Mu2蛋白亚细胞结构定位及其相互作用蛋白的筛选  

Subcellular localization of Mu2 protein and screening of its interacting proteins

作　　者：霍云龙[1] 王春玉[2] 赵越[2] 

机构地区：[1]中国医科大学盛京医院病理科,辽宁沈阳110003 [2]中国医科大学基础医学院染色质生物学研究室教育部医学细胞生物学重点实验室卫生部细胞生物学重点实验室,辽宁沈阳110122

出　　处：《吉林大学学报（医学版）》2016年第6期1054-1058,I0011,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(31271364;31401115);国家重点基础研究发展计划(973计划)项目资助课题(2013CB945200);教育部科学技术研究项目资助课题(v201308)

摘　　要：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。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 5components,including pif1 A,CG31782,mip120,CG31690,and G protein-sa60 A,may be included in FLAG-Mu2 complex.Conclusion：The FLAG-Mu2 protein mainly localizes in the nucleus of drosophila S2cells;a total of 5kinds of proteins are found to be potential components of FLAG-Mu2 protein complex.

关 键 词：Mu2蛋白 融合蛋白 亚细胞结构定位 相互作用蛋白筛选 

分 类 号：Q291[生物学—细胞生物学]