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研究生:陳彥愷
研究生(外文):Yen-Kai Chen
論文名稱:丙型肝炎病毒核心蛋白與核酸交互作用影響其結構變化
論文名稱(外文):Hepatitis C Virus core protein structure changes induced by protein/nucleic acid interactions
指導教授:劉哲文劉哲文引用關係
指導教授(外文):Je-Wen Liou
學位類別:碩士
校院名稱:慈濟大學
系所名稱:微生物學免疫學暨生物化學碩士班
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:41
中文關鍵詞:丙型肝炎病毒核心蛋白組裝核酸/蛋白質交互作用
外文關鍵詞:HCV core assemblynucletide/protein interaction
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丙型肝炎病毒(HCV)是造成慢性肝炎的致病原之一,目前尚未有疫苗及全效性的治療法能有效預防與治療HCV的感染。了解HCV的感染過程對於藥物開發及環境預防有很大的幫助。HCV為攜帶RNA遺傳物質的病毒,其基因組可分為兩部分:結構性基因與非結構性基因。結構性基因中的核心蛋白(core)基因所轉譯出的核心蛋白為組成病毒顆粒的單元之一,對HCV的感染力非常重要。為了瞭解病毒組裝過程中HCV病毒顆粒與RNA基因組包覆過程,我們利用大腸桿菌M15菌株成功地大量表現不同截短長度的HCV核心蛋白,並利用Tris-urea緩衝液分離純化出核心蛋白,再用His-tag親和性層析與膠體過濾法進一步純化核心蛋白。我們利用具有二級結構的tRNA模擬HCV基因體中的核醣體進入位置(IRES),與純化後的HCV核心蛋白1-73、1-116片段或1-191片段混合後使核心顆粒組裝後,利用流式細胞儀觀察核心蛋白顆粒,發現顆粒與標定上螢光染劑fluorescein的tRNA有交互作用。而螢光消光實驗中也證明核心顆粒能抑制碘化鉀對螢光標定的tRNA之消光作用。原子力顯微鏡觀察結果顯示:在1-116和1-191片段中,含有tRNA之核心蛋白顆粒會有直徑增加的現象。根據以上結果,我們推論tRNA會與HCV核心蛋白1-73、1-116片段及1-191片段之間產生交互作用、並且被包覆於顆粒中,而此交互作用會進而影響組成核心顆粒的大小。
Hepatitis C Virus (HCV) is one of the causes of chronic hepatitis. In spite of the serious problems caused by this virus, there is currently no effective vaccine or treatment to prevent or cure HCV infection. The core protein translated by HCV genome is assembled into virus cores and is important for viral infectivity. The virus core must interact with HCV genomic RNA and assemble to yield infectious virus particles. In order to understand the process of HCV core–RNA interactions, we applied Escherichia coli M15 strain to over-express HCV core proteins with different truncations. The truncated core proteins have been successfully purified with His-tagged affinity and gel filtration columns with tris-urea buffer system. The purified core protein 1-73, 1-116, and 1-191 are observed to interact with fluorescein-labeled transfer-RNA (tRNA), which is used to mimic the structured internal ribosomal entry site of HCV RNA as revealed by flow cytometry. Fluorescence quenching experiment using potassium iodide quenching demonstrated that labeled tRNA is packaged into core-like particles assembled by different truncated core proteins. The atomic force microscopy showed that the diameters of HCV core protein 1-116 and 1-191 formed particles were increased when tRNA was included. We propose that HCV core protein 1-73, 1-116, and 1-191 can interact with tRNA, and the tRNA is packed into the particle. These interactions indeed affect the quaternary structures of the proteins and thus change the morphology of nucleocapsid formed.
Abstract i
摘要 ii
1 Introduction 1
1.1 Hepatitis C Virus (HCV) 1
1.2 HCV Core Protein 2
1.3 HCV and Structural Nucleotides and the Aim of the Study 3
2 Materials and methods 4
2.1 Over-expression of HCV core proteins using E. coli 4
2.2 Purification of HCV core proteins 5
2.3 in vitro assembly of HCV core-like particles 6
2.4 Flow cytometry 6
2.5 Fluorescence quenching 7
2.6 AFM imaging of HCV core-like particles 7
2.7 Software 8
3 Results and Discussion 9
3.1 Expression and purification of recombinant HCV core protein 10
3.2 Flow cytometry reveals that core protein-formed particles were interacted with structural RNA 10
3.3 The interaction between HCV core proteins and fluorescein-labeled tRNA can protect the fluorescence from fluorescence quenching 11
3.4 Morphology of the core-like particles changed in the presence of structural RNA 13
4 Conclusion 16
References 17
Figures 20
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