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.
出处
《安徽医科大学学报》
CAS
北大核心
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)
