Monoclonal antibody for resisting porcine epidemic diarrhea virus N protein, antigen epitope peptide recognized by monoclonal antibody and application of monoclonal antibody

Abstract

The invention discloses a monoclonal antibody for resisting porcine epidemic diarrhea virus N protein, an antigen epitope peptide recognized by the monoclonal antibody and application of the monoclonal antibody, and belongs to the technical field of monoclonal antibodies. A heavy chain variable region of the monoclonal antibody 8G2 comprises a CDR1 of which the amino acid sequence is NHGMN, a CDR2 of which the amino acid sequence is WINTNSGEPTYAEFKG and a CDR3 of which the amino acid sequence is EGYYP; the light chain variable region comprises a CDR1 of which the amino acid sequence is KSSQSLLDSDGQTYLN, a CDR2 of which the amino acid sequence is LVSKLDS and a CDR3 of which the amino acid sequence is WQGTHFPL. And the amino acid sequence for recognizing the epitope is DLKDIPEWR. The 8G2 monoclonal antibody prepared by the invention is wide in application, can be used in laboratories and clinics, and lays an important foundation for clinical detection of PEDV and research of pathogenic mechanism of PEDV.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody, and particularly to a monoclonal antibody against the N protein of porcine epidemic diarrhea virus, an antigenic epitope peptide recognized thereby, and applications thereof. Background Art

[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 incidence rate and is one of the most serious diseases endangering the aquaculture industry at present, causing huge economic losses to the global pig industry every year. PEDV can infect pigs of all ages. Adult pigs mainly show symptoms such as weight loss and malnutrition, but the lethality rate for suckling piglets within one week of age is as high as 80 - 100%.

[0003] PEDV belongs to the genus Coronavirus of the family Coronaviridae and is an enveloped single-stranded positive-sense RNA virus. Its genome length is about 28 kb, including a 5'-cap and a 3'-polyadenylate, and has 7 coding open reading frames (ORFs), namely 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 highly immunogenic, and a large number of antibodies against the N protein can be induced in the early stage of virus infection. Therefore, the N protein can be used as an early detection target for PEDV infection and has important application value in clinical research.

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

[0005] The object of the present invention is to provide a monoclonal antibody against the N protein of porcine epidemic diarrhea virus, an antigenic epitope peptide recognized thereby, and applications thereof, so as to solve the problems existing in the above-mentioned prior art. The 8G2 monoclonal antibody prepared by the present invention has wide applications and can be used in laboratories and clinical applications, laying an important foundation for the clinical detection of PEDV and its N protein antigens.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present 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 as shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of the monoclonal antibody 8G2 is as shown in SEQ ID NO.2.

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

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

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

[0012] Optionally, the monoclonal antibody 8G2 has a specific immune reaction 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 application of the monoclonal antibody 8G2 or the gene or the expression cassette or 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, and the product contains the monoclonal antibody 8G2 or the gene or the expression cassette or the recombinant vector or the host bacterium.

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

[0018] The present invention provides a monoclonal antibody 8G2 against the nucleocapsid protein of porcine epidemic diarrhea virus (PEDV). This monoclonal antibody can specifically recognize PEDV and can be used in applications such as ELISA, WB, IFA, IHC, and flow cytometry. After identification, the heavy chain subtype is IgG1 and the light chain subtype is Kappa. The amino acid sequence of the antigenic epitope it recognizes is 274-DLKDIPEWR-282. The monoclonal antibody 8G2 prepared by the present invention has a wide range of applications and can be used in laboratories and clinical applications, providing new ideas and methods for the prevention and control of PEDV. Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 It is the detection result of the titer of the purified antibody;

[0021] Figure 2 It is the IFA detection of the N protein expression at different times after infecting with PEDV;

[0022] Figure 3 It is the WB detection of the N protein expression in PEDV-infected cells at different times; 1: blank cells; 2: inoculated with virus for 6 h; 3: inoculated with virus for 12 h; 4: inoculated with virus for 24 h; 5: inoculated with virus for 36 h; 6: inoculated with virus for 48 h; 7: inoculated with virus for 60 h; 8: inoculated with virus for 72 h;

[0023] Figure 4 It is the result graph of the positive rate detected by flow cytometry at different time points in PEDV-infected cells;

