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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳媺玫 | |
dc.contributor.author | Mu-Jung Tu | en |
dc.contributor.author | 杜牧融 | zh_TW |
dc.date.accessioned | 2021-06-16T10:23:44Z | - |
dc.date.available | 2016-08-23 | |
dc.date.copyright | 2013-08-23 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-15 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60622 | - |
dc.description.abstract | 錦鯉疱疹病毒 (Koi herpesvirus, KHV ) 的爆發造成養殖業者的極大損失,目前KHV被分類為Alloherpesviridae 下的鯉魚疱疹病毒第三型 (Cyprinid herpesvirus 3, CyHV-3),具封套、雙股DNA,核酸大小約為295kbp,對於初次感染的家鯉或錦鯉有極高的死亡率,耐過錦鯉疱疹病毒攻擊的鯉魚會成為帶原魚,而其持續感染的機制目前尚不清楚。潛伏感染是持續感染的一種方式,其為疱疹病毒科的特色。除了常見的哺乳類之疱疹病毒科 (Herpesviridae) 會潛伏感染外,Alloherpesviridae中的河鯰病毒 (Channel catfish virus)、鯉魚疱疹病毒第一型 (Cyprinid herpesvirus-1) 也證實會產生潛伏感染。而KHV可因為溫度變化而造成潛伏感染,其潛伏位置包含很多器官以及周邊血球。無症狀的帶原鯉魚溫度改變或遭受壓力時,疾病就會再度爆發。因此,本研究想了解錦鯉疱疹病毒持續感染的機制,根據文獻指出病毒持續感染的機制可能是由於病毒DNA插入宿主染色體、病毒產生環狀的游離基因 (episome)、細胞產生干擾素或是病毒產生與潛伏相關的轉錄 (LATs)。本實驗以帶原錦鯉進行初代細胞培養,建立兩株帶原KHV之持續性細胞株。從生長速度、染色體數目等細胞特性分析顯示其與健康細胞株有差異,且該細胞不接受水生動物來源之病毒重複感染。其釋出之病毒力價較低,攻毒實驗魚不造成死亡,用原位雜合法僅在實驗魚之鰓及腎偵測到陽性訊號。以此兩株細胞進行持續感染機制探討,首先比較帶原細胞株釋出之病毒與標準病毒的力價,以及於實驗魚體內分布狀況、帶原細胞株是否會分泌干擾素、帶原細胞的病毒有無形成環狀的游離基因體、或是病毒基因嵌入細胞染色體之可能。目前結果顯示帶原細胞株干擾素的分泌或病毒嵌入宿主染色體可能都是造成KHV於此二株細胞內持續感染的機制。 | zh_TW |
dc.description.abstract | Koi herpesvirus (KHV) infection cause severe financial losses. KHV is a member of Alloherpesviridae, and newly designated species Cyprinid herpesvirus 3. The virion have an outer envelope and an icosahedral capsid contains a single, linear, double-stranded DNA of 295 kbp. Common carp (Cyprinus carpio carpio) and Koi carp(Cyprinus carpio koi) are the only species known to be affected by KHV. The survivors will become carrier fishes, but persistent infection mechanism of KHV is not clear. One of the unique features of Herpesviridae is latency which had been confirmed in Channel catfish virus and Cyprinid herpesvirus infection model. KHV can become latent in the peripheral white blood cells and various tissues, and will reactivation while temperature shifting, or been forced. The aim of this study is trying to find out persistent infection mechanism of KHV. Persistent infection mechanism in tissue culture includes interferon, intranuclear episome, viral DNA insertion, or virus producing latency-associated transcripts. We established two KHV persistently infected cell lines from carrier fishes through primary cell culture. Persistent infection cells characteristics such as growth rate and chromosome analysis are different from healthy cells. These two cells do not accept superinfection of virus from aquatic sources. The virus titer of persistently infected cells is fluctuation, and have no ability to induce fish mortality. The results of in situ hybridization showed the KHV positive signals in gill and kidney. In this study, virus produced by the two persistently infected cells are compared to the wild KHV, such as virus titer and organ distribution. Current results showed that the mechanism of KHV persistent infection is relate to interferon releasing and virus genome insertion. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:23:44Z (GMT). No. of bitstreams: 1 ntu-102-R00629017-1.pdf: 4464615 bytes, checksum: 8c69d747072eb2dbd40ff6c3abb383a9 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 目錄
