TACI-Fc融合蛋白的基因构建、原核表达及生物活性鉴定  

Construction and Prokaryotic Expression of TACI - Fc Fusion Protein

作　　者：冀丽军[1] 白乌仁图雅[1] 柴琳[1] 孙剑[1] 

机构地区：[1]天津大学药物科学与技术学院,天津300072

出　　处：《生物技术》2013年第3期24-28,共5页Biotechnology

基　　金：国家自然科学基金项目("新型BAFF拮抗剂在系统性红斑狼疮中治疗作用的研究";81273308)资助;2012天津大学大学生创新创业训练计划项目;天津大学第五批实验教学改革项目的资助~~

摘　　要：目的:构建sTACI胞外区与人IgG1 Fc的重组基因,并进行原核表达和纯化具有生物活性的融合蛋白。方法:通过普通PCR和重叠PCR法,获得sTACI-Fc重组基因,进而构建重组子pET28a-sTACI-Fc,并转入E.coli BL21(DE3)中进行融合蛋白的表达,采用蛋白A凝胶亲和柱进行纯化以及ELISA法检测其生物活性。结果:成功获得了sTACI-Fc的重组基因,pET28a-sTACI-Fc重组子,通过表达和纯化得该融合蛋白,且其能够与BAFF特异性结合,结合活力呈现剂量依赖性,浓度5ng/μl时,两者的吸附达饱和。结论:成功构建了基因大小为1 000bp的sTACI-Fc重组基因,并在原核系统中进行了可溶性表达,得大小约为40kDa的融合蛋白sTACI-Fc,体外实验证实在其浓度为5ng/μl时与BAFF的结合达饱和,为下一步的研究工作奠定了基础。Objeetive：Construction of the recombinant gene of the extracellular domain of sTACI and human IgG1 Fc, and prokaryotic ex- pression,purification and activity identification of the fusion protein, nethod：TACI - Fc recombinant gene was gained by PCR and over- lapping PCR,following by cloning into pET28a to construct pET28a - sTACI - Fc. After pET28 （a） - sTACI - Fc was transformed intoE. coli BI21 （DE3）, the fusion protein was expressed and purified by protein A affinity column. And, the fusion protein was identified by ELISA. Result：The target sequences of sTACI - Fc was successful constructed and cloned into pET28a. After expression and purification, the fusion protein was gained. Also, it had been identified by ELISA that sTACI - Fc could bind to BAFF specifically in a dose - dependent manner,and the saturated concentration was 5ng/μl. Conclusion ：The recombinant gene sTACI -Fc of 1 000bp was obtained. The fusion protein of 40 kDa was successfully expressed in E. coli BL21 （DE3） and could bind with BAFF at the concentration of 5ng/μl saturately, which would be a standard material for the further study.

关 键 词：TACI B细胞活化因子 原核表达 融合蛋白 

分 类 号：R392.2[医药卫生—免疫学]