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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.
AFP is an important embryonic stage and hepatocellular carcinoma related protein. In order to further study its role in the proliferation of hepatocellular carcinoma cells, AFP gene shRNA expression plasmid was constructed, and a stably transfected hepatoma cell clone . The shRNA was designed by bioinformatics method. The expression plasmids were constructed by restriction enzyme digestion and antibiotic screening. Plasmids were verified by restriction enzyme digestion, agarose gel electrophoresis and sequencing. Lipofectin was transfected into SMMC-C -7721 cells were selected and the stably transfected cell clones were screened by Puromycin in the presence of high-specific shRNA design results. ShRNA expression plasmids targeting human AFP gene were constructed and cell clones stably transfected with the plasmids were obtained.