Recombinant chicken infectious bursal disease virus VP2 protein and application thereof

Abstract

The invention discloses a vaccine composition, an immunogen of the vaccine composition is a fusion protein containing a VP2 protein of a new isolate of chicken infectious bursal disease and an auxiliary protein, and the immunogen can be independently assembled into subvirus particles. The vaccine composition has good safety to chickens, the titer of an antibody generated by immunizing the chickens is high, and the challenge protection effect of the immunized chickens is good. The vaccine has an application prospect.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products and relates to a recombinant chicken infectious bursal virus VP2 protein and its application. Background Technology

[0002] Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive disease caused by the infectious bursal disease virus (IBDV), primarily infecting chickens aged 3–6 weeks. The disease mainly affects the bursa of Fabricius; IBDV proliferates in the B lymphocytes of the bursa and damages them, easily leading to secondary infections or vaccine failure. The disease spreads rapidly, has a high morbidity and mortality rate, and a short course, causing enormous losses to the poultry industry worldwide.

[0003] IBDV is a member of the family DiRNAviridae and the genus Avian DiRNAvirus. The virus is non-enveloped and exhibits icosahedral symmetry. The genome of IBDV consists of two segments of double-stranded RNA, A and B, which encode five proteins: VP1, VP2, VP3, VP4, and VP5. VP2 is the most important structural protein of IBDV and is closely related to viral virulence, pathogenicity, and antigenic drift. Furthermore, VP2 is mainly exposed outside the nucleocapsid and is involved in biological functions such as the induction and recognition of viral neutralizing antibodies. It accounts for 51% of the total structural proteins and, together with another major structural protein, VP3, forms the nucleocapsid backbone. It is the protein of choice for developing genetically engineered subunit vaccines against chicken IBD.

[0004] There are many methods for expressing the VP2 gene in exogenous systems, such as E. coli expression systems and yeast expression systems, but all of them have problems such as low expression levels, low activity, and unsatisfactory immune effects. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8 and P9;

[0006] P1: Combinatorial protein

[0007] The amino acid sequence of the combined protein is shown in positions 1-504 of SEQ ID NO.4;

[0008] P2: Fusion protein

[0009] The fusion protein includes the peptide segments and additional peptide segments of the combined protein described in P1;

[0010] The additional peptide is selected from signal peptides and labeled peptides;

[0011] P3: RNA

[0012] The RNA can be translated into the combined protein described in P1 or the fusion protein described in P2;

[0013] P4: Genes

[0014] The coding sequence of the gene can encode the combinatorial protein described in P1 or the fusion protein described in P2;

[0015] P5: Gene Expression Kit

[0016] The gene expression product in the gene expression cassette assembly is the RNA described in P3;

[0017] The promoter of the gene expression cassette is a constitutive expression promoter or an artificially inducible promoter;

[0018] P6: Gene Engineering Vector

[0019] The genetic engineering vector encodes the expressible RNA described in P3;

[0020] P7: Cells

[0021] The cells contain the gene engineering vector described in P6;

[0022] P8: Composition

[0023] The composition contains the combined protein described in P1, the fusion protein described in P2, the RNA described in P3, the gene engineering vector described in P6, or the cell described in P7.

[0024] P9: Reagent Kit

[0025] The kit contains the combined protein described in P1, the fusion protein described in P2, the RNA described in P3, the gene engineering vector described in P6, or the cell described in P7.

[0026] In some implementations, the choice is any one of the following A1, A2, A3, A4, and A5;

[0027] A1: The labeled peptide is a tag peptide used for protein isolation and purification;

[0028] A2: The fusion protein has a linker arm or random amino acid sequence of 1-15 amino acids between different domains that does not affect the functional independence of the domains;

[0029] A3: The backbone of the genetic engineering vector is selected from pUC57 plasmid and pFastBac1 vector;

[0030] A4: The host cells of the cells are selected from Escherichia coli JM109 strain, Escherichia coli DH10Bac strain, sf9 cells and insect cell High 5 strain;

[0031] A5: The composition also contains therapeutically active substances, immunologically active substances, inert substances, excipients, or unavoidable impurities.

[0032] In some embodiments, the composition contains an immunogen and the excipients; the immunogen is the combined protein described in P1 or the fusion protein described in P2.

[0033] In some embodiments, the composition comprises the immunogen, an emulsifier, and an oil phase mixture;

[0034] The volume ratio of the immunogen, the emulsifier, and the oil phase mixture is 100:3-9:150-450.

[0035] The amount of immunogen used is calculated based on the immunogen having a potency of 1:4096 as determined by the agarose gel diffusion precipitation method.

[0036] In some implementations, the choice is any one of B1, B2 and B3 below;

[0037] B1: The emulsifier is Tween-80;

[0038] B2: The oil phase mixture contains white oil, Span-80 and aluminum stearate, and the weight ratio of the white oil, the Span-80 and the aluminum stearate is 100:2-6:1-3;

[0039] B3: The immunogen is an inactivated immunogen.

[0040] The second aspect of this invention provides the use of the biomaterial described in the first aspect of this invention in the preparation of formulations for use alone, for use in combination with other immunizing agents and / or drugs, or as a component of a compound formulation composed of other immunizing agents and / or drugs to prevent, mitigate, and / or control infectious bursal disease in chickens. Attached Figure Description

[0041] Figure 1The electrophoresis results of the rBac-VP2F recombinant shuttle plasmid are shown. Lane M represents the results of DNA marker DL15000; Lane 1 shows the product of VP2-specific primer amplification of the shuttle plasmid rBac-VP2F; Lane 2 shows the product of M13 universal primer amplification of the rBac-VP2F shuttle plasmid; Lane 3 shows the product of pFastBac1 vector transformed into DH10Bac competent cells amplified by M13 universal primer; Lane 4 shows the product of DH10Bac strain amplified by M13 universal primer.

