A monoclonal antibody against porcine epidemic diarrhea virus N protein, a hybridoma cell strain and application thereof

Abstract

The application discloses a kind of anti-swine epidemic diarrhea virus N protein monoclonal antibody, hybridoma cell strain and its application, belong to biotechnology field.The monoclonal antibody 16F6 provided by the application has brand-new CDR amino acid sequence, and the antigen epitope identified is PEDV N protein 401-EEAIYDDV-408 region, and the hybridoma cell strain PEDV-N-16F6 secreting the antibody is preserved in China typical culture preservation center, and the preservation number is CCTCC NO:C2026112.The application further provides the application of the above-mentioned monoclonal antibody in the preparation of test strip or kit for detecting swine epidemic diarrhea virus.In immunochromatographic detection, it has excellent performance and good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody against the N protein of porcine epidemic diarrhea virus, a hybridoma cell line, and their applications. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is an important infectious enteric pathogen in pigs, belonging to the genus *A* coronaviruses in the family Coronaviridae. Pigs of all ages are susceptible to PEDV infection, with mortality rates approaching 100% in newborn piglets under 7 days old. The high mortality rate and decreased herd productivity caused by PEDV result in significant economic losses to the pig farming industry. Therefore, establishing a rapid, accurate, and clinically applicable PEDV detection method is crucial for the early diagnosis and control of PEDV infection.

[0003] The PEDV nucleocapsid protein (N protein) is one of the important structural proteins of the virus. It is expressed at high levels during viral infection and exhibits strong immunogenicity and good sequence conservation, making it a commonly used target protein in PEDV antibody preparation and antigen detection. Antibodies that specifically recognize the PEDV N protein can be used to capture or detect viral antigens. Therefore, the performance of PEDV N protein monoclonal antibodies directly affects the sensitivity, specificity, and stability of related immunological detection methods.

[0004] Currently, various monoclonal antibodies targeting the PEDV N protein have been developed, and some have been applied to enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays, and immunochromatographic assays. However, due to differences in the amino acid composition of the heavy and light chain variable regions and the structure of their complementarity-determining regions, different monoclonal antibodies may exhibit significant variations in their recognition ability of the PEDV N protein, their reactivity with natural viral antigens, their cross-reactivity with other porcine viruses, and their performance in different detection systems. Therefore, obtaining novel monoclonal antibodies that can stably and specifically recognize the PEDV N protein remains a crucial foundation for developing immunological detection methods for PEDV.

[0005] Existing PEDV detection technologies mainly include virus isolation and culture, nucleic acid detection, and immunological detection. Virus isolation and culture have a long operation cycle and require high experimental conditions; nucleic acid detection technologies such as RT-PCR and real-time quantitative PCR have high sensitivity and specificity, but they have certain requirements for instruments and operating conditions. In contrast, colloidal gold immunochromatographic test strips have advantages such as simple operation, fast detection speed, no need for large instruments, and ease of field application, making them more suitable for rapid screening of samples from grassroots farms and clinical sites. However, the detection performance of immunochromatographic methods depends to a large extent on the antigen-binding ability, specificity, and pairing effect between antibodies used. Existing PEDV immunochromatographic detection methods still have room for improvement in terms of their ability to detect samples with low viral loads.

[0006] Therefore, obtaining a monoclonal antibody with a novel variable region sequence, capable of specifically recognizing PEDV N protein, exhibiting good distinguishing ability from other common porcine enteric coronaviruses, and suitable for application in colloidal gold immunochromatographic detection systems is of great significance for improving the sensitivity and specificity of rapid PEDV detection and developing rapid PEDV detection reagents suitable for field applications. Summary of the Invention

[0007] To address the aforementioned issues in the prior art, the present invention aims to provide a monoclonal antibody against porcine epidemic diarrhea virus (PEDV) N protein, a hybridoma cell line, and their applications. This addresses the problems of insufficient sequence diversity, concentrated epitope distribution, and limited possible antibody pairing combinations in existing anti-PEDV N protein monoclonal antibodies. To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this invention provides a monoclonal antibody (named 16F6) against porcine epidemic diarrhea virus (PEDV) N protein. The antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes three complementarity-determining regions (CDRs): VLCDR1, VLCDR2, and VLCDR3. The amino acid sequence of VLCDR1 is shown in SEQ ID NO. 5, the amino acid sequence of VLCDR2 is KVS, and the amino acid sequence of VLCDR3 is shown in SEQ ID NO. 11. The heavy chain variable region includes three CDRs: VHCDR1, VHCDR2, and VHCDR3, whose amino acid sequences are shown in SEQ ID NO. 18, SEQ ID NO. 22, and SEQ ID NO. 26, respectively. These CDR sequences constitute the structural basis for the antibody's specific recognition of the PEDV N protein.

[0008] Furthermore, the full-length amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.15, and the full-length amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.30. Epitope identification revealed that the antigenic epitope recognized by this monoclonal antibody is amino acids 401-408 of the PEDV N protein, with the sequence EEAIYDDV. This epitope is located in the C-terminal region of the N protein, and spatial structure analysis shows that it is exposed on the surface of the N protein, exhibiting good antigen accessibility, which is beneficial for the antibody to effectively recognize and bind to viral antigens under native conformation conditions.

[0009] In a second aspect, this invention provides a hybridoma cell line, PEDV-N-16F6, that secretes the aforementioned monoclonal antibody. This hybridoma cell line was obtained by immunizing BALB / c mice with recombinant PEDV N protein, fusing spleen cells from the immunized mice with SP2 / 0 myeloma cells, and then screening the resulting cells. This hybridoma cell line was deposited on May 25, 2026, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: C2026112, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, and classified as hybridoma cell line PEDV-N-16F6. This hybridoma cell line can be stably passaged and continuously secretes the target monoclonal antibody, providing a stable and reliable biological material for the large-scale preparation of antibodies.

