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目的研究血管内皮细胞一氧化氮合酶(eNOS)启动子的功能和eNOS基因表达的调控机制 ,利用红色荧光蛋白构建在哺乳动物细胞中表达的人eNOS启动子全长及其不同区段DNA序列驱动的红色荧光蛋白报告基因载体 ,并对它们在哺乳动物细胞的表达规律作了观察。方法从人脐静脉内皮细胞基因组DNA中克隆eNOS启动子全长( -1～ -1600bp)基因序列(GenBandTM,AccessionNumbe:AF387340)将其构建到红色荧光蛋白载体pDsRed1 -1上 ,经PCR、酶切和DNA测序鉴定正确后命名为pDse_NOSRed。然后以重组pDseNOSRed载体为模板 ,通过DNA序列删除法将eNOS启动子的不同区段DNA序列亚克隆至红色荧光蛋白载体pDsRed1 -1上。这些区段分别是F1033 ,主要删除AP1( -1524～ -1530)、SRE1( -1379～ -1385)和AP2( -1155～ -1163)元件 ;F494 ,主要删除SSRE( -994～ -1000)、AP1( -656～ -662)和RCE( -857～ -863,-812～ -817 ,-711～ -716)元件 ;F166 ,主要删除CACCC( -366～ -370)和GATA( -226～ -231)元件 ,并保留SP1( -95～ -104)元件。所用上游引物分别为5’ -gaagatctatctgatgctgcctgtcaccttgaccctgag-3’,5’-ccagatctccgtttctttcttaaact_3’、5’_cgagatctgaggtgaaggagagaac_3’和5’_caagatctgtggagctgaggcttt_3’,均含有Bg1Ⅱ酶切位点(黑体和下线所?
Objective To study the function of eNOS promoter and the regulation of eNOS gene expression in vascular endothelial cells. The full-length human eNOS promoter expressed in mammalian cells and its different segment DNA sequences were constructed by using red fluorescent protein Driven red fluorescent protein reporter gene carrier, and their expression in mammalian cells were observed. Methods The full length (-1 ~ -1600bp) cDNA sequence of eNOS promoter was cloned from human umbilical vein endothelial cells (GenBandTM, AccessionNumbe: AF387340) and cloned into the red fluorescent protein vector pDsRed1 -1. DNA sequencing and identification of the correct named pDse_NOSRed. Then, the different segment DNA sequence of eNOS promoter was subcloned into the red fluorescent protein vector pDsRed1 -1 using the recombinant pDseNOSRed vector as a template by DNA sequence deletion method. These sections are F1033, which mainly delete AP1 (-1524 ~ -1530), SRE1 (-1379 ~ -1385) and AP2 (-1155 ~ -1163). F494 mainly delete SSRE (-994 ~ -1000) AP1 (-656 ~ -662) and RCE (-857 ~ -863, -812 ~ -817, -711 ~ -716); F166, mainly delete CACCC (-366 ~ -370) and GATA 231) components, and retain SP1 (-95 ~ -104) components. The upstream primers used were 5 ’-gaagatctatctgatgctgcctgtcaccttgaccctgag-3’, 5’-ccagatctccgtttctttcttaaact_3 ’, 5’_caagatctgaggtgaaggagagaac_3’ and 5’_caagatctgtggagctgaggcttt_3 ’, both containing the Bg1II restriction sites (blackbody and off-line).