of the recognized antigenic epitope is 274-DLKDIPEWR-282. The 8G2 monoclonal antibody prepared by this invention has wide applications in both laboratory and clinical settings, providing new ideas and methods for the prevention and control of PEDV. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The results are for the titer of the purified antibody.

[0021] Figure 2 To detect N protein expression at different time points after PEDV infection using IFA;

[0022] Figure 3 Western blot analysis was used to detect the expression of N protein in PEDV-infected cells at different time points; 1: blank cells; 2: 6 h after infection; 3: 12 h after infection; 4: 24 h after infection; 5: 36 h after infection; 6: 48 h after infection; 7: 60 h after infection; 8: 72 h after infection;

[0023] Figure 4 Figure showing the positive rate of PEDV-infected cells at different time points as detected by flow cytometry.

[0024] Figure 5 A quantitative statistical graph showing the positive rate of PEDV-infected cells at different time points as detected by flow cytometry;

[0025] Figure 6 To detect the distribution of PEDV virus in the jejunal tissue of PEDV-infected piglets using IHC;

[0026] Figure 7 For the identification of mouse monoclonal antibody subtypes;

[0027] Figure 8 A strategy for truncating the PEDV N protein sequence;

[0028] Figure 9For the identification of the smallest antigenic epitope and determination of key amino acids for antibody recognition; A: Preliminary identification of Mab 8G2 antigenic epitope; BD: Identification of the smallest antigenic epitope; E: Determination of key amino acids. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Example 1

[0035] 1. Virus strains, bacterial species, cells, laboratory animals, and reagents

[0036] PEDV CV777 strain was isolated and preserved by the Animal Molecular Pathogen Laboratory of Henan Agricultural University; pET-32a(+) plasmid, Vero cells and SP2 / 0 cells were also preserved by the Animal Molecular Pathogen Laboratory of Henan Agricultural University; BALB / c mice were purchased from Henan Provincial Experimental Animal Center; mouse monoclonal antibody subtype identification kit was purchased from Proteintech.

[0037] 2. PCR amplification

[0038] 2.1 Primer Design and Synthesis

[0039] Referring to the gene sequence of porcine epidemic diarrhea virus strain CV777 in GenBank (GenBank accession number: KT323979.1), a pair of specific primers for amplifying the PEDV N protein sequence were designed using CE Design software according to the homologous recombination method. The forward and reverse primers were respectively given downstream homologous arms and EcoRI and HindIII restriction sites (underlined). The primer sequences are shown in Table 1.

[0040] Table 1 PCR amplification primers

[0041]

[0042] 2.2 PCR amplification

[0043] Total RNA was extracted from PEDV using the Trizol method, and cDNA was synthesized by reverse transcription using PEDV RNA as a template. The PCR reaction system is shown in Table 2.

[0044] Table 2 PCR reaction system

[0045]

[0046] After the reaction, 1% agarose gel electrophoresis and other detection and analysis were performed. If the results met expectations, the target fragment was purified using the Tiangen Gum Recovery Kit and stored at -20℃ for subsequent experiments.

[0047] 3. Construction of recombinant plasmid pET-32a-PEDV-N

[0048] The empty pET-32a vector plasmid was double-digested using EcoRI and HindIII fast digesters for subsequent construction of prokaryotic expression plasmids. The digestion system is shown in Table 3.

[0049] Table 3 Double enzyme digestion system

[0050]

[0051]

[0052] After mixing the prepared system, incubate it in a metal bath at 37℃ for 40 min. Use the Tiangen Gum Recovery Kit to recover and purify the enzyme digestion product, measure the concentration of the recovered product, and store it at -20℃ for subsequent experiments.

[0053] The purified target product and the linearized vector pET-32a were ligated using the Novitane source recombination kit. The reaction system is shown in Table 4.

[0054] Table 4 Connection System

[0055]

[0056] Gently pipette and mix well. Briefly centrifuge to collect the reaction solution to the bottom of the tube. React at 37°C for 30 minutes. Cool to 4°C or immediately place on ice to cool.

