【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Ni²+-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。更多还原

【Abstract】 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.更多还原