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研究生:李宗樺
研究生(外文):Zong Hua Li
論文名稱:研究與A型流感病毒NS1相關蛋白質交互作用之宿主細胞蛋白質
論文名稱(外文):Identification of Host Proteins Interacted with Influenza A Virus NS1-related Proteins by Proteomic Analysis
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:2015
畢業學年度:103
論文頁數:92
中文關鍵詞:A 型流感病毒NS1液相層析質譜儀14-3-3εPrp19CPSF2CPSF7
外文關鍵詞:Influenza A virus NS1LC MS/MS14-3-3εPrp19CPSF2CPSF7
相關次數:
  • 被引用被引用:0
  • 點閱點閱:134
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  • 下載下載:0
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致謝 iii
摘要 iv
Abstract v
目錄 vi
圖目錄 ix
表目錄 xi
一、 前言 - 1 -
1. A型流感病毒 - 1 -
2. A型流感病毒引起第一型干擾素反應(Type I interferon) - 2 -
3. A型流感病毒非結構蛋白質1 (NS1; Non-structural protein 1) - 3 -
4. A型流感NS1作用之蛋白質 (附錄一) - 4 -
5. A型流感病毒NS1 mRNA第二個轉譯起始點產生之蛋白質 - 5 -
6. 蛋白質體學與流感病毒 - 5 -
7. 14-3-3 protein epsilon (14-3-3ε,Gene name:YWHAE) - 6 -
8. Pre-mRNA-processing factor 19 (Prp19,Gene name:PRPF19) - 6 -
9. Cleavage and polyadenylation specificity factor subunit 2 and 7 (CPSF2 and 7,Gene name:CPSF2 and 7) - 7 -
10. 研究假說 - 7 -
11. 特定目標 - 8 -
二、 材料與方法 - 10 -
1. 細胞 - 10 -
2. 病毒株與病毒感染 - 10 -
3. 小RNA干擾 (siRNA)轉染 - 11 -
4. 病毒斑試驗 (Plaque assay) - 12 -
5. Real-time RT-PCR - 12 -
6. 質體 - 13 -
7. 質體轉染 - 13 -
8. 細胞蛋白質萃取 - 14 -
9. Dual-luciferase assay - 15 -
10. 免疫沉澱法(IP; Immunoprecipitation) - 15 -
11. 銀染(Silver stain) - 16 -
12. Colloidal blue stain - 17 -
13. 膠體內酵素水解(In gel digestion) - 17 -
14. 西方墨點法(Western blot) - 19 -
15. 生物資訊分析 - 19 -
三、 實驗結果 - 20 -
1. MALDI-TOF MS分析免疫沉澱後產物 - 20 -
2. LC MS/MS分析PR8 NS1相關蛋白質之免疫沉澱後產物 - 21 -
3. LC MS/MS分析H7N9 NS1的免疫沉澱後產物 - 24 -
四、 討論 - 28 -
五、 參考文獻 - 31 -
六、 圖表 - 35 -
七、 附錄 - 79 -
附錄一、A型流感NS1作用之蛋白質 - 79 -


圖目錄
圖一、檢測FLAG-NS1蛋白質表現量 (PR8) - 35 -
圖二、銀染 - 36 -
圖三A、檢測FLAG-NS1蛋白質表現量 (PR8) - 37 -
圖三B、Colloidal blue stain (PR8) - 38 -
圖四、宿主蛋白STRING 10分析 (PR8 NS1) - 39 -
圖五、宿主蛋白STRING 10分析 (PR8 tNS1) - 40 -
圖六A、檢測14-3-3ε蛋白質與NS1之交互作用 - 41 -
圖六B、驗證在細胞中過表現Myc-1433ε與流感病毒NS1相關蛋白質之結合 - 42 -
圖七、探討PR8 NS1對RIG-I及14-3-3ε所引起的第一型干擾素反應之影響 - 43 -
圖八、檢測Prp19蛋白質與NS1之交互作用 - 44 -
圖九、探討Prp19對於流感病毒生長之影響 - 45 -
圖十A、檢測FLAG-NS1蛋白質表現量 (H7N9) - 47 -
圖十B、Colloidal blue stain (H7N9) - 48 -
圖十一、宿主蛋白STRING 10分析 (H7N9 NS1) - 49 -
圖十二、檢測CPSF2及CPSF7蛋白質與H7N9 NS1之交互作用 - 50 -
圖十三、探討H7N9病毒感染對於宿主mRNA polyadenylation機制之影響 - 51 -

表目錄
表一、MALDI-TOF MS分析結果 - 52 -
表二、LC MS/MS分析結果 (PR8 NS1) - 53 -
表三、LC MS/MS分析結果 (PR8 tNS1) - 57 -
表四、LC MS/MS分析結果 (Overlap) - 60 -
表五、DAVID-Biological Process (PR8 NS1) - 61 -
表六、DAVID-Biological Process (PR8 tNS1) - 63 -
表七、宿主蛋白STRING 10分析總表 (PR8 NS1) - 65 -
表八、宿主蛋白STRING 10分析總表(PR8 tNS1) - 67 -
表九、LC MS/MS分析結果 (H7N9 NS1, ratio>3 P&;lt;0.05) - 69 -
表十、DAVID-Biological Process (H7N9 NS1) - 71 -
表十一、宿主蛋白STRING 10分析總表(H7N9 NS1) - 74 -


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