表 1、本研究所用之引子序列
Name/Categories Sequence (5’to3’) Usage
Construction
wssv108-BamHI-F CGGGATCCATGGACGTTTCTTCCTAT
pDHSP-wssv108-V5/His
108-5’UTR-H3-F CCAAGCTTGTCAGTTCCAGAAGAAGAG
pDHSP-5’UTR-wssv108-V5
pGL3-wssv304-(-36~+82)
GGGCATCTCGCCCACACAGAAA Probe, (-104 nt/-36 nt)
wssv304-93~64F
CCACACAGAAACAGGATATGA-TATCATAG
Cold probe, (-93 nt/-64 nt)
wssv304-93~64R CTATGATATCATATCCTGTTTCTGTGTGG Cold probe, (-93 nt/-64 nt)
wssv304-63~36F
ATTTCTGGGAAGAGGGTGTTTTTT-GTGCC
Cold probe, (-63 nt/-36 nt)
wssv304-63~36R
GGCACAAAAAACACCCTCTTCCCAGAA AT
Probe, (-104 nt/-36 nt) Cold probe, (-63 nt/-36 nt)
wssv304-78~50F ATATGATATCATAGATTTCTGGGAAGAG Cold probe, (-78 nt/-50 nt) wssv304-78~50R CTCTTCCCAGAAATCTATGATATCATAT Cold probe, (-78 nt/-50 nt) AP1 binding site-1 CGC TTG ATG AGT CAG CCG GAA Cold probe, AP1 site AP1 binding site-2 TTC CGG CTG ACT CAT CAA GCG Cold probe, AP1 site STAT binding site-1 TTA TTC CTA GAA ATG Cold probe, STAT site STAT binding site-2 CAT TTC TAG GAA TAA Cold probe, STAT site
bio-tin-wssv304-(-65~-46 )-F
Biotin-GATTTCTGGGAAGAGGGTGT Probe, (-65 nt/-46 nt)
wssv304-(-65~-46)-R ACACCCTCTTCCCAGAAATC
Probe, (-65 nt/-46 nt) Cold probe, (-65 nt/-46 nt) wssv304-(-65~-46)-F GATTTCTGGGAAGAGGGTGT Cold probe, (-65 nt/-46 nt)
wssv304-(-55~-36)-F AAGAGGGTGTTTTTTGTGCC Cold probe, (-55 nt/-36 nt) wssv304-(-55~-36)-R GGCACAAAAAACACCCTCTT Cold probe, (-55 nt/-36 nt)
圖 1、WSSV108 蛋白質在秋夜盜蛾細胞 （Sf9） 中的表現
將 wssv108 蛋白質表現質體 pDHSP-wssv108-V5/His 轉染進 Sf9 細胞中，培養 72 小時後收取細胞，利用 SDS-PAGE 分離樣品，並以抗 His 抗體進行偵測，lane 1 為轉入 pDHSP-V5/His 的細胞萃取物；Lanes 2、3 分別為轉入帶有 5’UTR 之 pDHSP-5’UTR-wssv108-V5/His，以及 pDHSP-wssv108-V5/His 表現質體。
圖 2、WSSV108 蛋白質在 E. coli BL21(DE3) 中的表現
(A) 以送入質體 pGEX-4T1 的 E. coli BL21(DE3) 所表現蛋白質作為對照 (lane 1)，送入質體 pGEX-4T1-wssv108，並以 IPTG 誘導 5 小時表現蛋白質 (lane 2)。樣品經 SDS-PAGE 分離後，以抗 GST 抗體進行偵測。(B) 以送入質體 pET32a(+) 的 E. coli BL21(DE3) 所 表 現 蛋 白 質 作 為 對 照 組 (lane 1) ， 送 入 質 體 pET32a-wssv108，並以 IPTG 誘導 5 小時表現蛋白質 (lane 2)。樣品經 SDS-PAGE 分離後，以抗 His 抗體進行偵測。
