臺灣博碩士論文加值系統

同源病毒干擾 (homologous interference) 現象為一種病毒感染細胞後會對於其同源病毒再次感染同一個細胞產生抑制的干擾現象，而此現象存在於許多病毒中。根據實驗室先前的研究顯示登革病毒無論在初次感染或持續感染細胞的情況下，均會干擾第二種病毒的再次感染 (superinfecrion)，而此干擾現象不僅存在於哺乳動物細胞中亦發生於白線斑蚊 (Aedes albopictus) 腸道細胞C6-36中。本實驗將從宿主細胞本身來探討，由黃質病毒感染所造成之同源病毒干擾現象的機制為何。由於黃質病毒RNA複製時會產生大量的雙股RNA (double strand RNA) ，而此可能會誘發細胞內抗病毒的RNA interference (RNAi) 反應，參與黃質病毒感染所造成的同源病毒干擾現象。首先，我們探究Den2是否具有抑制RNAi的功能，以專一性抑制pEGFP-C1的shRNA系統，我們觀察到Den2單一基因或Den2感染均無法抑制RNAi。所以，我們利用已被報導的RNAi 抑制者 (suppressor) 送進有病毒持續性感染的細胞中，希望將細胞內的RNAi抑制後，觀察後續感染的黃質病毒是否可成功複製，結果發現黃質病毒感染所造成之干擾現象並沒有因RNAi 抑制者的存在而被破壞。另一方面，在已被病毒持續性感染的細胞中，利用間接免疫螢光染色法 (IFA) 觀察個別黃質病毒蛋白質的表現情形，我們發現各病毒蛋白質的表現並不會受到持續感染的影響；反之，先轉染單一黃質病毒的基因進入細胞後再感染同源的黃質病毒，也觀察到相似的結果，此結果可能為單一病毒基因不足以造成干擾現象；然而JEV NS5蛋白質似乎可促進Den2感染與病毒擴散，而JEV的NS1與NS2A蛋白質則不具有此功能，顯示JEV NS5蛋白質對於黃質病毒的感染可能扮演著促進的角色。由於protein tyrosine phosphatases (PTPs)的抑制劑-原釩酸鈉 (sodium orthovanadate；Na3VO4) 可阻斷JEV NS5蛋白質對於干擾素 (Interferon；IFN) 訊號傳遞的作用，我們探討Na3VO4是否影響黃質病毒感染所產生的同源病毒干擾現象，結果發現Na3VO4對病毒感染所產生之同源病毒干擾現象似乎不具影響力。綜合以上，本實驗結果顯示黃質病毒之同源干擾似未涉及RNAi的反應，故其干擾機制需進一步研究。

Homologous interference is a phenomenon where a cell infected with a virus is resistant to superinfection by the same or similar virus, and such interference has been observed for a number of viruses. According to our previous research, Dengue virus could resist to a superinfection by homologous virus in a primary and persistent infection in mammalian and C6-36 (Aedes albopictus) cells. In this study, we investigated what causes homologous interference in flavivirus. During flavivirus productive replication, double-stranded RNA (dsNRA) intermediates and stem-loop secondary structures derived from certain regions of ssRNA genome are believed to be substantially generated in cytoplasm, which may induce cellular RNAi as a nucleic acid-based immunity leading to homologous interference. First, we found that Den2 replication could not suppress RNAi. Second, when use known RNAi suppressor to block induced RNAi, we observed that this strategy could not break homologous interference. Third, we found persistent infection did not affect the expression of individual flaviviral genes by transient transfection, and conversely, the transient expression flaviviral gene had no effects on the following flavivirus infection, indicating flavivirus single gene is not sufficient to cause homologous interference. JEV NS5 protein alone seems to promote Den2 virus spread and infection. Finally, we observed that protein tyrosine phosphatases (PTPs) inhibitor- sodium orthovanadate could not inhibit flavivirus interference, the result shown that homologous interference didn’t break by PTPs inhibitor. To sum up, our results suggest flavivirus interference may not involve the cellular RNAi response.

誌謝
目錄 I
圖目錄 III
中文摘要 IV
英文摘要 V
第一章 前言 1
第二章 材料與方法 4
一、細胞培養 4
二、病毒株 4
三、試劑、藥品、酵素 5
四、引子 (Primer) 5
五、本論文所使用之抗體 5
六、HIV-1 Tat基因與VV E3L基因之構築與表現 6
七、微量質體DNA的製備 6
八、質體DNA的大量製備 7
九、聚合酵素鏈鎖反應 7
十、細胞內病毒RNA的抽取 7
十一、反轉錄反應 8
十二、利用LipofectamineTM2000進行細胞之轉染反應 (Transfection) 8






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項目 頁數
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十三、間接免疫螢光染色 (Indirect immunofluoresent assay) 8
十四、西方墨點法 (Western blot) 9
十五、登革第二型病毒持續性感染B2-5細胞株之建立 (B2-5D2) 9
十六、Den2之增殖 9
十七、JEV RP9之增殖 10
十八、病毒效價之測定 (virus titration) 10
十九、流式細胞儀分析 (Flow cytometry) 10
第三章 結果 11
一、探討Den2基因是否能抑制RNAi 11
二、探討RNAi是否涉及黃質病毒持續感染所產生之同源干擾現象 12
三、持續感染細胞的黃質病毒對後續單一黃質病毒蛋白質表現之影響 13
四、探討單一黃質病毒蛋白質表現對於後續黃質病毒感染之影響 14
五、探討Protein tyrosine phosphatases (PTPs) 抑制劑對黃質病毒感染所產生之同源病毒干擾現象之影響 16
第四章 討論 18
第五章 圖表 22
第六章 附錄 51
參考文獻 56

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