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研究生:黃鵬年
研究生(外文):Peng Nien Huang
論文名稱:腸病毒71型5端非轉譯區位於真實起始子附近的區域扮演之角色
論文名稱(外文):Roles of "Spacer region" Near Authentic Start Codon in 5' Untranslated Region of Enterovirus 71
指導教授:施信如施信如引用關係
指導教授(外文):S. R. Shih
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
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:2008
畢業學年度:96
論文頁數:74
中文關鍵詞:腸病毒71型5端非轉譯區
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腸病毒71型 (EV-71)利用病毒基因體5端非轉譯區(5’UTR)中,internal ribosome entry site (IRES)來啟動轉譯。5’UTR有六個stem-loops,在stem-loop VI和真實起始子間的區域(n.t. 636-745),稱為spacer region,在病毒中扮演的角色仍不清楚。本論文分兩部分探討spacer region對病毒所扮演之角色,第一部分是cis-regulation,藉由改變spacer region長度,觀察對病毒的影響。利用dicistronic reporter system,插入不同長度spacer region的5’UTR,觀察對IRES活性的影響。當刪除spacer region後半部(△692-745),病毒的生長減緩且IRES活性下降至30.1%。第二部份是trans-regulation,細胞中的蛋白質也會影響IRES活性。因此用競爭試驗發現far upstream element binding protein (FBP)、insulin-like growth factor II mRNA-binding protein 1 (IMP-1)、及heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1)會和EV-71 5’UTR有專一性的相互作用。再利用biotin RNA-protein pull-down assay證實此三個蛋白質會與spacer region相互作用。利用siRNA抑制這三種蛋白質表現,用35S-Met labeling觀察對病毒蛋白質合成之影響。結果顯示當FBP表現量受抑制,會影響病毒生長及蛋白質合成。本篇論文發現,n.t. 692-745對IRES活性是重要的,且FBP與spacer region產生專一性相互作用促進病毒生長與蛋白質合成。
The 5’ untranslated region (5’UTR) in the genomic RNA of Enterovirus 71 (EV-71) contains an internal ribosomal entry site (IRES) that directs the initiation of viral protein translation. Some cellular proteins affect IRES activity. The 5’UTR has six stem loops and a spacer region (n.t. 637-745 between stem-loop VI and authentic start coden). The role of this region in viral infection has not yet been identified. This study, truncated this region into different lengths of infectious plasmids, including full-deletion, half-deletion and duplication. The infectious plasmid with different lengths of spacer regions can produce infectious virus. The IF-△692-745 truncated virus grows slowly in RD cells. A dicistronic reporting system was used to analyze the effects of this region different lengths on IRES activity. The region was truncated on dicitronic report plasmid. The IRES activity of the reporter plasmid pRHF-△692-745 decreased to 30.1%. Further, some cellular proteins interacting with EV-71 5’UTR were noted, some of which are important for viral protein translation. Competition assay and biotinylated RNA mapping, identified of far upstream element-binding protein (FBP), insulin-like growth factor II mRNA-binding protein 1 (IMP-1), and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as the spacer region specific associated proteins. Viral yields were lower, and protein synthesis was decreased significantly in FBP siRNA knock-down cells. These analytical results demonstrated that n.t. 692-745 affects IRES activity, FBP interacts with spacer region to facilitate viral protein synthesis.
指導教授推薦書 i
口試委員會審定書 ii
博碩士論文電子檔案上網授權書 iii
誌 謝 v
中文摘要 vi
英文摘要 vii
目 錄 viii
圖表目錄 xi
第一章 前言 - 1 -
1.1 腸病毒的分類 - 1 -
1.2 腸病毒的流行病學 - 2 -
1.3 腸病毒的一般特性 - 2 -
1.4 腸病毒的顆粒結構 - 2 -
1.5 腸病毒的生活史 - 3 -
1.6 腸病毒的基因體 - 4 -
1.7 腸病毒的5’端非轉譯區域 - 4 -
1.8 Internal ribosomal entry site (IRES) - 5 -
1.9 小RNA病毒科轉譯相關的細胞蛋白質 - 6 -
1.10本實驗室在腸病毒71型5’端非轉譯區域的研究結果 - 7 -
1.11 研究目的 - 8 -
第二章 實驗材料與方法 - 9 -
2.1 建立不同spacer region 長度EV-71 infectious plasmid- 9 -
2.2 In vitro RNA transcription - 9 -
2.3 病毒RNA的轉染試驗 - 10 -
2.4 病毒斑試驗 (plaque assay) - 11 -
2.5 病毒生長曲線 - 11 -
2.6 建立不同長度spacer region的dicistronic reporter system表現載體 - 12 -
2.7 dicistronic reporter system - 13 -
2.8 Cellular protein extraction - 13 -
2.9 In vitro RNA transcription - 13 -
2.10 Affinity Extractions Using Biotin-Tagged RNAs- 14 -
2.11 FBP、IMP-1、及hnRNP A1 siRNA 轉染試驗 - 15 -
2.12 35S-Met labeling - 15 -
第三章 結果 - 17 -
3.1 △692-745 infectious virus的plaque size減小 - 17 -
3.2 Spacer region後半部刪除的EV-71 (△692-745)生長曲線最為緩慢 - 18 -
3.3 Spacer region 後半部會影響IRES啟動轉譯 - 18 -
3.4 FBP, IMP-1, and hnRNP A1與spacer region產生specific interaction - 20 -
3.5 EV-71在FBP knocked-down cell中生長情形較為緩慢 - 21 -
3.6在FBP knocked-down cell中病毒蛋白質合成量減少 - 21 -
第四章 討論 - 23 -
參考文獻 - 32 -
圖表 - 40 -
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