臺灣博碩士論文加值系統

　　2013年於美國及台灣相繼爆發嚴重Porcine epidemic diarrhea disease (PED) 疫情，造成嚴重的經濟損失。然而造成本次疫情嚴重性上升的因子尚不明憭。在過去的研究中顯示，冠狀病毒的5’UTR及3’UTR是病毒的cis-acting RNA elements，並且在病毒的基因表達中扮演了重要的角色。因此本研究先分析台灣PEDV的5’UTR及3’UTR的序列，再將分析的序列與CV777比較，以探討台灣PEDV UTR的序列是否有變異，並分析此變異對於病毒基因表達的影響。我們發現：（i）與原毒株 (prototype) CV777 Europe1980 (CV777) 相比，台灣的PEDV在5’UTR及3’UTR有所改變。（ii）在UTR的相似性及親緣樹分析中我們發現，台灣的PEDV與美國較為相近。(iii) 在病毒的基因表達分析方面，我們發現，相較於CV777，台灣的PEDV在轉譯效率上並沒有明顯差異，但在複製效率方面，台灣PEDV則高出CV777十倍以上。由以上結果可以推測，台灣PEDV於UTR的改變可能近年來造成台灣PED疫情嚴重性增加的因子之一。

　　The outbreak of porcine epidemic diarrhea (PED) has resulted in considerable economic losses both in the United States and Taiwan in 2013; however, the factors leading to the increased severity of the disease remain to be elucidated. The sequence and structures located in the 5’untranslated region (UTR) and 3’UTR have been demonstrated to be cis-acting elements required for coronavirus gene expression. We, therefore, in the present study seek to determine whether the sequence variation in UTRs of PEDV Taiwan strain (PEDV TW) is one of potential factors causing the increased virulence of the virus. The findings are as follows. (i) There are several sequence variations occurred in the 5’ UTR and 3’UTR of PEDV TW in comparison with those of prototype PEDV CV777. (ii) Phylogenetic analyses of UTRs revealed that PEDV strains in Taiwan are similar to the major strains in USA. (iii) Functional analyses suggested that there is no significant difference between PEDV TW and PEDV CV777 in translation efficiency; however, replication efficiency of PEDV TW is ten folds higher than that of CV777. Accordingly, the alternations of sequence in UTRs may be one of the factors for the increased severity of PED in Taiwan.

