The Research Of The Protein Subunit Vaccine Based On VP2 Of Chicken Infectious Bursal Disease Virus

Infectious Bursal Disease(IBD)of chicken is a highly contagious immunosuppressive disease caused by Infectious Bursal Disease Virus(IBDV).At present,traditional inactivated vaccines and live attenuated vaccines are mainly used to prevent the occurrence and treatment of the disease,but have problems such as complicated operation,strong virulence,and the continuous mutation of IBDV reduces the effect of prevention.Therefore,it is urgent to develop safe and efficient new vaccines.The popular new vaccines of IBD mainly include viral vector vaccines,DNA vaccines,virus-like particles(VLPs)and protein vaccines.Protein subunit vaccines have become the focus of research due to their high safety and low costs.The capsid protein VP2 is an important structural protein of IBDV,which contains multiple host-protected neutralizing epitopes,and it can also self-assemble to form VLPs with a diameter of 25 nm during expression.Therefore,VP2 is a key object of IBD protein subunit vaccines.It has been reported that VP2 protein has successfully expressed in Escherichia coli,Pichia pastoris,and insect cells,eg.However,VP2 protein has not only low expression,but also cannot form the correct conformation,which leads to high cost,poor immunogenicity and so on.Therefore,solving this problem is the key to the development of VP2 protein vaccines.Ferritin is a nanoparticle with high biocompatibility,low immunogenicity,and adjuvant effect.Displaying antigens on the outer surface of ferritin may promote their recognition and uptake by antigen-presenting cells,thereby inducing a stronger immune response.Therefore,this study intends to use different expression systems,combined with chicken ferritin(Chicken ferritin,CF)nanoparticle antigen presentation platform and other strategies to improve the expression level and immunogenicity of VP2-related proteins,which may be helpful for the development of the IBD subunit vaccine with high immunogenicity.The main contents are as follows:1.Heterologous expression of full-length VP2 protein: First,the full-length VP2 protein was expressed in Pichia pastoris GS115 using the p PIC9 K vector.The results showed that expressed VP2 protein was not secreted but remained in the cell.Intracellular VP2 protein was purified and observed via transmission electron microscope(TEM),and VLPs were observed in the photographs.Next,VP2 protein was expressed in E.coli BL21(DE3)using the p ET28 a vector,and the results found that the expressed VP2 protein was slightly soluble,and most of it existed in the formation of inclusion bodies.VP2 was purified from the bacterial supernatant,and the yield was only 2.16 mg/ L bacterial solution,and the VP2 protein assembly efficiency and the binding force with IBDV specific antibody are very low.2.Acquisition of VP2-CF through trans-cleavage via Intein: p ET28a-SC-In C-CF and p SJ2-MBP-3C-VP2-In N expression vectors were constructed respectively.After expressed in Escherichia coli BL21(DE3),SC-In C-CF and VP2-In N were purified and then trans-cleaved by Npu Dna E Intein.Thus VP2-CF fusion protein was successfully obtained,and the VP2 protein was displayed on the surface of CF nano particle.Through indirect ELISA,It was found that the binding force of VP2-CF to IBDV-specific antibody was 1.93 times higher than that of VP2,indicating that CF can promote the immunospecificity of VP2 protein.3.The Research about the ferritin VP2 epitope vaccine: three VP2 epitopes were selected and connected in series to form 3Es.3Es was fused to the N-terminal,Loop region and Cterminal of CF respectively(3Es-CFN,3Es-CFL,3Es-CFC).3Es-CFN,3Es-CFL,3Es-CFC were all soluble expressed in E.coli BL21(DE3),among which 3Es-CFL has the highest soluble expression level,and 3Es-CFN has the lowest expression level.After purification,all of them can be self-assembled into nanoparticles,and successfully displayed on the surface of nanoparticles.The binding ability of the three kinds of nanoparticles and IBDV-specific antibody is basically the same.Chickens immunized by 3Es-CFL began to produce antibodies on the 17 th day of immunization,and the antibody titer reached the highest on the 24 th day,which was 1:3978,which was slightly lower than the commercial live vaccine(1:4999);Chickens immunized by 3Es-CFN and 3Es-CFC produced antibodies on the 24 th day,and the titers were 1:751 and 1:436 respectively.It shows that 3Es-CFL can effectively stimulate the immune response in chickens.Among the three epitope vaccines,3Es-CFL has the highest expression level,the best assembly effect and the best immunogenicity.In conclusion,the heterologous expression of full-length VP2 protein and its immune specificity are not ideal.The fusion of VP2 protein with CF protein via trans-cleavage enhances the binding of VP2 and IBDV specific antibody,but the operation of protein fusion in vitro is complicated and the assembly efficiency is relatively low,which cannot be used for the vaccine production.However,the method of fusion expression of VP2 epitope and CF in Escherichia coli was used in this study to obtain a protein subunit vaccine with good immunogenicity.In this research,CF was used in the development of IBD vaccine for the first time,and it was confirmed that CF can enhance the immunospecificity of VP2 protein.In addition,the fusion method of tandem epitopes and CF can not only rapidly prepare polyvalent subunit vaccines,but also provide reference for the application of ferritin nanoparticles in the development of other viral protein vaccines.