AFP基因shRNA表达质粒稳定转染肝癌细胞克隆的构建

甲胎蛋白(AFP)是一种重要的胚胎期及肝癌相关蛋白,为了深入研究其在肝癌细胞增殖中的作用,构建了针对AFP基因的shRNA表达质粒,拟建立可稳定转染的肝癌细胞克隆;利用生物信息学方法设计shRNA,经酶切连接和抗生素筛选构建表达质粒;通过酶切、琼脂糖凝胶电泳及测序对质粒加以验证;优化转染条件后采用脂染法转染肝癌SMM C-7721细胞,利用Purom yc in筛选稳定转染的细胞克隆.成功获得高特异的shRNA设计结果,构建了针对人类AFP基因的shRNA表达质粒,并获得该质粒稳定转染的细胞克隆.

Stable Suppression of AFP Gel Expression by RNAi in SMMC-7721 Cells

Although the serum-marker capacity of alpha-fetoprotein(AFP) has long been exploited in the diagnosis of human hepatocellular carcinoma,less is known of the biological activities of this oncofetal protein during cancinoma cell proliferation.For proof of concept we developed a vector-derived short hairpin RNA(shRNA)expression system that is based on the alpha-fetoprotein mRNA and eukaryotic RNA polymerase III promoters(U6) to selectively inhibit expression of alpha-fetoprotein in human hepatocellular carcinoma cell.After identified by sequencing and gel electrophoresis,the shRNA expressing vector was used to transfect SMMC-7721 cells by lipofetin.SMMC-7721 cell clones, which the vector stably integrated into,were successfully selected by puromycin screening.This means that our vector-derived shRNA expression system can be used to study further alpha-fetoprotein's function in cell proliferation of hepatocellular carcinoma.