本文選題：近日節(jié)律 + hPER1�。� 參考：《四川大學》2006年博士論文

【摘要】： 目的: 本研究的主要目的是尋找人腦組織cDNA文庫中與hPER1蛋白相互作用的新蛋白,完善Per1相關的近日節(jié)律鐘控系統(tǒng)的詳細機制和信號傳導通路。研究分為三個部分:1、人腦組織中與hPER1相互作用新蛋白的篩選與鑒定;2、新蛋白與hPER1相互作用的關鍵結構域的確定;3、新蛋白與hPER1的功能聯(lián)系的研究。 方法與結果: 第一部分:人腦cDNA文庫中與hPER1相互作用蛋白的篩選方法:采用BD Clontech公司提供的文庫構建試劑盒(BD Matchmaker? Library Construction Screening Kits)構建人腦cDNA文庫。通過RT-PCR擴增hPER1-PAS結構域的編碼序列,獲得的片斷與質(zhì)粒載體pGBKT7連接重組為誘餌質(zhì)粒pGBKT7/hPer1PAS。采用順序轉(zhuǎn)染法構建酵母雙雜交系統(tǒng)(MATCHMAKER GAL4 Two-Hybrid System,BD Clontech),篩選人腦cDNA文庫中能與誘餌蛋白hPER1-PAS結構域相互作用的蛋白。并且采用營養(yǎng)缺陷培養(yǎng)基、β-半乳糖苷酶印膜法檢測報告基因的表達;免疫共沉淀實驗確證蛋白間的相互作用。陽性克隆子經(jīng)PCR擴增,進行測序和比對分析。 結果:成功構建以pGBKT7/hPer1PAS為誘餌蛋白的表達質(zhì)粒;構建人腦cDNA文庫;成功構建pGBKT7/hPer1PAS和pGADT7-Rec/cDNA的酵母雙雜交系統(tǒng),并經(jīng)過營養(yǎng)缺陷篩選、β-半乳糖苷酶印膜法檢測以及免疫共沉淀實驗,總共篩選到
[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
【學位授予單位】：四川大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R346

【參考文獻】

中國期刊全文數(shù)據(jù)庫 前2條

1 馬洪波,杜堅;酵母雙雜交系統(tǒng)的研究進展與應用[J];中國國境衛(wèi)生檢疫雜志;2004年02期

2 徐東剛;酵母雙雜交系統(tǒng)的應用研究進展[J];國外醫(yī)學.臨床生物化學與檢驗學分冊;2001年06期

，

本文編號：1924701