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

[0025] Figure 6 It is the IHC detection of the virus distribution in the jejunum tissue of PEDV-infected piglets;

[0026] Figure 7 It is the identification of the subtype of the mouse monoclonal antibody;

[0027] Figure 8 It is the strategy of the truncated sequence of the PEDV N protein;

[0028] Figure 9For the identification of the minimum antigenic epitope recognized by the antibody and the determination of key amino acids; A: Preliminary identification of the antigenic epitope of Mab 8G2; B-D: Identification of the minimum antigenic epitope; E: Determination of key amino acids. Detailed implementation mode

[0029] The various exemplary implementation modes 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 should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the present invention specification, which are obvious to those skilled in the art. Other implementation modes obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] 1. Strains, strains of bacteria, cells, experimental animals and reagents

[0036] The PEDV CV777 strain was isolated and preserved by the Laboratory of Animal Molecular Etiology, Henan Agricultural University; the pET-32a(+) plasmid, Vero cells and SP2 / 0 cells were all preserved by the Laboratory of Animal Molecular Etiology, Henan Agricultural University; BALB / c mice were purchased from the Henan Experimental Animal Center; the 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 the porcine epidemic diarrhea virus CV777 strain on GenBank (GenBank accession number: KT323979.1), a pair of specific primers for specifically amplifying the PEDV N protein sequence was designed using CE Design software according to the homologous recombination method. The forward and reverse primers were added with downstream and upstream homologous arms and the restriction enzyme cleavage sites of EcoRⅠ and HindⅢ (underlined) at the 5' end respectively. The primer sequences are shown in Table 1.

[0040] Table 1 PCR Amplification Primers

[0041]

[0042] 2.2 PCR Amplification

[0043] The total RNA in PEDV was extracted using the Trizol method, and cDNA was reverse transcribed using the RNA of PEDV 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 carried out. If the results met the expectations, the target fragment was purified using the Tiangen gel recovery kit and stored at -20°C for subsequent experiments.

[0047] 3. Construction of Recombinant Plasmid pET-32a-PEDV-N

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

[0049] Table 3 Double Digestion System

[0050]

[0051]

[0052] After mixing the prepared system evenly, incubate it in a metal bath at 37°C for 40 min. Use the Tiangen Gel Extraction Kit to recover and purify the digested product, measure the concentration of the recovered product, and store it at -20°C for subsequent experiments.

[0053] Ligate the purified target product and the linearized vector pET-32a using the Novoprotein homologous recombination kit. The reaction system is shown in Table 4.

[0054] Table 4 Ligation system

[0055]

[0056] Gently pipette to mix evenly, briefly centrifuge to collect the reaction solution at the bottom of the tube, and react at 37°C for 30 min; cool to 4°C or immediately place on ice.

[0057] Thaw the cloning competent cells on ice. Take 10 μL of the ligation product and add it to 100 μL of the competent cells. Gently flick to mix evenly and then let it stand on ice for 30 min. Also, the transformation volume of the recombinant product should not exceed 1 / 10 of the volume of the DH5α competent cells. Then, perform a heat shock at 42°C in a water bath for 45 sec and quickly ice-bath for 2 - 3 min. After that, add 900 μL of LB medium without antibiotics, shake the bacteria at 37°C, 200 - 250 rpm for 1 h. At the same time, preheat the ampicillin-resistant LB solid medium plate at 37°C. After shaking the bacteria, centrifuge at 5000 rpm for 5 min, discard 900 μL of the supernatant, resuspend the bacterial cells and spread them on the plate with the correct resistance, and incubate them upside down at 37°C for 12 - 16 h.

[0058] Randomly pick 3 - 4 white round single colonies from the solid medium and culture them in a liquid medium containing ampicillin resistance for 6 - 7 h; use the bacterial liquid as a template to perform bacterial liquid PCR identification of the recombinant plasmid.

[0059] Send the positive bacterial liquid to Shangya Biotechnology Co., Ltd. for sequencing. Use the snapgene software to compare the sequencing results with the PEDVCV777 strain sequence for homology to determine whether the target gene is correctly inserted into the vector and to check for mutations at the same time. Name the plasmid with correct sequencing results as pET-32a-PEDV-N. Extract the plasmid from the positive bacterial liquid according to the instructions of the Tiangen Plasmid Mini Extraction Kit and store it at -20°C for subsequent experiments.

