摘要
新近报道糖皮质激素诱导的肿瘤坏死因子受体的配体(GITRL)具有抑制前体破骨细胞的作用,故命名为Osteostat,为深入研究其功能和机制,本研究原核表达人GITRL胞外段并进行活性分析。利用限制性内切酶Eco31I获得大肠杆菌偏嗜性GITRL的胞外段cDNA序列,构建了基于pQE-30Xa的原核表达载体,并在M15[pREP4]菌株中经IPTG诱导表达带有His融合标签的GITRL重组蛋白,主要以包涵体形式存在。经体外包涵体变性、复性及纯化后,采用SDS-PAGE和Western blotting进行分析和鉴定。同时建立了报告基因技术检测GITRL重组蛋白生物活性的方法,简单便捷、灵敏而且周期短,利用此方法分析了重组GITRL胞外段表达蛋白的生物活性。
GITRL （Glucocorticoid-induced tumor necrosis factor receptor ligand） has been receiftly identified as a novel inhibitor of osteoclastogenesis and hence called Osteostat. In this study, we expressed recombinant extracellular domain of GITRL protein in Escherichia coli and analyzed its bioactivity. Using an Eco311 enzyme-based restriction and ligation method, we obtained an E. coli-preferred DNA sequence coding for the extracellular domain of human GITRL. The DNA was cloned into expression vector pQE-30Xa that encodes a fusion tag of 6xHis before the insert. The resultant recombinant expression vector pQE/GITRL was subsequently transformed into E. coli strain M15[pREP4]. After induction with Isopropyl ^-D-Thiogalactoside （IPTG）, the cells produced the fusion protein mainly in the form of inclusion bodies as identified by SDS-PAGE. The recombinant protein was purified by affinity chromatography through Ni-NTA column and recognized by anti-His polyclonal antibody using Western blotting analysis. Moreover, we established a simple, efficient and sensitive reporter gene-based method to detect the activity of the recombinant protein. The results showed that the target protein was biologically active.
出处
《生物工程学报》
CAS
CSCD
北大核心
2009年第5期708-713,共6页
Chinese Journal of Biotechnology
基金
国家自然科学基金项目(No.30300171)
天津市高等学校科技发展基金项目(No.20060207)资助~~
