人内皮细胞一氧化氮合酶红色荧光蛋白报告基因载体的构建与表达

Construction and expression of red fluorescent protein reporter gene vector containing human eNOS promoter

目的 研究血管壁切应力诱导的人血管内皮细胞内皮细胞一氧化氮合酶 (eNOS)基因表达的调控机制 ,构建在哺乳动物细胞中表达的eNOS红色荧光蛋白报告基因载体。方法 从人脐静脉内皮细胞基因组DNA中克隆eNOS启动子 ,将其构建到红色荧光蛋白载体pDsRed1 1上 ,经PCR、酶切和DNA测序鉴定后将重组载体转染 2 93细胞 ,在荧光显微镜下观察其在细胞内的表达和分布情况。结果 PCR、酶切和DNA测序结果均表明重组载体pDseNOSRed的构建正确 ,该载体能在 2 93细胞中高效表达。由eNOS启动子驱动表达的红色荧光蛋白大部分均匀分布于整个细胞中 ,转染后 1 2h开始出现 ,36～ 48h为表达高峰 ,1 2 0h后红色荧光几乎全部消失。结论 成功构建了eNOS红色荧光蛋白报告基因载体 ,该载体能在哺乳动物细胞中高效表达 。

Objective To construct the red fluorescent protein reporter gene vector containing human eNOS promoter sequence to study the mechanism of regulating the expression of eNOS gene by vessel wall shear stress. Methods The genomic DNA of endothelial cells from fetal umbilical vein was drawn. The gene sequence of eNOS promoter gene therein was cloned by PCR technique and constructed into the red fluorescent protein vector, pDsRed 1. The reconbinant vector, pDseNOSRed was then transfected into 293 cells, human fetal renal epithelial cells. Blank vector, pDsRed 1, was transfected into 293 cells as controls. The expression and distribution of the reporter gene were observed by fluorescent microscopy. Results PCR and double restriction enzyme digestion showed that the recombinant vector, pDseNOSRed, was constructed correctly. This vector was highly expressed in the 293 cells. Expression of red fluorescence, evenly distributed in whole cells, occurred since 12 hours after transfection, reached the peak concentration 36~48 h after transfection, and dissapeared almost completely 120 h after. No red fluorescence was observed in the control cells. Conclusion A red fluorescent protein reporter gene vector containing human eNOS promoter sequence and expressed highly in mammalian cells has been constructed successfully, thus providing an important and convenient tool to study the mechanism mechanism of regulating the expression of eNOS gene by vessel wall shear stress.