Infectious bursal disease virus suitable for full suspension culture and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses an infectious bursal disease virus suitable for full suspension culture and application of the infectious bursal disease virus. The invention discloses a bursal disease virus strain, the bursal disease virus strain is named as IBDV-DHN and preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: V202572. The bursal disease virus IBDV-DHN strain is obtained through separation and screening, and the virus strain can adapt to full suspension culture and can be used for large-scale production of bursal disease vaccines. Meanwhile, the strain can adapt to reverse genetic modification, and the pathogenicity, the interspecific transmission capacity and the virus variation mechanism of the IBDV are explored from the molecular level. The virus strain can be used as a strain skeleton to construct vaccine candidate strains with different specificity, provides more specific immune protection for immunizing poultry, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an infectious bursal virus strain adapted to whole suspension culture and its applications. Background Technology

[0002] Infectious bursal disease (IBD) is caused by the infectious bursal disease virus (IBD virus). Infectious Bursal Disease Virus Infectious bursal disease (IBDV) is an acute, highly contagious disease that primarily damages the immune system of poultry, leading to immunosuppression and inducing secondary infections with other viral or bacterial diseases. This directly or indirectly affects the survival rate and productivity of poultry, causing significant harm to the poultry industry. IBDV comprises two serotypes: serotype I and serotype II, with only serotype I being pathogenic to chickens. Wild-type IBDV strains are further classified into classic, variant, and super-virulent strains; the super-virulent strain exhibits the strongest pathogenicity, the widest impact, and the greatest economic damage. Over the past 30 years, advancements in IBDV vaccine research and improvements in husbandry management have effectively controlled the incidence of IBD in poultry farming. Currently, IBDV vaccines are mainly categorized into chicken embryo vaccines and cell-based vaccines based on their preparation source. Chicken embryo vaccines suffer from high production costs, long production cycles, complex production processes, unstable potency, and susceptibility to contamination by avian pathogens. Cell-based vaccines effectively circumvent these drawbacks, but their preparation presents numerous challenges. First, most infectious bursal disease (IBD) virus strains isolated at present lack cell adaptability, with a few strains only adapting to specific avian cells, including primary chicken embryo fibroblasts (CEF) and passaged chicken embryo fibroblasts (DF1). Second, IBD virus strains are adapted to adherent cells, which involve cumbersome propagation procedures, low growth density, high risk of contamination, and high culture costs, thus hindering their use for large-scale vaccine production. Therefore, to address the shortcomings of the aforementioned vaccine products, it is urgent to actively isolate IBD virus strains and seek more cell-adaptable strains. Simultaneously, further research and development of suspension culture techniques to cultivate IBD viruses adaptable to full suspension culture may be of great significance for the large-scale, efficient production of IBD vaccines. Summary of the Invention

[0003] This invention aims to solve at least one of the technical problems existing in the prior art. This invention provides an infectious bursal virus strain adapted to whole suspension culture. This strain can be adapted to reverse genetic modification and can be used for vaccine production. It has advantages such as simple culture process, low difficulty in scale-up production, low risk of contamination, and good stability of the produced vaccine. It effectively solves the problem of large-scale production difficulties of IBDV cell vaccines, while greatly reducing production costs, and has broad application prospects.

[0004] The first aspect of the present application aims to provide a bursa virus strain.

[0005] The second aspect of the present application aims to provide the application of the bursa virus strain of the first aspect of the present application in the research of the pathogenicity, interspecies transmission ability and virus variation mechanism of the bursa virus.

[0006] The third aspect of the present application aims to provide a bursa virus mutant strain.

[0007] The fourth aspect of the present application aims to provide the application of the bursa virus strain of the first aspect of the present application or the bursa virus mutant strain of the third aspect of the present application in the preparation of a product for preventing and / or treating bursa disease.

