LNGG 的後方之蛋白酶活性相印證(Thiel et al., 2003; Lindner et al., 2005;
Barretto et al., 2005)，故可推論此重組 SARS PLpro 蛋白是具有活性、功 能性的。
(Reporter gene assay)中，SARS PLpro 能抑制 Interferon α 刺激細胞內 ISRE(interferon-stimulated response element)訊息之表現，使 ISRE 訊息表 現程度下降。並以Real-time RT-PCR 測定 ISRE 其下游基因 PKR 之表現， 上升，但SARS PLpro 表現細胞在同樣經過 Interferon β 刺激後其 Interferon α 之表現量僅有 Mock cell 之 50%，即 SARS PLpro 蛋白之表現使 Interferon β 促使之細胞 Interferon α 表現量受到抑制，故可證實 SARS PLpro 不但抑 制細胞中第一型干擾素誘導之下游訊息及基因之表現，也抑制第一型干 擾素誘導之蛋白表現，與先前文獻發現SARS PLpro 能水解 Interferon 所 誘導之蛋白 ISG15(Barretto et al., 2005)對照之下，發現 SARS PLpro 與 Interferon 之間相關聯性越顯重要。
另外，當 SARS PLpro 表現細胞經過 LPS 刺激，以促使發炎反應過 後，其 NFκB 之訊息表現程度僅為 Mock cell 經 LPS 刺激後之 33%，即 SARS PLpro 能抑制細胞中發炎相關轉錄因子 NFκB 之訊息表現。
病毒藉由抑制 Interferon 以達到破壞細胞之免疫系統作用為常見之模
式，在其他文獻中發現，如：流行性感冒病毒(influenza virus)之 NS1 蛋 白會藉由與double-stranded RNA 結合達到抑制細胞之干擾素誘導之免疫 反應(Bergmann, M. et al., 2000；Hatada, E. et al., 1992 Lu, Y. et al., 1995)；
伊波拉病毒(Ebola virus)所轉譯出的蛋白 VP35 會藉由結合並隔離開 double-stranded RNA 以達到抑制 Interferon 之作用，而蛋白 VP24 則藉由 妨礙活化的STAT1 進入細胞核以達到抑制 Interferon 之作用(Basler, C. F.
et al., 2000 Reid, S. P. et al., 2006)；尼帕病毒(Nipah virus)會轉譯出 3 種蛋 白去中和、抑制干擾素上游STAT1 以達到逃避細胞免疫反應之作用(Park, M. S. et al., 2003)。而 SARS 之 ORF3b、ORF6 及核蛋白衣(nucleocapsid) 被發現具有能中和Interferon 之功效(Sarah A. et al., 2007) ，因此我們推論 SARS PLpro 具有中和 Interferon 之功效，能使細胞之免疫能力下降，保 護SARS 病毒不受感染細胞之免疫攻擊。
以二維電泳檢測細胞全面性的蛋白質表現，初步以 Mock cell 加入 Interferon α 為 Reference gel 比對其他膠體發現，蛋白質點 697、635、691 與SARS PLpro 表現細胞之同位置蛋白質點有表現量之差異，經由 MASS 分析蛋白質點分別為：Heat shock 27 kDa protein、Chloride intracellular channel protein 1、Myosin-9 與 NADH dehydrogenase iron-sulfur protein 3，
其中Heat shock 27 kDa protein 已知與 anti-apoptosis 有關，在受到第一型 干擾素刺激下會增加表現(M. Caraglia1 et al., 1999) ；Chloride intracellular
channel 可調控細胞內外之電位平衡、液體傳送以及維持細胞體積之功能 (Michael Eisenhut et al., 2006) ；而 NADH dehydrogenase 為粒線體之前驅 物質，在受到第一型干擾素刺激下會增加其表現，但在本研究之二維電 泳分析結果卻發現其表現量為下降。其它二維電泳分析之Interferon 相關 蛋白質點表現之改變尚在分析中。
在未來的分析中，希望藉由從二維電泳分析出差異性之蛋白質點為 何，並從中探討SARS PLpro 所影響、改變之蛋白表現與訊息途徑，架構
出整體蛋白質表現網絡系統，以用於將來開發SARS 診斷及治療之新方
向。
SARS-PLP(1332 bp )
PLpro
A. B.
HRP 0.1ug
圖4-4 SARS PLpro 活性試驗(Horseradish Peroxidase, HRP )
不同濃度的 SARS PLpro 與相同濃度的 HRP(0.1μg)在 37℃下反應 1 小時後，藉由ABTS/H2O2 呈色，再以 O.D.(405nm)測其吸光值。當 PLpro 濃度增加時，HRP 吸光值則相對下降，可得知 PLpro 具有活性。
(p<0.05)
圖4-5 SARS PLpro 切割 HRP 活性試驗
利用 HRP(Horseradish Peroxidase)作為 substrate，取 5 μg 與不同濃度 之PLP(0、2、4、6 μg/ml)混合，於 37℃下反應 6 小時，以 Native gel 將 蛋白展開後，加入HRP 呈色劑 TMB membrane peroxidase substrate 產生 藍色區域，並以Density meter 換算出藍色區域減少之百分比。
A. B.
PCR：
SARS-PLP-HSV ( 1332 bp )
PLpro PLpro
M M
Cutting：
SARS-PLP-HSV ( 1332 bp )
