关键词：启动子 白蛋白 增强子 甲胎蛋白 转录调控
【摘要】 目的 克隆人肝癌细胞特异的甲胎蛋白增强子(EAFP)—白蛋白启动子(PALB)联合转录调控序列。方法 用聚合酶链反应(PCR)和分子克隆方法，从人染色体基因组中扩增白蛋白启动子，将其融合到甲胎蛋白增强子的下游，此融合序列与白细胞介素-2(IL-2)cDNA连接，定向克隆入真核表达质粒pcDNA-3的多克隆位点区。用磷酸钙共沉淀法将质粒转染HepG2和HeLa细胞，用MTS/PMS法测定细胞培养液中IL-2的活性。结果 PCR扩增的白蛋白启动子长406bp，DNA序列分析发现含有典型的TATA box、CAAT box和GC box。与甲胎蛋白增强子融合后的片段(EAFP-PALB)长度为822bp，它能调控IL-2在HepG2细胞中的表达，平均IL-2活性为782U/106细胞，而在转染的HeLa细胞培养液中未测得IL-2活性。结论 甲胎蛋白启动子-白蛋白增强联合转录调控序列能特异地调控目的基因在肝癌细胞中的表达，这为肝癌基因治疗研究提供了一有效手段。
THE CLONING OF COMBINEDTRANSCRIPTIONAL REGULATORY SEQUENCES OF α -FETOPROTEIN ENHANCER AND ALBUMIN PROMOTER
He Ping, Ye Shenglong, Tang Zhaoyou. Liver Cancer Institute, Shanghai Medical University, Zhongshan Hospital, Shanghai200032
【Abstract】 Objective To clone the specific a-fetoprotein enhancer(EAFP)-albumin promoter(PALB) combinated transcriptional regulatory sequences ofhuman hepatoma cells. Methods An albumin promoter was amplified fromhuman genomic DNA by PCR. This promoter was fused into the down-stream of a-fetoproteinenhancer, ligated with human interleukin-2 cDNA, and then inserted into polyclonal site ofeukaryotic expression vector pcDNA-3. The recombinant plasmid was transfected into HepG2and Hela cells by calcium phosphate transfection. The activity of IL-2 in culture mediumwas measured by MTS/PMS method. Results The 406bp EAFP was confirmed byDNA sequencing that it contains typical TATA box, CAAT box and GC box. The fused EAFP-PALBcould regulate the expression of IL-2 gene in HepG2 cells, and the activity of IL-2 in theculture medium of transfected HepG2 is 782U/106 cells, but not in transfected Hela cells. Conclusion The combined transcriptional regulatory sequences of EAFP-PALB can regulate specificallythe expression of target genes in hepatoma cells.
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