蝦白點症病毒 wssv 044 基因表現之研究

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標題:	蝦白點症病毒 wssv 044 基因表現之研究 The study of shrimp white spot syndrome virus –wssv 044 gene expression
作者:	蔡佳怡 Chia-Yi Tsai
貢獻者:	田乃月嘉南藥理科技大學：生物科技研究所
關鍵字:	蛋白質-蛋白質交互作用轉染作用轉型作用 wssv044 基因 protein-protein interaction transfection wssv genes transformation
日期:	2008
上傳時間:	2009-03-11 11:42:10 (UTC+8)
摘要:	造成蝦養殖產業極大損失的白點症病毒，隸屬Nimaviridae病毒科(Whispovirus病毒屬)，具大型雙股 DNA基因體，其開放譯讀區能轉譯出釵h獨特性蛋白質，但是至今大部分的基因弁鉬P特性均尚未明瞭。蝦白點症病毒 ( 臺灣分離株 ) 的wssv044基因，其全長為 201 個核苷酸，可轉譯成 67 個胺基酸的蛋白產物；本論文主要應用大腸桿菌原核系統與昆蟲細胞真核系統，進行該基因的表現分析與弁鈺敦Q。另外利用 pull-down assay探討 WSSV蛋白質與宿主蝦體組織蛋白質的交互作用關係。以 IPTG 誘導在大腸桿菌 BL21( DE3 ) 內表現 His6-WSSV044 融合性蛋白質，並以anti-His 抗體確認所產蛋白質；另以 Ni-NTA 樹脂純化該蛋白質，發現 His6-WSSV044 融合性蛋白質在 37℃、1 mM IPTG 及3小時內的培養條件下，主要是以包涵體形式產生。在 Sf9昆蟲細胞中，則以綠色螢光蛋白 (EGFP) 共同融合wssv基因，分別生產 EGFP-WSSV 或 WSSV-EGFP 等型態的蛋白質，應用倒立螢光顯微鏡觀察綠螢光表現狀況，以了解wssv基因的表現情形；此外透過染核試劑 ( bisBenzimide H 33342 trithydrochloride, Hoechst # 33342 ) 協助，發現不同的 EGFP-WSSV融合性蛋白質在昆蟲細胞中有不同表現差異，主要在細胞質表現，但部分蛋白質明顯會在細胞中表現出顆粒性亮點型態。另外藉由 Pull-down assay 方式，以大腸桿菌生產的蝦白點症病毒的融合性蛋白質與蝦體組織內蛋白質進行交互作用，期望探討 WSSV044 蛋白質之生物弁遄C White spot syndrome virus (WSSV), genus Whispovirus, family Nimaviridae, is a major shrimp pathogen that is highly virulent in penaeid shrimp and can also infect most species of crustacean. Because of the large size of WSSV genome and the uniqueness of viral proteins , only a few WSSV genes have been studied beyond sequence analysis. In this study, the ORF of wssv044 gene contained 201 nucleotides, encoding 67 a.a. The wssv044 gene was expressed in E. coli with His6-WSSV044 fusion protein ( 11-kDa ). The His6-WSSV044 were confirmed using Western bolt analysis with mouse anti-His antibodies. After 1mM IPTG and 37℃ induction for 3 hours, we found the His6-WSSV044 fusion proteins were express with inclusion body form. Subsequently, the expression of WSSV genes by fusing enhanced green fluorescent protein (EGFP) gene as a reporter gene were investigated in the insect Sf9 cells. First, we constructed and isolated the recombinant plasmids containing different WSSV genes respectively, which fused with N-terminal or C-terminal of EGFP. Then these recombinant plasmids were transfected into Sf9 cells and observed under a inverted fluorescence microscope. The results revealed that the fusion proteins containing different WSSV genes might be expressed diversely in transfected insect cells. A His pull-down assay has been used further to confirm the interactions between WSSV044 and shrimps proteins.
關聯:	校內校外均不公開
顯示於類別:	[生物科技系(所)] 博碩士論文

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