臺灣博碩士論文加值系統

摘要
C型肝炎病毒為主要輸血性及偶發性感染的非A非B型肝炎毒病。它是一個屬於Flaviviridae family的單股正性的RNA病毒。如同對其他RNA病毒感染患者的治療方法，目前臨床上是以干擾素來進行治療HCV的病患，但是只有10~15%的病人可治癒。而目前全世界有1~2%的人口感染過HCV，這些感染的病患又有70~80%會變成長性感染。已知肝硬化及肝癌與長期感染具有高度的相關性。所以發展治療C型肝炎的方法是為迫切需要的。HCV的基因體會轉譯成一單一多蛋白（polyprotein），再經由宿主細胞的siganl peptidase及病毒的NS3 protease分別對結構及非結構蛋白的部份進行切割，產生至少十個成熟的蛋白。這表示HCV的NS3可以當作發展治物藥物的標的。為了建立NS3的protease的活性測試系統，在此我們構築了三種主要的質體：（1）pδ-5A/5B-GFP；（2）pFBD-5A/5B-AD；（3）pFNS34-SEAP。在第一個方法我們使用HDV的delta antigen（1-163）具有NLS會分佈在核仁的特性，所以可在顯微鏡下觀察到GFP分佈在核仁。一旦NS3 protease切割delta與GFP之間的NS5A/5B切處點，將會使得GFP散佈在細胞質中。不過以Western blotting來看，並沒有發現δ-5A/5B-GFP這個融合蛋白會被NS3切動。在第二個方法，我們是將HCV的NS5A/5B jucntion插入GAL4的DNA binding domain（BD）與activation domain（AD）之間。完整的GAL4會活化其reporter gene的轉錄，若BD與AD因被HCV NS3 protease切開而分離，則reporter gene的表現就會下降。在這個方法中，我們並沒有得到BD-5A/5B-AD被切割的結果。在第三個方法中，我們將HCV的NS34與SEAP（secreted alkaline phosphotase）融合，兩者之間則為4A/4B junction。利用NS3的自我切割，使得SEAP的signal sequence曝露，於是SEAP便會被分泌出細胞外。在這個方法我們可在medium測到SEAP活性，且由西方點墨法確認NS34-SEAP這個融合蛋白的確有被切割的情形。之後我們利用NS4A/4B junction衍生來的peptide當作HCV NS3的inhibitor，以2.5mM處理暫時表現NS34-SEAP的細胞18小時，可測得的SEAP活性明顯地下降。在我們測試的三種方法裡，只有第三種可行，這可能意味著在切割處兩旁的胺基酸序列可能會造成蛋白的結構改變並使得切割處無法被NS3蛋白辨認到。

Hepatitis C virus, a major pathogen of post-transfusional non-A, non-B hepatitis, is a single positive sense RNA virus. After long-term treatment of interferonα-2b alone or combination with ribavirin, the usual therapy to RNA virus, only few patients (10~15%) were curable. With worldwide distribution (1~2 %) and high frequency to become chronic infection, which often progresses to liver cirrhosis and hepatocellular carcinoma, it is urgent to establish assay systems for screening of inhibitors of HCV. The HCV encoded a polyprotein with the structure proteins at the N-terminus and the non-structure proteins at the C-terminus, which has to be processed by the host signal pepetidase and viral NS3 protease, respectively. This suggests that NS3 protease is a suitable target for anti-HCV drug development. To establish an assay system for HCV protease in vivo, three main plasmids were constructed: (i) pδ-HCV5A/B-GFP; (ii) pFBD-HCV5AB-AD; (iii) pFNS34-SEAP. The first one used the HDV delta protein, which has an NLS that will target the δ-HCV5A/B-GFP fusion protein to the nucleoli. If the HCV5A/B junction is cleaved by the NS3 protease, GFP will disperse through the cytoplasm rather than targeting to the nucleoli. But our results showed that no cleavage by protease was observed by western blotting with the anti-δ antibody. The strategy for creating pBD-HCV5AB-AD is using the GAL4 transcription activator inserted with an NS3 protease cleavage site between the DNA binding domain (BD) and the transcription activation domain (AD). The GAL4 activated expression of reporter gene, GFP, will diminish if GAL4 fusion protein is cleaved by HCV NS3 protease. However, no positive result was obtained in this system. The third plasmid pFNS34-SEAP was constructed with the 4A/4B cleavage site inserted between NS34 and SEAP (secreted alkaline phophotase). The SEAP will be secreted out once the NS3 auto-cleaves the immediately downstream 4A/4B junction and expose the signal sequence of SEAP. By the chemiluminescent method, the activity of SEAP in culture medium was measured. Positive results were obtained and this system allowed us to screen potential inhibitors. Treating transiently NS34-SEAP expressing cells with a NS4A/4B junction deriving inhibitor, the SEAP activity of cell medium decreased apparently when the concentration of the inhibitor was used in 2.5mM. Three systems were tested in this study, only one succeeded. It leaded to a conclusion that the flanking sequence may change the protein conformation and is important for protein function.

目次
英文摘要……………………………………………１
中文摘要……………………………………………３
緒論…………………………………………………５
實驗方法……………………………………………１５
實驗材料……………………………………………２０
結果…………………………………………………２６
討論…………………………………………………３０
參考文獻……………………………………………３５
圖表…………………………………………………４６

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