分枝杆菌融合表达ICL-GFP穿梭质粒的构建及鉴定

目的:构建融合表达ICL-GFP的E.coli-分枝杆菌穿梭载体,在耻垢分枝杆菌(MS)中融合表达结核分枝杆菌(MTB)潜伏感染相关蛋白异柠檬酸裂合酶(ICL)及绿色荧光蛋白(GFP),为抗MTB ICL的表达及抗MTB潜伏感染的药物筛选奠定基础. 方法:采用PCR法从MTB H37Rv基因组DNA中扩增出icl基因,克隆入pcDNA3.1( ), 构建重组质粒pcDNA-icl. 用PCR法自pUV15中扩增出gfp基因,克隆入pcDNA-icl中icl基因片段的下游,构建重组质粒pCDIG. 鉴定无误后,将icl-gfp融合基因从pCDIG中亚克隆入pUV15,构建融合表达ICL-GFP的穿梭质粒pUVIG. 将pUVIG电转化MS,经潮霉素B筛选后,抽提质粒用PCR法鉴定. 培养MS的阳性转化子,在荧光显微镜下观察GFP的表达,Western-blot法检测ICL的表达并检测ICL-GFP融合蛋白的ICL活性. 结果:所克隆的icl序列出现一个碱基突变,为无义突变. gfp序列完全正确. 重组质粒pUVIG能在E.coli-MS间进行穿梭,并在MS中表达ICL-GFP融合蛋白. 该融合蛋白同时具有GFP的自发荧光功能和ICL的酶活性. 结论:成功构建能在MS中表达ICL-GFP融合蛋白的E.coli-分枝杆菌穿梭质粒.

Construction and identification of shuttle plasmid expressing ICL-GFP fusion protein in Mycobacterium

AIM: To construct the E. coli-Mycobacterium 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 pcDNA-icl. The gfp gene fragment was amplified by PCR from plasmid pUV15 and inserted into the downstream of icl gene fragment in pCDNA-icl to obtain recombinant plasmid pCDIG. The recombinant shuttle plasmid expressing ICL-GFP fusion protein was obtained by subcloning the icl-gfp fusion gene fragment from pCDIG into E.coli-Mycobacterium 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. ICL-GFP fusion protein was identified by fluorescence microscope and western-blot. 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. coli-Mycobacterium shuttle plasmid pUVIG replicated in both E.coli and M. smegmatis, and expressed ICL-GFP fusion protein in M. smegmatis. The fusion protein ICL-GFP expressed in M. smegmatis maintained both the irradiance characteristic of GFP and the enzyme activity of ICL. CONCLUSION: The E. coli-Mycobacterium shuttle plasmid is constructed successfully, which expresses ICL-GFP fusion protein in M. smegmatis and maintains the native function of both GFP and ICL.