摘要
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.
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 in- hibit 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.
出处
《南开大学学报（自然科学版）》
CAS
CSCD
北大核心
2006年第5期58-62,共5页
Acta Scientiarum Naturalium Universitatis Nankaiensis
基金
天津市科委资助项目(05gfjmjc08100)
关键词
甲胎蛋白
RNA干扰
短发卡RNA
alpha-fetoproteins
RNA interference
short hairpin RNA （shRNA）