[0042] Figure 2 The results of Western blot identification are shown. Lane M represents the results of protein molecular weight labeling; Lane 1 represents the electrophoresis results of lysates of SF9 cells infected with P3 generation of recombinant baculovirus rVP2F; Lane 2 represents the results of lysates of SF9 cells infected with wild-type baculovirus; and Lane 3 represents the results of lysates of normal SF9 cells.

[0043] Figure 3 The results of the agar gel diffusion assay show the recombinant VP2 subviral particle solution. Wells 7 and 14 show the results after adding IBDV positive serum. Wells 1-6 show the results after adding recombinant baculovirus rVP2 to SF9 cells for 72 hours, yielding recombinant VP2 protein at a 1:2 ratio. 7 ~12 Dilution results (dilution increases sequentially with increasing label); wells 8-13 contain recombinant VP2 protein harvested 96 hours after infection of SF9 cells with recombinant baculovirus rVP2 at a 1:2 ratio. 7~12 The results of dilution (the dilution increases sequentially with the number).

[0044] Figure 4 This image shows a negative-stained electron microscope image of recombinant VP2 subviral particles. The cell lysate was harvested 72 h after SF9 cells were infected with recombinant baculovirus rVP2. Under electron microscopy, the recombinant VP2 particles were seen to assemble into well-formed particles with a diameter of about 20 nm. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0046] Materials and instruments not described in this invention are conventional materials and instruments in the art. Operational details not described in this invention are conventional operations in the art. The software used in this invention is operated by conventional methods in accordance with the software provider's instructions. The reagent kits used in this invention are operated by conventional methods in accordance with the reagent kit's instruction manual.

[0047] The nucleic acid sequences shown in this invention are all written from left to right in the direction from 5' to 3', and the protein sequences are written from left to right in the direction from N-terminus to C-terminus.

[0048] The materials used in this invention are described below: The nucleic acid extraction kit was purchased from TransGen Biotech Ltd.; the reverse transcription kit and gel extraction kit were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; DNA Taq enzyme was purchased from Novizan Biotech Ltd.; ampicillin, kanamycin, tetracycline, and gentamicin were purchased from Beijing Solarbio Science & Technology Co., Ltd.; and lipo 3000 was purchased from Thermo Fisher Scientific China Co., Ltd. The baculovirus expression system (including the transfer vector plasmid pFastBac1, the E. coli DH10Bac strain containing the baculovirus shuttle plasmid, and Spodoptera frugierda ovarian tissue cells Sf9 and High5, etc.) was a product of Invitrogen. IBDV positive serum was prepared by immunizing SPF chickens with the inactivated IBDV variant YK3. HRP-labeled goat anti-chicken IgG was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. The M13 universal primers were the sequences in the Bac-to-Bac system instructions (Invitrogen) and were synthesized by Jilin Kumei Biotechnology Co., Ltd. Grace's insect cell culture medium used for Sf9 cell culture is a Gibco product. The serum-free culture medium used for High 5 cell culture is IB905 insect cell suspension medium from Yishengke (Shenzhen) Co., Ltd.

[0049] Unless otherwise specified, all instruments and reagents used in the experiments of this invention are commercially available products.

[0050] Example 1: Obtaining the YK3 strain

[0051] I. Case Study

[0052] A 30-day-old broiler chicken at a chicken farm showed clinical signs of lethargy, and necropsy revealed severe atrophy of the infectious bursa of Fabricius. Infectious bursal disease was suspected.

[0053] II. Pathogen Processing and Pathogen Identification

[0054] The bursal disease samples from infected chickens were ground with an appropriate amount of sterile PBS, subjected to five freeze-thaw cycles, and centrifuged at 6000 rpm for 10 min. 0.2 mL of the supernatant was inoculated through the chorioallantoic membrane into 10-day-old SPF chicken embryos and incubated at 37°C. Embryos that died within 24 hours were discarded, while those that died after 24 hours were placed at 4°C, and allantoic fluid was collected. The same method was then used for five blind passages. Allantoic fluid from the fifth-generation embryos was collected to obtain allantoic fluid containing suspected infectious bursal disease virus (allantoic fluid a).

[0055] Total RNA was extracted from allantoic fluid a. Using the reverse transcription product cDNA as a template, the cDNA was amplified by PCR using primers F1 (SEQ ID NO.1) and R1 (SEQ ID NO.2) of the chicken infectious bursal virus VP2 gene.

[0056] The F1 sequence is as follows: 5'-GATCGCAGCGATGACGAACCTGCAA-3'

[0057] The R1 sequence is as follows: 5'-AGCGGCGGGTGGGAACAATGTAGAT-3'

[0058] The PCR amplification results were subjected to Sanger sequencing. The protein sequence obtained was converted from the coding sequence and compared with all infectious bursal virus (IBDV) VP2 protein sequences in GenBank using BLAST. The IBDV HK46 strain (GenBank: AAC06017.1) VP2 protein showed the highest homology, with a homology of 96.70%. This indicates the presence of an IBDV strain in the allantoic fluid. This strain was named IBDV YK3 strain, or simply YK3 strain.