[0008] The fifth aspect of the present application aims to provide the application of the bursa virus strain of the first aspect of the present application or the bursa virus mutant strain of the third aspect of the present application in the preparation of a diagnostic reagent.

[0009] The sixth aspect of the present application aims to provide a vaccine.

[0010] The seventh aspect of the present application aims to provide a culture method of the bursa virus.

[0011] The eighth aspect of the present application aims to provide a method for constructing an animal model infected with the bursa disease virus.

[0012] In order to achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows: The first aspect of the present application provides a bursa virus strain, which is named IBDV-DHN, and is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: V202572.

[0013] In some embodiments of the present application, the nucleotide sequence of the A segment gene of the bursa virus strain is shown in SEQ ID NO: 9, and the nucleotide sequence of the B segment gene is shown in SEQ ID NO: 10.

[0014] The second aspect of the present application provides the application of the bursa virus strain of the first aspect of the present application in the research of the pathogenicity, interspecies transmission ability and virus variation mechanism of the bursa virus.

[0015] The bursa virus strain provided by the present application can adapt to reverse genetic modification, and the pathogenicity, interspecies transmission ability and virus variation mechanism of the bursa virus can be researched through reverse genetic modification.

[0016] A third aspect of the present invention provides a mutant strain of infectious bursal virus, which is obtained by reverse genetic rescue of the infectious bursal virus strain of the first aspect of the present invention.

[0017] In some embodiments of the present invention, the reverse genetic rescue includes the following steps: Using the cDNA of the infectious bursal virus strain of the first aspect of the present invention as a template, segments A and B of the infectious bursal genome with homologous arms were amplified respectively, and used as insert fragments for recombinant plasmids. Linearized vectors were obtained by digesting PHW2000 plasmid with Bsmb I restriction endonuclease using PHW2000 plasmid as a template. The recombinant plasmid was obtained by homologous recombination of the insert fragment and the linearized vector in competent cells using a seamless cloning method. The recombinant plasmid was transfected into the cells and cultured to obtain the infectious bursal virus strain.

[0018] In some embodiments of the present invention, the seamless cloning method includes mixing the insert fragment and the linearized vector, and then introducing them into competent cells (e.g., after mixing with competent cells and then subjecting them to heat shock).

[0019] The seamless cloning technique requires only a single specific restriction endonuclease to construct vectors, effectively reducing construction costs. Furthermore, it eliminates the need for specific restriction sites on the chosen vector; any restriction site can be selected, allowing the target fragment to be inserted into any site within any vector. No additional sequences are introduced between the ligated fragments, ensuring precise directional ligation and increasing gene fidelity. The homologous recombination reaction takes only 15 minutes, significantly less than traditional restriction enzyme digestion and ligation methods. Moreover, the entire construction process eliminates the need for PCR product digestion and gel recovery, making it faster, simpler, and more efficient.

[0020] A fourth aspect of the present invention provides the use of the infectious bursal virus strain of the first aspect of the present invention or the infectious bursal virus mutant strain of the third aspect of the present invention in the preparation of products for the prevention and / or treatment of infectious bursal disease.

[0021] In some embodiments of the present invention, the product includes a vaccine or a drug.

[0022] In some embodiments of the present invention, the vaccine is selected from a monovalent vaccine, a multivalent vaccine, or a combination vaccine.

[0023] The monovalent vaccine refers to a vaccine containing only the viral serum of the infectious bursal disease virus strain of the present invention; the multivalent vaccine refers to a vaccine prepared by mixing the viral serum of the infectious bursal disease virus strain of the present invention with one or more other viral strains that can prevent and / or treat infectious bursal disease; the combined vaccine refers to a vaccine prepared by mixing the viral serum of the infectious bursal disease virus strain of the present invention with several pathogenic microorganisms or serums that can prevent and / or treat other viruses.

[0024] In some embodiments of the present invention, the vaccine includes an inactivated vaccine.

