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研究生:林誼萍
研究生(外文):Yi-Ping Lin
論文名稱:鴨環狀病毒核酸疫苗與重組次單位疫苗之研發
論文名稱(外文):Development of DNA Vaccine and Recombinant Subunit Vaccine for duck circovirus
指導教授:張伯俊
指導教授(外文):Poa-Chun Chang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫微生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:55
中文關鍵詞:鴨環狀病毒滾輪式擴增法
外文關鍵詞:circovirusRCA
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鴨環狀病毒(duck circovirus, DuCV)與鵝環狀病毒(goose circovirus, GoCV)為目前新興之水禽傳染病原體。此兩種病毒於台灣近年造成大量水禽的感染,並造成羽毛脫落、體重減輕、生長遲緩等臨床症狀出現並導致水禽養殖業者的嚴重損失。在近年的研究中多以聚合酶連鎖反應(polymerase chain reaction;PCR)作為水禽環狀病毒的主要的檢測方法。本實驗之主要目標為研發鴨環狀病毒之核酸疫苗以及次單位疫苗,藉以控制鴨環狀病毒之感染。核酸疫苗之製作方式,是將鴨環狀病毒之外殼蛋白(cap)之基因選殖至真核系統表現之載體pcDNA3.1/TOPO中,而重組次單位疫苗之製作方式,則是利用原核系統表現重組之病毒cap蛋白,免疫方式是將核酸疫苗以及重組次單位疫苗,分別以肌肉以及皮下注射入正番鴨隻中之後再進行攻毒試驗,實驗結果顯示,無論在免疫核酸疫苗以及重組次單位疫苗,皆無法給予其鴨隻明顯之保護。目前實驗還在進行中,希望藉由提升免疫劑量來改善其兩種疫苗之效率。另一方面,本實驗分別利用聚合酶連鎖反應以及滾輪式擴增法來偵測鴨環狀病毒之DNA,發現聚合酶連鎖反應之敏感度高於滾輪式擴增法。
Duck circovirus (DuCV) and goose circovirus infections are an emerging diseases of waterfowls. These viruses could invade lymphoid tissues and lead to immunosuppression of infected ducks. There is no in vitro culture system for waterfowl circovirus, and thus polymerase chain reaction (PCR) is the most important method for diagnosis of these viruses. The goal of this study was to develop the DNA vaccine and recombinant subunit vaccine for control of DuCV infections. The DNA vaccine was constructed by cloning the gene encoding the capsid protein of DuCV into the vector pcDNA3.1/TOPO. The subunit vaccine was prepared from the recombinant capsid protein expressed in E. coli. Muscovy duck was immunized with DNA vaccine or recombinant capsid protein and then challenged with a field strain of DuCV. The preliminary result showed that both DNA vaccine and recombinant subunit vaccines did not confer significant protection against challenge with DuCV. Experiments are underway to improve the efficacy of these two vaccines. This study has developed a multiply primed rolling-circle amplification (RCA) method for detection of DuCV. The sensitivity of RCA for detection of DuCV was found to be less than that of PCR.