[0059] Example 2: Construction of expression vector

[0060] I. Sequence Design and Synthesis

[0061] Based on the codon preference of insect cells, the VP2 gene sequence of YK3 strain was optimized, and the VP2 fusion gene fragment (named VP2F) was synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0062] The nucleotide sequence of VP2F is as follows (SEQ ID NO.3):

[0063]

[0064] Positions 4-9 are Bam HI restriction sites, positions 10-1377 are the VP2 protein coding sequence, positions 1378-1521 are auxiliary sequences, positions 1522-1539 are the histidine tag coding sequence, and positions 1543-1548 are the Hind III restriction sites.

[0065] The protein sequence encoded by VP2F is as follows (SEQ ID NO.4):

[0066] MANLQDQTQQIVPSIRSLLMPTTGPASIPDDTLEKHTLRSETSTYNLTVGDTGSGLIVFFPGFPGSVVGAHYTLQSSGNYKFDQMLLTAQNLPASYNYCRLVSRSLTVRSSTLPGGVYALNGTINAV TFHGSLSELTDVSYNGLMSATANINDKIGNVLVGEGVTVLSLPTSYDLGYVRLGDPIPAIGLDPKMVATCDSSDRPRVYTITAADDYQFSSQYQPGGVTITLFSANIDAITSLSVGGELVFKTSVQGL VLGATIYLIGFDGTAVITRAVAADNGLTTGTDNLLPFNLVIPTNEITQPITSMKLEIVTSKSGGQAGDQMSWSSAGSLAVTIHGGNYPGALRPVTLVAYERVATGSVVTVAGVSNFELIPNPELAKN LVTEYGRFDPGAMNYTKLILSERDRLGIKTVWPTREYTDFREYFMEVADLNSPLKIAGAFGFKDIIRAIRRVERGGGGSGGGGSGGGGSRMKQIEDKIEEILSKIYHIENEIARIKKLVGERHHHHHH

[0067] Among them, positions 1-456 are VP2 protein, positions 457-504 are accessory protein, and positions 505-510 are histidine tags.

[0068] The VP2F and pUC57 plasmids were double-digested with restriction endonucleases BamHI and Hind III, respectively, and ligated using T4 DNA ligase. The ligation product was transformed into E. coli JM109 competent cells, and the plasmid was extracted. The plasmid was amplified using universal primers for the pUC57 plasmid, and successful nucleic acid insertion was confirmed by gel electrophoresis. The recombinant plasmid was named pUC57-VP2F.

[0069] II. Construction of Recombinant Baculovirus Shuttle Plasmid

[0070] The recombinant plasmid pUC57-VP2F and the pFastBac1 vector were double-digested with Bam HI and Hind III, respectively. The reaction system was as follows: 2 μL 10×M Buffer, 2 μL recombinant plasmid pUC57-VP2F, 1 μL Bam HI, 1 μL Hind III, 12 μL ddH2O, for a total volume of 20 μL. The digestion products were detected by 1% agarose gel electrophoresis, and the VP2F and pFastBac1 vector fragments were recovered. 6 μL of the digested VP2F gene fragment, 2 μL of the digested pFastBac1 vector, 1 μL 2×Ligation Buffer, and 1 μL T4 DNA Ligase were added sequentially to a 0.2 mL centrifuge tube, mixed well, and ligated at 16℃ for 20 h to insert the VP2F gene fragment into pFastBac1. The recombinant plasmid was named pFastBac1-VP2F.

[0071] The ligation product was transformed into E. coli JM109 competent cells, and a single clone was obtained by screening on LB agar containing ampicillin. This single clone contained the positive plasmid pFastBac1-VP2F. The pFastBac1-VP2F plasmid was extracted from this single clone, and then transformed into competent DH10Bac cells, followed by three blue-white colony selections. The steps for each blue-white colony screening are as follows: The bacterial culture is plated on solid LB agar plates containing gentamicin (7 μg / ml), kanamycin (50 μg / ml), tetracycline (10 μg / ml), IPTG (40 μg / ml), and X-gal (100 μg / ml) and incubated for 24–48 h. White colonies are then picked and inoculated into liquid LB medium containing gentamicin (7 μg / ml), kanamycin (50 μg / ml), and tetracycline (10 μg / ml) and incubated for 12 h. Colonies obtained after three blue-white colony screenings are then subjected to PCR detection using universal primers M13 and VP2-specific primers (F2 and R2), respectively. Positive clones are selected, thus yielding the strain containing the recombinant baculovirus shuttle plasmid rBac-VP2F containing the VP2 fusion gene.

[0072] F2 sequence (SEQ ID NO.5): 5'-ATGGCTAACCTGCAAGATCAGACTC-3'

[0073] R2 sequence (SEQ ID NO.6): 5'- TTAGTGGTGATGGTGATGGTGTCTC-3'

[0074] See the results of the recombinant identification. Figure 1 .from Figure 1 As can be seen, the amplification product using VP2 primers contains a specific band of approximately 1500 bp, the amplification product using M13 universal primers contains a specific band of approximately 3800 bp, and the product of positive clones obtained by amplifying the pFastBac1 vector transformed into DH10Bac competent cells using M13 universal primers contains a specific band of 2300 bp. This indicates that the plasmid pFastBac1-VP2F has undergone transposition recombination with Bacmid in DH10Bac to form the viral shuttle plasmid rBac-VP2F.