[0025] A fifth aspect of the present invention provides the use of the infectious bursal virus strain of the first aspect of the present invention or the infectious bursal virus mutant strain of the third aspect of the present invention in the preparation of diagnostic reagents.

[0026] A sixth aspect of the present invention provides a vaccine, the active ingredient of which comprises the infectious bursal virus strain of the first aspect of the present invention or the infectious bursal virus mutant strain of the third aspect of the present invention.

[0027] In some embodiments of the present invention, the vaccine further includes pharmaceutically acceptable excipients.

[0028] In some embodiments of the present invention, the excipients include at least one of a diagnostic agent, a carrier, a protective agent, an adjuvant, an excipient, or a diluent.

[0029] In some embodiments of the present invention, the immunization method of the vaccine includes nasal drops, injection, oral administration, soaking, or eye drops; nasal drops are preferred.

[0030] In some embodiments of the present invention, the vaccine includes an inactivated vaccine.

[0031] In some embodiments of the present invention, the method for preparing the vaccine includes uploading and culturing the infectious bursal virus strain of the first aspect of the present invention or the infectious bursal virus mutant strain of the third aspect of the present invention in cells (such as LMH suspension cells), collecting the virus fluid, and adding excipients to obtain the vaccine.

[0032] A seventh aspect of the present invention provides a method for culturing infectious bursal virus, the method comprising the following steps: inoculating the infectious bursal virus strain of the first aspect of the present invention into LMH suspension cells and culturing them to obtain infectious bursal virus.

[0033] In some embodiments of the present invention, the culture conditions are 35~38℃, 4%~6%CO2, and 100~120rpm.

[0034] An eighth aspect of the present invention provides a method for constructing an animal model of infectious bursal disease virus infection, comprising introducing an infectious bursal disease virus strain of the first aspect of the present invention or an infectious bursal disease virus mutant strain of the third aspect of the present invention into an animal to induce disease in the animal.

[0035] In some embodiments of the present invention, the animal is poultry.

[0036] In some embodiments of the present invention, the animal is a chicken.

[0037] The beneficial effects of this invention are: This invention obtained an infectious bursal disease virus (IBDV)-DHN strain through isolation and screening. This strain can adapt to whole suspension culture and can be used for large-scale production of IBDV vaccines. Simultaneously, this strain is adaptable to reverse genetic engineering, allowing for the investigation of IBDV pathogenicity, interspecies transmissibility, and viral mutation mechanisms at the molecular level. This strain can serve as a skeletal framework for constructing vaccine candidate strains with different specificities, providing more specific immune protection for immunized poultry, and has broad application prospects. Attached Figure Description

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The image shows the agarose gel electrophoresis results of the IBDV-DHN strain isolation and screening. In the figure, M is the DL2 000 DNA Marker, and 1 is the positive band.

[0039] Figure 2 The image shows the agarose gel electrophoresis results of the IBDV-DHN strain whole genome amplification. In the figure, M1 is the DL2000 DNA Marker, M2 is the DL5000 DNA Marker, 1 is fragment A1, 2 is fragment A2, and 3 is fragment B.

[0040] Figure 3 The results show the biological characteristics (cytopathic response) of the IBDV-DHN strain, where A represents Ctrl (no inoculation) and B represents the IBDV-DHN strain.

[0041] Figure 4 The image shows the agarose gel electrophoresis results of recombinant plasmids containing segments A and B of the infectious bursa of Fabricius genome with homologous arms. In the figure, M is the DL5000 DNA Marker, 1 is IBDV-B, 2 is IBDV-A, and 3 is the PHW2000 enzyme digestion product. Detailed Implementation

[0042] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0043] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0044] Experimental materials and main reagents Cells, virus strains, and main experimental materials: DF1 cells, LMH cells, BHK cells, MDCK cells, HEK-293T cells, IBDV GT strain, IBDV GX strain, and PHW2000 vector were preserved by Zhaoqing Dahua Agricultural Biopharmaceutical Co., Ltd.; SPF chicken embryos were purchased from Wenshi Dahua Agricultural Biotechnology Co., Ltd.

