目的 构建携带绿色荧光蛋白的ABCE1基因的siRNA表达载体,通过将载体转染入小细胞肺癌细胞株NCI-H446,筛选稳定转染细胞株.方法 根据ABCE1的DNA序列设计3对siRNA引物,构建可用于筛选阳性克隆且带有绿色荧光的ABCE1基因siRNA表达载体ABCE1-pRNAT-U6.1/Neo-siRNA-1、ABCE1-PRNAT-U6.1/Neo-siRNA-2及ABCE1-pRNAT-U6.1/Neo-siRNA-N,采用FUGENE-HD法将载体转染入NCI-H446细胞中,并以G418进行阳性克隆筛选,筛选ABCE1基因沉默的稳定转染细胞系.结果 经酶切和DNA测序验证,证实ABCE1-pRNAT-U6.1/Neo-siRNA表达栽体构建成功.将ABCE1基因siRNA表达载体成功转染小细胞肺癌细胞NC1-H446后,小细胞肺癌细胞内可见绿色荧光,通过G418筛选,获得了ABCE1基因沉默的小细胞肺癌细胞系.该细胞系的ABCEl水平明显低于未转染siRNA的小细胞肺癌细胞.结论 成功的构建了ABCE1基因siRNA表达载体ABCE1-pRNAT-U6.1/Neo-siRNA.筛选出ABCE1基因沉默的稳定转染小细胞肺癌细胞系,为今后应用ABCE1基因siRNA表达载体研究ABCE1基因的作用机制提供实验基础.%Objective To construct siRNA expression vector of ABCE1,which carry green fluorescent protein gene,and transfect the vector into small cell lung cancer cell line NCI-H446 then screen positive clones.Methods According to the sequence of ABCE1 in GenBank, three target sequences were designed and synthesized for construction of siRNA vector ABCE1- pRNAT-U6.1/Neo-siRNA-1,ABCE1- pRNAT- U6.1/Neo-siRNA-2,ABCE1-pRNAT-U6.1/Neo-siRNA-N.The new vectors carry green fluorescent gene and can be used for screening positive clones.Then we transfected siRNA vector into NCI-H446 cells,using G418 (400 μg/ml) for positive screening,then screened ABCE1 gene silencing stable transfection cell lines.Results By restriction endonuclease and sequencing,eukaryotic expression plasmid of ABCE1 pRNAT-U6.1 /Neo-siRNA were proved to be successfully constructed.After ABCE1-pRNAT-U6.1/Neo-siRNA was transfected into small cell lung cancer cells successfully,green fluorescence could be seen in those cells.Through G418 screening,we got ABCE1 gene silencing stable transection cell lines of small cell lung cancer,the ABCE1 level of the cell lines was less than that of the untransfected cell lines.Conclusion The ABCE1-pRNAT-U6.1/Neo-siRNA expression vector have been successfully constructed.We got ABCE1 gene silencing stable transfection cell lines of small cell lung cancer successfully.Our results may provide some ideas for the gene therapy in small cell lung cancer in future.
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