[0075] III. Obtaining Recombinant Baculoviruses Expressing VP2

[0076] 6 μl of recombinant baculovirus shuttle plasmid rBac-VP2F was transfected into a monolayer of Sf9 cells in logarithmic growth phase using the Lipo 3000 transfection reagent, and the cells were cultured statically at 27°C. After more than 80% of the cells swelled and ruptured, the cells and supernatant were collected and centrifuged at 1000 rpm for 10 min. The supernatant was collected to obtain the first generation of recombinant baculovirus, named recombinant baculovirus rVP2F. The recombinant baculovirus rVP2F was inoculated into Sf9 cells and multiplied to the 5th generation (P5). Cells and supernatants from each generation were harvested, repeatedly frozen and thawed, and then centrifuged at 1000 rpm for 10 min. The supernatant was collected and stored. The TCID of the 5th generation recombinant baculovirus rVP2F was determined using the Reed-Muench method. 50 10 8.2 TCID 50 / 0.1ml.

[0077] Example 3: Protein Expression

[0078] I. Growth conditions of insect cells and expression of recombinant VP2 protein

[0079] High 5 insect cells at 1.5 × 10 5 Cells were resuspended at a density of cells / ml in serum-free IB905 medium and cultured at 27°C for 24 h. Cells were then collected and the cell density was adjusted to 1.8 × 10⁶ cells / ml with fresh IB905 medium. 6 Cells / ml were inoculated with P5 generation recombinant baculovirus rVP2F at an inoculation rate of 0.5 MOI. The culture medium was at pH 6.2 and cultured at 27°C and 80 rpm for 100 h. The cell suspension was harvested, and after three freeze-thaw cycles, it was lysed by ultrasound and then incubated at 4°C for 24 h to obtain a solution containing self-assembled recombinant VP2 subviral particles (solution 1).

[0080] II. Western blot analysis for the identification of recombinant VP2 expression

[0081] Following the method described in Section 1 of this embodiment, P5 generation recombinant baculovirus rVP2F was inoculated into suspension-cultured High 5 cells. Cell culture was collected 96 h after infection, subjected to three freeze-thaw cycles, lysed by sonication, and centrifuged at 4°C, 2000 rpm for 10 min. The supernatant was collected. A negative control (wild-type baculovirus inoculated into High 5 cells, with the same infection dose and operating parameters) and a blank control (normal High 5 cells, uninoculated) were also included. Western blot was performed according to standard procedures, with the primary antibody being IBDV-positive chicken serum (1:300 dilution) and the secondary antibody being HRP-labeled goat anti-chicken IgG (1:50000 dilution). Western blot results ( Figure 2 The results showed a specific band at approximately 54 kDa, consistent with the expected size, indicating that the VP2 protein was successfully expressed and could bind to IBDV-positive serum, meaning that the recombinant VP2 protein has good reactivity.

[0082] 3. Agar diffusion assay to identify the reactivity of recombinant VP2

[0083] Following the method described in Section 1 of this embodiment, P5 generation recombinant baculovirus rVP2F was inoculated into High 5 suspension culture cells and cultured at 27°C for 96 hours. Samples were taken every 24 hours, subjected to three freeze-thaw cycles, and lysed by sonication. The samples were then centrifuged at 4°C and 2000 rpm for 10 minutes, and the supernatant was collected for agar gel diffusion precipitation (AGP) assay (referring to DB13T 500-2004 standard, Technical Specifications for Prevention and Control of Infectious Bursal Disease in Commercial Broilers). The central well contained IBDV positive serum, and the peripheral wells contained serial dilutions of each sample (2...). 1 2 2 2 3 …2 12 (Serial dilutions). Results showed that cell cultures harvested at 24h and 48h did not react with IBDV-positive serum; cell cultures harvested at 72h and 96h reacted with IBDV-positive serum at a 1:2 ratio. 1 ~1:2 12 Clear precipitation lines were visible after dilution. Figure 3 This indicates that the recombinant VP2 protein has good immunoreactivity, and the highest AGP titer of the obtained recombinant VP2 subviral particle solution can reach 1:2. 12 (1:4096).

[0084] IV. Negative staining electron microscopy observation of recombinant VP2 subviral particles

[0085] Following the method described in Section 1 of this embodiment, P5 generation recombinant baculovirus was inoculated into High 5 suspension cells and cultured at 27°C for 96 hours. The supernatant after cell lysis was harvested. This supernatant was concentrated by sucrose gradient density centrifugation (approximately 20-fold concentration), and the self-assembly morphology of the recombinant VP2 protein was observed using transmission electron microscopy. The results are as follows: Figure 4 As can be seen, under an electron microscope, regularly shaped particles with a diameter of about 25 nm can be observed, indicating that the recombinant VP2 protein can self-assemble into subviral-like particles smaller than IBDV virus particles.

[0086] Example 4: Vaccine Preparation (I) Preparation of Recombinant VP2 Subviral Particle Vaccine

[0087] A method for preparing a recombinant VP2 subviral particle vaccine includes the following steps:

[0088] (1) Preparation of antigen solution and determination of AGP titer: The antigen solution used in the preparation of the vaccine was a solution (solution 1) containing self-assembled recombinant VP2 subviral particles prepared according to the method in Section 1 of Example 3. The AGP titer was then determined. The results showed that the AGP titer was ≥1:2. 12 .