[0045] Main reagent: Lipofectamine TM LTX Reagent with PLUS TM Reagent transfection reagent, DMEM cell culture medium, Opti-MEM TM Reduced Serum Medium was purchased from Thermo Fisher Scientific; TransStart® FastPfu Fly DNA Polymerase and pEASY®-Basic Seamless Cloning and Assembly Kit were purchased from Beijing TransGen Biotech Co., Ltd.; Plasmid Extraction Kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; Gel Recovery Kit was purchased from OMEGA; Low Melting Point Agarose was purchased from Qingke Biopharmaceutical Co., Ltd.; HiScript IV 1stStrand cDNA Synthesis Kit (+gDNA wiper) and FastPure Viral DNA / RNA Mini Kit Pro were purchased from Nanjing Novizan Biotechnology Co., Ltd.; DL2000 DNA Marker, DL5000 DNA Marker, and DH5α Competent Cells were purchased from Baori Medical (Dalian) Biological Products Co., Ltd.; BsmbⅠ was purchased from New England Biotechnology (Beijing) Co., Ltd.; Infectious Bursal Disease Virus VP2 Protein ELISA Antibody Detection Kit was purchased from Adway (Qingdao) Biotechnology Co., Ltd.

[0046] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0047] Example 1: Isolation and Identification of Viruses 1. Isolation of IBDV-DHN strain Pathogenic samples collected from diseased chickens at a chicken farm in Guangdong Province were placed in a sterilized mortar, and an appropriate amount of silica and sterile physiological saline solution were added and ground thoroughly. The mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. Penicillin and streptomycin were added to achieve a final concentration of 1%, and the virus was inoculated into the chorionic allantoic cavity of 9-day-old SPF chicken embryos at a rate of 0.2 mL / embryo. The embryos were incubated at 37°C, and chicken embryos that died within 24 hours were discarded. After the chicken embryos died, they were refrigerated at 4°C for 12 hours. The dead and live embryos were dissected and the lesions were observed. Chicken embryos with obvious lesions were ground into a homogenate, and sterile physiological saline solution at a volume ratio of 1:5 was added and mixed well. The mixture was subjected to three freeze-thaw cycles and centrifuged at 6000 rpm for 8 minutes. The supernatant was collected and stored at -20°C for later use. The supernatant was diluted 1000-fold, and 0.2 mL of the diluted solution was inoculated into 9-day-old SPF chicken embryos. This process was repeated three times. Allantoic fluid from the last dead chicken embryo was collected for nucleic acid extraction. After reverse transcription into cDNA, PCR identification was performed using detection primers. The detection primer sequences were 5'-GCATGCGGTATGTGAGGCTTGGTGAC-3' (SEQ ID NO:1) and 5'-GAATTCGATCCTGTTGCCACTCTTTC-3' (SEQ ID NO:2).

[0048] Electrophoresis results showed a positive band ( Figure 1 The positive sample (denoted as IBDV-DHN strain) was stored as a seed virus at -80℃.

[0049] 2. Determination of biological characteristics of IBDV-DHN strain IBDV-DHN strain was inoculated into DF1 cells (passivable chicken embryo fibroblasts) using 10-fold serial dilutions. Cytopathic effects were observed from 24 hours post-inoculation until 120 hours post-inoculation. The median infection dose (TCID) of the cell culture tissue was calculated using the Reed-Muench method. 50 ).

[0050] The results showed that after IBDV-DHN strain infected cells, cytopathic effects initially appeared at 24 hours, and after reaching the plateau phase, the viral titer could reach 10. 9.0 TCID 50 / mL ( Figure 2 ).