圖 3、WSSV108 蛋白質在 Sf9 細胞中的分布
將控制組載體 pDHSP-GFP-V5/His 與 GFP-WSSV108 融合蛋白質的質體 pIB-GFP-wssv108-V5/His 分別轉染至昆蟲細胞 Sf9 中。細胞核使用 DAPI 染色，並 以螢光顯微鏡觀察蛋白質分布狀況。
圖 4、WSSV108 蛋白質在 Sf21 細胞中具有調控轉錄活化的能力
(A) 實驗所使用之質體，轉錄起始點以+1 表示。DBD 為結合 GAL4 site 之 DNA binding domain。(B) 將報導載體 G5p35BAS-Luc 與表現載體 GAL4-DBD、
GAL4-IE1 及 GAL4-WSSV108 分別共轉染至昆蟲細胞 Sf21 中，48 小時後偵測冷 光活性數值。倍率為各組所測得之冷光數值除以轉染 GAL4-DBD 之冷光數值。數 據以 Student's t-test 分析，星號 (*) 表示與負控制組相比具有顯著的差異 (p value<0.05)。實驗每次進行二重複。數值為三次的平均值，誤差線為標準差 (standard deviation, SD)。
圖 5、極早期蛋白質 WSSV108 對極早期與早期基因啟動子轉錄活性的影響 將表現載體 pIB-V5/His、pIB-5’UTR-wssv108-V5/His 與白點症病毒基因啟動 子報導質體共轉染到 Sf21 細胞中，48 小時後偵測冷光活性數值。倍率為各組轉 染 pIB-5’UTR-wssv108-V5/His 所測得之冷光數值除以轉染 pIB-V5/His 之冷光數 值。數據以 Student's t-test 分析，星號 (*) 表示與負控制組相比具有顯著的差異 (p value<0.05)。實驗每次進行二重複。數值為三次的平均值，誤差線為標準差 (standard deviation, SD)。
圖 6、極早期蛋白質 WSSV108 對 wssv304 啟動子片段轉錄活性的影響
將表現載體 pIB-V5/His、pIB-5’UTR-wssv108-V5/His 與帶有不同長度的啟動 子報導質體共轉染到 Sf21 細胞中，48 小時後偵測冷光活性數值。倍率為各組轉 染 pIB-5’UTR-wssv108-V5/His 所測得之冷光數值除以轉染 pIB-V5/His 之冷光數 值。數據以 Student's t-test 分析，星號 (*) 表示與負控制組相比具有顯著的差異 (p value<0.05)。實驗每次進行二重複。數值為三次的平均值，誤差線為標準差 (standard deviation, SD)。
圖 7、WSSV108 結合在 wssv304 基因啟動子上 -63 nt 至 -36 nt 區域
以電泳流動性移轉分析實驗 (EMSA) 進行分析，(A) 使用在 EMSA 中的核 苷酸序列。(B) Lane 1 為只含有 Biotin 標記的探針 (-104 nt/-36 nt)。Lane 2 為 Sf9 細 胞 原 生 核 萃 取 物 與 Biotin 標 記 的 探 針 。 Lane 3 為 轉 染 質 體 pDHSP-5’UTR-wssv108-V5/His 至 Sf9 細胞 72 小時後的核蛋白質萃取物與 Biotin 標記的探針，並形成蛋白質-DNA 複合體 (rWSSV108-DNA)。Lanes 6~10 為 Biotin
標記的探針與無 Biotin 標記的競爭者序列，共同競爭重組蛋白質 WSSV108，分 別為 wild type wssv304 promoter、wild type wssv304 promoter 前段 (-93 nt/-64 nt)、
中段 (-78 nt/-50 nt)、後段 (-63 nt/-36 nt)、非專一性的 STAT binding site 和 AP1 site 序列。競爭者的濃度為探針的 5 倍。Lane 4 使用抗 His 抗體，Lane 5 使用抗 V5 進行 supershift 分析。
圖 8、WSSV108 結合在 wssv304 基因啟動子上 -65 nt 至 -46 nt 區域
