Chicken infectious bursal disease virus VP2 protein, VP2 protein virus-like particle vaccine and preparation method thereof

Abstract

The invention discloses a chicken infectious bursal disease virus VP2 protein, a VP2 protein virus-like particle vaccine and a preparation method thereof. The method comprises the following steps: firstly, optimally designing an IBDV VP2 protein; secondly, a pCold II-SUMO prokaryotic expression vector is obtained by adding SUMO protein on the basis of an escherichia coli cold shock expression vector pCold II, the prokaryotic soluble expression efficiency of the VP2 protein is improved, and the concentration of the purified VP2 protein with the SUMO label can reach 1.00 mg / mL; after the SUMO label is removed, the VP2 protein can be 100% assembled into VLPs in a specific solution, and after emulsification-demulsification, the VP2 protein still keeps a complete VLPs structure instead of a VP2 monomer, the stability is good, and experimental results show that the VLPs vaccine can 100% protect SPF chicken from IBDV virulent virus attack. Besides, the preparation method of the VP2 protein is simple, molecular sieve chromatography purification with high cost is avoided, complex processes such as ultracentrifugation are not needed, the recovery rate of two-step affinity purification is close to 60%, the purity of the purified VP2 protein is about 80%, and industrial-scale preparation of IBDV structural protein and IBDV VLPs vaccines becomes possible.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a chicken infectious bursal disease virus VP2 protein, a VP2 protein virus-like particle vaccine and a preparation method thereof. BACKGROUND

[0002] Infectious bursal disease (IBD) is an immunosuppressive infectious disease caused by infectious bursal disease virus (IBDV), which mainly destroys the bursa of young chickens. IBDV infection often leads to failure of vaccination against avian influenza and Newcastle disease, causing significant economic losses to the poultry industry. The virus belongs to the avian double-stranded RNA virus of the Birnaviridae family, has a single coat without envelope structure, and the virus particles are icosahedral symmetry with a diameter of about 60 nm. According to the pathogenicity, it can be divided into classic strain and variant strain: the classic strain will cause chicken bursa congestion, hemorrhage and acute death; the variant strain usually does not cause obvious lesions of the bursa, and has no typical clinical symptoms, but will damage the immune function of bursa lymphocytes, leading to failure of prevention and control of other diseases.

[0003] In China, vaccination is the preferred measure for preventing and controlling IBD. Virus-like particles (VLPs) are a safe and effective subunit vaccine platform, which has a similar morphology and structure to virus particles. Compared with virus particles, VLPs do not contain nucleic acids, neither infectious nor transmissible, and there is no risk of virus spread. The IBDV genome encodes five proteins (VP1-VP5), among which VP2 is the main antigenic protein, which can self-assemble into icosahedral VLPs with a diameter of about 20 nm, and has good immunogenicity, and is a safe and effective candidate for developing IBDV vaccine. However, due to the low assembly efficiency and poor stability of VLPs (especially whether the structure remains intact after emulsification is unclear), there is only IBDV VP2 subunit vaccine on the market, and no IBDV VLPs vaccine has been launched, so it is urgent to develop IBDV VLP vaccine with high assembly efficiency and good stability, in order to better immunogenicity to protect chickens from avian infectious bursal disease virus strong strain.

[0004] Escherichia coli expression system has become a widely used protein expression platform due to its low cost, short cycle, easy to scale up production and other advantages. However, the Escherichia coli expression system is prone to form insoluble inclusion bodies, which leads to poor biological activity of target protein and limits its further development and utilization. The IBDV VP2 crystal shows that its secondary structure contains a high proportion of β-sheet, which is not conducive to the soluble expression of prokaryotic system. Huang Jianfei et al. cloned VP2 gene into pSYno-1 vector with MBP fusion tag to obtain pSYno-1-VP2 prokaryotic expression vector, which can soluble express VP2 protein, but after cutting off the fusion tag, the loss of target protein after secondary purification is great, and the final concentration is only 0.1 mg / mL, which is difficult to realize large-scale application. Wang Yulong et al. expressed VP2 protein with extremely low expression amount, which can only be detected by Western blotting before ammonium sulfate enrichment, and high-purity VLPs can be obtained after further purification by molecular sieve after ammonium sulfate enrichment. SUMMARY

