- 學位論文
蝦白點病毒極早期表現蛋白WSSV IE1在白蝦原酚氧化酶活化系統上的角色探索
The Role of WSSV IE1 in Prophenoloxidase Activating System in Penaeus vannamei
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摘要
本論文在探討白點症病毒 (White Spot Syndrome Virus, WSSV) 極早期表現蛋白WSSV IE1於白蝦原酚氧化酶活化系統 (prophenoloxidase activating system) 所扮演之角色。以蛋白質層析儀進行WSSV IE1純化，發現含有10% glycerol的緩衝液A能穩定WSSV IE1重組蛋白。首先進行遠端西方墨點法分析，發現IE1 能與白蝦之原酚氧化酶活化系統的上游蛋白相互作用，包括轉谷氨酰胺酶 (transglutaminase)、原酚氧化酶 (prophenoloxidase) 及血藍素 (hemocyanin)。於注射白點症病毒顆粒後分析白蝦酚氧化酶的活性變化，發現白蝦在注射白點症病毒顆粒4小時後，白蝦酚氧化酶的活性能被激活，但於注射後8-12小時其活性則急速降低。依據白蝦酚氧化酶活性分析結果，發現WSSV IE1能抑制酚氧化酶的活性，然負控制組WSSV364則未能抑制酚氧化酶活性。實驗結果證明WSSV IE1會抑制白蝦酚氧化酶活性，使得經LPS活化劑處理後的白蝦酚氧化酶活性與控制組無顯著差異。推測IE1可能具有抑制原酚氧化酶活化途徑的功能，關於其作用機制猶待進一步之實驗分析以確認之。
並列摘要
In the thesis, we explored the role of white spot syndrome virus (WSSV) immediate early gene (IE1) in prophenoloxidase activating system. The SDS-PAGE results revealed that 10% glycerol Buffer A could stabilize WSSV IE1 purification. After far-western blotting analysis, it revealed that WSSV IE1 could interact with transglutaminase, prophenoloxidase and hemocyanin. We validate temporal phenoloxidase activity in Penaeus vannamei after WSSV viral particle injection, it indicated that phenoloxidase activity in Penaeus vannamei could be upregulated in 4 hours and downregulated in 8-12 hpi. According to the phenoloxidase activity assay, it revealed that WSSV IE1 could suppress phenoloxidase activity in Penaeus vannamei; WSSV364 was used as negative control in phenoloxidase activity assay. According to the phenoloxidase activity in Penaeus vannamei with WSSV IE1 pretreatment, it could be concluded that IE1 inhibited prophenoloxidase activating pathway. It suggested that IE1 could have suppressive function in prophenoloxidase activating pathway.
參考文獻
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