核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

目的 构建谷胱甘肽转硫酶（GST）与EGFP相融合的新型蛋白质示踪载体--pGST-EGFP,以用于蛋白质细胞亚定位信号序列的深入分析.方法 以质粒pEGFP-N1为骨架,融合从pGEX-2TK载体中扩增的GST编码序列,构建成pGST-EGFP融合表达质粒;再插入人工合成的已知核定位蛋白SV40的核定位序列（NLS）,构建成pGST-EGFP-SV40 NLS作为阳性对照;另外,构建小分子量蛋白TNNI2在pGST-EGFP的融合表达质粒.将对照pEGFP-N1和各重组质粒分别用脂质体介导,瞬时转染HeLa细胞,荧光显微镜下观察蛋白的核定位情况.结果 单独表达的EGFP呈全细胞分布,而GST-EGFP融合蛋白只存在于细胞浆;SV40 NLS能将GST-EGFP融合蛋白带进细胞核.虽然TNNI2-EGFP融合蛋白的细胞亚定位呈现核内丰度更高的特点,但TNNI2-GST-EGFP融合蛋白仅限定于胞浆分布,提示TNNI2不能主动定位到细胞核中.结论 成功构建了蛋白质细胞亚定位示踪载体--pGST-EGFP.作为核定位信号分析系统,其对小分子蛋白细胞亚定位的示踪效果优于传统的pEGFP载体,更适用于科研工作中小分子量蛋白质核定位信号序列的研究.     

Heat-induced alterations in the localization of HSP72 and HSP73 as measured by indirect immunohistochemistry and immunogold electron microscopy.

The heat shock proteins are a family of stress-inducible proteins that act as molecular chaperones for nascent proteins and assist in protection and repair of proteins whose conformation is altered by stress. HSP72 and HSP73 are two major cytosolic/nuclear stress proteins of mammalian cells, with extensive sequence homology. HSP73 is constitutively expressed, whereas HSP72 is highly stress-inducible. However, it is unclear why two isoforms are expressed and whether these two proteins have different functions in the cell. To assist in the delineation of function, we have completed a detailed study of the localization of HSP72 and HSP73 in the cell before and after heat stress, using two different methods of detection. By indirect immunohistochemistry, the localization of these two proteins is similar, cytoplasmic and nuclear in nonstressed cells with a translocation to nucleoli immediately after heat. By the more sensitive immunogold electron microscopy technique, differences in localization were noted. In nonstressed cells, HSP72 was primarily nuclear, localized in heterochromatic regions and in nucleoli. HSP73 was distributed throughout the cell, with most cytoplasmic label associated with mitochondria. Mitotic chromosomes were also heavily labeled. After stress, HSP72 concentrated in nuclei and nucleoli and HSP73 localized to nuclei, nucleoli, and cytoplasm, with increased label over mitochondria. These differences in localization suggest that the HSP72 and HSP73 may associate with different proteins or complexes and hence have different but overlapping functions in the cell.