[0060] 4. Exploration of the expression conditions of the recombinant protein

[0061] Transfer the positive plasmid into Rosetta(DE3) competent cells and expand the culture in LB liquid medium containing ampicillin. Wait until the OD 600nmWhen the value is around 0.6, IPTG is added for induction expression to make its final concentration 1 mmol / L, and induction is carried out at 37 °C for 4 h to confirm the successful expression of the recombinant protein. Then, the optimal induction conditions are explored, which are set as 37 °C, 4 h; 37 °C, 5 h; 37 °C, 6 h; and 26 °C, 10 h respectively. After induction, centrifuge at 6000 r / min for 10 min, resuspend the cell pellet with sterile PBS, and fully disrupt it with an ultrasonic cell disruptor (power 300 W, working for 3 s and stopping for 3 s, disrupting for 20 min). Centrifuge at 6000 r / min for 10 min at 4 °C to collect the supernatant and precipitate after disruption. Take 10 μL of the sample, add 6× protein loading buffer, boil in a water bath for 10 min, and then perform SDS-PAGE protein electrophoresis verification. The remaining samples are used for subsequent protein purification.

[0062] 5. Purification and Identification of Recombinant Protein

[0063] (1) Sample preparation: Obtain 200 mL of positive bacterial solution, and take the supernatant after ultrasonic disruption and centrifugation.

[0064] (2) Nickel column purification: Mix and load the Ni-Agarose Resin packing material into the column, balance it with TBS containing 10 mM imidazole, bind the supernatant to the nickel column at 4 °C for 2 h, and collect the flow-through at a rate of one drop every 3 s; elute the impurities with TBS containing low-concentration imidazole (10, 20, 50 mM), and then elute the target protein with TBS containing 100 mM and 250 mM imidazole and collect the eluate; wash the nickel column with deionized water and store it with 20% anhydrous ethanol.

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

[0066] (4) Dialysis and storage: Pretreat the dialysis bag with a pore size of 8000 D, load the eluate containing PEDV N protein, place it in 0.01 M PBS buffer at 4 °C overnight, measure the concentration, and then aliquot and store at -80 °C.

[0067] (5) Identify the immunogenicity of the purified protein by SDS-PAGE and WB.

[0068] 6. Screening of Positive Hybridoma Cell Lines

[0069] Select 6- to 8-week-old healthy female BALB / c mice, adjust the concentration of the purified recombinant protein to 1 mg / mL, and the immunization process is as follows: For the primary immunization, emulsify the antigen with Freund's complete adjuvant at a ratio of 1:1, and subcutaneously inject 200 μL of the emulsion containing 60 μg of antigen per mouse; for the booster immunizations on days 14 and 28, emulsify the antigen with Freund's incomplete adjuvant at a ratio of 1:1, and subcutaneously inject 200 μL of the emulsion containing 60 μg of antigen per mouse. After the titer reaches the required level, give a boost immunization, emulsify PBS and the antigen at a ratio of 1:1, and intraperitoneally inject 200 μL of the emulsion containing 60 μg of antigen.

[0070] Detect the antibody titer using the indirect enzyme-linked immunosorbent assay (ELISA). Three days before cell fusion, select the mouse with the highest ELISA titer for boost immunization. Sterilely collect and isolate the spleen cells of the above mice, and fuse them with mouse myeloma cells SP2 / 0 at a ratio of 5:1 - 10:1. Culture the fused cells in HAT medium for 7 days and 10 days, and then perform the first and second medium replacements respectively. Detect the cell supernatant by indirect ELISA to screen for positive clones. At the same time, perform four subclonings by the limiting dilution method. When each well of the hybridoma cells is a single cell mass and all the supernatant detection results are positive, screen for the cell line that stably secretes antibodies and has the strongest antibody secretion ability, name it 8G2, and store it in a liquid nitrogen tank.