[0010] In a third aspect, this invention provides the application of the aforementioned monoclonal antibody in the preparation of products for detecting porcine epidemic diarrhea virus (PEDV). Based on this antibody, immunochromatographic test strips or enzyme-linked immunosorbent assay (ELISA) kits for PEDV detection can be prepared. Specifically, the test strip uses a conjugate of the monoclonal antibody and colloidal gold as a detection probe, a polyclonal antibody against PEDV N protein as a detection line capture antibody, and a goat anti-mouse antibody as a quality control line, employing a double-antibody sandwich method to achieve rapid detection of PEDV antigen. The kit uses an enzyme-labeled version of the monoclonal antibody as the detection antibody and can be used for serological detection of PEDV antibodies.

[0011] Compared with the prior art, the present invention has the following beneficial effects: (1) The monoclonal antibody 16F6 provided by the present invention has a novel CDR amino acid sequence. Its heavy chain and light chain variable region sequences are different from those of the anti-PEDV N protein monoclonal antibodies disclosed in the prior art, which enriches the sequence diversity of antibodies for PEDV diagnosis.

[0012] (2) The antigenic epitope recognized by the monoclonal antibody 16F6 of the present invention is the PEDV N protein 401-EEAIYDDV-408 region. This epitope is located at the C-terminus of the N protein. Spatial structure analysis shows that it is exposed on the protein surface and has good antigen accessibility.

[0013] (3) The monoclonal antibody 16F6 of the present invention has excellent specificity. As verified by IFA and Western blot, it only reacts with PEDV and has no cross-reaction with common porcine enteric coronaviruses such as porcine PDCoV and transmissible gastroenteritis virus (TGEV).

[0014] (4) The monoclonal antibody 16F6 of this invention has high affinity and an ELISA titer of up to 1:25600. It can effectively bind antigens at low antibody concentrations, which provides a guarantee for the establishment of a high-sensitivity detection method.

[0015] (5) The monoclonal antibody 16F6 of the present invention performs excellently on the colloidal gold immunochromatographic platform, and has good pairing effect with the anti-PEDV N protein polyclonal antibody. The detection limit for PEDV virus solution can reach about 78 TCID. 50 It has a concentration of 1000 mg / mL and shows no cross-reactivity with PDCoV, TGEV, PSV, etc., indicating good potential for clinical application. Attached Figure Description

[0016] Figure 1 This is an IFA diagram of the 16F6-specific reaction identification of the monoclonal antibody in the examples.

[0017] Figure 2 This is a Western blot image of the cross-reactivity identification of monoclonal antibody 16F6 in the examples.

[0018] Figure 3 This is a Western blot image of the antigenic epitope identified by monoclonal antibody 16F6 in the examples.

[0019] Figure 4 The above are hydrophilicity analysis diagrams of monoclonal antibody 16F6 and epitope localization diagrams of the recognized antigens in the examples.

[0020] Figure 5 This is a model diagram of the spatial structure of the N protein antigen epitope recognized by antibody 16F6 in the embodiment.

[0021] Figure 6 The results are from the ELISA titer assay of purified antibody 16F6 in the examples.

[0022] Figure 7 This involves determining the optimal dilution factor for the enzyme-labeled antibody in the examples.

[0023] Figure 8 This involves determining the optimal coating conditions in the embodiments.

[0024] Figure 9 This refers to the determination of the sample diluent in the examples.

[0025] Figure 10 This refers to the determination of the color development time in the embodiments.

[0026] Figure 11 It is a sensitivity test of PEDV colloidal gold test strips.

[0027] Figure 12 It is a specific detection method for PEDV colloidal gold test strips. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The specific embodiments of the present invention will be further described in detail below. The following detailed descriptions are illustrative and intended to provide further explanation of this application, not to limit the scope of the invention.

[0030] The materials used in the examples are from the following sources: Porcine epidemic diarrhea virus strain CV777, PDCoV HNZK-02 (GenBank: MH708123), and TGEV HN-2012 (GenBank: OP434397), pET-32a(+) plasmid, and Vero cells were all preserved by the Animal Molecular Pathogen Laboratory of Henan Agricultural University; SP2 / 0 cells were purchased from Shanghai Lianmai Biotechnology Co., Ltd.; competent cells DH5α and BL21(DE3) were purchased from Qingke Biotechnology Co., Ltd.; and BALB / c mice were purchased from Henan Provincial Experimental Animal Center.

[0031] The reagents used in the examples were prepared using the following methods: (1) 0.06M acetate buffer: Prepare solution A (0.18M sodium acetate), dissolve 2.45g of sodium acetate in deionized water and bring the volume to 100mL; prepare solution B (0.18M acetic acid), add 2mL of glacial acetic acid to deionized water and bring the volume to 70mL. Mix A:B in a ratio of 5.9:4.1, and add twice the volume of deionized water to the mixture to obtain 0.06M acetate buffer with pH 4.8.

[0032] (2) Saturated sodium sulfate solution: Add excess ammonium sulfate granules to 1L of deionized water at room temperature, stir to dissolve until the solid granules at the bottom no longer dissolve.

[0033] (3) 0.1M NaIO4 solution: Weigh 241mg sodium periodate and dissolve it in 10mL of high-pressure deionized water. After complete dissolution, store at 4℃ and prepare fresh before use.

[0034] (4) 1mM sodium acetate buffer solution: Dilute solution A in (2) above by 180 times with deionized water.

