魚類神經壞死病毒造成多種高經濟魚種的死亡。本篇研究以龍膽石斑神經壞死病毒(DGNNV)之似病毒顆粒(wt-VLPs)以及將殼蛋白的N端截短25個氨基酸之似病毒顆粒(ΔN25-VLPs)為題材。經由拆解及組裝的機制，證明wt-VLPs能包裹大小為608 bp 的DNA。純化過程中不同酸鹼值會影響VLPs的沈降速率，酸鹼值越高沈降速率越小；而純VLPs的穩定性則不受酸鹼值的影響。以電子顯微鏡觀察wt-VLPs在酸鹼值不同下之形態，觀察到偏鹼性時 wt-VLPs 則呈現鬆散且不規則的形狀，並且會有空心的形態產生，而偏酸性的 wt-VLPs 呈現緊實的形態。以西方點墨法分析wt-VLPs 與溶菌酶的抗體抗原結合反應，結果顯示 wt-VLPs 與 lysozyme 有共沈澱之反應。

Fish nodavirus causes the death of several high economic fish. For studying the package and stability of dragon grouper necrosis virus (DGNNV), wild type virus-like particles (wt-VLPs) and ΔN25-VLPs were employed. After experiments of disassembly and assembly, the result showed that DNA of 608 bp was able to be packaged by wt-VLPs. The sedimentation of VLPs was affected by different pH buffers during the process of purification: the VLPs at alkaline conditions behaved faster than those at the acidic. The stability of pure VLPs was pH-independent. The micrographs of the wt-VLPs in alkaline buffers showed that particles were rough and irregular in shapes, some of which were stain-permeable. However, the wt-VLPs in acidic buffers were morphologically indistinguishable from the untreated VLPs. The Western blotting for gradiently purified miture of VLPs and lysozyme revealed that lysozyme was co-precipitated with wt-VLPs.

謝誌 I
摘要 II
Abstract III
圖目錄 VI
壹、前言 1
一、 台灣石斑魚現今概況： 1
二、 魚類神經壞死病毒： 3
三、 感染Betanodavirus之臨床病徵： 4
四、 Betanodavirus的傳播： 4
五、 神經壞死病毒的感染途徑： 5
六、 Nodavirus基因體及其遺傳物質之特性: 7
七、 魚類神經壞死病毒之防疫： 11
八、 似病毒顆粒(Virus-like particles, VLPs): 12
九、 研究目的： 16
貳、材料與方法： 17
一、 似病毒顆粒之製備與純化： 17
二、 似病毒顆粒之拆解與包裹： 18
三、 DNA 的萃取： 18
四、 DNA 電泳： 19
五、 蛋白質定量(Lowry method)： 19
六、 不同酸鹼值的緩衝溶液： 20
七、 SDS-PAGE蛋白質電泳: 20
八、 VLPs和抗溶菌酶抗體之結構相似度分析: 21
九、 抗VLP之小鼠血清製備： 21
十、 抗Lysozyme之小鼠血清製備: 22
十一、 西方點墨法(Western blotting): 22
十二、 流式分光光度計及自動分樣器： 23
十三、 總RNA的抽取： 24
十四、 RNA 電泳： 24
十五、 負染色： 25
參、 結果 26
一、 純化DGNNV wt-VLPs (似病毒顆粒): 26
二、 VLPs包裹遺傳物質： 26
三、 在純化過程中酸鹼值對 DGNNV wt-VLPs的影響： 26
四、 DGNNV wt-VLPs 在不同酸鹼度下之穩定性： 27
五、 酸鹼值對似病毒顆粒形態的影響： 28
六、 純化ΔN25-VLPs： 29
七、 在純化過程中酸鹼值對ΔN25-VLPs產量的影響： 29
八、 ΔN25-VLPs 在不同酸鹼值下之穩定性： 30
九、 溶菌酶(Lysozyme) 和wt-VLPs之共沈澱反應： 30
肆、 參考文獻 37
伍、 圖表 46

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