人核迁移蛋白shRNA慢病毒载体的构建及转导  

Construction and transduction of hNUDC shRNA interference lentiviral vector

作　　者：庞实锋[1] 廖霞[1] 郑克勤[1] 周汝滨[1] 梁朋[1] 曹定国[1] 张国平[1] 李文荣[1] 

机构地区：[1]广东医学院生物学教研室,广东湛江524023

出　　处：《细胞与分子免疫学杂志》2012年第7期688-690,695,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：教育部科学技术研究重点项目(210156);广东省卫生厅青年基金(B2010235);广东医学院博士启动基金(XB1007);广东省大学生创新实验项目(KY1003);湛江市科技攻关项目(2010C3111005)

摘　　要：目的:通过绿色荧光蛋白(GFP)标记,构建可示踪的人核迁移蛋白(hNUDC)慢病毒RNA干扰载体,并实现其对靶细胞的转导。方法:通过分子克隆技术将人核迁移蛋白的发夹RNA(shNUDC-F)克隆至慢病毒表达载体中,构建成靶向针对hNUDC的RNA干扰载体,通过磷酸钙转染法,将获得的慢病毒高纯度质粒转染进低次代293T细胞中进行包装,通过高速离心收获慢病毒颗粒,经滴度测定后,对Dami巨核细胞进行转导实验。用显微镜观察转导效率,用Westernblot检测RNA干扰效果。结果:慢病毒携带的shNUDC-F片段对Dami巨核细胞能高效转导,并使其内源hNUDC基因发生基因沉默,干扰效果较好。结论:成功构建出慢病毒hNDUC RNA干扰载体,并在293T细胞中实现包装并成功转入Dami细胞。AIM： To construct a lentiviral vector carrying human nuclear distribution protein C （hNUDC）-specific shRNA （sh-NUDC-F） and knock down hNUDC expression in Dami cells by infection of the lentivirus. METHODS： After labeling of green fluorescent protein （GFP）, sh-NUDC-F was cloned into lentiviral vector pRRL-cPPT-CMV-X-PRE- SIN, and the high-quality plasmid was transfected into 293T cells to produce lentiviral particles by the calcium phosphate method. After high-speed centrifugation, lentiviral particles were collected and determined for its titer. The high-titer lentiviral particles were then transduced into Dami cells. By detecting the expression of GFP in lentiviral vector using a microscope, the transduction efficiency was readily figured out. And hNUDC protein level was detected by Western blotting. RESULTS： hNUDC was totally knocked down by the efficient transduction of Dami cells with the lentivirus carrying sh-NUDC-F. CONCLUSION： Lentiviral vector containing sh-NUDC-F can be successfully packaged in 293T cells and then efficiently transduced into Dami cells to silence hNUDC gene.

关 键 词：慢病毒 RNA干扰 发夹RNA 巨核细胞 

分 类 号：Q78[生物学—分子生物学]