以電泳流動性移轉分析實驗 (EMSA) 進行分析，(A) 使用在 EMSA 中的核
苷酸序列。(B) Lane 1 為只含有 Biotin 標記的探針 (-65 nt/-46 nt)。Lane 2 為 Sf9 細 胞 原 生 核 萃 取 物 與 Biotin 標 記 的 探 針 。 Lane 3 為 轉 染 質 體 pDHSP-5’UTR-wssv108-V5/His 至 Sf9 細胞 72 小時後的核蛋白質萃取物與 Biotin 標記的探針，並形成蛋白質-DNA 複合體 (rWSSV108-DNA)。Lanes 5~10 為 Biotin 標記的探針與無 Biotin 標記的競爭者序列，共同競爭重組蛋白質 WSSV108，分 別 為 wild type wssv304 promoter 、 六 點 核 酸 突 變 之 序 列 (-64/-59)mut 、 (-58/-53)mut、(-52/-47)mut、wild type wssv304 promoter (-55 nt/-36 nt)片段、非專 一性的 STAT site 序列。競爭者的濃度為探針的 5 倍。Lane 4 使用抗 His 抗體，Lane 5 使用抗 V5 進行 supershift 分析。
圖 9、WSSV108 結合位突變影響 WSSV108 對 wssv304 啟動子的調控
(A) wssv304 promoter 啟動子片段報導質體示意圖，轉錄起始點以+1 表示。(B) 將表現載體 pIB-V5/His、pIB-5’UTR-wssv108-V5/His 與 WSSSV108 的結合範圍突 變的啟動子報導質體共轉染到 Sf21 細胞中，48 小時後偵測冷光活性數值。倍率 為各組轉染 pIB-5’UTR-wssv108-V5/His 所測得之冷光數值除以轉染 pIB-V5/His 之冷光數值。數據以 Student's t-test 分析，星號 (*) 表示與負控制組相比具有顯 著的差異 (p value<0.05)。實驗每次進行二重複。數值為三次的平均值，誤差線為 標準差 (standard deviation, SD)。
圖 10、WSSV108 蛋白質形成多聚體及與 LvYY1 直接結合
將 glutathione-Sepharose agarose 加入含有 GST-WSSV108 的 E. coli BL21(DE3) 之細菌溶裂液中。共同作用一小時後加入能表現 His-WSSV108 (lane 3)與含有 His-LvYY1 (lane 4) 的 E. coli BL21(DE3) 細菌溶裂液，並以 pET32a(+) 作為負控 制組 (lane 2)，在 4°C 共同作用一小時並清洗後，以抗 His 抗體進行西方點墨法 分析，偵測結合於 GST-WSSV108-glutathione Sepharose 膠體的蛋白質 (lanes 5、
6、7)，以抗 GST 抗體偵測結合於膠體的 GST 融合蛋白質 (lane 1)。
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附錄
附錄 1、白點症病毒之外觀型態
(9a) 感染白點症病毒的白蝦外殼，出現典型白色斑點。(9b) 利用穿透式電子 顯微鏡拍攝白點症病毒的白蝦血淋巴中病毒顆粒，可發現有一個類似尾巴的結構 由外殼延伸出來。(9c) 利用穿透式電子顯微鏡拍攝白點症病毒的核鞘。(9d) 簡單 的示意圖關於白點症病毒的外膜、核鞘以及其相關的蛋白質 (節錄自 Lightner, 2011)。
附錄 2、WSSV108 蛋白質之結構預測
以線上資料庫 CFSSP 進行 WSSV108 蛋白質的二級結構預測。
附錄 3、WSSV108 蛋白質之進核序列預測
以線上資料庫 NucPred 進行 WSSV108 蛋白質的進核序列預測 (Brameier et al., 2007)，顯示 WSSV108 在第 73 到 82 個胺基酸間可能存在一個蛋白質進核序 列。
附錄 4、WSSV108 蛋白質之進核序列預測示意圖
以蛋白質預測建模網站 SCRATCH 與 Pymol 軟體進行 WSSV108 蛋白質的三
級結構模型建構，以紅色標示出α 螺旋體結構，而以黑色字體標示出預測所得之
進核序列。