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RNA干扰CA916798基因真核表达载体的构建与鉴定

, PP. 23-26

Keywords: CA,RNA干扰,真核表达载体,构建,转染

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Abstract:

目的构建CA916798基因RNA干扰(RNAi)的真核细胞表达载体。方法以CA916798为靶基因,以pGenSil-l质粒为载体,设计构建重组体,根据GenBank数据库提供的CA916798基因核苷酸序列,按照Tuschl设计原则,选择设计2条带发夹结构的核苷酸序列,克隆到空载体pGenSil-l中,转化DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析。用脂质体2000将重组质粒转染A549/CDDP细胞并用G418筛选,MTT法测定药物敏感性并绘制细胞增殖曲线。结果构建针对CA916798的pRNAi-CA916798shRNA,经限制性内切酶酶切、PCR和DNA测序证实与设计完全一致;在荧光显微镜下观察到A549/CDDP细胞表达绿色荧光蛋白(GFP)证实重组质粒己转染入细胞并用G418剔除了未转染质粒的细胞,1.0μg/ml顺铂作用后pRNAi-CA916798shRNA转染组细胞增殖受到明显抑制(P<0.01)。结论成功构建CA916798基因的shRNA表达载体,并筛选出转染了CA916798shRNA的A549/CDDP细胞。

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