构建过表达/干扰多梳蛋白2的人胶质瘤细胞系

Construction and Expression of Overexpressing/Interfering Polycomb-like 2 Recombinant Lentivirus

目的构建过表达/干扰多梳蛋白2(polycomb-like 2,PCL2)基因重组慢病毒并稳转胶质瘤U251细胞系。方法过表达PCL2重组慢病毒载体pLVX-hPCL2-3flag-ZsGreen-Puro和干扰PCL2重组慢病毒载体pLVX-shRNA2-Puro-hPCL2通过克隆技术成功构建,在293T细胞中导入质粒载体完成慢病毒包装,经过干预HEK293获得转染效率,病毒滴度采用逐孔稀释梯度法测得。将构建好的hPCL2/shRNA PCL2重组慢病毒转染胶质瘤U251细胞,倒置显微镜下观察荧光强度及范围,Western blot检测PCL2蛋白的表达情况。结果成功构建hPCL2/shRNA PCL2重组慢病毒载体,Western blot结果显示,相比对照组和空载组,目的蛋白在转染过表达PCL2重组慢病毒U251细胞中表达明显升高,在转染干扰PCL2重组慢病毒U251细胞中表达降低(P均<0.05)。结论成功构建hPCL2/shRNA PCL2重组慢病毒的U251细胞系。

Objective The recombinant lentivirus expressing/interfering polycomb-like 2(PCL2)gene was constructed and stably transformed into glioma U251 cell line.Methods The recombinant lentivirus vector pLVX-hPCL2-3flag-ZsGreen-Puro which overexpressed PCL2 and the recombinant lentivirus vector pLVX-shRNA2-Puro-hPCL2 which interfered with PCL2 were successfully constructed by cloning technology.After the plasmid vector was introduced into 293T cells to complete the packaging of lentivirus,the transfection efficiency of HEK293 was obtained after intervention,and the virus titer was measured by the poor-by-hole dilution gradient method.The constructed recombinant lentivirus was transfected into U251 cells.The fluorescence intensity and range were observed under inverted fluorescence microscope,and the expression of PCL2 gene was measured by Western blot.Results The recombinant lentivirus vector of overexpressing/interfering PCL2 was successfully constructed.Western blot results showed that the expression of the target gene was significantly higher in the transfected U251 cells with overexpressing PCL2 than in the control group and the no-load group,and significantly lower in the transfected U251 cells with interfering PCL2(P all<0.05).Conclusion U251 cell line of overexpressed/interfered with PCL2 recombinant lentivirus was successfully constructed.