白點症病毒wssv162基因的重組蛋白質表現系統之探討

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標題:	白點症病毒wssv162基因的重組蛋白質表現系統之探討 The study of recombinant protein expression systems for white spot syndrome virus wssv162 gene
作者:	陳俞汎
貢獻者:	嘉南藥理科技大學：生物科技系暨研究所田乃月
關鍵字:	秋夜盜蛾細胞株-Sf9 轉染白點症病毒增強型綠色螢光蛋白 wssv162 基因 Enhancer green fluorescent protein (EGFP) Transfection wssv162 gene Sf9 cell line White spot syndrome virus
日期:	2009
上傳時間:	2011-10-27 14:43:07 (UTC+8)
摘要:	白點症病毒(White Spot Syndrome Virus, WSSV)是隸屬於Nimaviridiae病毒科及Whispovirus 病毒屬，主要感染宿主為水產甲殼類，會造成養殖蝦從感染到病發只需2-10 天，且死亡率可高達100%。目前已知有三株病毒分離株，分別來自於中國、泰國、臺灣，本研究主要探討WSSV 的台灣分離株當中一段未知功能性的開放譯讀區-wssv162，核苷酸片段大小為231bp，可能轉譯出含77 個胺基酸的蛋白質產物。為提高wssv162 在真核系統的基因表現性，利用Dhsp70 (Drosophila heat shock protein 70) 與OpIE2 (Orgyia pseudotsugata immediate-early 2)啟動子分別驅動egfp、egfp-wssv162 及wssv162-egfp 在秋夜盜蛾細胞株-Sf9 (Spodopter frugiperda, Sf9 cell line) 中進行基因表現。研究結果顯示，OpIE2 啟動子及Dhsp70 啟動子在Sf9 細胞中均能驅動表現出WSSV162-EGFP 或EGFP-WSSV162 融合蛋白質，但Dhsp70 啟動子的驅動效率較OpIE2 啟動子佳。另一發現Sf9 細胞單獨表現EGFP 蛋白的效率遠高於WSSV162-EGFP 或EGFP-WSSV162 融合蛋白，目前原因未明但值得深入研究探討。而在WSSV162 的探討上，則期望以原核系統生產His6-WSSV162 水溶性融合蛋白，且能藉蛋白質交互作用技術中的Pull-down assay 來推測可能IV與His6-WSSV162 產生直接或間接作用的蛋白質。以原核系統進行WSSV162蛋白質表達及純化，其結果顯示，以IPTG 在37℃下對大腸桿菌進行His6-WSSV162 融合蛋白誘導，在胞內所生產的His6-WSSV162 融合蛋白皆為非水溶性蛋白質，因此無法直接用在Pull-down assay 實驗中，必須要先解除掉包涵體的狀態。而在嘗試以降低誘導溫度以及改變誘導劑減緩大腸桿菌生產His6-WSSV162 融合蛋白的速率，依然無法產生水溶性蛋白質。但在實驗過程中發現，以IPTG 在37℃的誘導條件下，胞外液有少許的水溶性蛋白產生，因此，可以藉由大量培養來取得足夠的水溶性His6-WSSV162 融合蛋白質。 White spot syndrome virus (WSSV) the virus genus Whispovirus and family Nimaviridiae, is an important pathogen of aquatic crustaceans and cultured shrimp. The shrimps were infected and dead with in 2 to 10 days, the mortality can be reached l00%. 　　There are three virus isolates from China, Thailand, Taiwan, this study focused on the WSSV Taiwan isolate were functional for some one unknown ORF-wssv162, it has a nucleotide size of 231bps, a possible translation containing 77 amino acids. To improve wssv162 in eukaryotic gene expression of the system, using Dhsp70 (Drosophila heat shock protein 70) and OpIE2 (Orgyia pseudotsugata immediate-early 2) the respective promoter-driven egfp, egfp-wssv162 and wssv162-egfp in the autumn Pirates of the moth cells strain-Sf9 (Spodopter frugiperda, Sf9 cell line) carried out gene expression. 　　The reporter plasmid caused EGFP expression in transfected insect Sf9 cell lines in gene expression. This result suggested that the OpIE2 promoter and Dhsp70 promoter in Sf9 cells can could express WSSV162-EGFP or EGFP-WSSV162 fusion protein, but Dhsp70 promoter more efficient than OpIE2 . Another found that the performance of EGFP protein in Sf9 cells alone is much higher than the efficiency of WSSV162-EGFP or EGFP-WSSV162 fusion protein, at present is unknown but it is worth exploring in-depth study. 　　In the WSSV162 discussion on the expectations of the original production of His6-WSSV162 nuclear systems, water-soluble fusion protein, the protein interaction technology and can use the pull-down assay to speculate may be related to His6-WSSV162 have a direct or indirect role of the protein. 　　We also purified the IPTG　induced His6-WSSV162　products under 37　, but the His6-WSSV162 fusion protein in the E.coli were expressed as insoluble form. Therefore, we couldn’t be directly pull-down assay. In the attempt to lower the temperature, as well as induced by lower dose often inducetion rate of the His6-WSSV162 fusion protein, it still was unable to produce in soluble form. However, in the experiment we found little increase of soluble fraction in extracellular region at 37℃ using IPTG. So we could obtain a large amount of the His6-WSSV162 by large-scale production, so could a lot of training to obtain sufficient soluble fusion protein His6-WSSV162.
關聯:	校內外均一年後公開，學年度：97，79 頁
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