Hybridoma cell strain of porcine epidemic diarrhea virus, monoclonal antibody and preparation method and application thereof

Abstract

This invention belongs to the field of molecular biology, specifically relating to a hybridoma cell line for porcine epidemic diarrhea virus (PEDV), a monoclonal antibody, its preparation method, and its applications. The hybridoma cell line 7AH-11H, with accession number CCTCC NO: C202679, was deposited at the China Center for Type Culture Collection on April 16, 2026. This invention optimizes the N protein gene sequence, expresses and purifies it to obtain a high-purity immunogen, and through immunization of mice, cell fusion, and subclonal screening, obtains a stable antibody-secreting hybridoma cell line, thereby preparing a high-affinity, broad-spectrum monoclonal antibody. This monoclonal antibody can be used to prepare kits for detecting PEDV, such as Western blotting, IFA, and IHC kits, providing important tool support for the clinical diagnosis and basic research of PEDV.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a hybridoma cell line of porcine epidemic diarrhea virus, a monoclonal antibody, its preparation method and application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious disease caused by the porcine epidemic diarrhea virus. It is characterized by vomiting, watery diarrhea, dehydration, and anorexia in piglets, causing huge economic losses to the global pig industry.

[0003] Porcine epidemic diarrhea virus (PEDV) is a positive-sense RNA virus belonging to the family Coronaviridae, genus Alphacoronavirus. Its genome is approximately 28 kb in length, including 7 open reading frames (ORFs) encoding 16 non-structural proteins, 4 structural proteins, and the accessory protein ORF3. The PEDV nucleocapsid protein, or N protein, is one of the structural proteins of PEDV and a core component of coronaviruses. The N protein exhibits high conservation and high abundance expression across different isolates, participating not only in viral genome packaging and replication regulation but also serving as a core target for virus detection, epidemiological investigation, and pathogenic mechanism research. Therefore, the N protein is a preferred protein for developing molecular diagnostic tools for PEDV.

[0004] Currently, antibody tools are crucial for PEDV detection technology and basic research. Monoclonal antibodies, in particular, are widely used in immunohistochemistry, ELISA kit development, and viral particle localization due to their advantages such as high specificity, high uniformity, and scalability. However, existing PEDV N protein monoclonal antibodies have several shortcomings: First, some antibodies target epitopes with insufficient conservation, only recognizing classic PEDV strains and failing to effectively bind to currently prevalent variants, thus limiting their applicability. Second, traditional antibody preparation processes suffer from low antigen purity, unreasonable immunization protocols, and limited screening methods, resulting in low antibody titers and weak affinity, making it difficult to meet the demands of high-sensitivity detection. Third, existing antibody identification systems are incomplete, lacking comprehensive validation of antibody specificity, broad spectrum, and functionality, and cannot guarantee their stability in different application scenarios. Meanwhile, in PEDV VLP vaccine development and virus-host interaction mechanism research, highly specific and broad-spectrum N protein monoclonal antibodies are urgently needed as core reagents for viral antigen quantification, dynamic tracking of viral infection, and analysis of related protein interactions. Therefore, providing a hybridoma cell with a high-affinity and broad-spectrum monoclonal antibody targeting the conserved epitope of PEDV N protein is of great significance for improving the clinical diagnostic efficiency of PEDV and promoting the vaccine development process. It is also a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a hybridoma cell line of porcine epidemic diarrhea virus, which solves the problems existing in the prior art.

[0006] The technical solution adopted in this invention is: This invention provides a hybridoma cell line 7AH-11H, which has the accession number CCTCC NO: C202679 and was deposited at the China Center for Type Culture Collection on April 16, 2026.

[0007] A second aspect of the present invention provides a monoclonal antibody against porcine epidemic diarrhea virus, said monoclonal antibody being secreted by the hybridoma cell line 7AH-11H.

[0008] A third aspect of this invention provides a method for preparing the monoclonal antibody, comprising the following steps: Mice were immunized with an immunogen to obtain immune spleen cells; the nucleotide sequence encoding the immunogen is shown in SEQ ID NO.2; The immune spleen cells were fused with myeloma cells and screened to obtain the hybridoma cell line 7AH-11H. Monoclonal antibodies were prepared using the hybridoma cell line 7AH-11H.

[0009] Preferably, the ratio of immune spleen cells to myeloma cells is 2:1.

