王金和Wang, Ching-Ho臺灣大學:獸醫學研究所林佳瑤Lin, Chia-YaoChia-YaoLin2010-05-042018-07-092010-05-042018-07-092008U0001-2107200813052400http://ntur.lib.ntu.edu.tw//handle/246246/178898目前有關家禽網狀內皮增生症病毒 (Avian Reticuloendotheliosis Virus, REV)研究以雞與火雞為主，與鵝相關的研究很少，本研究的目的是了解台灣現場鵝隻REV疾病現況，利用聚合酶鏈反應 (PCR) 以及血清中和試驗來檢測REV感染情形。於一家白羅曼鵝場病例中將buffy coat接種到DF-1細胞，分離到九株REV分離株，完成其中一株全段前病毒基因定序，將此株REV與台灣雞REV分離株共四株與國外REV進行親源性分析，比較國內外REV的基因序列差異。結果顯示此分離株與其他株的gag跟pol基因相似度分別是96.9到99.9%以及96.7到99.9%之間，env基因相似度為95.9到100%之間，LTR是在REV基因序列中差異最大的區域，此分離株與其他株LTR基因相似度為74.9到99.8%之間，因為在LTR區域中含有較大片段的插入和缺失。根據以上序列比對以及親源性分析之結果，顯示此分離株與其他REV毒株相當接近，表示REV在不同品種的禽類宿主基因的變異性很小。塗鍍濃縮純化的REV全病毒搭配標示過氧化氫酶之單株抗體以架構阻斷型酵素連結免疫吸附反應 (ELISA)，以用來檢測水禽REV抗體，實驗結果顯示每孔需要塗鍍600 ngREV全病毒搭配50倍稀釋標有過氧化氫酶之抗體才能達到OD值接近於1之效果，但此條件不符合經濟效應。以PCR檢測四場鵝隻血液，結果顯示血液REV核酸陽性率為28.8%，以血清中和試驗檢測抗體，其陽性率為60.3%，顯示台灣鵝隻REV感染情況為相當普遍。Many reticuloendotheliosis virus (REV) associated studies have been focused on chicken or turkey, but rarely on goose. The purpose of this study was to investigate theEV occurrence in geese in Taiwan. PCR and serum neutralization test were used to detect REV infection. Nine REV isolates from a white Roman goose farm were isolated by inoculating buffy coat onto the DF-1 cell line. The complete proviral sequence of one REV was determined. The relationship of gene sequences of four Taiwanese REVs were compared with the reference REVs by the phylogenetic analysis. The percent of nucleotide identity of gag and pol ranged from 96.9 to 99.9% and 96.7 to 99.9%, respectively. The percent of nucleotide identity of env ranged from 95.9 to 100%. The LTR was the most divergent region of genome with 74.9 to 99.8% nucleotide identity containing deletion and insertion. It indicates that this isolate is closely related to other REVs and the gene diversity among REV isolates in different avian hosts is minimal. For developing a blocking enzyme-linked immunosorbent assay (ELISA) to detect antibody in waterfowls, the concentrated and purified REV was used as coating antigen and monoclonal antibody was labeled with horseradish peroxidase as tracer. The optical density value was close to 1 on the condition that 600 ng of REV per well was coated with 50-fold diluted tracer. The REV detection by PCR showed 28.8% positive rate and antibody detection by serum neutralization test showed 60.3% positive rate in four goose farms. Thus REV infection is common in geese in Taiwan.口試委員會審定書-------------------------------------------------------------------------- Ι謝-------------------------------------------------------------------------------------------- Π文摘要-------------------------------------------------------------------------------------- Ш文摘要-------------------------------------------------------------------------------------- IV錄-------------------------------------------------------------------------------------------- VI次-------------------------------------------------------------------------------------------- XI次-------------------------------------------------------------------------------------------- XII錄-------------------------------------------------------------------------------------------- XII一章 緒言-------------------------------------------------------------------------- 1二章 文獻探討-------------------------------------------------------------------- 3一節 簡介-------------------------------------------------------------------------- 3二節 歷史背景-------------------------------------------------------------------- 3三節 病毒特性-------------------------------------------------------------------- 3-3.1 病毒分類-------------------------------------------------------------------- 3-3.2 病毒形態-------------------------------------------------------------------- 4-3.3 病毒基因體----------------------------------------------------------------- 4-3.4 致癌基因-------------------------------------------------------------------- 5-3.5 病毒基因的轉錄與複製-------------------------------------------------- 5-3.6 病毒蛋白及其功能-------------------------------------------------------- 6-3.7 病毒族群-------------------------------------------------------------------- 6-3.8 病毒複製之宿主特異性-------------------------------------------------- 7-3.9 病毒物理化學特性-------------------------------------------------------- 8四節 臨床症狀與病理變化----------------------------------------------------- 8-4.1 潛伏期----------------------------------------------------------------------- 8-4.2 臨床症狀-------------------------------------------------------------------- 9-4.3 病理變化-------------------------------------------------------------------- 9-4.3.1 雞隻消耗性症狀----------------------------------------------------------- 9-4.3.2 