Neutralizing epitope of chicken infectious bursal disease virus VP3, monoclonal antibody, hybridoma cell strain and application of neutralizing epitope, monoclonal antibody and hybridoma cell strain

Abstract

The invention belongs to the technical field of biology, and relates to identification of chicken infectious bursal disease virus, in particular to a neutralizing effect of chicken infectious bursal disease virus VP3, an epitope, a monoclonal antibody, a hybridoma cell strain and application of the chicken infectious bursal disease virus VP3. The epitope regions identified by the mAb 19D8 are 19 to 24 AA, 25 to 30 AA, 37 to 42 AA and 49 to 54 AA, and the key amino acids are F20A, K21A, T23A and E25A. The invention lays a material basis for the establishment of a novel IBDV detection method, and lays a foundation specifically for the synergistic treatment and effective prevention and control of IBDV.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the preparation of monoclonal antibodies against the VP3 protein of infectious bursal disease virus of chickens. Background Art

[0002] Infectious bursal disease (IBD) of chickens is a highly contagious viral disease caused by infectious bursal disease virus (IBDV), which mainly affects poultry, especially chickens. The bursa of Fabricius plays an important role in the development of the avian immune system. IBDV infection can cause damage to the bursa of Fabricius, thereby weakening the immune ability of poultry and causing immunosuppression. IBDV belongs to the genus Avibirnavirus of the family Birnaviridae, and its genome consists of two double-stranded RNA segments, A and B. Segment A encodes VP2, VP3, VP4, and VP5 proteins, and segment B encodes VP1 protein. The outermost layer of the IBDV virion is not coated with an envelope, and the particle diameter is 65-70 nm. It has an icosahedral structure with cubic symmetry. The VP3 protein is highly conserved and is called the "scaffolding protein", which is essential for the formation of the virus capsid and the integrity of the virus morphology. The VP3 protein interacts with the virus genome, VP1 protein, and VP2 protein, playing roles in stabilizing the virus genome structure, weakening the recognition and cleavage of the virus genome by nucleases, and promoting the packaging of virus particles. The C-terminus of the VP3 protein binds to the VP1 protein to form the virus nucleocapsid and participates in the replication of the IBDV genome. In addition, changes in the C-terminus of the VP3 protein, especially the valine at position 235 (235Val), will affect the virus virulence and antigenicity. The VP3 protein also plays an important role in regulating cell programmed apoptosis and maintaining IBDV replication.

[0003] The VP3 protein plays a crucial role in the life cycle of IBDV, but the specific mechanisms of its viral pathogenicity and immune escape have not been fully elucidated. The application with the publication number CN 112626032 A discloses a monoclonal antibody and a hybridoma cell line based on the VP3 protein of infectious bursal disease virus of chickens, but it can only recognize the antigenic epitope with the polypeptide sequence of SEEQILRAATSIYGA. The monoclonal antibody in this patent mainly focuses on the detection of infectious bursal disease virus (IBDV) of chickens. Although it has a high reaction titer, its ability to neutralize virus activity is not mentioned. Limited epitope coverage: The antigenic epitope recognized by the monoclonal antibody mentioned in this patent is only limited to amino acids 196 - 210 of the VP3 protein, and other potential antigenic epitopes of the VP3 protein have not been explored in depth. Insufficient adaptability to virus variants: As IBDV continuously generates variants, this monoclonal antibody may not be able to effectively recognize and respond to these new variants, thus affecting its wide application in diagnosis and prevention and control. Currently, the in-depth research on the VP3 protein is relatively limited, restricting its application potential in vaccine development and immunotherapy. Especially in the face of highly variable virus strains, the immune effects of existing vaccines are often not satisfactory. Therefore, our research group has conducted in-depth research. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a neutralizing antigenic epitope, monoclonal antibody and hybridoma cell line of VP3 of infectious bursal disease virus of chickens and their applications.