[0010] Preferably, the fusion agent used for cell fusion is PEG4000.

[0011] The fourth aspect of the present invention provides the application of the hybridoma cell line 7AH-11H and / or the monoclonal antibody, wherein the application refers to the preparation of a detection reagent for detecting porcine epidemic diarrhea virus.

[0012] Preferably, the detection reagent is suitable for IFA detection, IHC detection and / or Western Blot detection.

[0013] Preferably, the detection reagent also includes an enzyme-labeled secondary antibody or a fluorescently labeled secondary antibody.

[0014] Preferably, the enzyme-labeled secondary antibody includes a horseradish peroxidase-labeled secondary antibody or an alkaline phosphatase-labeled secondary antibody; The fluorescently labeled secondary antibody was a FITC-labeled secondary antibody.

[0015] The preservation information for biological material samples involved in this invention is as follows: Hybridoma cell line 7AH-11H was deposited at the China Center for Type Culture Collection (CCTCC) on April 16, 2026, with accession number CCTCC No: C202679, at Wuhan University, Wuhan, China.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a hybridoma cell line 7AH-11H, whose accession number is CCTCC NO: C202679, and which was deposited at the China Center for Type Culture Collection on April 16, 2026. This invention obtains a hybridoma cell line by optimizing the N protein gene sequence, preparing an immunogen using a mammalian expression system, and combining a reasonable immunization procedure and screening strategy. The hybridoma cell line 7AH-11H and its secreted monoclonal antibody provided by this invention systematically solve the problems of insufficient epitope conservation, low antibody titer, poor broad-spectrum activity, and imperfect identification system in existing technologies. It possesses high specificity, high sensitivity, broad-spectrum cross-reactivity, and multi-platform applicability, and has significant application value in the epidemiological surveillance, clinical diagnosis, pathological research, and basic research of PEDV. Attached Figure Description

[0017] Figure 1 Coomassie Brilliant Blue analysis for protein purification.

[0018] Figure 2 Western blot analysis for purified protein.

[0019] Figure 3 The antibody titer in the serum of mice after secondary immunization.

[0020] Figure 4 These are hybridoma cells after fusion.

[0021] Figure 5 To determine the potency of monoclonal antibodies derived from ascites fluid.

[0022] Figure 6 Western blot identification of monoclonal antibodies.

[0023] Figure 7 For IFA identification of monoclonal antibodies.

[0024] Figure 8 IHC identification of monoclonal antibodies. Detailed Implementation

[0025] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0026] The inventive concept of this invention is as follows: This invention, through optimizing the N protein gene sequence, preparing the immunogen using a mammalian expression system, and combining a rational immunization and screening strategy, obtained a hybridoma cell line and its secreted monoclonal antibody. Compared with existing technologies, this invention has the following advantages:

[0027] 1. More natural antigen conformation: The mammalian expression system ensures the correct folding and modification of the N protein, effectively inducing highly specific antibodies against the native conformation epitope, avoiding epitope loss or conformational abnormalities caused by the lack of post-translational modification in prokaryotic expression products.

[0028] 2. Higher antibody titer: Immunization with the native conformation of N protein significantly enhances the intensity of the immune response, resulting in a serum antibody titer as high as 1:64000, and the titer of the fused antibody is 1:6400, providing higher sensitivity for subsequent detection applications.

[0029] 3. Broader cross-reactivity: This monoclonal antibody can simultaneously recognize multiple circulating PEDV genotypes, overcoming the limitations of existing antibodies that can only recognize a single or a few strains, and providing a powerful tool for the broad-spectrum detection and diagnosis of PEDV.

[0030] 4. Enhanced Detection Applicability: The antibody is compatible with three mainstream immunological detection methods: Western Blot, IFA, and IHC. It can achieve virus localization at the cellular level and in situ detection in tissue sections, meeting multiple needs for qualitative, localization, and quantitative detection, and significantly improving the flexibility and breadth of application scenarios.

[0031] In summary, the monoclonal antibody provided by this invention has the advantages of high specificity, high sensitivity, broad-spectrum cross-reactivity, and multi-platform applicability, and has important application value in the epidemiological surveillance, clinical diagnosis, pathological research and basic research of PEDV.

[0032] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. In the description of this invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0033] The list of abbreviations for this invention is shown in Table 1.

