Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31647
標題: 利用矮南瓜黃化嵌紋病毒載體表現西瓜和花生頂芽壞疽病毒之核鞘蛋白及單株抗體之製備
Production of monoclonal antibodies against Zucchini yellow mosaic virus -expressed nucleocapsid proteins of Watermelon bud necrosis virus and Peanut bud necrosis virus
作者: 林貞璇
Lin, Chen-Hsuan
關鍵字: ArrayBunyaviridae nucleocapsid protein monoclonal antibodies
核鞘蛋白
西瓜銀斑病毒
血清群
單株抗體
出版社: 植物病理學系
摘要: 番茄斑萎病毒屬 (Tospovirus) 是Bunyaviridae科中唯一感染植物之一屬,為具有三條單股線狀負極性或雙極性的RNA分子和脂質蛋白套膜 (lipoprotein envelope) 之球型病毒。依其寄主範圍、媒介薊馬種類及核鞘蛋白 (nucleocapsid protein, NP) 序列相似度等分為17個病毒種 (species)。根據核鞘蛋白血清學上的親緣關係又可分為三個血清群 (serogroup) 和四個血清型 (serotype)。而於台灣所分離到的西瓜銀斑病毒 (Watermelon silver mottle virus, WSMoV) 即為番茄斑萎病毒屬之一員,與分離自印度的花生頂芽壞疽病毒 (Peanut bud necrosis virus, PBNV) 和西瓜頂芽壞疽病毒 (Watermelon bud necrosis virus, WBNV)、澳洲的辣椒黃化病毒 (Capsicum chlorosis virus, CaCV)、美國的大岩桐病毒 (high temperature-recovered gloxinia tospovirus ) 及台灣新發現的海芋黃化斑點病毒 (Calla lily chlorotic spot virus, CCSV) 同屬於WSMoV血清群。由於此類病毒極不穩定,難以傳統方式純化,故一般多藉由細菌表現系統來生產並純化之。本實驗乃利用研究室所開發之矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus, ZYMV) 載體系統表現兩個外來病毒的核鞘蛋白做為製備多元與單株抗體之用。先將PBNV和WBNV的核鞘蛋白轉譯架構 (open reading frame) 各自插入ZYMV載體之P1和HC-Pro基因之間,並於感染之系統性寄主矮南瓜上表現蛋白,利用層差離心與膠體電泳方法於100克的感病葉片中純化出1.25 ~ 1.5 mg的WBNV核鞘蛋白或0.1~0.2 mg的PBNV核鞘蛋白。由二者純化的蛋白所製備之抗體皆可與各自的抗原反應良好。其中,WBNV核鞘蛋白的單株抗體可與WSMoV和HT-1反應良好,但不與CCSV及PBNV的核鞘蛋白反應。而PBNV核鞘蛋白的單株抗體除了與CCSV反應較微弱外,跟上述其他病毒的核鞘蛋白皆有反應。由實驗結果可知,利用ZYMV病毒載體表現系統配合簡單的純化程序,可獲得高產量且穩定的番茄斑萎病毒屬之核鞘蛋白,所製備的WBNV核鞘蛋白單株抗體更可用於區別PBNV和WBNV,解決在印度此二病毒混淆的問題。
Tospovirus, the only plant-infecting genus in the family Bunyaviridae, is characterized by quasi-spherical enveloped particles containing three single-stranded negative or ambisense RNA segments. Seventeen distinct species are established by characteristics such as the host range, vector specificity and amino acid identity of nucleocapsid protein (NP). Based on the serological relationships of NPs, tospoviruses are classified into three serogroups and four distinct serotypes. Watermelon silver mottle virus (WSMoV) and Calla lily chlorotic spot virus (CCSV) from Taiwan, Peanut bud necrosis virus (PBNV) and Watermelon bud necrosis virus (WBNV) from India, Capsicum chlorosis virus (CaCV) from Australia, and high temperature-recovered gloxinia tospovirus (HT-1) from the United State were clustered in the same WSMoV serogroup. Due to the instability of virus particles, it is difficult to purify viral proteins from infected plants. In many previous reports, tospoviral proteins were frequently expressed by the bacterial expression systems for purification. In this study, two tospoviral NPs were expressed by the engineered Zucchini yellow mosaic virus (ZYMV) vector in our laboratory for the production of polyclonal antibody (PAb) and monoclonal antibodies (MAbs). The NP ORFs of PBNV and WBNV were individually introduced in between the P1 and HC-Pro genes of the ZYMV vector for protein expression in the infected squash plants. The infected squash leaves were harvested for purification of the expressed NPs by a method involving differential centrifugation and slab gel electrophoresis. The yields of the purified NPs of WBNV and PBNV from 100 g infected tissues were approximately 1.25 ~ 2.5 mg and 0.1~0.2 mg, respectively. PAbs and MAbs produced to the purified ZWBNV NP and MAb produced to ZPBNV NP strongly with the homologous antigens. In addition, MAbs to WBNV NP strongly reacted with the NPs of WSMoV and HT-1, but did not react with those of PBNV and CCSV. The MAb to PBNV NP and the aniserum to WBNV NP strong cross-reacted with the NPs of the WSMoV, HT-1 and WBNV, and cross-reacted weakly reacted with that of CCSV in immunoblotting. Our results demonstrate that high amounts of stable tospoviral NPs can be expressed by the ZYMV expression system and purified by simple procedures. Moreover, the produced WBNV NP MAb is a unique tool to distinguish WBNV from PBNV, both originated in India.
URI: http://hdl.handle.net/11455/31647
Appears in Collections:植物病理學系

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