[0005] The technical solution of the present invention is realized as follows:

[0006] Through monoclonal antibody preparation technology, the present invention innovatively focuses on the antigenicity of the VP3 protein. The screened hybridoma cell line 19D8 and the monoclonal antibody secreted by it have a neutralizing effect, and a new neutralizing antigenic epitope has been successfully identified. By identifying the neutralizing epitope, it helps to reveal the molecular mechanism of the interaction between the virus and host cells. This discovery not only provides an important material basis for the establishment of a new detection method for IBDV, but also lays a theoretical foundation for the development of safe and efficient synergistic treatment for IBDV.

[0007] The monoclonal antibody mAb 19D8 of VP3 of infectious bursal disease virus of chickens in this application recognizes the epitope regions of 19 - 24AA, 25 - 30AA, 37 - 42AA and 49 - 54AA, and the key amino acids are F20A, K21A, T23A and E25A.

[0008] Furthermore, the heavy chain of the above monoclonal antibody is IgG1, and the light chain is Kappa chain.

[0009] Preferably, the above monoclonal antibody is secreted by the hybridoma cell line IBDV-VP3-19D8.

[0010] On the one hand, this application requests protection for the use of the above monoclonal antibody in the preparation of a diagnostic reagent that specifically recognizes infectious bursal disease virus of chickens.

[0011] And the use of the above monoclonal antibody in the preparation of a kit that specifically recognizes infectious bursal disease virus of chickens.

[0012] This application also requests protection for a diagnostic reagent containing the above monoclonal antibody.

[0013] And a kit containing the above monoclonal antibody.

[0014] This application also discloses a hybridoma cell line, which is classified and named as hybridoma cell line IBDV-VP3-19D8 (Hybridoma cell line IBDV-VP3-19D8), with the deposit number of CCTCC NO: C202534, the deposit date of January 8, 2025, and the deposit address of Wuhan University, Wuhan, China.

[0015] And the use of the above hybridoma cell line in the preparation of monoclonal antibodies.

[0016] The present invention has the following beneficial effects:

[0017] 1. This application provides a hybridoma cell line 19D8 against VP3 protein and the monoclonal antibody secreted by it. A monoclonal antibody with neutralizing effect has been screened, and the ELISA titer is up to 3.9×10 8 . Through the neutralization experiment, mAb19D8 shows effective neutralizing activity against IBDV infection, making it a promising candidate for further functional and therapeutic research. And a new neutralizing antigenic epitope has been successfully identified, which can lay a foundation for subsequent vaccines.

[0018] 2. The present invention innovatively focuses on the antigenicity of VP3 protein through monoclonal antibody preparation technology. The screened monoclonal antibody 19D8 has a neutralizing effect, and a new neutralizing antigenic epitope has been successfully identified. The epitope regions recognized by mAb 19D8 are 19-24AA, 25-30AA, 37-42AA, and 49-54AA, and the key amino acids are F20A, K21A, T23A, and E25A. The antigenic epitope mAb 19D8 of this application may inhibit the virus from attaching to host cells, providing a mechanism different from that of targeted antibodies.

[0019] 3. The present application has successfully screened out the monoclonal antibody mAb 19D8 with neutralizing effect, which can effectively neutralize IBDV infection, providing a new potential means for the treatment of infectious bursal disease in chickens and filling the gap in the treatment application of this patent. Identification of new neutralizing epitopes: The present application not only determines that the epitope regions recognized by mAb 19D8 are 19-24AA, 25-30AA, 37-42AA, and 49-54AA of the VP3 protein, but also precisely locates the key amino acids through point mutation experiments, broadening the understanding of the antigenic epitopes of the IBDV VP3 protein and providing more targets for the development of more comprehensive diagnostic and treatment means. Based on the recognition of multiple regions of the VP3 protein and the verification of epitope conservation, the monoclonal antibody of the present application is more likely to maintain good recognition ability for different IBDV variants, thus having more advantages in the face of virus variation and contributing to improving the effectiveness of diagnostic and prevention and control measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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 the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 For the construction of the pET-32a-IBDV-VP3 recombinant vector.

[0022] Figure 2 For the prokaryotic expression and bioactivity verification of the IBDV VP3 protein.

[0023] Figure 3 For the identification of the monoclonal antibody of the VP3 protein.

[0024] Figure 4 For the screening, identification and neutralization verification of the monoclonal antibody of the VP3 protein.

[0025] Figure 5 For the identification of the antigenic epitope of the VP3 protein.

