中文摘要
瑪拉巴石斑腦神經壞死病毒（MGNNV ）是屬於
Piscinodavirus (Betanodavirus)屬的魚類病毒，感染此科的病毒會引起病毒性神經壞死症或病毒性腦及視網膜病變症。從活體所分離出來的MGNNV 病毒可於SSN-1 細胞株增殖，此細胞株是從蛇頭鱧Channa striatus 仔魚所建立的細胞株。於繼代五次，其TCID50ml- 1值從104個到10
8個，顯示出SSN-1 對MGNNV的高敏感性和高生產力。在MGNNV 熱穩定性的分析中，病毒保存的最適溫度為25 ℃以下。而將MGNNV 注射於吳郭魚的眼睛玻璃體後，於腦組織可偵測出MGNNV ，說明了病毒在魚體中從眼睛感染到腦的現象。至於在MGNNV-結合蛋白的實驗中，顯示SSN-1 、輪蟲、瑪拉巴石斑受精卵及各器官的細胞抽出物與MGNNV 結合，各會產生不同的MGNNV-結合蛋白。SSN-1 細胞抽出物中病毒結合蛋白為100 kDa 和56 kDa ，輪蟲細胞抽出物中病毒結合蛋白為60 kDa 、43 kDa 、36 kDa 和30
kDa ，石斑魚受精卵膜之病毒結合蛋白為62 kDa 、50 kDa 、25 kDa和20 kDa 。而各個組織之細胞抽出物中，腦的病毒結合蛋白為80 kDa 和43kDa ，眼為60 kDa 、45 kDa 、43 kDa 和23 kDa，肝為80 kDa 和18 kDa ，胰為60 kDa 、45 kDa 、43 kDa 和30 kDa，脾為45 kDa 和24 kDa ；腎為80 kDa ，卵巢為100 kDa 、95 kDa 、80 kDa 、48 kDa 、45 kDa 和30 kDa 。

Abstract
Nervous necrosis virus of Epinephelus malabaricus (MGNNV) belongs to the genus of Piscinodavirus (Betanodavirus) that causes vacuolating encephalopathy and retinopathy or viral nervous necrosis. MGNNV propagated in SSN-1 cells that were derived from fry tissue of striped snakehead fish, Channa striatus. SSN-1 was highly permissive to MGNNV infection and production, in which the TCID50 per ml increased from the
magnitude of 10 4 to 10 8 after 5 passages. The thermal stability analysis unveiled that the optimal activity of MGNNV can be resumed at the
temperature from frozen to less than 25 ℃. Oreochromis mossambica was highly susceptible to MGNNV only when injecting the virus into the vitreous body of fish eye, via which the virus could propagate in the fish brain. Employing the technique of immobilizing virus on itrocellulose, several cellular MGNNV-binding proteins were detected. Two MGNNV-binding proteins of 100 and 56 kDa were found in SSN-1 cell lysate; four proteins of 60, 43, 36, and 30 kDa in rotifer lysate; four proteins of 62, 50, 20 and 25 kDa
on the membrane of grouper fertilized eggs. In different organ lysates of Epinephelus malabaricus, two MGNNV-binding proteins of 80 and 43 kDa were found in brain; four MGNNV-binding proteins of 60, 45, 43 and 23 kDa were found in eye; two MGNNV-binding proteins of 80, 18 kDa were found in liver; four MGNNV-binding proteins of 60, 45, 43 and 30 kDa were found in pancreas; two MGNNV-binding proteins of 45 and 24 kDa were found in spleen; one MGNNV-binding proteins of 80 kDa were found in kidney; six MGNNV-binding proteins of 100, 95, 80, 48, 45 and 30 kDa were found in ovary.

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、 石斑魚 ----------------------------------- 1
二、 台灣養殖石斑魚之現況 --------------------- 2
三、 魚類病毒 --------------------------------- 3
四、 Piscinodavirus --------------------------- 4
五、 溫度對Piscinodavirus 感染力的影響 -------- 5
六、 Nodavirus 的基因體 ----------------------- 6
七、 Nodavirus 複製機制 ----------------------- 7
八、 建立增殖Piscinodavirus 之細胞株 ---------- 8
九、 關於結合蛋白的研究 ---------------------- 10
十、 本研究目的 ------------------------------ 10
貳、材料與方法
一、 Total RNA 的抽取 ------------------------ 11
二、 RNA 電泳 -------------------------------- 11
三、 反轉錄聚合連鎖反應(RT-PCR) -------------- 12
四、 DNA 電泳 -------------------------------- 12
五、 從檢體製備病毒液 ------------------------ 13
六、 從SSN-1 細胞株製備病毒液 ---------------- 13
七、 SDS-PAGE 蛋白質電泳 --------------------- 13
八、 銀染 ------------------------------------ 14
九、 TCID50 定量病毒 ------------------------- 15
十、 SSN-1 增殖NNV --------------------------- 15
十一、超高速離心純化病毒 --------------------- 16
十二、細胞膜的製備 --------------------------- 16
十三、製作細胞抽出物 ------------------------- 17
十四、藻類培養 ------------------------------- 17
十五、MGNNV 感染藻類Isocrysis galbana -------- 17
十六、感染吳郭魚Oreochromis mossambica ------- 18
十七、不同細胞抽出物與MGNNV 之結合 ----------- 19
參、結果
一、 建立增殖MGNNV 的方法 -------------------- 20
二、 MGNNV 繼代增殖的倍增量 ------------------ 20
三、 不同狀態的細胞皆含有MGNNV --------------- 21
四、 純化MGNNV ------------------------------- 21
五、 MGNNV 之熱穩定性 ------------------------ 22
六、 MGNNV 感染吳郭魚 ------------------------ 22
七、 MGNNV 固定於消化纖維膜 ------------------ 23
八、 不同生物之MGNNV-結合蛋白 ---------------- 24
九、 Blocking reagent 之選取 ----------------- 24
十、 瑪拉巴石斑不同組織之MGNNV-結合蛋白 ------ 25
肆、討論
一、 MGNNV 繼代增殖的倍增量 ------------------ 26
二、 不同狀態的細胞皆含有MGNNV --------------- 26
三、 MGNNV 之熱穩定性 ------------------------ 27
四、 MGNNV 感染吳郭魚 ------------------------ 28
五、 不同生物之MGNNV-結合蛋白 ---------------- 29
伍、參考文獻 --------------------------------- 31
陸、圖表 ------------------------------------- 36
附錄A ---------------------------------------- 54
附錄B ---------------------------------------- 59
附錄C ---------------------------------------- 64
附錄D ---------------------------------------- 65
附錄E ---------------------------------------- 67
附錄F ---------------------------------------- 68

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