摘要 i
Abstract ii
目次 iii
表次 vi
圖次 vii
第一章 前言 1
第二章 文獻探討 2
第一節 冠狀病毒之基本簡介 2
1.1. 冠狀病毒之背景 2
1.2. 冠狀病毒之分類 2
1.3. 冠狀病毒之基因體結構 2
第二節 Cis-acting RNA elements 在病毒基因表達的重要性 3
2.1. Cis-acting RNA elements的特性 3
2.2. 冠狀病毒5’UTR之cis-acting RNA elements 4
2.3. 冠狀病毒3’UTR之cis-acting RNA elements 4
第三節冠狀病毒的轉譯、複製及轉錄 4
3.1. 冠狀病毒的生活史 4
3.2. 冠狀病毒複製相關蛋白質之轉譯 5
3.3. 冠狀病毒基因體的複製（正股基因體的合成） 5
3.4. 冠狀病毒次級基因體RNA的轉錄 6
第四節 利用冠狀病毒的天然缺陷株RNA （defective interfering RNA，DI RNA） 研究cis-acting RNA elements對於轉譯及複製的功能 6
4.1. 冠狀病毒天然缺陷株RNA （DI RNA） 6
4.2. 以DI RNA作為研究模組之原因 6
4.3. 利用冠狀病毒的DI RNA研究病毒的轉譯 7
4.4. 利用冠狀病毒的DI RNA研究病毒的複製 7
第三章 材料與方法 8
第一節 台灣PEDV 5’UTR及3’UTR序列分析 8
1.1. PEDV檢體來源 8
1.2. 自檢體萃取RNA 8
1.3. 互補DNA (cDNA) 合成 8
1.4. 以聚合酶連鎖反應增幅5’UTR及3’UTR片段 8
1.5. 以軟體分析台灣PEDV與各國PEDV於UTR之序列差異 9
1.6. 以軟體分析台灣PEDV與各國PEDV 於UTR之相似性及親緣性 9
1.7. 以軟體分析5’UTR與3’UTR之二級結構與自由能 9
第二節 台灣PEDV DI RNA及CV777 DI RNA質體之構築 9
2.1. 台灣PEDV DI RNA之構築 9
2.2. 以台灣PEDV DI RNA為模板構築CV777之DI RNA的質體 10
第三節 PEDVDI RNA片段的轉殖及增殖 11
3.1. 利用XL-TOPO Vector (Invitrogen) 系統將突變之PCR DNA 片段進行選殖 11
3.2. 利用PCR篩選內含突變序列的菌落 12
3.3. 質體DNA之萃取 12
3.4. 以PCR增幅DI RNA質體之DNA產物 12
3.5. DNA產物沈澱 13
3.6. DNA濃度測定 13
第四節 PEDV DI RNA的體外反轉錄反應及定量 13
4.1. 體外轉錄反應 (In vitro transcription) 13
4.2. RNA之純化 14
4.3. RNA濃度測定 14
第五節 確認PEDV DI RNA的複製能力 14
5.1. 細胞及病毒 14
5.2. 豬流行性下痢病毒DI RNA之轉染 15
5.3. 自Vero細胞萃取RNA 15
5.4. 互補DNA (cDNA) 合成 15
5.5. 以PCR偵測DI RNA訊號 16
第六節 台灣PEDV DI RNA及CV777 PEDV DI RNA之轉譯效率分析 16
6.1. 台灣PEDV DI RNA及CV777 PEDV DI RNA之體外轉譯 16
6.2. 蛋白質電泳之分析 16
6.3. 以軟體分析蛋白質轉譯效率 17
第七節 利用即時定量聚合酶連鎖反應偵測 PEDV DI RNA合成 17
7.1. 細胞及病毒 17
7.2. 豬流行性下痢病毒DI RNA之轉染 17
7.3. 自Vero細胞萃取RNA 17
7.4. 互補DNA (cDNA) 合成 17
7.5. 即時定量聚合酶連鎖反應Standard 建立 17
7.6. 即時定量聚合酶連鎖反應 17
第八節 統計分析 18
第四章 結果 19
第一節 台灣PEDV的5’UTR之序列、相似性與親原性、二級結構與自由能分析 19
1.1. 台灣PEDV 的5’UTR序列分析 19
1.2. 台灣PEDV 與其它國家在5’UTR序列相似度之分析 19
1.3. 台灣PEDV 的5’UTR的親緣樹分析 20
1.4. 台灣PEDV 的5’UTR 二級結構分析 20
1.5. 台灣PEDV 的5’UTR 自由能分析 21
第二節 台灣PEDV的3’UTR之序列、相似性與親緣性、二級結構與自由能分析 21
2.1. 台灣PEDV 的3’UTR 序列分析 21
2.2. 台灣PEDV的3’UTR相似性分析 22
2.3. 台灣PEDV 的3’UTR 親緣樹分析 22
2.4. 台灣PEDV 的3’UTR 二級結構分析 23
2.5. 台灣PEDV 的3’UTR自由能分析 23
第三節 PEDV DI RNA系統之建立 24
3.1. PEDV DI RNA 之基因體結構 24
3.2. 以RT-PCR偵測PEDV DI RNA之訊號 24
第四節 利用invitro translation 比較台灣PEDV DI RNA及CV777 DI RNA的轉譯效率 24
4.1. 以in vitro translation 分析DI RNA的轉譯效率 24
第五節 比較台灣PEDV DI RNA及CV777 DI RNA的複製效率 25
5.1. 利用qRT-PCR 比較台灣PEDV DI RNA及CV777 DI RNA的複製效率 25
第五章 討論 26
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