[0005] Based on the above, the purpose of the present application is to provide a chicken infectious bursal disease virus VP2 protein, a VP2 protein virus-like particle vaccine and a preparation method thereof, which improves the prokaryotic soluble expression of IBDV VP2 by further introducing a small ubiquitin-like fusion protein (SUMO) into an Escherichia coli cold shock expression vector, and then purifies and assembles to obtain VLPs vaccine of IBDV structural protein VP2 with good immunogenicity. The vaccine still maintains the intact VLPs structure instead of VP2 monomer after emulsification and demulsification, and can protect 100% of SPF chickens from IBDV strong virus attack after immunization.

[0006] To achieve the purpose, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides an IBDV structural protein VP2, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0008] The nucleotide sequence of the gene encoding the above-mentioned IBDV structural protein VP2 is shown in SEQ ID NO. 2.

[0009] The above-mentioned IBDV structural protein VP2 can be used for preparing IBDV vaccine. Preferably, the vaccine is a subunit vaccine. More preferably, it is a VLPs vaccine without nucleic acid, neither infectious nor transmissible, without the risk of virus spread, and with good immunogenicity.

[0010] In a second aspect, the present application provides a prokaryotic expression vector for soluble expression of the IBDV structural protein VP2 in the first aspect, which is obtained by inserting a small ubiquitin-like fusion protein SUMO after the His tag of the E. coli cold expression vector pCold II, and the nucleotide sequence of the multiple cloning site of the prokaryotic expression vector is shown in SEQ ID NO. 3.

[0011] In a third aspect, the present application provides a method for preparing the IBDV structural protein VP2 by using the prokaryotic expression vector in the second aspect, which comprises: expressing the prokaryotic expression vector by an expression system, and then purifying to obtain the IBDV structural protein VP2; and the expression system is selected from any one of an insect cell-baculovirus expression system, a mammalian expression system, a prokaryotic expression system or a yeast expression system.

[0012] Preferably, the method comprises the following steps:

[0013] (1) constructing the prokaryotic expression vector pCold II-SUMO by inserting a small ubiquitin-like fusion protein after the His tag of the E. coli cold expression vector pCold II;

[0014] (2) cloning the gene sequence encoding the IBDV structural protein VP2 into the prokaryotic expression vector pCold II-SUMO by a molecular cloning technique to obtain a recombinant expression plasmid pCold II-SUMO-IBDV-VP2;

[0015] (3) transforming the recombinant expression plasmid pCold II-SUMO-IBDV-VP2 in step (2) into a competent E. coli for culture to obtain a genetically engineered bacterium;

[0016] (4) performing fermentation culture and inducing expression on the genetically engineered bacterium to obtain the IBDV structural protein VP2 with a SUMO tag;

[0017] (5) performing affinity chromatography purification on the IBDV structural protein VP2 with the SUMO tag, and then performing SUMO enzyme cleavage and affinity chromatography purification to obtain the VP2 protein without the SUMO tag.

[0018] In a fourth aspect, the present application provides an IBDV structural protein VLPs vaccine, which is obtained by self-assembly of the IBDV structural protein VP2 prepared by the method in the third aspect in an assembly buffer.

[0019] Preferably, the assembly buffer is 50mM Tris-HCl, 500mM NaCl, and pH is 8.0.

[0020] By adopting the above technical solutions, the present application has the following beneficial effects:

