王金和臺灣大學：獸醫學研究所王朝正WANG, Chao-ChengChao-ChengWANG2007-11-282018-07-092007-11-282018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/60021Reticuloendotheliosis virus (REV) is an avian gamma-retrovirus which results in immunosuppression, runting disease and lymphomas. Furthermore, REV has been found as a contaminant of Marek's disease and fowl pox vaccines (FPV). Since REV is well known to integrate into host DNA, the purpose of this study was to detect endogenous REV (eREV) gene in chickens and geese and in a FPV. One REV strain isolated from chickens was used as a template for PCR with specific primers to amplify pol, gag, env, and LTR genes, labeling with Digoxigenin as a probe for detecting eREV in chickens and geese and in one FPV by southern blot hybridization analysis. The DNA samples extracted from 10 Leghorn layers , 10 Arbor Acres broiler breeders, 10 Avian broiler breeders and 10 White Roman geese were used for eREV detection. We isolated REV 3337/05 from chicken. Furthermore, The result showed that no eREV was detected in chickens or in geese. However, one REV 5’ LTR fragment integrated into the FPV in the region between ORFs 201 and 203 was found. This LTR-integration shows no damage to chickens after vaccination clinically because only the FPV carrying a near intact REV is likely to cause tumors in vaccinated chickens. Since only one eREV copy integrating in the FPV was found, this vaccine could be used clinically.目錄------------------------------------------------Ⅳ 表次------------------------------------------------Ⅺ 圖次------------------------------------------------Ⅻ 附錄------------------------------------------------ⅫⅠ 中文摘要------------------------------------------------ⅪV 英文摘要------------------------------------------------XV 第一章 緒言------------------------------------------------------------------------------ 1 第二章 文獻探討------------------------------------------------------------------------ 2 第一節 簡介------------------------------------------------------------------------------ 2 第二節 歷史背景------------------------------------------------------------------------ 2 第三節 臨床症狀------------------------------------------------------------------------ 2 2-3.1 一般臨床症狀------------------------------------------------------------------ 2 2-3.2 消耗性症狀--------------------------------------------------------------------- 2 2-3.3 慢性腫瘤------------------------------------------------------------------------ 3 2-3.4 急性腫瘤------------------------------------------------------------------------ 3 第四節 病理變化------------------------------------------------------------------------ 3 2-4.1 一般病理變化------------------------------------------------------------------ 3 2-4.2 分類------------------------------------------------------------------------------ 4 第五節 流行病學------------------------------------------------------------------------ 4 2-5.1 分布------------------------------------------------------------------------------ 4 2-5.2 宿主特異性--------------------------------------------------------------------- 4 2-5.3 病毒的傳染途徑--------------------------------------------------------------- 4 2-5.3.1 水平傳播------------------------------------------------------------------------ 4 2-5.3.2 垂直傳播------------------------------------------------------------------------ 5 2-5.3.3 污染的生物製劑--------------------------------------------------------------- 5 2-5.4 臺灣地區情形------------------------------------------------------------------ 6 第六節 分類------------------------------------------------------------------------------ 6 2-6.1 一般分類------------------------------------------------------------------------- 6 2-6.2 依病毒顆粒分類---------------------------------------------------------------- 6 2-6.3 依病毒抗原性及基因分類---------------------------------------------------- 7 2-6.4 病毒族群------------------------------------------------------------------------- 7 第七節 病毒特性------------------------------------------------------------------------- 7 2-7.1 型態、結構與生物性----------------------------------------------------------- 7 2-7.2 病毒基因體---------------------------------------------------------------------- 8 2-7.3 致癌基因------------------------------------------------------------------------- 8 2-7.4 病毒蛋白及其功能------------------------------------------------------------- 8 2-7.5 病毒基因的轉錄與複製------------------------------------------------------- 9 2.7.5.1 外源性---------------------------------------------------------------------------- 9 2.7.5.2 