[0034] Table 1. List of abbreviations for this invention Example 1 The hybridoma cell line, monoclonal antibody, and preparation method of porcine epidemic diarrhea virus are detailed below: 1. Expression and purification of PEDV N protein.

[0035] Based on the gene sequence SEQ ID NO.1 of PEDV N protein (AFQ37602.1) registered with NCBI, the gene sequence was optimized according to the codon preference of mammals. EcoRI and XhoI restriction sites were added to both ends of the sequence, and a 3×Flag sequence was added to the C-terminus to obtain SEQ ID NO.2. It was sent to Anhui Bio-General Company for full sequence synthesis and sequencing to obtain the recombinant plasmid pcDNA3.1(+)-N.

[0036] The recombinant plasmid pcDNA3.1(+)-N was transfected into HEK 293T cells using Lipofectamine 2000 at a ratio of 3:1 to form a transfection complex. The cell supernatant containing the N protein was collected after 48 hours. Protein purification was performed using an Anti-Flag gel. The specific procedure was as follows: 40 μL of gel was placed in a 1.5 mL centrifuge tube. 500 μL of pre-chilled PBS was added to the gel, and the mixture was gently mixed. The gel was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 1.8 mL of protein supernatant labeled with N protein was added to each tube. The tubes were incubated at 4°C for 3 h, then centrifuged at 2000 rpm for 10 min. The gel was collected and eluted. 100 μL of elution buffer (pH 4.5) was added to each centrifuge tube, and the tubes were incubated at room temperature for 10 min. The gel was then separated by centrifugation at 2000 rpm for 5 min. The supernatant containing N protein was retained. 20 μL of neutralization buffer (pH 8.8) was added to each 100 μL of elution buffer to neutralize the low pH, yielding purified PEDV N protein. The purified protein was then analyzed by Coomassie Brilliant Blue and Western Blot.

[0037] (1) Coomassie Brilliant Blue Detection.

[0038] After SDS-PAGE electrophoresis, the purified N protein was stained with Coomassie stain using rapid staining. The specific procedure is as follows: After the protein is pulled apart from the separating gel, remove the electrophoresis tank, place the gel in distilled water and heat to boiling. Discard the distilled water, add Coomassie Brilliant Blue staining solution to cover the gel, heat to boiling, discard the staining solution, add destaining solution, and heat to boiling again. Repeat the destaining process three times. At this point, the target band of approximately 55 kDa can be observed. The results are as follows. Figure 1 As shown. Figure 1In the diagram, lane purification N-1 represents N protein purified by Flag gel (L-1013 kit), and lane purification-2 represents N protein purified by Flag gel (L-1109 kit).

[0039] (2) Western Blot detection.

[0040] The purified N protein was expressed and electrophoresed by SDS-PAGE. The N protein was then transferred to a PVDF membrane, blocked with 5% skim milk at 37°C for 1 hour, and incubated overnight at 4°C and 60 rpm with Flag antibody at a 1:1000 ratio. After incubation, the membrane was washed three times with TBST at room temperature and 80 rpm for 10 minutes each time. The secondary antibody Goat anti-Mouse IgG was added at a 1:20000 ratio, and the membrane was incubated at 37°C and 60 rpm for 1 hour. Finally, ECL staining was performed. The results are as follows: Figure 2 As shown, a clear target protein band is visible at 55 kDa, indicating successful expression of the target protein. Lane N-1 represents N protein purified using Flag gel (L-1013 kit), and lane N-2 represents N protein purified using Flag gel (L-1109 kit).

[0041] 2. Preparation of PEDV N protein monoclonal antibody.

[0042] (1) Antibody titer detection method.

[0043] PEDV N protein was diluted to 1.25 μg / mL using protein coating buffer, and 100 μL / well was added to a 96-well ELISA plate. The plate was incubated overnight at 4°C, washed three times with TBST, and then blotted dry. 300 μL / well blocking buffer was added, and the plate was blocked at 37°C for 2 h. After discarding the blocking buffer, the plate was washed three times with TBST. Samples were diluted at dilutions of 1:500, 1:1000, 1:2000...1:128000, and added to each well of the 96-well ELISA plate at 100 μL. Positive and negative serum controls were also included. The plate was incubated at 37°C for 1 h, washed three times with TBST, and then blotted dry. Goat anti-Mouse IgG-HRP was diluted 1:2000 using secondary antibody dilution buffer, and 100 μL / well was added to each well of the 96-well ELISA plate. The plate was incubated at 37°C for 1 h, washed three times with TBST, and then blotted dry. Add 100 μL of TMB enzyme substrate reaction solution to each well and incubate at 37°C for 10 min. Finally, add 100 μL of 2M H2SO4 stop solution to each well. Immediately after adding the stop solution, use a microplate reader to detect the OD450 value of each well and record the data. If the OD450 value of the well is greater than or equal to 2.1 times that of the negative control (P / N value > 2.1), it is considered positive.