M：Marker
Lane 1：SARS PLpro PCR 產物 Lane 2：Cutting 之 SARS PLpro
圖4-6 構築 pcDNA3.1 His C 作為細胞表現 PLpro 蛋白表現系統。
(A) 以設計帶有 HSV-tag 之反股 primer，由 pET 系列構築出之 SARS PLpro 質體中，經PCR 得到之 PLpro 片段。
(B) 將篩選到帶有 SARS PLpro 質體之大腸桿菌 Top 10，抽取其質體 做限制酵素水解試驗。
A. B. C.
D. E. F.
圖4-7 以 Anti His-tag 為抗體做免疫螢光染色之細胞內蛋白表現確認 將細胞以福馬林固定、甲醇打洞後，以 Anti His-tag 為第一抗體，在 37℃下反應 1 小時，再以 Anti mouse Rhodamin 為第二抗體，37℃下反應 1 小時，最後以螢光顯微鏡觀察
A~C 圖為以 9：1 比例轉染入 pcDNA3.1 + pEGFP 之 HL-CZ 細胞。
D~F 圖為以 9：1 比例轉染入 SARS PLpro-pcDNA3.1 + pEGFP 之 HL-CZ 細胞。
A、D 圖為在可見光下觀察到之細胞型態，B、E 圖為在螢光激發下觀察 到 pEGFP 之綠光，C、F 圖為在螢光下觀察到載體 pcDNA3.1 因帶有 His-tag，經由染色所觀察到之紅色螢光。
C. 到 SARS PLpro 因帶有 HSV-tag，經由染色所觀察到之紅色螢光，C、G
圖為在可見光下觀察到之細胞型態，D、H 圖則為綠光、紅光與可見光之
重疊圖。
Anti HSV tag conjugate nano gold
ISRE +IFN-a
0 50 100 150 200 250
IFN-a 0U/ml IFN-a 3000U/ml
Relative luciferase activity
HLCZ-pEGFP HLCZ-PLpro
(p<0.05)
圖4-10 Single-reporter assay(Luciferase assay)測試 ISRE 訊息
將 Mock cell(HL-CZ-pEGFP+pcDNA3.1)與 HL-CZ-SARS-PLpro 分別 再以 9：1 比例共同轉染入 pISRE-Luc.與 pRenilla-Luc。兩天後再加入 Interferon α(3000U/ml)反應四小時後，收取細胞以測其冷光量值。
NFkB + LPS
圖4-11 Single-reporter assay(Luciferase assay)測試 NFκB 訊息
將 Mock cell(HL-CZ-pEGFP+pcDNA3.1)與 HL-CZ-SARS-PLpro 分別 再以 9：1 比例共同轉染入 pNFκB-Luc.與 pRenilla-Luc。兩天後再加入 LPS( Lipopolysaccharide, 10μg/ml)反應四小時後，收取細胞以測其冷光量 值。
RT-PCR PKR
HLCZ HLCZ+IFN α PLpro PLpro+IFN α
Relative folds of PKR mRNA
圖4-12 即時定量聚合酶連鎖反應偵測 Interferon α 誘發之下游基因表現 將 Mock cell(HL-CZ-pEGFP)與 HL-CZ-PLpro cell 以 1×107 個為一 組，各取兩組。其中一組Mock cell 與 HL-CZ-PLpro cell 分別加入 Interferon α 3000U/ml，另一組則維持不變，放入培養箱培養四小時後抽取其細胞全 mRNA，再轉錄成為 cDNA。之後取等量 cDNA 加入基因 PKR 之成對引 子及探針後反應，將所得到之Ct 值與 Control(GAPDH)之 Ct 值作換算，
繪製成圖。
(p<0.05) Interferon α 為一抗，反應兩小時，再以 anti-mouse Co AP 為二抗，反應兩 小時，最後測其O.D.405nm 之吸光值。
(A) (B)
pEGFP+pcDNA3.1 pEGFP+pcDNA3.1 Treat IFN a
(C) (D)
PLP-HSV/pcDNA3.1+pEGFP PLP-HSV/pcDNA3.1+pEGFP Treat IFN a
圖4-14-1 二維電泳
在經過 interferon α treat 前後，mock cell 與 HL-CZ-PLpro 個別都有點 的數量及濃度上的差異。
HL-CZ-pEGFP+pcDNA3.1 treat interferon α
HL-CZ-SARS PLpro-pcDNA3.1+pEGFP treat interferon α
圖4-14-2 二維電泳差異之蛋白質點
HL-CZ-pEGFP+pcDNA3.1 treat interferon α 與 HL-CZ-SARS
PLpro-pcDNA3.1+pEGFP treat interferon α 表現具差異量之蛋白質點。
Referance
表 4-2 以質譜儀分析出差異之蛋白質點
將比對後蛋白表現量具有差異之蛋白質點以質譜儀分析，並依其數值對照出可能之蛋白質點為何。
Table 4-2
Spot ID Protein Identification MW(kDa)/pI Score Peptide Sequence Match coverage(%) Up-regulatory in PLpro-expressing cells treated interferon α compared to mock cells treated interferon α
Mock+IFN α-697 Heat shock 27 kDa protein 22.8/5.98 173 11 32 Mock+IFN α-635 Chloride intracellular 26.9/5.09 170 11 30 channel protein 1
Down-regulatory in PLpro-expressing cells treated interferon α compared to mock cells treated interferon α Mock+IFN α-691 NADH dehydrogenase 30.2/6.99 93 6 10 iron-sulfur protein 3
Down-regulatory in mock cells treated interferon α compared to PLpro-expressing cells treated interferon α PLpro+IFN α -59 Heat shock cognate 70.9/5.37 775 11 33
71 kDa protein
PLpro+IFN α-663 Ran-specific GTPase-activating 23.3/5.19 167 3 17 protein (Ran-binding protein 1)
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