[0026] Figure 6 For the bioinformatics analysis of the antigenic epitope of the VP3 protein.

[0027] Figure 7 For the display of the antigenic epitope of the VP3 protein on its structure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0029] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0030] The tumor cell line SP2 / 0 is preserved by this laboratory; BALB / c mice are purchased from Henan Experimental Animal Center; positive IBDV mouse serum is preserved by this experiment.

[0031] Embodiment

[0032] 1 Construction and identification of prokaryotic expression vector

[0033] Referring to the sequence information of the Hb06t VP2 gene strain (accession number: MW795727.1) published on GenBank, Sal I and Not I restriction enzyme sites were introduced at the upstream and downstream. A pair of primers for amplifying its full length was designed using SnapGene software (Table 1) and sent to Sangon Biotech for synthesis.

[0034]

[0035] The primers of the VP3 gene sequence of the IBDV strain (accession number: MW795727.1) were sent to Sangon Biotech for synthesis, and the target gene was amplified using viral cDNA as a template. The pET-32a vector was digested with Sal I and Not I, and the amplified target gene was purified and ligated to the pET-32a vector by homologous recombination to obtain the pET-32a-IBDV-VP3 plasmid. The ligation product was transformed into competent Escherichia coli DH5α cells, then inoculated on LB solid medium, placed in an oscillating shaker, and cultured overnight at 37°C. The correct plasmid was sent to Sangon Biotech for sequencing after identification by colony PCR and double digestion.

[0036] PCR amplification was performed using the pET-32a-IBDV-VP3 positive plasmid as a template. After nucleic acid electrophoresis detection, a specific band was found at about 750 bp. The size of the VP3 protein gene is 770 bp, and this band is the target band ( Figure 1 A). The target gene was ligated to the pET-32a empty vector and cultured overnight. The colonies were picked, and the results of colony PCR showed that all the picked single colonies were positive ( Figure 1 B). The single colonies were cultured on a large scale to extract the plasmid, and double digestion was performed with Sal I and Not I for identification. The band sizes were correct (Figure 1 C). The sequencing results of the positive recombinant plasmid were consistent with the expected results, indicating that the prokaryotic expression vector pET-32a-IBDV-VP3 was successfully constructed.

[0037] 2 Induced expression of the recombinant vector

[0038] The constructed plasmid pET-32a-IBDV-VP3 was transformed into Escherichia coli BL21. The transformed positive colonies were cultured in LB medium containing 1000 μg / ml ampicillin. When the OD600 reached 0.6 - 0.8, IPTG (isopropyl-β-D-thiogalactopyranoside) solution was added at 37 °C to make its final concentration 0.5 mM. After 12 h of induction, the bacterial liquid precipitate was collected by centrifugation, resuspended with PBS, and the bacteria were lysed using a low-temperature high-pressure homogenizer. The supernatant and precipitate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The target protein was affinity purified through a nickel column, and the immunogenicity of the VP3 protein was identified by Western Blotting using His antibody and positive serum.

[0039] For the specific procedure of Western Blotting, the protein samples were subjected to SDS-PAGE electrophoresis on a 12.5% polyacrylamide gel, and then the proteins were transferred onto a PVDF membrane. The PVDF membrane was incubated in 5% skim milk at room temperature for 1 h, then the membrane was soaked in a 5% skim milk powder solution containing the primary antibody and incubated at room temperature for 1 h, and washed 4 times with TBST. The secondary antibody was commercial HRP-goat anti-mouse IgG (1:3000) or HRP-goat anti-mouse IgG (1:5000), incubated at room temperature for 1 h, washed 4 times with TBST, and developed with ECL.

[0040] The prokaryotic expression vector pET-32a-IBDV-VP3 was transformed into Escherichia coli BL21 competent cells, expanded in culture, and induced with an inducer for expression. The results showed the expression of the VP3 protein, and the VP3 was successfully purified ( Figure 2 A), detected by HIS-tag antibody ( Figure 2 B) and IBDV positive serum ( Figure 2 C) by Western Blotting, indicating that the recombinant IBDV VP3 protein had good antigenicity.

