Soluble Expression Purification And Immunogenicity Analysis Of VP3 Protein Of Bluetongue Virus Type?

Bluetongue is a non-contact infectious disease of domestic or wild ruminants caused by the Bluetongue virus(BTV).It is transmitted by Culicoides biting midges.Animals infected with BTV usually show clinical symptoms such as fever,mucosal edema,and ulcers.BTV virus is an icosahedral symmetric RNA virus with 27different serotypes.BTV consists of 7 structural proteins(VP1?VP7)and 5non-structural proteins(NS1,NS2,NS3/NS3a,NS4,NS5).The VP3 protein encoded by the L3 gene is relatively high conserved structural protein,and constitute the inner symmetric icosahedral core shell of virus particle.In this study,the VP3 protein of bluetongue type I virus was correctly expressed and purified by prokaryotic expression system to study the protective effect and specific antibody production of this protein.Three recombinant vectors,p ET-32a-VP3,p ET-28a-VP3 and p ET-28a-Nus A-VP3,were constructed.Later,these plasmids were transformed into E.coli BL21(DE3),and p ET-28a-VP3 were transformed into BL21(DE3)(Tf16).Then L-arabinose and IPTG were used to induce the expression.After analysis the result,we selected p ET-28a-VP3 as the expression plasmid and co-expression with a chaperon p Tf16 to increase the soluble recombinant protein.At last,optimized the incubate condition as follow IPTG 0.3mmol/L L-arabinose 3.5 mg/m L,for 12 hours at 20?.Under the most optimized induction conditions,the expression level of recombinant protein VP3 in the soluble form was twice as high as that in the absence of molecular chaperone.The protein was purified by nickel affinity chromatography.The purified BTV VP3 had a purity of 75%and at the concentration of 0.32 mg/m L.Two Balb/c mice were immunized with the purified VP3 recombinant protein at a dose of 30?g per mouse.After the third immunization,the serum titer was determined by ELISA,and the value was 1:1.28×10~5.In the Dot-ELISA assay,the recombinant protein VP3 could react with the mouse anti-BTV-1 polyclonal sera.The results of IFA showed that the antibody produced by immunized mice with recombinant protein VP3 could react with the VP3 protein expressed in 293T cell.In conclusion,the recombinant protein VP3 with the same conformation as eukaryotic expression system and had same immunogenicity with inactivated virus was expressed,which laid a foundation for further study on the structure and function of BTV.