[0051] 3. Gene sequencing of IBDV-DHN strain (1) Primer design and synthesis The full gene sequences of IBDV A and B segments published in GenBank were searched, and the specific sequences were analyzed. Primers were designed based on their relatively conserved sequence information to amplify the A and B segments of the infectious bursal disease virus strain. The A segment was amplified in two parts, resulting in a total of 3 PCR products. The synthesis of the above primers was completed by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequence information is shown in Table 1.

[0052] Table 1 Primer sequence information

[0053] (2) Acquisition and reverse transcription of the genome of infectious bursal virus (IBDV-DHN strain) The bursa of Fabricius genome of the obtained IBDV-DHN strain was extracted according to the FastPure Viral DNA / RNA Mini Kit Pro instructions. Using the obtained genome as a template, cDNA was obtained by reverse transcription using the HiScript IV 1st StrandcDNA Synthesis Kit (+gDNA wiper). The specific procedures are as follows: Prepare the RNA template denaturation reaction system (Total RNA 10pg~5μg, RNase-Free ddH2O to 8μL). Heat the reaction system in a PCR instrument at 65℃ for 5 min, then quickly place it on ice to cool, and let it stand on ice for 2 min.

[0054] Prepare the genomic DNA removal reaction system (8 μL of the mixture from the previous step, 2 μL of 5 × gDNA wiper Mix), mix thoroughly by pipetting after preparation, and heat in a PCR instrument at 42°C for 2 min.

[0055] Prepare the first-strand cDNA synthesis reaction solution according to Table 2. After preparation, mix well by pipetting and place in a PCR instrument at 42℃ for 15 min, then at 85℃ for 5 s to obtain the corresponding cDNA.

[0056] Table 2 First-strand cDNA Synthesis Reaction System

[0057] (3) Amplification of the whole viral genome Using the obtained cDNA as a template, and employing detection primers (SEQ ID NO: 1-2), a PCR system was prepared according to the TransStart® FastPfu Fly DNA Polymerase instructions (Table 3) to amplify segments A and B of the bursa of Fabricius genome. PCR reaction conditions: 98℃, 1 min; 98℃, 10 s; 58℃, 5 s; 72℃ (with extension time set at 6 kb / min), 34 cycles; 72℃, 1 min, 4℃, infinite cycles. All products were tested by 1% agarose gel electrophoresis. Figure 3 ), and specific fragments were recovered by gel electrophoresis for subsequent TA cloning.

[0058] Table 3 PCR reaction system

[0059] (4) Construction of recombinant plasmids by TA cloning and sequencing Using the PCR product obtained from gel recovery and the pMD18-T vector as templates, the ligation reaction system was prepared according to the Mighty TA-cloning Kit instructions (Table 4). After mixing the reaction system by pipetting, it was placed in a PCR instrument and heated at 16°C for 30 min. After the reaction was completed, it was placed on ice for incubation.

[0060] Table 4 Connection Reaction System

[0061] Remove the purchased DH5α competent cells from the -80℃ freezer and incubate on ice for 5 min, then allow to thaw. Take a 1.5 mL EP tube, add 50 μL of the revived competent cells and 5 μL of ligation reaction mixture, mix well by pipetting, and incubate on ice for 30 min. Set the water bath to 42℃, heat shock the mixture for 45 s, and incubate again on ice for 3 min. Add 500 μL of antibiotic-free LB liquid medium to the 1.5 mL EP tube, mix well by pipetting, and incubate on a shaker at 37℃ and 200 rpm for 1 h. Take 50 μL of the incubated culture medium and spread it evenly on an LB agar plate containing ampicillin. Place the plate in an incubator upright for 10 min, then invert and incubate overnight. Five to ten single colonies were picked from each of the overnight LB plates. Positive clones were initially selected using identification primers. The selected positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The recombinant plasmid was then tested to obtain the complete gene sequence information of the isolated infectious bursal virus.

[0062] The nucleotide sequence of the IBDV-DHN strain is as follows:

[0063]

[0064] The IBDV-DHN strain was deposited on October 12, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: V202572, named Infectious Bursal Disease IBDV-DHN, and located at Wuhan University, Wuhan, China.