[0035] (5) 0.2M carbonate buffer solution (CB): Weigh 1.59g of sodium carbonate and 2.93g of sodium bicarbonate and dissolve them in 250mL of deionized water. After complete dissolution, store at 4℃ for later use. Dilute with deionized water 4 times to obtain 0.05M carbonate buffer solution (pH=9.6).

[0036] (6) 4 mg / mL NaBH4 solution: Weigh 4 mg NaBH4 and dissolve it in 1 mL of deionized water. After it is completely dissolved, store it at 4 °C and prepare it immediately before use.

[0037] (7) 0.1M Tris hydrochloric acid buffer solution (TB, pH=7.5): Weigh 12.1g Tris and add it to 1L of deionized water, then add concentrated hydrochloric acid until the pH is 7.5.

[0038] (8) 2% BSA: Weigh 2g of bovine serum albumin (BSA) and dissolve it in 80mL of 0.01M PBS. After it is completely dissolved and mixed, bring the volume up to 100mL and store at 4℃ for later use.

[0039] Example 1: Expression and purification of recombinant PEDV N protein 1. Selection of the dominant region of PEDV N protein antigen The antigenicity, hydrophilicity, and surface accessibility of the N protein of PEDV CV777 strain (GenBank accession number: AF353511) were analyzed using DNA Star software. Based on the comprehensive analysis of these parameters, the antigenic dominant region 1-442 aa was selected as the target fragment for recombinant expression.

[0040] 2. Construction of the pET-32a-PEDV-N recombinant vector Total RNA was extracted from PEDV CV777 strain using the Trizol method and transcribed into cDNA using a reverse transcription kit (PrimeScript™ RTMaster Mix, TaKaRa). Primers for amplifying the full-length N gene were designed based on the N gene sequence of PEDV CV777 strain. F:GGCTGATATCGGATCCGAATTCATGGCTTCTGTCAGCTTTCA (SEQ ID NO:1) R:TCGAGTGCGGCCGCAAGCTTTCCTGTGTCGAAGA (SEQ ID NO: 2) PCR reaction system (total 50 μL): KOD Taq Mix 25 μL, ddH2O 19 μL, forward and reverse primers 2 μL each, cDNA 2 μL.

[0041] PCR reaction program: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 68℃ extension for 10 s, for a total of 35 cycles; final extension at 72℃ for 1 min.

[0042] The gene fragment and pET-32a(+) vector were digested and amplified using endonucleases EcoRI and HindIII, ligated using homologous recombination, and transformed into E. coli DH5α competent cells. Single colonies were picked for bacterial PCR identification. The positive strain (pET-32a-PEDV-N) was sent to Shangya Biotechnology Co., Ltd. for sequencing. The strains with correct sequences were retained for subsequent experiments.

[0043] 3. Inducible expression of recombinant PEDV N protein The frozen pET-32a-PEDV-N expression strain was inoculated 1:100 into liquid LB medium containing 100 μg / mL ampicillin and cultured at 37°C with shaking at 220 rpm until OD500 was reached. 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 1 mmol / L, and expression was induced at 37℃ for 4 h. After induction, the bacterial cell pellet was collected by centrifugation at 4℃ and 8000 rpm for 10 min.

[0044] 4. Purification of recombinant PEDV N protein After resuspending the bacterial cell pellet in PBS, it was sonicated in an ice bath (300 W, 5 s on, 5 s off, total 15 min). The lysate was centrifuged at 12000 rpm for 30 min at 4°C. The supernatant was collected, filtered through a 0.45 μm filter, and purified using a nickel-ion affinity chromatography column (HisTrap™ HP, GE Healthcare). The specific steps were as follows: the column was equilibrated with PBS containing 20 mM imidazole, impurities were washed with PBS containing 50 mM imidazole, and the target protein was eluted with PBS containing 250 mM imidazole. The elution peak was collected.

[0045] The purified protein solution was placed in a dialysis bag (molecular weight cutoff 14 kDa) and dialyzed overnight in PBS (pH 7.4) at 4°C, with the dialysate changed three times during the process. After dialysis, the protein concentration was determined using a BCA protein assay kit, and the purity was identified by SDS-PAGE. The protein was then aliquoted and stored at -80°C for later use.

[0046] Example 2: Preparation and screening of hybridoma cell lines 1. Mouse immunization Three 6-8 week old SPF-grade female BALB / c mice were selected and immunized with the PEDV-N recombinant protein purified in Example 1. A total of three immunizations were administered, with each immunization spaced two weeks apart.

[0047] First immunization: 60 μg of recombinant protein was emulsified with an equal amount of Freund's complete adjuvant and administered via multiple subcutaneous injections on the back.

[0048] Second immunization (14 days after the first immunization): Take 60 μg of recombinant protein and emulsify it thoroughly with an equal amount of Freund's incomplete adjuvant. The immunization route is the same as the first immunization.

[0049] Third immunization (14 days after the second immunization): Take 60 μg of recombinant protein and emulsify it thoroughly with an equal amount of Freund's incomplete adjuvant. The immunization route is the same as the first immunization.

[0050] Three days before fusion, a booster immunization was administered at a dose of 60 μg per animal, without adjuvant, via intraperitoneal injection.

[0051] Blood was collected from the tail of mice on day 7 after each immunization, and serum was separated. Serum titer was detected using an indirect ELISA method. Mice with the highest serum titer were selected for subsequent cell fusion experiments.

[0052] 2. Cell fusion One week before fusion, SP2 / 0 myeloma cells were revived and expanded in RPMI-1640 complete medium containing 20% ​​fetal bovine serum to ensure that the cells were in the logarithmic growth phase and the cell viability was >95% on the day of fusion.