[0089] (2) Inactivation of recombinant VP2 subviral particles: Add 10% formaldehyde aqueous solution (mass percentage concentration) to the recombinant VP2 subviral particle solution obtained in step (1) above, and stir evenly to obtain an inactivated recombinant VP2 subviral particle solution. The amount of 10% formaldehyde aqueous solution (mass percentage concentration) added is 0.1% of the volume of the recombinant VP2 subviral particle solution.

[0090] The specific implementation method is as follows: Place the recombinant VP2 subviral particle solution in an inactivation vessel, and add formaldehyde aqueous solution while stirring in a specific ratio to ensure thorough mixing. Then, raise the temperature to 37°C and continue inactivation for 24 hours (start timing when the temperature reaches 37°C). After inactivation is stopped, take a sample from the vessel for inactivation testing. The inactivated recombinant VP2 subviral particle solution should be stored in the dark at 2–8°C for no more than 3 months.

[0091] (3) Test for complete inactivation of recombinant VP2 subviral particles: Normally growing sf9 cells were inoculated with an inactivated recombinant VP2 subviral particle solution, and the changes in cell growth were observed to test whether the recombinant VP2 subviral particle solution was completely inactivated. The specific method was as follows: The inactivated recombinant VP2 subviral particle solution was inoculated into a culture flask containing a well-grown monolayer of sf9 cells. The inoculation volume was 5% of the culture medium volume in the flask. The cells were incubated at 27°C for 3 days, and the cell growth status was observed. After 3 days, the culture supernatant was transferred to another well-grown monolayer of sf9 cells and incubated at 27°C for 3 days, and the cell growth status was observed. No cytopathic effects were observed in the sf9 cells during both generations of culture, indicating complete inactivation.

[0092] (4) Sterility test: The solution containing self-assembled recombinant VP2 subviral particles was tested for sterility according to Appendix 28 of the Veterinary Pharmacopoeia of the People's Republic of China (2020 edition, Part III). The test result was no bacterial growth.

[0093] (5) Preparation of oil phase: Prepare the oil phase by mixing 94 parts by weight of white oil for injection (see Appendix 54 of the 2020 edition of the Veterinary Pharmacopoeia of the People's Republic of China, Part III), 4 parts by weight of Span-80, and 2 parts by weight of aluminum stearate. Take aluminum stearate, mix it with a small amount of white oil for injection, heat it until it is translucent, then mix it evenly with the full amount of Span-80 and the remaining white oil for injection, sterilize it at 121°C for 15 min, and cool it to room temperature to obtain the oil phase.

[0094] (6) Aqueous phase preparation: Take the qualified antigen solution (containing the self-assembled recombinant VP2 subvirus particles), add 6% of the antigen solution volume of Tween-80, and stir at 1000 rpm for 2 min to obtain the aqueous phase.

[0095] (7) Emulsification: The oil phase prepared in step 5 above is first added to the emulsification tank and stirred at 200 rpm. While stirring, the aqueous phase prepared in step (6) above (the volume ratio of aqueous phase to oil phase is 1:2.5) is added and stirred for 20 min to obtain a uniformly mixed emulsion. Then, at a temperature of about 25°C, the above mixed emulsion is stirred at 15000 rpm for 5 min. This high-speed stirring is performed twice to obtain a fully emulsified recombinant VP2 subviral particle inactivated vaccine. Take 10 ml of the emulsified recombinant VP2 subviral particle inactivated vaccine and place it in a 15 ml centrifuge tube. Centrifuge at 3000 rpm for 15 minutes. No stratification should occur. The recombinant VP2 subviral particle inactivated vaccine should be stored at 2-8°C, sealed and protected from light.

[0096] To test the safety and immunogenicity of the prepared recombinant VP2 subviral particle inactivated vaccine, three batches of vaccine were prepared according to the above method, namely VPs01, VPs02 and VPs03.

[0097] Example 5: Vaccine Safety Testing

[0098] (a) Safety of recombinant VP2 subviral particle vaccine (prepared in Example 4)

[0099] Safety tests were conducted on the three batches of recombinant VP2 subviral particle inactivated vaccines VPs01, VPs02, and VPs03 prepared in Example 4. The specific procedures were as follows: Forty 14-day-old SPF chickens were divided into four groups of ten each. Three groups were injected with the three batches of vaccine mentioned above, at a dose of 0.3 ml / chicken; the other group of ten served as a negative control, injecting 0.3 mL / chicken of physiological saline. Forty 7-day-old broiler chickens were also divided into four groups of ten each. Three groups were injected with the three batches of vaccine mentioned above, at a dose of 0.3 ml / chicken; the other group of ten served as a negative control, injecting 0.3 mL / chicken of physiological saline. All vaccines and physiological saline were injected subcutaneously in the neck. Chickens were observed continuously for 21 days post-injection. Daily observations included feed and water intake, systemic and local reactions, activity and mental state, and other clinical symptoms. On days 7, 14, and 21 post-immunization, the injection sites were visually and physically examined for redness, swelling, or other local injection reactions. Twenty-one days after immunization, all experimental chickens were culled, and the absorption of the vaccine at the injection site and the presence of tissue lesions at the injection site were examined. The results showed that none of the three batches of vaccine caused adverse reactions at the injection site or throughout the entire experimental observation period. During the entire observation period, the experimental chickens ate and drank normally. Autopsy 21 days after immunization showed that, except for one SPF chicken with a small amount of vaccine residue, there was no vaccine residue at the injection site and no lesions in the body tissues of the other experimental chickens, proving that the vaccine is safe for subcutaneous immunization of target animals (chickens) (Table 1).