[0071] 7. Ascites preparation and purification

[0072] Select healthy BALB / c mice with appropriate body weights, inject 500 μL of paraffin oil into the abdominal cavity of each mouse. Seven days later, inject the screened positive hybridoma cells into the mice by intraperitoneal injection. The cell amount injected into each mouse is generally 10 6 to 10 7 cells. After one week, when the abdominal cavity of the mouse is swollen, the ascites can be extracted. First, centrifuge at low speed to remove red blood cells in the ascites, and then centrifuge at high speed to remove fat. Aliquot it into centrifuge tubes and store it at -40 °C.

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

[0074] (2) Precipitate miscellaneous proteins with caprylic acid: Dropwise add caprylic acid at a ratio of 33 μL of caprylic acid / mL of ascites, and continuously stir for 30 min. Let it stand at 4 °C for 2 h, then centrifuge at 4 °C and 12,000 rpm for 30 min, collect the supernatant and filter it through a 0.45 μm filter membrane.

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

[0076] (4) Antibody precipitation and recovery: Centrifuge the overnight sample at 4°C and 12,000 rpm for 30 min, discard the supernatant, and gently resuspend the precipitate in 0.01 M PBS (pH = 7.4).

[0077] (5) Dialysis for desalting: Load the resuspended solution into a pre-treated dialysis bag (dialysis molecular weight 8,000 - 14,000 D), place it in 1 L of PBS, and dialyze overnight at 4°C, changing the dialysis fluid every 4 h. After dialysis, aliquot and store at -80°C for long-term. The obtained monoclonal antibody is named Mab-8G2.

[0078] 8. Identification of monoclonal antibody reactivity

[0079] 8.1 ELISA verification of the titer of the purified antibody

[0080] Coat PEDV virus (10 4 TCID 50 / 0.1 mL; diluted 1:3), 100 μL per well, and incubate at 37°C for 3 h. Discard the coating solution, wash 3 times with PBST, add 1.5% BSA blocking solution, 150 μL per well, and incubate at 37°C for 2 h. Discard the blocking solution, wash 3 times with PBST, add the mouse serum to be tested and the control diluted in PBS in a serial dilution (1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400), 100 μL per well, and incubate at 37°C for 1 h. Discard the primary antibody, wash 5 times with PBST, add HRP-goat anti-mouse IgG secondary antibody diluted in PBS (diluted 1:8000), 100 μL per well, and 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 per well, and react in the dark at 37°C for 10 min. Add the stop solution to terminate the reaction, 50 μL per well, and measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader to determine the results. The results show that the titer of Mab-8G2 can reach 102,400 or more ( Figure 1 ).

[0081] 8.2 IFA verification of antibody reactivity

[0082] Vero cells were infected with PEDV, and the cells at different time points after PEDV infection were fixed, with untreated cells as the negative control. Mab-8G2 was used as the primary antibody for IFA detection. The results showed that the PEDV N protein began to be expressed at 6 h after infection, and with the increase of the infection time, the expression of the PEDV N protein increased, and cell detachment and death became more severe ( Figure 2 ).

[0083] 8.3 WB verification of antibody reactivity

[0084] Vero cells were inoculated into a 6-well cell culture plate. When the cells grew confluent, the PEDV CV777 strain with an MOI of 1 was inoculated to cover the cells. Adsorb at 37°C for 1 h, wash twice with D-Hank's, and then add 2 mL of maintenance medium (2 mL of DMEM medium containing 2.5% trypsin), and place it in a 37°C, 5% CO₂ cell culture incubator for culture. After collecting the cell protein samples at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h respectively, use the prepared Mab-8G2 as the primary antibody for WB detection.

[0085] The results showed that the PEDV N protein began to be expressed at 6 h post-infection, and the expression level increased with the increase of the infection duration until the cells began to die massively at 60 h. This indicated that the prepared Mab-8G2 could be used for WB assay to detect PEDV-infected cells ( Figure 3 ).

[0086] 8.4 Verification of antibody reactivity by flow cytometry

[0087] Vero cells were infected with PEDV. Single-cell suspensions were prepared from the cells infected for 6 h, 12 h, and 24 h. The cells were fixed and permeabilized, and the untreated cells were used as negative controls. Use Mab-8G2 as the primary antibody to verify whether Mab-8G2 could be used for flow cytometry to detect virus infection. The results showed that the percentage of positive cells increased with time, indicating that Mab-8G2 could be used for flow cytometry to detect virus infection ( Figures 4 - 5 ).

