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

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1100bv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。

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

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 1100bp 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 cytomegalovirus(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.