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研究生:林宜潔
研究生(外文):Yi-Chieh Lin
論文名稱:EB病毒Rta蛋白質刪減片段對野生型Rta功能之影響
論文名稱(外文):Effects of the truncated Rta mutants of Epstein-Barr virus on the biological function of wild-type Rta
指導教授:許翠瑛
指導教授(外文):Tsuey-Ying Hsu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:52
中文關鍵詞:EB病毒Rta蛋白
外文關鍵詞:RtaBRLF1Epstein-Barr virus
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Rta是由EB病毒裂解期特早期基因BRLF1所表現的產物。其可以藉由直接結合到DNA上、或者經由細胞因子作用的方式,轉活化許多EB病毒裂解期基因及細胞基因的表現,進而促使EB病毒由潛伏期轉變成裂解期。Rta的胺基酸序列上具有三個重要的功能區段,包括N端的雙體形成區段、DNA結合區段、以及C端的轉活化區段。本實驗將各種Rta刪減片段質體--包括R1-119 (Rta N端第1-119個胺基酸)、R1-333、R1-441、R100-333、R100-605、R364-441、R401-441及R401-605,單獨轉染入NA細胞,發現任何形式的刪減片段均無法促進EB病毒裂解期發生;且去除C端164個胺基酸的刪減片段(R1-441),會抑制全長Rta所誘導的裂解期蛋白質如Zta、EA-D、DNase、主要DNA結合蛋白(major DNA binding protein, mDBP)、BHRF1、以及BGLF4的表現,且抑制Rta轉活化BGLF5、BRLF1、及BZLF1啟動子的能力;因此可將R1-441作為一個具有顯性抑制效果的Rta突變。經由雙向免疫沈澱法以及更細部的刪減片段分析,顯示R1-441會與Rta形成異雙體,而有抑制Rta的功能;而當N端99個胺基酸刪減後,所得之R100-441則無法和Rta形成異雙體,也不具有抑制效能。但實驗結果也發現,另一個刪減片段R1-333,同樣也能與Rta形成異雙體,且缺少C端轉活化區段,卻不具有顯性抑制效能。
Rta, encoded by Eptein-Barr virus (EBV) immediate early gene BRLF1, is a transcription activator that mediates the switch from latency to lytic viral replication through direct DNA binding or through cellular factors. Three major functional domains have been defined on the Rta protein, including an amino-terminal dimerization domain, a DNA binding domain, and a carboxyl- terminal transactivation domain. Here we show that each deletion mutant of Rta, including R1-119 (aa 1 to 119), R1-333, R1-441, R100-333, R100-605, R364-441, R401-441 and R401-605 alone cannot induce EBV lytic cycle when transfected into NA cells. However, R1-441, deleted 164 amino acids from the C terminus of Rta, is able to function as a dominant negative mutant, inhibiting the Rta-induced expression of Zta, EA-D, DNase, mDBP (major DNA binding protein), BHRF1, and BGLF4, and the transactivation ability of wild-type Rta on BGLF5, BRLF1, and BZLF1 promoter. By co-immunoprecipitation analysis and further deletion of Rta, we find that R1-441 can dimerize with wild-type Rta and inhibit Rta’s function. When the N-terminal 99 amino acid is deleted, the R100-441 cannot dimerize with wild-type Rta and cannot interfere with Rta’s function. Another C-terminal truncated mutant, R1-333, however, is also able to interact with wild-type Rta, but cannot interfere the transactivating ability of Rta.
目錄

中文摘要………………………………………………………………1
英文摘要………………………………………………………………2
序論……………………………………………………………………3
實驗材料……………………………………………………………11
實驗方法……………………………………………………………17
結果……………………………………………………………………23
討論………………………………………………………………28
附錄……………………………………………………………………33
參考文獻………………………………………………………………43
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