內源性---------------------------------------------------------------------------- 9 2-7.6 病毒的增殖與培養------------------------------------------------------------- 9 第八節 診斷方法------------------------------------------------------------------------- 10 2-8.1 抗原及抗體檢測---------------------------------------------------------------- 10 2-8.2 區別診斷------------------------------------------------------------------------- 11 第九節 預防與治療---------------------------------------------------------------------- 11 第十節 污染的生物製劑---------------------------------------------------------------- 12 2-10.1 禽痘病毒污染之研究---------------------------------------------------------- 12 2-10.2 馬立克病毒污染之研究------------------------------------------------------- 13 第十一節 內源性反轉錄病毒------------------------------------------------------------- 14 2-11.1 反轉錄病毒---------------------------------------------------------------------- 14 2-11.2 內源性反轉錄病毒------------------------------------------------------------- 14 第三章 材料與方法---------------------------------------------------------------------- 16 第一節 外源性 REV病毒分離及序列分析----------------------------------------- 16 3-1.1 細胞培養------------------------------------------------------------------------- 16 3-1.1.1 細胞------------------------------------------------------------------------------- 16 3-1.1.2 解凍細胞------------------------------------------------------------------------- 16 3-1.1.3 培養細胞------------------------------------------------------------------------- 17 3-1.1.4 細胞計數------------------------------------------------------------------------- 17 3-1.1.5 冷凍細胞------------------------------------------------------------------------- 17 3-1.1.6 胎牛血清之生長測試---------------------------------------------------------- 18 3-1.2 病毒分離------------------------------------------------------------------------- 18 3-1.2.1 血液樣本採集------------------------------------------------------------------- 18 3-1.2.2 分離病毒------------------------------------------------------------------------- 19 3-1.2.3 偵測病毒------------------------------------------------------------------------- 19 3-1.2.3.1 人工引子------------------------------------------------------------------------- 19 3-1.2.3.2 RNA萃取------------------------------------------------------------------------ 20 3-1.2.3.3 RT-PCR-------------------------------------------------------------------------- 20 3-1.2.3.4 RT-PCR產物的確認----------------------------------------------------------- 21 3-1.2.4 前病毒核酸檢測---------------------------------------------------------------- 21 3-1.2.4.1 人工引子------------------------------------------------------------------------- 21 3-1.2.4.2 DNA萃取------------------------------------------------------------------------ 21 3-1.2.4.3 PCR------------------------------------------------------------------------------- 22 3-1.2.4.4 PCR產物的確認---------------------------------------------------------------- 22 3-1.2.5 保存病毒------------------------------------------------------------------------- 22 3-1.2.6 測定病毒力價------------------------------------------------------------------- 23 3-1.2.6.1 接種病毒------------------------------------------------------------------------- 23 3-1.2.6.2 DNA萃取------------------------------------------------------------------------ 23 3-1.2.6.3 PCR------------------------------------------------------------------------------- 23 3-1.2.6.4 PCR產物的確認---------------------------------------------------------------- 23 3-1.3 基因選殖與序列分析---------------------------------------------------------- 23 3-1.3.1 載體之選擇---------------------------------------------------------------------- 23 3-1.3.2 設計人工引子------------------------------------------------------------------- 24 3-1.3.3 病毒增殖------------------------------------------------------------------------- 25 3-1.3.4 DNA萃取------------------------------------------------------------------------ 26 3-1.3.5 PCR------------------------------------------------------------------------------- 26 3-1.3.6 PCR產物的確認---------------------------------------------------------------- 26 3-1.3.7 PCR產物之純化---------------------------------------------------------------- 26 3-1.3.8 純化產物的確認---------------------------------------------------------------- 27 3-1.3.9 接合反應------------------------------------------------------------------------- 27 3-1.3.10 勝任細胞的製備---------------------------------------------------------------- 27 3-1.3.11 轉型作用------------------------------------------------------------------------- 28 3-1.3.12 