靶向DAL-1基因的shRNA表达载体的构建与鉴定

Construction and identification of specific shRNA interference plasmid vector targeted to DAL-1 gene mRNA

目的:建立DAL-1稳定抑制的非小细胞肺癌(NSCLC)细胞实验模型.方法:构建靶向DAL-1的shRNA表达载体转染NSCLC细胞株,筛选出阳性克隆,在mRNA和蛋白质水平鉴定其抑制效率,最终得到DAL-1稳定抑制的细胞株.结果:通过双酶切鉴定和测序鉴定构建的shRNA表达载体序列正确,T4表达载体在mRNA和蛋白质水平能有效抑制DAL-1的表达,抑制率分别为(87.4±2.0)%和(82.7±2.1)%.结论:成功设计并构建了靶向DAL-1的shRNA表达载体,建立了DAL-1稳定抑制的NSCLC细胞株,为进一步研究DAL-1在NSCLC细胞中的作用提供了实验模型.

AIM： To build the experimental NSCLC cellular model in which expression of DAL-1 is steadily inhibited. METHODS： Specific shRNA interference plasmid vectors targeted to DAL-1 mRNA was constructed. The positive clones were screened by double-enzyme digestion and DNA sequencing. The expression at levels of DAL-I mRNA and protein was detected with Q-PCR and Western blot to screen the clones in which DAL-1 was steady inhibited. RESULTS： The sequences of the constructs were confirmed by double-enzyme digestion and DNA sequencing. The expressions of DAL-1 mRNA and DAL-1 protein of T4 group decreased significantly and the inhibition rate was （87.4 ± 2.0 ）% and （ 82. 7 ± 2. 1 ） % respectively. CONCLUSION： Specific shRNA interference plasmid vector targeted to DAL-1 gene mRNA is successfully designed and constructed. NCI-H460/T cell line with stable inhibition of DAL-1 expression is established successfully, which provides experimental models for further study of the roles of DAL-A in NSCLC cells.