DKK1基因过表达慢病毒载体构建及其在大鼠肾上腺嗜铬细胞瘤细胞PC12中的表达

摘要

目的 构建DKK1基因过表达慢病毒载体,并探讨其是否能在大鼠肾上腺嗜铬细胞瘤细胞PC12中高效、稳定表达.方法 PCR法扩增DKK1基因序列,将其转入质粒并整合到载体上,构建GV358-DKK1慢病毒表达载体;将构建成功的目的质粒和辅助包装质粒共转染293T细胞对慢病毒进行包装,荧光显微镜观察基因在293T细胞中的表达,用病毒梯度稀释法测定病毒滴度.将培养好的PC12细胞随机分为三组,空载体阴性对照组、PC12-DKK1组分别用制备的空载体慢病毒及GV358-DKK1颗粒进行感染,空白对照组常规培养.用qRT-PCR和Western blotting法分别检测PC12细胞中DKK1 mRNA与蛋白的相对表达量.结果 限制性内切酶鉴定及测序结果提示GV358-DKK1慢病毒表达载体构建成功,转染293T细胞获得高滴度的病毒.PC12-DKK1组DKK1 mRNA及蛋白相对表达量均较空载体组、空白对照组高(P均＜0.05).结论 DKK1基因过表达慢病毒载体构建成功,且其可在PC12细胞中稳定表达.%Objective To construct the lentiviral vector with DKK1 gene over-expression and to explore whether it can be efficiently and stably expressed in rat adrenal pheochromocytoma PC12 cells.Methods PCR was used to amplify DKK1 gene sequence.Plasmid was transferred and integrated into the vector and GV358-DKK1 lentiviral expression vector was constructed.The 293T cells were co-transfected with the constructed target plasmids and the helper packaging plasmids to package the lentivirus.Fluorescence microscopy was used to observe the expression of genes in 293T cells.The virus titer was determined by virus gradient dilution method.The cultured PC12 cells were randomly divided into three groups:the empty vector negative control group,the PC12-DKK1 group which was infected with the empty vector lentivirus and GV358-DKK1 particles,respectively,and the blank control group which was routinely cultured.The relative expression of DKK1 mRNA and protein in the PC12 cells were detected by qRT-PCR and Westernblotting,respectively.Results The results of restriction endonuclease digestion and sequencing indicated that the recombinant lentiviral vector of GV358-DKK1 was successfully constructed and transfected into 293T cells to obtain high-titer virus.The relative expression of DKK1 mRNA and protein in the PC12 cells of the PC12-DKK1 group was higher than that of the the empty vector negative control group and the blank control group (all P ＜ 0.05).Conclusion The lentiviral vector overexpressing DKK1 gene is constructed successfully,meanwhile,DKK1 gene can be stably expressed in the PC12 cells.