人血管内皮生长因子和端粒酶催化亚基复合调控序列的构建及转录活性的研究

Construction of Multiplicity Transcriptional Regulatory Elements of Vascular Endotrelial Growth Factor Enhancer and Telomerase Catatytic Subunit Core Promoter and Research of Transcription Activity

以人肝癌细胞基因组DNA为模板,应用PCR方法扩增VEGF增强子和hTERT核心启动子序列,该序列与NCBI数据库参考序列相比,同源性达到99%以上。将VEGF增强子和hTERT核心启动子定向克隆到pBluescriptⅡSK载体中,构建VEGF增强子和hTERT核心启动子复合调控序列。酶切去除表达载体pECFP-C1的启动子CMV,并将所制备的复合调控序列连接到CMV位置,构建含该复合调控序列并可表达绿色荧光蛋白报告基因的重组质粒,转染人肺癌细胞和正常人肝细胞。结果显示,在人肺癌细胞中观察到绿色荧光,而在人肝细胞中没有检测到。由此说明,具有肿瘤特异性的VEGF增强子和hTERT核心启动子复合转录调控序列可以驱动绿色荧光蛋白基因在人肺癌细胞中表达,但不能驱动该蛋白在正常人肝细胞中表达。

Vascular endothelial growth factor enhancer and telomerase catalytic subunit core promoter purpose fragments were amplified by genome from human liver cancer cell. Sequence analysis showed that the fragment obtained had 99 % homology with previously reported fragments in Genehank. The purpose fragments were cloned into pBluescript Ⅱ SK vector directly, getting a multiplicity transcriptional regulatory sequence （VEGF-hTERT）. The promoter CMV of green fluorescent protein expression vector PECFP-C1 was replaced by the obtained multiplicity transcriptional regulatory sequence to construct the recombinant plasmid PECFP-CL-VEGF-hTERT. The constructed plasmid was transfected into the human lung cancer cell A549 and normal human liver cell lines. Then ECFP expression in lung cell line A549 was observed but not in normal liver cell line.