[0053] On the third day after booster immunization, immunized mice were euthanized by exsanguination through the orbital fossa. The spleen was removed under aseptic conditions and placed in a sterile Petri dish. The spleen was washed three times with serum-free RPMI-1640 medium. The spleen was then placed on a 200-mesh stainless steel sieve and gently ground using a syringe plunger to collect the spleen cell suspension. The spleen cells were washed twice with serum-free RPMI-1640 medium and counted.

[0054] SP2 / 0 cells and spleen cells were mixed at a ratio of 1:5. 1 mL of 50% PEG 4000 (pH 8.0), pre-warmed to 37°C, was added dropwise over 1 minute with gentle stirring. Then, 10 mL of serum-free RPMI-1640 medium was slowly added over 2 minutes to terminate the fusion reaction. Serum-free medium was then added to bring the total volume to 50 mL. The mixture was centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The cell pellet was resuspended in HAT selective medium, and the cell density was adjusted to 2 × 10⁶ cells / mL. 6 Cells / mL were seeded into 96-well cell culture plates at a rate of 200 μL per well and incubated at 37°C in a 5% CO2 incubator.

[0055] 3. Screening of positive hybridoma cells On day 7 post-fusion, half the medium was replaced with HAT medium. On days 10-14 post-fusion, when the hybridoma cell colonies grew to approximately 1 / 10-1 / 5 of the well bottom area, the cell culture supernatant from each well was collected and positive selection was performed using an indirect ELISA method.

[0056] The indirect ELISA method is as follows: (1) Coating: The recombinant PEDV N protein purified in Example 1 was diluted to 1 μg / mL with 0.05 M carbonate buffer (pH 9.6), and 100 μL was added to each well of a 96-well microplate. The plate was coated overnight at 4°C.

[0057] (2) Washing: Discard the coating solution, wash 3 times with PBST (PBS containing 0.05% Tween-20) for 3 min each time, and pat dry.

[0058] (3) Blocking: Add 150 μL of PBS blocking solution containing 5% skim milk powder to each well and block at 37°C for 2 h. Wash 3 times and pat dry.

[0059] (4) Sample addition: Add 100 μL of hybridoma cell culture supernatant to each well. SP2 / 0 cell supernatant was used as a negative control, PBS as a blank control, and positive mouse serum as a positive control. Incubate at 37℃ for 1 h. Wash 3 times and pat dry.

[0060] (5) Add enzyme-labeled secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse IgG (1:5000 dilution) to each well and incubate at 37℃ for 1 h. Wash 3 times and pat dry.

[0061] (6) Color development: Add 100 μL of TMB single-component color development solution to each well and develop color at room temperature in the dark for 10 min.

[0062] (7) Termination: Add 50 μL of ELISA stop solution to each well and read the OD value using a microplate reader. 450 value.

[0063] Hybridoma cells from positive wells (OD value ≥ 2.1 times that of the negative control) were subcloned using a limiting dilution method. Cells were diluted to a density of 0.5–1 cells per well and seeded into 96-well plates. After monoclonal cell colonies formed, indirect ELISA was performed again. Subcloning was repeated 2–3 times until all cells from positive wells secreted specific antibodies, thus obtaining a stable hybridoma cell line secreting monoclonal antibodies.

[0064] After the above screening, a hybridoma cell line that stably secretes monoclonal antibodies was obtained, named 16F6, and cryopreserved in a liquid nitrogen tank.

[0065] This hybridoma cell line was deposited at the China Center for Type Culture Collection (CCTCC) on May 25, 2026, with accession number CCTCC NO: C2026112. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. It is classified and named as hybridoma cell line PEDV-N-16F6.

[0066] Example 3: Preparation and purification of monoclonal antibody 16F6 1. Preparation of monoclonal antibody ascites Healthy BALB / c mice aged 8-10 weeks were selected, and each mouse was pretreated by intraperitoneal injection of 500 μL of liquid paraffin one week before the injection of hybridoma cells.

[0067] The hybridoma cell line PEDV-N-16F6 obtained in Example 2 was resuscitated from liquid nitrogen and cultured to the logarithmic growth phase. The cells were washed twice with serum-free RPMI-1640 medium, and the cell density was adjusted to 2 × 10⁶ cells / year. 6 Cells / mL. Each mouse was intraperitoneally injected with 0.5 mL of cell suspension (containing 1×10⁶ cells / mL). 6 (cells).

[0068] 7-10 days after injection, when the mouse abdomen is noticeably distended, the peritoneal cavity is punctured with a 16-gauge needle to collect ascites. The collected ascites is centrifuged at 3000 rpm for 15 min at 4°C to remove the fat layer and cell debris. The supernatant is aliquoted and stored at -40°C for later use.

[0069] 2. Purification of monoclonal antibodies Monoclonal antibodies in ascites fluid were purified using the caprylic acid-ammonium sulfate precipitation method. The specific steps are as follows: (1) Take ascites fluid and slowly add 2 volumes of 0.06 M acetate buffer (pH 4.8) to 1 volume of ascites fluid at a ratio of 1:2, and stir to mix well.

[0070] (2) Add 33 μL of caprylic acid per milliliter of ascites fluid, while stirring and slowly drop the caprylic acid, and let it stand at 4°C for 2 h.

[0071] (3) Centrifuge at 4℃ and 12000 rpm for 30 min, take the supernatant and filter it with double-layer filter paper to remove residual lipids.

[0072] (4) Add 1 / 10 volume of 0.01 M PBS (pH 7.4) to the supernatant and adjust the pH to 7.4 with 1 M NaOH.

