Prokaryotic expression and identification on the ag85a and mpb70 fusion gene of Mycobacterium bovis
BIOTECHNOLOGY, CHEMICAL AND MATERIALS ENGINEERING III, PTS 1 AND 2(2014)
Abstract
Based on splicing by overlapping extension(SOE) polymerase chain reaction(PCR),the ag85a and mpb70 were amplified and the fusion gene ag85a-mpb70 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-85a-70. pMD-85a-70 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified ag85a-mpb70 fusion gene was subcloned into the expression vector pET28a(+),and the prokaryotic expression vector pET-85a-70 was constructed Plasmid containing pET-85a-70 was transformed into competence Escherichia coli BL21(DE3).The bacterium was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 49 KDa fusion protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that Ag85A-MPB70 was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of novel vaccine against bovine tuberculosis.
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Key words
Mycobacterium bovis,ag85a gene,mpb70 gene,Fusion gene,Prokaryotic expression
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