High-level Expression And Immunogenicity Of Infectious Bursal Disease Virus VP2 Protein

Infectious Bursal Disease(IBD)is an acute and highly contagious immunosuppressive disease caused by Infectious Bursal Disease virus(IBDV).IBDV mainly infects the chicken's central immune organ bursal which rapidly proliferates on its B lymphocytes and severely damages the function of B lymphocytes,then resulting in decreased immune function,moreover causing immunosuppression and increasing other infectious diseases to infect on chicks,thus making it immune to failure.The IBDV major structural protein VP2 contains a conformation-specific antigenic determinant that can induce neutralizing antibody production.Its induced neutralizing antibody can passively protect the host from IBDV infection and is the major protective antigen of IBDV.Due to its specificity,it has become the main protein for the preparation of a genetic engineering subunit vaccine for IBD.The current prevention of IBD is mainly through vaccination.Although many types of vaccines have been developed at present,the prevention situation is still not optimistic and it is urgent to develop new vaccines to strengthen the prevention of IBD.The purpose of this study was aimed to achieve efficient and soluble expression of VP2 protein in IBDV B87 strain in E.coli,determining the purification protocol and identifying the formation of IBDV virus-like particles(VLPs),In addition,validating the immunoprotective effect of VP2 protein.Therefore laying the foundation for the preparation of IBD subunit vaccines.1.Screening of IBDV VP2 soluble protein high-expression strain and optimization of inducedexpression conditionsThe recombinant plasmid p ET28a-VP2 inserted into IBDV B87 strain VP2 gene was transformed into different Escherichia coli genetic engineering strains,and inducing VP2 exogenous protein by IPTG induction.The high-level expression strains were screened using a rapid test strip of IBD antigen,Finally,BL21(DE3)was identified as a highly efficient expression strain of VP2 soluble protein.SDS-PAGE and Western blot analysis showed that VP2 achieved soluble expression and had good reactogenicity.The expression conditions were optimized.The results showed that at the induction temperature of 30 °C,IPTG concentration was 0.6 mmol/L,and the expression level of VP2 was the highest when the expression was induced for 8 h.Thus,this study obtained a highly efficient expression strain of VP2 protein and determined the optimal conditions for soluble expression.2.Determination of IBDV VP2 purification conditionsBased on the soluble expression of VP2 protein in BL21(DE3),the purification scheme was explored.It was finally determined that when the VP2 protein was purified by nickel-column affinity chromatography,the target protein with high purity could be recovered under the elution conditions of PBS+100 mmol/L Na Cl p H=7;the protein was purified by Q Sepharose Fast Flow anion exchange chromatography.During purification,the higher purity target protein can be recovered under the elution conditions of 50 mmol/L Tris-HCl+50 mmol/L Na Cl p H=7.In addition,by TEM and DLS analysis,it was found that the VP2 protein obtained by nickel column affinity chromatography can form VLPs structure with a size of 25 nm,and the uniformity is also good.This study shows that IBDV VP2 protein expressed by prokaryotic expression system can effectively form VLPs,providing new reference information for the development of subunit vaccines and VLP vaccines.3.SPF chicken immune evaluationThe soluble soluble VP2 protein was analyzed by agar diffusion(AGP).The results showed that the VP2 protein AGP titer can reach 1:64.The animal immunoassay protocol was determined was confirmed,and the expressed recombinant protein was filtered and transformed into a tangential flow ultrafiltration system.The 50 KD membrane filter group,the 50 KD concentrated group,the refolding group,and the disrupted protein supernatant group were set.For the experimental group,the commercial vaccine group and the PBS group were also used as a control group to immunize 2 weeks old SPF chickens.The results showed that the titer of the supernatant of the recombinant protein fragment was up to 1:16 for antiserum AGP,suggesting that the recombinant protein can act on SPFs.Chickens were challenged to protect against infection;in addition,the 50 KD concentrated group and the fragmented protein supernatant group had a good immune protective effect on SPF chickens,indicating that the prepared subunit vaccine can effectively stimulate the humoral immune response of the chicken body and be infectious.A solid foundation has been laid for the research and preparation of a subunit vaccine for genetic engineering of IBD.