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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/16735

Title: 點帶石斑魚Mx基因之特性及其抗病毒之研究
Characterization of Epinuphelus coioides Mx genes and its anti-viral activity
Authors: Chih-Hung Lin
林志鴻
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 點帶石斑魚;Mx;神經壞死病毒
orange-spotted grouper;Mx;nodavirus
Date: 2006
Issue Date: 2011-06-30T08:41:40Z
Abstract: 以雙股RNA或病毒處理可誘導細胞產生干擾素(Interferon),此干擾素進一步誘導細胞表現抗病毒蛋白質-Mx蛋白質。因此,我們從台灣的經濟魚種-石斑魚(grouper,Epinephelus coioides)經由poly I:C,LPS,與福馬林不活化YGNNV和GIV病毒處理後所建構的石斑魚腎臟cDNA基因庫中篩選出27個Mx陽性選殖株,經序列比對分析後歸納出三種基因型,命名為MxI,MxII和MxIII。經由轉譯的蛋白質序列的比對分析後,顯示這三型Mx蛋白質皆具有保守性相當高的GTP結合區,以及dynamin家族的特徵與leucine zipper motif。在演化分析上明顯區分出魚類、哺乳類與鳥類三個分支,顯示魚類Mx在演化上已與其他兩分支獨立出來。 在石斑魚組織特異性分析上,以poly I:C處理石斑魚後,純化腦、心臟、肝臟、脾臟、腎臟、腸與鰓組織的總體RNA來檢測Mx mRNA的表現,與未處理組比較,腦、脾臟、腎臟與鰓皆可受到poly I:C的誘導表現Mx基因,其中腎臟Mx基因表現最高,脾臟次之,腦與鰓則表現較少,但是,肝臟與腸組織則無法被poly I:C誘導表現Mx基因。在real time PCR的結果中發現,YGNNV病毒攻毒的結果與poly I:C處理後的結果相呼應,但是卻發現GIV病毒亦可誘導腎臟組織表現MxI基因,而且其表現比YGNNV還高。但是,在脾臟組織中GIV病毒則不會誘發石斑魚三種基因型的Mx基因的表現。 藉由建立的對神經壞死病毒(Nodavirus)敏感性的石斑魚腦組織細胞株-GB3細胞株,來進一步研究石斑魚三型Mx蛋白質的抗病毒活性。將石斑魚三型Mx基因構築於pcDNA3CF表現載體並轉殖於GB3細胞中,從免疫化學染色法的結果中發現,表現的石斑魚三型Mx重組蛋白質皆只局部化在細胞質中;經由G418篩選GB3細胞穩定表現Mx的選殖株後發現可降低神經壞死病毒的10至100倍的感染力。另外,再透過免疫螢光化學染色法發現,Mx蛋白質表現量高的細胞可以抑制神經壞死病毒的增殖。因此,本研究顯示石斑魚三型Mx基因所表現之蛋白質皆具有抑制神經壞死病毒的活性,且藉由此結果進一步設計有效的DNA疫苗來賦予魚類早期非專一性的抗病毒能力。
Mx proteins are interferon induced, anti-viral proteins, expressed in response to treatment with double stranded RNA or virus infection. Here we report the cloning, sequencing and antiviral property of three forms of Mx genes, MxI, MxII and MxIII from grouper (Epinephelus coioides) kidney cDNA library that was treated by poly I:C, LPS, or fomalin inactivated YGNNV and GIV. Multiple comparison of grouper Mx amino acid sequences revealed the conservation of Mx putative GTP-binding domain, dynamin family signature and leucine zipper motif. Phylogenetic analysis revealed that these grouper Mxs along with that of other fish form separate branches independent of avian and mammalian homologues, reflecting a distant evolutionary relationship from birds and mammals. On tissue specific expression for Mx, Northern blot demonstrates the constitutive expression of Mx transcripts in grouper tissues such as, heart, spleen, kidney and gill and induced expression in brain, spleen, kidney and gill in response to the poly I:C. But, poly I:C failed to up-regulate the Mx expression in liver and intestine. On real time PCR for Mx, the results of YGNNV infection is similar to poly I:C treatment. Surprising, GIV infection induces the expression of MxI gene, and is higher than YGNNV infection. However, the expression of Mx genes is not induced in spleen by GIV infection. We had established a new cell line from grouper brain (GB3 cell line) and prepared stable clones expressing Flag-epitope tagged grouper MxI, MxII and MxIII. Transient transfection and Immunostaining shows that all the three grouper Mx proteins are localized in the cytoplasm. To examine the antiviral activity of grouper Mx proteins, these stable clones were infected by a nodavirus, yellow grouper nervous necrosis virus (YGNNV) and the results showed that all the three Mx isoforms have the efficiency of reducing the titre of virus from 10 to 100 fold. Moreover, through immunocytochemistry we demonstrated that Mx protein can inhibit the YGNNV propagation in GB3 cells. Taken together, this study demonstrates that grouper Mx proteins have efficient inhibitory activity against nodavirus, the most endangered virus of fish and this information would be helpful to design effective DNA vaccines that can confer an early non-specific antiviral protection.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0D88330005
http://ntour.ntou.edu.tw/ir/handle/987654321/16735
Appears in Collections:[水產養殖學系] 博碩士論文

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