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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.author | 戴君如 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:12:27Z | - |
| dc.date.available | 2021-07-01T08:12:27Z | - |
| dc.date.issued | 2000 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75269 | - |
| dc.description.abstract | 傳染性胰臟壞死病毒（Infectious Pancreatic Necrosis Virus; IPNV）是兩段雙股之RNA病毒，屬於兩段雙股核醣核酸病毒科。在魚類傳染疾病中，是一種分佈廣泛的水生生物病毒的傳染病，在臺灣曾造成多種經濟性魚類的死亡。本實驗室之IPNV-E1S是由病鰻中分離鑑定後，屬於Ab型之水生兩段雙股核醣核酸病毒。 在最近的研究報告中指出，細胞凋亡現像是誘發魚類細胞受IPNV感染後產生病變效應的主要因素，而CHSE-214細胞株可明顯表現出細胞凋亡的外觀及生化之特徵。IPNV所引發之細胞凋亡現像是由何基因所調控，推測是由其VP3伴演了此角色。為了瞭解細胞凋亡現象是否與VP3有關，便利用反意VP3核醣核酸（A2, A4，及A7, 包括nt 501-711, 285-711及1-711）之穩定轉殖CHSE-214細胞作測試。三株轉殖細胞對IPNV所引起之細胞死亡有抑制的功能，由分析結果中可知VP3的表現量受到壓制，而Mcl-1的表現量則下降得較緩慢，及在反意基因表現的初期可使DNA Fragmentation量降低。 由以上的結果推測VP3可受反意RNA的抑制，而VP3可能為IPNV引發之細胞凋亡調控因數之一。 | zh_TW |
| dc.description.abstract | IPNV (Infectious Pancreatic Necrosis Virus) is a double- stranded RNA viruse which has been shown to be a major cause of many contagious and widespread fish diseases. IPNV belongs to Birnaviridae virus family and is also a pathogen of many economically important fishes in Taiwan. The IPNV-E1S (Eel No.1 Spleen) virus strain was isolated from the spleen of diseased Japanese eel (Anguilla japonica) and is an Ab serotype of Aquatic Birnavirus. Recent study has established apoptosis as a major cause for the observed IPNV-induced cytopathic effects in infected fish cells. IPNV-infected CHSE-214 cells displayed all previously known morphological and biochemical hallmarks of apoptosis. At present, the apoptosis-inducing activity of IPNV has not been definitively assigned to any IPNV factors. Nevertheless, there was evidence implicating VP3 as such a factor. To identify the unknown apoptosis-inducing element of IPNV, CHSE-214 cells stably expressing various antisense RNAs (A2, A4, and A7, complement to nt 501-711, 285-711, and 1-711, respectively) against VP3 were established. All antisense RNA expressing cells showed increased resistance to IPNV-induced apoptosis in viral challenge experiments (MOI 1). Further analyses showed that VP3 protein expression was suppressed, Mcl-1 expression was longer sustained, and DNA fragmentation was reduced in these antisense RNA expressing cells during early viral replication cycle. These results suggest that the antisense RNAs could knock down the VP3 expression and VP3 is an apoptosis-inducing factor responsible for the IPNV-induced apoptotic cell death. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:12:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2000 | 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簡介. . . . . . . . . . . . . .2 （一）傳染性胰臟壞死病毒的發現歷史. . . . . . . . . . . . . .2 （二）傳染性胰臟壞死病毒的分類. . . . . . . . . . . . . .3 （三）傳染性胰臟壞死病毒的生長與生化特性. . . . . . . . . . . 3 （四）傳染性胰臟壞死病毒的遺傳物質與蛋白質之研究. . . 4 （五）傳染性胰臟壞死病毒的轉錄與複製. . . . . . . . . . . . . .6 （六）傳染性胰臟壞死病毒之轉譯. . . . . . . . . . . . . .6 （七）傳染性胰臟壞死病毒蛋白質及其功能. . . . . . . . . . . . 8 （八）傳染性胰臟壞死病毒的宿主種類與臨床症狀. . . . . 10 （九）臺灣水生兩段核醣核酸病毒之研究. . . . . . . . . . . . 10 二、反意核酸之簡介及發展利用. . . . . . . . . . . . . .12 （一）反意核酸的發現. . . . . . . . . . . . . .12 （二）反意核酸的簡介. . . . . . . . . . . . . .12 （三）反意核酸的類型. . . . . . . . . . . . . .15 （四）反意核酸作用的原理. . . . . . . . . . . . . .17 （五）反意核酸應用於基因調控與疾病治療的研究. . . . .17 三、細胞凋亡之簡介. . . . . . . . . . . . . .20 （一）細胞凋亡之生理功能. . . . . . . . . . . . . .20 （二）細胞凋亡與細胞壞死之外觀現象. . . . . . . . . . . . . .20 （三）細胞凋亡之訊息傳遞與調控. . . . . . . . . . . . . .20 貳、實驗材料. . . . . . . . . . . . 22 一、生物材料. . . . . . . . . . . . 22 二、儀器與器材. . . . . . . . . . . . . .24 三、反應試劑. . . . . . . . . . . . . .25 參、實驗方法. . . . . . . . . . . . . .32 一、構築反意VP3 RNA表現載體. . . . . . . . . . . . . .32 （一）聚合酵素連鎖反應. . . . . . . . . . . . . .32 （二）聚合酵素連鎖反應產物之分離. . . . . . . . . . . . . .32 （三）銜接反應與轉型作用. . . . . . . . . . . . . .33 （四）小量質體的抽取. . . . . . . . . . . . . .33 （五）定序反應. . . . . . . . . . . . . .34 （六）大量質體DNA製備. . . . . . . . . . . . . .34 二、轉殖反意質體細胞株之分析. . . . . . . . . . . . . .35 （一）穩定轉CHSE-214細胞株之建立. . . . . . . . . . . . . .35 （二）轉殖細胞內反意基因表現之偵測. . . . . . . . . . . . . .36 （三）轉殖細胞抗病毒感染之活存率測試. . . . . . . . . . . . . .38 （四）病毒蛋白質VP3受抑制後表現量之評估. . . . . . . . .38 （五）轉殖細胞對抗病毒感染後細胞凋亡現象之測試. . . 40 （六）病毒抑制感染之實驗. . . . . . . . . . . . . .41 肆、結果. . . . . . . . . . . . . .42 一、構築反意核酸表現質體. . . . . . . . . . . . . .42 二、細胞試驗. . . . . . . . . . . . . .43 （一）反意核酸轉殖試驗之偵測. . . . . . . . . . . . . .43 （二）轉殖細胞抗病毒感染之活存率測試. . . . . . . . . . . . . .44 （三）病毒蛋白質抑制試驗. . . . . . . . . . . . . .45 （四）DNA Fragmentation分析試驗. . . . . . . . . . . . . .47 （五）轉殖細胞所產之病毒力價試驗. . . . . . . . . . . . . .47 伍、討論. . . . . . . . . . . . . .49 陸、結論. . . . . . . . . . . . . .55 染、參考文獻. . . . . . . . . . . . . .56 附圖. . . . . . . . . . . . . .78 | |
| dc.language.iso | zh-TW | |
| dc.title | 以傳染性胰臟壞死病毒之VP3反意核醣核酸表現抑制本病毒所誘發之細胞凋亡 | zh_TW |
| dc.title | The Inhibitions of IPNV Induced Apoptosis by Its VP3 Antisense RNA Expression in Fish Cell Line | en |
| dc.date.schoolyear | 89-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 99 | |
| 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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