Smad4基因RNAi慢病毒载体的构建与鉴定

Construction and identification of lentiviral vector of RNA interference of Smad4 gene

目的:构建Smad4基因RNAi慢病毒载体.方法:针对已经筛选确定的Smad4基因RNAi有效靶序列,合成靶序列的OligoDNA,退火形成双链DNA,与经MluI和ClaI酶切后的pLVTHM载体[含H1启动子和绿色荧光蛋白(GFP)]连接产生LVshSmad4慢病毒载体,PCR筛选阳性克隆,测序鉴定.用LVshSmad4,pCMVdR8.74和pMD2G3质粒共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度.结果:PCR和测序证实,成功构建Smad4shRNA的慢病毒载体LVshSmad4.包装慢病毒,浓缩病毒悬液的滴度为5×1010pfu/L.结论:成功构建人Smad4基因RNAi慢病毒载体.

AIM： To construct a lentiviral vector of RNA interference （RNAi） of Smad4 gene. METHODS： The effective sequence of siRNA targeting Smad4 gene was confirmed in our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pLVTHM vector, which contained H1 promoter and green fluorescent protein （GFP）. The resulting lentiviral vector containing Smad4 shRNA was named LVshSmad4, and it was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector LV-shSmad4 ,pCMVdR8.74 and pMD2G. All virus stocks were produced by calcium phosphate-mediated transfection. The titer of virus was tested according to the expression level of GFP. RESULTS： PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of Smad4 （LV-shSmad4） producing Smad4 shRNA was constructed successfully. The titer of concentrated virus was 5 × 10^10pfu/L. CONCLUSION： The lentivirus RNAi vector of Smad4 was constructed successfully.