核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

Construction and Identification of a Nuclear Localization Signal Anal- ysis Vector--GST-EGFP Fusion Protein Expression Vector

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.

Objective To construct a mammalian fusion expression vector GST-EGFP for sublocalization analysis of a certain protein. Methods The GST encoding sequence was amplified from pGEX-2TK vector by PCR and inserted into pEGFP-N1 to construct pGST-EGFP mammalian fusion expression vector. The identified nuclear localization SV40 nuclear localization signal （NLS） was synthesized and sequentially inserted into pGST-EGFP to construct a system positive control vector. TNN12, a low molecular protein without any known NLS, was also inserted into the con- structed GST-EGFP vector. The subcellular localization of EGFP or any of the GST-EGFP-eontaining fusion proteins in transfected HeLa cells was determined under the fluorescence microscopy. Results Fluorescence microscopy revealed that EGFP was distributed in the entire cells, and GST-EGFP fusion protein was only expressed in cytoplasm. With SV40 NLS, EGFP-GST could be translocated from cytoplasm into nucleus. As compared with TNN12-EGFP which was expressed mainly in nucle-us, TNNI2-GST-EGFP was apparently only localized in cytoplasm. Conclusion The recombinant plasmid pGST-EGFP was established and might be employed in the identification of NLS and study of cytoplasm-nucleus translocation mechanism, especially for some low molecular weight proteins.