Fusion, Expression and Identification on the mpb51 and mpb63 Genes of Mycobacterium bovis

Abstract:

The DNA fragments of mpb51 and mpb63 were fused by splicing by overlapping extension(SOE) polymerase chain reaction(PCR),and the fusion gene mpb51-63 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-51-63. pMD-51-63 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified mpb51-63 fusion gene was subcloned into the expression vector pET28a(+),and the prokaryotic expression vector pET-51-63 was constructed. Plasmid containing pET-51-63 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 43ku 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 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 subunit vaccine, DNA vaccine and diagnostic reagents against bovine tuberculosis.

• Periodical: Applied Mechanics and Materials (Volume 319)
• Pages: 140-145
• DOI: https://doi.org/10.4028/www.scientific.net/AMM.319.140
• Online since: May 2013
• Authors: Yun Hang Gao, Chun Fang Wang, Xiu Yun Jiang, Hong Xia Ma, Da Ming Gao, Yan Ru Zheng, Jia Ning Guan, Jia Ming Lin, Shuang Hou, Lie Liu, Bing Jie Li, Lu Lu Li, Fei Ren
• Keywords: Cloning, Fusion Gene, Mycobacterium bovis, Prokaryotic Expression

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