C型肝炎病毒(Hepatitis C virus, HCV)屬於黃熱病毒科肝炎病毒屬，是ㄧ個具有單股正向RNA基因體的病毒。HCV感染會引起急性或慢性肝炎，更嚴重的會演變成肝硬化或肝癌。HCV的基因體可以轉譯一條大約3000個胺基酸的多蛋白，再藉由細胞產生的蛋白水解酶或是病毒本身製造的蛋白水解切割為10個具功能的病毒蛋白。HCV感染全球大約1億七千萬人口。一直以來因為缺少了ㄧ個有效自然的HCV感染模式，也因此對於研究HCV的生活史以及宿主與病毒之間的交互作用關係必須依賴HCV病毒株的建立以及細胞培養系統。在本篇研究中，主要目標是建立不同長度的HCV subgenomic clones.分別含有從非結構性蛋白(NS)2或NS3開始一直到最後的3’端非轉譯區。為了方便篩選以及分析，每一個HCV subgenomic clone 會接上一個報導
基因，包含抗藥性基因neomycin phosphotransferase gene、 加強型綠色螢光蛋白基因(enhanced green fluorescent protein gene)或是螢火蟲冷光酵素基因(firefly luciferase gene).同時我們也在實驗室中建立了對HCV RNA具高度容忍性的細胞株。將帶有HCV RNA會自我複製的Huh-7細胞經過67天的干擾素(IFN-α)的治療後，HCV RNA會被完全的清除，稱為cured Huh-7細胞株。Huh-7或是cured Huh-7細胞溶解液中的冷光酵素活性在72小時內持續的被偵測，結果顯示帶有螢火蟲冷光基因的HCV subgenomic HCV RNA 可以在cured Huh-7 細胞中有效複製，但是在Huh-7細胞中則不行。而且為了讓此複製系統穩定，我們更進一步的調整宿主細胞的狀態以及
分析的條件，使達到最佳化。因此，本篇研究成功的在培養的肝癌Huh-7細胞株中建立了暫時表現的C型肝炎病毒報導系統，這個系統為研究HCV複製相關機制或是與宿主細胞之間的交互作用提供了ㄧ個很好的工具。

Hepatitis C virus (HCV) belongs to the Hepacivirus genus in Flaviviridae family and is a single positive-stranded RNA virus that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. HCV genome encodes a polyprotein of about 3,000 amino acids, which can be processed into 10 mature viral proteins by cellular or viral proteases. It affects an estimated 170 million people worldwide. Because of lacking efficiently native infection models, investigation of HCV life cycle and host-virus interaction mostly depended on the establishment of HCV molecular clones. In this study, we aimed to establish HCV subgenomic clones with different length, starting from nonstructural protein (NS) 2 or NS3 to the 3’ untranslation region. In order to facilitate clone selection and further analysis, each of HCV subgenomic clone was constructed to carry one of the following reporter genes, including neomycin phosphotransferase gene, enhanced green fluorescent protein gene and firefly luciferase gene. We also set up highly permissive cell line (cured Huh-7) for subgenomic HCV RNA replication by prolonged treatment with IFN-α. After 67 days, the self-replicating subgenomic RNA could be eliminated from Huh-7 cells. Luciferase activity in Huh-7 and cured Huh-7 cell extracts was measured over 72 h and the results showed that the bicistronic, subgenomic HCV RNA containing firefly luciferase gene replicated well in cured Huh-7 cells but not Huh-7 cells. Furthermore, we optimize the cellular environment for HCV replication and analytic conditions. Thus, the transient reporter HCV replication system was established in cured Huh-7. This in vitro HCV replicative system provides a tool for researching HCV replication.

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