人端粒酶催化亚单位启动子驱动的EGFP真核表达载体的构建及在肺癌细胞的表达

Construction of eukarytic expression vector of enhanced green fluorescence protein driven by telomerase catalytic subunit promoter and its expression targeted in human lung cancer cells

目的:构建人端粒酶催化亚单位(hTERT)启动子驱动的增强绿色荧光蛋白(EGFP)真核表达载体,研究其调控EGFP在肿瘤细胞中的靶向表达。方法:从含hTERT启动子序列的重组质粒pGL3-hTERTp上,酶切获取约1100bp的启动子片段,克隆至无启动子的EGFP质粒载体pEGFP-1的多克隆位点中,构建pEGFP-hTERTp真核表达载体。用含有巨细胞病毒(CMV)启动子的质粒pEGFP-N1作为阳性对照,pEGFP-1作为阴性对照,脂质体转染法分别转染人肺癌细胞95D,NCI-H446,A2,A549,LTEP-a-2,YTMLC和人正常细胞MRC-5,荧光显微镜下观察各细胞中EGFP转录表达情况。结果:双酶切和单酶切均显示载体pEGFP-hTERTp构建成功。细胞转染结果显示:在转染了pEGFP-hTERTp质粒的肺癌细胞中EGFP都有不同程度的表达,而在MRC-5中无EGFP表达;转染pEGFP-N1的肺癌细胞和正常细胞中均可清晰地观察到EGFP强荧光表达。结论:hTERT启动子能调控EGFP在肺癌细胞中靶向性转录表达。CMV启动子调控的EGFP在肺癌细胞和正常细胞中的表达不具有特异性。hTERT启动子有可能作为肿瘤靶向性基因治疗的调控元件。

Objective：To construct an eukaryotic expression vector of enhanced green fluorescence protein（EGFP） gene driven by telomerase catalytic subunit （hTERT） gene promoter and observe the specific expression of EGFP in lung cancer cell lines. Methods ：The 1 100 bp promoter fragment was obtained by enzyme digestion from a recombinant plasmid of pGL3-hTERTp containing the hTERT promoter. The hTERT promoter was then subcloned into the upstream of the report gene EGFP of pEGFP-1 without promoter. The expression vector pEGFP-hTERTp was successfully constructed. The vector pEGFP-N1 containing eytomegalovirus（CMV） promoter was used as a positive control. The vector pEGFP-1 without promoter was used as a negative control. The vectors were transfected into human lung cancer cell lines 95D, NCI-H446, A2, A549, LTEP-a-2, YTMLC and normal MRC-5 through lipofectamine respectively. EGFP expression was detected under the fluorescence microscope. Results：pEGFP-hTERTp was confirmed by enzyme digestion with correct result. That the EGFP expression was detected in all of eight lung cancer cells transfected with pEGFP-hTERTp, but not in MRC cells. By contrast, high intensity EGFP expression was observed in both lung tumor cells and normal cells, which were transfected with pEGFP-N1. Conclusion ：The EGFP controlled by hTERT promoter can be expressed specifically in lung cancer cell lines, hTERT promoter may be used as an excellent regulation element in tumor-targeting gene therapy.