[0041] 3 Immunization of mice and titer detection

[0042] Mix 50 μg of the purified VP3 protein with Freund's complete adjuvant. After complete emulsification, immunize mice by subcutaneous multi-point injection. Fourteen days later, mix the protein with Freund's incomplete adjuvant for the second immunization. According to the procedure of the second immunization, conduct the third immunization 14 days later. After the third immunization, collect blood and separate the serum, and detect the serum titer of mice by the indirect ELISA method.

[0043] For the specific procedure of the indirect ELISA test, dilute the VP3 protein with 0.05 M CB to a concentration of 2 μg / mL, coat it at 37 °C for 3 h, and block it with 1.5% BSA in an incubator at 37 °C for 2 h. Dilute the mouse serum at ratios of 1 / 200, 1 / 400, 1 / 800, 1 / 1600, 1 / 3200, 1 / 6400, 1 / 12800 with PBS. Add the diluted mouse serum as the primary antibody into the wells, react at 37 °C for 1 h, wash 4 times with PBST, add the secondary antibody HRP-goat anti-mouse IgG diluted at 1 / 12000, react at 37 °C for 1 h, wash 4 times with PBST, and then perform color development and reading. The results show that the ELISA titer of the mouse serum after immunization is above 1 / 12800 ( Figure 3 A).

[0044] Transfect the eukaryotic expression plasmid pET-32a-IBDV-VP3 into 293T cells. After 24 hours, collect the cell samples and detect the reactivity of the mouse serum with the eukaryotic VP3 protein by Western Blotting. The results show that the mouse serum reacts well with the VP3 protein and does not react with the empty vector ( Figure 3 B,C), indicating that the mouse serum has good specificity for the VP3 protein and can continue with the preparation of subsequent monoclonal antibodies.

[0045] Preparation and identification of monoclonal antibodies against VP3 protein

[0046] Select the mouse with the highest serum titer for booster immunization with 50 μg of the protein. Three days later, isolate the mouse spleen and fuse it with SP2 / 0 cells under the action of 50% PEG1450, and add HAT to the culture medium to screen the fused hybridoma cells. Screen out the monoclonal antibodies that can react with the IBDV VP3 protein by methods such as indirect ELISA, Western Blotting, and IFA, and identify the subtype of the monoclonal antibody using the antibody subtype identification kit from Proteintech.

[0047] The specific procedure of the IFA test: Transfect the eukaryotic expression plasmid pET-32a-IBDV-VP3 into DF-1 cells. After 20 hours, fix with 4% paraformaldehyde at room temperature for 20 min, permeabilize with PBS solution containing 0.1% Triton X-100 at room temperature for 10 min, and block with PBS solution containing 5% BSA at room temperature for 1 h. Add the supernatant of the screened positive cell line, incubate at room temperature for 1 h, and wash three times with PBST. Use FITC-goat anti-mouse IgG antibody (diluted 1 / 500 times) as the secondary antibody, incubate at room temperature for 1 h, and wash three times with PBST. Dropwise add 4',6-diamidino-2-phenylindole (DAPI) to stain the cell nuclei at room temperature for 8 minutes, and observe the cells with a fluorescence microscope. The blue fluorescence is the nucleus, and the green fluorescence is the IBDV VP3 protein.

[0048] After fusion with hybridoma cells, monoclonal cell lines mAb 1H9, mAb 2H11, mAb 3E6, mAb 6F9, mAb 10C8, mAb 15F4, mAb 19D8, and mAb 20G6 that can react with the prokaryotically expressed VP3 protein were screened out by ELISA test Figure 4 A), and the results showed that the heavy chain subtype of mAb 3E6 was IgG2b. The heavy chain subtypes of the other seven antibodies, namely mAb 1H9, mAb 2H11, mAb 6F9, mAb 10C8, mAb 15F4, mAb 19D8, and mAb 20G6, were identified as IgG1. The light chain subtype of all 8 mAbs was kappa. Figure 4 B). The ELISA titer of 19D8 ascites against the VP3 protein was detected by ELISA experiment to be above 3.9×10^8 Figure 4 C). The IFA results showed that 8 monoclonal antibodies could bind to the eukaryotic VP3 protein with strong specificity, and there was no fluorescence in the control wells transfected with the empty vector Figure 4 D). The neutralization ability of 8 monoclonal antibodies against HD11 cell samples infected with IBDV was detected by neutralization test. The results showed that mAb 19D8 had an obvious neutralization effect. Figure 4 E). In view of the neutralization effect of mAb 19D8, the neutralization mechanism was further studied. The initial stage of virus replication includes virus adsorption, internalization, and membrane fusion. To determine the exact stage at which the antibody neutralizes early virus replication, this study investigated the effects of antibody 19D8 on the virus adsorption and internalization stages. The results showed that the viral RNA level of virus particles adsorbed on the cell membrane and internalized into the cells after treatment with 19D8 was significantly lower than that of the control group Figure 4 F). These findings suggest that the neutralization mechanism of mAb 19D8 may involve inhibiting the adsorption of the virus to host cells.

