臺灣博碩士論文加值系統

病毒性神經壞死症（Viral nervous necrosis disease, VNN disease），嚴重衝擊石斑魚養殖產業發展並造成重大的經濟損失，其病原為神經壞死病毒主要感染魚苗及幼魚，並引起高達 80-100％的死亡率。因此，疾病的預防與控制遂成為一重要課題，除了養殖環境的改善、種魚篩選及優質種苗生產外，疫苗的開發成為有效控制疾病的策略之ㄧ。本研究以大腸桿菌系統來表現石斑魚神經壞死病毒的外鞘蛋白基因，利用基因重組方式生產該病毒的外鞘蛋白，期望能作為製成有效次單位疫苗的應用。
本研究從石斑魚神經壞死病毒中選殖之外鞘蛋白基因長度為 1017 bp，並在 5’ 端及 3’ 端分別設計 Hind? 切位及 XhoΙ 切位，經定序比對與石斑魚 Epinephelus coioides nervous necrosis virus truncated coat protein gene 相似度高達 99％。將此基因片段與 pET-32a (+)（Novagen）表現載體進行接合後，轉型至 E. coli（BL21（DE3）pLysS），以完成構築。
將構築完成的菌株，於 LB/Ampcillin broth, 37℃，培養至 OD600=0.6，加入濃度為 1 mM IPTG 進行誘導，分別經不同時間點誘導後，收集總蛋白、可溶性蛋白及不可溶性蛋白，以 Sodium dodecyl sulfate－polyacrylamide gel 電泳分析，結果發現在 IPTG 誘導 2 小時其外鞘蛋白表現量最高，並且會以包涵體之形式存在，分子量大小為37 kDa。另外以同樣培養方式於不同濃度的 IPTG 培養液培養，經 2小時誘導後，發現表現蛋白在 0.8 mM IPTG 誘導 2 小時其表現量最高，因此可以在此條件下以鎳金屬螯合層析法進行純化，本研究結果可做為未來次單位疫苗開發及疾病檢測之應用。

Viral nervous necrosis disease may cause serious economic threat to grouper culture industry. The disease may cause high mortality（80-100％）particularly in larva and juvenile. Therefore, the control and prevention of this disease is very important. Except improving culture conditions, screening broodstock fish or raising healthy fry. Using vaccine is an effective method to control the disease. The present study used E coli system to express grouper nervous necrosis virus coat protein gene for further production of recombinant coat protein as a possible candidate for efficient prophylactic subunit vaccine development.
The coat protein gene was cloned from grouper nervous necrosis virus with a fragment size of 1017 bp. The 5’ end and 3’ end were designed as Hind? site and XhoΙ site, respectively. The nucleotide sequence of the cloned gene were compared with Epinephelus coioides nervous necrosis virus truncated coat protein gene and exhibited 99％ similarity. The gene of coat protein was cloned into a pET-32a (＋) expression vector and expressed in E coli (BL21 (DE3) pLysS).
The recombinant E coli was cultured in LB/Ampcillin broth at 37℃. When the bacteria grown to OD600=0.6, 1 mM IPTG was added to induce its expression. Total protein, soluble protein and insoluble protein were collected at various time, then sodium dodecyl sulfate－polyacrylamide gel electrophoresis was used for identification. The result revealed that the use of IPTG to induce 2 hours exhibited high coat protein expression with the formation of inclusion body. The coat protein expressed was a 37 kDa protein as measured by SDS-PAGE. Furthermore, the use of 0.8 mM IPTG to induce 2 hours exhibited high coat protein expression. Metal chelate chromatography was found to be useful for recombinant protein purification. The results revealed that the 37 kDa recombinant protein could be used as a candidate for future development of subunit vaccine and application of disease diagnosis.

謝辭……………………………………………………………………....i
中文摘要…………………………………………………………………iii
英文摘要………………………………………………………………....v
目錄………………………………………………………………………vii
圖目錄…………………………………………………………………….?
一、 前言……………………………………………………....………....1
二、 文獻整理………………………………………………………..…. 3
三、 材料方法…………………………………………………………...22
3.1石斑魚神經壞死病毒增殖及確認……………………………..….22
3.1.1石斑魚神經壞死病毒來源……………………………………..22
3.1.2石斑魚神經壞死病毒增殖……………………………………..22
3.1.2.1 石斑魚腦細胞（GBC1）之培養…………………………..22
3.1.2.2 石斑魚腦細胞（GBC1）的冷凍保存……………………..22
3.1.2.3 石斑魚腦細胞（GBC1）的解凍…………………….........23
3.1.2.4 石斑魚神經壞死病毒的增殖……………………................23
3.1.3 石斑魚神經壞死病毒的確認…………………………………..23
3.1.3.1 病毒核酸進行RT-PCR及Nested PCR確認……………..23
3.1.3.1.1 NNV RNA 的萃取……………………...........................23
3.1.3.1.2 反轉錄-聚合?連鎖反應…………………….................24
3.1.3.1.3 巢式聚合?連鎖反應（nested-PCR）………………...25
3.1.3.2 病毒核酸定序………………...............................................25
3.1.3.2.1 膠體純析（Gel elution）..……………………….……..25
3.1.3.2.2 定序載體的構築（Construction）與選殖（Selection）
………………….…………………………………………………26
3.1.3.2.3 接合作用（Ligation）..……………………….………26
3.1.3.2.4 轉型作用（Transformation）..………………………..26
3.1.3.2.5 篩選（Selection） ..…………………………………..27
3.1.3.2.6 重組質體 DNA 的萃取（Plasmid extraction）..……27
3.1.3.2.7 限制?酵素切割（Restriction enzyme digestion）..…28
3.1.3.2.8 定序及序列比對..…….………………………………..28
3.1.3.3 病毒顆粒層次的確認..……………………………………..28
3.1.3.3.1 穿透式電子顯微鏡負染色觀察……………………….28
3.2石斑魚神經壞死病毒外鞘蛋白基因選殖..……………………….28
3.2.1 石斑魚神經壞死病毒外鞘蛋白基因專一性引子之設計…….28
3.2.2 石斑魚神經壞死病毒外鞘蛋白基因片段序列選殖…….........29
3.2.3 石斑魚神經壞死病毒外鞘蛋白基因片段定序…….................29
3.3 石斑魚神經壞死病毒外鞘蛋白基因表現載體的構築……..........29
3.3.1 勝任細胞的製備…….................................................................29
3.3.2 石斑魚神經壞死病毒外鞘蛋白基因表現載體的構築…….....30
3.4 石斑魚神經壞死病毒重組外鞘蛋白的表現與純化……..............30
3.4.1 石斑魚神經壞死病毒重組外鞘蛋白的誘導表現……..............31
3.4.1.1 細胞萃取液（cell extract）製備...........................................31
3.4.1.2 可溶性蛋白與不可溶性蛋白的收集…….............................31
3.4.1.3 蛋白質膠體電泳及染色…….................................................31
3.4.1.4 NCP最佳的誘導條件測試….................................................32
3.4.2以Ni2+-NTA親合性管柱純化石斑魚神經壞死病毒重組外鞘蛋白…...............................................................................................33
3.4.2.1鎳金屬螯合層析法（metal chelate chromatography）.........33
四、 結果…………………………………………………………………34
五、 討論…………………………………………………………………37
六、 參考文獻……………………………………………………………42
圖…………………………………………………………………………57
附錄……………………………………………………………………....70

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