[0073] (5) While stirring, slowly add saturated ammonium sulfate solution (pH 7.4) to make the final concentration of ammonium sulfate 45%, and let it stand at 4℃ for 12-16 h.

[0074] (6) Centrifuge at 4℃ and 12000 rpm for 30 min, discard the supernatant, and resuspend the precipitate in an appropriate amount of 0.01 M PBS.

[0075] (7) Place the resuspension into a dialysis bag (molecular weight cutoff 8-14 kDa), place it in 0.01 M PBS (pH 7.4), and dialyze at 4°C for 12-16 h, changing the dialysis solution 3 times during the process to fully remove ammonium sulfate.

[0076] (8) After dialysis, the antibody solution was centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant was collected and the antibody concentration was determined using a Nanodrop2000 spectrophotometer. The solution was then aliquoted and frozen at -80°C.

[0077] SDS-PAGE was used to identify the purity of the purified antibody. The results showed that the light chain (approximately 25 kDa) and heavy chain (approximately 55 kDa) bands of the antibody were clear, and the purity was >90%.

[0078] Sequencing of the CDR and FR sequences of the variable region of monoclonal antibody 16F6 (mAb 16F6) was performed, and the results are as follows: mAb 16F6 antibody light chain: FR1 amino acid (SEQ ID NO:3): DVLMTQTPLSLPVSLGDQASISCRSS; FR1 nucleotide (SEQ ID NO:4): gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagt; VLCDR1 amino acid (SEQ ID NO:5): QSIVHSNGNTY; VLCDR1 nucleotide (SEQ ID NO:6): cagagcattgtacatagtaatggaaacacctat; FR2 amino acid (SEQ ID NO:7): LEWYLQKPGQSPKLLIY; FR2 nucleotide (SEQ ID NO:8): ttagaatggtacctgcagaagccaggccagtctccaaagctcctgatctac; VLCDR2 amino acids (sequence with fewer than 4 amino acids that do not meet specific significance and are not included in the sequence listing): KVS; VLCDR2 nucleotides (the sequence contains fewer than 10 specifically defined nucleotides and is not listed in the sequence listing): aaagtttcc; FR3 amino acid (SEQ ID NO:9): NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYC; FR3 nucleotide (SEQ ID NO:10): aaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggaatttattactgc; VLCDR3 amino acid (SEQ ID NO:11): FQTSHVPPT; VLCDR3 nucleotide (SEQ ID NO:12): tttcaaacttcacatgttcctccgacg; FR4 amino acid (SEQ ID NO:13): FGGGTKLEIK; FR4 nucleotide (SEQ ID NO:14): ttcggtggaggcaccaagctggaaatcaaa; The amino acid sequence of mAb 16F6-light chain (SEQ ID NO:15): DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQTSHVPPTFGGGTKLEIK.

[0079] mAb 16F6 antibody heavy chain: FR1 amino acid (SEQ ID NO:16): EVKLVDSGGGLVQPGGSLRLSCVTS; FR1 nucleotide (SEQ ID NO:17): gaggtgaagctggtggactctggaggaggcttggtacagcctgggggttctctgagactctcctgtgtgacttct; VHCDR1 amino acid (SEQ ID NO:18): GFTFTDYY; VHCDR1 nucleotide (SEQ ID NO:19): gggttcaccttcactgattactac; FR2 amino acid (SEQ ID NO:20): MSWVRQPPGKALEWLGF; FR2 nucleotide (SEQ ID NO:21): atgagctgggtccgccagcctccaggaaaggcacttgagtggttgggtttt; VHCDR2 amino acid (SEQ ID NO:22): IRNKANGYTT; VHCDR2 nucleotide (SEQ ID NO:23): attachaaacaaagctaatggttacacaaca; FR3 amino acid (SEQ ID NO:24): EYSASVKGRFTISRDNSQNILYLQMNTLTPEDSATYYC; FR3 nucleotide (SEQ ID NO:25): gagtacagtgcatctgtgaagggtcggttcaccatctccagagataattcccagaacatcctctatcttcaaatgaataccctgacacctgaggacagtgccacttattactgt; VHCDR3 amino acid (SEQ ID NO:26): ARDSDWAEFAY; VHCDR3 nucleotide (SEQ ID NO:27): gcaagagattctgactgggccgagtttgcttac; FR4 amino acid (SEQ ID NO:28): WGQGTLVTVSA; FR4 nucleotide (SEQ ID NO:29): tggggccaagggactctggtcactgtctctgca; The amino acid sequence of mAb 16F6-heavy chain (SEQ ID NO:30): EVKLVDSGGGLVQPGGSLRLSCVTSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISRDNSQNILYLQMNTLTPEDSATYYCARDSDWAEFAYWGQGTLVTVSA.

[0080] Example 4: Specificity identification of monoclonal antibody 16F6 1. Identification by indirect immunofluorescence assay (IFA) Vero cells were seeded in 24-well cell culture plates and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the cell density reached approximately 80%. Then, PEDV CV777 virus strain was inoculated (MOI=1). Vero cells without virus inoculation were set up as a negative control.

[0081] 24 h after inoculation, discard the culture medium and wash the cells three times with PBS. Add 1 mL of pre-cooled anhydrous ethanol (4°C) to each well and fix at 4°C for 4 h. After fixation, discard the ethanol and wash three times with PBST.

[0082] Add 1 mL of PEDV monoclonal antibody 16F6 diluted 1:1000 with PBS to each well and incubate at 37°C for 1 h. After washing three times with PBST, add FITC-labeled goat anti-mouse IgG (1:500 dilution) to each well and incubate at 37°C in the dark for 1 h. After washing three times with PBST, add a mounting medium containing DAPI to each well and observe and photograph using an inverted fluorescence microscope.

