人胰岛素样生长因子腺病毒表达载体构建及其在C17.2神经干细胞的表达

Construction of adenoviral expression vector carrying human insulin-like growth factor-1 and its expression in C17.2 neural stem cells

目的:构建人胰岛素样生长因子腺病毒表达载体,并观察其在C17.2神经干细胞的表达。方法:实验于2004-03/11在中山大学附属第二医院林百欣医学中心完成。将胰岛素样生长因子克隆入复制缺陷性腺病毒穿梭质粒得到含胰岛素样生长因子的腺病毒穿梭质粒,再与腺病毒骨架质粒在大肠杆菌BJ5183中进行同源重组得到含胰岛素样生长因子的整合质粒,在HEK293A细胞中包装加膜,聚合酶链反应鉴定,大量扩增病毒。将含目的基因的病毒转染C17.2神经干细胞,原位杂交检测胰岛素样生长因子mRNA,蛋白印迹检测胰岛素样生长因子蛋白的表达,酶联免疫吸附实验检测胰岛素样生长因子蛋白的表达曲线。结果:经酶切鉴定及聚合酶链反应鉴定证实腺病毒载体构建正确。原位杂交检测到胰岛素样生长因子mRNA,蛋白印迹分析检测到胰岛素样生长因子蛋白在转染了重组腺病毒的C17.2神经干细胞内表达。酶联免疫吸附实验结果显示胰岛素样生长因子蛋白在病毒转染5～7d达高峰,13d仍高于转染前。结论:成功构建了含胰岛素样生长因子的重组腺病毒载体,转染了该腺病毒的C17.2神经干细胞可稳定表达胰岛素样生长因子。

AIM:To construct the adenoviral expression vector carrying insulin like growth factor 1 and observe its expression in C17.2 neural stem cells.METHODS:The experiment was finished in the Second Affiliated Hospital of Sun Yat sen University from March to November 2004.Insulin like growth factor 1 was inserted into adenovirus Padtrack CMV to obtain the recombinant plasmid Padtrack CMV insulin like growth factor 1;then homologous recombination was carried in BJ5183 bacteria with adenovirus backbone plasmid padeasy,and integrated plasmid pAdeasy CMV insulin like growth factor 1 was obtained.The adenovirus vector was then packed in HEK293A cells.After identified with polymerase chain reaction,the adenovirus vector was amplified in HEK293A cells.The adenoviral vector was transfected into C17.2 neural stem cells.The insulin like growth factor 1 mRNA,expression of insulin like growth factor 1 protein and its curve in C17.2 neural stem cells were detected by using in situ hybrization,Western blot and enzyme linked immunoadsordent assay,respectively.RESULTS:Restriction analysis and polymerase chain reaction verified the successful construction of adenoviral vector carrying insulin like growth factor 1.In situ hybrization and Western blot verified the expression of insulin like growth factor 1 mRNA and protein in the C17.2 neural stem cells transfecting this recombinant adenoviral vector.Enzyme linked immunoadsordent assay showed that the peak of the expression of insulin like growth factor 1 was in 5- 7 days after transfection,and was still higher at the 13th day as compared with before transfection.CONCLUSION:The adenoviral vector carrying insulin like growth factor 1 is successfully constructed.Insulin like growth factor 1 can be expressed stably in C17.2 neural stem cells transfecting this adenoviral vector.