病毒性病源是水產養殖產業上疾病爆發的主因之一，往往造成魚體大量死亡與嚴重的經濟損失。Myxovirus-resistance(Mx)是魚類先天免疫組成的一員，受第一型干擾素(interferon; IFN)誘導表現且扮演抵抗病毒活性的功能。本研究以日本稻田魚Medaka (Oryzias latipes)作為神經壞死病毒(nervious necrosis virus; NNV)感染的動物模式，研究Mx基因對NNV感染的免疫反應以及抗病毒功效。本研究由日本稻田魚選殖出Mx與Mx-like基因，Mx基因全長為2286 bp，包含88 bp的5’端非轉譯區 (5’ untranslated region; 5’ UTR)、323 bp的3’端非轉譯區以及1875 bp的開放閱讀框(open reading frame; ORF)，可轉譯出624個胺基酸；Mx-like基因含1479 bp的ORF，轉譯出492個胺基酸。胺基酸序列分析顯示Mx蛋白N端為GTPase domain，C端為GTPase effector domain與連接兩端的Middle domain三個功能區，而Mx-like蛋白則缺少GTPase effector domain。以即時聚合酶鏈鎖反應(Real-time PCR)分析Mx與Mx-like基因於日本稻田魚胚胎發育與各組織的表現情況，結果顯示Mx與Mx-like基因的表現會隨胚胎的發育而有增加的趨勢，Mx基因於腎臟與脾臟有較高的表現量；Mx-like基因則於脾臟與腸道有較高的表現量。魚體以NNV病毒感染後24~48小時，Mx與Mx-like基因於腦、鰓、脾臟與腎臟等組織分別被誘導表現。細胞實驗中，將表現IFN-a、IFN-d、Mx與Mx-like之載體分別轉染至稻田魚OLHE-131細胞中，結果顯示Mx與Mx-like皆會受到IFN-a與IFN-d的誘導表現，且Mx具有抗病毒活性與抑制NNV感染所造成的細胞凋亡現象，然而Mx-like並無抑制病毒的功能，此結果說明Mx與Mx-like基因於免疫系統所扮演的功能不同。本研究證實稻田魚Mx基因抗神經壞死病毒的功能，此結果可作為一平台技術，利用Mx基因的誘導表現應用篩選抗病毒物質，有助於加速水產養殖上病毒性疾病防治方法的開發。

Viral pathogens is one of major cause of disease outbreaks in aquaculture. It often causes a large amount of death and severe economic loss in aquaculture. Myxovirus-resistance (Mx), which induced by type 1 Interferon (IFN), is one of innate immune members and acts important reole in antiviral function. In the present study, medaka (Oryzias latipes) was used as an animal model to study the immune response and antivial function of Mx genes for NNV infection. In this study, two Mx genes, Mx and Mx-like genes, were cloned from medaka. The full-length of Mx gene is 2286 bp consist of 88 bp of 5’ UTR (5’ untranslated region), 323 bp of 3’ UTR and 1875 bp coding region, which putative encode 624 amino acids. The coding region of Mx-like gene comprises 1479 bp which putative encoded 492 amino acids. Amino acid sequence aligment revraled that Mx comprised GTPase domain in N terminus, GTPase effector domain in C terminus and Middle domain; however Mx-like lack GTPase effector domain in C terminus. Expressional profile of Mx genes in diverse developmental stage and tissue distribution were determined by real- time PCR. Results showed that the expressions of Mx and Mx-like were increased accompanied with embryonic development. Mx gene was aboundant expressed in kidney and spleen, and higher expression of Mx-like gene in spleen and intestine. Mx and Mx-like genes were induced in diverse tissues of medaka by NNV infedction suggesting their antiniral function in innate immune system. In vitro, Mx and Mx-like genes were induced respectively in medaka OLHE-131 cells after IFN-a and IFN-d transfection suggesting the expression of Mx and Mx-like were regulated by IFN-a and IFN-d. Functional assay showed that OLHE-131 cells transfected with Mx rxpressing plasmid suppress NNV-induced apoptosis, whereas Mx-like without anti-apoptosis function. This result suggested the Mx and Mx-like may play distinct finction in immune system against NNV infection. The results presented in this study can be used as a platform to screen antiviral substances based on the induction of Mx expression. It is helpful to accelerate the development of biocontrol approaches for viral-induced disease problems in aquaculture.

摘要 I
Abstract III
第一章 文獻回顧 1
一、神經壞死病毒 1
1. 神經壞死病毒簡介 1
2. 神經壞死病毒的傳播 3
3. 神經壞死病毒的病理表現 3
4. 神經壞死病毒的預防 4
二、魚類免疫反應與免疫因子 4
1. 魚類免疫系統 4
2. 神經壞死病毒造成的免疫反應 5
3.干擾素(Interferon) 6
4. Mx蛋白 7
三、稻田魚細胞與動物模式 8
1. 稻田魚簡介 8
2. 實驗動物與細胞模式 9
四、研究動機 10
五、實驗架構 10
第二章 實驗材料與方法 11
一、實驗材料 11
1. 實驗動物 11
2. 載體與質體 11
3. 實驗細胞 11
4. 實驗菌株 11
5. 培養基 11
6. 試劑(reagent) 12
7. 試劑套組(kit) 12
8. 儀器設備 13
9. 限制酵素(restriction enzyme) 14
10. 引子 14
二、實驗方法 15
1、Mx 與Mx-like基因選殖 15
1-1. Total RNA抽取 15
1-2. 反轉錄酶反應(Reverse transcription,RT) 15
1-3. 聚合酶反應(Polymerase Chain Reaction, PCR) 16
1-4. DNA膠體電泳分析 16
1-5. DNA回收 16
1-6. 接合反應(ligation) 17
1-7. 轉型作用(transformation)與藍白篩選(Blue white screen) 17
1-8. 小量質體抽取 18
1-9. cDNA末端快速增幅(Rapid Amplification of cDNA Ends, RACE) 18
2. Mx 與Mx-like基因表現分析 21
2-1. 稻田魚產卵 21
2-2. 稻田魚發育Mx與Mx-like基因表現分析 21
2-3. 稻田魚各組織Mx與Mx-like基因表現分析 21
2-4.稻田魚被NNV感染後Mx與Mx-like基因表現分析 22
2-5. 即時聚合酶鏈鎖反應(Real-time PCR , qPCR) 22
3-1. 細胞繼代 22
3-2. 細胞解凍與保存 23
3-4. 病毒培養與保存 23
3-5. 病毒力價計算 24
4. Mx與Mx-like特性分析 24
4-1. 細胞轉染與分析 24
4-2. 細胞凋亡分析 25
4. 統計分析 25
第三章 結果 26
一、稻田魚Mx 與Mx-like cDNA的選殖與序列 26
二、親緣關係與胺基酸序列分析 26
三、Mx與Mx-like在發育階段與不同組織的表現 27
四、神經壞死病毒攻毒後Mx與Mx-like基因的表現 28
五、OLHE-131細胞轉染Interferon誘導Mx與Mx-like基因的表現 29
六、Mx與Mx-like抗病毒能力分析 29
第四章 討論 31
一、稻田魚Mx與Mx-like cDNA的選殖 31
二、Mx與Mx-like在發育階段與不同組織的表現 32
三、神經壞死病毒後攻毒Mx與Mx-like基因表現 33
四、Mx與Mx-like功能分析 34
參考文獻 37

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