[0021] Firstly, the present application is based on the sequence of the IBDV strain popular in China in recent years, and the genetic evolution analysis of the current domestic IBDV popular strain is carried out through epidemiological investigation, the gene sequence of the structural protein VP2 is optimized, and an IBDV VP2 protein is obtained, which improves the stability, immunogenicity and solubility of the prokaryotic protein; secondly, the pColdII-SUMO prokaryotic expression vector is obtained by further adding the small ubiquitin-like fusion protein to the basis of the E. coli cold shock expression vector pColdII, which further improves the solubility expression efficiency of the VP2 protein, and then the concentration of the VP2 target protein with a fusion tag SUMO can reach 1.00 mg / mL by using E. coli expression and affinity chromatography purification; finally, the VP2 protein after removing the fusion tag SUMO can be assembled into VLPs vaccine in a specific solution, the Native-PAGE identification result shows that the assembly efficiency is about 100%, and after emulsification and demulsification, the VLPs structure is still maintained instead of VP2 monomer, which has good stability and better immunogenicity than VP2 monomer. The experimental results show that the antibodies produced by the VLPs vaccine immunized SPF chickens can have an agar diffusion reaction with the popular strain of chicken infectious bursal disease virus, and can protect 100% of the SPF chickens from IBDV strong virus attack. In addition, the preparation method of the VP2 protein of the present application is simple, avoids the high-cost molecular sieve chromatography purification, does not need to use complex processes such as ultracentrifugation, the recovery rate of two-step affinity purification is close to 60%, and the purity of the VP2 protein after purification is about 80%, which makes it possible to prepare IBDV structural protein and IBDV VLPs vaccine on an industrial scale. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is the construction result of the prokaryotic expression vector pColdII-SUMO-IBDV required by the present application; A is IBDV VP2 gene specific amplification; in B, lane 1 is pColdII-SUMO vector, and lane 2 is linearized pColdII-SUMO vector with restriction endonuclease XhoI and SalI; C is colony PCR identification of homologous recombination of the constructed prokaryotic expression vector pColdII-SUMO-IBDV-VP2, lane 1 is pColdII-SUMO-IBDV-VP2 positive transformation monoclonal colony, and lane 2 is DH5α monoclonal empty colony;

[0023] Figure 2 It is the SDS-PAGE identification result of the fusion small ubiquitin-like modified protein (SUMO) tag chicken infectious bursal disease virus structural protein obtained by the present application; lane 1 is whole bacteria before induction; lane 2 is supernatant after lysis of whole bacteria after induction, and the loading amount is 10 μL;

[0024] Figure 3SDS-PAGE identification result of the SUMO-tagged chicken infectious bursal disease virus structural protein purified by His-tag affinity chromatography obtained in the application after SUMO enzyme digestion; wherein, M is molecular weight Marker; 1 is the SUMO-tagged chicken infectious bursal disease virus structural protein after affinity chromatography purification; 2 is the chicken infectious bursal disease virus protein without SUMO tag after SUMO enzyme digestion, the loading amount is 10 μL;

[0025] Figure 4 Transmission electron microscope observation result of the IBDV protein self-assembled in the application;

[0026] Figure 5 Assembly efficiency result chart of the IBDV VP2 assembled into VLPs in a specific buffer; A is the efficiency of VP2 protein assembled into VLPs detected by Native Page; B is the particle size of VLPs detected by zeta particle size instrument;

[0027] Figure 6 Native Page detection result of the VLPs sample before and after emulsification by oil adjuvant, lane 1: before demulsification, lane 2: after demulsification. DETAILED DESCRIPTION

[0028] The above scheme is further described in combination with specific examples. It should be understood that these examples are used to illustrate the application and do not limit the scope of the application. The implementation conditions used in the examples can be further adjusted according to the specific manufacturer's conditions, and the implementation conditions not mentioned are usually the conditions in the conventional experiments.

[0029] The experiments described in the following examples obtained the biosafety license:

[0030] According to the relevant requirements of biosafety level 3 laboratory (BSL-3) and foot-and-mouth disease related biosafety, the Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences reported to the biosafety committee of Lanzhou Veterinary Research Institute, the experimental animal ethics committee, the biosafety committee of Chinese Academy of Agricultural Sciences, the experimental animal ethics committee of Lanzhou Veterinary Research Institute, and the biosafety committee of Lanzhou Veterinary Research Institute, obtained the license for carrying out IBDV pathogen and animal research, and has been recorded in the Ministry of Agriculture and Rural Affairs, which meets the requirements of national biosafety level.

