臺灣博碩士論文加值系統

禽白血病J亞群 (subgroup J avian leukosis virus) 在控制上主要以撲滅計畫為主，因此最重要的是區分出感染和未感染的雞隻，在檢測方面主要是偵測病毒抗原、核酸，或是做抗體的檢測，不過目前所用的方式都有一些限制存在。因此本研究欲試圖發展更好的診斷方法。本研究利用基因選殖技術將本實驗室ALV-J分離株2921的部分gp85基因 (含vr2、hr1及hr2三個高變異區) 選殖出來，進行重組蛋白 ”gp85N”的表現並針對選殖部分的胺基酸片段發展單株抗體，再同時利用重組蛋白gp85N發展間接ELISA來偵測ALV-J 特異性抗體及使用單株抗體發展一三明治ELISA來偵測ALV-J抗原。其中偵測ALV-J 特異性抗體間接ELISA使用上有非特異性反應。發展單株抗體方面一共獲得2株單株抗體mAb14及mAb22，進一步利用單株來偵測ALV-J抗原，結果mAb14及mAb22都可成功偵測到重組蛋白gp85N，但是只有mAb22可以偵測到ALV-J病毒。另外，國內的有色雞族群為了增加產能，多以台灣本地雞與進口肉種雞做雜交以改良後代，因而將ALV-J引入台灣有色雞群中，但詳細的感染狀況卻仍不明，因此針對有色雞群做血清學調查，共調查10個種用雞群與40個肉用雞群，其中公母各半，種用雞群不論公母，全部為ALV-J抗體陽性；肉用雞群方面，公雞群有15% (3/20) 為ALV-J抗體陽性，母雞群則有10% (2/ 20) 為ALV-J抗體陽性，結果顯示ALV-J普遍存於台灣有色雞族群中。此外，在2002至2004年間我們仍陸續自白肉種雞與蛋種雞病例見有腫瘤發生且分離出ALV-J，顯示除了有色雞族群外，在白肉種雞與蛋種雞族群間ALV-J感染問題仍然存在。

Control of subgroup J avian leukosis virus (ALV-J) is mainly through elimination, and the first challenge is to differentiate between the infected and noninfected chickens. Methods for direct detection of ALV-J include the detection of viral RNA and viral antigen, or indirect detection for antibodies. However all of the currently available diagnostic methods have some limitation. The specific aim of this study is to develop more useful diagnostic methods for detecting ALV-J. In this study, partial gp85 gene, which included three variable regions: vr3, hr1 and hr2, was cloned into pRSET B and expressed in BL21(DE3). The expressed protein gp85N was used to develop indirect ELISA for detecting ALV-J specific antibody but this indirect ELISA had nonspecific reaction. The same gene fragment was used to prepare monoclonal antibodies. Two monoclonal antibodies, mAb14 and mAb22, were obtained and used to detect ALV-J antigen. The results indicate that both mAb14 and mAb22 react with gp85N, but only the later could detect ALV-J virus. In order to increase economic benefits, the poultry industries hybrid broiler breeder and native chicken in Taiwan and ALV-J was introduced into Taiwan by this way. But the situation of ALV-J infection in hybrid native chickens was unclear. The serological investigation was done for hybrid native chickens. In this study, 10 flocks of breeder and 40 flocks of meat-type chicken were investigated. Breeder flocks was 100% (10/10) ALV-J antibody positive no matter the male or female, meat-type-chicken flocks was 15% (3/20) ALV-J antibody positive in male and 10% (2/ 20) ALV-J antibody positive in female. The results of serological investigation indicate ALV-J is widespread among hybrid native chickens. Furthermore, tumors induced by ALV-J were seen in broiler breeder and layer breeder, and viruses were isolated from these clinical cases during 2002-2004 reveal that ALV-J still exists in broiler breeder and layer breeder flocks.

目錄----------------------------------------------------------------------------------------------Ⅰ
圖次----------------------------------------------------------------------------------------------Ⅳ
表次----------------------------------------------------------------------------------------------Ⅵ
中文摘要----------------------------------------------------------------------------------------Ⅶ
英文摘要----------------------------------------------------------------------------------------Ⅷ

