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目的构建人抗原R(HuR)的绿色荧光真核表达载体,初步探讨其生物学功能。方法克隆构建pEGFP-HuR(绿色荧光蛋白HuR)真核表达载体,并使用脂质体转染的方法将pEGFP-HuR转染入NIH 3T3细胞,Western blotting(WB)检测其表达,并给予热休克处理,利用荧光显微镜观察其在细胞内定位及与应激小体的共定位情况。结果 pEGFP-HuR真核表达载体构建成功,转染入细胞后,利用WB技术及荧光显微镜技术均能观察到其在细胞内高表达,并主要定位于细胞核内,给予热休克处理后,HuR向胞外移出并形成颗粒状小体,与应激小体的主要成分G3BP1蛋白存在共定位。结论 HuR主要定位于细胞核内,给予热休克处理后,HuR向胞外移动并与应激小体形成共定位,提示HuR定位的改变可能在应激时具有重要意义。
Objective To construct a green fluorescence eukaryotic expression vector of human antigen R (HuR) and to explore its biological function. Methods The eukaryotic expression vector pEGFP-HuR (green fluorescent protein HuR) was constructed and transfected into NIH 3T3 cells by lipofectamine 2000. Western blotting (WB) was used to detect the expression of pEGFP-HuR and heat shock Treatment, the use of fluorescence microscopy to observe its location in the cell and the co-localization of stress bodies. Results The eukaryotic expression vector pEGFP-HuR was constructed successfully. After transfected into the cells, the expression of pEGFP-HuR was observed in the cells by WB and fluorescence microscopy. The expression of pEGFP-HuR was mainly localized in the nucleus. After heat shock treatment, Extracellular movement and the formation of granular bodies, and stress the body’s main component G3BP1 protein co-localization exists. Conclusion HuR mainly locates in the nucleus. After heat shock treatment, HuR migrates extracellularly and co-locates with the stressor, suggesting that the localization of HuR may play an important role in stress.