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Purification and serology of the nucleocapsid proteins of four distinct tospoviruses
|摘要:||Tospovirus是Bunyaviridae科內唯一可感染植物的一病毒屬,目前 Tospovirus有三個主要血清型,分別為TSWV,INSV及WSMV型.一種從花生上 分離類似番茄斑萎病毒(tomato spotted wilt virus,TSWV)於本研究中被 稱為Tospo-P.由於tospovirus為一套膜(envelope)包被的病毒粒子 ,在生 體外極不穩定,且在純化過程中難以去除寄主成份,因此製備高專一性血清 頗為困難.為了解Tospo-P與其它Tospoviruses之血清關係,本實驗發展一 簡易迅速純化核鞘(nucleocapsids)方法,並進一步分離其核鞘蛋白(NP), 製備高專一性血清,利用免疫擴散分析,酵素聯結血清反應及西方漬染等主 要血清方法,釐清本省花生分離株和不同tospoviruses間之血清學關係.四 種不同之Tospoviruses,包括Tospo-P自花生分離的 Tospo-PD2,西瓜銀斑 病毒西瓜分離株,鳳仙花疽斑病毒鳳仙花分離株和番茄斑萎病毒紐約分離 株接種至奎藜(Chenpodium quinoa)後,4-6天採集其葉片以TB緩衝液萃取, 利用低速離心,及加入1﹪Triton X-100淨化處理後,再經蔗糖墊底沉降濃 縮及 35%硫酸銫等密度離心純化所得之病毒核鞘(nucleocapsids)以SDS解 離後,經一次12% polyacrylamide膠體電泳,可得到純化之四種不同分子量 核鞘蛋白,分別為WSMV-W NP(32kDa ),Tospo-P NP (31 kDa),TSWV-NY NP(30 kDa)及INSV-M NP (29 kDa),其每 100 克植物組織可得純化蛋白量 約為1-5 mg。此四種蛋白分別免疫紐西蘭白兔,而得其相對之四種抗血清 。 Tospo-P是最近在本省中部花生栽培區所發現類似TSWV的病毒病害,由 其純化之核鞘蛋白所製備之抗血清,利用免疫擴散反應,間接酵素聯結血清 反應及西方漬染分析,結果清楚顯示Tospo-P與WSMV,INSV,TSWV多個病毒分 離株之純化核鞘蛋白及感病粗汁液中之抗原皆無血清關係, 故 Tospo-P為 Tospovirusg屬之一新血清型並可能為該屬之一新種。 A TSWV-like virus previously isolated from peanut was designated as Tospo-P in this invesigation. In order to clarify the serological relationships between a paracircular Tospovirus- like virus, isolate PD2 of Tospo-P from peanut and other tospoviruses,the nucleocapsid proteins of four tospoviruses including the Tospo-PD2, A watermelon isolate of watermelon silver mottle virus (WSMV-W) , an impatiens isolate of impatiens necrotic spot virus (INSV-M) , a TSWV isolate from New York (TSWV-NY) were purified from leaf tissues of Chenopodium quinoa infected with each virus. The leaves tissues of C.quinoa. 4-6 days after inoculation with the four individual tospoviruses were extracted with TB buffer. The supernatant after centrifugation was treated with Triton X-100 and followed by high-speed centrifugation through 20% sucrose cushion. The pellets were resuspended and further separated by 35% cesium sulfate isopycnic centrifugation. The nucleocapside zones were drawn and the NPs were purified by SDS-polyacryamide gel electrophoresis. The yields of the purified NPs were 1-5 mg per 100 g tissue. Specific antisera were produced by injecting each antigen into individual New Zealand white rabbits. In the immunodiffusion, Western blot and indirect ELISA tests, the four antisera against NPs only reacted to their homologous purified antigens and crude antigens in plant extracts; and no cross reactions in the heterologous combinations were noticed. These results of homologous and heterologous assays with the four antisera against NPs of the different tospoviruses clearly indicated that the NPs of TSWV, TNSV, WSMV and Tospo-P are serologically unrelated.Thus,tospo-P is considered a new serotype and possible new member of the genus Tospovirus.|
|Appears in Collections:||植物病理學系|
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