[0044] (2) Hybridoma cell preparation.

[0045] Purified PEDV N protein was emulsified with Freund's adjuvant at a volume ratio of 1:1. The dosage of N protein was 30 μg per mouse, and mice were immunized by intramuscular injection in the hind limb. Freund's complete adjuvant was used for the initial immunization and the first booster immunization, while Freund's incomplete adjuvant was used for subsequent booster immunizations, with an immunization interval of 2 weeks. Three mice were used in each group. Mice injected with an equal volume of PBS and adjuvant served as negative controls. One week before cell fusion, mice received a second booster immunization. After the second immunization, mouse serum was collected for antibody titer testing. Figure 3 The serum titer of positive mice can reach 1:64000.

[0046] The day before cell fusion, a healthy, unimmunized mouse was euthanized by cervical dislocation. The mouse was placed supine on the operating table, and the abdominal wall was disinfected with 75% alcohol. The abdominal skin was opened with a scalpel, ensuring the peritoneum remained intact. The peritoneum was grasped with forceps, and 10 mL of DMEM incomplete culture medium was injected into the peritoneal cavity. The mouse was gently shaken by its tail, and the mixed culture medium fluid in the peritoneal cavity was collected. After centrifugation, feeder cells were obtained and resuspended in HAT complete culture medium at a ratio of 1×10⁻⁶. 5 / mL was seeded into 96-well plates. On the day of cell fusion, blood was collected from the ophthalmic plexus vein of boosted mice, and the mice were euthanized by cervical dislocation. Serum was collected as a positive control. The spleen of the mice was removed and placed on a sterile dish. Then, an appropriate amount of DMEM medium was drawn up with a syringe, and the spleen was repeatedly ground to prepare a spleen cell suspension. Subsequently, the spleen cell suspension and SP2 / 0 cells were seeded at a ratio of 2.9 × 10⁶ cells / mL. 7 With 1.45×10 7 Cells were fused at a 2:1 ratio using PEG4000. Cells were gently resuspended in HAT selective medium and added at 100 μL / well to feeder cells prepared one day prior. The cells were then incubated at 37°C in a 5% CO2 incubator. Cell status was observed approximately 4 days later; the fused hybridoma cells appeared as follows: Figure 4 .

[0047] (3) Subcloning screening.

[0048] When the hybridoma cells reached 30% of the bottom area, positive wells with strong antibody-secreting ability were screened using an indirect ELISA method. Subcloning was performed using limiting dilution, and after five subcloning processes, five stable antibody-secreting cell lines were obtained: 7AH-12G, 7AH-8G, 7AH-10G, 7AH-11H, and H11-9G. Among them, 7AH-11H showed the best effect, and this invention has been deposited; the deposit information is as follows:

[0049] Hybridoma cell line 7AH-11H was deposited at the China Center for Type Culture Collection (CCTCC) on April 16, 2026, with accession number CCTCC No: C202679, and the deposit address is Wuhan University, Wuhan, China.

[0050] Example 2 The application of hybridoma cell lines and monoclonal antibodies against porcine epidemic diarrhea virus is detailed below: Large-scale preparation of monoclonal antibodies against PEDV N protein was carried out using an in vivo ascites induction method: One week prior to preparation, two BALB / c mice were sensitized by intraperitoneal inoculation with 0.5 mL of Freund's adjuvant. Hybridoma cells in logarithmic growth phase were resuspended and counted, and each mouse was intraperitoneally injected with 1 × 10⁻⁶ cells. 6 Cells. Ascites fluid was collected and identified approximately 10 days later. The titers of the purified ascites fluid were detected by indirect ELISA at 1:1600 and 1:6400, respectively. Figure 5 .

[0051] (1) Western Blot identification of monoclonal antibodies.

