hPER1相互作用蛋白的筛选及与近日节律关系的研究

发布时间：2018-05-23 12:22

  本文选题：近日节律 + hPER1　； 参考：《四川大学》2006年博士论文

【摘要】： 目的: 本研究的主要目的是寻找人脑组织cDNA文库中与hPER1蛋白相互作用的新蛋白,完善Per1相关的近日节律钟控系统的详细机制和信号传导通路。研究分为三个部分:1、人脑组织中与hPER1相互作用新蛋白的筛选与鉴定;2、新蛋白与hPER1相互作用的关键结构域的确定;3、新蛋白与hPER1的功能联系的研究。 方法与结果: 第一部分:人脑cDNA文库中与hPER1相互作用蛋白的筛选方法:采用BD Clontech公司提供的文库构建试剂盒(BD Matchmaker? Library Construction Screening Kits)构建人脑cDNA文库。通过RT-PCR扩增hPER1-PAS结构域的编码序列,获得的片断与质粒载体pGBKT7连接重组为诱饵质粒pGBKT7/hPer1PAS。采用顺序转染法构建酵母双杂交系统(MATCHMAKER GAL4 Two-Hybrid System,BD Clontech),筛选人脑cDNA文库中能与诱饵蛋白hPER1-PAS结构域相互作用的蛋白。并且采用营养缺陷培养基、β-半乳糖苷酶印膜法检测报告基因的表达;免疫共沉淀实验确证蛋白间的相互作用。阳性克隆子经PCR扩增,进行测序和比对分析。 结果:成功构建以pGBKT7/hPer1PAS为诱饵蛋白的表达质粒;构建人脑cDNA文库;成功构建pGBKT7/hPer1PAS和pGADT7-Rec/cDNA的酵母双杂交系统,并经过营养缺陷筛选、β-半乳糖苷酶印膜法检测以及免疫共沉淀实验,总共筛选到
[Abstract]:Objective: The main purpose of this study is to search for new proteins interacting with hPER1 protein in human brain cDNA library and to improve the detailed mechanism and signal transduction pathway of circadian clock control system related to Per1. The study was divided into three parts: 1, screening and identification of new proteins interacting with hPER1 in human brain tissues, identification of key domains of interaction between new proteins and hPER1, and functional relationship between new proteins and hPER1. Methods and results: Part one: screening method of interacting proteins with hPER1 in human brain cDNA library: using the library provided by BD Clontech to construct the kit of BD match maker? Library Construction Screening Kits) was used to construct the human brain cDNA library. The coding sequence of hPER1-PAS domain was amplified by RT-PCR. The obtained fragment was ligated with plasmid vector pGBKT7 and recombined into bait plasmid pGBKT7 / hPer1PAS. A yeast two-hybrid GAL4 Two-Hybrid system, BD Clontecha, was constructed by sequential transfection to screen proteins that interact with bait protein hPER1-PAS domain in human brain cDNA library. The expression of the reporter gene was detected by 尾 -galactosidase imprinting and the interaction between proteins was confirmed by co-immunoprecipitation. The positive clones were amplified by PCR, sequenced and compared. Results: the expression plasmid using pGBKT7/hPer1PAS as bait protein was successfully constructed, the human brain cDNA library was constructed, the yeast two-hybrid system of pGBKT7/hPer1PAS and pGADT7-Rec/cDNA was successfully constructed, and the screening of nutritional deficiency, 尾 -galactosidase membrane assay and co-immunoprecipitation test were carried out. Total filtered
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346

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本文编号：1924701