Smad4基因RNAi慢病毒载体构建及鉴定.docVIP

Smad4基因RNAi慢病毒载体构建及鉴定 　 作者：季国忠，张发明，翟惠虹，范志宁，樊代明，王学浩 【关键词】 慢病毒 Construction and identification of lentiviral vector of RNA interference of Smad4 gene 　　【Abstract】 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 LVshSmad4，pCMVdR8.74 and pMD2G. All virus stocks were produced by calcium phosphatemediated 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 (LVshSmad4) producing Smad4 shRNA was constructed successfully. The titer of concentrated virus was 5×1010 pfu/L. CONCLUSION: The lentivirus RNAi vector of Smad4 was constructed successfully. 　　【Keywords】 RNA interference; Smad4; Neoplasms; Lentivirus 　　【摘要】 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Oligo DNA，退火形成双链DNA，与经Mlu I和Cla I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pMD2G 3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Smad4 shRNA的慢病毒载体LVshSmad4. 包装慢病毒，浓缩病毒悬液的滴度为5×1010 pfu/L. 结论：成功构建人Smad4基因RNAi慢病毒载体. 　　【关键词】 RNA干扰；Smad4；肿瘤；慢病毒 　　0引言 　　转化生长因子β(TGFβ)Smad信号途径在肿瘤发生机制中发挥重要作用. Smad4（deleted in pancreatic carcinoma locus 4, DPC4）在信号传输途径中处于中枢地位，活化的RSmads与Smad4结合成寡聚物，将TGFβ的信号传递到核内，调节靶基因的转录并诱导下游基因的表达［1］. 近年来，RNA干扰（RNA interference, RNAi）技术的出现，为研究内源性基因功能和信号转导途径提供了强有力的研究工具. 我们在已经获取Smad4基因的RNAi有效靶序列的基础上，设计、构建和鉴定表达Smad4 siRNA的慢病毒载体，为后期研究Smad4基因在肿瘤细胞中的作用机制和基因治疗打下基础. 　　1材料和方法 　　1.1材料采用慢病毒的包装细胞293T细胞株（中科院上海细胞所）、大肠杆菌菌