[0083] The results are as follows Figure 1 As shown, a distinct green fluorescent signal was observed in PEDV-infected Vero cells, with the fluorescence mainly distributed in the cytoplasm; while no specific fluorescent signal was observed in the uninoculated negative control cells. These results indicate that monoclonal antibody 16F6 can specifically recognize the N protein in PEDV-infected cells.

[0084] 2. Western blot to identify cross-reactivity Vero cells were inoculated with PEDVCV777, PDCoV HNZK-02 (GenBank: MH708123), and TGEV HN-2012 (GenBank: OP434397), respectively, with uninoculated Vero cells serving as a negative control. Twenty-four hours after infection, cells were collected and lysed on ice for 10 min using RIPA lysis buffer containing PMSF. Cells were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. 5×SDS loading buffer was added, and the cells were denatured in a boiling water bath for 10 min.

[0085] Prepare a 12% SDS-PAGE gel, add 20 μL of denatured protein sample to each well, and perform electrophoresis (stacking gel 80 V, separating gel 120 V). After electrophoresis, transfer the protein to a PVDF membrane (0.45 μm pore size) using a semi-dry transfer method. Transfer conditions: constant current 200 mA, transfer for 60 min.

[0086] After transfer, the PVDF membrane was placed in TBST blocking buffer containing 5% skim milk powder and blocked on a shaker at room temperature for 2 h. After blocking, the membrane was washed three times with TBST for 5 min each time. The membrane was then incubated overnight at 4°C in PEDV monoclonal antibody 16F6 diluted in PBS (1:5000 dilution). After three TBST washes, the membrane was placed in HRP-labeled goat anti-mouse IgG secondary antibody (1:10000 dilution) and incubated at room temperature for 1 h. After three TBST washes, the membrane was developed using ECL chemiluminescence immunoassay and photographed using an ImageQuant 800 imager.

[0087] The results are as follows Figure 2 As shown, monoclonal antibody 16F6 was detected only in PEDV-infected cell lysate samples with a specific band of approximately 55 kDa (consistent with the molecular weight of PEDV N protein), while no specific band was observed in PDCoV- and TGEV-infected cell lysates or uninfected cell lysates. These results indicate that monoclonal antibody 16F6 has good specificity and no cross-reactivity with porcine enteric coronaviruses such as PDCoV and TGEV.

[0088] Example 5: Identification of antigenic epitopes recognized by monoclonal antibody 16F6 1. Design and expression of truncated fragments of PEDV N protein To precisely locate the antigenic epitope recognized by monoclonal antibody 16F6, three overlapping truncated fragments covering the full length (1-442 aa) of the N protein were designed (N1: 1-150 aa, N2: 150-300 aa, N3: 290-442 aa). Primers were designed based on the gene sequences of each truncated fragment (primer sequences are shown in Table 1). PCR amplification was performed using pET-32a-PEDV-N plasmid as a template. The corresponding recombinant expression vector was constructed according to the method in Example 1, transformed into BL21(DE3) competent cells, and IPTG was used to induce the expression of the recombinant protein. After SDS-PAGE to identify the expression status, Western blot analysis was performed according to the method described in Example 4.

[0089] Western blot results showed that monoclonal antibody 16F6 reacted only with the N3 truncated protein (290-442 aa) and not with the N1 and N2 truncated proteins, suggesting that the antigenic epitope is located in the 290-442 aa region of the N protein.

[0090] To further refine the localization, the N3 region (290-442 aa) was divided into seven overlapping segments (N3-1 to N3-7, corresponding to 290-320 aa, 310-340 aa, 330-360 aa, 350-380 aa, 370-400 aa, 390-420 aa, and 410-442 aa, respectively). Primers were designed (primer sequences are shown in Table 1), and expression and Western blot identification were performed according to the methods described above.

[0091] Western blot results showed that monoclonal antibody 16F6 reacted with the N3-6 truncated protein (390-420 aa), suggesting that the antigenic epitope is located in the 390-420 aa region.

[0092] 2. Precise identification of antigenic epitopes The N3-6 region (390-420 aa) was further divided into four overlapping segments (N3-6-1 to N3-6-4), corresponding to 390-405 aa, 395-410 aa, 400-415 aa, and 405-420 aa, respectively. Primers were designed (primer sequences are shown in Table 1), and recombinant plasmids were constructed for expression and identified by Western blot.

[0093] The results showed that monoclonal antibody 16F6 reacted with both N3-6-1 (390-405 aa) and N3-6-2 (395-410 aa) truncated proteins, suggesting that the antigenic epitope is located in the overlapping region of the two, namely 395-405 aa.

[0094] The overlapping regions of the N3-6-1 and N3-6-2 genes were truncated into nine overlapping fragments (N3-6-1-1 to N3-6-1-9, corresponding to 395-405 aa, 396-406 aa, 397-407 aa, 398-408 aa, 399-409 aa, 400-410 aa, 401-411 aa, 402-412 aa, and 403-413 aa, respectively). Primers were designed (primer sequences are shown in Table 1), and expression and Western blot identification were performed.

[0095] Western blot results showed that ( Figure 3 The monoclonal antibody 16F6 reacted with truncated proteins of N3-6-1-4 (398-408 aa), N3-6-1-5 (399-409 aa), and N3-6-1-6 (400-410 aa), suggesting that the core epitope is located in a common region of these fragments.

[0096] Further verification through dot blot hybridization using synthesized overlapping peptides confirmed that the antigenic epitope targeted by monoclonal antibody 16F6 is 401-EEAIYDDV-408.