[0100] Table 1. Immunological safety trials of three batches of recombinant VP2 subviral particle vaccine

[0101] vaccine batch experimental chickens Age in days Quantity (pieces) Dosage (ml) Foraging and drinking Injection site Whole body reaction VPs01 SPF Chicken 14 10 0.3 normal No residue, no swelling, no lumps none VPs01 Broiler chickens 7 10 0.3 normal No residue, no swelling, no lumps none VPs02 SPF Chicken 14 10 0.3 normal One small amount of residue, no swelling, no lumps none VPs02 Broiler chickens 7 10 0.3 normal No residue, no swelling, no lumps none VPs03 SPF Chicken 14 10 0.3 normal No residue, no swelling, no lumps none VPs03 Broiler chickens 7 10 0.3 normal No residue, no swelling, no lumps none control group SPF Chicken 14 10 0.3 normal No swelling or lumps none control group Broiler chickens 7 10 0.3 normal No swelling or lumps none

[0102] Example 6: Determination of vaccine immunogenicity

[0103] Immunogenic efficacy of recombinant VP2 subviral particle vaccine (prepared in Example 4).

[0104] The immunogenicity of the three batches of recombinant VP2 subviral particle inactivated vaccines VPs01, VPs02 and VPs03 prepared in Example 4 was tested in immunization test chickens.

[0105] I. Standards for Testing the Immunogenicity of Recombinant VP2 Subviral Particle Inactivated Vaccine

[0106] The immunogenicity testing standards for the recombinant VP2 subviral particle inactivated vaccine are as follows: After immunizing 15-day-old SPF chickens with one dose (0.3 mL) of the recombinant VP2 subviral particle inactivated vaccine for 28 days, 80% of the immunized test chickens should have a serum agar-agar antibody level not lower than 1:8 (Note: an agar-agar antibody level not lower than 1:8 is considered positive), and the average serum virus neutralizing titer should not be lower than 1:5120, thus qualifying as a qualified serological efficacy test. In the challenge protection test, more than 80% of the immunized test chickens should be protected, and at least 80% of the control test chickens should develop the disease. The cysteine ​​ratio in the challenge test chickens should not differ significantly from that in the control test chickens, thus qualifying as a qualified challenge protection test. The aforementioned qualified immunogenicity test requires that the immunogenicity tests of all three batches of vaccine meet the above criteria.

[0107] II. Methods for Testing the Immunogenicity of Recombinant VP2 Subviral Particle Inactivated Vaccine

[0108] The specific implementation of the immunogenicity testing of three batches of recombinant VP2 subviral particle inactivated vaccine VPs01, VPs02, and VPs03 was as follows: Ninety 15-day-old SPF chickens were divided into four groups of 20 chickens each. Three groups were injected with one dose (0.3 mL) of recombinant VP2 subviral particle vaccine (referred to as recombinant VP2 vaccine). The third group served as a non-immunized challenge control, and another 10 SPF chickens served as a non-immunized, non-challenge control (blank control). Wing vein serum was collected from each group at 14, 21, and 28 days post-immunization. Agar agar antibody and virus serum neutralizing antibody levels were measured according to the methods in the *Veterinary Pharmacopoeia of the People's Republic of China* (2020 edition, Part III) to evaluate the level of infectious bursal disease (IBDV) antibodies in chicken serum. Twenty-eight days after immunization, the standard IBDV strain BC6 / 85 was administered at a dose of 0.2 mL / chicken (containing 10 EIDs). 50 Eye-drop challenge was used on chickens. The challenge test included chickens immunized with three batches of recombinant VP2 vaccine and a control group that was not immunized. 72 hours post-challenge, each chicken was necropsydraught with lesions in the pectoral muscles, leg muscles, gizzard, proventriculus, and bursa of Fabricius. Infection was diagnosed if any of the typical lesions of infectious bursa of Fabricius were observed, including brush-like hemorrhages in the pectoral and leg muscles, hemorrhage at the junction of the gizzard and proventriculus, hemorrhage in the bursa of Fabricius, or bursal edema. Simultaneously, each chicken and its dissected bursa of Fabricius were weighed, and the bursa-to-body ratio (%) was calculated (bursa weight / body weight * 100).

[0109] III. Immunopotency Test Results of Three Batches of Recombinant VP2 Subviral Particle Vaccine

[0110] Twenty-eight days after immunization with the three batches of recombinant VP2 subviral particle vaccine, the serum neutralizing antibody level for infectious bursal disease was 1:10513, and the antibody titer of each batch reached 1:8 or higher in 100% of the batches, meeting the serological test qualification standards. After the challenge experiment, each batch achieved over 95% (19 / 20) protection, while the non-immunized challenge control group showed 100% (20 / 20) infection and disease. The bursal-to-body ratio in the challenged chickens (0.23) was not significantly different from that in the non-immunized, non-challenge control group (0.22), meeting the protection qualification standards for the challenge experiment. Based on the above results, all three batches of recombinant VP2 subviral particle inactivated vaccine met the quality standards. Specific results are shown in Tables 2, 3, and 4.

