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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張麗冠 | |
dc.contributor.author | Ya-Yun Chu | en |
dc.contributor.author | 朱雅筠 | zh_TW |
dc.date.accessioned | 2021-06-15T01:24:29Z | - |
dc.date.available | 2012-07-27 | |
dc.date.copyright | 2009-07-27 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-23 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42814 | - |
dc.description.abstract | Epstein-Barr virus (EBV) 進入溶裂循環需要病毒極早期基因BRLF1與BZLF1轉錄出Rta與Zta蛋白質協同啟動溶裂循環。Zta可以直接結合上BRLF1的啟動子,Rta卻無法直接結合至BZLF1的啟動子,而是須藉由MAPK、JNK或ERK路徑活化ATF2,讓ATF2結合至ZII區域,促進BZLF1的轉錄。本實驗室先前發現MCAF1能做為Rta與Sp1的中介者,活化基因的轉錄,且在老鼠體內MCAF1的異構物mAM也被證實能與ATF2結合,因此本研究的目的是要探討Rta、MCAF1與ATF2之間的關係。首先利用免疫共沉澱分析與GST pull-down發現MCAF1與ATF2能直接結合,讓Rta透過MCAF1與ATF2形成複合體,且其中ATF2的第1至第119的胺基酸位置與第324到505的胺基酸位置能和MCAF1上的功能性胺基酸區域 Domain 1與Domain 2 結合。然後利用CHIP assay證明Rta-MCAF1-ATF2複合體會結合至含有AP-1結合序列的DNA片段上。接著使用冷光活性分析發現Rta能活化具AP-1序列啟動子的轉錄,且冷光值會隨著ATF2或MCAF1蛋白質表現量增加而上升;而利用dominant negative的ATF2或siRNA抑制MCAF1表現,則Rta活化AP-1序列的能力也被降低。接著透過冷光活性分析發現ATF2或MCAF1的異常表現也會影響Rta活化BZLF1啟動子的轉錄活性,證明Rta確實能以此複合體的形式,結合至BZLF1啟動子的ZII區域,進而促進BZLF1的轉錄。另外,Rta也能促進宿主細胞內BIC mRNA的表現,影響宿主細胞的生理功能。因此本篇研究結果證實Rta能藉由與MCAF1結合影響受ATF2轉錄因子調控基因的轉錄活性,可能對於誘導EBV進入溶裂循環相當重要。 | zh_TW |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:24:29Z (GMT). No. of bitstreams: 1 ntu-98-R96b47403-1.pdf: 11924087 bytes, checksum: 968d174e8975df4d45c3fe54de064326 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 目錄
口試委員會審定書………………………………………………………… i 誌謝………………………………………………………………………… ii 中文摘要…………………………………………………………………… iii 英文摘要…………………………………………………………………… iv 壹、前言……………………………………………………………………… 1 一、 Epstein-Barr virus ( EBV ) 的發現……………………………………1 二、 EBV的結構……………………………………………………………1 三、 與EBV相關的疾病……………………………………………………2 四、 EBV的生活史…………………………………………………………2 五、 Rta與Zta蛋白質………………………………………………………3 六、 EBV極早期基因 BRLF1與BZLF1的轉錄調控……………………5 七、 MBD1-containing chromatin-associated factor 1 (MCAF) ……………8 八、 Activating transcription factor-2 (ATF2) ………………………………8 九、 BIC 與 microRNA-155 (miR-155) …………………………………10 貳、研究目的………………………………………………………………………11 參、材料方法…………………………………………………………………………12 一、菌種與細胞株…………………………………………………………12 二、質體的建構……………………………………………………………12 三、勝任細胞的製備……………………………………………………………12 四、質體DNA的萃取…………………………………………………………13 五、RNA的萃取………………………………………………………………13 六、大腸桿菌的轉型作用………………………………………………………13 七、蛋白質的誘導表現…………………………………………………………13 