重组人截短型胰岛素样生长因子1在大肠杆菌中的高效表达

High level expression of human truncated insulin like growth factor 1 in E.coli

目的:建立人截短型胰岛素样生长因子1的原核高效表达系统。方法:利用基因重组技术将人截短型胰岛素样生长因子1〔Des(13)IGF1〕的cDNA片段克隆到融合蛋白表达载体pMTY4中,用离子交换层析法纯化蛋白并经SDS聚丙烯酰胺凝胶电泳、放射免疫检测、N末端前16位氨基酸序列测定及生物活性检测等方法对所获蛋白进行了鉴定。结果:获得含人Des(13)IGF1基因的重组质粒,在大肠杆菌中高效表达出含凝血酶识别序列的MS2Des(13)IGF1融合蛋白,该蛋白经凝血酶消化后纯化可获得与天然型一致的人Des(13)IGF1。结论:该方法是获得人重组Des(13)IGF1的有效途径,对进一步研究Des(13)IGF1有重要意义。

Objective: To establish an efficient expression system for human truncated insulin like growth factor 1 〔Des(1 3)IGF1〕as fusion protein in Escherichia coli(E.coli). Methods: The cDNA of Des(1 3)IGF1 was cloned into an fusion protein expression plasmid, pMTY4, using gene recombinant technique. The protein was purified by ion exchange chromatography and identified by SDS polyacrylamide gel electrophoresis, radioimmunoassay(RIA), N terminal amino acid sequence and biological activity. Results: A prokaryotic expression vector was constructed and the fusion protein containing MS2 polymerase fragment, thrombin recognition site and human Des(1 3)IGF1 was expressed in E.coli at high level. It was showed that the purified recombinant Des(1 3)IGF1 released from the fusion protein after digestion with thrombin was identical to the native Des(1 3)IGF1.Conclusion:This is an effective method for obtaining human recombinant Des(1 3)IGF1 and it is very important for further study of Des(1 3)IGF1.