绵羊Nanog启动子克隆及其表达活性的检测

Molecular cloning and expression activity detection of sheep Nanog promoter

通过比对人、鼠、兔和牛的Nanog启动子保守区设计引物,PCR扩增绵羊Nanog基因启动子序列并分析其调控位点。将其连接到去掉自身CMV启动子的pAcGFP1-N1载体上,构建一个由Nanog启动子带动的真核表达载体(SNanog-pAcGFP1-N1),即构建一个能由该启动子序列带动并产生绿色荧光蛋白(GFP)的真核表达载体,并用脂质体法转染入终末分化细胞(小鼠成纤维细胞和绵羊成纤维细胞)、胚胎干细胞和小鼠胚胎,检测其表达活性。结果显示:成功克隆出1 836bp Nanog启动子序列,通过分析发现该序列含有SP1结合位点、CAAT框、TATA框等功能区;当转染SNanog-pAcGFP1-N1重组质粒后,小鼠胚胎和小鼠胚胎干细胞中能检测到绿色荧光,且在转染72h后达到最大荧光强度,而在小鼠成纤维细胞和绵羊成纤维细胞中不能观察到GFP。结果表明:本试验成功克隆获得绵羊Nanog启动子,所构建的真核表达载体具有在多能干细胞中特异性表达活性。

In order to clone the sheep Nanog gene promoter and detect its expression activity, Nanog promoter sequence（SNanog） was amplified by specific primer designed from conserved sequence of human,rat,rabbit and cattle. SNanog was linked with pAcGFP1-N1 which was removed its CMV promoter,and eukaryotic expression vector SNanog-pAcGFP1 N1 was constructed. Terminal dif- ferentiation cell（MEF or SEF）,embryonic stem cell and mouse embryos were respectively transfected by the SNanog-pAcGFP1-N1 using liposome transfection technique. The results showed as followed：the 1 836 bp Nanog promoter sequence was successfully cloned,and contained SP1 hinding site,CAAT box,TATA box and other cis-acting elements. GFP could be detected in mouse embryonic stem cell and mouse embryos which were transfected with the recombinant plasmid fluorescence intensity was maximized after being transfected 72 h,but did not detected in mouse or sheep fibroblasts. The results showed that sheep Nanog promoter was successfully obtained and the eukaryotic expression vector SNanog-pAcGFP1-N1 had specific expression activity in pluripotent stem cells.