雞隻華氏囊腫瘤----------------------------------------------------------- 10-4.3.3 雞隻非華氏囊腫瘤-------------------------------------------------------- 10-4.3.4 其他禽種之淋巴肉瘤----------------------------------------------------- 10-4.4 致病機制-------------------------------------------------------------------- 11-4.4.1 消耗性症狀----------------------------------------------------------------- 11-4.4.2 慢性淋巴瘤----------------------------------------------------------------- 11-4.4.3 急性網狀細胞瘤----------------------------------------------------------- 11五節 流行病學-------------------------------------------------------------------- 12-5.1 宿主特異性----------------------------------------------------------------- 12-5.2 傳染途徑-------------------------------------------------------------------- 12-5.2.1 水平傳播-------------------------------------------------------------------- 12-5.2.2 垂直傳播-------------------------------------------------------------------- 13-5.2.3 污染的生物性物質-------------------------------------------------------- 14-5.3 臺灣地區情形-------------------------------------------------------------- 14六節 診斷方法-------------------------------------------------------------------- 14-6.1 病毒抗原檢測-------------------------------------------------------------- 14-6.2 抗體檢測-------------------------------------------------------------------- 15-6.3 區別診斷-------------------------------------------------------------------- 15七節 預防與治療----------------------------------------------------------------- 16三章 材料與方法----------------------------------------------------------------- 18一節 REV 病毒核酸檢測------------------------------------------------------- 18-1.1 病材採樣-------------------------------------------------------------------- 18-1.1.1 血液樣本採樣-------------------------------------------------------------- 18-1.1.2 腫瘤組織採樣-------------------------------------------------------------- 18-1.1.3 乳劑製作-------------------------------------------------------------------- 18-1.1.4 採樣來源-------------------------------------------------------------------- 18-1.2 前病毒核苷酸檢測-------------------------------------------------------- 18-1.2.1 DNA 萃取------------------------------------------------------------------- 18-1.2.2 引子 (primer)-------------------------------------------------------------- 19-1.2.3 聚合酶鏈反應 (PCR)----------------------------------------------------- 19-1.2.4 洋菜膠電泳分析----------------------------------------------------------- 20-1.2.5 PCR 產物核苷酸定序確認----------------------------------------------- 20二節 病毒分離-------------------------------------------------------------------- 20-2.1 細胞培養-------------------------------------------------------------------- 20-2.1.1 細胞-------------------------------------------------------------------------- 20-2.1.2 解凍細胞-------------------------------------------------------------------- 21-2.1.3 培養細胞-------------------------------------------------------------------- 21-2.1.4 細胞計數-------------------------------------------------------------------- 21-2.1.5 冷凍細胞-------------------------------------------------------------------- 22-2.2 病材採樣-------------------------------------------------------------------- 22-2.2.1 血液樣本採樣-------------------------------------------------------------- 22-2.2.2 腫瘤組織採樣-------------------------------------------------------------- 22-2.2.3 乳劑製作-------------------------------------------------------------------- 22-2.2.4 採樣來源-------------------------------------------------------------------- 23-2.3 分離病毒-------------------------------------------------------------------- 23-2.4 偵測病毒-------------------------------------------------------------------- 23-2.4.1 病毒RNA萃取------------------------------------------------------------- 23-2.4.2 反轉錄聚合酶鏈反應 (RT-PCR)--------------------------------------- 24-2.4.3 洋菜膠電泳分析----------------------------------------------------------- 24-2.5 病毒增殖-------------------------------------------------------------------- 25-2.6 病毒力價測定-------------------------------------------------------------- 25-2.6.1 接種病毒-------------------------------------------------------------------- 25-2.6.2 聚合酶鏈反應-------------------------------------------------------------- 25-2.6.3 洋菜膠電泳分析----------------------------------------------------------- 26-2.6.4 計算病毒力價-------------------------------------------------------------- 26-2.7 病毒濃縮與純化----------------------------------------------------------- 26-2.8 蛋白質濃度測定----------------------------------------------------------- 27-2.9 抗原性分析----------------------------------------------------------------- 27-2.9.1 單株抗體-------------------------------------------------------------------- 27-2.9.2 免疫墨點法 (Immunodot blot assay)----------------------------------- 