Cloning and Expression two recombinant proteins of Mycobacterium bovis

Introduction: Mycobacterium bovis is agent of bovine tuberculosis that mainly separated from bovidae. The consequences of disease are widely spread including decrease in production, early death and economic losses. Primary diagnosis based on tuberculin skin test that may have false positive. ESAT-6 and CFP-10 are important proteins that secreted in early stage of disease. Also these proteins are eliminated in process of bovine PPD (PPD-B) production. To investigate the functions of that, two genes esat-6 and CFP-10 are cloned and expressed.Material and method: Sequence of esat6 & cfp10 of Mycobacterium bovis obtained from bovilst. First, primers designed with restriction enzymes BamHI and EcoRI and after that synthetize at Macrogen co. After that, Polymerase Chain Reaction(PCR) was performed to amplify these genes. In parallel DH5α-pET23a (+) replicated and then isolated the vector. Both vector and PCR product are digested, ligation was done at 16˚C then cloned vector transform to DH5α. After being identified with sequencing, cloned vector transformed to BL21. Expressed proteins optimized and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western Blotting (WB).Results: The esat6 and cfp10 genes were amplified successfully. Both of them were cloned into expression vector. After identified by sequencing, genes were expressed. Analyzed of proteins expressed showed the molecular weight about 10 KDa and identified by WB techniqueConclusion: The esat6 and cfp10 genes were successfully cloned and expressed in E. coli.