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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張麗冠(Li-Kwan Chang) | |
dc.contributor.author | Yu-Chun Lee | en |
dc.contributor.author | 李宇群 | zh_TW |
dc.date.accessioned | 2021-06-16T22:58:27Z | - |
dc.date.available | 2017-08-28 | |
dc.date.copyright | 2012-08-28 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-08 | |
dc.identifier.citation | Adamson, A. L. 2005. Effects of SUMO-1 upon Epstein-Barr virus BZLF1 function and BMRF1 expression. Biochem Biophys Res Commun 336 (1):22-28.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64747 | - |
dc.description.abstract | Epstein-Barr病毒 (EBV)屬於人類皰疹病毒科的一種,會感染B細胞或是上皮細胞,其生活史包含了潛伏期與溶裂期。當病毒在宿主細胞內受到刺激便會由潛伏期進入溶裂期,並在約24小時後開始大量複製以產生病毒顆粒,在其溶裂期的生活史中,極早期基因的產物Rta及Zta能夠促使早期基因的活化,而這類早期基因主要功能皆與病毒DNA的複製相關,此外,Rta、Zta亦能夠在Zta Response Element (ZRE)與宿主蛋白質MCAF1形成複合體,幫助病毒協同活化早期基因。本研究的目的是想要了解MCAF1,是否會參與EB病毒的溶裂複製。首先以免疫螢光染色的結果發現,當細胞在加藥誘導進入溶裂期後的12小時開始,MCAF1便與Rta或Zta位於細胞核內相同的位置,而這樣的現象直到加藥後的60小時依然可見;除此之外,在加藥後36小時觀察到Rta與MCAF1和病毒早期蛋白質BBLF2/3、BALF2和BBLF4位於細胞核內相同位置。另外,經由共免疫沉澱分析顯示,MCAF1會與病毒早期基因BALF2和BBLF2/3產生交互作用。最後,利用siRNA將細胞內的MCAF1抑制之後發現,雖然EB病毒早期基因EA-D的表現未受影響,但病毒複製的能力仍因MCAF1表現量的降低而有所下降。綜合上述結果,MCAF1除了能夠在病毒溶裂期的極早期幫助Rta、Zta活化早期基因外,亦藉由與早期蛋白質的交互作用參與EB病毒DNA的複製。 | zh_TW |
dc.description.abstract | Epstein-Barr virus (EBV) is a human gammaherpesvirus which infects B lymphocytes and epthilial cells with two distinct life cycle, latency and lytic cycle. When a host cell is suffered from stresses, EBV will reactivate from latency and amplify its genome via lytic replication rapidly in 24 hrs. During the lytic cycle, EBV espresses two immediate early proteins, Rta and Zta, which play a key role in the regulation of early genes that are associated with viral DNA replication. In addition, Rta and Zta form a complex via MCAF1 on the Zta response element (ZRE) to accelerate activation of early genes synergistically. This study is to demonstrate whether MCAF1 involves in EBV lytic replication. Firstly, immunofluorescence analysis showed that MCAF1 colocalizes with Rta and Zta after lytic induction for 12 hr - 60 hr. Moreover, Rta and MCAF1 also colocalize with BBLF2/3, BALF2 and BBLF4 at 36 hr after lytic induction. Additionally, MCAF1 interactes with BALF2 and BBLF2/3 by coimmunoprecipitation assay. Furthermore, RNAi assay demonstrated that knock down of MCAF1 decreases EBV lytic replication, although the expression of EA-D remains unchanged. Hence, MCAF1 not only participates in the activation of early gene with Rta and Zta, but also involves in EBV lytic replication by interacting with replication proteins. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T22:58:27Z (GMT). No. of bitstreams: 1 ntu-101-R99b22011-1.pdf: 8569447 bytes, checksum: abde64aba5e6143429c7e8d981b7587e (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 摘要 i
