雌二醇依赖性的细胞表达载体的研究  

Construction of Expression Vectors for Detection of β Estradiol

作　　者：杨桂香[1] 彭险峰[1] 曾振灵[1] 陈杖榴[1] 

机构地区：[1]华南农业大学兽医学院/广东省兽药研制与安全评价重点实验室,广州510642

出　　处：《中国农业科学》2005年第5期1046-1051,共6页Scientia Agricultura Sinica

摘　　要：根据雌激素类化合物在机体内的作用机理,以增强型绿色荧光蛋白(EGFP)基因为报告基因构建了4个启动子不同的表达载体(pERE-SV-EGFP,pERE-TA-EGFP,pERE-CMV-EGFP,pERE-TK-EGFP)。以这些表达载体瞬时转染MCF-7细胞,报告基因的基础表达与启动子的强度相关。以17β-雌二醇(E2,10-9mol·L-1)诱导表达载体瞬时转染的MCF-7细胞,发现在表达载体pERE-TA-EGFP、pERE-TK-EGFP、pERE-SV-EGFP转染的MCF-7细胞中,E2能诱导2倍以上的报告基因的表达。这表明在这些瞬时表达系统中报告基因的表达是雌激素依赖性的,因此这些表达载体可用于稳定转染以建立稳定表达分析系统。The estrogenic endocrine disrupting compounds whether from environment and food chains or from chemicals illegally used in animal production pose a serious threat to the safety of animal derived products. Because of the huge diversities of these chemical structures, it is necessary to establish high-throughput screening and detecting methods to screen them. In this study, 4 recombination expression vectors with enhanced GFP as reporter gene were constructed. These constructs, namely, pERE-SV-EGFP, pERE-TA-EGFP, pERE-CMV-EGFP, pERE-TK-EGFP, only differ in promoters. Then the four expression vectors were transiently transfected into MCF-7 cells with liposome transfection reagent Tfx-20. The constitutive expressions of EGFP in transient transfected cells were related to the activity of the promoters in the expression vectors. And the fluorescence intensity treated by 17- βestradiol (10-9 mol·L-1) was two-fold higher than that treated by DMSO (control) in cells transiently transfected with pERE-SV-EGFP, pERE-TA-EGFP and pERE-TK-EGFP. These results indicated that the expression of reporter gene in ERE-SV-EGFP, pERE-TA-EGFP and pERE-TK-EGFP was estrogenic chemical dependent, and could be used for stable transfection to establish stable cell lines for estogenics screen.

关 键 词：雌二醇 依赖性 表达载体 报告基因 瞬时表达系统 增强型绿色荧光蛋白 药物残留 动物性食品 

分 类 号：Q782[生物学—分子生物学]