鄭登貴臺灣大學：畜產學研究所謝禮安Hsieh, LianLianHsieh2007-11-292018-06-292007-11-292018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/63606內部核糖體進入位(internal ribosome entry site)目前已知被認為是特定某一類病毒如脊髓灰質炎病毒在宿主細胞當中進行轉譯複製作用之重要利用機轉，然而，在酵母菌(Saccharomyces cerevisiae)當中發現的一段小RNA－抑制型RNA(inhibitor RNA，IRNA)其具有能夠抑制宿主細胞當中病毒IRES-mediated轉譯作用的進行，且不影響正常帽依賴型轉譯作用(cap-dependent translation)的進行。針對IRNA結構上的分析結果更指出IRNA為具有2個loop構造、7個鹼基長的骨架構造與一個大的bugle區域，長度為71bp的小RNA分子。基於上述之研究結果，科學家們更進一步嘗試應用IRNA策略作為治療某些特定病毒性感染疾病的可行性。 本研究之目的旨在進一步確認IRNA是否能夠進一步對於其他IRES-mediated轉譯作用之病毒具有抑制之效果，更希望應用基因顯微注射技術，產製帶有並能持續表現IRAN之轉基因小鼠，希望藉由此動物模式，進一步驗證IRNA能夠使動物個體產生抵禦病毒感染之能力。 試驗首先建構IRNA之表現載體，IRNA之表現主要透過H1啟動子來進行轉譯表現，而表現載體上的EGFP與Neomycin ORF同時可作為報導基因與進行穩定細胞株篩選之用，為初步證實架構之表現載體pSuper-Neo+GFP-IRNA能夠表現IRNA，利用微脂體轉染法將表現載體送入Vero cell當中，再進行腸病毒71型之接種，實驗結果證明在病毒感染後40小時，可發現IRNA表現組其螢光表現細胞型態較為健康而完整，相對於對照組而言細胞出現細胞病變(cytopathic effect)之現象。 進一步試驗應用原核顯微注射技術建立帶有能持續產生IRNA轉基因小鼠之動物模式，暨能使小鼠具有抵禦特定病毒性疾病之感染之能力，試驗結果顯示，業經顯微注射之866個ICR小鼠原核胚，共獲得83隻仔小鼠，至目前為止，產下之仔小鼠尚待進一步PCR分析與確定攜帶有目標基因與否當中，待其轉基因小鼠基因型確定與品系建立之後，準備進行抗病毒能力之試驗。It is well recoginized that the internal ribosome entry site (IRES) existed within the host cells is essentially required for translation in some species of viral RNA infected and this is particularly true in those of poliovirus. Moreover, there was a group of small RNA found within the yeast and these small RNA were characterized as an inhibitor RNA (IRNA) by showing capability in a selective-blocking the IRES-mediated translation of the infected viral RNA without resulting in interference of the cap-dependent translation in the host cells. Studies of using single-strand- and double-strand specific nucleases have predicted the secondary structure of IRNA consisting two loops, a 7-base long stem, and a large bugle region. Based on the fact that IRNA inhibits viral IRES-mediated translation can also be achieved by sequestering noncanonical transacting factors and canonical factors, both essential for IRES-mediated translation, attempts of the present studies were made to investigate the feasibility of using IRNA as a strategy for prevention the diseases suffer from specific viral infections. To meet the purpose described above, a fragment length in 71 bp of the IRNA sequences driving by a H1 promoter was constructed into the pSuper-Neo+GFP vector to allow the transgene equipped with both the Neomycin ORF and the EGFP ORF. The transgene, named as pSuper-Neo+GFP-IRNA, after construction was first subjected to transfection into Vero cells and the efficiency of IRNA against to the IRES-mediated translation was evaluated after the transient transfected Vero cells had been challenged with enterovirus 71. Evidences from cell morphology examination appeared that majority of the control Vero cells showing remarkable cytopathic effect after they had been challenged enterovirus 71 when comparisons were made to those of transient transfected Vero cells, indiciating that IRNA did express their inhibitory effect on the IRES-mediated viral translation and subsequently resulting in inhibition of the enterovirus replication. Therefore, further studies were made to use the transgenic mice harboring the IRNA-transgene as an animal model for elucidation the function of IRNA in IRES-mediated viral translation in vivo and the generation of transgenic mice was conducted by microinjecting the IRNA transgene into pronucleus of newly fertilized eggs. Of these studies, a total 866 mouse embryos after gene injection were reimplanted into the recipients and 83 newborn mice have so far been obtained. Further verification of the transgene integrated within their genomic DNA and also the ability of the potential transgenic mice possess resistance of those disease suffered from specific viral infections are now in progression.目錄 頁次 目錄……………………………………………………………I 表次……………………………………………………………II 圖次……………………………………………………………III 中文摘要………………………………………………………1 前言……………………………………………………………3 文獻檢討 一、病毒的生活史…………………………………………4 (一) 病毒生活史簡介 (二) 真核細胞生物轉譯作用 (三) 病毒基因表現對真核細胞生物轉譯作用的調控 (四) 病毒mRNA轉譯作用機制與調控 (五) 內部核糖體進入位(internal ribosome entry site) 二、IRNA的歷史－the discovery of IRNA….………………22 (一) IRNA(inhibitor RNA)的發現 (二) IRNA抑制作用機轉 (三) 截至目前為止研究成果 三、研究緣由…………………………………………………26 (一) 其他類病毒之抗病毒能力試驗 (二) 動物模式的建立 (三) 應用之技術及方法的背景介紹 材料與方法 一、轉殖基因載體之架構…………………………………… 27 二、試驗動物之飼養管理與超級排卵處理………………… 30 三、基因轉殖小鼠之分析…………………………………… 32 四、IRNA抗病毒能力測試......................... 34 結果與討論 一、IRNA表現載體之構築…………………………………… 39 二、暫時性表現IRNA之細胞株其抗病毒能力測試………… 39 三、攜帶抑制型IRNA序列轉基因小鼠之產製與分析……… 40 結論…………………………………………………………… 52 參考文獻……………………………………………………… 53 英文摘要……………………………………………………… 61 作者小傳……………………………………………………… 632126652 bytesapplication/pdfen-US抑制型RNA腸病毒IRNAIRES-mediated translationenterovirus[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/63606/1/ntu-94-R89626003-1.pdf