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目的 :为了研究人血管内皮细胞一氧化氮合酶 (eNOS)启动子的功能 ,构建在哺乳动物细胞中表达的人eNOS启动子不同区段驱动的红色荧光蛋白报告基因载体。方法 :从重组pDseNOSRed载体上将eNOS启动子的不同长度DNA序列亚克隆至红色荧光蛋白载体pDsRed1 - 1上 ,经PCR、酶切和DNA测序鉴定 ,将重组载体pDsF1 0 33Red ,pDsF494Red和pDsF1 66Red转染NIH3T3细胞 ,在倒置荧光显微镜下观察它们在细胞内的表达情况。结果 :PCR、酶切和DNA测序结果均表明重组载体pDsF1 0 33Red ,pDsF494Red和pDsF1 66Red的构建正确 ,这些载体能在NIH3T3细胞中有效表达。由eNOS启动子不同区段驱动表达的 95 %以上的红色荧光蛋白均匀分布于整个细胞 ,转染后 48- 60h开始出现 ,96 - 1 4 4h为红色荧光蛋白 (RFP)的表达高峰 ,RFP的荧光在 1 4 4h最强 ,1 68h后红色荧光逐渐消退 ,2 1d后仍有很少量红色荧光残留。RFP的表达量和荧光强度明显低于强启动子pCMVIE驱动的RFP。结论 :成功构建了eNOS启动子不同区段驱动的红色荧光蛋白报告基因载体 ,它们能在哺乳动物细胞中有效表达 ,静息状态下呈现弱转录活性 ,为研究人eNOS启动子不同区及其顺式调控元件的作用提供了实用而又方便的工具
AIM: To investigate the function of nitric oxide synthase (eNOS) promoter in human vascular endothelial cells, a red fluorescent protein reporter vector driven by different segments of human eNOS promoter expressed in mammalian cells was constructed. Methods: Different length DNA sequences of eNOS promoter were subcloned into the red fluorescent protein vector pDsRed1 - 1 from recombinant pDseNOSRed vector. The recombinant vectors pDsF1 033Red, pDsF494Red and pDsF1 66Red were subcloned into pDsRed1 - 1 by PCR, restriction enzyme digestion and DNA sequencing. NIH3T3 cells were stained and their expression in the cells was observed under an inverted fluorescence microscope. Results: The results of PCR, restriction enzyme digestion and DNA sequencing indicated that the recombinant vectors pDsF1 0 33Red, pDsF494Red and pDsF1 66Red were constructed correctly. These vectors can be efficiently expressed in NIH3T3 cells. More than 95% of red fluorescent protein driven by different segments of eNOS promoter distributed evenly throughout the whole cell, 48- 60h after transfection began to appear, 96- 144h was the expression peak of red fluorescent protein (RFP), RFP Fluorescence was the strongest in 114h, red fluorescence gradually subsided after 68h, and there was still a small amount of red fluorescence residue after 21d. RFP expression and fluorescence intensity was significantly lower than the strong promoter pCMVIE-driven RFP. CONCLUSION: The red fluorescent protein reporter gene vectors driven by different segments of eNOS promoter were successfully constructed, which can be efficiently expressed in mammalian cells and showed weak transcriptional activity in resting state. In order to study the different regions of eNOS promoter and its cis The role of regulatory elements provides a practical and convenient tool