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標題: A型流行性感冒病毒非結構蛋白1A在活細胞分佈情形之研究
Intracellular localization of influenza virus A/PR/8/34 NS1A protein in live cells
作者: 蔡傳馥
Tsai, Chuan-Fu
關鍵字: influenza virus;流感病毒;NS1A;FlAsH;live cells;非結構蛋白1A;FlAsH;活細胞
出版社: 生物科技學研究所
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A型流行性感冒病毒屬於正黏液病毒屬,具有八條單股負極性分節的核醣核酸基因體,可轉錄出十一個病毒蛋白質。其中第八條分節的核醣核酸基因體可以轉譯出兩個病毒非結構性蛋白質,NS1A和NEP。前人研究發現A型流行性感冒病毒NS1蛋白質在病毒感染時具有許多功能,主要扮演抑制細胞中免疫反應以及訊息核醣核酸的成熟和出核,並且幫助病毒基因體的複製與轉譯。在本論文的研究中,為了能夠即時觀測在病毒感染時NS1A的分佈,我們在NS1A蛋白質核酸結合區域與因子結合區域間之連接區段接合tetracysteine-tag,再利用逆向遺傳系統 (reverse genetic system)生產出病毒,經由病毒感染細胞後,以FlAsH螢光標定,利用共軛焦顯微鏡觀測NS1A在活細胞中分佈情形。經過逆向遺傳系統產生的突變株病毒 (NS1-tc-linker virus),在感染細胞後可以利用西方點墨法確認NS1A蛋白質在病毒感染的細胞中的表現量,與野生型病毒之NS1A的表現量相似。並且利用融菌斑試驗確認突變株病毒具有和野生型病毒相似的感染力價。在試管內FlAsH螢光接合試驗中,突變株病毒之NS1A蛋白質能有效率且具有專一性的與FlAsH結合。將突變株病毒感染細胞後,以FlAsH螢光標定,再利用NS1A抗體進行免疫螢光染色觀察NS1A蛋白質在細胞中的分佈情形,證實FlAsH螢光的位置與利用抗體標定之NS1A蛋白質位置具有相同的分佈情形。最後利用共軛焦顯微鏡觀測經過病毒感染的細胞中,被FlAsH標定的NS1A蛋白質的分佈,發現在病毒感染1.5小時後,NS1A蛋白質會分佈於粒線體中;而在感染後4小時與8小時,NS1A蛋白質則會主要分佈在細胞核並且形成類似核仁的螢光亮點。本研究中建立了利用FlAsH螢光標定,在病毒感染的過程中,病毒蛋白質在活細胞的分佈情形,將成為深入了解病毒生活史的有利工具。

The influenza A viruses are the prototype of the family Orthomyxoviridae. They contain eight single-stranded, negative-sense, segmented viral RNA (vRNA), which encode 11 known proteins. The vRNA segment eight encodes two nonstructural proteins, NS1A and NEP. The NS1 protein is a multifunctional protein that counteracts host cell antiviral responses and inhibits host cell pre-mRNA processing. In order to study the dynamics of localization of NS1 protein during virus infection in live cells, tetracysteine-tag (TC-tag) which can bind the membrane permeable bis-arsenical fluorescein FlAsH with fluorescent was fused with NS1 at the linker region between RNA-binding domain and effector domain and the loop region inside the effector domain. The construct containing TC-tag at the linker region of NS1A is cotransfected with all other plasmids of reverse genetic system into 293 cells to generate the recombinant virus. The mutant virus shows similar the infectivity with that of wild type. In vitro labeling the mutant NS1-tc-linker protein with FlAsH was successful and specific. The specific localization of NS1 in infected cells can be visualized after FlAsH labeling by confocal microscopy. NS1 was found to localize in mitochondria at very early time point of infection (1.5 hpi); and at 4 hpi and 8hpi, it was localized predominantly in nucleus at 4 hpi and 8 hpi and formed a granular pattern. These results suggested that the biarsenical labeling technique is successfully established and it can be an important strategy to visualize the location of target proteins dynamically in live cell.
其他識別: U0005-2701201003073400
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