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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.author | Wei-Lun Wang | en |
dc.contributor.author | 汪惟倫 | zh_TW |
dc.date.accessioned | 2021-07-01T08:11:21Z | - |
dc.date.available | 2021-07-01T08:11:21Z | - |
dc.date.issued | 1999 | |
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Acids Res. 23: 1192-1196. 洪健睿 (1999):感染性胰臟壞死病毒所誘發寄主細胞凋亡及分子機制。國防醫學院生命科學研究所博士論文,臺北。 徐慧雯 (1996):抗感染性胰臟病毒核醣核酸酵素之研究。國立臺灣大學漁業科學研究所碩士論文,臺北。 蕭亦何 (1999):吳郭魚卯巢細胞內表現抗感染性胰臟病毒鎚頭型核醣核酸?抑制該病毒之感染力。國立臺灣海洋大學水產生物技術研究所碩士論文,臺北。 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75013 | - |
dc.description.abstract | 傳染性胰臟壞死病毒(Infectious Pancreatic Necrosis Virus, IPNV)為兩段雙股核醣核酸科(Birnaviridae) 病毒。其為一傳染性高且分佈廣泛的魚類病毒,在臺灣的水產養殖上造成相當大的損失。因此如何防治此病毒的危害為當前相當重要的課題。在IPNV的基因組中,A段基因體(segment A)於5'端有一個444 bp的small ORF,可轉譯出一17 kDa的病毒蛋白VP5,在病毒感染寄主細胞初期表現,以延後細胞自戕(apoptosis)現象,其功能可能與 anti-apoptosis有關。本實驗嘗試在魚類最適生長溫度下,設計對VP5 RNA進行切割之鎚頭型核醣核酸醃,並針對其切割作用活性加以研究。 以GCG之MFold程式預測VP5 RNA的二級結構,共設計了16個鎚頭型核醣核酸醃。於37℃時共有5個核醣核酸?可對VP5 RNA進行切割反應,其中RZ-1、-5、-7、-8位於套環區,而RZ-16則位於榦狀區。將切割反應溫度降至25℃, RZ-5、-7、-8仍具切割活性。鎂離子在切割作用中為一重要輔助因數,而RZ-5與RZ-8於3 mM低濃度鎂離子時仍具相當切割活性。二價金屬離子除Mg2+外包括Ca2+、Mn2+、Cu2+、Co2+與Cd2+皆可於進行切割作用時當做輔助因數。於反應中添加PEG-6000及Urea或formamide皆可增加切割反應之效率。反應之切割效果與溫度的提升呈正相關,而RZ-5與RZ-8兩組核醣核酸?皆可於17℃的低溫下作用,顯示其未來在ex vivo或in vivo之應用潛力。 | zh_TW |
dc.description.abstract | Infectious pancreatic necrosis virus (IPNV) is one of the contagious and widespread fish diseases. IPNV belongs to the Bimaviridae family of virus, which has bisegmented ds-RNA genomes and often causes seriously damages to the aquaculture in Taiwan. VP5 is encoded from the small ORF located at 5'end of A segment. VP5 protein had been demonstrated to play a dual role on the regulation of virus replication and up-regulation of the survival factor Mcl-1 during IPNV infection. In this study, we designed highly specific hammerhead ribozymes to test the cleavages of VP5 RNA. Sixteen predicted cleavage sites (14 in loop and 2 in stem) of hammethead ribozyme were se1ected according to the secondary structure of VP5 cDNA sequence by GCG Mfold program . The ribozyme RNAs and VP5 RNA (Partial sense RNA of IPNV) were both synthesized by in vitro transcription. Five (4 in loop and 1 in stem) out of 16 hammethead ribozymes (predicted cleavage site) possess the trans-cleavage activity at 37℃ in vitro. The trans-cleavage activities of RZ-5 and RZ-8 are concentration-dependent ofdivalent-cation (Mg++) fashion at 25℃ in vitro. Divalent-cations including Ca++, Co++, Mn++, Cd++,and Cu++ as Mg++ act as cofactors in trans-cleavage reaction of hammethead ribozymes. The addition of chemical reagents (PEG-6000, urea, and formamide) can improve the cleavage of VP5 RNA by hammethead ribozymes (RZ-5 & RZ-8). The trans-cleavage capabilities of RZ-5 and RZ-8 at low temperature ( 17℃ and 25℃), revealing that these two hammethead ribozymes have strong potential for ex vivo or in vivo trans-cleavage in fish cells to prevent IPNV infection. | en |
dc.description.provenance | Made available in DSpace on 2021-07-01T08:11:21Z (GMT). No. of bitstreams: 0 Previous issue date: 1999 | en |
dc.description.tableofcontents | 目 錄 I 中文摘要 Ⅲ 英文摘要 Ⅳ 壹、前言 1 (一) 傳染性胰臟壞死病毒之基本特性 1 (1)傳染性胰臟壞死病毒之發現史與分類 (2)傳染性胰臟壞死病毒宿主種類與臨床症狀 (3)傳染性胰臟壞死病毒之血清型分類與分類情形 (4)傳染性胰臟壞死病毒之生長與生化特性 (5)傳染性胰臟壞死病毒遺傳物質之研究 (6)傳染性胰臟壞死病毒的蛋白質及其功能 (7)傳染性胰臟壞死病毒之複製與轉錄 (8)傳染性胰臟壞死病毒之轉譯 (9)台灣地區傳染性胰臟壞死病毒之相關研究 (二)具催化功能的RNA 之基本特性 13 (1)具催化活性的RNA 發現歷程 (2) Large catalytic RNA (I)RbomicleaseP (Ⅱ)Group I introns (Ⅲ) Group II introns (3)Small catalytic RNA (三)鎚頭型核醣核酸醃之基本簡介 23 (l)鎚頭型核醣核酸晦的發現史 (2)鎚頭型核醣核酸醃催化區作用機制 (3)鎚頭型核醣核酸醃的活性與專一性 (4)鎚頭型核醣核酸醃標的序列之選擇 (5)鎚頭型核醣核酸嗨之運送形式 (6)鎚頭型核醣核酸醃於動力學上之計算方式 (7)核醣核酸醃於細胞株上的研究 (8)核醣核酸醉於病毒上之應用 貳、研究方向 30 三、實驗材料 31 (一)生特材料 31 (二)反應試劑 33 (三)反應溶液與緩衝溶液 36 (四)實驗器材 40 肆、實驗方法 41 (一)表現載體之構築 41 一、構築活體外受質 RNA 表現載體 二、構築細胞內受質 RNA 表現載體 (二) 活體外轉錄作用合成 RNA 47 一、活體外轉錄作用合成受質 RNA 二、活體外轉錄作用合成核醣核酸醃 RNA (三) 活體外核醣核酸醃 RNA 切割受質 RNA 反應 50 伍、實驗結果 54 (一) 鎚頭型核醣核酸醃標的位置之選擇 54 (二) 活體外轉錄作用 54 (三) 活體外鎚頭型核醣核酸醃切割反應 55 (l)測試各個核醣核酸醃之切割情形 (2)反應濃度對核醣核酸醃切割作用的影響 (3)溫度對核醣核酸醃切割作用的影響 (4)鎂離子濃度對核醣核酸醃切割作用的影響 (5)其他二價陽離子對核醣核酸醃切割作用的影響 (四) 改善活體外切割反應的效果 59 (l)添加 PEG 對核醣核酸醃切割作用的影響 (2)添加變性劑對核醣核酸醃切割作用的影響 陸、討論 61 柒、結論 69 捌、參考文獻 70 玖、圖表 85 | |
dc.language.iso | zh-TW | |
dc.title | 鎚頭型核醣核酸醃於低溫下切割傳染性胰臟壞死病毒之VP5 RNA | zh_TW |
dc.title | Hammethead ribozymes on the cleavage of VP5 RNA in infectious pancreatic necrosis Virus( IPNV ) at low temperature. | en |
dc.date.schoolyear | 87-2 | |
dc.description.degree | 碩士 | |
dc.relation.page | 131 | |
dc.rights.note | 未授權 | |
dc.contributor.author-dept | 生命科學院 | zh_TW |
dc.contributor.author-dept | 漁業科學研究所 | zh_TW |
顯示於系所單位: | 漁業科學研究所 |
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