[0088] 8.5 Verification of antibody reactivity by IHC

[0089] Jejunum tissues from negative and PEDV-challenged piglets were taken to explore the reactivity of this antibody with PEDV-infected intestinal samples. Using Mab-8G2 as the primary antibody, staining showed a large number of positive epithelial cells in the jejunum of PEDV-challenged pigs ( Figure 6 ). This indicated that Mab-8G2 could be used to analyze clinical samples infected with PEDV.

[0090] 9. Identification of monoclonal antibody subtypes

[0091] The obtained monoclonal antibodies were identified for antibody subtypes according to the operation instructions of the mouse monoclonal antibody subclass identification kit from Proteintech. The heavy chain subtype of the monoclonal antibody Mab-8G2 was IgG1, and the light chain subtype was Kappa ( Figure 7 ).

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

[0093] Truncate the PEDV N protein according to the method of equally dividing and cross-overlapping 10 amino acids. The truncation sequence strategy of the PEDV N protein is as Figure 8 shown. Truncate multiple cross-overlapping truncated proteins on the PEDV N protein. The longer peptide fragments are ligated into the digested pET-32a using a homologous recombination kit, and the shorter peptide fragments are ligated using T4 DNA ligase. Sequence the recombinant plasmid, and then transform the recombinant plasmid into Escherichia coli BL21(DE3) cells. After inducing with 0.7 mM IPTG at 37 °C for 4 h, detect the reactivity of the truncated protein with the monoclonal antibody by WB. The antigenic epitope peptide targeted by the monoclonal antibody Mab-8G2 is amino acids aa274-DLKDIPEWR-282( Figure 9 A-D in

[0094] 11. Site-directed mutagenesis assay

[0095] To identify the key amino acids in the aa274-282 epitope, each residue is sequentially substituted with alanine as described above. In this example, the mutant epitope is expressed through a prokaryotic expression system. Detect the reactivity of Mab-8G2 with the mutant aa274-282 epitope by SDS-PAGE and WB. The key amino acids are 277A, 279P, 281W( Figure 9 E in

[0096] 12. PCR amplification of the variable regions of monoclonal antibodies

[0097] Amplify the light chain variable region gene and the heavy chain variable region gene from the cDNA of 8G2 hybridoma cells. After gel extraction and recovery, clone them into the pMD19-T vector for sequencing. Compare the sequencing results with the antibody gene bank (IMGT). The sequencing results confirm that the amplified sequences are the complementary determining regions (CDRs) of the heavy chain variable region and the light chain variable region of the monoclonal antibody. Specifically, the amino acid sequence of the heavy chain variable region of Mab-8G2 is as shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of Mab-8G2 is as shown in SEQ ID NO.2. The DNA sequence encoding the heavy chain variable region of Mab-8G2 is as shown in SEQ ID NO.3; the DNA sequence encoding the light chain variable region of Mab-8G2 is as shown in SEQ ID NO.4. The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain and light chain variable regions of monoclonal antibody 8G2 are shown in Table 5 below.

[0098] Table 5 Amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain and light chain variable regions 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] gacattcagctgacccagtctccactcactttgtcggttacccttggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggacagacatatttgaattggttgttacataagccaagccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttccgctcacgttcggtgctgggaccaagcggagc。

[0108] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined 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 comprises 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 comprises 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 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 8G2 is as shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of the monoclonal antibody 8G2 is as shown in SEQID NO.

2.

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

4.

4. An expression cassette or recombinant vector containing the gene according to claim 3.

5. A host bacterium containing the recombinant vector according to claim 4.

6. The monoclonal antibody 8G2 according to claim 1, characterized in that, The monoclonal antibody 8G2 has a specific immune reaction with porcine epidemic diarrhea virus or its N protein antigen.

7. The monoclonal antibody 8G2 according to claim 1, wherein, The amino acid sequence of the antigenic epitope peptide recognized by the monoclonal antibody 8G2 is DLKDIPEWR.

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

9. Use of the monoclonal antibody 8G2 according to claim 1, or the gene according to claim 3, or the expression cassette or recombinant vector according to claim 4, or the host bacterium according to claim 5 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 according to claim 1, or the gene according to claim 3, or the expression cassette or recombinant vector according to claim 4, or the host bacterium according to claim 5.

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