作　　者： ; ; ;

机构地区： 暨南大学生命科学技术学院

出　　处： 《现代生物医学进展》 2011年第14期2601-2604,2623,共5页

摘　　要： 目的:筛选与氧化固醇结合蛋白相关蛋白8(Oyxterol binding protein related protein 8,ORP8)相互作用的蛋白质,为揭示ORP8在细胞中的功能及其机制提供线索,并根据相互作用蛋白质初步探索ORP8的功能。方法:构建重组诱饵质粒pGBKT7-ORP8m,利用GAL4酵母双杂交系统筛选人Universal cDNA文库,筛选出与ORP8相互作用的蛋白质,通过GSTPull-down以及免疫共沉淀(Co-immunoprecipitation,Co-IP)验证蛋白质相互作用,并通过流式细胞仪检测过表达ORP8对HepG2细胞周期的影响。结果:酵母双杂交筛选得到了精子相关抗原5(Homo sapiens sperm associated antigen 5,SPAG5),体外GSTpull-down和Co-IP实验确证了ORP8-SPAG5的相互作用,流式细胞术显示ORP8过表达后与空载体相比,人肝癌细胞系HepG2的S期细胞数增加,G1期细胞数减少。结论:SPAG5是与ORP8相互作用的蛋白质,ORP8过表达影响人肝癌细胞系HepG2的细胞周期,可能通过SPAG5起作用。 Objective： To investigate the function of oxyterol binding protein related protein 8（ORP8）and to provide clues for better understanding its function and mechanism.Methods： Yeast two-hybrid system screening human universal cDNA library was used to obtained proteins interacting with ORP8.Putative interactions were further confirmed by GST Pull-down and Co-IP experiments.The effects of ORP8 on cell cycle were detected by flow cytometry in HepG2 cells.Results： ORP8 interacted with SPAG5 in two-hybrid screen using ORP8 as bait.ORP8-SAPG5 interaction was further confirmed in GST Pull-down and Co-IP experiments.Transient over-expression of ORP8 in HepG2 cells decreased cell population of G0/G1-phase and increased S-phase compared with that in control.Conclusion： ORP8 physically interact with SPAG5 and influence the cell cycle of HepG2 cell lines,probably through a mechanism in-volving SPAG5.ORP8-SPAG5 interaction provides clues to further explore the function of ORP8 and its mechanism.

关 键 词： 酵母双杂交 蛋白质相互作用 细胞周期

领　　域： [生物学] [生物学]