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Construction and identification of shuttle plasmid expressing ICL-GFP fusion protein in Mycobacterium
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 1
- DOI:
- 10.3321/j.issn:1000-2790.2005.01.003
- Key Word:
- 结核分枝杆菌;异柠檬酸裂合酶;绿色荧光蛋白;融合蛋白;穿梭质粒
Abstract： 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.
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