中文摘要.............................................I
英文摘要.............................................II
目 錄..............................................III
圖目錄...............................................VI
第一章 緒言..........................................1
第二章 文獻探討......................................2
第一節 環狀病毒之歷史背景............................2
第二節 環狀病毒之分類與特徵..........................3
2.2.1 環狀病毒之分類.................................3
2.2.2 病毒型態與結構.................................4
2.2.3 物理化學特性...................................4
2.2.4 環狀病毒之DNA複製..............................4
第三節 環狀病毒之基因體介紹..........................5
第四節 環狀病毒之致病性..............................6
2.4.1 豬環狀病毒(PCV)之致病性........................6
2.4.2 雞貧血病毒 (CAV) 之致病性......................6
2.4.3 鸚鵡喙羽病毒 (PBFDV) 之致病性..................7
2.4.4 鵝環狀病毒 (GoCV) 之致病性.....................7
2.4.5 鴨環狀病毒 (DuCV)之致病性......................7
第五節 環狀病毒之實驗室診斷..........................8
2.5.1 環狀病毒之臨床診斷.............................8
2.5.2 PCR診斷.......................................8
2.5.3 點漬雜合法 (Dot blot hybridzation).............8
2.5.4 滾輪式擴增法(rolling circle ampification,RCA).9
2.5.5 定量PCR (Quantitative PCR).....................9
第六節 環狀病毒之感染控制............................9
2.6.1 環狀病毒之疫苗控制.............................10
2.6.2 核酸疫苗.......................................10
第三章 材料與方法....................................12
第一節 重組次單位疫苗表現質體之構築..................12
3.1.1載體之製備......................................12
3.1.2 引子之設計.....................................12
3.1.3聚合酶連鎖反應增幅Cap基因.......................12
3.1.4 TOPO TA Cloning................................13
3.1.5 質體的轉型作用.................................13
3.1.6 接合反應.......................................13
3.1.7重組質體之轉型作用..............................14
第二節 重組次單位疫苗之表現..........................14
3.2.1 重組蛋白之表現.................................14
3.2.2 表現蛋白之純化.................................14
3.2.3 重組蛋白之電泳分析與確認.......................15
3.2.4 測蛋白濃度與濃縮...............................16
3.2.5 動物實驗.......................................16
3.2.6 免疫方式.......................................16
3.2.7 臟器乳劑之製備.................................17
3.2.8 聚合酶連鎖反應檢測臟器中鴨環狀病毒.............17
3.2.9 PCR產物之電泳分析.............................18
第三節 核酸疫苗之製備與效力評估......................19
3.3.1 病毒來源與核酸之萃取...........................19
3.3.2 核酸疫苗之構築.................................19
3.3.3 真核表現載體之構築.............................20
3.3.4 轉型作用.......................................20
3.3.5 抽取大量質體 ...................................20
3.3.6 質體DNA之定量..................................21
3.3.7 動物實驗.......................................21
3.3.8 實驗設計.......................................21
3.3.9 免疫方式.......................................22
3.3.10 臟器乳劑之製備................................22
3.3.12 PCR產物之電泳分析............................22
第四節 滾輪式擴增法..................................23
3.4.1 材料來源.......................................23
3.4.2 聚合酶增幅環狀病毒之基因.......................23
3.4.3 限制酵素之切割.................................23
第四章 結果..........................................25
第一節 鴨環狀病毒重組次單位疫苗之效力評估............25
4.1.1 鴨環狀病毒重組次單位疫苗表現質體之構築.........25
4.1.2 重組外殼蛋白(Cap)之表現........................25
4.1.3 重組次單位疫苗之動物實驗.......................25
4.1.4 以PCR分析免疫與未免疫鴨隻在攻毒後之胸腺與華氏囊DuCV的DNA含量..............................................25
4.1.5 以multiscreen Western blot 檢測正番鴨中抗重組Cap蛋白之抗體.................................................26
第二節 鴨環狀病毒核酸疫苗之效力評估..................26
4.2.1 以PCR分析核酸疫苗免疫組與對照組鴨隻在攻毒後之胸腺與華氏囊中鴨環狀病毒的DNA含量............................27
4.2.2 以multiscreen western blot分析正番鴨以核酸疫苗免疫後之抗體.................................................27
4.2.3 抽取PBMC之DNA以PCR檢測DuCV.....................27
第三節 以滾輪式擴增法與聚合酶連鎖反應檢測DUCV之檢測敏感度比較...................................................28
4.3.1 樣本之選取.....................................28
4.3.2 聚合酶連鎖反應之敏感性測試.....................28
4.3.3 滾輪式擴增法之敏感性測試.......................28
第五章 討論..........................................47
第六章 參考文獻......................................50
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