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研究生:黃怡綸
研究生(外文):Ie-Lun
論文名稱:利用酵母菌雙雜合系統尋找與RhoGDIβ結合的蛋白質
論文名稱(外文):Searching for proteins that bind to RhoGDIβ by yeast two-hybrid system
指導教授:林育誼
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:口腔醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:65
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先前由中山醫學大學楊肇基老師實驗室將ZAK蛋白質利用酵母菌雙雜交系統尋找與ZAK相關之蛋白質,其中找到了與調節small GTPases功能有關的RhoGDIβ。Rho GDP-dissocation inhibitors(RhoGDIs)為一內生性(endogenous)的Rho GTPase的抑制物(inhibitor),在調節生物活性(biological activity)上扮演著舉足輕重的角色。目前已知RhoGDIs以三種形式存在,分別為RhoGDIα (RhoGDI,RhoGDI-1)、RhoGDIβ (D4/LyGDI,RhoGDI-2)及RhoGDIγ (RhoGDI-3)而 RhoGDIβ大多表現於造血細胞(Haematopoetic cell)中。在我們實驗室中發現RhoGDIβ表現在心臟細胞時會造成心肌細胞的肥大,為了想更進一步的了解RhoGDIβ在心臟細胞中的功能便利用藉由酵母菌雙雜交系統的方式來尋找人類心臟細胞內可與RhoGDIβ結合的蛋白質來了解RhoGDIβ在心臟細胞中所扮演的角色。藉由酵母菌雙雜交系統所得到酵母菌菌落,送至定序後獲得二個與RhoGDIβ相關的蛋白質,分別為Rho A及 MYBPC3,這二個蛋白質都能與RhoGDIβ蛋白質相結合,接著我們將更進一步的去確認這二個蛋白質在in vivo中是否能真正的與RhoGDIβ結合及RhoGDIβ是否能調節此二蛋白質的功能。

To search effectors of ZAK and to study the ZAK signaling cascade, our laboratory used a yeast two-hybrid system to isolate ZAK associated proteins from a human heart cDNA library. One of the isolated cDNAs encoded Rho GDP dissociation inhibitor beta (RhoGDIβ). RhoGDIβ, also known as Ly-GDI or D4-GDI, which belongs to a family of Rho GDP dissociation inhibitors that includes RhoGDIα, RhoGDIβ and RhoGDIγ is thought to regulate many biological activities in cells and also regulate the activity and localization of Rho family proteins. RhoGDIβ is almost exclusively expressed in hematopoietic lineages. In the lab, we discovered that RhoGDIβ induced hypertrophic growth of a cultured rat cardiac cell line, H9c2. In order to study the mechanisms of RhoGDIβ to regulate hypertrophic growth, we used a yeast two-hybrid system to determine RhoGDIβ associated protein from a human heart cDNA library. Only four yeast colonies were growing in selection plates and we isolated these cDNAs. We then identified two possible RhoGDIβ associated proteins, RhoA and MYBPC3, after sequencing these cDANs. We set to determine the association of these proteins with RhoGDIβ in vivo in this study.

章節目錄
章節目錄          1
圖次                  4
附錄文次                6
英文摘要                    7
中文摘要                8      
第壹章 前言              9 
一. Rho GDP-dissocation inhibitors(RhoGDIs)介紹
二. 酵母菌雙雜合系統原理(Yeast two-hybrid system)
第貳章 研究動機           16
第參章 實驗材料 17
一. Kit
二. Buffer
三. 酵素(Enzyme)
四. 抗生素(Antibiocin)
五. 抗體(Antibody)
六. 細胞株
七. 菌株
八. 藥品
第肆章 實驗材料配製與方法 22
一. 藥品的配置與實驗方法
二. Yeast Two-Hybrid assay 操作方法
三. 共同轉染(Co Transfection)
四. 共同免疫沉澱分析 (Coimmunorecipitate Assay)
五.西方點墨法(Westernblot)

第伍章 實驗結果 40
一. RhoGDIβ的製備
二. 質體DNA轉植入酵母菌
三. 轉殖完的酵母菌圖在缺少特定胺基酸的培養基
四. 酵母菌交配
五. 酵母菌菌株篩選
六. 酵母菌內質體DNA的萃取及聚合酵素連鎖反應
七. 人類心臟細胞基因庫DNA定序及進入基因資料庫中比對
八. 設計primer找出所要全長DNA序列
九. 共同轉染及共同免疫沉澱分析檢測新基因與RhoGDIβ之間的關係
第陸章 實驗討論 51

第柒章 參考文獻 54

附錄 57


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