杀伤细胞受体KIR3DS1胞外区的表达及纯化

目的表达并纯化杀伤细胞受体KIR3DSl胞外区。方法采用定向克隆的方法将KIR3DS1胞外区基因连接到pET28a-DsbA载体上,成功构建pET28a-DsbA/KIR3DS1重组质粒。将重组质粒导入大肠杆菌BL21（DE3）,IPTG诱导DsbA-KIR3DS1融合蛋白表达,溶解于8M尿素中的包涵体经Ni-NTA琼脂糖亲和层析纯化,成功进行复性,再经Su-perdex75凝胶层析柱进一步纯化,SDS-PAGE和Western blot鉴定目的蛋白的表达及纯化。结果表达产物呈部分可溶性表达,包涵体纯化后证实获得纯度〉95%的DsbA-KIR3DS1融合蛋白。结论 KIR3DS1胞外区的成功表达及纯化,为进一步研究KIR3DS1与相应配体之间相互作用奠定了基础。

Expression and purification of killer immunoglobulin-like receptor KIR3DS1 extracelluar domain

Objective To express and purify killer immunoglobulin-like receptor KIR3DS1 extracelluar domain.Methods KIR3DS1 extracelluar domain was amplified by polymeras chain reaction（PCR） and cloned into into the prokaryotic expression vector pET28a-DsbA to construct the recombinant vector pET28a-DsbA/KIR3DS 1.The recombinant plasmid was transformed into E.coll.BL21（DE3）,and induced with IPTG.Bacterial pellets were resuspended in 8M urea and centrifuged to remove insoluble material.The crude extract was purified by passing over a Ni-NTA-agarose column.After the inclusion body flowing through the Ni-NTA-agarose affinity chromatography was refolded successfully,it was purified by Superdex75 gel filtration and the purification effects of the fusion protein were identified by SDS-PAGE and western blot.Results Some fusion protein was expressed in the supematant,and the others were expressed in the form of inclusion bodieS.The punty of fusion protein was over 95%aner purincati on under denaturing condition.Conclusion The highly efficient expression of KIR3DS1 extracelluar domain laid the foundation for the further studies on exploration of the mechanism of immunization recognition between KIR3DS1 and itsligand.