[0111] Table 2. Serum AGP antibody titers after immunization with three batches of VP2 subviral particle inactivated vaccine

[0112] vaccine 14-day AGP positivity rate (%) 21-day AGP positivity rate (%) 28-day AGP positivity rate (%) <![CDATA[14-day AGP average (nlog2)]]> <![CDATA[21-day AGP average value (nlog2)]]> <![CDATA[28-day AGP average value (nlog2)]]> VPs01 80(16 / 20) 100(20 / 20) 100(20 / 20) 3.13±1.35 4.43±1.05 5.18±0.52 VPs02 85(17 / 20) 90(18 / 20) 100(20 / 20) 3.27±1.24 4.36±1.19 5.13±1.07 VPs03 80(16 / 20) 95(19 / 20) 100(100 / 100) 3.16±1.04 4.46±1.05 5.23±1.27 Challenge control group 0 0 0 0 0 0 Blank control group 0 0 0 0 0 0

[0113] Table 3. Neutralizing antibody levels after immunization with three batches of VP2 subviral particle inactivated vaccine

[0114] vaccine 14 days 21 days 28 days VPs01 10.63（1:1584） 12.35(1:5220) 13.36(1:10513) VPs02 10.72（1:1686） 12.37(1:5293) 13.58(1:12245) VPs03 10.60（1:1552） 12.58(1:6122) 13.68(1:13214) Challenge control group 0 0 0 Blank control group 0 0 0

[0115] Table 4. Protection rate against viral challenge after immunization with three batches of VP2 subviral particle inactivated vaccine

[0116] vaccine Body weight (g) Bag weight (g) Cyst-to-body ratio (%) Number of cases Death count Number of cystic lesions Protection rate (%) VPs01 1183.49 2.82 0.23 0 / 20 0 / 20 1 / 20 95 VPs02 1166.08 2.85 0.24 0 / 20 0 / 20 1 / 20 95 VPs03 1133.55 2.61 0.23 0 / 20 0 / 20 0 / 20 100 control group 1084.92 4.42 0.41 14 / 20 12 / 20 20 / 20 0

[0117] Note: The average blastocyst-to-whole ratio (B / W) of the control group (non-immunized, non-challenged) was 0.22.

[0118] Example 7: Duration of Immunity Test

[0119] Duration of immunity study of recombinant VP2 subviral particle vaccine (prepared in Example 4).

[0120] The duration of immunity was determined by immunizing test chickens with the three batches of recombinant VP2 subviral particle inactivated vaccines VPs01, VPs02 and VPs03 prepared in Example 4.

[0121] The specific implementation of the immunity duration testing for three batches of recombinant VP2 subviral particle inactivated vaccines VPs01, VPs02, and VPs03 is as follows: 160 15-day-old SPF chickens were divided into 4 groups of 40 chickens each. Three groups were injected with one dose (0.3 mL) of recombinant VP2 subviral particle vaccine (referred to as recombinant VP2 vaccine), while the other group served as a non-immunized challenge control. Wing vein blood was collected from each group at 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, and 5 months post-immunization. Agar agar antibody and virus serum neutralizing antibody levels were measured according to the methods in the *Veterinary Pharmacopoeia of the People's Republic of China* (2020 edition, Part III) to evaluate the level of infectious bursal disease antibodies in chicken serum. On day 28 post-immunization, 20 immunized test chickens were simultaneously challenged with the control group, following the procedure in step 2 of Example 5.

[0122] The results of the immunity duration tests for three batches of recombinant VP2 subviral particle inactivated vaccines (VPs01, VPs02, and VPs03) are as follows: 28 days after immunization, the serum neutralizing antibody levels of infectious bursal disease (IBD) in chickens from all three batches of the recombinant VP2 subviral particle vaccine were greater than 1:5120, and the antibody titer agar-agar expanded at 100% of the batches reached 1:8 or higher, meeting the serological test qualification standards. After challenge experiments, all batches achieved 100% (20 / 20) protection, and the non-immunized challenge control group showed 100% (20 / 20) infection and disease incidence, meeting the challenge test protection qualification standards. At 2, 3, 4, and 5 months after immunization, the serum neutralizing antibody levels of IBD in chickens from all three batches were above 1:5480; the antibody titer agar-agar expanded at 90% of the batches reached 1:8 or higher, meeting the serological test qualification standards. Based on the above results, all three batches of recombinant VP2 subviral particle inactivated vaccine met the quality standards. Specific results are shown in Tables 5, 6, 7, and 8.

[0123] Table 5. Duration of AGP Antibody After Immunization with Three Batches of Recombinant VP2 Subviral Particle Inactivated Vaccine

[0124] vaccine 2 weeks 3 weeks 1 month 2 months 3 months 4 months 5 months VPs01 3.12±1.18(1:8.69) 4.43±1.05(1:21.56) 5.26±0.66(1:38.32) 5.22±0.64(1:37.27) 4.52±0.88(1:22.94) 4.17±1.13(1:18.0) 4.07±0.95(1:16.79) VPs02 3.19±1.10(1:9.13) 4.48±1.05(1:22.32) 5.21±0.98(1:37.01) 5.22±1.14(1:37.27) 4.76±0.93(1:27.09) 4.23±1.04(1:18.76) 4.17±1.03(1:18.0) VPs03 3.18±1.14(1:9.06) 4.47±1.10(1:22.16) 5.34±1.05(1:40.50) 5.35±1.05(1:40.79) 4.79±1.07(27.66) 4.24±1.04(1:18.89) 3.96±1.07(1:15.56) control group 0 0 0 0 0 0 0