[0097] Table 1: Primer sequences for each truncated gene of PEDV N Example 6: Prediction of the spatial structure of antigenic epitopes To determine the spatial location of the epitope recognized by monoclonal antibody 16F6 on the PEDV N protein, the three-dimensional structure of the PEDV N protein was predicted using Swiss-model software. The predicted three-dimensional structure was edited and visualized using RCSB-PDB software, and the spatial location and surface accessibility of the epitope (401-EEAIYDDV-408) recognized by monoclonal antibody 16F6 within the N protein's three-dimensional structure were analyzed.

[0098] The results are as follows Figure 5 As shown, the antigenic epitope recognized by monoclonal antibody 16F6 is exposed on the surface of PEDV N protein in a three-dimensional structure, which has good surface accessibility and facilitates antibody recognition and binding.

[0099] Example 7: Determination of the titer of purified antibody 16F6 The titer of the purified monoclonal antibody 16F6 from Example 3 was determined using an indirect ELISA method. The specific steps are as follows: (1) Coating: The recombinant PEDV N protein purified in Example 1 was diluted to 1 μg / mL with 0.05 M carbonate buffer (pH 9.6), and 100 μL was added to each well of a 96-well microplate. The plate was coated overnight at 4°C.

[0100] (2) Washing: Discard the coating solution, wash 3 times with PBST, and pat dry.

[0101] (3) Blocking: Add 150 μL of PBS containing 5% skim milk powder to each well and block at 37°C for 2 h. Wash 3 times and pat dry.

[0102] (4) Sample addition: The purified monoclonal antibody 16F6 was serially diluted with PBS (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200), and 100 μL was added to each well. SP2 / 0 cell culture supernatant was used as a negative control, and PBS as a blank control. The cells were incubated at 37℃ for 1 h. The cells were washed three times and patted dry.

[0103] (5) Add enzyme-labeled secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse IgG (1:5000 dilution) to each well and incubate at 37℃ for 1 h. Wash 3 times and pat dry.

[0104] (6) Color development: Add 100 μL of TMB single-component color development solution to each well and develop color at room temperature in the dark for 10 min.

[0105] (7) Termination: Add 50 μL of ELISA stop solution to each well and read the OD value using a microplate reader. 450 value.

[0106] The results are as follows Figure 6 As shown, a positive result is defined as an OD value higher than 0.3, and the purified 16F6 antibody ELISA titer can reach 1:25600. It can bind efficiently to the PEDV N protein.

[0107] Example 8: Performance evaluation of clonal antibody 16F6 in PEDV detection I. Establishment of Antibody Detection Method Based on Blocking ELISA 1. HRP labeling of monoclonal antibodies The monoclonal antibody 16F6 was HRP-labeled using the sodium periodate method. NaIO4 was used to oxidize the sugar molecules on the HRP surface to aldehyde groups, which then bound to the amino groups on the IgG. The steps are as follows.

[0108] (1) Weigh 5 mg of horseradish peroxidase (HRP) and dissolve it in 1 mL of distilled water.

[0109] (2) Add 200 μL of freshly prepared 0.1 M NaIO4 solution to the above solution and stir at room temperature in the dark for 20-30 min.

[0110] (3) The above solution was placed into a dialysis bag (dialysis molecular weight 8000-14000D) and dialyzed in a 1mM sodium acetate buffer solution with pH=4.4 at 4℃ for 12h.

[0111] (4) Add 200 μL of 0.2 M (pH=9.6) CB solution to the dialysis solution, adjust the pH of the aldehyde HRP to about 9.5 using 1 M NaOH, and then quickly add 10 mg of purified antibody (concentration of 1.4 mg / mL, solvent of 0.01 M PBS), and stir gently at 4 °C in the dark for 12-16 h.

[0112] (5) Add 235 μL of freshly prepared 4 mg / mL NaBH4 solution (47 μL of NaBH4 per 1 mg HRP) to the above solution, mix well and let stand at 4℃ for 2 h.

[0113] (6) Add an equal volume of saturated ammonium sulfate solution to the above solution, precipitate at 4℃ for 30 min, and centrifuge at 4℃ and 12000 r / min for 30 min.

[0114] (7) Discard the supernatant, resuspend the precipitate in 0.01M PBS, and dialyze it in 0.01M PBS solution at 4℃ for 12-16h.

[0115] (8) Centrifuge the dialysis solution at 12000r / min for 10min. The supernatant is the HRP-labeled monoclonal antibody 16F6 (enzyme-labeled antibody). Aliquot and store at -80℃.

[0116] 2. Optimization of optimal reaction conditions for blocking ELISA (1) Determination of antigen coating concentration and serum dilution factor The optimal antigen coating concentration and serum dilution factor were determined using a square matrix method. The recombinant PEDVN protein purified in Example 1 was diluted with 0.05 M carbonate buffer (pH 9.6) to 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and 0.0625 μg / mL, respectively, and coated onto the microplate at 100 μL per well, incubating overnight at 4°C. After washing and blocking, standard positive and negative sera were diluted with PBS at dilutions of 1:5, 1:10, 1:50, and 1:500, respectively, and added to the microplate at 100 μL per well, with two replicates for each dilution. After incubation at 37°C for 1 h, the plates were washed three times, and 100 μL of HRP-labeled goat anti-mouse IgG (1:5000 dilution) was added to each well, incubating at 37°C for 1 h. After washing, TMB chromogenic buffer was added, and the OD was read after termination. 450 The optimal conditions are a negative serum OD value greater than 1.0, a positive serum OD value less than 0.3, and a minimum P / N ratio.