[0065] Example 2: Suspension culture of IBDV-DHN strain This embodiment is used to investigate whether the IBDV-DHN strain isolated in this invention can be cultured in suspension, as detailed below: IBDV-DHN strain was inoculated using LMH suspension cells, BHK suspension cells, and MDCK suspension cells, and passaged continuously. LMH suspension cells were cultured at 37℃, 5% CO2, and 110 rpm; virus inoculation was performed at a concentration of 0.5% (according to an MOI range of 0.25–1.25). At passage 10, cell-virus suspensions of different culture durations were taken, freeze-thawed once, and RNA was extracted using an automated nucleic acid extractor. The assay was prepared according to the HiScript li U+ One StepqRT-PCR Probe Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number Q222-CN). The detection volume was 20 μL, with 1 μL of extraction template added. Detection was performed using a Gentier 96C fully automated PCR analysis system from Xi'an Tianlong Technology Co., Ltd. The primer and probe sequences are as follows: IBDV-F: 5'-GTTCCGTTCATA CGGAGCC-3' (SEQ ID NO:11), IBDV-R: 5'-CCCCACAGTCAAATTGTAGGTCG-3' (SEQ ID NO:12), IBDV-P: 5'-FAM-GAAGCACACTCTCAGGTCAGAG-BHQ1-3' (SEQ ID NO:13).

[0066] The IBDV-DHN strain was found to be incompatible with BHK and MDCK suspension cells, and the Ct values ​​of samples taken at different culture time points did not change significantly, indicating that the virus did not amplify. However, the IBDV-DHN strain was compatible with LMH suspension cells, and it reached a plateau phase after 72 hours of growth in LMH suspension cells, with the Ct value eventually reaching 18.5.

[0067] Example 3: Evaluation of the effectiveness of the IBDV-DHN strain This embodiment is used to investigate whether the IBDV-DHN strain isolated in this invention can be used as a vaccine to prevent infectious bursal disease, as detailed below: The efficacy of the IBDV-DHN strain was evaluated through animal experiments. Thirty one-day-old SPF chickens were randomly divided into three groups: Group 1 was the vaccine immunization experimental group (n=10), Group 2 was the vaccine positive control group (n=10), and Group 3 was the blank control group (n=10). At one day of age, the chickens in Group 1 were immunized with the candidate vaccine strain IBDV-DHN via nasal or ocular drops at a dose of 2.0 × 10⁻⁶. 4 PFU / bird; Group 2 was inoculated with the same volume and dose of IBDV-GT strain, while Group 3 received no treatment. Blood was collected from all chickens at 21, 28, and 35 days post-immunization, and serum was separated. IBDV serum antibodies were detected using an ELISA kit (purchased from Adway (Qingdao) Biotechnology Co., Ltd., catalog number 99-44300).

[0068] The results showed that after immunization, the vaccine-immunized experimental group and the immunized positive control group SPF chickens produced similar antibody levels (specific measurement data are shown in Table 5), suggesting that the IBDV-DHN strain isolated in Example 1 can be used to prepare a vaccine for the prevention of infectious bursal disease.

[0069] Table 5 Antibody titers at different times after immunization

[0070] Example 4: Reverse genetic rescue of IBDV-DHN strain This embodiment is used to investigate whether the IBDV-DHN strain isolated in this invention can be used for reverse genetics rescue. Details are as follows: (1) Primer design and synthesis Primers for constructing the reverse genetic expression vector were designed based on the sequenced IBDV-DHN strain (i.e., SEQ ID NO:9 and SEQ ID NO:10) and the full gene sequence of IBDV GX strain B segment (AY705393.2) published in GenBank. The specific primer sequences are shown in Table 6.

