C端带多聚组氨酸标签的重组小鼠基质细胞衍生因子-1α原核系统的表达及分离纯化  

Bacterial Expression System and Purification System of Recombinant Mouse SDF-1a with C Terminal Tagged 6 Poly-Histidine

作　　者：蔡绍晖[1] 杜军[2] 任先达[1] 谭毅[3] 李新平[3] 

机构地区：[1]暨南大学药学院,广州510632 [2]日本名古屋大学大学院医学部保健学科医疗检查系 [3]重庆大学生物工程学院教育部生物力学与组织工程重点实验室,重庆400044

出　　处：《高技术通讯》2003年第9期47-52,共6页Chinese High Technology Letters

基　　金：国家自然科学基金(30271519);高等学校重点实验室访问学者基金资助项目。

摘　　要：在构建SDF-1原核表达系统的基础上,探索对其活性表达产物分离纯化的技术路线。将从小鼠骨髓组织克隆出的SDF-1α成熟肽基因插入原核表达质粒pET101/D-TOPO,用以转化大肠杆菌BL21Star^(TM)菌株,经IPTG进行诱导表达。SDS-PAGE和Western Blot检测证实:其表达产物在约11.6kD处有明显条带显示,其大小与预测的重组SDF-1α/His分子量一致。采用Ni^2+-Resin固相亲和层析法对由该系统表达的C端带6Xhis标签的重组SDF-1α/His进行分离纯化,纯度达92％。体外活性检测结果表明:重组小鼠SDF-1α/His对T细胞有明显趋化和促生存作用;能有效诱导Jurkat细胞ERK磷酸化。The aim of this study is to investigate an effectual technologic itinerary for separating and purifying active SDF-la based on establishing a high efficient bacterial expression system of SDF-1α. SDF-1α gene without signal peptide was cloned from bone marrow cells of mice and inserted into a bacterial expressive vector, pET101/D-TOPO and transformed competent E. coil BL21StarTM. Western blot with specific 6 poly-histidines antibody revealed that the BL21 stimulated by IPTG had a strong band with molecular weight of about 11. 6kDa that was approximately to the size of this recombinant chiasmic peptide, SDF-1α/His. The purity of the purified product was about 92% , which was separated and purified by using Ni2+-Resin immobile affinity chromatograph. The biologic activity identification proved that the recombinant SDF-1α/His appeared obvious effects on promoting T cell survival and chemoatractment in vitro, and inducing the phosphorylation of ERK in Jurkat cells.

关 键 词：多聚组氨酸 基质细胞衍生因子-1Α 原核系统 表达 分离 纯化 趋化因子 基因重组 固相亲和层析 基因转染 

分 类 号：R346[医药卫生—基础医学]