菌株之篩選---------------------------------------------------------------------- 28 3-1.3.13 PCR產物的確認---------------------------------------------------------------- 29 3-1.3.14 菌種之超低溫保存------------------------------------------------------------- 29 3-1.3.15 質體純化------------------------------------------------------------------------- 29 3-1.3.16 核酸序列測定------------------------------------------------------------------- 29 3-1.3.17 核酸序列比對及親緣樹分析------------------------------------------------- 30 第二節 內源性REV基因檢測---------------------------------------------------------- 30 3-2.1 雜合反應樣品------------------------------------------------------------------- 30 3-2.1.1 樣品採集------------------------------------------------------------------------- 30 3-2.1.2 病毒------------------------------------------------------------------------------- 31 3-2.1.3 細胞------------------------------------------------------------------------------- 31 3-2.1.4 大量DNA萃取------------------------------------------------------------------ 31 3-2.1.5 PCR------------------------------------------------------------------------------- 31 3-2.1.6 PCR產物的確認---------------------------------------------------------------- 31 3-2.1.7 限制內切核酸酶切割反應---------------------------------------------------- 32 3-2.2 合成探針------------------------------------------------------------------------- 32 3-2.2.1 人工引子------------------------------------------------------------------------- 32 3-2.2.2 合成探針之模板---------------------------------------------------------------- 32 3-2.2.3 合成寡鏈核酸探針------------------------------------------------------------- 32 3-2.2.4 寡鏈核酸探針的確認---------------------------------------------------------- 33 3-2.3 南方雜合反應------------------------------------------------------------------- 33 3-2.3.1 洋菜膠電泳法------------------------------------------------------------------- 33 3-2.3.2 毛細轉漬作用------------------------------------------------------------------- 33 3-2.3.3 雜合反應------------------------------------------------------------------------- 34 3-2.3.4 呈色反應------------------------------------------------------------------------- 34 第三節 禽痘病毒污染REV------------------------------------------------------------- 35 3-3.1 病毒顆粒型態觀察------------------------------------------------------------- 35 3-3.1.1 疫苗來源------------------------------------------------------------------------- 35 3-3.1.2 增殖病毒------------------------------------------------------------------------- 35 3-3.1.3 病毒之濃縮與純化------------------------------------------------------------- 35 3-3.1.4 電子顯微鏡之病毒型態------------------------------------------------------- 36 3-3.2 病毒性狀與基因分析---------------------------------------------------------- 36 3-3.2.1 人工引子------------------------------------------------------------------------- 36 3-3.2.2 增殖病毒------------------------------------------------------------------------- 36 3-3.2.3 絨毛尿囊膜接種---------------------------------------------------------------- 36 3-3.2.4 DNA萃取------------------------------------------------------------------------ 37 3-3.2.5 PCR------------------------------------------------------------------------------- 37 3-3.2.6 PCR產物的確認---------------------------------------------------------------- 37 3-3.3 基因選殖與序列分析---------------------------------------------------------- 37 3-3.3.1 載體之選擇---------------------------------------------------------------------- 37 3-3.3.2 增殖病毒------------------------------------------------------------------------- 37 3-3.3.3 DNA萃取------------------------------------------------------------------------ 37 3-3.3.4 聚合酶鏈反應------------------------------------------------------------------- 38 3-3.3.5 PCR產物的確認---------------------------------------------------------------- 38 3-3.3.6 PCR產物之純化---------------------------------------------------------------- 38 3-1.3.7 純化產物的確認---------------------------------------------------------------- 38 3-3.3.8 接合反應------------------------------------------------------------------------- 38 3-3.3.9 轉型作用------------------------------------------------------------------------- 38 3-3.3.10 菌株之挑選---------------------------------------------------------------------- 38 3-3.3.11 PCR產物的確認---------------------------------------------------------------- 38 3-3.3.12 菌種之超低溫保存------------------------------------------------------------- 38 3-3.3.13 質體純化------------------------------------------------------------------------- 38 3-3.3.14 