28-2.9.3 聚丙烯醯胺膠片電泳 (SDS-PAGE)----------------------------------- 28-2.9.4 酵素連結免疫吸附分析 (ELISA)-------------------------------------- 29三節 病毒基因全長定序與序列分析----------------------------------------- 29-3.1 病毒來源-------------------------------------------------------------------- 29-3.2 前病毒DNA 萃取--------------------------------------------------------- 29-3.3 引子 (primer)-------------------------------------------------------------- 30-3.4 聚合酶鏈反應 (PCR)----------------------------------------------------- 31-3.5 洋菜膠電泳分析----------------------------------------------------------- 31-3.6 PCR 產物之純化----------------------------------------------------------- 31-3.7 基因選殖-------------------------------------------------------------------- 32-3.7.1 載體 (vector)--------------------------------------------------------------- 32-3.7.2 接合反應 (ligation) ------------------------------------------------------ 32-3.7.3 轉形作用 (transformation)----------------------------------------------- 32-3.7.4 菌株之篩選----------------------------------------------------------------- 33-3.8 質體純化-------------------------------------------------------------------- 33-3.9 核苷酸定序----------------------------------------------------------------- 34-3.10 核酸序列比對及親緣樹分析-------------------------------------------- 34四節 血清學檢測----------------------------------------------------------------- 35-4.1 血液樣本採集-------------------------------------------------------------- 35-4.1.1 採樣方式-------------------------------------------------------------------- 35-4.1.2 採樣來源-------------------------------------------------------------------- 36-4.2 血清中和試驗-------------------------------------------------------------- 36-4.2.1 中和反應-------------------------------------------------------------------- 36-4.2.2 聚合酶鏈反應-------------------------------------------------------------- 37-4.2.3 洋菜膠電泳分析----------------------------------------------------------- 37-4.2.4 抗體力價判定-------------------------------------------------------------- 37-4.3 血液樣本之REV 前病毒核酸檢測------------------------------------- 37-4.3.1 DNA 萃取------------------------------------------------------------------- 37-4.3.2 引子 (primer) ------------------------------------------------------------- 38-4.3.3 聚合酶鏈反應 (PCR)----------------------------------------------------- 38-4.3.4 洋菜膠電泳分析----------------------------------------------------------- 38五節 阻斷型ELISA 之開發---------------------------------------------------- 38-5.1 病毒來源-------------------------------------------------------------------- 38-5.2 單株抗體-------------------------------------------------------------------- 38-5.3 單株抗體純化-------------------------------------------------------------- 38-5.4 過氧化氫酶標示單株抗體----------------------------------------------- 39-5.5 阻斷型ELISA 最佳化---------------------------------------------------- 39四章 結果-------------------------------------------------------------------------- 40一節 REV 病毒核酸檢測------------------------------------------------------- 40-1.1 病例介紹-------------------------------------------------------------------- 40-1.2 聚合酶鏈反應-------------------------------------------------------------- 40-1.3 PCR產物核苷酸定序確認----------------------------------------------- 41二節 病毒分離-------------------------------------------------------------------- 41-2.1 偵測病毒核酸-------------------------------------------------------------- 41-2.2 病毒力價測定-------------------------------------------------------------- 41-2.3 病毒濃縮與純化----------------------------------------------------------- 41-2.4 蛋白質濃度測定----------------------------------------------------------- 42-2.5 抗原性分析----------------------------------------------------------------- 42-2.5.1 免疫墨點法----------------------------------------------------------------- 42-2.5.2 酵素連結免疫吸附分析-------------------------------------------------- 42三節 病毒基因全長定序與序列分析----------------------------------------- 42-3.1 聚合酶鏈反應-------------------------------------------------------------- 42-3.2 基因選殖之菌株篩選----------------------------------------------------- 42-3.3 病毒基因全長定序-------------------------------------------------------- 43-3.4 核酸序列比對及親緣樹分析-------------------------------------------- 43-3.5 胺基酸序列比對及親緣樹分析----------------------------------------- 45四節 血清學檢測----------------------------------------------------------------- 46-4.1 肉鵝血清學檢測----------------------------------------------------------- 46-4.2 肉鵝REV抗原檢測-------------------------------------------------------- 46-4.3 資料分析 46五節 阻斷型ELISA 之開發---------------------------------------------------- 47-5.1 單株抗體純化-------------------------------------------------------------- 47-5.2 過氧化氫酶標示單株抗體----------------------------------------------- 47-5.3 阻斷型ELISA 最佳化---------------------------------------------------- 47五章 討論與結論----------------------------------------------------------------- 48六章 參考文獻-------------------------------------------------------------------- 54次igure 1. Schematic diagram of a retrovirus virion indicating various structures and proteins.