[0057] Thaw the cloning competent cells on ice. Add 10 μL of ligation product to 100 μL of competent cells, gently mix, and incubate on ice for 30 min. The transformation volume of the recombinant product should not exceed 1 / 10 of the DH5α competent cell volume. Then, heat shock at 42°C for 45 sec, followed by immediate ice incubation for 2-3 min. Next, add 900 μL of antibiotic-free LB medium and incubate at 37°C and 200-250 rpm for 1 h. Simultaneously, preheat the ampicillin-resistant LB agar plate at 37°C. After incubation, centrifuge at 5000 rpm for 5 min, discard 900 μL of supernatant, resuspend the cells, and plate them onto plates containing the correct antibiotic. Incubate upside down at 37°C for 12-16 h.

[0058] Randomly select 3-4 white, round single colonies from the solid culture medium and culture them in a liquid medium containing ampicillin for 6-7 hours; use the bacterial culture as a template for bacterial culture PCR identification of recombinant plasmids.

[0059] The positive bacterial culture was sent to Shangya Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the PEDVCV777 strain sequence using SnapGene software to determine if the target gene was correctly inserted into the vector and to check for mutations. The plasmid with correct sequencing results was named pET-32a-PEDV-N. The plasmid was extracted from the positive bacterial culture according to the instructions of the Tiangen plasmid mini-extraction kit and stored at -20℃ for subsequent experiments.

[0060] 4. Exploration of expression conditions for recombinant proteins

[0061] The positive plasmid was transformed into Rosetta(DE3) competent cells and cultured in LB broth medium containing ampicillin until OD was reached. 600nmWhen the pH value was around 0.6, IPTG was added to induce expression, bringing the final concentration to 1 mmol / L. Induction was performed at 37℃ for 4 h to confirm successful expression of the recombinant protein. The optimal induction conditions were then determined, set at 37℃ for 4 h, 37℃ for 5 h, 37℃ for 6 h, and 26℃ for 10 h. After induction, the cells were centrifuged at 6000 rpm for 10 min, and the bacterial pellet was resuspended in sterile PBS. The cells were then thoroughly disrupted using an ultrasonic cell disruptor (300W power, 3-second operation, 3-second pause, 20-min disruption). The cells were then centrifuged at 4℃ at 6000 rpm for 10 min, and the supernatant and pellet were collected. 10 μL of each sample was added to a 6× protein loading buffer, boiled in a water bath for 10 min, and then subjected to SDS-PAGE protein electrophoresis for verification. The remaining sample was used for subsequent protein purification.

[0062] 5. Purification and identification of recombinant proteins

[0063] (1) Sample preparation: Obtain 200 mL of positive bacterial culture, sonicate and centrifuge, and take the supernatant.

[0064] (2) Ni column purification: Ni-Agarose Resin packing material was mixed and packed into the column, equilibrated with TBS containing 10 mM imidazole, and the supernatant was bound to the nickel column at 4 °C for 2 h. The flow-through was collected drop by drop every 3 seconds. Impurities were eluted with TBS containing low concentrations of imidazole (10, 20, 50 mM), followed by elution with TBS containing 100 mM and 250 mM imidazole and collection of the eluent. The nickel column was washed with deionized water and then stored with 20% anhydrous ethanol.

[0065] (3) Electrophoresis detection: Take 10 μL of flow-through buffer, washing buffer and elution buffer, treat with 6× protein loading buffer, boil for 10 min and then perform SDS-PAGE electrophoresis for identification.

[0066] (4) Dialysis preservation: Pre-treat 8000D dialysis bags, fill them with elution buffer containing PEDV N protein, place them in 0.01M PBS buffer at 4℃ overnight, measure the concentration, and then aliquot and store at -80℃.

[0067] (5) SDS-PAGE and WB were used to identify the immunogenicity of the purified protein.

[0068] 6. Screening of positive hybridoma cell lines

[0069] Healthy female BALB / c mice aged 6–8 weeks were selected. The concentration of purified recombinant protein was adjusted to 1 mg / mL. The immunization procedure was as follows: For the primary immunization, the antigen was emulsified 1:1 with Freund's complete adjuvant, and each mouse was subcutaneously injected with 200 μL of emulsion containing 60 μg of antigen. Booster immunizations were performed on days 14 and 28, with the antigen and Freund's incomplete adjuvant emulsified 1:1, and each mouse was subcutaneously injected with 200 μL of emulsion containing 60 μg of antigen. After reaching the required titer, a pulse immunization was performed, with PBS emulsified 1:1 with the antigen, and 200 μL of emulsion containing 60 μg of antigen was injected intraperitoneally.

