臺灣博碩士論文加值系統

冠狀病毒擁有一全長約 30kb 的正股單鏈 RNA 基因 (gRNA)，能夠轉譯出許多參與病毒轉錄及複製的非結構蛋白。組成病毒顆粒的結構蛋白則是以冠狀病毒獨特的不連續轉錄機制 (discontinuous transcription mechanism) 所產生的若干段亞基因 (sgmRNAs) 表現出。此機制是藉由轉錄調控序列 (transcription regulatory sequence, body TRS) 與前導轉錄調控序列 (leader TRS) 在轉錄時產生的模板切換 (template switch) 而調控，進而使產生的所有亞基因以及基因皆為共終端 (co-terminal)。
我們先前的研究確認了病毒核殼蛋白受 GSK-3 磷酸化後，擁有調控不連續轉錄機制進行的功能。磷酸化後的核殼蛋白能夠吸引宿主 RNA 解旋酶 DDX1 至核殼蛋白所在的聚合體中，進一步促進模板的讀續 (template read through) 而增加較長片段亞基因和基因之表現。本研究的目的在於建立缺陷干擾 RNA (defective interfering RNA, DI-RNA) 系統，以此探討 DDX1/pN 在病毒基因中的目標雙鏈 RNA，進而推測其促進連續轉錄的機制。另外，也會一併測試能夠阻斷此機制的有效抑制劑。
有鑑於 DDX RNA 解旋酶家族通常只能解旋少於 15 個鹼基對的雙鏈 RNA，故我們推論 DDX1/pN 的作用目標在於 body TRS 與 leader TRS 形成互補鹼基對的雙鏈 RNA。我們以基因上擁有 leader TRS 以及 body TRS 7 的 MHV DI-RNA 來驗證此假說，結果顯示當我們縮短 TRS 間的互補序列後，亞基因 7 的表現量也因此大幅度的下降，此結論支持 DDX1 是以 TRS 間形成的互補雙鏈 RNA 為目標，但還尚需進一步的實驗以確認此結果。接著，我們利用數種 GSK-3 抑制劑抑制核殼蛋白磷酸化的程度進而干擾病毒之生長，比較其抑制效率後，確認出其中一種化合物最具有對抗病毒生長的潛能。

碩士論文口試委員會審定書 I
誌謝 II
摘要 III
Abstract IV
目錄 VI
圖表目錄 VIII
第一章、序論 1
1.1 冠狀病毒的歷史 1
1.2 冠狀病毒的分類 1
1.3 冠狀病毒的傳播及致病性 2
1.4 冠狀病毒的基因結構 3
1.4.1冠狀病毒亞基因之結構 3
1.5 冠狀病毒的生活史 4
1.6 冠狀病毒之不連續轉錄現象及其調控機制 5
1.6.1 病毒基因體 5’ 端和 3’ 端序列之靠近 5
1.6.2病毒轉錄過程中基因模板切換步驟之調控 6
1.6.2.1 病毒自身RNA解旋酶可能參與促進模板切換步驟之調控 7
1.6.2.2 宿主 DDX1 RNA解旋酶交互作用可能參與減緩模板切換步驟之調控….………………………………………………………………………...7
1.7 冠狀病毒核殼蛋白 (nucleocapsid) 和宿主 DDX1 蛋白之交互作用 7
1.8 冠狀病毒之缺陷干擾 RNA (defective interfering RNA, DI-RNA) 8
實驗假說與目的 10
實驗目的 10
第二章、材料與方法 11
2.1 質體轉形與製備 (Transform and plasmid DNA extraction) 11
2.2 PCR recombination mutagenesis 11
2.3 體外轉錄 (In vitro transcription) 11
2.4 細胞培養 (Cell culture) 12
2.5 病毒感染 (Virus infection) 12
2.6 細胞轉染 RNA (PR6 RNA transfection) 13
2.7 樣本準備以及定量 (Sample preparation and quantification) 13
2.8 西方墨點法 (Western blotting) 13
2.9 北方墨點法 (Northern blotting) 14
2.10 病毒斑分析 (Plaque assay) 14
2.11 病毒液萃取 RNA 之 RT-qPCR 15
2.12 LD50 determination for GSK-3 inhibitors 15
2.13 化學品以及抗體 (Chemicals and antibodies) 15
第三章、結果 17
3.1 PR6 定序結果及 body TRS 序列突變 17
3.2 利用 PR6 RNA 轉染以及 PR6 病毒感染建立表現系統 18
3.3 縮短 TRS 序列的 PR6 突變型 sgRNA 的表現量大幅下降 19
3.4 GSK-3 抑制劑處理對於野生型與突變型 PR6 病毒感染的影響 19
3.5 GSK3 inhibitors 對於抑制 JHMV 感染的有效濃度測試 20
第四章、討論 22
圖表 25
參考文獻 38

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