臺灣博碩士論文加值系統

感染性胰臟壞死病毒(infectious pancreatic necrosis virus, IPNV) 屬於雙股RNA病毒科(Birnaviridae) ，為一高度傳染性的魚類病毒，具有廣泛分佈性，世界各地都有感染病例出現。在臺灣，感染性胰臟壞死病毒常因大量感染，造成養殖業嚴重損失。因此如何有效防治感染性胰臟壞死病毒所造成的損害，即為一重要之課題。
在本篇論文中，利用鎚頭型核醣的表現，來研究是否可抑制病毒蛋白的表現。首先經由GCG 的foldRNA程式預測VP2 之cDNA的二級結構，以七個位於特定結構區域的核醣標的位置設計七個鎚頭型核醣並根據標的位置命名為RZ56、RZ78、RZ114、RZ187、RZ209、RZ227及RZ255，並將其雙股DNA構築在pcDNA3及pTracer-CMV2載體內T7和CMV起動子下游。先將VP2之cDNA融合入pCMV-luc載體冷光基因下游，當與核醣表現載體一同在轉譯/轉錄合併系統內表現後，經由冷光值的減少，可評估核醣抑制效益。結果顯示，在與不同核醣一同表現後，冷光值減少百分比由3%至35%。其中RZ209抑制最好達到35%。進一步將各核醣表現載體送入持久性感染的吳郭魚卵巢細胞，經抗生素Zeocin連續篩選後，每一個核醣皆挑選5個次細胞株(subclones)。經北方式雜合反應結果篩選核醣穩定表現細胞株。接著利用西方式雜合反應分析核醣抑制病毒蛋白的表現，結果顯示核醣穩定表現細胞株中有抑制情形的有細胞株56C, 56A, 78C, 78D, 114F, 114C, 209A, 209B, 209C, 209E, 209F, 227A, 227C, 227E, 255E及 255F，其中RZ209的所有次細胞株皆能抑制病毒蛋白的表現。進一步比較RZ209的所有次細胞株與對照組的CPE情形，發現皆有不同程度延遲CPE的現象。因此in vitro及ex vivo實驗結果顯示，鎚頭型核醣RZ209的表現可抑制病毒蛋白。

Infectious pancreatic necrosis virus (IPNV) is one kind of the contagious and widespread fish diseases. IPNV belongs to the Birnaviridae family of virus, which has bisegmented ds-RNA genomes and often causes sever damage to the aquaculture industry in Taiwan.
The purpose of this study was to investigate whether the expression of hammerhead ribozyme in persistent Tilapia ovary-2 (TO-2) cells can protect the cells from viral replication. Seven target sites of hammerhead ribozyme were selected according to secondary structure of VP2 cDNA sequence predicted by GCG foldRNA program. Their target sites were 56, 78, 114, 187, 209, 227 and 255 respectively. The dsDNA of hammerhead ribozyme was synthesized by PCR and constructed into pcDNA3 and pTracer-CMV2 under cytomegalovirus and T7 promoter driving. VP2 cDNA was constructed into pCMV-luc upstream luciferase gene. When ribozyme and VP2-luciferase fusion gene co-expressed in transcription /translation coupled system, the decrease of luciferase activity was used to determine the inhibition activity of ribozyme. The result showed that the ribozyme RZ209 could inhibit the expression of VP2-luciferase fusion protein up to 35%. The plasmids p56, p78, p114, p187, p209, p227 and p255 were introduced into persistent TO-2 cells by lipofection. Five stable transfected subclones were selected for each ribozyme, respectively. After the northern blotting, most transfected cells could express hammerhead ribozyme especially subclones of RZ209. By using IPNV-E1S polyclonal antibody in the western blotting, the viral protein decreased in some ribozyme-expressing clones comparing to control clone after 24hr incubation. The ribozyme-expressing clones 56C, 56A, 78C, 78D, 114F, 114C, 209A, 209B, 209C, 209E, 209F, 227A, 227C, 227E, 255E and 255F had the inhibition activity of viral protein expression. All subclones of ribozyme RZ209 had inhibition activity of viral protein. In addition, all subclones of ribozyme RZ209 had less the viral cytopathic effect (CPE) compared to the control group. These results indicate that the expression of ribozyme RZ209 can inhibit viral protein expression in vitro and ex vivo.

中文摘要 i
英文摘要 ii
壹、前言 3
貳、文獻回顧 4
一、感染性胰臟壞死病毒之基本特性 4
(一)、感染性胰臟壞死病毒的臨床症狀與宿主種類 4
(二)、感染性胰臟壞死病毒的帶原及存留感染情形 6
(三）、感染性胰臟壞死病毒的發現史 7
(四）、感染性胰臟壞死病毒的生物物理與生化特性 8
(五）、感染性胰臟壞死病毒的遺傳物質與病毒蛋白 8
(六）、感染性胰臟壞死病毒的血清分類 10
(七）、台灣感染性胰臟壞死病毒之分佈及其特性 11
二、鎚頭型核醣之基本簡介 13
(一）、選擇核醣標的位置 14
(二）、RNA結合蛋白對核醣功能的影響 15
(三）、表現核醣載體的選擇 17
(四）、核醣表現系統的運用 18
(五）、核醣在細胞培養中的應用 19
(六）、抑制HIV病毒之研究 20
(七)、核醣應用於其他病毒的例子 21
參、實驗材料 23
一、 生物材料 23
二、 反應試劑 24
三、 反應溶液及緩衝溶液 26
肆、實驗方法 30
一、 構築表現質體 31
二、 建立TNT系統 35
三、 細胞內核醣活性測試 36
伍、結果 42
一、標的序列的選擇 42
二、構築表現核醣之質體 43
三、TNT快速篩選系統中核醣抑制受質蛋白表現情形 44
四、吳郭魚卵巢細胞內核醣抑制感染性胰臟壞死病毒 45
陸、討論 48
柒、參考文獻 50
捌、圖表

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