人tau融合蛋白的原核表达及纯化  

Expression and purification of human tau fusion proteins

作　　者：梁燕[1] 王海萍[2] 冯利杰[1] 李琪[1] 沈玉先[1] 

机构地区：[1]安徽医科大学教育部"重要遗传疾病基因资源利用"重点实验室(省部共建),合肥230032 [2]安徽医科大学临床药理研究所

出　　处：《安徽医科大学学报》2007年第4期367-370,共4页Acta Universitatis Medicinalis Anhui

基　　金：教育部新世纪人才支持计划资助项目(编号:NCET-04-0589);教育部重点项目(编号:206067)

摘　　要：目的构建人His-tau和GST-tau融合蛋白表达质粒并在大肠杆菌中诱导表达及纯化。方法以质粒pEGFP-tau为模板通过PCR扩增出人tau全长cDNA,并构建到原核表达载体pET28a和pGEX-5X-1中,挑选阳性重组子,经限制性内切酶鉴定后转化到大肠杆菌BL21中,然后用IPTG诱导表达,通过SDS-PAGE染色及Western-blot方法鉴定表达的融合蛋白。分别使用His Bind Resin和Glutathione Sepharose 4B与融合蛋白结合来纯化融合蛋白。结果成功构建原核表达质粒pET28a-tau和pGEX-5X-1-tau并在大肠杆菌BL21中诱导其大量表达。经His Bind Resin和Glutathione Sepharose 4B纯化后,可以得到较纯化的His-tau和GST-tau融合蛋白。SDS-PAGE及Western-blot分析显示,特异性的抗tau单克隆抗体(tau-5)所识别的融合蛋白分子量与理论值相近。结论构建了人tau两种原核表达的融合蛋白质粒,并高效表达和纯化了该蛋白,为进一步的tau与其它功能性蛋白的相互作用研究奠定了基础。Objective To construct the plasmids encoding human His-tau and GST-tau proteins in bacteria, to express and purificate them. Methods Utilizing pEGFP-tau plasmid as template, human tau cDNA was amplified by polymerase chain reaction（PCR）. The expression plasmid was constructed by inserting tau cDNA into pET28a（ ＋ ） or pGEX-5X-1 （ ＋ ）. The positive recombinants were identified by restriction endonuclease digestion and expressed in E. coli BL21 induced by isopropyl-beta D-thiogalactopyranoside（IPTG）. The desired fusion proteins were confirmed by SDS-PAGE and Western blot. The expressed products were purified by affinity chromatography using His and GST fusion protein purification beads. Results Human tau cDNA was cloned into pET28a（ ＋ ） and pGEX-SX- 1 （ ＋ ） vectors and expressed in E. coli BL21 successfully. SDS-PAGE analysis and Western blot showed the molecu-lar weight of the fusion protein recognized by the specific monoclonal antibody tau-5 was that predicted. Additionally, the interest proteins were purificated prolifically. Conclusion The human tau protein is obtained by prokaryotic expression, which lays the foundation for study of its function.

关 键 词：tau蛋白质类/遗传学 大肠杆菌 基因表达 质粒 

分 类 号：R341.3[医药卫生—基础医学] R378