製備西瓜銀斑病毒複製酶之單株抗體偵測複製酶在植物土之表現

Generation of Monoclonal Antibody against the Replicase of Watermelon silver mottle virus and Its Application on the Detection of L Protein Expression in Planta

張賀雄(H.-H. Chang) ； 曾獻嫺(H.-H. Tseng) ； 葉錫東(S.-D. Yeh) ； 古新梅(H.-M. Ku) ； 詹富智(F.-J. Jan)

《植物病理學會刊》 18卷4期 (2009/12) Pp. 237-246

https://doi.org/10.6649/PPB.200912_18(4).0005

摘要

西瓜銀斑病毒(Watermelon silver mottle virus, WSMoV)屬於Bunyaviridae科Tospovirus屬植物負極性RNA病毒(negative-strand RNA virus)，具有三條基因體RNA，其中最大的L RNA以病毒反義股(anti-genomic sense)的方向編譯著WSMoV最大的病毒蛋白，L蛋白；目前已知L蛋白是WSMoV的複製酶(RNA-dependent RNA polymerase, replicase)，且L蛋白、核鞘蛋白(nucleocapsid protein, N protein)與病毒基因體RNA總繞成核酸蛋白復合體(ribonucleoprotein complex, RNP)；根據動物bunyaviruses的研究指出，鑑定出具感染力RNP的所有組成成分，是建立逆向遺傳系統(reverse genetics system)不可或缺的門檻。本研究的目的爲產生WSMoV L蛋白的單株抗體(monoclonal antibody)，並應用於感病植株上L蛋白表現的偵測。利用細菌表現系統表現L蛋白C端部分(L-c)，經過實驗鼠免疫注射、細胞融合及篩選培養，獲得表現所需抗體之融合瘤細胞株，並注射實驗鼠使產生含有高濃度單株抗體之腹水(ascites fluid)。此舉株抗體對於WSMoV的L蛋白具有專一性，不與其他三個tospoviruses血清群番茄斑萎病毒(Tomato spotted wilt virus)、鳳仙花壞疽斑點病毒(Impatiens necrotic Spot virus)、花生輪點病毒(Groundnut ringspot virus)發生反應，且可以西方轉漬法偵測到感病圓葉菸草(Nicotiana benthamiana)上L蛋白的表現依接種後經過天數不同而有消長現象；將此單株抗體結合蛋白A膠金標記(protein A-gold labeling)電子顯微鏡技術，可以觀察到纏繞著N蛋白、L蛋白的RNP結構。本研究所產生的L蛋白單株抗體，未來搭配WSMoV逆向遺傳系統的建立將可進行更深入地研究應用。

關鍵字

西瓜銀斑病毒 ； 核酸蛋白複合體 ； 細菌表現系統 ； 融合瘤細胞 ； 蛋白A膠金標定電子顯微鏡

並列摘要

Watermelon silver mottle virus (WSMoV), belonging to the Tospovirus genus of the Bunyaviridae, has a segmented-genome designed as S (small), M (medium) and L (large) RNA. L RNA, a negative-sense RNA, encodes the largest protein (L protein) of WSMoV. The L protein is a RNA-dependent RNA polymerase of WSMoV, and forms the ribonucleoprotein complex (RNP) with the nucleocapsid protein and the genomic RNA. According to the studies of animal bunyaviruses, identifying the components of RNP is the key step for developing a reverse genetics system. The purpose of this study is to generate the monoclonal antibody against L protein, and utilize it for the detection of L protein in virus-infected plants. The C terminus of WSMoV L protein (L-c) was purified from a bacterial expression system and used to immunize BALB/c mice. Ascites fluid containing the monoclonal antibody against the L protein was collected from the mice intraperitoneally injected with hybridoma cells. This monoclonal antibody specifically reacts with the L protein of WSMoV, but not with those of the other three serogroups of tospoviruses including Tomato spotted wilt virus, Impatiens necrotic spot virus, or Groundnut ringspot virus. While detecting WSMoV-infected Nicotiana benthamiana plants with the monoclonal antibody, a growth and decline profile in the L protein expression level was observed. The RNPs were observed in electron microscope with the monoclonal antibody and protein A-gold labeled 2(superscript nd) antibody. The L protein monoclonal antibody developed in this study has high specificity and sensitivity, and will be a powerful tool for virus functional genomic study in reverse genetics system.

並列關鍵字

Watermelon silver mottle virus ； ribonucleoprotein complex ； bacterial expression system ； hybridoma ； protein A-gold labeling electron microscopy