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標題: 西瓜銀斑病毒複製酶之單元抗體製備及具感染力核鞘之特性分析
Generation of monoclonal antibody against the replicase of Watermelon silver mottle tospovirus and characterization of the infectious nucleocapsids
作者: 曾獻嫺
Tseng, Hsien-Hsien
關鍵字: WSMoV
出版社: 植物病理學系所
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摘要: 西瓜銀斑病毒(Watermelon silver mottle tospovirus, WSMoV)是屬於以節枝動物傳播之Bunyaviridae科Tospovirus屬之植物病毒。依據在bunyaviruses的研究結果指出,由病毒感染之細胞所分離得到之核鞘(nucleocapsids)包含有三條病毒基因體RNA,大量核鞘蛋白(nucleocapsid protein, N protein)及少量聚合酶蛋白(polymerase, L protein);且Bunyaviruses的轉錄及複製最少需要其三條基因體RNA和N及L蛋白同時存在才能進行,而在屬於Bunyaviridae科的幾個動物病毒的逆向遺傳系統(reverse genetics system)上也曾證明此一結論。目前植物負極性RNA病毒如Tospovirus的逆向遺傳系統尚未建立完備,具感染力最少之病毒組成份與其核鞘特性也為曾有系統的研究。本研究之目的在於探討WSMoV具有感染力核鞘之成分及特性,而為了偵測在具感染力之核鞘內的tospovirus蛋白組成,我們製備了能辨識WSMoV之L蛋白之單元抗體。罹病組織經萃取及1%的Triton X-100處理後,以20%蔗糖緩衝液為墊層(sucrose cushion)進行高速離心,最後再以硫酸銫(cesium sulfate)密度梯度離心進行純化核鞘,並針對硫酸銫密度梯度離心前後的粗純化WSMoV核鞘做分析。感染力的測試乃利用粗純化之核鞘溶液接種於奎藜(Chenopodium quinoa)上觀察其單斑的形成,結果顯示在未經硫酸銫密度梯度離心之前的粗純化核鞘溶液均具有感染力。利用西方轉漬法以抗體分析於具感染力與不具感染力之核鞘上的相關蛋白成分,結果卻發現兩者都能偵測到WSMoV的四種蛋白L , N, NSs及NSm。因此,為了獲得較純淨的具感染力核鞘,於是我們使用膠體管柱過濾法進一步分離由20%蔗糖墊層超高速離心下所得之粗純化核鞘。經膠體管柱過濾分離所得的具感染力核鞘則僅能偵測到L 及N蛋白的存在而沒有NSm及NSs蛋白。此純化的核鞘再進一步以抗體抑制分析法分析其中所含病毒L及N蛋白是否參與影響核鞘的感染力,接種活性測試指出個別L與N的抗體均能抑制核鞘的感染力。總合上述,結果說明了WSMoV具感染力的核鞘中,除病毒RNA外,最少需包含有L與N蛋白的存在。這些結果對於我們嘗試建立第一個植物負極性RNA病毒的逆向遺傳系統提供重要的資訊。
Watermelon silver mottle tospovirus (WSMoV) is a species of plant viruses belonging to the genus Tospovirus within the arthropod-born Bunyaviridae. The virions of bunyaviruses contain ribonucleoproteins (RNPs, nucleocapsids) comprising of three linear, negative-sense single-stranded RNA tightly encapsidated by the nucleocapsid protein (N protein) and a few copies of the viral RNA-dependent RNA polymerase (RdRp, L protein). The viral genomic RNAs, N and L proteins are necessary for bunyaviruses to complete their transcription and replication. Similar conclusions have been also reported in several animal viruses belonging to Bunyaviridae using reverse genetics system. However, the reverse genetics systems of plant negative-sense RNA viruses such as tospoviruses have not yet been developed. Also, the minimal components conferring tospoviral infectivity and the characteristics of tospoviral nucleocapsids have not yet been systemically studied. This study was dedicated to develop monoclonal antibodies against the L protein of WSMoV and characterize the components of WSMoV nucleocapsids. In order to detect the tospoviral proteins in the infectious nucleocapsids, the monoclonal antibody against the bacterium-expressed L-c protein of WSMoV was prepared in this thesis. The WSMoV nucleocapids were purified serially by high-speed centrifugation, 1% Triton X-100 treatment, ultra-high speed centrifugation through 20% sucrose cushion and followed by 35% cesium sulfate isopycnic centrifugation. The infectivity of the purified nucleocapsids was analyzed by bioassay with Chenopodium quinoa. Our results showed that the partially purified nucleocapsids in any purifying steps prior to the cesium sulfate isopycnic centrifugation were still infectious to C. quinoa. Proteins associated with either the infectious or noninfectious nucleocapsids were tested by western blotting using antibodies against L, N, NSs or NSm proteins of WSMoV. L, N, NSs and NSm proteins were detected in both infectious and noninfectious nucleocapsids. To obtain pure and clean infectious nucleocapsids, partially purified nucleocapsids derived from the sucrose cushion centrifugation were further purified by gel filtration chromatography. Infectious nucleocapsids were monitored in the first exfluid which contains the N and L but not NSs and NSm proteins. Antibody inhibition assays were performed to explore the tospoviral proteins involving in the infectivity of nucleocapsids. The infectivity of nucleocapsids were inhibited by the incubation with antibody against N or/and L proteins. Taken together, our results indicate that the infectious nucleocapsids of WSMoV required the L and N proteins in addition to genomic RNA. These results are important to develop the first reverse genetics system of plant negative sense RNA virus.
其他識別: U0005-2308200615470700
Appears in Collections:植物病理學系



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