1. 緒言-------------------------------------------------------------------------------------------1
2. 文獻探討-------------------------------------------------------------------------------------3
2.1 歷史背景-------------------------------------------------------------------------------------3
2.2 家禽白血病病毒介紹----------------------------------------------------------------------4
2.2.1 家禽白血病病毒分類-------------------------------------------------------------------4
2.2.2 家禽白血病病毒的形態與特性-------------------------------------------------------5
2.2.3 家禽白血病病毒的基因體結構與病毒蛋白----------------------------------------5
2.3 家禽白血病病毒致腫瘤機制-------------------------------------------------------------6
2.4 家禽白血病J亞群病毒--------------------------------------------------------------------7
2.4.1 外源性家禽白血病J亞群病毒--------------------------------------------------------7
2.4.2 內源性家禽白血病J亞群病毒--------------------------------------------------------8
2.5 家禽白血病之流行病學-------------------------------------------------------------------9
2.5.1 臨床症狀與病理變化-------------------------------------------------------------------9
2.5.2 病毒的傳播------------------------------------------------------------------------------10
2.6 家禽白血病病毒之實驗室診斷--------------------------------------------------------11
2.7 家禽白血病之預防與控制--------------------------------------------------------------12
3. 材料與方法---------------------------------------------------------------------------------14
3.1 血漿樣本採集與血清學檢測-----------------------------------------------------------14
3.1.1 血漿樣本採集---------------------------------------------------------------------------14
3.1.1.1 採樣方式-------------------------------------------------------------------------------14
3.1.1.2 樣本來源-------------------------------------------------------------------------------14
3.1.2 血清學檢測------------------------------------------------------------------------------14
3.1.2.1 抗體檢測-------------------------------------------------------------------------------14
3.1.2.2 抗原檢測-------------------------------------------------------------------------------14
3.2 細胞培養-----------------------------------------------------------------------------------15
3.3 病毒增殖與病毒核酸偵測--------------------------------------------------------------15
3.3.1 病毒來源---------------------------------------------------------------------------------15
3.3.2 病毒增殖---------------------------------------------------------------------------------15
3.3.3 病毒之濃縮與純化---------------------------------------------------------------------16
3.3.4 病毒力價測定-------------------------------------------------------------------------16
3.3.5 病毒核酸偵測---------------------------------------------------------------------------16
3.3.5.1 病毒RNA之萃取---------------------------------------------------------------------16
3.3.5.2 細胞之DNA萃取--------------------------------------------------------------------17
3.3.5.3 反轉錄反應 (RT)--------------------------------------------------------------------17
3.3.5.4 聚合酵素鏈反應 (PCR)------------------------------------------------------------18
3.4 聚合酵素鏈反應產物之選殖 (cloning)-----------------------------------------------18
3.4.1 聚合酵素鏈反應產物之純化---------------------------------------------------------18
3.4.2 聚合酵素鏈反應產物之選殖---------------------------------------------------------18
3.5 建構表現載體-----------------------------------------------------------------------------19
3.5.1 目標基因之增幅------------------------------------------------------------------------19
3.5.2 構築表現載體---------------------------------------------------------------------------19
3.6 重組蛋白之表現與純化-----------------------------------------------------------------20
3.6.1 勝任細胞的製備與表現載體的轉殖------------------------------------------------20
3.6.2 質體小量抽取法 (miniprep)----------------------------------------------------------20
3.6.3 限制酶切割------------------------------------------------------------------------------21
3.6.4 重組蛋白之表現------------------------------------------------------------------------21
3.6.5 重組蛋白之純化------------------------------------------------------------------------21
3.6.6 重組蛋白之確認------------------------------------------------------------------------22
3.6.7 重組蛋白之定量------------------------------------------------------------------------22
3.7 重組蛋白之應用--------------------------------------------------------------------------23
3.7.1 gp85N塗鍍ELISA平盤之製備與應用-Indirect ELISA--------------------------23
3.7.1.1 不同gp85N塗鍍量對抗體偵測結果之影響-------------------------------------23
3.7.1.2 不同血清種類與血清稀釋倍數對抗體偵測結果之影響----------------------23
3.7.2 單株抗體之生產與應用---------------------------------------------------------------24
3.7.2.1 單株抗體之生產----------------------------------------------------------------------24
3.7.2.2 單株抗體之確認----------------------------------------------------------------------24
3.7.2.2.1 Immunodot blot assay--------------------------------------------------------------24
3.7.2.2.2 免疫螢光分析 (immunofluorescence assay, IFA)-----------------------------24
3.7.2.3 單株抗體之應用-Sandwich ELISA------------------------------------------------25
4. 結果------------------------------------------------------------------------------------------26
4.1 台灣有色雞群之血清學調查-----------------------------------------------------------26
4.2 2002年至2004年肉雞、蛋種雞與有色雞種雞之家禽白血病J亞群病毒分離情形----------------------------------------------------------------------------------------------26
4.3 建構表現載體-----------------------------------------------------------------------------29
4.3.1 表現載體之確認------------------------------------------------------------------------29
4.4 重組蛋白之表現與純化-----------------------------------------------------------------30
4.4.1 重組蛋白之表現------------------------------------------------------------------------30
4.4.2 重組蛋白之純化------------------------------------------------------------------------30
4.5 重組蛋白之應用--------------------------------------------------------------------------30
4.5.1 應用gp85N塗鍍ELISA平盤偵測ALV-J抗體------------------------------------30
4.5.1.1 不同gp85N塗鍍量對抗體偵測結果之影響-------------------------------------30
4.5.1.2 不同血清種類與血清稀釋倍數對抗體偵測結果之影響----------------------30
4.5.1.3 使用gp85N塗鍍ELISA平盤測試SPF雞隻血清樣本-------------------------31
4.5.2 單株抗體抗原性之確認---------------------------------------------------------------31
4.5.2.1 Immunodot blot assay-----------------------------------------------------------------31
4.5.2.2 西方墨點法----------------------------------------------------------------------------31
4.5.2.3 免疫螢光分析 (immunofluorescence assay, IFA)-------------------------------31
4.5.3 單株抗體之力價測定------------------------------------------------------------------31
4.5.4 Sandwich ELISA之開發---------------------------------------------------------------31
5. 討論------------------------------------------------------------------------------------------67
6. 參考文獻------------------------------------------------------------------------------------71

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