[0125] Table 6. AGP antibody positivity rate after immunization with three batches of recombinant VP2 subviral particle inactivated vaccine

[0126] vaccine 2 weeks 3 weeks 1 month 2 months 3 months 4 months 5 months VPs01 75%(15 / 20) 100%(20 / 20) 100%(20 / 20) 100%(20 / 20) 100%(20 / 20) 100%(20 / 20) 90%(18 / 20) VPs02 80%(16 / 20) 100%(20 / 20) 100%(20 / 20) 100%(20 / 20) 100%(20 / 20) 100%(20 / 20) 95%(19 / 20) VPs03 85%(17 / 20） 100%(20 / 20) 100%(20 / 20) 100%(20 / 20) 100%(20 / 20) 100%(20 / 20) 95%(19 / 20) control group 0 0 0 0 0 0 0

[0127] Table 7. Duration of serum neutralizing antibodies after immunization with three batches of recombinant VP2 subviral particle inactivated vaccine

[0128] vaccine 2 weeks 3 weeks 1 month 2 months 3 months 4 months 5 months VPs01 10.63(1:1584) 12.40(1:5404) 13.36(1:10513) 13.72(1:13493) 13.59(1:12330) 13.23(1:9607) 12.44(1:5556) VPs02 10.65(1:1606) 12.33(1:5148) 13.24(1:9674) 13.27(1:9877) 13.23(1:9607) 12.85(1:7383) 12.59(1:6165) VPs03 10.48(1:1428) 12.39(1:5367) 13.36(1:10513) 13.45(1:11190) 13.24(1:9674) 12.67(1:6517) 12.42(1:5480) control group 0 0 0 0 0 0 0

[0129] Table 8. Protection rate against virus challenge after immunization with three batches of recombinant VP2 subviral particle inactivated vaccine

[0130] vaccine attacking the virus strain Infectious drug dosage Number of cases Death count Number of cystic lesions Protection rate VPs01 BC6 / 85 10BID50 0 / 20 0 / 20 0 / 20 100% VPs02 BC6 / 85 10BID50 0 / 20 0 / 20 0 / 20 100% VPs03 BC6 / 85 10BID50 0 / 20 0 / 20 0 / 20 100% control group BC6 / 85 10BID50 15 / 20 10 / 20 20 / 20 0

[0131] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A biomaterial, said biomaterial being any one of the following P1, P2, P3, P4, P5, P6, P7, P8 and P9; P1: Combinatorial protein The amino acid sequence of the combined protein is shown in positions 1-504 of SEQ ID NO.4; P2: Fusion protein The fusion protein includes the peptide segments and additional peptide segments of the combined protein described in P1; The additional peptide is selected from signal peptides and labeled peptides; P3: RNA The RNA can be translated into the combined protein described in P1 or the fusion protein described in P2; P4: Genes The coding sequence of the gene can encode the combinatorial protein described in P1 or the fusion protein described in P2; P5: Gene Expression Kit The gene expression product in the gene expression cassette assembly is the RNA described in P3; The promoter of the gene expression cassette is a constitutive expression promoter or an artificially inducible promoter; P6: Gene Engineering Vector The genetic engineering vector encodes the expressible RNA described in P3; P7: Cells The cells contain the gene engineering vector described in P6; P8: Composition The composition contains the combined protein described in P1, the fusion protein described in P2, the RNA described in P3, the gene engineering vector described in P6, or the cell described in P7. P9: Reagent Kit The kit contains the combined protein described in P1, the fusion protein described in P2, the RNA described in P3, the gene engineering vector described in P6, or the cell described in P7.

2. The biomaterial as described in claim 1, characterized in that, Choose from any one of the following: A1, A2, A3, A4, and A5; A1: The labeled peptide is a tag peptide used for protein isolation and purification; A2: The fusion protein has a linker arm or random amino acid sequence of 1-15 amino acids between different domains that does not affect the functional independence of the domains; A3: The backbone of the genetic engineering vector is selected from pUC57 plasmid and pFastBac1 vector; A4: The host cells of the cells are selected from Escherichia coli JM109 strain, Escherichia coli DH10Bac strain, sf9 cells and insect cell High 5 strain; A5: The composition also contains therapeutically active substances, immunologically active substances, inert substances, excipients, or unavoidable impurities.

3. The biomaterial as described in claim 2, characterized in that, The composition contains an immunogen and the excipients; the immunogen is the combined protein described in P1 or the fusion protein described in P2.

4. The biomaterial as described in claim 3, characterized in that, The composition comprises the immunogen, emulsifier, and oil phase mixture; The volume ratio of the immunogen, the emulsifier, and the oil phase mixture is 100:3-9:150-450. The amount of immunogen used is calculated based on the immunogen having a potency of 1:4096 as determined by the agarose gel diffusion precipitation method.

5. The biomaterial as described in claim 3, characterized in that, Choose from any one of B1, B2, and B3 below; B1: The emulsifier is Tween-80; B2: The oil phase mixture contains white oil, Span-80 and aluminum stearate, and the weight ratio of the white oil, the Span-80 and the aluminum stearate is 100:2-6:1-3; B3: The immunogen is an inactivated immunogen.

6. Use of the biological material according to any one of claims 1-5 in the preparation of formulations for use alone, for use in combination with other immunizing agents and / or drugs, or as a component of a compound formulation composed of other immunizing agents and / or drugs to prevent, mitigate and / or control infectious bursal disease in chickens.

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