乳铁蛋白基因启动子元件调控效应分析

本试验先后进行2轮载体构建及转染试验,对牛乳铁蛋白基因启动子元件调控效应进行了分析。首先进行第1轮载体构建及转染试验：采用PCR方法,利用4对引物从牛乳铁蛋白基因5′调控区-1 799向3′端依次缺失500bp进行调控序列扩增,将获得的扩增片段替换pEGFP-N1的CMV启动子,构建了4个重组载体,将重组载体转染牛乳腺上皮细胞（BMEC）,经筛选获得稳定的单克隆细胞,测定并比较各组细胞的荧光强度。在以上试验基础上,为进一步精确确定牛乳铁蛋白基因启动子顺式调控元件序列,进行第2轮载体构建及转染试验,方法同第1轮。结果表明,牛乳铁蛋白基因上游调控序列-1 323～-1 372存在负调控元件,-1 372～-1 560存在正调控元件。

Regulation effect analysis of lactoferrin gene promoter elements

Two rounds vector reconstruction and stably transfection experiments were carried out to analyse the regulation effect of bovine lactoferrin gene promoter elements.In the first round,adopted PCR,using 4 pairs of primers,500 bp was deleted in turn to amplify regulatory sequence from bovine lactoferrin gene 5 ′ flanking region-1 799 to 3′.pEGFP-N1 CMV promoter was replaced by the amplified fragment and four recombinant vectors were constructed and then transfected into bovine mammary epithelial cells(BMEC).By screening,we obtained stable monoclonal cells to test and compare the fluorescence intensity of cells in each group.On the basis of the above experiment,in order to further locate the position of bovine lactoferrin gene promoter cis-element sequence in a smaller range,we carried out a second round vector reconstruction and stably transfection experiments using similar approach to the first round.The results showed that bovine lactoferrin gene upstream regulatory sequence-1 323——1 372 has negative regulatory elements;-1 372——1 560 has pasitive regulatory elements.