表現系統
非結構蛋白NS1 已可利用 E.coli 系統大量表現（Das et al., 2009），因此 欲表現DV2 之 NS1 蛋白以探討蛋白質表現的結果。
NS1-HAHis 的分子量大小預期大小約為 44.7。皆可於上清液與 pellet 偵測到大小分別為50 kDa、45 kDa、40 kDa 的蛋白質，45kDa 的大小較符 合預期。此三條蛋白質於C41（DE3）菌株 pellet 中也能以 Coomassie blue staining 偵測到，蛋白質表現量似乎較 NS2、NS4 與 E 來的多。
為了確認在表現NS1-HAHis 蛋白質所出現的不專一性片段是否與 C41
之NS1 可經由 E.coli BL21(DE3)進行蛋白質大量表現及純化，Western blot 結果顯示並無雜片段的形成（Das et al., 2009），因此將 pET 5T△ -D2NS1 -HAHis 轉型到 BL21 菌株作蛋白質表現，就 Coomassie blue staining 的結果 觀察，pET 5T△ -D2NS1-HAHis 在 C41（DE3）與 C43（DE3）菌株無法觀 察出明顯蛋白質表現，但在BL21（DE3）菌株則可觀察出源於 NS1-HAHis 所產生的雜片段，Western blot 的結果則與 C41（DE3）和 C43（DE3）菌株 表現一樣，在 50 kDa、45 kDa、40 kDa 的大小可以偵測到蛋白質，其中 45 kDa 大小的片段符合預期，但是相較於 50 kDa、40 kDa 等雜片段，並無法 以Coomassie blue staining 測得，換言之，其雜片段的表現量較佳，雜片段 的問題還是無法加以解決。
由於有三條蛋白質片段，為了加以確認 NS1-HAHis 蛋白質表現的結
果，將 NS1 基因於 3’端的部分截短 336 bp，並建構 pET 5T△ -D2NS1(d)- HAHis 質體，想觀察蛋白質大小位移的狀況。其表現出的蛋白質為 NS1(d) -HAHis，預期的分子量約 32.1 kDa。利用 Western blot 偵測，可於上清液及 pellet 偵測到分子量大小約 40 kDa、33 kDa、26 kDa 的蛋白質，以 33 kDa 大小的片段較符合預期結果。其結果呈現與pET 5T△ -D2NS1-HAHis 表現結 果相似，而40kDa 與 26kDa 的蛋白質同樣也可於 Coomassie blue staining 偵 測到，相較於pET 5T△ –D2NS1-HAHis 在 C41（DE3）和 C43（DE3）菌株 中的表現，其pET 5T△ –D2NS1(d)-HAHis 表現量較佳，推測截短其 C 端疏 水性片段將有助於在 E.coli 系統蛋白質大量表現。
將pET 5T△ -D2NS1-HAHis 與 pET 5T△ -D2NS1(d)-HAHis 做蛋白質表現 比較後發現，前者所表現出的蛋白質片段分別為50 kDa、45 kDa、40 kDa，
後者則為40 kDa、33 kDa、26 kDa，截短後皆縮小了約 10 kDa 左右。推測 此三條截短蛋白質與NS1 基因 3’端截短有密切關係。
為了探討片段的形成是否與載體pET 5T△ -HAHis 有關？由於 pET 5T△ - HAHis 載體是由 pET-30a(+)載體修飾而成【圖 3.1】，因此我將 PL046 strain 中之NS1 基因重新建構於未經修飾之 pET-30a(+)載體，並於 NS1 基因 3’端 建構HAHis tag 以利於 Western blot 抗體偵測，建構完成之質體為 pET-30a
-D2NS1-HAHis，並同樣轉型於 C41（DE3）和 C43（DE3）菌株中表現，
由於所轉錄之NS1-HAHis 包含 pET-30a(+)載體之 5’端 His tag 與 S tag，因 此NS1-HAHis 的分子量大小約 50 kDa，以 Western blot 抗體偵測的結果與 pET 5T△ -D2NS1-HAHis 在 C41（DE3）與 C43（DE3）菌株中表現結果類 似，分別在55 kDa、50 kDa 與 45 kDa 的位置可以偵測到蛋白質，50 kDa 符合預期大小，但雜片段的現象並沒有因更換pET-30a(+)載體而有所改善，
因此雜片段和蛋白質表現量似乎與pET-30a(+)載體 5’端修飾沒有直接相關。
此三條蛋白質中，以pET 5T△ -D2NS1-HAHis 來看，分子量 50 kDa 的 蛋白質較預期的44.7 kDa 來的大，由於 NS1 之 N 端具有 heterologous signal peptide 與 hemagglutinin epitope，推測可能其 N 端與胞內之物質鍵結，導致 分子量變大（Noisakran et al., 2007）。較預期分子量略小的蛋白質片段（約 40 kDa），推測與 NS1-HAHis 之 N 端修飾有關，由於其 N 端有 linear epitopes，可能較容易被細菌胞內之 protease 水解，導致分子量略為變小
（Falconar et al., 1994）。
伍、結 論
本研究運用大腸桿菌之蛋白質表現系統 C41（DE3）與 C43（DE3），
表現登革熱病毒DV-2 PL046 strain 及 DV-3 H87 strain 之 HA-His 複合蛋白
（influenza hemagglutinin-polyhistidine protein），包括非結構蛋白 NS2A、
NS2B、NS4A、NS4B 及外膜蛋白（E），以及 PL046 strain 之 NS1 非結構 蛋白，其基因接於 pETΔ5T-HAHis 載體中皆可成功的被表現出來，不過僅 可於Western blot 中可偵測到蛋白質表現。
大腸桿菌C41（DE3）與 C43（DE3）菌株通常用於大量表現膜蛋白或 對宿主細胞有毒害的蛋白質並能避免宿主細胞死亡（Dumon-Seignovert et al., 2004），尤其可以改善 PL046 strain 之 NS2A-HAHis 蛋白質在 BL21（DE3）
中無法以Western blot 中可偵測到蛋白質表現的情形（楊馥嘉, 2006, 交大碩 士論文），但 C41（DE3）與 C43（DE3）在蛋白質的表現中卻仍然無法以 Coomassie blue 的染色方式觀察到蛋白質表現的情形，顯示蛋白質的表現量 仍然相當微量（＜10-6 mg），並有蛋白質結合或水解等現象。此外，文獻指 出Dengue virus Type-1 之 NS1 可經由 E.coli BL21(DE3)進行蛋白質大量表現
（Das et al., 2009），因此本研究不能大量表現 NS1 的原因就值得進一步探 討。根據前人研究，如能將大腸桿菌大量培養加以純化濃縮，才能得到適 量的蛋白質以利於進行Coomassie blue 染色觀察（Khromykh et al., 1996）。
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