[0071] Table 6 Primer sequence information

[0072] (2) Linearization of the recombinant vector and acquisition of the inserted fragment Using PHW2000 plasmid as a template, the enzyme digestion reaction system was prepared according to the instructions of BsmbⅠ restriction endonuclease (Table 7). The reaction system was heated at 55℃ for 1 h in a PCR instrument to obtain the linearized vector of the recombinant vector.

[0073] Table 7 Enzyme digestion reaction system

[0074] Using the primers designed and synthesized in Table 6, PCR fragment amplification was performed under the conditions for amplification of the whole viral genome in Example 1 (3), and the corresponding fragment fusion was completed to obtain the A and B segments of the bursa of Fabricius genome with homologous arms, thereby obtaining the insert fragment of the recombinant plasmid. All the above products need to be tested by 1% agarose gel electrophoresis. Figure 4 ), and specific fragments were recovered using gel electrophoresis for subsequent construction of recombinant plasmids.

[0075] (3) Seamless cloning to construct recombinant plasmids Using the obtained linearized vector and insert fragment as templates, a seamless cloning reaction system was prepared according to the pEASY®-Basic Seamless Cloning and Assembly Kit instructions (Table 8). After mixing the reaction system by pipetting, it was placed in a PCR instrument at 50°C for 15 min. After the reaction, it was incubated on ice. Simultaneously, DH5α competent cells purchased from a -80°C freezer were taken out and incubated on ice for 5 min, then allowed to thaw. A 1.5 mL EP tube was taken, and 50 μL of the revived competent cells and 5 μL of the seamless cloning reaction mixture were added. After mixing by pipetting, it was incubated on ice for 30 min. The water bath was set to 42°C, and the mixture was heat-shocked for 45 s, followed by incubation on ice for another 3 min. 500 μL of antibiotic-free LB liquid medium was added to the 1.5 mL EP tube after the above operations, and after mixing by pipetting, it was incubated on a shaker at 37°C and 200 rpm for 1 h. Take 50 μL of the incubated culture medium and spread it evenly on an LB agar plate containing ampicillin. Place the plate in a constant temperature incubator, invert it for 10 min, and then incubate it overnight.

[0076] Table 8 Seamless Cloning Reaction System

[0077] (4) Identification of recombinant plasmids Five to ten single colonies were picked from each of the overnight LB plates. Positive clones were initially selected using identification primers. The selected positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Strains with correct sequencing were expanded and plasmids were extracted using a plasmid miniprep kit for later use, thus obtaining the IBDV-A expression plasmid and different IBDV-B modified plasmids (IBDV-B-Mu1, IBDV-B-Mu2).

[0078] (5) Reverse genetics rescue of IBDV-DHN modified strains HEK-293T (human embryonic kidney cells) and DF1 cells were cultured. When the cell confluence reached approximately 80%, the cells were digested, and the two cell types were transferred to 35mm culture dishes at a 1:1 ratio. Once the mixed cell confluence reached 80% again, the medium was replaced with Opti-MEM. Lipofectamine was used. TM LTX Reagent with PLUS TM Reagent transfection reagent was used for transfection, co-transfecting the previously constructed IBDV-A expression plasmid and different IBDV-B modified plasmids (IBDV-B-Mu1, IBDV-B-Mu2) at a 1:1 ratio. The total mass of plasmids transfected in a single 35mm culture dish was uniformly 6μg. A control group was also included. The specific transfection steps are as follows: Solution A was Lipofectamine diluted with Opti-MEM medium. TM LTXReagent reagent mixture; Solution B is a DNA premix prepared by diluting plasmid DNA with Opti-MEM medium, and then adding PLUS. TM Mix the Reagent reagent thoroughly; after mixing solutions A and B, incubate each for 5 min. Then, mix equal volumes of solutions A and B and incubate for 15 min. Add the mixture to a 35 mm culture dish and gently shake to ensure thorough mixing with the culture medium. 8–12 h after transfection, wash twice with PBS, replace with 5% FBS DMEM medium, and continue culturing for 48 h. After two freeze-thaw cycles, harvest the cell mixture, aliquot into 2 mL EP tubes, and store at -80°C for later use.

