FDFT1基因shRNA和过表达载体的构建及在牛胎儿成纤维细胞中的验证

Construction of overexpression shRNA vectors of FDFT1 gene and its verification in bovine fetal fibroblasts

构建了带有绿色荧光标记的法尼基二磷酸法尼基转移酶1（farnesyl—diphosphate farnesyltransferase1，FDFT1）基因shRNA干扰载体以及过表达载体，将其转染牛胎儿成纤维细胞（bovine fetal fibroblasts，BFF），采用荧光定量PCR和Westernblot的方法，检测FDFT1基因mRNA和蛋白的表达水平，在细胞水平验证了shRNA干扰载体的干扰效率和过表达载体的有效性。结果显示：shRNA组FDFT1-Bos-520的FDFT1基因mRNA表达水平降低至shRNANC组的40．7％（P〈0．01），蛋白质表达水平降低至shRNANC组的39．61％（P〈0．01）；而FDFT1基因pBI-CMV3-FDFT1过表达组FDFT1基因mRNA的表达水平为pBI-CMV3空载体组的137．9倍（P〈0．01），蛋白表达水平升高至空载体组的2．34倍（P〈0．01）。本试验通过荧光定量PCR和Western blot的检测方法验证了FDFT1基因过表达载体及干扰栽体转染BFF后，FDFT1基因表达量的变化，为进一步研究FDFT1基因对胆固醇代谢和脂质代谢的影响和作用机制提供了实验材料及分子依据。

FDFT1 gene （farnesyl -diphosphate farnesyltransferase 1） is an important gene in the metabolic pathway of cholesterol. To understanding the function of FDFT1 in cholesterol metabolism and lipid metabolism overexpression vectors of FDFT1 gene and shRNA vector with green fluorescent protein （GFP） were constructed transferred into bovine fetal fibroblasts （BFF） and detected the expression levels of mRNA and protein. The result showed that compared with shRNA NC group expression levels of mRNA in shRNA group were downregulated 40.7% （P〈0.01） and protein were downregulated by 39.61% （P〈0. 01）;in pBI-CMV3-FDFT1 overexpression group expression levels of mRNA were upregulated by 137.9 times more than pBI-CMV3 （P〈0.01） and protein were only 2.34 times （P〈0.01）. This study detected the mRNA and protein expression levels by RT-PCR and western blot after transfection and verified the effectiveness of shRNA and overexpression vectors of bovine FDFT1 gene and provided an molecular basis for further study of FDFT1 in cholesterol metabolism and lipid metabolism.