[0049] Verify the neutralization mechanism of mAb 19D8 against IBDV at the adsorption and invasion stages through qPCR experiments. The neutralization effect is more obvious at the adsorption stage. Figure 4 G)

[0050] Then, detect the DF-1 cell samples infected with IBDV using 8 monoclonal antibodies, with the VP3 protein monoclonal antibody as a control, and analyze the ability of mAbs to recognize viral proteins through western blotting. Consistently, all 8 mAbs can recognize and bind to IBDV Figure 4 H).

[0051] Identification of 5 antigenic epitopes

[0052] Construction of eukaryotic mutant plasmids of VP3 protein. Design primers for VP3 protein mutants using SnapGene software (Table 2). Mutate every 6 amino acids into alanine, and mutate amino acids that are already alanine into glycine, resulting in a total of 39 eukaryotic expression plasmids. When mutating 6 amino acids, the primer extends approximately 20 bp towards the mutation site. When performing single-point mutations, the primer extends approximately 20 bp towards both ends at the mutation site, and the primer length should be greater than 35 bp. Add 20 μL of PCR product to 1 μL of Dpn I enzyme, digest at 37 °C for 1 h, transform into Escherichia coli DH5α competent cells, and after culturing on solid LB medium for 12 h, select 2 monoclonal colonies for sequencing identification.

[0053] Verify the expression of VP3 protein mutants and their reactivity with mAb 19D8 through Western Blotting experiments to determine the epitope region recognized by mAb 19D8. After determining the epitope region recognized by the monoclonal antibody, use the alanine scanning method of point mutation to identify the key amino acids that react with mAb 19D8. The primers were sent to Sangon Biotech for synthesis (Table 2).

[0054]

[0055]

[0056]

[0057]

[0058] By constructing VP3 proteins with different mutants Figure 5 A), identify the antigenic epitopes recognized by mAb 19D8. The results of the 6-amino-acid mutants showed that the epitope regions recognized by mAb 19D8 were 19-24AA, 25-30AA, 37-42AA, and 49-54AA Figure 5B). The results of point mutations showed that the key amino acids of the epitope recognized by mAb 19D8 were F20A, K21A, T23A, and E25A( Figure 5 C).

[0059] 6 Bioinformatics analysis of the antigenic epitope of IBDV VP3 protein

[0060] By downloading the amino acid sequences of the VP3 protein of IBDV strains from different countries around the world and different regions of China in NCBI, and using MEGA7 to perform homology alignment analysis on these sequences, the conservation of the antigenic epitope of mAb 19D8 was analyzed (Table 3).

[0061]

[0062] By aligning the VP3 protein sequences of 20 IBDV strains from all over the world, the results showed that the antigenic epitope recognized by mAb 19D8 was conserved among the 20 IBDV strains. This indicates that the biological properties of mAb 19D8 are very excellent( Figure 6 ).

[0063] 7 Prediction of the 3D structure of IBDV VP3 protein

[0064] To identify the structure of the VP3 protein and the position of the antigenic epitope on IBDV VP3. The structure of the VP3 protein was predicted through an online website (https: / / seq2fun.dcmb.med.umich.edu / / I-TASSER / ), and then its antigenic epitope was displayed on the VP3 protein structure through the software POMOL. The results showed that the antigenic epitope was located on the α-helix on the surface of the VP3 protein, with good hydrophilicity and antigenicity( Figure 7 A).

