蝦白點症病毒三種基因表現之研究

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Title:	蝦白點症病毒三種基因表現之研究 Expression of three white spot
Authors:	黃明宏 Ming-Hung Huang
Contributors:	田乃月嘉南藥理科技大學：生物科技研究所
Keywords:	基因表現白點症病毒轉染作用 transfection gene expression WSSV
Date:	2005
Issue Date:	2008-10-31 16:15:46 (UTC+8)
Abstract:	白點症病毒的基因體是目前無脊椎動物病毒中最大的，已被證實為雙股環狀DNA所組成，其宿主範圍包括蝦類和其他淡水和海水的甲殼類。此病毒被分類歸屬在新病毒科Nimaviridae，Whispovirus屬的一員，在病毒顆粒的一端具有尾狀突起的特殊構造 (tail-like projection)。目前已經建立三個不同地區的白點症病毒基因體庫，然而，此病毒大部分的開放譯讀區(open reading frames-ORF)所表現的蛋白質與現今已知的蛋白質弁鈰洇C相似性甚低。本研究針對白點症病毒台灣分離株(GenBank Accession NO. AF440570)基因體，根據WSSV感染之病蝦mRNA抽取物的RT-PCR及microarray分析顯示，三段新型基因的ORFs (WSSV071；WSSV302；WSSV338)可能與末期基因有關聯性。在本實驗中，構築含有三種此病毒特定基因之重組質體，經由轉型作用在大腸桿菌BL-21(DE3)中，分別順利地合成含有His-tagged的融合性蛋白質，並且純化、預計生產抗體。另構築含有上述三種病毒基因之重組質體，轉染送入昆蟲Sf9細胞中表現出含有強化螢光蛋白EGFP之融合蛋白，利用螢光顯微鏡觀察螢光表現情形，藉以作為判斷病毒基因表現與否的依據。 White Spot Syndrome Virus (WSSV) is a large, circular dsDNA virus , which may infect shrimps and other crustaceans. This virus is a member of the genus Whispovirus within a new virus family called Nimaviridae, referred to the thread-like polar extension on the virus particle. The complete sequences of the WSSV genomes have now been published as three different isolates. However, most of WSSV open reading frames , which can encode proteins, have no homology to any known proteins or motifs. With in the genome of the Taiwan WSSV isolate, RT-PCR and microarry analyses showed that these ORFs (WSSV071, WSSV302,WSSV338) were putative as very late genes. Therefore, in this study, we try to construct the recombinant plasmids containing the ORFs, and to express them by transformation into Escherichia coli, BL-21(DE3). Until now, the His-tagged fusion proteins containing the WSSV071, WSSV302 and WSSV338 respectively were expressed successfully and purified to produce antiserums for Western blot analysis. In addition, we construct the recombinant plasmids containing the ORFs, and to express them by transfection into Spodoptera frugiperda (Sf9) insect cells .The efficiency of transfection were detected with EGFP expression using the fluorescent microscopy.
Relation:	校內校外均不公開
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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