摘要…………………………………………………………………………….………...I Abstract………………………………………………………………………….………II 目錄………………………………………………………………...…………………..III 圖目錄…………………………………………………………………………………VII 表目錄………………………………………………………………………………..VIII 第一章 緒論……………………………………………………………………...……..1 第二章 文獻回顧………………………………………………………………...……..3 第一節 錦鯉疱疹病毒之特性……………………………………………...……..3 1.1病毒之分類………………………………………………………………..3 1.2疱疹病毒之複製…………………………………………………………..4 1.3 宿主範圍與傳播方式…………………………………………...……….6 1.4 致病機制………………………………………………………...……….7 1.5 臨床症狀及病理變化…………………………………………...……….8 1.6 診斷方式…………………………………………………………...…….9 1.7 KHV之持續感染…………………………………………...…………...10 第二節 病毒之持續感染…………………………………………………...……11 2.1 持續感染定義………………………………..…………………...…….11 2.2 持續感染的分期……………………………………………………......12 2.3 細胞培養之病毒持續感染機制………………………………...……...13 第三節 疱疹病毒的持續感染機制…………………………………...…………14 3.1 疱疹病毒的持續感染機制…………………………………...………...14 3.2 Alphaherpesvirinae………………………………………...…………….14 3.3 Betaherpesvirinea…………………………………………...…………...17 3.4 Gammaherpesvirinea…………………………………………...………..18 3.5 Alloherpesviridea……………………………………………………….. 19 第三章 材料與方法……………………………………………………………...……22 第一節 實驗設計………………………………………………………...………22 第二節 建立初代細胞…………………………………………………………...23 2.1實驗材料…………………………………………………………..…..…23 2.2初代細胞培養…………………………………………………..……..…24 第三節 初代細胞株病毒持續感染檢測…………………………………...……24 3.1 病毒去氧核醣核酸萃取…………………………………………...…...25 3.2 聚合酶鏈鎖反應 (PCR)…………………………………..……...…….25 3.3 瓊脂醣凝膠電泳……………………………………………...………...26 3.4 穿透式電子顯微鏡切片及觀察記錄………………………...………...27 3.5 健康細胞攻毒試驗…………………………………………...………...28 3.6 健康錦鯉攻毒試驗……………………………...…………………...…29 第四節 細胞特性分析…………………………………...………………………30 4.1 生長曲線………………………………………...……………………...30 4.2 染色體分析……………………………………………...……………...30 4.3 細胞種植率分析 (Plating efficiency)………………...……..…………31 第五節 病毒力價監測……………………………………………………...……32 5.1 組織培養細胞半數感染量…………………………………………......32 5.2 病毒斑試驗………………………………………………………...…...32 第六節 以免疫染色法偵測帶原細胞中的病毒分布…………………………...33 6.1 免疫細胞化學染色法 (Immunocytochemistry stain)…………….……33 6.2 免疫螢光染色法 (Immunofluorescence assay)………………….…….34 第七節 原位雜合………………………………………………………….…......35 7.1 探針合成………………………………………………………….…….35 7.2原位雜合…………………………………………………….…………...36 第八節 干擾素與抗病毒基因偵測………………………………….…………..38 8.1細胞處理…………………………………………………….…………...38 8.2 RNA萃取………………………………………………………………..38 8.3 cDNA的合成……………………………………………………………39 8.4 反轉錄-聚合酶鏈鎖反應 (RT-PCR)……………………..….………....40 第九節Gardella Gel Analysis………………………………………….…………41 9.1 實驗材料……………………………………………………….……….41 9.2 實驗方法…………………………………………………...…….……..41 第十節 南方墨點法 (Southern blot)……………………………………….……42 10.1 實驗材料……………………………………………………….……...42 10.2 實驗方法………………………………………………………….…...43 第四章 實驗結果……………………………………………………………………...46 第一節 初代細胞培養…………………………………………………………...46 1.1初代細胞分離……………………………………………………..……..46 1.2 初代細胞繼代培養…………………………………………………......46 第二節 初代細胞株病毒持續感染檢測………………………………………...46 2.1 偵測KHV病毒核酸……………………………………………………46 2.2 健康細胞攻毒試驗…………………………………………...………...47 2.3 健康錦鯉攻毒試驗…………………………………………………......47 第三節 細胞特性分析…………………………………………………………...48 3.1生長曲線…………………………………………………………………48 3.2 染色體數目分析……………………………………………………......48 3.3細胞種植率分析…………………………………………………………49 第四節 病毒力價監測………………………………………………………….49 第五節 以免疫染色法偵測帶原細胞中的病毒分布………………………….50 第六節 重複感染……………………………………………………………….50 第七節 持續感染機制之研究…………………………………………………...51 7.1 持續感染細胞釋出之KHV病毒與強毒株比較……………………….51 7.2 病毒基因嵌入宿主染色體………………………………………...…...51 7.3 宿主細胞干擾素分泌………………………………………………......51 第五章 討論…………………………………………………………………………...53 第六章 參考文獻……………………………………………………………………...82 | |
dc.language.iso | zh-TW | |
dc.title | 錦鯉疱疹病毒潛伏感染機制之探討 | zh_TW |
dc.title | Study of Koi Herpesvirus Persistent Infection Mechanism | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃金城,蔡信雄,齊肖琪 | |
dc.subject.keyword | 錦鯉疱,疹病毒, 持續感染, 干擾素, 原位雜合, 病毒基因嵌入染色體, | zh_TW |
dc.subject.keyword | Koi herpesvirus, persistent infection, interferon, in situ hybridization, viral genome insertion, | en |
dc.relation.page | 97 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-16 | |
dc.contributor.author-college | 獸醫專業學院 | zh_TW |
dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
顯示於系所單位: | 獸醫學系 |
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