重组人LIF融合蛋白表达纯化及其活性鉴定  

Expression,Purification and Bioactivity Identification of Recombinant Human Leukemia Inhibitory Factor(hLIF) Fusion Protein

作　　者：孙静[1] 王斌[1] 段志青[1] 胡凝珠[1] 李建芳[1] 李彦涵[1] 胡云章[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《中国生物工程杂志》2013年第5期50-55,共6页China Biotechnology

基　　金：协和医学院青年科研基金“microRNA诱导人胚肺二倍体细胞(KMB17)向肝细胞转分化研究”项目资助

摘　　要：目的:原核表达重组hLIF融合蛋白并进行诱导表达、纯化及活性鉴定。方法:将hLIF基因克隆至pThioHisA载体,构建融合表达载体pThioHisA-hLIF,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经亲和层析后,Western blot检测目的蛋白的特异性。用小鼠胚胎干细胞脱饲养层培养对纯化后的重组hLIF融合蛋白进行生物活性的鉴定。结果:降低诱导温度和延长诱导时间能增加hLIF融合蛋白的可溶性表达,纯化后的重组蛋白纯度大于95%,Western blot检测显示了良好的特异性。在脱饲养层细胞培养条件下,添加纯化的hLIF融合蛋白能够有效的维持小鼠胚胎干细胞的未分化状态。结论:重组hLIF融合蛋白可在大肠杆菌中高效表达,具有良好的特异性,为干细胞研究及hLIF蛋白的其他功能研究奠定了基础。Objective： To express recombinant human Leukemia inhibitory factor （hLIF） fusion protein in prokaryotic cells, purify and identify the bioactivity of expressed product. Methods： The hLIF gene was cloned into vector pThioHisA, and the constructed recombinant plasmid pThioHisA-hLIF was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. After the expression product waspurified by affinity chromatography, the fusion protein was tested for its specificity by western blot detection. To assess the bioactivity of the hLIF fusion protein expressed in E. coli, it was tested to maintain the pluripotency of the mouse embryonic stem cells in feeder-independent culture system. Results： Lower the induction temperature and prolong the induction time can increase soluble hLIF fusion protein expression. The recombinant protein reached a purity of more than 95% after purification, and its specificity was successfully proved by western blot detection. In feeder-independent culture system, adding hLIF fusion protein can effectively maintain the mouse embryonic stem cells in an undifferentiated state. Conclusion： The recombinant hLIF fusion protein was highly expressed in E. coli and showed good specificity, which lay the foundation for stem cells research and further research on hLIF biological functions

关 键 词：重组人LIF融合蛋白 原核表达 纯化 小鼠胚胎干细胞 生物活性鉴定 

分 类 号：Q786[生物学—分子生物学]