臺灣博碩士論文加值系統

中文摘要
C型肝炎病毒非結構蛋白質NS5B是以RNA為模板的RNA聚合酶，在C型肝炎病毒的複製上扮演一個重要的角色。已有文獻報導指出，當C型肝炎病毒進行複製時會有細胞因子與病毒本身的蛋白質參與其中，然而目前對於參與C型肝炎病毒複製複合體形成的因子了解有限。本論文利用GST pull down系統，以C型肝炎病毒的RNA聚合酶NS5B蛋白質做餌，在肝癌細胞株Huh 7中尋找與NS5B交互作用的蛋白質，經由質譜儀分析及西方墨點法確認後，確定候選蛋白質為脂肪酸合成酶。進一步以共同免疫沉澱法證明脂肪酸合成酶與NS5B在細胞內交互作用的關係。此外，利用脂肪酸合成酶的抑制劑C75作用在具有C型肝炎病毒複製系統的細胞中，發現此抑制劑可以使C型肝炎病毒的病毒蛋白質及RNA表現量減少。本研究證明脂肪酸合成酶在C型肝炎病毒複製機轉上具有其重要性存在，將進一步探討脂肪酸合成酶在病毒複製時所扮演的角色。

Abstract
Hepatitis C virus (HCV) nonstructure protein 5B (NS5B) is a RNA-dependent RNA polymerase that acts as a key player in the HCV replication complex. Some viral and cellular factors were reported to involve in HCV replication. However, the components of the HCV replication complex are still not yet completely understood. In this study, the HCV NS5B was used as the bait in a pull down assay to screen for NS5B-interacting proteins present in Huh 7 hepatoma cells. After mass spectrophotometric analysis, a putative lipogenic enzyme, fatty acid synthase (FAS), was identified to interact with NS5B. Co-immunoprecipitation analysis further confirmed the direct binding between the cellular protein and HCV NS5B. In addition, the expression of viral proteins and RNA in HCV subgenome replicon cells was decreased by treatment with C75, a specific FAS inhibitor. Together, these results suggest a critical role of FAS in the regulation of HCV infection through the modulation of HCV replication complex.

目錄
一. 文獻探討 4
1.1 C型肝炎病毒的歷史 4
1.2 C型肝炎病毒的基因體及構造組成 5
1.3 C型肝炎病毒複製相關因子 9
1.4 C型肝炎病毒複製模擬系統 10
1.5 脂肪酸合成酶 (fatty acid synthase ; FAS) 12
二. 材料與方法 14
2.1實驗室提供的質體 14
2.2 細胞培養方法 14
2.3 細胞轉染試驗 15
2.4 GST pull down試驗 15
2.5 蛋白質銀染法 17
2.6 西方墨點法 17
2.7 共同免疫沉澱法 18
2.8免疫螢光染色法 19
2.9 Membrane flotation assay 20
2.10脂肪酸合成酶活性抑制試驗 21
2.11 RNA 的萃取 21
2.12 反轉錄即時定量聚合酶反應 21
三. 結果 24
3.1 利用 GST pull down 系統尋找與HCV NS5B交互作用的蛋白質。 24
3.2 MALDI-TOF mass spectrometry 質譜儀進行蛋白質定序鑑定具交互作用的蛋白質。 24
3.3 藉由西方墨點法確認NS5B蛋白質交互作用的結果。 25
3.4 探討在細胞內脂肪酸合成酶與HCV NS5B交互作用的情形。 26
3.5脂肪酸合成酶在replicon cell中與HCV proteins的交互作用。 26
3.6探討C型肝炎病毒蛋白NS5B與脂肪酸合成酶在細胞內的相對位置。 27
3.7脂肪酸合成酶並未與C型肝炎病毒複製複合體ㄧ同存在detergent resistant membrane 中。 28
3.8脂肪酸合成酶抑制劑可以抑制C型肝炎病毒複製系統的表現。 28
四.討論 30
五.附圖 33
圖 1. HCV NS5B蛋白質可與細胞內蛋白質產生交互作用。 33
圖 2. 與HCV NS5B產生交互作用的胞內蛋白質為脂肪酸合成酶。 34
圖 3. 與HCV NS5B產生交互作用的蛋白質為脂肪酸合成酶。 35
圖 4. HCV NS5B與細胞內源性的脂肪酸合成酶(FAS)共存在於細胞質。 36
圖 5. 轉染表現HCV NS5B於HEK293 細胞亦可與脂肪酸合成酶產生交互作用。 37
圖 6. 脂肪酸合成酶在replicon cell中可與NS5B和NS5A產生交互作用。 38
圖 7. 脂肪酸合成酶不會與單獨表現在HEK293的NS5A蛋白質產生交互作用。 39
圖 8. 脂肪酸合成酶並非與C型肝炎病毒複製複合體一同存在detergent resistant membrane。 40
圖 9. 脂肪酸合成酶抑制劑可以降低C型肝炎病毒蛋白質的表現。 41
圖10. 以即時定量反轉錄PCR偵測HCV total RNA。 42
圖 11. 脂肪酸合成酶抑制劑可以降低C型肝炎病毒總RNA的表現。 43
六. 結論 44
七. 參考文獻 45

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