[0070] Antibody titers were detected using indirect enzyme-linked immunosorbent assay (ELISA). Three days before cell fusion, mice with the highest ELISA titers were selected for a shock immunization. Spleen cells from these mice were aseptically collected and isolated, and fused with mouse myeloma cells SP2 / 0 at a ratio of 5:1 to 10:1. The fused cells were cultured in HAT medium for 7 and 10 days, followed by the first and second medium changes. The cell supernatant was detected by indirect ELISA to screen for positive clones. Simultaneously, four subclonings were performed using the limiting dilution method. When each well contained a single cell cluster of hybridoma cells and all supernatant results were positive, the cell line that stably secreted antibodies and had the strongest antibody secretion capacity was selected and named 8G2, and cryopreserved in liquid nitrogen.

[0071] 7. Preparation and purification of ascites fluid

[0072] Healthy, appropriately weighed BALB / c mice were selected, and each mouse was injected intraperitoneally with 500 μL of paraffin oil. Seven days later, the selected positive hybridoma cells were injected into the mice intraperitoneally, with each mouse typically receiving 10 cells. 6 Up to 10 7 One week later, when the mouse's abdominal cavity is swollen, ascites fluid can be extracted. First, remove red blood cells from the ascites fluid by low-speed centrifugation, then remove fat by high-speed centrifugation. Aliquot into centrifuge tubes and store at -40°C.

[0073] (1) Ascites pretreatment: Add 2 times the volume of 0.06M acetate buffer to the ascites and mix thoroughly by magnetic stirring.

[0074] (2) Octyl acid precipitation of impurities: Octyl acid was added dropwise at a ratio of 33 μL octanoic acid / mL ascites fluid, and the mixture was stirred continuously for 30 min. After standing at 4℃ for 2 h, the mixture was centrifuged at 4℃ and 12000 rpm for 30 min, and the supernatant was collected and filtered through a 0.45 μm filter membrane.

[0075] (3) Ammonium sulfate precipitation of antibodies: Place the filtrate on a magnetic stirrer and slowly add 1 / 10 volume of pre-cooled PBS (pH=7.4) to adjust the pH to neutral. Add saturated ammonium sulfate solution dropwise to a final concentration of 45% saturation and let stand overnight at 4°C (12-16h).

[0076] (4) Antibody precipitation recovery: Centrifuge the overnight sample at 4℃ and 12000rpm for 30min, discard the supernatant, and gently resuspend the precipitate in 0.01MPBS (pH=7.4).

[0077] (5) Dialysis for desalting: The resuspension was placed in a pretreated dialysis bag (dialysis molecular weight 8000-14000D), placed in 1L PBS, and dialyzed overnight at 4°C, changing the dialysis buffer every 4 hours. After dialysis, the solution was aliquoted and stored at -80°C for long-term storage. The obtained monoclonal antibody was named Mab-8G2.

[0078] 8. Monoclonal antibody reactivity identification

[0079] 8.1 ELISA validation of purified antibody titer

[0080] PEDV virus (10) was coated with CB coating solution. 4 TCID 50 0.1 mL (1:3 dilution), 100 μL / well, incubate at 37°C for 3 h. Discard the coating buffer, wash 3 times with PBST, add 1.5% BSA blocking buffer, 150 μL / well, incubate at 37°C for 2 h. Discard the blocking buffer, wash 3 times with PBST, add serially diluted mouse serum and controls (1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400), 100 μL / well, incubate at 37°C for 1 h. Discard the primary antibody, wash 5 times with PBST, add HRP-goat anti-mouse IgG secondary antibody (1:8000 dilution) diluted in PBS, 100 μL / well, incubate at 37°C for 1 h to allow the secondary antibody to bind to the antibody bound to the antigen. Discard the secondary antibody, wash 5 times with PBST, add TMB substrate (100 μL / well), and incubate at 37°C in the dark for 10 min. Stop the reaction by adding stop solution (50 μL / well), and determine the result by measuring absorbance at 450 nm using a microplate reader. Results showed that the Mab-8G2 titer could reach over 102400 (…). Figure 1 ).