[0052] When Vero cells reached 90% confluence, they were infected with PEDV G2a, G2b, and G2c circulating strains at 0.01 MOI. After 12 hours, the supernatant was discarded, the cells were washed three times with PBS, and RIPA lysis buffer was added to lyse the cells and obtain the supernatant for Western blotting. A high-titer purified antibody against strain 7AH-11H was selected as the primary antibody, diluted 1:200, and incubated overnight at 4°C. The colorimetric results are shown below. Figure 6 A clear target band was observed at 55 kDa, demonstrating that the monoclonal antibody can bind to PEDV subtypes G2a, G2b, and G2c respectively, and its specific binding effect with G2a and G2b strains is better than that with G2c strain.

[0053] (2) IFA identification of monoclonal antibodies.

[0054] When Vero cells reached 90% confluence, they were infected with PEDV virus. After 12 hours, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 20 minutes, followed by blocking with fluorescent rapid blocking buffer for 1 hour. A high-titer purified antibody against strain 7AH-11H was used as the primary antibody, diluted 1:200, and incubated overnight at 4°C. After discarding the primary antibody, Goat-anti-Mouse Alexa Fluor 488 secondary antibody was added and incubated at 37°C for 1 hour. Then, DAPI staining was performed, the secondary antibody was discarded, and PBS was added. The results were observed using a fluorescence inverted microscope. Figure 7 As shown, the monoclonal antibody of the present invention can bind to PEDV G2a, G2b, and G2c subtypes respectively; according to the Western Blot detection results, the antibody has a better specific binding effect on G2a and G2b than on G2c.

[0055] (3) IHC identification of monoclonal antibodies.

[0056] PEDV-infected porcine jejunal tissue was paraffin-embedded, cut into 6 μm slices, and adsorbed onto glass slides. The slides underwent dewaxing, 1× sodium citrate antigen retrieval, 5% goat serum blocking, and then overnight incubation with 1:100 PEDV N monoclonal antibody. Afterward, DAB staining was performed for 5 min, terminated with tap water, followed by dehydration using a gradient of alcohols. After clearing with xylene, the slides were mounted with neutral resin. The results are shown below. Figure 8 The negative control showed no specific brownish-red positive signal, only a pale blue background stained with hematoxylin. Compared to the negative control group, after incubation with the N protein monoclonal antibody, a large number of specific brownish-red positive staining signals appeared in the cytoplasm of porcine small intestinal villi epithelial cells. The positive signals were continuously and diffusely distributed in the small intestinal mucosal epithelial layer.

[0057] SEQ ID NO.1:

[0058] SEQ ID NO.2:

[0059] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0060] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A hybridoma cell line 7AH-11H, characterized in that, The hybridoma cell line 7AH-11H, with accession number CCTCC NO: C202679, was deposited at the China Center for Type Culture Collection on April 16, 2026.

2. A monoclonal antibody against porcine epidemic diarrhea virus, characterized in that, The monoclonal antibody is secreted by the hybridoma cell line 7AH-11H as described in claim 1.

3. The method for preparing monoclonal antibodies as described in claim 2, characterized in that, Includes the following steps: Mice were immunized with the immunogen to obtain immune spleen cells; the nucleotide sequence encoding the immunogen is shown in SEQ ID NO.2; The immune spleen cells were fused with myeloma cells and screened to obtain the hybridoma cell line 7AH-11H. Monoclonal antibodies were prepared using the hybridoma cell line 7AH-11H.

4. The preparation method according to claim 3, characterized in that, The ratio of immune spleen cells to myeloma cells is 2:

1.

5. The preparation method according to claim 3, characterized in that, The fusion agent used for cell fusion is PEG4000.

6. The application of the hybridoma cell line 7AH-11H according to claim 1 and / or the monoclonal antibody according to claim 2, characterized in that, The application refers to the preparation of diagnostic reagents for detecting porcine epidemic diarrhea virus.

7. The application as described in claim 6, characterized in that, The detection reagents are suitable for IFA detection, IHC detection and / or Western Blot detection.

8. The application as described in claim 6, characterized in that, The test reagent also includes enzyme-labeled secondary antibodies or fluorescently labeled secondary antibodies.

9. The application as described in claim 8, characterized in that, The enzyme-labeled secondary antibody includes horseradish peroxidase-labeled secondary antibody or alkaline phosphatase-labeled secondary antibody; The fluorescently labeled secondary antibody was a FITC-labeled secondary antibody.

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