我國石斑魚魚苗產值高達三億五千萬元；然而，神經壞死病毒感染卻造成其仔稚魚死亡率達100％，對養殖漁業造成嚴重的經濟損失。使用單株抗體可對病毒顆粒的表面結構做定位分析模擬病毒之主抗原區，以了解感染機制。本研究利用龍膽石斑神經壞死病毒及其似病毒顆粒(7種選殖株)，以腹腔及皮下方式施打Balb/c小鼠來製作融合瘤，初步評估多株和單株抗體在製備和產量的優劣勢。研究結果顯示，融合後之融合瘤細胞之一，hAb_VLP8在七週內有顯著地生產抗體的能力，但在七至九週之間生產力開始下降，九週後已無生產抗體的能力。而正控制組中能生產單株抗體的細胞及其腹水，在九週後都穩定地生產抗體，腹水的抗體力價高於細胞生產的1200倍，而正控制組細胞的生產量又比hAb_VLP8高出100倍。所以在尚未選出單株之前，可以在融合後六到七週細胞初步穩定時，可先以腹水的方式生產抗體。

The groupers are vital fish in the market of over 350 million dollars, while grouper nervous necrosis virus (NNV) has caused mass mortality at about 100% in larvae and juveniles, which impacts on economic of marine cultured fish. The monoclonal antibody is one of the best methods to identify the epitopes on the 3D structure. For evaluation, the Balb/c mice were injected with DGNNV and virus-like particles (VLPs) in this study. The results showed that ascite of mAb-cells produced 1200 times higher than the cell secretion in the medium whereas our best clone hAb_VLP8 can only produced 100 times less antibody than the cell secretion. In the meantime before the monoclonal producer is established, the hAb_VLP8 could be used for ascite production to gain high antibody production.

謝誌 -------------------------------------------------------------------------- I
中文摘要 ------------------------------------------------------------------- Ⅱ
Abstract --------------------------------------------------------------------- Ⅲ
目錄 ------------------------------------------------------------------------- Ⅳ
表目錄 ---------------------------------------------------------------------- Ⅵ
圖目錄 ---------------------------------------------------------------------- Ⅶ
壹、前言
一、台灣石斑養殖現況 ------------------------------------------------------------- 1
二、魚類神經壞死病毒(Piscine nervous necrosis virus) ----------------------- 3
三、三、增殖Betanodavirus之細胞株 ----------------------------------------- 5
四、似病毒顆粒(virus-like particles, VLPs) -------------------------------------- 8
五、抗體的特性 ---------------------------------------------------------------------- 14
六、多株抗體 ------------------------------------------------------------------------- 16
七、單株抗體的發展優勢 --------------------------------------------------------- 17
八、抗體工程的發展 --------------------------------------------------------------- 20
九、研究目的 ---------------------------------------------------------------------- 23
貳、材料與方法
一、質體的抽取 ---------------------------------------------------------------------- 26
二、勝任細胞 (Competent cell) 的製作 ----------------------------------------- 26
三、細胞轉型 ------------------------------------------------------------------------- 26
四、似病毒顆粒之製備與純化 ---------------------------------------------------- 27
五、流式分光光度計及自動分樣器 ---------------------------------------------- 28
六、負染(Negative stain)及電子顯微鏡 ------------------------------------------ 29
七、SDS-PAGE蛋白質電泳 ------------------------------------------------------- 29
八、脾臟組織Total RNA的抽取 -------------------------------------------------- 30
九、細胞Total RNA的抽取 ------------------------------------------------------- 31
十、RT-PCR --------------------------------------------------------------------------- 31
十一、DNA電泳 --------------------------------------------------------------------- 32
十二、SSN-1細胞培養 ------------------------------------------------------------- 32
十三、DGNNV的生產 ------------------------------------------------------------- 33
十四、The 50% tissue culture infectious dose (TCID50) 之測定 ------------- 33
十五、蛋白質定量 (Lowry method) ---------------------------------------------- 34
十六、抗VLP之小鼠血清製備 --------------------------------------------------- 35
十七、Kohler細胞融合法 ---------------------------------------------------------- 35
十八、Modify Kohler法 ------------------------------------------------------------ 40
十九、Kohler法之酵素免疫吸附法 ---------------------------------------------- 44
二十、Modify Kohler法之酵素免疫吸附法 ------------------------------------ 45
二一、14日長週期之細胞培養 --------------------------------------------------- 46

參、結果
一、融合瘤細胞所需高純度之DGNNV與VLP --------------------------------- 47
二、Kohler免疫法與Modify Kohler免疫法 -------------------------------------- 49
三、融合所需之細胞 ---------------------------------------------------------------- 50
四、Kohler細胞融合法與Modify Kohler細胞融合法 ------------------------ 51
五、融合型態與表現 ---------------------------------------------------------------- 52
六、腹水製備抗體與細胞培養生產抗體 ---------------------------------------- 55
肆、討論 ----------------------------------------------------------------------- 56
一、抗原的選擇 ----------------------------------------------------------------------- 56
二、免疫動物 -------------------------------------------------------------------------- 58
三、取得融合用脾臟之處理 -------------------------------------------------------- 59
四、融合瘤細胞之融合反應 -------------------------------------------------------- 61
五、融合用藥劑之比較 -------------------------------------------------------------- 62
六、培養環境與培養基添加物對融合瘤細胞之影響 -------------------------- 64
七、抗體的篩選 ----------------------------------------------------------------------- 68
八、未來發展 -------------------------------------------------------------------------- 72
伍、參考文獻 --------------------------------------------------------------- 74
陸、圖表 --------------------------------------------------------------------- 101
附錄A ------------------------------------------------------------------------- 144
附錄B ------------------------------------------------------------------------- 146
附錄C ------------------------------------------------------------------------- 147
附錄D ------------------------------------------------------------------------- 154

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