Prokaryotic Expression and Identification on the ag85a and mpb70 Fusion Gene of Mycobacterium bovis

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-β-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.

• Periodical: Advanced Materials Research (Volumes 884-885)
• Pages: 498-502
• DOI: https://doi.org/10.4028/www.scientific.net/AMR.884-885.498
• Online since: January 2014
• Authors: Xin Liu, Chun Fang Wang, Hong Xia Ma, Yun Hang Gao, Jia Ning Guan, Jia Ming Lin, Bao Feng Luo, Bing Ai, Xiu Yun Jiang
• Keywords: ag85a Gene, Fusion Gene, mpb70 Gene, Mycobacterium bovis, Prokaryotic Expression

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