Mu2蛋白亚细胞结构定位及其相互作用蛋白的筛选

目的：采用果蝇S2细胞表达FLAG-Mu2蛋白，观察其亚细胞结构定位并纯化鉴定FLAG-Mu2质粒蛋白复合物，为Mu2蛋白的研究提供依据。方法：FLAG-Mu2转染果蝇S2细胞后表达FLAG-Mu2融合蛋白，共聚焦激光扫描显微镜下观察FLAG-Mu2蛋白的亚细胞定位，并通过核浆分离蛋白提取实验观察细胞核提取物中FLAG-Mu2蛋白的表达情况；采用免疫沉淀技术沉淀FLAG-Mu2复合物，并对其进行纯化及质谱测序以确定蛋白复合物的组分。结果：共聚焦激光显微镜下观察，FLAG-Mu2蛋白主要表达于细胞核中。核浆分离蛋白提取检测，融合蛋白相对分子质量约为140 000，主要存在于细胞核提取物中。纯化FLAG-Mu2蛋白复合物并进行蛋白质质谱分析，FLAG-Mu2复合物中可能含有pif1A、CG31782、mip120、CG31690和G protein-sa60A。结论：FLAG-Mu2蛋白在果蝇S2细胞中主要定位于细胞核，FLAG-Mu2蛋白复合物中有5种可能的蛋白组分。

Subcellular localization of Mu2 protein and screening of its interacting proteins

Objective:To express the FLAG-Mu2 protein in drosophila S2 cells,and to investigate its subcellular localization and to purify and identify the FLAG-Mu2 protein complex, and to provide basis for the study of Mu2 protein. Methods:The vector was transfected into drosophila S2 cells. The subcellular localization of FLAG-Mu2 protein was tested by confocal laser scanning microscope. The expression levels of FLAG-Mu2 protein in nuclear extractions were examined using nuclear and cytoplasmic protein extraction method. The FLAG-Mu2 complex was preciptated by precipitation technology and purified by chromatographic steps and the components were analyzed by mass spectrometry. Results:The confocal laser scanning microscope results showed that the FLAG-Mu2 protein mainly expressed in nucleus.The nuclear and cytoplasmic protein extraction results showed that the fusion protein, with a relative molecular weight of 140 000,mainly existed in nuclear extraction. The FLAG-Mu2 complex was purified and analyzed by mass spectrometry,and its results showed that there were 5 components,including pif1A, CG31782,mip120,CG31690,and G protein-sa60A,may be included in FLAG-Mu2 complex. Conclusion:The FLAG-Mu2 protein mainly localizes in the nucleus of drosophila S2 cells;a total of 5 kinds of proteins are found to be potential components of FLAG-Mu2 protein complex.