【摘要】 目的:表达和纯化人TRIM5α嵌合体蛋白[TRIM5αH(R328-332)],并探讨该蛋白和HIV-1gag间的相互作用。方法:将构建的TRIM5α嵌合体pET28aTRIM5αH(R328-332),转化大肠杆菌BL21(DE3),获得重组表达质粒pETTRIM5αH(R328-332),30℃下经IPTG诱导,该融合蛋白在大肠杆菌BL21(DE3)中表达。获得的重组的蛋白再经过纯化,SDS-PAGE分析重组蛋白,并用免疫共沉淀技术和ELISA等检测重组蛋白与HIV-1gag间的相互作用。结果:构建的重组质粒在大肠杆菌中获得表达,重组TRIM5αH(R328-332)蛋白经纯化复性后,通过免疫共沉淀和ELISA等技术,证明TRIM5αH(R328-332)蛋白能够与HIV-1gag间的相互作用。结论:在大肠杆菌表达系统中成功表达了重组TRIM5αH(R328-332)蛋白,并且证实其在体外与HIV-1gag有结合作用。更多还原

【Abstract】 Objective:To express and purify the TRIM5α chimaera[TRIM5α H(R328-332)] protein and to explore the interaction between the TRIM5α H(R328-332)and HIV-1gag. Methods:The plasmid pET28aTRIM5α H(R328-332) was transformed to E.coli BL21 (DE3) strain ,and the expression of TRIM5α H(R328-332) protein was induced by IPTG,purified with Ni2+ chromatography.The expression and purification of TRIM5α H(R328-332) were analyzed by SDS-PAGE and Western blot,and the interaction between TRIM5α H(R328-332) and HIV-1gag was detected by co-immunoprecipitation,His pull-down and ELISA. Results:The recombinant plasmid pET28aTRIM5α H(R328-332) was successfully expressed in E.coli. The results showed that the purified full length TRIM5α H(R328-332) interacted with HIV-1gag protein. Conclusion:The human TRIM5α chimaera was expressed successfully in vitro,and the study demonstrates that the human TRIM5α chimaera interacts with HIV-1 gag in vitro.更多还原