Expression of Integrated Bluetongue Vp7vp5 Proteins In Prokaryotic System

Abstract

This study describes the expression of integrated VP7 (group specific) VP5 (Type specific) recombinant protein of BTV16. The primers were designed to amplify complete VP7 and VP5 encoding gene of Bluetongue virus serotype 16 (BTV-16). The suitable RE sites were included in the primers for pET32a insertion. Both genes were amplified by reverse transcription polymerase chain reaction (RT-PCR) using gene specific BTV16 VP5 and VP7 primers. Complete VP7 of 1109bp and VP5 gene of 1625bp was inserted into pET32a vector. Recombinant plasmid DNA was integrated into the genome of Escherichia coli (E. coli) DH5 alpha cells by CaCl2 method. Colonies were selected on LB plate containing 50μg/ml ampicillin. Selected colonies were confirmed through colony PCR and Restriction Enzyme analysis. Conformed positive recombinant BTVVP7VP5 plasmid was expressed in a prokaryotic expression system, BL21 (DE3) pLysS E. coli . The expressed protein was separated on 12% SDS PAGE, which showed 117kDa band. The actual size of integrated protein VP7+VP5 is 98kDa. The extra 20 kDa proteins are from the fusion tag of the vector comprising thioredoxin (Trx) tag and GSSG-6 His tag. The protein was confirmed by Western Blot Analysis