[0031] Explanation and interpretation of related terms in the application:

[0032] The term "prokaryotic expression system" refers to a system consisting of E. coli (strain) and a vector, wherein the E. coli (strain) is derived from commercially available, by way of example but not limited to: BL21(DE3), BL21(DE3)pLysS, B834(DE3), BLR(DE3), JM109, XL1 Blue, ER2566, Rosetta, GI698, preferably Rosetta(DE3).

[0033] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted, and wherein the protein is expressed. A vector can be used to transform, transduce or transfect a host cell, so that the genetic material elements carried by the vector are expressed in the host cell. By way of example, vectors include: plasmids; bacteriophages; cosmids; and the like.

[0034] The term "vaccine" refers to a biological preparation that provides a protective response in an animal, wherein the vaccine has been delivered and does not cause serious illness. The vaccine of the present application is a genetically engineered IBDV structural protein VP2 VLPs vaccine.

[0035] The vaccine of the present application, further optionally comprises one or more adjuvants, excipients, carriers and diluents. The adjuvant can be any suitable adjuvant, including biochemical class immunological adjuvants such as thymic peptides, transfer factor, interleukins and the like. The preferred adjuvant can be Freund's adjuvant.

[0036] The vaccine of the present application can also be used in combination vaccines, such as in combination with vaccines for Newcastle disease, infectious bronchitis, viral arthritis and the like, to prepare bivalent, trivalent and the like. The viral subunit vaccine of the present application is preferably administered to the animal by subcutaneous injection in the neck. The vaccine can be administered in a single dose, or can be administered in a "prime-boost" manner, to stimulate the body to produce higher levels of antibodies or to extend the period of protection. For example, a susceptible animal can be primed and boosted after a period of time (e.g. 14 days) after the first immunization.

[0037] Example 1: Preparation of IBDV structural protein VP2

[0038] The nvIBDV gene sequence is selected in the present application, and the sequence at positions 943-975 is replaced with the sequence of the vvIBDV-SD10LY01-2014 strain, to obtain a gene encoding the IBDV structural protein VP2, and the nucleotide sequence is shown in SEQ ID NO. 2.

[0039] The amino acid sequence of the IBDV structural protein VP2 is shown in SEQ ID NO. 1.

[0040] 1. Construction of IBDV structural protein recombinant expression vector

[0041] (1) Constructing prokaryotic expression vector pCold II-SUMO containing small ubiquitin-like modifier (SUMO) tag, that is, cloning the SUMO gene fragment synthesized by Xi'an Qikexizixi Biological Technology Co., Ltd. into pCold II prokaryotic expression vector by molecular cloning technology;

[0042] (2) Cloning the VP2 fusion gene fragment synthesized by Xi'an Qikexizixi Biological Technology Co., Ltd. into pCold II-SUMO vector by molecular cloning technology, obtaining the recombinant pCold II-SUMO-IBDV-VP2 prokaryotic expression vector after sequence identification, and the vector construction process is as shown in Figure 1 .

[0043] (3) Transforming the above-mentioned pCold II-SUMO-IBDV-VP2 prokaryotic expression vector into competent E. coli BL21 (Rosseta), coating on solid LB medium resistant to ampicillin, and incubating at 37℃ for 10-12 h until single colonies are clear. Picking single colonies into 4 mL liquid LB medium containing ampicillin, and incubating at 37℃, 220 r / min for 12 h, and taking 1 mL bacterial liquid to be stored at -80℃.

[0044] 2. Prokaryotic expression of IBDV structural protein VP2

[0045] (1) Taking E. coli strains with the recombinant expression vector pCold II-SUMO-IBDV-VP2 from the -80℃ refrigerator, and inoculating into 10 mL LB liquid medium resistant to ampicillin, 220 r / m, 37℃, and culturing for about 12 h, and then transferring into 1 L LB liquid medium, and culturing at 37℃, and adding IPTG with a final concentration of 1 mM when the OD value reaches 0.6-0.8, and inducing expression at 16℃ for 12 h. 600

[0046] The SDS-PAGE identification result of the fusion small ubiquitin-like modifier (SUMO) tag IBDV structural protein obtained in the application is as shown in Figure 2 , wherein M is molecular weight Marker, 1 is whole bacteria before induction, and 2 is supernatant after whole bacteria lysis after induction, loading, 10 μL / well. The experimental results show that the above chicken infectious bursal disease virus structural protein with SUMO tag can be solubly co-expressed in E. coli.