八、細胞的轉染………………………………………………………………14 1、Electroporation…………………………………………………………14 2、Lipofectamine transfection……………………………………………14 九、EBV溶裂循環的誘導……………………………………………………14 十、細胞體內免疫沉澱分析…………………………………………………14 十一、細胞體外免疫沉澱分析………………………………………………15 十二、Glutathione S-transferase (GST) pull-down assay……………………15 十三、西方點墨法……………………………………………………………16 十四、染色質免疫沉澱分析…………………………………………………16 十五、冷光酵素分析…………………………………………………………17 十六、反轉錄聚合酶連鎖反應………………………………………………17 肆、結果……………………………………………………………………………19 一、Rta 與 ATF2 在細胞體內的結合……………………………………19 二、ATF2與MCAF1在細胞體外的直接結合與其結合位置…………19 1、ATF2與MCAF1在細胞體外直接結合………………………………20 2、ATF2上與MCAF1結合的位置分析…………………………20 3、MCAF1上與ATF2結合的區域分析…………………………20 三、Rta-MCAF1-ATF2複合體與AP-1序列的結合……………………21 四、Rta對具有AP-1結合序列啟動子活性的影響……………………21 五、 Rta活化具有AP-1結合序列啟動子時需要ATF2的參與………22 六、 MCAF1對Rta活化具有AP-1結合序列啟動子的影響…………23 1、MCAF1蛋白質大量表現對Rta活化啟動子的影響…………23 2、抑制MCAF1蛋白質表現對Rta活化啟動子的影響…………23 七、Rta對極早期基因BZLF1的活化需要MCAF1與ATF2的參與………24 1、ATF2對Rta活化Zp的影響………………………………………24 2、MCAF1對Rta活化Zp的影響……………………………………25 八、Rta對BIC基因轉錄的活化……………………………………………25 伍、討論……………………………………………………………………………27 陸、圖表集…………………………………………………………………………32 圖1:EBV的結構。…………………………………………………………32 圖2:EBV的生活史…………………………………………………………33 圖3:EBV在宿主細胞內的基因體構型。…………………………………34 圖4: EBV在溶裂期表現與誘導的基因。………………………………35 圖5:調控因子結合至BZLF1啟動子-220至+1中不同的區域。………36 圖6:ATF-2的結構與特性。………………………………………………37 圖7:Rta與ATF2在細胞體內的結合。…………………………………38 圖8:MCAF1與ATF2在細胞體外的結合分析。………………………39 圖9:ATF2上與MCAF1結合的區域。…………………………………40 圖10:MCAF1上與ATF2結合的區域。……………………………………41 圖11:Rta、MCAF1與ATF2結合在Zp啟動子上的ZII序列。…………42 圖12:Rta對含有AP-1序列的啟動子的活化。 ………………………………43 圖13:Rta活化具AP-1序列的啟動子需要ATF2的參與。………………44 圖14:MCAF1對於Rta調控含有AP-1序列啟動子的影響。……………45 圖15:Rta活化Zp需要ATF2的參與。……………………………………46 圖16:Rta活化Zp需要MCAF1的參與。…………………………………47 圖17:Rta促進BIC mRNA的表現。………………………………………48 附表:質體的建構。…………………………………………………………49 柒、參考文獻………………………………………………………………………53 | |
dc.language.iso | zh-TW | |
dc.title | EB病毒Rta蛋白質活化ATF-2所調控基因的轉錄活性 | zh_TW |
dc.title | Activation of ATF2-Mediated Transcription by Rta of Epstein-Barr Virus | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 劉世東,蔡錦華,洪義人,陳俊任 | |
dc.subject.keyword | EB病毒,病毒極早期蛋白質Rta,MCAF1,ATF2,轉錄調控, | zh_TW |
dc.subject.keyword | EBV,Rta,MCAF1,ATF2,transcription regulation, | en |
dc.relation.page | 68 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-24 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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