Construction and identification of shuttle plasmid expressing ICLª²GFP fusion protein in Mycobacterium

    LI Junª²Ming, ZHU Daoª²Yin, YI Zhengª²Jun, JIANG Shan, LUO Xuª²Dong

    Department of Microbiology, Chongqing University of Medical Sciences, Chongqing 400016, China

    ¡¾Abstract¡¿ AIM: To construct the E. coliª²Mycobacterium shuttle vector expressing Mycobacterium tuberculosis (MTB) latent infection related protein, isocitrate lyase (ICL) and green fluorescence protein (GFP) for future screening of drugs against the ICL expression and the latent infection of MTB. METHODS: The gene fragment encoding ICL was amplified by PCR from the genome of MTB H37Rv strain and inserted into plasmid pcDNA3.1(+) to obtain recombinant plasmid pcDNAª²icl. The gfp gene fragment was amplified by PCR from plasmid pUV15 and inserted into the downstream of icl gene fragment in pCDNAª²icl to obtain recombinant plasmid pCDIG. The recombinant shuttle plasmid expressing ICLª²GFP fusion protein was obtained by subcloning the iclª²gfp fusion gene fragment from pCDIG into E.coliª²Mycobacterium shuttle plasmid pUV15, named pUVIG. pUVIG was transformated into Mycobacterium smegmatis by electroporation, screened on 7H10 medium containing hygromycin B, and then identified by PCR. ICLª²GFP fusion protein was identified by fluorescence microscope and westernª²blot. The enzyme activity of ICL was assayed. RESULTS: A nonsense mutation occurred in icl sequence amplified by PCR. The amplified sequence of gfp was identical to the sequence reported in GenBank by sequence analysis. The recombinant E. coliª²Mycobacterium shuttle plasmid pUVIG replicated in both E.coli and M. smegmatis, and expressed ICLª²GFP fusion protein in M. smegmatis. The fusion protein ICLª²GFP expressed in M. smegmatis maintained both the irradiance characteristic of GFP and the enzyme activity of ICL. CONCLUSION: The E. coliª²Mycobacterium shuttle plasmid is constructed successfully, which expresses ICLª²GFP fusion protein in M. smegmatis and maintains the native function of both GFP and ICL. ......