Expression And Immunological Analysis Of VP5 Protein Of Bluetongue Virus Type 1

Bluetongue(BT)is a non-contact infectious disease caused by Bluetongue virus(BTV).BTV is listed by the World Organisation for Animal Health as a mandatory animal infectious disease.BTV mainly infects ruminants such as sheep and cattle,and has been sporadic outbreaks in carnivores.BTV spreads through insects such as Clam and Aedes between ruminants.BTV has seven structural proteins(VP1-VP7)and four non-structural proteins(NS1,NS2,NS3/NS3a and NS4).VP2 can induce the production of neutralizing antibodies which can be enhanced by VP5.VP2 and VP5have been used for the assembly of virus-like particles and genetic engineering vaccine research.In this study,the prokaryotic expression system was utilized to express VP5of BTV1,and immunogenicity of Recombinant VP5 protein was analyzed.The prokaryotic recombinant plasmid pET-28a-VP5 was constructed and transformed into competent cells BL21(DE3).Recombinant bacteria pET-28a-VP5-BL21 was induced with IPTG.After optimizing prokaryotic expression conditions,the optimum inducement time and concentration of IPTG was determined.SDS-PAGE,Western-blot and Dot-ELISA were performed to identify the immunoreactivity of purified recombinant VP5 protein subsequently.The results showed that the molecular weight of recombinant VP5 protein was about 62kDa,which was consistent with the expectations.High expression of recombinant protein was obtained by inducing0.4mmol/L IPTG in 2×YT medium at 25°C for 6 hours.Balb/c mices were immunized with purified recombinant protein VP5 at 40?g and20?g,respectively,meanwhile a negative control was set.The titer of antibody was detected by ELISA after three immunizations in mice.The result shows that the highest titer was up to 1:2.048×10~5.VP5 antibodycan react specifically with BTV1 virus by Dot-ELISA assay.This study provides an important experimental material,which can be used to analysis the mechanism about the role of VP5.At the same time,this study laid a foundation for the development of polyvalent subunit vaccine and polypeptide vaccine.