人胰岛素样生长因子1的真核细胞表达及其鉴定

目的 构建人胰岛素样生长因子１（ＩＧＦ－１）的真核细胞表达质粒。方法 用ＰＣＲ方法从人的肝细胞ｃＤＮＡ文库中克隆出ＩＧＦ－１ｃＤＮＡ，然后定向插入真核细胞表达载体ｐｃＤＮＡ３中，并用脂质体方法转染ＣＯＳ７细胞。用ＥＬＩＳＡ法和人胚肺纤维母细胞以及ＮＩＨ３Ｔ３纤维细胞增殖法分别测定转染细胞上清液中ＩＧＦ－１的含量和生物活性。结果 重组的真核细胞表达质粒ｐｃＤＮＡ３－ＩＧＦ－１所含的ＩＧＦ－１ｃＤＮＡ序列和插入方向均正确，其转染的ＣＯＳ７细胞分泌较高浓度的ＩＧＦ－１，并且具有明显促进纤维细胞增殖的能力。结论 本实验所构建的重组真核细胞表达质粒ｐｃＤＮＡ３－ＩＧＦ－１能够高效表达有活性的ＩＧＦ－１，对进一步研究ＩＧＦ－１体内表达的生理和病理作用有一定意义。

Expression of human insulin like growth factor 1 in eukaryotic cell and detection of its activity

ObjectiveTo construct eukaryotic expression plasmid for human insulin like growth factor 1(IGF 1) Methods IGF 1 cDNA was cloned by PCR from the human liver cDNA library, and inserted into eukaryotic expression vector pcDNA3 with gene recombinant technique, forming recombinant pcDNA3 IGF 1 plasmid The latter was transfected into COS7 cells with liposome mediated method.Concentration and activity of IGF 1 in the supernatant were detected by ELISA and fibro blast proliferation assays respectively Results IGF 1cDNA sequence and its insertion direction in recombinant pcDNA3 IGF 1 plasmid were are correct The COS7 cells transfected with the pcDNA3 IGF 1 plasmid secreted high concentration of IGF 1,which markedly induced the proliferation of human fibroblast and NIH3T3 cells Conclusion The recombinant pcDNA3 IGF 1 plasmid can express active IGF 1 with high efficiency, which facilitates the study of its physical and pathological roles in vivo