NK细胞免疫球蛋白样受体KIR3DL1胞外区的表达及纯化

Expression and purification of killer immunoglobulin-like receptor KIR3DL1 extracelluar domain

目的表达并纯化NK细胞免疫球蛋白样受体KIR3DL1胞外区。方法以pUC57-KIR3DL1为模板,PCR扩增KIR3DL1胞外区序列,与pGEM-T载体进行A-T克隆;测序正确后,采用定向克隆的方法将KIR3DL11胞外区基因连接到pET28a-DsbA载体上,成功构建了pET28a-DsbA/KIR3DL1重组质粒。将重组质粒导入大肠杆菌BL21(DE3),IPTG诱导DsbA-KIR3DL1融合蛋白表达,溶解于8mol/L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Superdex75凝胶层析柱进一步纯化,SDS-PAGE和Westernblot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%以上的DsbA-KIR3DS1融合蛋白。结论KIR3DL1胞外区的成功表达及纯化为进一步研究KIR3DL1与相应配体之间的相互作用奠定了基础。

Objective To express and purify the killer immunoglobulin like receptor KIR3DIA extracelluar domain. Methods pUC57 KIR3DL1 was used as template, KIR3DL1 extracelluar domain was amplified by polymerase chain reaction （PCR） and cloned into pGEM-T vector with A-T cloning technique. After DNA sequence analysis, the target fragment was insetted into the prokaryotic expression vector pET28a-DsbA to construct the recombinant vector pET28a DsbA/KIR3DL1. The recombinant plasmid was transformed into E coli BI.21 （DE3）, and induced with IPTG. Bacterial pellets were resuspended in 8M urea and centrifuged to remove the insoluble mate rial. 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. Result Some fusion proteins were expressed in the supernatant, and the others were expressed in the form of inclusion bodies. The purity of fusion protein was over 95%0 after purification under denaturing condition. Conclusion The highly efficient expression of KIR3DL1 extracelluar domain laid the foundation for the further studies on exploration of the mechanism of immunization recognition between KIR3DL1 and its ligand.