Interactions between penaeus monodon cell surface receptors and white spot syndrome virus structural proteins / 曲君平
曲君平、張雲祥
E-mail: [email protected]
ABSTRACT
White spot syndrome virus (WSSV; genus Whispovirus, family Nimaviridae) is a widely occurring virus which attacks cultured shrimp and many other crustaceans and has caused severe mortalities and huge economic losses for the global shrimp farming industry. Currently, no effective method is available for treating this disease caused by WSSV infection. However, recent studies have reported host cell proteins related to WSSV infection. This study, cloned full length of gene. Furthermore, pmgC1qR (penaeus monodon C1q receptor) and other 2 cell surface receptors, including pmRab7 (P. monodon Rab7 protein) and Lamr (laminin receptor) interacted with WSSV structural proteins, were globally analyzed using on a WSSV structural protein yeast two-hybrid library. Yeast two-hybrid screening revealed that the pmgC1qR, pmRab7 and Lamr interacted with 19, 24, and 42 viral structure proteins, respectively. Co-immunoprecipitation assays confirmed the interactions of pmgC1qR with WSSV VP52A, VP19 and VP26; between pmRab7 with WSSV VP52B and VP56; and Lamr with WSSV VP31, VP264C and VP26. The study results may contribute to clarifying the mechanism of WSSV infection and the developing an anti-viral strategy. Key Words: White spot syndrome virus, Receptor, Penaeus monodon, virus-host interaction, protein-protein interaction
Keywords : White spot syndrome virus、Receptor、virus-host interaction、protein-protein interaction、Penaeus monodon Table of Contents
封面內頁 簽名頁 中文摘要... iii 英文摘
要... iv 誌謝... v 目錄... .. vi 圖目
錄... ix 表目錄...
xi 1. 前言... 1 1.1 白點症病 毒... 1 1.2 白點症病毒結構性蛋白 質... 2 1.3 細胞膜受體於病毒感染之重要
性... 3 1.4 細胞膜受體Rab7... 4 1.5 細 胞膜受體gC1qR... 5 1.6 細胞膜受體laminin receptor (Lamr)
... 5 1.7 研究目的... 7 2. 材料方 法... 9 2.1 草蝦細胞膜受體基因選殖與分
析... 9 2.1.1 RNA萃取... 9 2.1.2 cDNA末端快速擴增法(rapid-amplification of cDNA ends, RACE) ... 9 2.1.3 反轉錄聚合?鏈鎖反
應... 11 2.1.4 聚合?鏈鎖反應... 12 2.1.5 pmgC1qR基因親緣分析... 13 2.2 酵母菌雙雜交基因庫篩選分
析... 14 2.2.1 白點症病毒結構性蛋白質酵母菌基因庫建
構... 14 2.2.2 質體DNA構築... 14 2.2.3 酵母 菌轉形... 15 2.2.4 轉形酵母菌蛋白質表現分
析... 15 2.2.5 酵母菌自體激活測試... 16 2.2.6 酵母菌雙雜交基因庫篩選受體與結構性蛋白質交互作用... 17 2.3 共免疫沉澱分
析... 18 2.3.1 質體DNA構築...
18 2.3.2 Sf9細胞蛋白表現分析... 19 2.3.3 共免疫沉澱分
析... 20 3. 實驗結果... 22 3.1 pmgC1qR與白點症病毒結構性蛋白質交互作用分析... 22 3.1.1 pmgC1qR基因選
殖... 22 3.1.2 pmgC1qR基因特性與親緣關係分 析... 22 3.1.3 pmgC1qR於酵母菌基因庫蛋白質表現分
析... 23 3.1.4 pmgC1qR於酵母菌基因庫自體激活分析...
23 3.1.5 pmgC1qR與白點症病毒結構性蛋白質交互作用篩選... 24 3.1.6 pmgC1qR於秋行軍蟲
細胞株Sf9蛋白表現分析... 24 3.1.7 pmgC1qR與白點症病毒結構性蛋白質共免疫沉澱分 析... 25 3.2 pmRab7與白點症病毒結構性蛋白質交互作用分 析... 25 3.2.1 pmRab7基因選殖... 25 3.2.2 pmRab7於酵母菌基因庫蛋白質表現分析... 26 3.2.3 pmRab7於酵母菌基因庫自體激活分 析... 26 3.2.4 pmRab7與白點症病毒結構性蛋白質交互作用篩
選... 27 3.2.5 pmRab7於秋行軍蟲細胞株Sf9蛋白表現分析...
27 3.2.6 pmRab7與白點症病毒結構性蛋白質共免疫沉澱分析... 27 3.3 Lamr與白點症病毒結構 性蛋白質交互作用分析... 28 3.3.1 Lamr基因選
殖... 28 3.3.2 Lamr於酵母菌基因庫蛋白質表現分
析... 28 3.3.3 Lamr於酵母菌基因庫自體激活分析...
28 3.3.4 Lamr與白點症病毒結構性蛋白質交互作用篩選... 29 3.3.5 Lamr於秋行軍蟲細胞 株Sf9蛋白表現分析... 29 3.3.6 Lamr與白點症病毒結構性蛋白質共免疫沉澱分
析...30 4. 結論... 31 參考文
獻... 71 附錄... 77 REFERENCES
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