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研究生: 呂祖皜
Lu, Tsu-Hau
論文名稱: 以DNA報導系統探討日本腦炎病毒RNA元件對轉譯的調控
Utilizing a DNA-based reporter system to investigate how RNA elements within Japanese encephalitis virus regulate translation
指導教授: 余佳益
Yu, Chia-Yi
學位類別: 碩士
Master
系所名稱: 醫學院 - 微生物及免疫學研究所
Department of Microbiology & Immunology
論文出版年: 2023
畢業學年度: 111
語文別: 中文
論文頁數: 64
中文關鍵詞: 黃病毒RNA元件RNA轉譯DNA報導系統日本腦炎病毒
外文關鍵詞: Flavivirus RNA elements, RNA translation, DNA reporter system, Japanese encephalitis virus
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  • 中文摘要 I 英文延伸摘要 II 致謝 VI 目錄 VII 壹、 緒論 1 一、 真核生物的轉譯起始機制 1 二、 真核生物中調控轉譯效率的機制 1 三、 RNA病毒轉譯起始機制 2 四、 黃病毒轉譯策略 3 五、 黃病毒高度保留RNA元件 4 六、 病毒元件在分子生物學上的應用實例 5 七、 研究動機 6 貳、 材料與方法 8 一、 質體DNA 8 二、 細胞株與培養方式 14 三、 細胞轉染 15 四、 大量DNA質體製備 15 五、 聚合酶鍊連鎖反應 16 六、 反轉錄 & 即時聚合酶連鎖反應 16 七、 勝任細胞製備 17 八、 免疫螢光染色法 17 九、 西方墨點法 18 十、 冷光蛋白酶分析 18 十一、 體外轉錄及轉譯實驗 (In vitro transcription& translation assay, TNT assay) 19 十二、 體外轉錄實驗 (In vitro transcription, IVT) 19 十三、 RNA二級結構預測 20 十四、 引子 20 參、 實驗結果 22 一、 建立探討轉譯效率的 DNA 報導系統 22 1. 篩選JEV RNA元件的研究範圍 22 2. DNA 報導系統的設計 22 3. P2A肽有效分離病毒蛋白殘基 23 4. HDVr達成高轉錄終結效率 23 5. CMV promoter最小化轉錄效率差異 24 二、 初始模板J(650+1000) 的轉譯能力顯著優於JR2A 24 三、 同時具有JEV 5’和3’兩端序列顯著提升轉譯能力 25 四、 JEV 3’基因組末端UTR以外區域不影響轉譯效率 25 五、 延長JEV 5’基因組末端可提升轉譯能力且主要位於197個核苷酸內 26 六、 刪除JEV 5’基因組197個核苷酸內元件導致轉譯能力下降 27 七、 JEV 3’UTR的轉譯能力需要DB1結構但不需要Domain III的參與 28 八、 此系統結果與RNA結果的差異 29 九、 結論 30 肆、 討論 32 一、 DNA系統設計的討論 32 二、 JEV RNA元件的討論 33 三、 DNA與RNA系統結果差異的討論 36 四、 研究限制 38 伍、 參考文獻 40 陸、 圖示 46 圖一、建立探討轉譯效率的 DNA 報導系統 47-48 圖二、初始模板J(650+1000) 的轉譯能力顯著優於JR2A 49-50 圖三、同時具有JEV 5’和3’兩端序列顯著提升轉譯能力 51-52 圖四、JEV 3’基因組末端UTR以外區域不影響轉譯能力 53-55 圖五、延長JEV 5’基因組末端可提升轉譯能力且主要位於197個核苷酸內 56-57 圖六、刪除JEV 5’基因組197個核苷酸內元件導致轉譯能力下降 58-59 圖七、JEV 3’UTR的轉譯能力需要DB1結構但不需要Domain III的參與 60-61 圖八、此系統結果與RNA結果的差異 62-64

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