Objective To express and purify the killer immunoglobulin-like receptor KIR3DL1 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 inserted into the prokaryotic expression vector pET28a-DsbA to construct the recombinant vector pET28a-DsbA /KIR3DL1.The recombinant plasmid was transformed into E.coli BL21(DE3),and induced with IPTG.Bacterial pellets were resuspended in 8M urea and centrifuged to remove the 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.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% 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.

目的:初步证实咖啡豆α-半乳糖苷酶基因在大肠杆菌JM83中可溶性表达、纯化及活性.方法:通过PCR扩增得到咖啡豆α-半乳糖苷酶基因克隆到载体pGEM-T easy,目的片段与预期的大小一致.咖啡豆α-半乳糖苷酶基因与分泌性原核表达载体pAS18连接,转化到宿主菌E.coli JM83.经过IPTG诱导表达并通过SDS-PAGE电泳分析,Western-blot印迹证实蛋白表达的特异性.结果:PCR扩增得到目的片段测序结果正确.SDS-PAGE电泳分析得到目的蛋白41 kD,经过Western blot鉴定正确.ELISA检测证实咖啡豆α-半乳糖苷酶具有生物活性.结论:优化诱导条件,初步证实重组咖啡豆α-半乳糖苷酶基因具有生物活性.

酶的体外定向进化是目前酶学工程发展的一个新技术,本文综述了易错PCR、DNA重组等策略的进展,该项技术为医学、工业、农业等方面提供了广阔的前景.