大鼠热休克蛋白70的融合表达及纯化鉴定

Fusion Expression and Purification of Rat Heat Shock Protein 70

目的:在大肠杆菌中高效表达并纯化大鼠热休克蛋白(HSP)70与麦芽糖结合蛋白(MBP)的融合蛋白,以进一步研究细胞外HSP70的生物学功能。方法:用RT-PCR方法扩增目的基因,并将其克隆到原核表达载体pMAL-c2X中,酶切鉴定并进行DNA测序;将该重组表达载体转化大肠杆菌BL21,用IPTG在不同温度及时间下进行诱导表达,建立最佳诱导表达条件;采用Amylose树脂预装柱对目的蛋白进行亲和纯化,并对不同表达条件下的产物进行SDS-PAGE及Western blot分析。结果:克隆出目的基因,构建了融合表达载体pMAL-c2X/hsp70;诱导表达后经SDS-PAGE检测表明获得了目的条带,并纯化出纯度较高的融合蛋白;免疫印迹鉴定表明其具有抗原活性。结论:在大肠杆菌中高效表达并纯化了融合蛋白MBP-HSP70,为进一步研究细胞外HSP70的生物学效应提供了有用的材料。

Objective： To obtain high-level expression and purification of fusion protein of rat heat shock protein（HSP） 70 and （maltose-binding protein, MBP）, and laid foundations for studying biological function of extracellular HSP70. Methods： The genes of rat HSP70 by RT-PCR method was ligated, and was subcloned into pMAL-c2X which was then transformed into competent E.coli BL21. MBP-HSP70 fusion protein expressed in recombinant E.coli BL21 was induced by IPTG in different temperature and time, and was purified by Amylose sepharose affinity chromatography. The fusion protein was detected with SDS-PAGE and Western blot. Results： The pMAL-c2X/hsp70 recombinant plasmids and purified fusion protein were successfully obtained. Western blot analysis showed that the fusion protein was expressed. Conclusion： The recombinant expressing vector constructed could produce MBP-HSP70 fusion protein, and the high purified fusion protein was obtained. This study provides useful materials for investigation into extracellular HSP70 biological effect.