[0117] The results are shown in Table 2. When the PEDV N protein coating concentration was 0.125 μg / mL and the serum dilution factor was 1:500, the negative serum OD value was higher and the positive serum OD value was lower, and the P / N value was the smallest. These were determined to be the optimal coating concentration and serum dilution factor.

[0118] Table 2: Optimal antigen coating concentration and standard positive and negative serum dilution factors for determination by the square matrix method (2) Determination of the optimal dilution factor for enzyme-labeled antibodies The HRP-labeled monoclonal antibody 16F6 prepared in Example 8 was diluted with PBS at ratios of 1:1000, 1:2000, and 1:4000, respectively. The ELISA plate was coated with the optimal antigen coating concentration (0.125 μg / mL), and standard positive and negative sera were treated with the optimal serum dilution (1:500). After adding the enzyme-labeled antibody, the ELISA procedure was performed as usual. OD values ​​were read. 450 The optimal dilution factor for enzyme-labeled antibody is determined by minimizing the P / N value.

[0119] The results are as follows Figure 7 As shown, the P / N value of the enzyme-labeled antibody is lowest when diluted at 1:4000. Therefore, the optimal dilution ratio for the enzyme-labeled antibody is 1:4000.

[0120] (3) Determination of optimal coating conditions After coating the microplate with the optimal concentration of PEDV N protein (0.125 μg / mL), the plates were incubated at 4°C for 20–24 h and at 37°C for 3 h, respectively. All other steps were performed according to the optimized blocking ELISA conditions, with the temperature and time resulting in the lowest P / N ratio considered the optimal coating conditions.

[0121] The results are as follows Figure 8 As shown, the P / N value of coating at 4℃ for 20 h is lower than that of coating at 37℃ for 3 h. Therefore, coating at 4℃ for 20 h is selected as the optimal coating condition.

[0122] (4) Determination of the optimal sample diluent After coating and blocking, standard positive and negative sera were diluted with PBS and PBST (PBS containing 0.05% Tween-20) at the optimal dilution factor (1:500), respectively. The remaining steps were performed as optimized conditions for the blocking ELISA assay. The optimal sample dilution was determined by the lowest P / N value.

[0123] The results are as follows Figure 9 As shown, the P / N value of the test group diluted with PBS was lower than that of the test group diluted with PBST, therefore PBS is more suitable as a sample diluent.

[0124] (5) Determination of the optimal color development time ELISA assays were performed under optimized conditions. After the enzyme-labeled antibody reaction, the cells were washed five times with PBST. Then, 100 μL of TMB single-component chromogenic solution was added to each well, with incubation times set to 5 min, 8 min, 10 min, and 15 min, respectively. OD values ​​were read after adding stop solution. 450 The optimal color development time is determined by minimizing the P / N value.

[0125] The results are as follows Figure 10 As shown, the P / N value is lowest when the color development time is 8 min, which is determined to be the optimal color development time.

[0126] II. PEDV antigen detection based on immunochromatographic test strips A detection probe was prepared by conjugating monoclonal antibody 16F6 with colloidal gold. A double-antibody sandwich immunochromatographic test strip was established using goat anti-mouse antibody as the control line (C line) and PEDV N protein polyclonal antibody as the detection line (T line). After optimization, the optimal conditions for the colloidal gold test strip were: T line pAb PEDV N coating concentration of 1.5 mg / mL, 10 μL of colloidal gold-labeled antibody added per mL, 8 μL of probe used per 150 μL of sample, and a reaction time of 10 min.

[0127] PEDV virus fluid (10) 4 TCID 50 A series of serial dilutions were performed, and the results were detected using test strips. The results showed ( Figure 11 When the PEDV virus solution is diluted to 1:128 (i.e., 2...), 7 Even after dilution (by several times), a distinct band still appears on the test strip. Therefore, the detection limit for PEDV of this invention is approximately 7.8 × 10⁻⁶. 1 TCID 50 / mL.

[0128] Furthermore, specific detection was performed using PEDV, PDCoV, TGEV, and PSV viral solutions as samples. The test strips only reacted with PEDV and showed color development, with no cross-reaction with other viruses. Figure 12 It has high specificity.

[0129] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. A monoclonal antibody against the N protein of porcine epidemic diarrhea virus, characterized in that, The monoclonal antibody against porcine epidemic diarrhea virus N protein comprises VLCDR1 with the amino acid sequence shown in SEQ ID NO:5, VLCDR2 with the amino acid sequence KVS, and VLCDR3 with the amino acid sequence shown in SEQ ID NO:11; and VHCDR1 with the amino acid sequence shown in SEQ ID NO:18, VHCDR2 with the amino acid sequence shown in SEQ ID NO:22, and VHCDR3 with the amino acid sequence shown in SEQ ID NO:

26.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 15, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.

30.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the antigenic epitope recognized by the monoclonal antibody is EEAIYDDV.

4. A hybridoma cell line PEDV-N-16F6, characterized in that, The hybridoma cell line PEDV-N-16F6 secretes the monoclonal antibody of claim 1. The hybridoma cell line PEDV-N-16F6 was deposited at the China Center for Type Culture Collection on May 25, 2026, with accession number CCTCC NO: C2026112, and the deposit address is Wuhan University, Wuhan, China.

5. The use of the monoclonal antibody according to claim 1 in the preparation of products for detecting porcine epidemic diarrhea virus.

6. The application according to claim 5, characterized in that, The product in question is a test strip.

7. The application according to claim 6, characterized in that, The test strip comprises a detection probe, a detection line, and a control line; the detection probe is a conjugate of monoclonal antibody and colloidal gold as described in claim 1, the detection line is coated with a polyclonal antibody against porcine epidemic diarrhea virus N protein, and the control line is coated with goat anti-mouse antibody.

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