臺灣博碩士論文加值系統

台灣為世界三大水產養殖國之一，而台灣的養殖技術，尤其是魚苗的孵化，更是整個東南亞第一，是魚苗與幼魚的重要輸出國。在台灣和東南亞國家中，石斑魚（Epinephelus spp.）是主要之海水養殖魚類，但是在近幾年來，因為養殖的石斑魚苗經常受到石斑魚神經壞死病毒（grouper nervous necrosis virus, GNNV）的感染，而且死亡率也相當高，因此造成了經濟上的嚴重損失；除了感染石斑魚以外，神經壞死病毒也造成其他多種養殖魚類的疾病，其中以鱸魚、河豚、比目魚、鸚哥魚、鰺魚等，而造成養殖漁業的嚴重損失，目前對NNV的檢測方法主要是RT-PCR，但操作較為繁複、費時；另一方面，由於至今仍然沒有一個有效的NNV防治方法，因此發展一個較快速簡便的檢驗方法和一個有效的疫苗來對抗神經壞死病毒對於養殖產業的發展極為重要。
神經壞死病毒屬於野田病毒科(Nodaviridae)的betanodavirus屬，會引起魚類的病毒性神經壞死症(viral nervous necrosis, VNN)，致死率極高，根據先前對NNV的研究得知，神經壞死病毒的鞘蛋白(coat protein)具有些微免疫的效果，再參考1999年由 Nishizawa T.等人利用單株抗體分析鰺魚的神經壞死病毒(SJNNV)所提出的兩個抗原決定區(epitope)，分別一般型和具基因型專一性的epitope，我們再用電腦比對NNV鞘蛋白胺基酸與核酸序列的結果，然後自NNV鞘蛋白分別挑出兩段各含十個胺基酸序列的胜月太(peptide)來做為疫苗的候選，以此作為基礎利用TR-PCR方法來建構出LAE( linear array epitopes)的抗原蛋白，若此抗原能夠有效誘發免疫反應，那麼它就有機會可以發展成檢驗試條和疫苗，進一步地可應用於檢測NNV和防治由NNV所引起的病害上。

Taiwan is one of the three best aquaculture countries in world and has the best aquaculture techniques especially for fry hatch in Southeast Asian. Taiwan is an important country for fry and larvae export. Grouper, Epinephelus spp., is a major fish species of mariculture in Taiwan and other Southeast Asian countries. In recent years, maricultured grouper has come to be easily infected by grouper nervous necrosis virus, GNNV, resulting in high mortalities among the fish stock. This has caused severe economic loss. Furthermore, the nervous necrosis virus has been recognized as the cause of severe diseases among other fish, such as sea bass, jack, parrotfish, pufferfish and, flatfish etc, resulting in further severe economic loss. NNV’s diagnosis method up to now has been RT-PCR, but which is complicated and time-consuming. Also as there is not yet a method to prevent or cure viral nervous necrosis caused by NNV, We feel we must develop both a diagnosis method to handle the problem faster and more easily, as well as develop an avaliable vaccine to combat NNV.
Nervous necrosis virus, NNV belongs to Nodaviridae, betanodavirus. It usually infects fish and causes viral nervous necrosis with a high mortality rate. NNV coat protein can induce an immune response and provide some protection. 1999 Nishizawa T. used monoclonal antibody to analyze striped jack NNV and other genotype NNV coat proteins. His finding indicated that there are two epitopes, NNV general epitope, and NNV genotype specific epitope. We will then utilize multiple alignments to compare both NNV coat protein amino acid and nucleic acid sequences. We selected two peptide fragments containing ten amino acids that included two epitopes, respectively. Then we used these two peptides as candidates for vaccine development. If an NNV antigen construct containing LAE (linear array epitopes) selected from the conserved region around the epitope of coat protein is able to induce an immune response, it might be develop to a strip for diagnosis. We expect that this will prove beneficial in the treatment of most nervous necrosis virus diseases.

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致謝‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧Ⅰ
目錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧Ⅱ
圖表目錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧Ⅲ
附錄目錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧Ⅳ
中文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧1
英文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧2
一、前言‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧3
二、材料與方法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧10
三、結果‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧21
四、討論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧28
圖表‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧35
參考文獻‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧52
附錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧59

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