SA/TNFα双功能融合蛋白的克隆、表达及活性研究  

Generation and Characterization of SA/TNFα Bifunctional Fusion Proteins

作　　者：李金龙[1] 徐翠香[1] 胡志明[1] 高基民[1] 

机构地区：[1]南方医科大学生物技术学院生物治疗研究所,广东广州510515

出　　处：《生物技术》2010年第3期36-39,共4页Biotechnology

基　　金：国家"863"计划项目(No2006AA02Z4C4);广州市白云区科技计划项目(2009-SZ-40)资助

摘　　要：目的:制备链亲合素标记的TNFα双功能融合蛋白,并对其活性进行研究。方法:构建原核表达质粒pET24a-SA-TNFα和pET21a-TNFα-SA;转化大肠杆菌BL21(DE3),IPTG诱导表达,Ni-NTA亲合层析纯化后进行透析复性;流式细胞仪检测融合蛋白对生物素化MB49细胞的锚定活性,L929细胞杀伤实验检测融合蛋白的TNFα活性。结果:成功制备了两种链亲合素标记的TNFα双功能融合蛋白SA-TNFα和TNFα-SA;其表达量分别约占总蛋白的30%和23%,纯化效率均达90%以上;两种融合蛋白均能有效地锚定于生物素化的MB49细胞表面,其锚定效率分别为95%和92%;L929细胞杀伤实验显示SA-TNFα具备TNFα活性,但TNFα-SA不具备TNFα活性。结论:成功制备了具备双功能活性的链亲合素标记的TNFα融合蛋白,TNFα在该融合蛋白中的位置与其活性有重要的关系。Objective：To generate streptavidin（SA）-tagged human TNFα bifunctional fusion proteins and examine their bioactivity.Method：TNFα and streptavidin genes were cloned by RT-PCR,and cloned into pET24a or pET21a to get pET24a-SA-TNFα and pET21a-TNFα-SA plasmids.The fusion proteins were expressed in BL21（DE3） bacteria,purified through the Ni-NTA affinity chromatography,and refolded by dialysis.The bifunctionality of the fusion proteins-biotin-binding function and TNFα activity-was analyzed by flow cytometry and cytotoxic effect on L929 cells,respectively.Result：SA-TNFα and TNFα-SA fusion protein were expressed at about 30% and 23% of the total cellular protein respectively,and finally prepared at high purity（above 90%）.Both fusion proteins could efficiently immobilize on the surface of biotinylated MB49 cell（95% for SA-TNFα and 92% for TNFα-SA）.L929 cytotoxic assay showed that SA-TNFα but not TNFα-SA had TNFα activity.Conclusion：SA-tagged TNFα fusion proteins were successfully prepared,and TNFα activity was dependent on the location of TNFα in the fusion protein.

关 键 词：人肿瘤坏死因子Α 链亲和素 融合蛋白 纯化 

分 类 号：R392.5[医药卫生—免疫学]