人胰岛素样生长因子-l的原核表达和纯化

目的构建高表达、易纯化的人胰岛素样生长因子-1(hIGF-1)工程菌。方法采用pET32a(+)质粒构建带羟胺裂解部位的表达载体,转入大肠杆菌DH5α进行诱导表达。表达产物经镍离子-亲和柱色谱分离后进行羟胺裂解。裂解产物纯化后用MALDI-TOF-MS法测定相对分子质量(Mr)。结果重组质粒序列完全正确。工程菌可表达预计Mr的融合蛋白,经Western blot证实有hIGF-1抗原活性。经镍离子-亲和柱色谱分离、羟胺裂解后得到的肽的Mr为7 640,和理论值相符。结论成功构建了表达hIGF-1的工程菌,为开发hIGF-1奠定了基础。

Expression and purification of human insulin-like growth factor-1 in bacteria

Purpose This study was aimed at producing human insulin-like growth factor-1(hIGF-1) in E.coli.Methods The hIGF-1 DNA was cloned into expression vector pET32a(+).After induction,the expressed fusion protein was purified by Ni2+ affinity chromatography and subjected to NH2OH cleavage.The mass of resulting peptide was determined by MALDI-TOF-MS.Results The sequence of the recombinant DNA fragment was correct.The produced fusion protein showed the expected molecule weight on SDS-PAGE and was confirmed by Western blot.After Ni affinity purification and NH2OH cleavage,the released hIGF-1 was confirmed by MALDI-TOF-MS.Conclusion Our results demonstrate that hIGF1 expression vector has been established.