【摘要】 目的寻找与节律蛋白hPeriod1(hPer1)相互作用的蛋白,并对筛选出的层粘连蛋白受体Ⅰ(Lamr1)在节律系统中的功能作初步研究。方法分别构建pGAD rec脑/cDNA文库和pGBKT7/hPer1bH LH-PAS重组诱饵蛋白质粒,酵母双杂交筛选下丘脑里与hPer1相互作用的蛋白。利用RT-PCR技术检测新蛋白在脑、肺、心、肝、肾组织中的表达及在SH-SY5Y细胞中的节律性质。通过RNA干扰技术研究新蛋白与hPer1的关系。结果通过酵母双杂交筛选,证明Lamr1能与hPer1蛋白发生相互作用。该蛋白在脑、肺、心、肝、肾组织广泛表达,灰度比较显示经马血清诱导后的SH-SY5Y细胞的hPer1表达呈现近日节律,而Lamr1表达无节律变化。RNA干扰显示hPer1表达下调不影响Lamr1的表达。结论Lamr1能与hPer1蛋白发生相互作用,但其表达在RNA水平无节律变化。Lamr1参与细胞的黏附、转移和侵袭。其前体蛋白作为核蛋白参与细胞蛋白的合成。Lamr1可能参与hPer1相关的生物节律系统的信号传出。更多还原

【Abstract】 Objective To find the proteins which can interact with Per1 and to study the founction of Laminin receptor 1(Lamr1) in circadian rhythm.Method The GAL4 AD-brain fusion library was produced by cotransforming yeast with BD SMART brain ds cDNA and small-linearized pGADT7-Rec vector with the Matchmaker Library Construction & Screening Kits.The proteins interacting with Per1 were found using the yeast two-hybrid system with the bHLH-PAS domain of human hPer1 as bait.The expression of novel protein was observed with RT-PCR in brain,lung,heart,liver,and kidney samples.The relationship between hPer1 and novel protein with the RNA interfering plasmid of hPer1(pTER/hPer1)was studied.Result The results showed that Lamr1 could interact with hPer1.RT-PCR result showed that Lamr1 mRNA could be transcribed widely in brain,lung,heart,liver,and kidney and there was no conspicuous difference in the expression of Lamr1 in different tissues.The expression of Lamr1 in SH-SY5Y cell was detected by RT-PCR at the time points described above,and the result showed that its expression pattern does not have a circadian rhythm.However,the expression of hPer1 appeared to have a circadian rhythm.The hPer1 RNA interfering test showed that hPer1 was almost unexpressed in the SH-SY5Y cells after transfection with pTER/hPer1 plasmid,but the level of Lamr1 expression is not down regulated with hPer1.Conclusion Lamr1 can interact with hPer1.hPer1 is a critical protein of circadian system,and some investigators suggest it may be related to the output signaling pathway of the clock.This suggests Lamr1 is an important candidate for the output signaling pathway of the circadian clock.更多还原