核酸序列測定------------------------------------------------------------------- 39 3-3.3.15 核酸序列比對及親緣樹分析------------------------------------------------- 39 3-3.4 雜合反應樣品------------------------------------------------------------------- 39 3-3.4.1 病毒------------------------------------------------------------------------------- 39 3-3.4.2 細胞------------------------------------------------------------------------------- 39 3-3.4.3 大量DNA萃取------------------------------------------------------------------ 39 3-3.4.4 限制內切核酸酶切割反應---------------------------------------------------- 39 3-3.5 合成探針------------------------------------------------------------------------- 39 3-3.6 南方雜合反應------------------------------------------------------------------- 39 第四章 結果------------------------------------------------------------------------------- 40 第一節 外源性REV病毒分離--------------------------------------------------------- 40 4-1.1 細胞培養------------------------------------------------------------------------- 40 4-1.1.1 細胞------------------------------------------------------------------------------- 40 4-1.1.2 胎牛血清之生長測試---------------------------------------------------------- 40 4-1.2 病毒分離------------------------------------------------------------------------- 40 4-1.2.1 分離病毒與核酸檢測---------------------------------------------------------- 40 4-1.2.2 病毒保存------------------------------------------------------------------------- 41 4-1.2.3 病毒力價測定------------------------------------------------------------------- 41 4-1.3 基因選殖與序列分析---------------------------------------------------------- 41 4-1.3.1 病毒增殖------------------------------------------------------------------------- 41 4-1.3.2 基因選殖之聚合酶鏈反應---------------------------------------------------- 41 4-1.3.3 純化產物的確認---------------------------------------------------------------- 41 4-1.3.4 菌株之挑選及確認------------------------------------------------------------- 42 4-1.3.5 菌種之保存---------------------------------------------------------------------- 42 4-1.3.6 核酸序列比對及親緣樹分析------------------------------------------------- 42 第二節 內源性REV基因檢測---------------------------------------------------------- 43 4-2.1 雜合反應樣品------------------------------------------------------------------- 43 4-2.2 合成探針------------------------------------------------------------------------- 43 4-2.2.1 合成探針之模板---------------------------------------------------------------- 43 4-2.2.2 合成寡鏈核酸探針------------------------------------------------------------- 43 4-2.3 南方雜合反應------------------------------------------------------------------- 43 第三節 禽痘病毒污染REV------------------------------------------------------------- 44 4-3.1 病毒顆粒型態觀察------------------------------------------------------------- 44 4-3.1.1 增殖病毒------------------------------------------------------------------------- 44 4-3.1.2 病毒之濃縮與純化------------------------------------------------------------- 44 4-3.1.3 電子顯微鏡之病毒型態------------------------------------------------------- 44 4-3.2 病毒性狀與基因分析---------------------------------------------------------- 44 4-3.2.1 增殖病毒------------------------------------------------------------------------- 44 4-3.2.2 絨毛尿囊膜接種---------------------------------------------------------------- 45 4-3.2.3 聚合酶鏈反應------------------------------------------------------------------- 45 4-3.3 基因選殖------------------------------------------------------------------------- 45 4-3.3.1 PCR------------------------------------------------------------------------------- 45 4-3.3.2 PCR產物之純化---------------------------------------------------------------- 45 4-3.3.3 菌株之挑選與確認------------------------------------------------------------- 45 4-3.3.4 菌種之超低溫保存------------------------------------------------------------- 46 4-3.3.5 核酸序列比對及親緣樹分析------------------------------------------------- 46 4-3.4 南方雜合反應------------------------------------------------------------------- 46 第五章 討論與結論---------------------------------------------------------------------- 47 第一節 分離病毒------------------------------------------------------------------------- 47 第二節 內源性REV--------------------------------------------------------------------- 48 第三節 禽痘病毒污染REV------------------------------------------------------------ 50 第六章 參考文獻------------------------------------------------------------------------- 525528150 bytesapplication/pdfen-US家禽網狀內皮增生症病毒內源性反轉錄病毒reticuloendotheliosis virusendogenousretrovirus[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/60021/1/ntu-96-R94629003-1.pdf