[0065] Analysis of implementation effects

[0066] IBDV poses a serious threat to the poultry farming industry. Its infection not only causes direct economic losses but also indirectly affects the health and production performance of chicken flocks through immunosuppression. At present, several proteins with good immunogenicity have been discovered and can be used for the serological diagnosis of IBDV. The viral structural proteins VP2 and VP3 can be used to develop blocking or double-antigen sandwich ELISA to detect antibodies against IBDV with high specificity and stability. The VP3 protein is a structural protein of IBDV. The development of an indirect ELISA assay for VP3 can be used for clinical detection, study the structure and antigenicity of the IBDV structural protein, and is of great significance for establishing effective vaccine evaluation criteria and new immuno-diagnostic techniques.

[0067] In this study, the HIS-tagged protein was expressed in Escherichia coli, and the recombinant protein showed a strong reaction with the anti-IBDV antibody serum, indicating that the VP3 protein has good antigenicity and is a potential target for IBDV detection. Eight VP3 monoclonal antibodies were identified by hybridoma technology, and the specificity of the eight VP3 monoclonal antibodies was analyzed by ELISA, Western blot, and IFA. All of them could specifically recognize the VP3 protein, and through neutralization experiments, mAb 19D8 showed significant neutralizing activity.

[0068] There are few reports on the epitopes of the IBDV VP3 protein. Therefore, it is crucial to identify the epitopes of IBDV VP3 by preparing monoclonal antibodies and then explore the function of the protein. In this study, alanine scanning mutagenesis experiments were carried out, and site-specific mutations were constructed every six amino acids to identify the epitopes. Alanine is the amino acid with the shortest side chain among amino acids. Mutating amino acid residues to alanine can change the surface residues of the protein without changing the three-dimensional structure of the protein. By examining whether the function of the mutant protein is lost or changed due to this mutation, the amino acid residues that have a key impact on the function of the protein can be located. The results showed that the epitope regions recognized by mAb 19D8 were 19-30AA, 37-42AA, and 49-54AA( Figure 5 B). The key amino acids of the epitope recognized by mAb19D8 were F20A, K21A, T23A, and E25A.

[0069] This study generated a monoclonal antibody mAb 19D8 against IBDV VP3 with neutralizing activity and identified new neutralizing epitopes. The results of this study not only provided a monoclonal antibody tool for further research on the function of VP3 but also provided new ideas for vaccine development and the establishment of serological diagnostic methods. It has important value in virus mechanism research, vaccine design, treatment strategies, and diagnostic tool development, and helps to promote the progress of medicine and biology.

[0070] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody against chicken infectious bursal disease virus VP3, characterized in that: The epitope regions recognized by the monoclonal antibody are 19-24AA, 25-30AA, 37-42AA, and 49-54AA.

2. The monoclonal antibody against infectious bursal disease virus VP3 according to claim 1, characterized in that: The key amino acids of the monoclonal antibody are F20A, K21A, T23A, and E25A.

3. The monoclonal antibody according to claim 2, characterized in that: The heavy chain of the monoclonal antibody is IgG1, and the light chain is the Kappa chain.

4. The monoclonal antibody according to claim 3, wherein: The monoclonal antibody is secreted by the hybridoma cell line IBDV-VP3-19D8.

5. Use of the monoclonal antibody according to claim 3 in the preparation of a diagnostic reagent for specifically recognizing infectious bursal disease virus of chickens.

6. Use of the monoclonal antibody according to claim 3 in the preparation of a kit for specifically recognizing infectious bursal disease virus of chickens.

7. A diagnostic reagent, characterized in that: Contains the monoclonal antibody according to claim 3.

8. A kit, characterized in that: Contains the monoclonal antibody according to claim 3.

9. A hybridoma cell line, classified and named as hybridoma cell line IBDV-VP3-19D8, with a deposit number of CCTCC NO: C202534, a deposit date of January 8, 2025, and a deposit address of Wuhan University, Wuhan, China.

10. Use of the hybridoma cell line according to claim 9 in the preparation of the monoclonal antibody according to claim 3.

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