Cloning and expression of ESAT-6 Gene of Mycobacterium bovis in Escherichia coli

摘要

The gene encoding ESAT-6 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR), the PCR product was approximately 288bp DNA segment. The PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-ESAT-6 was constructed successfully. The purified ESAT-6 gene was subcloned into the expression vector pGEX-4T-3, and the prokaryotic expression Plasmid pGEX-4T-3-ESAT-6 was constructed. Plasmid containing pGEX-4T-3-ESAT-6 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 34kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic reactivity of M. bovis. The results were expected to lay foundation for further studies on the subunit vaccine, DNA vaccine and diagnostic reagents of ESAT-6 gene in their prevention against bovine tuberculosis.