----------------------------------------------------- 67igure 2. Photograph of gross pathology of case 3410/06.----------------------- 68igure 3. Photograph of microscopic pathology of case 3410/06.--------------- 69igure 4. PCR products (291 bp) of LTR fragment amplified from the genomes of goose blood and tissue samples.---------------------------- 71igure 5. PCR products (1867 bp) of env amplified from the genomes of goose blood and tissue samples.------------------------------------------ 71igure 6. RT-PCR products (291 bp) of LTR fragment. RNA was extracted from the supernatants of infected cells.---------------------------------- 72igure 7. RT-PCR products (1867 bp) of env.-------------------------------------- 72igure 8. Virus titration of REV 3410/06 by PCR.-------------------------------- 73igure 9. Virus purification (isolate 3410/06).-------------------------------------- 73igure 10. Immunodot blot assay using different concentration of REV chicken isolate 3295/04 and goose isolate 3410/06 reacting with monoclonal antibodies 1, 2 and 3.---------------------------------------- 74igure 11. Binding ability of REV isolates 3295/04 and 3410/06 to REV monoclonal antibodies tested by ELISA.-------------------------------- 75igure 12. PCR products of different fragments amplified from the provirus of 3410/06 by different primer pairs.---------------------------------------- 76igure 13. Electrophoresis of colony PCR of env amplified by the primer pair M13-F/M13-R. The size of insert was 1867 bp. ------------------------ 77igure 14. Electrophoresis of colony PCR of partial gag-pol gene amplified by the primer pair M13-F/M13-R. The size of insert was 800 bp.------- 77igure 15. Electrophoresis of colony PCR of partial pol gene amplified by the primer pair M13-F/M13-R. The size of insert was 1776 bp.---------- 78igure 16. The proviral genome of REV 3410/06.----------------------------------- 78igure 17. Phylogenic tree of complete genome of the REV Taiwan isolate chicken/3337/05, goose/3410/06, and published isolates.------------- 79igure 18. Phylogenic tree of the nucleotide sequences of env gene among several REV isolates.------------------------------------------------------- 79igure 19. Phylogenic tree of the nucleotide sequences of LTR among REV isolates.------------------------------------------------------------------------ 80igure 20. Nucleotide sequence alignment of LTR region among REV isolates. 81igure 21. Alignment of amino acids of the predicted envelope protein among REV isolates.----------------------------------------------------------------- 84igure 22. SDS-PAGE analysis of purified monoclonal antibody 1 (mAb1) and HRP-conjugated mAb1.----------------------------------------------- 88igure 23. The optimum to develop blocking ELISA------------------------------- 88次able 1. primers used for PCR and sequencing------------------------------------- 89able 2. The identity of complete proviral sequences of REV isolates in Taiwan (goose/3410/06, chicken/3337/05) and published sequences.----- 91able 3. The identity of gag among Taiwan (goose/3410/06,hicken/3337/05) and published REV isolates. -------------------------- 92able 4. The identity of pol among Taiwan (goose/3410/06,hicken/3337/05) and published REV isolates.-------------------------- 93able 5. The identity of env among several Taiwan (goose/3410/06, chicken/3337/05, chicken/3122/03 and chicken/3295/04) and published REV isolates.----------------------------------------------------- 94able 6. The identity of LTR sequences among Taiwan (goose/3410/06, chicken/3337/05) and published REV isolates.-------------------------- 95able 7. The prevalence of antibody to REV and average titer of antibody.-- 96錄ppendix 1. The complete sequences of REV isolate goose/3410/06.--- 97ppendix 2. The prevalence of antibody to reticuloendotheliosis virus and detection of LTR region in geese. ---102ppendix 3. The results of PCR and Ab titer of goose blood samples tested.--- 103application/pdf2665351 bytesapplication/pdfen-US家禽網狀內皮增生症病毒序列分析病毒分離病毒中和試驗單株抗體鵝家禽腫瘤疾病avian reticuloendotheliosis virusphylogenetic analysisneutralization testmonoclonal antibodyvirus isolationgooseavian oncogenic diseasethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178898/1/ntu-97-R95629019-1.pdf