[0081] 8.2 IFA verification of antibody reactivity

[0082] Vero cells were infected with PEDV, and cells were fixed at different time points after PEDV infection, with untreated cells serving as a negative control. Mab-8G2 was used as the primary antibody for IFA detection. Results showed that PEDV N protein expression began 6 hours after infection, and increased with increasing infection time, leading to more severe cell separation and death. Figure 2 ).

[0083] 8.3WB to verify antibody reactivity

[0084] Vero cells were seeded into 6-well cell culture plates. Once the cells reached confluence, PEDV CV777 strain with an MOI of 1 was inoculated, covering the cells completely. The cells were incubated at 37°C for 1 hour for adsorption. After washing twice with D-Hank's solution, 2 mL of maintenance medium (2 mL of DMEM medium containing 2.5% trypsin) was added, and the plates were incubated at 37°C in a 5% CO2 cell culture incubator. Cell protein samples were collected at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, and Western blotting was performed using the prepared Mab-8G2 as the primary antibody.

[0085] The results showed that PEDV N protein expression began 6 hours after infection, and the expression level increased with the duration of infection until massive cell death began at 60 hours. This indicates that the prepared Mab-8G2 can be used for Western blotting to detect PEDV-infected cells. Figure 3 ).

[0086] 8.4 Flow cytometry to verify antibody reactivity

[0087] Vero cells were infected with PEDV, and single-cell suspensions were prepared at 6h, 12h, and 24h after infection. The cells were then fixed and perforated, with untreated cells serving as a negative control. Mab-8G2 was used as the primary antibody to verify its applicability for flow cytometry detection of viral infection. Results showed that the percentage of positive cells increased over time, indicating that Mab-8G2 can be used for flow cytometry detection of viral infection. Figures 4-5 ).

[0088] 8.5 IHC verification of antibody reactivity

[0089] Jejunal tissues were collected from piglets that were negative for PEDV and those attacked with PEDV to investigate the reactivity of the antibody with PEDV-infected intestinal samples. Mab-8G2 was used as the primary antibody, and staining showed that PEDV attacked a large number of positive epithelial cells in the pig jejunum. Figure 6 This indicates that Mab-8G2 can be used to analyze clinical samples infected with PEDV.

[0090] 9. Monoclonal antibody subtype identification

[0091] The obtained monoclonal antibody was identified as an antibody subclass according to the instructions of the Proteintech mouse monoclonal antibody subclass identification kit. The heavy chain subclass of monoclonal antibody Mab-8G2 was IgG1, and the light chain subclass was Kappa. Figure 7 ).

[0092] 10. Identification of antigenic epitopes recognized by monoclonal antibodies

[0093] The PEDV N protein was truncated by dividing it into 10 equal and overlapping amino acid segments. The PEDV N protein truncation strategy is as follows: Figure 8 As shown. Multiple overlapping truncated peptide fragments were truncated from the PEDV N protein. Longer peptide fragments were ligated into enzyme-digested pET-32a using a homologous recombination kit, and shorter peptide fragments were ligated using T4 DNA ligase. The recombinant plasmid was sequenced and then transformed into *E. coli* BL21(DE3) cells. After induction with 0.7 mM IPTG at 37°C for 4 h, the reactivity of the truncated protein with the monoclonal antibody was detected by Western blotting. The monoclonal antibody Mab-8G2 targets the antigenic epitope peptide aa274-DLKDIPEWR-282 (…). Figure 9 (Chinese AD).

[0094] 11. Site-directed mutagenesis test

[0095] To identify the key amino acids in the aa274-282 epitopes, each residue was sequentially substituted with alanine, as described above. In this example, the mutant epitopes were expressed using a prokaryotic expression system. The reactivity of Mab-8G2 with the mutant aa274-282 epitopes was detected by SDS-PAGE and Western blotting. The key amino acids were 277A, 279P, and 281W. Figure 9 (E).