Abstract ii 總目錄 iii 圖目錄 v 表目錄 vii 第一章 緒論 1 一、 Epstein-Barr 病毒的發現 1 二、 EB病毒的組成 1 三、 EB病毒與疾病 1 四、 EB病毒的生活史 2 1. EB病毒的潛伏期 2 2. EB病毒的再活化 3 3. EB病毒的溶裂期 3 五、 極早期蛋白質Rta與Zta 4 1. Rta蛋白質 4 2. Zta蛋白質 5 六、 EB病毒的溶裂期複製 7 七、 MBD1-containing chromatin-associated factor 1 (MCAF1) 8 第二章 材料方法 11 一、 菌種細胞株 11 二、 質體建構 11 三、 勝任細胞 (Competent cell)的製備 11 四、 大腸桿菌轉型作用( Transformation) 11 五、 質體DNA抽取 12 六、 細胞轉染 (Transfection) 12 1. 293T細胞 12 2. P3HR1細胞 12 七、 EB病毒的溶裂期誘導 12 八、 西方點墨法 (Western blot) 12 九、 免疫螢光染色 (Immunofluorescence, IF) 13 1. 玻片置備 13 2. 免疫螢光染色玻片製作 13 十、 免疫沉澱法 (Immunoprecipitation, IP) 14 十一、 RNAi實驗 14 十二、 定量PCR (qPCR) 14 十三、 數據統計 15 十四、 Glutathione S-Transferase (GST) pull-down assay 15 第三章 實驗結果 16 一、 MCAF1與Rta、Zta蛋白質在EB病溶裂期的結合 16 二、 Rta與EB病毒溶裂複製複合體的結合 16 三、 MCAF1與EB病毒DNA複製複合體之結合 17 四、 抑制MCAF1會影響EB病毒之溶裂複製 18 第四章 綜合討論與未來研究方向 20 第五章 圖表 25 第六章 參考文獻 53 圖目錄 圖1. EB病毒的DNA複製 28 圖2. EB病毒的複製複合體 29 圖3. Rta與Zta蛋白質在EB病毒溶裂期的表現。 30 圖4-1. 以免疫螢光染色法觀察Rta與MCAF1於誘導EB病毒進入溶裂期後12小時在細胞內的位置。 31 圖4-2. 以免疫螢光染色觀察Rta與MCAF1於誘導EB病毒進入溶裂期後24小時之變化。 32 圖4-3. 以免疫螢光染色觀察Rta與MCAF1於誘導EB病毒進入溶裂期後36小時之變化。 33 圖4-4. 以免疫螢光染色觀察Rta與MCAF1於誘導EB病毒進入溶裂期後48小時之變化。 34 圖4-5. 以免疫螢光染色觀察Rta與MCAF1於誘導EB病毒進入溶裂期後60小時之變化。 35 圖4-6. 以免疫螢光染色觀察Rta與MCAF1於誘導EB病毒進入溶裂期後72小時之變化。 36 圖5-1. 以免疫螢光染色觀察Zta與MCAF1於誘導EB病毒進入溶裂期後0小時之變化。 37 圖5-2. 以免疫螢光染色觀察Zta與MCAF1於誘導EB病毒進入溶裂期後12小時之變化。 38 圖5-3. 以免疫螢光染色觀察Zta與MCAF1於誘導EB病毒進入溶裂期後24小時之變化。 39 圖5-4. 以免疫螢光染色觀察Zta與MCAF1於誘導EB病毒進入溶裂期後36小時之變化。 40 圖5-5. 以免疫螢光染色觀察Zta與MCAF1於誘導EB病毒進入溶裂期後48小時之變化。 41 圖5-6. 以免疫螢光染色觀察Zta與MCAF1於誘導EB病毒進入溶裂期後60小時之變化。 42 圖5-7. 以免疫螢光染色觀察Zta與MCAF1於誘導EB病毒進入溶裂期後72小時之變化。 43 圖6. Rta與EB病毒複製蛋白質在溶裂期時位於細胞相同位置。 44 圖7. Rta與複製蛋白質BSLF1在溶裂期時位於細胞相同位置。 45 圖8. Rta與複製蛋白質BSLF1的結合。 46 圖9. MCAF1與EB病毒複製蛋白質於溶裂期在細胞內的相對位置。 47 圖10. MCAF1與病毒複製蛋白質的結合。 48 圖11. 抑制MCAF1會影響EB病毒OriLyt的複製。 49 表目錄 表1. 本研究中所使用之質體 25 附錄1. EB病毒的生活史(摘錄自Rev Med Virol. 2005, 15(1):3-15) 50 附錄2. OriLyt主要蛋白質結合區(摘錄自Journal of Virology. 2005, 79, 245-256) 51 附錄3. 複製蛋白質與Rta、Zta關係圖(摘錄自PLoS Pathog. 2010, 19;6 (8): e1001054) 52 | |
dc.language.iso | zh-TW | |
dc.title | MCAF1參與Epstein-Barr病毒溶裂期之複製 | zh_TW |
dc.title | Involvement of MCAF1 in Epstein-Barr Virus Lytic Replication | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 劉世東,張沛鈞,張世宗 | |
dc.subject.keyword | 病毒,溶裂期複製, | zh_TW |
dc.subject.keyword | MCAF1,EBV,lytic replication, | en |
dc.relation.page | 72 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-08 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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