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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。
OBJECTIVE: To construct a transducible target of lentiviral vector containing human nuclear transfer protein (hNUDC) by green fluorescent protein (GFP) labeling. Methods: Human hairpin RNA (shNUDC-F) of human nuclear transfer protein was cloned into the lentiviral expression vector by molecular cloning technology and constructed into RNA interference vector targeting to hNUDC. By using calcium phosphate transfection method, the obtained slow High-purity virus plasmids were transfected into low-generation 293T cells for packaging. Lentiviral particles were harvested by high-speed centrifugation. After titration, Dami megakaryocytes were transduced. The transduction efficiency was observed with a microscope, and the effect of RNA interference was detected by Western blot. Results: The shNUDC-F fragment carried by lentivirus efficiently transduced Dami megakaryocytes and silenced endogenous hNUDC gene. Conclusion: The lentivirus hNDUC RNA interference vector was successfully constructed and packaged in 293T cells and successfully transfected into Dami cells.