解偶联蛋白-2 RNA干扰慢病毒载体的构建  

Construction of RNA Interfering Lentivirus Vector Targeting Uncoupling Protein 2

作　　者：万赤丹[1] 程锐[1] 王春友[1] 王宏博[1] 王帅[1] 刘涛[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院普外科,武汉430022

出　　处：《中国康复》2008年第5期294-297,共4页Chinese Journal of Rehabilitation

基　　金：国家自然科学基金资助项目(30571764)

摘　　要：目的:构建解偶联蛋白-2(UCP-2)基因的RNA干扰慢病毒载体。方法:根据RNA干扰序列设计原则,针对UCP-2mRNA(NM_011671)设计3个RNA干扰靶点序列,合成含干扰序列的DNA双链,酶切后连入慢病毒转移质粒pGCL-GFP中。连接产物转化感受态细胞,所获克隆行PCR鉴定和序列测定。构建成功的RNA干扰质粒与UCP-2表达质粒共转染293T细胞,荧光显微镜下观测转染效果,Western blot检测UCP-2蛋白表达,筛选出干扰效果最好RNA干扰质粒。将该质粒与两种辅助包装原件载体质粒pHelper1.0(gag/pol元件)载体,Helper2.0(VSVG元件)共转染293T细胞包装成慢病毒并检测滴度。结果:PCR和测序结果显示针对3个靶点的RNA干扰质粒均构建成功;通过Westernblot检测获得最优干扰靶点,并成功包装成滴度为5×108TU/ml的慢病毒。结论:成功构建可供感染的解偶联蛋白-2RNA干扰慢病毒载体,为后续靶向抑制该基因表达以提高脂肪肝移植成功率的研究奠定物质基础。Objective： To construct RNA interfering （RNAi） lentivirus vector targeting uncoupling protein 2 （UCP-2）. Methods ： Three specific target sequences from UCP-2 mRNA sequence （NM_011671 ） were designed according to the principle of siRNA design. The double-strand DNA containing interfering sequence was designed, digested and linked with pGCL-GFP. The product was transfected into competent cells and the clones were subjected to PCR identifica- tion and sequencing. The successfully constructed RNAi ptasmid and UCP-2 expression plasmid were co-transfecteed into 293T cells. The transfection efficiency was observed under the fluorescence microscopy. Western blot was used to detect the expression of UCP-2 protein. RNAi plasmids with optimal interfering efficacy were screened, and co-transfeeted with pHelper 1.0 and Helper 2.0 into 293T cells to form lentivirus. Results：PCR and sequencing revealed that RNAi plasmids to three targets were successfully constructed. The optimal interfering targets were selected by Western blot. The lenti- virus with a titer of 5 × 108 TU/ml was successfully packed. Conclusion： RNAi lentivirus vector of UCP-2 was constructed successfully, which provides a foundation for targeting suppression of UCP-2 gene expression to increase the success rate of fatty liver transplantation.

关 键 词：解偶联蛋白-2 RNA干扰 慢病毒 

分 类 号：R657.3[医药卫生—外科学] R346[医药卫生—临床医学]