【摘要】 为寻找与G-CSF受体胞内区相互作用的蛋白,探讨其可能的信号传导机制,应用酵母双杂交技术从小鼠胎肝文库中筛选到与G-CSF受体胞内区相互作用的蛋白—细胞色素C氧化酶Ⅱ亚基（COXⅡ）;采用PCR技术从小鼠肌肉cDNA中克隆到全长COXⅡ,并应用哺乳动物细胞双杂交实验验证了COXⅡ与G-CSF受体胞内区相互作用;构建了一系列G-CSF受体胞内区短截体,应用细胞双杂交技术确定COXⅡ主要结合于G-CSF受体的Box3区;随后构建了COX Ⅱ-GFP融合表达载体,观察到COXⅡ在COS₇细胞中为全细胞分布。这些结果为揭示COXⅡ的新功能及发现G-CSFR新的信号传递通路提供了线索。更多还原

【Abstract】 To explore new pathways of G-CSF receptor signal transduction, a highly sensitive protein-protein interaction system, Yeast Two-hybrid System, was applied to screen the binding proteins of the intracellular domain of G-CSF receptor from mouse fetal liver library. Sequence analysis from one of the positive clones revealed that the cDNA insert encoded a protein identical to mouse cytochrome C oxidase subunit Ⅱ (COX Ⅱ). The full-length COX Ⅱ was amplified from mouse muscle cells by RT-PCR method, and the interaction between COX Ⅱ and G-CSFR were confirmed in vivo with mammalian two-hybrid assay. To delineate the region in G-CSFR required for association with COX Ⅱ, we made a series of G-CSFR deletion mutants and estimated their interaction with COX Ⅱ by using mammalian cell two-hybrid assays. The results clearly indicate that the Box3 region of G-CSFR is critical for the interaction with COX Ⅱ. The localization assays showed that COX Ⅱ dispersed in whole transfected COS7 cells. It is the first time to find that G-CSF receptor interacts with COX Ⅱ, this research in a certain sense may be important to understand the complexity of G-CSF receptor signal transduc-tion.更多还原