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研究生:林積成
研究生(外文):chi-cheng Lin
論文名稱:利用內源性第155號微小核醣核酸進行研究日本腦炎病毒感染的小鼠免疫細胞之致病角色
論文名稱(外文):Utilizing endogenous microRNA-155 to study pathogenic role of Japanese encephalitis virus in immune cells of the infected mice
指導教授:廖經倫
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:105
畢業學年度:98
語文別:中文
論文頁數:60
中文關鍵詞:第155號微小核醣核酸
外文關鍵詞:microRNA-155
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MicroRNAs (miRNAs) are endogenous non-coding RNAs that spatiotemporally modulate mRNAs in a post-transcriptional manner. The engineering of viruses by insertion of a tissue-specific miRNA recognition element (MRE) into viral mRNA can restrict viral tissue tropism. In this study we employed Japanese encephalitis virus (JEV) to investigate whether immune-enriched miRNAs are able to suppress JEV replication through the targeting of its nonpolyadenylated viral mRNA. Myeloid cells such as macrophages and dendritic cells are the primary targets for JEV infection; thus, likely contributing to viral pathogenesis. Expression of miR-155 can be enhanced in dendritic cells, macrophages/monocytes, B-cells and T-cells especially by inflammatory signals through Toll-like receptors upon exposure to antigen or by interferon stimulation. To create an immune cells-restricted JEV, we inserted MRE of miR-155 into the 3′-untranslated region (3′-UTR) of viral genome, and investigate whether endogenous miR-155 can diminish virulence of this recombinant JEV in the infected mice. This approach may not only allow us to study JEV pathogenesis involved particularly in viral tissue tropism but also provide an additional layer of biosafety, and thus having great potential for rational development of safer flavivirus vaccine。
目 錄 I
圖 表 目 錄 III
中文摘要 V
Abstract VI
前言 1
材料與方法 9
一、細胞培養及病毒株 10
二、動物實驗 10
三、本論文所使用的抗體 10
四、本論文所使用之引子 (Primers) 11
五、微量質體DNA的製備 12
六、勝任細胞 (Competent cell) 製備及其轉型作用 (Transformation)
12
七、利用LipofectamineTM 2000進行細胞的轉染反應 (Transfection)
13
八、病毒之擴增 (Virus amplification) 14
九、病毒效價測定 (Plaque-forming assay) 14
十、間接免疫螢光染色法 (Indirect immunofluorescent assay) 15
十一、聚合酵素連鎖反應 (Polymerase chain reaction; PCR) 16
十二、萃取細胞之RNA 16
十三、反轉錄聚合酵素連鎖反應 (RT-PCR) 17
十四、Dual-Luciferase repoter assay 17
實驗結果 19
一、 設計第155號微小核醣核酸靶位 (miR-155 MRE duplex),並構築日本腦炎病毒複製子 (reporter-containing JEV replicon, JR2A) 嵌入miR-155 MRE 20
二、評估嵌入2倍或者4倍miR-155 MRE的JR2A,利用Luciferase活性定量其在細胞中複製的程度 21
三、生產具有嵌入miR-155 MRE的重組日本腦炎病毒 21
四、分析以細胞製備回收 (recovery) 重組日本腦炎病毒 (rRP9/155pmT or rRP9/155scT) 過程中miR-155 MRE的完整性 22
五、分析日本腦炎病毒野生株 (RP9-XM) 與重組病毒株 (rRP9/155pmT or rRP9/155scT) 在細胞內複製的影響 23
六、日本腦炎病毒野生株 (RP9-XM) 與重組病毒株 (rRP9/155pmT or rRP9/155scT) 感染Raw264.7細胞的病毒生長曲線 24
七、以小鼠動物模式觀察日本腦炎病毒野生株 (RP9XM) 與重組病毒株 (rRP9/155pmT or rRP9/155scT) 之毒力分析 (virulence) 24
討論 26
參考文獻 30
圖 與 表 38
附 錄 46
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