[0047] (2) Correcting the pH electrode of the fermentation tank (Germany Sartorius CT5-2 fermentation tank), preparing 4 L medium, sterilizing at 121℃ for 30 min, correcting the dissolved oxygen electrode, and taking the un-aerated time after sterilization as zero point, and taking the initial stirring speed of 100 r / m after aeration before inoculation during fermentation as 100%.

[0048] ​(3) The next day, 400 mL of seed liquid was added to the fermenter, the temperature was 37°C, the pH value was 7.4, the stirring speed and the aeration rate were manually adjusted to maintain the dissolved oxygen above 40%. The glucose was fed at a rate of 30 mL / hour. The rotation speed was adjusted to control the dissolved oxygen in the fermenter at 30%. When the bacterial concentration reached OD 600 When the OD value reached 15, the culture temperature was reduced to 16°C, 1 mM IPTG was added to induce the culture for 15 hours. The final bacterial liquid concentration was OD 600 The fermentation was stopped when the OD value reached about 45, and the bacterial cells were collected by centrifugation, about 300 g.

[0049] 3. Affinity chromatography purification of IBDV structural protein with SUMO tag

[0050] The bacterial cells were resuspended at a ratio of 1 g of bacterial cells to 10 mL of equilibration buffer (20 mM Tris, 40 mM imidazole, 500 mM NaCl, pH 8.0), and the bacterial cells were broken by a homogenizer at a pressure of 700 bar for 2 times. The supernatant was collected by centrifugation at 30,000 g for 1 hour, and the target protein VP2 was detected by 12% SDS-PAGE gel electrophoresis. The supernatant was filtered with a 0.45 μm pore size filter, and then purified with a nickel affinity column (HisTrap FF, GE Healthcare Life Sciences).

[0051] Equilibration buffer: 20 mM Tris, 500 mM NaCl, 40 mM imidazole, pH 8.0;

[0052] Elution buffer: 20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 8.0;

[0053] Dialysis buffer: 20 mM Tris, 500 mM NaCl, 33 mM imidazole, pH 8.0;

[0054] Assembly buffer: 20 mM Tris-HCl, 500 mM NaCl, 0 mM imidazole, pH 8.0.

[0055] The sample was 1 L of E. coli cell supernatant after filtration with a 0.45 μm pore size filter and broken by a homogenizer.

[0056] The purification procedure was as follows: the protein was purified using a His medium preloaded column, the sample was loaded at a flow rate of 10 mL / min, after the loading was completed, the equilibration buffer was used to wash away the impurities, the elution buffer was used to elute and collect the target protein.

[0057] Take 100 μL of the sample purified by the method described in this embodiment, add 20 μL of 5× Loading Buffer and mix well. After incubating in a metal bath at 100℃ for 10 min, take 10 μL and electrophoresis it on a 12% SDS-PAGE gel. Subsequently, use Coomassie Brilliant Blue staining to visualize the electrophoretic bands. The obtained SUMO-tagged VP2 protein was digested with SUMOylase, and the SDS-PAGE identification results are as follows: Figure 3 As shown, M is the molecular weight marker, 1 is the SUMO-tagged VP2 protein purified by affinity chromatography, and 2 is the SUMO-untagged VP2 protein after SUMO enzyme digestion. The loading volume was 10 μL. SDS-PAGE identification results showed that the purification process yielded the fusion target protein of the expected size. Gray-scale analysis of the SDS-PAGE identification results showed that the purity of the purified VP2 protein was approximately 80%. The concentration of the purified SUMO-VP2 protein was determined to be approximately 1.00 mg / mL using a BCA protein quantification kit.

