驱动蛋白分子Kif18A各功能域细胞定位的研究  

Analyzing intracellular localization of structural domains in kinesin motor protein Kif18A

作　　者：王东秋[1] 高杰[1] 田明忠[1] 朱长军[2] 

机构地区：[1]山东大学医学院细胞学生物研究所,济南250012 [2]天津师范大学化学与生命科学院,天津300074

出　　处：《山东大学学报（医学版）》2010年第5期40-43,共4页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金面上项目(30971446);国家自然科学基金青年科学基金资助课题(30700396)

摘　　要：目的探讨人驱动蛋白分子Kif18A的各功能域在真核细胞中的定位。方法构建驱动蛋白分子Kif18A野生型及各功能域(马达区、杆状区、尾区和马达区含部分杆状区)的绿色荧光蛋白表达质粒,分别转染人乳腺癌细胞MCF7,运用聚丙烯酰胺凝胶电泳和免疫印迹法验证各蛋白的表达,免疫荧光染色法分别对细胞进行微管蛋白和DNA染色,荧光显微镜下观察各绿色荧光融合蛋白的具体亚细胞定位。结果有丝分裂间期,Kif18A蛋白分子的马达区绿色荧光沿微管分布,尾区在细胞核和微管上均有分布,杆状区则散在分布在胞质中;有丝分裂末期,Kif18A的马达区仍沿微管分布,而马达区含部分杆状区主要集中在中体部位。结论 Kif18A蛋白分子的马达区和尾区与微管共定位;尾区含核定位序列;马达区和杆状区共同作用才能使该蛋白在有丝分裂末期定位于中体。Objective To explore the intracellular localization of structural domains in kinesin motor protein Kif18A in eukaryotic cells.Methods The GFP-expressing plasmids of the Kif18A＇s wild type were construced and each domain including motor,stalk,tail and motor with part of stalk domain.Then these plasmids were transfected into the breast cancer cell MCF7.The GFP-fused proteins were detected by SDS-PAGE and Western blot.The immunofluorescence staining technique was used to stain microtubules and DNA in cells.The intracellular localization of GFP-fused proteins was determined under fluorescent microscopy.Results In interphase cells,the GFP-motor domain was distributed along microtubules.The GFP-tail domain was localized in the nucleus as well as along microtubules and the GFP-stalk domain was scattered throughout the cytoplasm.In telophase cells,the GFP-motor domain was also distributed along microtubules.In contrast,the GFP-motor with part of the stalk domain was concentrated on the mid-body.Conclusions The motor and tail domain of Kif18A can both associate with microtubules and the tail domain contained nuclear localization signal（NLS）.Only with part of the stalk region,is the motor domain of Kif18A able to localize on the mid-body.

关 键 词：人驱动蛋白分子Kif18A 功能域 人乳腺癌细胞MCF7 细胞定位 

分 类 号：R454.2[医药卫生—治疗学]