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研究生:謝佩璇
研究生(外文):Pei-shiuan Shie
論文名稱:抗病毒蛋白Mx在點帶石斑魚細胞之特性分析
論文名稱(外文):Characterization of Mx proteins for cellular function in orange-spotted grouper ( Epinephelus coioides )
指導教授:陳宗嶽
指導教授(外文):Tzong-yueh Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:80
中文關鍵詞:石斑魚神經壞死病毒gMx蛋白
外文關鍵詞:NNV(Nervous Necrosis Virus)GrouperMx protein
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  • 被引用被引用:2
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點帶石斑魚(Epinephelus coioides)是目前台灣的經濟養殖魚類之ㄧ,在整個養殖過程中幼魚受到神經壞死病毒(nervous necrosis virus)感染死亡情形相當嚴重,但在成魚則相對的沒那麼嚴重,因此點帶石斑魚似乎有一套防禦機制對抗神經壞死病毒的感染。脊椎動物的干擾素(interferon)防禦機制為抵抗病毒的第一道防線,它藉由啟動下游蛋白的合成來抑制病毒的複製,至少有三種以上的抗病毒蛋白受到干擾素的調控,而Mx蛋白就屬其中一種,Mx蛋白對於病毒複製的抑制特別是在RNA病毒方面被研究的相當透徹。在實驗室之前的工作中,點帶石斑魚的Mx cDNA已被選殖,全長1881bp並且轉譯出626個胺基酸,在結構上來看石斑魚Mx蛋白(gMx)跟哺乳類一樣都屬大型GTP結合蛋白。這裡我們探討石斑魚泳鰭細胞(GF-1)在分別轉染Mx-GFP及Mx-His融合蛋白並使其大量表現後感染NNV的細胞存活情形。此外並觀察NNV感染不同細胞株之後神經壞死病毒抗原表現情形及病毒產量。由實驗結果發現大量表現gMx蛋白之石斑魚細胞株在受到神經壞死病毒感染後:(1)存活率較一般細胞株高。(2)病毒抗原蛋白延遲出現且表現量較少。(3)病毒產量較低。綜合以上各點,我們認為Mx蛋白在石斑魚的確參與內生性免疫保護機制,具有抗病毒功能。且此機制可能透過跟NNV外殼蛋白結合,阻礙病毒組裝有關,因此Mx蛋白將可被利用在石斑魚抵抗神經壞死病毒的一大利器。
Grouper(Epinephelus coioides)is one of the economical fish in Taiwan. Because the virus infection in adult orange-spotted grouper is often light and unrecognized, grouper should have an efficient defense system against to nodaviruses. The defense system of interferon(IFN) in vertebrate provides an initial line of defense against viral infection by inducing the synthesis of proteins that inhibit virus replication. At least three different proteins mediate the antiviral response and one of them, Mx protein, specifically inhibits the replication of wide range of RNA virus has been well established. In the recent work, a homolog Mx (“OsgMx”) gene from orange-spotted grouper (Epinephelus coioides) had been cloned and sequenced. It contains an 1881 bp open reading frame region and encodes a protein with 626 amino acids. On protein structure, the grouper Mx protein is similar to the superfamily of large GTP-binding proteins. In this study, we transfected Mx-GFP and Mx-His fusion protein to GF1(grouper fin) cell line. Then we chose stable clones which can express Mx fusion proteins. We used MTT assay to observe the situation that the cell survives after NNV infection. Furthermore, we observed the NNV antigen expression and viral yields in cell lines after NNV infection. By the experimental results, we demonstrating that grouper Mx overexpression had an inhibitory effect on both coat protein and RNA dependent RNA polymerase (RdRp) of nodavirus antigens, resulting in reduced viral yields. We conclude that grouper Mx has a key role in cellular resistance to nodavirus infection. The antiviral activity through direct interaction, likely via the binding and perturbation of the intracellular localization of nodavirus coat protein.
圖目錄...................................................II
中文摘要..................................................i
英文摘要.................................................ii
致謝....................................................iii
前言......................................................1
材料與方法...............................................16
結果.....................................................34
討論.....................................................41
參考文獻.................................................49
圖.......................................................58
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