臺灣博碩士論文加值系統

石斑腦神經壞死病毒（MGNNV）是屬於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。本研究所建立的純化MGNNV實驗方法，可得到相當穩定的病毒濃縮液，以方便將來對MGNNV的研究。Betanodavirus可感染SSN-1細胞，判知在細胞中應有能與之結合的病毒接受子。酵素與VBP之關係的實驗中發現，以proteinase K與SSN-1細胞抽出物作用所得到的結合蛋白最少。接受子拮抗劑與VBP之關係的實驗中顯示，利用拮抗劑與接受子結合，可導致病毒無法經由接受子進入細胞進行感染，判斷serotonin 5-HT1A或α2 adrenergic的接受子可作為MGNNV的接受子。

Nervous necrosis virus of Epinephelus malabaricus (MGNNV) belongs to the genus of Betanodavirus that causes vacuolating encephalopathy and retinopathy and viral nervous necrosis. A method to purify MGNNV was proposed. The cellular components of SSN-1 reprent few viral receptors for Betanodavirus. Several cellular virus binding proteins (VBPs) were detected by employing the technique of immobilizing virus on nitrocellulose. The least VBPs were found in SSN-1 cell lysate that was treated with proteinase K. The approaches used receptor antagonists to identify the cell receptor. The antagonists are able to block the viral binding and thus potentially directly against the receptor. The results implied that the receptor of serotonin 5-HT1A or α2 adrenergic may act as the receptor of MGNNV.

目 錄
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 ----------------------------------------------------------------------2
二、台灣養殖石斑魚之現況 -------------------------------------------------3
三、魚類病毒 -------------------------------------------------------------------4
四、Betanodavirus -------------------------------------------------------------5
五、溫度對Betanodavirus感染力的影響 ---------------------------------7
六、Nodavirus的基因體 ------------------------------------------------------8
七、Nodavirus複製機制 ------------------------------------------------------9
八、 cDNA clones複製產生之病毒亦具有感染力 -----------------------10
九、建立增殖Betanodavirus之細胞株 ------------------------------------11
十、魚類的免疫系統 ---------------------------------------------------------- 14
十一、MGNNV的結合蛋白 -----------------------------------------------------15
十二、 魚類病毒接受子 ----------------------------------------------------------16
十三、關於血清素的接受子 ----------------------------------------------------18
十四、本研究目的 ----------------------------------------------------------------21
貳、材料與方法
一、Total RNA的抽取 --------------------------------------------------------22
二、RNA電泳 ------------------------------------------------------------------23
三、SSN-1細胞株的培養 -----------------------------------------------------23
四、SSN-1增殖NNV ----------------------------------------------------------24
五、從SSN-1細胞株製備病毒液 --------------------------------------------25
六、SDS-PAGE蛋白質電泳 -------------------------------------------------25
七、銀染 -------------------------------------------------------------------------26
八、TCID50定量病毒 ----------------------------------------------------------27
九、超高速離心純化病毒 ----------------------------------------------------28
十、Virus concentrate之蛋白質濃度定量 ----------------------------------29
十一、細胞全液之製備 ----------------------------------------------------------29
十二、Cooking Buffer的製備 ---------------------------------------------------30
十三、細胞全液之總蛋白與MGNNV之結合 -------------------------------30
十四、蛋白□處理完整細胞之膜蛋白 ----------------------------------------31
十五、去醣基酵素處理完整細胞之膜蛋白 ----------------------------------32
十六、Receptor antagonists處理完整細胞之膜蛋白 ------------------------32
十七、Anti-VLP抗血清的製備與純化 ----------------------------------------33
十八、Anti-MGNNV抗血清的製備與純化 ----------------------------------34
十九、抗體的保存 ----------------------------------------------------------------35
二十、西方墨點法（Western blotting） ---------------------------------------36
參、結果
一、MGNNV的純化 ----------------------------------------------------------38
二、Anti-VLP抗血清的製備 -------------------------------------------------39
三、Anti-MGNNV抗血清的製備 -------------------------------------------40
四、病毒萃取液（virus extract）之感染力價 -----------------------------41
五、MGNNV耐受蛋白□之分解 -------------------------------------------42
六、病毒的固定及脫附 -------------------------------------------------------42
七、VBP的脫附條件 ----------------------------------------------------------43
八、蛋白□對VBP的影響 ---------------------------------------------------44
九、Receptor antagonists與VBP的關係 ----------------------------------45
肆、討論
一、MGNNV的純化 ----------------------------------------------------------46
二、Anti-MGNNV與anti-VLP的交叉測試 -------------------------------47
三、Cooking Buffer對VBP的影響 -----------------------------------------47
四、蛋白□對VBP的影響 ---------------------------------------------------47
五、Receptor antagonists與VBP的關係 -----------------------------------49
伍、參考文獻 ---------------------------------------------------------------50
陸、圖表 ----------------------------------------------------------------------57
附錄A ------------------------------------------------------------------------82
附錄B ------------------------------------------------------------------------86
附錄C ------------------------------------------------------------------------87
附錄D ------------------------------------------------------------------------88
附錄E ------------------------------------------------------------------------89
附錄F ------------------------------------------------------------------------90
附錄G ------------------------------------------------------------------------91
表 目 錄
Table 1The potency of anti-MGNNV antiserum.---------------------57
Table 2Infectivity of the chloroform-extracting virus. ------------58
Table 3Comparative analysis of VBPs. ------------------------------59
圖 目 錄
Fig. 1Propagation of MGNNV in SSN-1 cell cultures. ---------------------60
Fig. 2Detrimental effects of detergent treatments on the MGNNV. -------61
Fig. 3Purification protocol for MGNNV. -------------------------------------62
Fig. 4Protein p40 purified from MGNNV-infected cultures. --------------64
Fig. 5Western blotting analysis of protein p40 during the propagation cultures. --------------------------------------------------------------------65
Fig. 6Western blotting of protein p40 using anti-VLP antiserum. ---------66
Fig. 7Western blotting analysis of p40 using anti-MGNNV antiserum. --67
Fig. 8Infectivity of the chloroform-extracting virus.. -----------------------68
Fig. 9Digesting MGNNV by the proteinases. --------------------------------69
Fig. 10Digesting MGNNV by Proteinase G. ----------------------------------70
Fig. 11Immobilization of MGNNV on nitrocellulose filter.. ----------------71
Fig. 12Western Blotting of immobilized MGNNV in nitrocellulose filter. 72
Fig. 13Immobilization of MGNNV on nitrocellulose filter after eluted by different cooking buffers. ------------------------------------------------73
Fig. 14Elution of VBP by different cooking buffers.----------------------------74
Fig. 15Western blotting analysis for the VBP in the elution by cooking buffers. ---------------------------------------------------------------------75
Fig. 16Protease treatments of SSN-1 membrane influence MGNNV binding. ---------------------------------------------------------------------76
Fig. 17The binding of the deglycosylated of VBP. ---------------------------79
Fig. 18Receptor antagonists inhibit the binding of virus and VBP. --------80
Fig. 19Western analysis for the effects of spiroxatrine on the binding of virus and VBP. ------------------------------------------------------------- 81

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