α平滑肌肌动蛋白红色荧光蛋白报告基因载体的构建及其在细胞中的表达

摘要

目的 构建α平滑肌肌动蛋白(α-SMA)启动子驱动的红色荧光蛋白报告基因载体,为研究细胞内α-SMA的基因表达调控提供重要工具.方法 克隆并构建含α-SMA启动子区的红色荧光报告质粒pDsRed-SMAp;用脂质体瞬时转染上皮细胞A549,观察α-SMA启动子对转化生长因子(TGF-β1)刺激的反应.结果 酶切和DNA测序证明所构建的红色荧光蛋白报告基因载体是pDsRed-SMAp重组载体；该表达载体在上皮细胞静息状态下表达水平很低,经TGF-β1刺激后,在荧光显微镜下可看到高亮度的红色荧光.结论 成功构建了α-SMA启动子驱动的红色荧光蛋白报告质粒pDsRed-SMAp,对生理相关刺激有很好的反应,可有效地用于α-SMA基因表达调控机制的研究.%Objective To construct a red fluorescent protein reporter gene which driven by α-smooth muscle acin (α-SMA) gene promoter,in order to provide important tools for study of α-SMA gene expression regulation in cells.Methods The red fluorescent protein reporter gene plasmid pDsRed-SMAp contained mouseα-SMA gene promoter was constructed by gene recombination technique.The plasmid was transiently transfected into A549 cells to observe their response to TGF-β1 stimulation.Results The pDsRed-SMAp plasmid was constructed correctly as verified by double enzyme digestion and sequence analysis.The plasmid was lowly expressed in resting A549 cells,but the expression level increased obviously after stimulation by TGF-β1.Conclusion A red fluorescent protein reporter gene plasmid driven by α-SMA gene promoter is constructed successfully with a sensitive response to physiological stimulation.This system can provide a convenient tool for the regulation mechanism study of α-SMA gene expression.