人胰岛素样生长因子-1真核表达载体的构建及在神经干细胞中的表达

目的:构建人胰岛素样生长因子-1(IGF-1)真核表达载体,并在人脐血神经干细胞(NSCs)中表达。方法:采用RT-PCR法从人胎肝中克隆IGF-1基因,将其导入真核表达载体pcDNA3.1中,用脂质体转染法将重组质粒转染人脐血NSCs。分别采用RT-PCR和免疫荧光细胞化学法检测转基因NSCs中IGF-1mRNA和蛋白的表达。结果:琼脂糖电泳显示RT-PCR扩增出462bp的条带;重组载体pcDNA3.1-IGF-1经HindⅢ与BamHⅠ双酶切后,得到5428bp和462bp的两个条带;目的基因转染的NSCs经RT-PCR扩增出一条与目的基因一致的条带;免疫荧光细胞化学检测到IGF-1蛋白的表达。结论:成功构建了人IGF-1基因的真核表达载体,转染后IGF-1重组蛋白在人脐血NSCs中能成功表达。

Construction of eukaryotic expression vector carrying human insulin-like growth factor-1 gene and expression in human neural stem cells

Aim:To construct eukaryotic expression vector for human insulin like growth factor-1(IGF-1),and observe its expression in human umbilical cord blood(HUCB) neural stem cells(NSCs).Methods:IGF-1 was cloned from human fetal liver by RT-PCR and then inserted into the eukaryotic expression vector pcDNA3.1.The recombinant plasmid pcDNA3.1-IGF-1 was transfected into HUCB NSCs with liposome-mediated method.IGF-1 mRNA and protein expressions in HUCB NSCs were detected by RT-PCR and immunocytochemistry,respectively.Results:A fragment of 462 bp was obtained by agarose electrophoresis after RT-PCR,and two fragments(5 428 bp and 462 bp) were obtained from the recombined plasmid after enzyme digestion.After transfection,a fragment that was consistent with the target gene was obtained,and the results of immunocytochemistry proved that there was expression of the transfected IGF-1 in HUCB NSCs.Conclusion:Eukaryotic expression vector containing IGF-1 has been successfully constructed.IGF-1 protein could be expressed in HUCB NSCs successfully.