臺灣博碩士論文加值系統

腸病毒71型（Enterovirus 71；EV 71）是小RNA病毒科（Picornaviridae）的成員，基因體5’端非轉譯區域（5’untranslated region；5’UTR）含有第一型內部核醣體進入位點（internal ribosome entry site；IRES），主要與帽蓋非依賴型轉譯作用（cap-independent translation）相關，本研究的目的是找出腸病毒71型真正具有功能的IRES區域。我們以M-fold軟體預測腸病毒71型5’UTR含六個stem-loops 及六個spacer regions的結構，構築含有不同區域的IRES片段並將之接入dicistronic 表現系統（pRHF質體），用Luciferase assay分析IRES活性，發現刪除193-228核苷酸位置（stem-loop III）使IRES活性降低至約50%左右，刪除91-167核苷酸位置（stem-loop II）、242-445核苷酸位置（stem-loop IV）、453-561核苷酸位置（stem-loop V）及566-636核苷酸位置（stem-loop VI）則使IRES活性完全喪失，可見stem-loop II、IV、V、VI對IRES的活性非常重要，而stem-loop III則較不重要。為了在細胞內進一步驗證這個推測，我們利用含有腸病毒71型全長基因體的infectious clone（IF）質體，構築含有不同區域IRES片段之IF，並利用in vitro transcription轉錄成RNA，轉染到細胞內，結果發現原始型（IF-WT）及刪除stem-loop III之突變型（IF-del III）皆具有產生病毒之能力，此兩株活病毒均影響細胞型態之改變，產生相似的病毒斑型態，病毒生長情形也非常相似，其他IF-del II、IF-del IV、IF-del V、IF-del VI則造成致死性突變（lethal mutation），此與Luciferase assay結果相符合。因此本研究證實91-636核苷酸（stem-loop II - VI）是IRES主要具有活性的區域，但是不包含193-228核苷酸位置（stem-loop III）。

Enterovirus 71 belongs to Picornaviridae family. The 5’untranslated region (5’UTR) of viral genome contains type I internal ribosome entry site (IRES). The main function of the IRES is cap-independent translation. This study attempts to identify the minimal functional sequences of EV71 IRES. The predicted secondary structure of EV71 IRES contains six stem-loops and six spacer regions by M-fold software. The different truncated forms of the IRES were inserted into the pRHF vector that contains a dicistronic expression system. The mutated DNA clone was transfected into HeLa cells, and the results from luciferase assay showed that the deletion of nucleotides 193 to 228 was decreased viral translation activity to about 50%. The deletion of nucleotides 91 to 167(stem-loop II), 242 to 445 (stem-loop IV), 453 to 561 (stem-loop V) and 566 to 636 (stem-loop VI) were loss almost entire IRES activity, suggesting that the stem-loop II, IV, V and VI are important for IRES activity but not the stem-loop III. However, recombinant viruses with deletion mutations generated from infectious clone caused lethality (IF-del II, IF-del IV, IF-del V and IF-del VI.) and deletion III mutation (IF-del III) was rescued. This virus exhibited the same CPE, plaque size and growth rates in comparison with that of wild-type. Taken together, our results suggest that nucleotides 91-636(stem-loop II-VI), but not nucleotides 193 to 228 (stem-loop III) is a major minimal functional region of IRES.

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口試委員會審定書
長庚大學博碩士論文著作授權書 iii
誌謝 iv
中文摘要 v
英文摘要 vii
目錄 viii
圖表目錄 x
第一章 緒論 1
1.1 腸病毒的分類 1
1.2 腸病毒的結構 1
1.3 腸病毒的基因體 2
1.4 腸病毒的生活史 3
1.5 腸病毒71型的流行病學 3
1.6 腸病毒轉譯的方式 4
1.7 腸病毒的5’端非轉譯區域 5
1.8 內部核醣體進入位點（IRES）的分類及特色 6
1.9 調控內部核醣體進入位點的分子 7
1.10 我們實驗室之前的研究 8
1.11 研究目的 9
第二章 材料與方法 10
2.1 pRHF系列不同刪除突變EV71 5’UTR質體之構築 10
2.2 Infectious clone（IF）系列刪除不同stem-loop 的 EV71-5’UTR質體之構築 11
2.3 取得不同長度5’UTR質體DNA 12
2.4 細胞培養 12
2.5 轉染質體DNA 到HeLa 細胞 13
2.6 Luciferase assay 13
2.7 試管內轉錄作用（In vitro transcription） 14
2.8 轉染RNA 到Vero細胞 14
2.9 病毒斑試驗（plaque assay） 15
2.10 病毒斑純化（plaque purified） 16
2.11 病毒培養（Virus amplication） 17
2.12 抽病毒RNA及反轉錄聚合酶鏈鎖反應（Reverse transcription- Polymerase chain reaction；RT-PCR) 17
2.13 time course 18
第三章 結果 19
3.1 含有不同長度IRES的Clone 19
3.2 腸病毒71型5’UTR不同二級結構序列構築片段之IRES具有不同轉譯活性 20
3.3 腸病毒71型5’UTR刪除不同stem-loop之infectious clone產生病毒 22
3.4 沒有造成CPE的突變序列沒有活病毒產生 23
3.5 比較腸病毒71型5’UTR原始型及刪除stem-loop III的病毒特性及對細胞的影響 24
第四章 討論 25
4.1 腸病毒71型5’UTR IRES區域與小兒麻痺病毒相似 25
4.2 腸病毒71型與小兒麻痺病毒5’UTR刪除stem-loop III的病毒特性相似 26
4.3 腸病毒71型IRES的改變可能影響細胞蛋白質的交互作用及IRES二級結構的折疊 28
4.4 IF-WT與IF-del III這兩株病毒於不同細胞及不同溫度可能有不同的生長曲線 28
第五章 參考文獻 31
第六章 圖表 37

表一 構築不同片段IRES所使用的引子（primers） 37
表二 不同stem-loop 刪除突變病毒的特性 38
圖一 腸病毒71型5’UTR的二級結構及dicistronic expression system 39
圖二 NotI 限制酵素切割確認接進去不同長度IRES片段大小 40
圖三 PCR確認接進去不同長度IRES片段正接與否 41
圖四 AflII及EcoRI 限制酵素切割確認改變infectious clone 5’UTR不同IRES二級結構之大小 42
圖五 EV71 5’UTR 5’端刪除突變及Luciferase assay測量IRES活性 43
圖六 EV71 5’UTR 3’端刪除突變及Luciferase assay測量IRES活性 44
圖七 EV 71 5’UTR 二級結構刪除突變及Luciferase assay測量IRES活性 45
圖八 確認刪除stem-loop II、IV、V、VI的突變株沒有病毒產生 46
圖九 EV71 5’UTR二級結構刪除突變病毒感染RD細胞株的CPE及病毒型態 47
圖十 EV71 5’UTR二級結構刪除突變病毒之生長曲線 48

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