[0096] 12. Monoclonal antibody variable region PCR amplification

[0097] The light chain variable region and heavy chain variable region genes were amplified from cDNA of 8G2 hybridoma cells, recovered by gel extraction, cloned into the pMD19-T vector, and sequenced. The sequencing results were compared with the antibody gene library (IMGT), confirming that the amplified sequences were the complementarity-determining regions (CDRs) of the heavy and light chain variable regions of the monoclonal antibody. Specifically, the amino acid sequence of the heavy chain variable region of Mab-8G2 is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of Mab-8G2 is shown in SEQ ID NO.2. The DNA sequence encoding the heavy chain variable region of Mab-8G2 is shown in SEQ ID NO.3; the DNA sequence encoding the light chain variable region of Mab-8G2 is shown in SEQ ID NO.4. The amino acid sequences of the heavy and light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody 8G2 are shown in Table 5 below.

[0098] Table 5. Amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the heavy and light chains of Mab-8G2.

[0099]

[0100] SEQ ID NO.1:

[0101] QSGAELMKPGETVKISCKASGYTFTNHGMNWVKQAPGKGLKWMGWINTNSGEPTYAEEFKGRFAFSLETSASTAYLHINNLKNEDTATYFCAREGYYPYWGQGTT;

[0102] SEQ ID NO.2:

[0103] DIQLTQSPLTLSVTLGQPASISCKSSQSLLDSDGQTYLNWLLHKPSQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKRS;

[0104] SEQ ID NO.3:

[0105] cagtctggagctgagctgatgaagcctggagagacagtcaagatctcctgcaaggcttctggatataccttcacaaaccatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacaccaacagtggagagccaacatatgctgaagagttcaagggacggtttgccttctctttggaaacctctgccagtactgcctatttgcacatcaacaacctcaaaaatgaggacacggctacatatttctgtgcaagagagggttactacccttactggggccaagggaccacg;

[0106] SEQ ID NO.4:

[0107] gacattcagctgacccagtctccactcactttgtcggttacccttggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggacagacatatttgaattggttgttacataagccaagccagtctccaaagcgcctaatctatctg gtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttccgctcacgttcggtgctgggaccaagcggagc.

[0108] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody 8G2 against the N protein of porcine epidemic diarrhea virus, characterized in that, The heavy chain variable region of the monoclonal antibody 8G2 includes CDR1 with the amino acid sequence NHGMN, CDR2 with the amino acid sequence WINTNSGEPTYAEEFKG, and CDR3 with the amino acid sequence EGYYP; the light chain variable region of the monoclonal antibody 8G2 includes CDR1 with the amino acid sequence KSSQSLLDSDGQTYLN, CDR2 with the amino acid sequence LVSKLDS, and CDR3 with the amino acid sequence WQGTHFPL.

2. The monoclonal antibody 8G2 as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 8G2 is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of the monoclonal antibody 8G2 is shown in SEQ ID NO.

2.

3. The monoclonal antibody 8G2 as described in claim 1, characterized in that, The monoclonal antibody 8G2 induces a specific immune response with porcine epidemic diarrhea virus or its N protein antigen.

4. The monoclonal antibody 8G2 as described in claim 1, characterized in that, The amino acid sequence of the antigenic epitope peptide recognized by the monoclonal antibody 8G2 is DLKDIPEWR.

5. The monoclonal antibody 8G2 as described in claim 1, characterized in that, The heavy chain subtype of the monoclonal antibody 8G2 is IgG1, and the light chain subtype is Kappa.

6. A gene encoding the monoclonal antibody 8G2 of claim 1, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8G2 is shown in SEQ ID No. 3, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8G2 is shown in SEQ ID No.

4.

7. An expression cassette or recombinant vector containing the gene of claim 6.

8. A host bacterium containing the recombinant vector of claim 7.

9. The use of the monoclonal antibody 8G2 of claim 1, the gene of claim 6, the expression cassette or recombinant vector of claim 7, or the host bacterium of claim 8 in the preparation of a detection product for porcine epidemic diarrhea virus or its N protein.

10. A product for detecting porcine epidemic diarrhea virus or its N protein antigen, characterized in that, The product comprises the monoclonal antibody 8G2 as described in claim 1.

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