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研究生:翁千惠
研究生(外文):Chian Huey Woung
論文名稱:研究受A型流感病毒PA蛋白質調控表現之細胞基因在宿主反應及病毒複製的角色
論文名稱(外文):Role of the cellular genes dysregulated by influenza A virus PA protein in host response and viral replication
指導教授:郭瑞琳郭瑞琳引用關係
指導教授(外文):R. L. Kuo
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術暨檢驗學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:90
中文關鍵詞:A型流感病毒PA蛋白質USP40USP34SOD2
外文關鍵詞:Influenza A virusPA proteinUSP40USP34SOD2
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目錄
指導教授推薦書
論文口試委員會審定書
中文摘要 i
英文摘要 ii
目錄 iii
圖目錄 vii
表目錄 ix
第一章、研究背景與文獻回顧 1
1.1 A型流感病毒 (Influenza A virus) 1
1.1.1 A型流感病毒之基因體 1
1.1.2 A型流感病毒之流行病學史 1
1.1.3 A型流感病毒之傳播途徑及臨床症狀 2
1.1.4 A型流感病毒之生活史 2
1.1.5 A型流感病毒PA蛋白質之功能及結構 3
1.2 粒線體 (Mitochondria) 與A型流感病毒之關聯 4
1.3 SOD2 (Superoxide dismutase [Mn], mitochondrial manganese SOD, MnSOD) 蛋白質 5
1.4 USP40 (Ubiquitin carboxyl-terminal hydrolase 40) 及USP34 (Ubiquitin carboxyl-terminal hydrolase 34) 蛋白質 6
第二章、研究動機與目的 8
2.1 研究動機 8
2.2 研究目的 8
2.2.1 尋找可能受PA蛋白質調控之宿主細胞蛋白或RNA 9
2.2.2 驗證PA蛋白質對目標宿主細胞基因之調控 9
2.2.3 探討目標宿主細胞基因對A型流感病毒複製之影響 9
第三章、實驗設計 11
3.1 尋找可能受PA蛋白質調控之宿主細胞蛋白或RNA 11
3.2 驗證PA蛋白質對目標宿主細胞基因之調控 12
3.3 探討目標宿主細胞基因對A型流感病毒複製之影響 13
第四章、實驗材料與方法 15
4.1 細胞株與細胞培養 15
4.2 病毒株 15
4.3 轉染 (Transfection) 15
4.4 免疫螢光染色 (Immunofluorescence assay, IFA) 16
4.5 Isobaric tags for relative and absolute quantitation mass spectrometry (iTRAQ-MS) 定量蛋白體技術 (由吳治慶老師實驗室操作) 17
4.6 RNA Sequencing (RNA-seq) 定量RNA技術 (由譚賢明老師實驗室操作) 18
4.7 小干擾RNA (siRNA) 及RNA干擾 (RNA interfering) 19
4.8 病毒感染 19
4.9 西方墨點法 (Western blot) 20
4.10 反轉錄即時聚合酶連鎖反應 (Quantitative Reverse Transcription PCR, RT-qPCR) 21
4.11 病毒斑試驗 (Plaque assay) 22
4.12 細胞存活率計數 (Cell viability test) 23
4.13 KOD-Plus-Mutagenesis 23
第五章、實驗結果 24
5.1 篩選受PA蛋白質調控之目標宿主細胞基因 24
5.2 確認目標基因之表現量變化 26
5.3 宿主細胞基因 USP40 及 USP34 可能受 PA 蛋白質之核酸內切酶影響而表現量下降 27
5.4 降低細胞中USP40基因內源性表現量會影響A型流感病毒之複製 28
5.5 降低細胞中USP34基因內源性表現量並不影響A型流感病毒之複製 28
5.6 SOD2蛋白質對於A型流感病毒複製之影響 29
第六章、討論 32
第七章、參考文獻 36
第八章、圖表 44
第九章、附錄 76









圖目錄
圖一、以iTRAQ篩選受PA蛋白質調控之宿主細胞蛋白 44
圖二、以RNA sequencing篩選受PA蛋白質調控之宿主細胞基因 46
圖三、共同受PA蛋白質調控表現量上升之宿主細胞基因及蛋白質 47
圖四、以RT-qPCR偵測目標基因於過表現PA、PB1、PB2之相對mRNA表現量 49
圖五、以RT-qPCR偵測目標基因於2 M.O.I.之Influenza A/PR8/34/H1N1 virus感染之相對mRNA表現量 50
圖六、宿主細胞基因USP40及USP34可能受PA蛋白質之endonuclease domain影響而下調控 52
圖七、藉由RNA干擾降低細胞之內源性USP40基因表現量可降低Influenza A/PR8/34/H1N1 virus之複製量 54
圖八、藉由RNA干擾降低細胞之內源性USP34基因表現量不影響Influenza A/PR8/34/H1N1 virus之複製量 55
圖九、藉由RNA干擾降低細胞之內源性SOD2蛋白質表現量可降低Influenza A/PR8/34/H1N1 virus之複製量 57
圖十、藉由質體轉染過表現SOD2蛋白質表現量可降低Influenza A/PR8/34/H1N1 virus之複製量 59
圖十一、SOD2蛋白質對於病毒複製之影響可能與IFN-β之mRNA表現量無關聯性 61





















表目錄
表一、iTRAQ鑑定受PA蛋白質調控而表現量上升之宿主細胞蛋白質於細胞中所在位置分析 63
表二、iTRAQ鑑定受PA調控表現量下降之82種宿主細胞蛋白質 67
表三、iTRAQ及RNA-seq共同鑑定受PA蛋白質調控而表現量上升之32個宿主細胞基因 71
表四、本實驗使用之qPCR primer序列 75
表五、本實驗使用之siRNA序列 75
第七章、參考文獻
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