臺灣博碩士論文加值系統

近年來台灣養殖魚類受到虹彩病毒的感染造成水產養殖上嚴重損失，目前已知會被虹彩病毒感染的魚種包括金目鱸(giant seaperch; Lates calcarifer)、龍膽石斑(king grouper; Epinephelus lanceolatus)、虎斑(Tiger grouper; Mycteroperca tigris)、點帶石斑(Orange-spotted grouper; Epinephelus coioides)、海鱺(Cobia; Rachycentron canadum)和嘉鱲(Red sea bream; Pagrus major)等魚種。此外養殖方式逐漸趨向高密度集約養殖，密度過高使魚隻容易擦傷且造成緊迫，且病毒性疾病無明顯症狀更加速了傳染病的發生及蔓延。虹彩病毒科(Iridoviridae)下共分Ranavirus、Lymphocystivirus、Megalocytivirus、Iridovirus及Chloriridovirus五屬。其中會感染魚類的虹彩病毒有三屬：如Ranavirus屬的GIV，Lymphocystivirus屬的LCDV和Megalocytivirus屬的TGIV。石斑魚虹彩病毒為雙股去氧核糖核酸病毒，病毒型態為20面體，直徑約170±10 nm，具外套膜。魚體在感染虹彩病毒之後會出現食慾不振、體色變黑、鰓絲蒼白，於鰓蓋及下顎、眼周圍、下腹部及鳍基部有出血情形。解剖時可以看到病魚的腎、脾腫大。虹彩病毒從魚苗到成魚各階段皆有感染病例發生，死亡率可達50%~100%。細胞株為研究及分離病毒極為重要的工具。本實驗以受精18小時的點帶石斑魚卵為材料，建立出對石斑魚虹彩病毒具有感受性的胚胎細胞株。此石斑魚胚胎(grouper embryo, GE)細胞株經單株選殖後的形態為表皮樣細胞，生長在含有5-20%胎牛血清的L-15培養基中，最適生長溫度為28 ℃。染色體數目分布以28、32居多。細胞已穩定繼代40代。GE細胞株對石斑魚虹彩病毒有高感受性，其感染力價可達108.5 TCID50/ml。以pEGFP-N1，pEGFP-N/GIV-Bcl和pDsRed2-Mito質體DNA轉染GE細胞，可觀察到螢光蛋白之表現。此結果顯示我們建立的石斑魚胚胎細胞株適合做為虹彩病毒之細胞學研究。

Iridovirus infection has been damaging devastatingly on Taiwan aquaculture in these few years. The fish species susceptible to iridovirus including giant seaperch Lates calcarifer, king grouper Epinephelus lanceolatus, Tiger grouper Mycteroperca tigris, Orange-spotted grouper Epinephelus coioides, Cobia; Rachycentron canadum and Red sea bream Pagrus major. The high density aquaculture manner not only causes the abrasion but also increases the tension and pressure of fish. And the insignificant symptom of viral diseases further speed the occurrence and spread of infectious diseases. Family Iridoviridae includes five genera: Ranavirus, Lymphocystivirus, Megalocytivirus, Iridovirus and Chloriridovirus. In which, three genera can infect fish: GIV belongs to genus Ranavirus, LCDV belongs to genus Lymphocystivirus and TGIV belongs to genus Megalocytivirus. Grouper iridovirus is a dsDNA virus, icosahedral, 170±10 nm in diameter and with envelop. Diseased fish will lack of appetite, dark color in body, pale color in gill filament, and hemorrhage in gill cover, lower jaw, around eyes, underbelly and gill base. Anatomy observation includes enlargement of spleen and kidney. Iridovirus can infect fish from fry throughout adult fish, and the mortality can reach 50%~100%. Cell line is an important tool for virus isolation and research. In this study, we used fertilized eggs of Orange-spotted grouper Epinephelus coioides as material and established iridovirus susceptible embryonic cell line. The morphology of grouper embryo, GE cell line was epidermal type after subcloning, and was kept in L-15 medium supplemented with 5-20% FBS at 28 ℃. The chromosome number distributed major in 28 and 32. Now the GE cell line has been passing for 40 generation. GE cell line has high susceptibility to grouper iridovirus, and the infection titer can reach 108.5 TCID50/ml. We also transfected pEGFP-N1, pEGFP-N/GIV-Bcl and pDsRed2-Mito into GE cells and found the expression of fluorescent protein. All these results suggest that the grouper embryo (GE) cell line we established is suitable for the cell biology study of iridovirus.

謝誌 I
目錄 II
中文摘要 V
英文摘要 VII
第一章文獻探討 1
1.1 石斑魚之簡介與台灣養殖概況 1
1.2 魚類虹彩病毒感染症之發現 4
1.3 虹彩病毒之特性 7
1.4 虹彩病毒之分類 10
1.5 魚類細胞株之簡介 12
第二章 材料與方法 14
2.1 初級細胞培養(primary cell culture) 14
2.2 繼代培養(subculture) 16
2.3 細胞單株化 17
2.4 病毒感受性篩選 19
2.5 細胞冷凍保存 21
2.6 病毒力價之測定 22
2.7 生長曲線分析 25
2.8 染色體分析 27
2.9基因轉染(gene transfection) 30
2.10 穿透式電子顯微鏡 31
第三章 結果 35
初級細胞培養(primary cell culture) 35
繼代培養(subculture) 35
細胞單株化 36
病毒感受性篩選 36
病毒力價之測定 37
染色體分析 38
基因轉染(gene transfection) 38
穿透式電子顯微鏡 (Transmission Electron Microscopy；TEM) 39
第四章 討論 40
參考文獻 43


圖表目錄
圖一、石斑魚胚胎組織初級培養 50
圖二、十五代繼代培養之石斑魚胚胎細胞 51
圖三、二十代繼代培養之石斑魚胚胎細胞 52
圖四、單株化之石斑魚胚胎細胞 53
圖五、GE-1細胞感染GIV之細胞病變效應 54
圖六、GE-1細胞染色體之Giemsa staining 55
圖七、GE-1細胞之染色體數目分布 56
圖八、GE-1細胞之轉染 57
圖九、GIV感染GE-1細胞之穿透式電子顯微鏡切片圖 58
表一、GE-1細胞感染不同GIV劑量之細胞病變效應 59

曾文陽 石斑魚養殖學. 前程出版社, 1998.
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