[0079] The above cell mixture was passaged three times in DF1 cells. Nucleic acid was extracted from the groups that showed obvious cytopathic reactions. After reverse transcription, the genetically modified regions were amplified by PCR using detection primers. The amplified products were recovered and sent for testing. The sequence alignment was consistent, confirming that the reverse genetic rescue was successful. The successfully rescued strains were named IBDV-DHN-Mu1 and IBDV-DHN-Mu2, respectively.

[0080] (6) Determination of biological characteristics of IBDV-DHN modified strains IBDV-DHN-Mu1 and IBDV-DHN-Mu2 strains were simultaneously inoculated into DF1 cells at 10-fold serial dilutions. Cytopathic effects were observed from 24 h post-inoculation until 120 h. The median infection dose (TCID) of the cell culture tissue was calculated using the Reed-Muench method. 50 ).

[0081] The results showed that both IBDV-DHN-Mu1 and IBDV-DHN-Mu2 strains initially showed cytopathic effects 24 hours after infecting cells, and the viral titer could reach 10 after reaching the plateau phase.9.0 TCID 50 / mL, indicating that the IBDV-DHN isolated in this invention can be rescued by reverse genetics and can be used to study the molecular mechanisms of IBDV infectivity and pathogenicity.

[0082] In summary, this invention provides an IBDV-DHN strain adapted for full suspension culture, which can be used as an important component of immunization vaccines for vaccine production, providing specific immune protection for immunized poultry. It boasts advantages such as simple culture process, low difficulty in scale-up production, low risk of contamination, and good vaccine stability, effectively solving the problem of large-scale production difficulties of IBDV cell-based vaccines. Simultaneously, it significantly reduces production costs and has broad application prospects. Furthermore, this strain can be rescued through reverse genetics, serving as a strain backbone for constructing vaccine candidate strains with different specificities. It also allows for the molecular-level exploration of IBDV pathogenicity, interspecies transmissibility, and viral mutation mechanisms, demonstrating broad application potential.

[0083] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A bursa virus strain, characterized in that, The bursa of fabricius virus strain is named IBDV-DHN, and is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: V202572.

2. The application of the bursa of fabricius virus strain of claim 1 in the research of pathogenicity, interspecies transmission ability, and virus variation mechanism of bursa of fabricius virus.

3. A mutant strain of bursa virus, characterized in that, The bursa of fabricius virus mutant strain is obtained by reverse genetic rescue of the bursa of fabricius virus strain of claim 1.

4. The application of the bursa of fabricius virus strain of claim 1 or the bursa of fabricius virus mutant strain of claim 3 in the preparation of products for preventing and / or treating bursa of fabricius disease.

5. Use according to claim 4, characterized in that, The products include vaccines or drugs.

6. The application of the bursa of fabricius virus strain of claim 1 or the bursa of fabricius virus mutant strain of claim 3 in the preparation of diagnostic reagents.

7. A vaccine, wherein the active ingredient of the vaccine comprises the bursa of fabricius virus strain of claim 1 or the bursa of fabricius virus mutant strain of claim 3.

8. The vaccine of claim 7, characterized in that, The vaccine further comprises at least one of a pharmaceutically acceptable adjuvant, a diagnostic agent, a carrier, a protective agent, an adjuvant, an excipient, or a diluent.

9. A method for culturing a bursa virus, characterized by, The culture method comprises the following steps: inoculating the bursa of fabricius virus strain of claim 1 to LMH suspension cells, and culturing, to obtain the bursa of fabricius virus.

10. A method for constructing an animal model of bursa of fabricius disease infection, comprising introducing the bursa of fabricius virus strain of claim 1 or the bursa of fabricius virus mutant strain of claim 3 into an animal, and causing the animal to be ill.

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