[0058] 4. Affinity chromatography purification of VP2 protein with SUMO tag removed

[0059] Take the VP2 protein elution sample from step 3, dialyze it with dialysate until the imidazole concentration in the buffer is 33 mM, then digest it with SUMO enzyme at 4°C for 12 h. Flow through a nickel column (HisTrap FF, GE Healthcare Life Sciences) and collect the flow-through. The SUMO tag binds to the nickel column, while VP2 protein without the SUMO tag remains in the flow-through.

[0060] Take 100 μL of the VP2 protein sample purified by the method described in this embodiment, add 20 μL of 5× Loading Buffer, mix well, and incubate at 100℃ for 10 min. Then, take 5 μL of each sample and perform electrophoresis on a 12% SDS-PAGE gel. Subsequently, use Coomassie Brilliant Blue staining to visualize the electrophoretic bands. The SDS-PAGE results after SUMOylase digestion are as follows... Figure 3 As shown, M is the molecular weight marker, 1 is the purified chicken infectious bursal virus structural protein VP2 with fusion tag, with a loading volume of 10 μL; 2 is the purified chicken infectious bursal virus structural protein VP2 without fusion tag, with a loading volume of 10 μL; SDS-PAGE identification results show that the target protein without fusion tag of the expected size was obtained after purification.

[0061] 5. Methods for IBDV structural proteins to self-assemble into VLPs

[0062] Collect the flow-through solution containing the structural protein VP2 in Step 4 above in the assembly solution (20 mM Tris-HCl, 500 mM NaCl, pH 8.0), assemble overnight at 4°C, and observe the self-assembly results of the IBDV structural protein by transmission electron microscopy using a FEI transmission electron microscope. Take 10 μL of the self-assembled protein solution, drop it onto a 200-mesh ultra-thin carbon film copper grid purchased from Zhongjing Keyi, stain it with 1% phosphotungstic acid for 20 s, and observe under the electron microscope after drying. The results are as Figure 4 shown. A large number of particles with a radius of about 20 nm can be observed by transmission electron microscopy. They are uniform in size and present a hollow morphology, which is consistent with the reported virus-like particle morphology.

[0063] The low assembly efficiency of VLPs is an important factor limiting their wide application in the vaccine field. In this example, through Native-Page and zeta particle size detection, it was found that the assembly efficiency of the IBDV VP2 protein described in this invention to form VLPs in a specific buffer is about 100% (as Figure 5 shown).

[0064] 6. Emulsification of VLPs

[0065] Emulsification is a conventional method to enhance the immunogenicity of vaccines. The method is as follows: Dilute the target VLPs protein with physiological saline to an agar diffusion titer of 1:64, 1:32, and 1:16 (the corresponding agar diffusion titers after emulsification are 1:32, 1:16, and 1:8). Weigh the protein and adjuvant at a mass ratio of adjuvant:protein of 7:3, stir with a high-shear emulsifier (brand FlUKO, model FA25model) at 10000 rpm, and add the antigen to the adjuvant while emulsifying. After all the antigen has been added, stir at 10000 rpm for 2 min. Increase the stirring speed to 19000 rpm, and continuously stir for 15 min under ice bath. Let it stand for 24 hours. Centrifuge at 3000 rpm for 15 min, and no water separation occurs in the vaccine.

[0066] The stability of VLPs is another factor limiting their application in the vaccine field, especially whether VLPs still remain unchanged after emulsification. In this example, through Native-PAGE identification, it was found that IBDV VLPs still exist in the form of VLPs after emulsification and then demulsification, rather than VP2 monomers (as Figure 6 shown).

[0067] Example 2 Evaluation of the Immunogenicity of VLPs Vaccine

[0068] 1. Serological method

[0069] SPF chickens of 3-8 weeks old were used, 20 in total, 10 of which were injected with the vaccine in the muscle or subcutaneously in the neck, 0.25 mL per chicken, 14 days after inoculation, another 10 were not inoculated as controls, and were raised in the same group. 21 days after inoculation, blood was collected, and serum was separated, and the IBD antibody titer was detected by agar diffusion test, 9 of the immunized chickens had an agar diffusion antibody titer of ≥1:8, and all the control chickens were negative.

