重组人胰岛素样生长因子Ⅰ的纯化及鉴定

Purification and identification of recombiant human IGF-Ⅰ

目的 :纯化在大肠杆菌中表达的重组人胰岛素样生长因子Ⅰ (rhIGF Ⅰ ) ,并对其性质进行鉴定。方法 :发酵表达菌株 ,提取包涵体 ,采用离子交换和凝胶过滤两步纯化 ,从相对分子质量、免疫学性质、荧光光谱、N端 15个氨基酸的序列分析等方面进行鉴定。另外 ,对重组蛋白进行了复性和生物学功能的检测。结果与结论 :纯化后rhIGF Ⅰ纯度可达 99%以上 ,相对分子质量、免疫印迹、荧光光谱及N端 15个氨基酸的分析结果与预计的相同 ,rhIGF Ⅰ促细胞生长的ED50 为 3～ 10ng/ml。

Objective:To obtain highly purified recombinant human IGF Ⅰ(rhIGF Ⅰ) and identify it.Methods:rhIGF Ⅰ Was purified through ion exchange chromatography and gel filtration chromatography after the inclusion bodies of rhIGF Ⅰ were extracted from Escherichia coli. The recombinant protein was characterized through molecular weight assay, Western blot, and fluorescent chromatography. The renaturation and biological assay of rhIGF Ⅰ were investigated. Results and Conclusions: The purity of rhIGF Ⅰ was higher than 99%. The analysis of molecular weight, Western blot, fluorescent chromatography and sequences of NH 2 terminal 15 amino acids were same as those anticipated. 3-10 mg/ml was the concentration of renatured rhIGF Ⅰ to support half maximal stimulation of cell proliferation with BALB/c 3T3 cells. [