摘要
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。
Purpose This study was aimed at producing human insulin-like growth factor-1 （hlGF-1） in E. coli. Methods The hlGF-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 NHEOH cleavage, the released hlGF-1 was confirmed by MALDI-TOF-MS. Conclusion Our results demonstrate that hlGF-1 expression vector has been established.
出处
《中国生化药物杂志》
CAS
CSCD
北大核心
2009年第4期226-229,共4页
Chinese Journal of Biochemical Pharmaceutics
基金
福建省自然科学基金重点项目(2008J0006)
福建省科技厅科技计划重点项目(2007Y0018)
福建医科大学教授学术发展基金(JS06027)资助