[0070] 2. Immunization and challenge

[0071] SPF chickens of 3-8 weeks old were used, 50 in total, 10 in each group, divided into 5 groups (G1-G5), G1, G2, and G3 were injected with the vaccine in the neck subcutaneously, 0.25 mL per chicken, with agar diffusion titers of 1:8, 1:16, and 1:32, respectively, G4 and G5 were not inoculated with the vaccine as the healthy control group and the challenge control group, respectively. 21 days after inoculation, all the immunized chickens and the challenge control chickens were challenged with IBDV BC6-85 strain virus 0.1 mL per eye (the actual virus content was ≥100 BID 50 ) per chicken. After the challenge, the chickens were observed for clinical manifestations every day, and the number of sick and dead chickens was recorded, the dead chickens were autopsied for bursa lesions, and the surviving chickens were killed by dissection 72-96 hours after the challenge, and were autopsied one by one to observe the bursa lesions. The results showed that all the immunized chickens were not sick, and the bursa had no lesions; all the control chickens were sick and had obvious bursa lesions (such as one or more of the following: striped bleeding in the pectoral muscle or leg muscle, bursa enlargement, atrophy, yellowing, and jelly-like secretions inside, etc.).

Claims

1. An IBDV structural protein VP2, characterized in that, The amino acid sequence of the IBDV structural protein VP2 is shown in SEQ ID NO.

1.

2. A gene encoding the IBDV structural protein VP2 as described in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. The application of the IBDV structural protein VP2 as described in claim 1 in the preparation of IBDV vaccines.

4. The application of the IBDV structural protein VP2 as described in claim 3 in the preparation of an IBDV vaccine, characterized in that, The vaccine in question is a subunit vaccine.

5. The application of the IBDV structural protein VP2 as described in claim 4 in the preparation of an IBDV vaccine, characterized in that, The subunit vaccine is a VLP vaccine.

6. A prokaryotic expression vector for soluble expression of the IBDV structural protein VP2 as described in claim 1, characterized in that, The prokaryotic expression vector was obtained by inserting the small ubiquitin-like fusion protein SUMO after the His tag of the Escherichia coli cold shock expression vector pColdⅡ. The nucleotide sequence of the multiple cloning site of the prokaryotic expression vector is shown in SEQ ID NO.

3.

7. The method for preparing IBDV structural protein VP2 using the prokaryotic expression vector as described in claim 6, characterized in that, The method includes: expressing the prokaryotic expression vector through an expression system, and then purifying it to obtain the IBDV structural protein VP2; the expression system is selected from any one of an insect cell-baculovirus expression system, a mammalian expression system, a prokaryotic expression system, or a yeast expression system.

8. The method for preparing IBDV structural protein VP2 as described in claim 7, characterized in that, The method includes the following steps: (1) A prokaryotic expression vector pColdⅡ-SUMO was constructed by inserting a small ubiquitin-like fusion protein after the His tag of the E. coli cold shock expression vector pColdⅡ. (2) The gene sequence encoding the IBDV structural protein VP2 was cloned into the prokaryotic expression vector pColdⅡ-SUMO using molecular cloning technology to obtain the recombinant expression plasmid pColdⅡ-SUMO-IBDV-VP2; (3) Transform the recombinant expression plasmid pColdⅡ-SUMO-IBDV-VP2 described in step (2) into competent Escherichia coli and culture it to obtain genetically engineered bacteria; (4) The genetically engineered bacteria were fermented and induced to express to obtain IBDV structural protein VP2 with SUMO tag; (5) The IBDV structural protein VP2 with SUMO tag was purified by affinity chromatography, and then digested with SUMO enzyme and purified by affinity chromatography to obtain VP2 protein without SUMO tag.

9. A vaccine containing IBDV structural protein VLPs, characterized in that, The VLPs vaccine is obtained by self-assembly of the IBDV structural protein VP2 prepared by the method of claim 7 or 8 in an assembly buffer.

10. The IBDV structural protein VLPs vaccine as described in claim 9, characterized in that, The assembly buffer solution consisted of 50 mM Tris-HCl, 500 mM NaCl, and a pH of 8.0.

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