摘要
目的构建GRIM-19及其截断体原核表达载体,在大肠杆菌中诱导表达并纯化融合蛋白。方法用RT-PCR法从HeLa细胞中扩增出带BamHⅠ、XhoⅠ酶切位点的GRIM-19及其截断体基因片段,将GRIM-19及其截断体基因片段克隆到pGEX-4T-3原核表达载体上,在大肠杆菌中诱导表达GST-GRIM-19及其截断体融合蛋白,用Glutathione Sepharose 4B纯化,纯化后蛋白经Western-blot鉴定。结果 pGEX-4T-3-GRIM-19及其截断体原核表达载体构建正确,并在大肠杆菌中成功诱导表达,IPTG诱导以浓度0.5mM,时间2h为宜,且通过Glutathione Sepharose4B成功纯化到融合蛋白。结论成功构建GRIM-19及其截断体原核表达载体,诱导表达并纯化GST-GRIM-19及其截断体融合蛋白。
Objective To construct the GRIM19 and its truncated body prokaryotic expression vector,express the Fusion protein in E.coli and purify the expressed product.Methods GRIM-19 Its Truncated body gene sequence with BamHⅠ and XhoⅠ,restriction enzyme cutting site were amplified from total RNA of HeLa cell line by reverse transcription-polymerase chain reaction（RT-PCR）,then the GRIM-19 its truncated body gene sequence was inserted into the prokaryotic expression vector pGEX-4T-3,induced express the fusion protein in E.coli and purified by Glutathione Sepharose 4B,Western-blot were used to test the fusion protein.Results The pGEX-4T-3-GRIM-19 its truncated body prokaryotic expression vector was constructed correctly and the fusion protein was correctly inducted expression,the optimal concentration and time of IPTG for induction was 0.5mM and 2h,and the fusion protein was successfully purified.Conclusion The pGEX-4T-3-GRIM-19 and its truncated body prokaryotic expression vector was successfully constructed,Inducible expression and purification of GST-GRIM-19 and its truncated body fusion protein.
出处
《中国实验诊断学》
2012年第2期191-194,共4页
Chinese Journal of Laboratory Diagnosis
基金
国家自然科学基金资助(81072127
81001168)
