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標題: 以轉基因菸草及病毒載體表現西瓜銀斑病毒非結構性NSs蛋白之單鏈抗體變異區
Expression of a single-chain variable fragment against the NSs protein of Watermelon silver mottle virus in tobacco plants by transgenic approach and Turnip mosaic virus vector
作者: 沈澤煌
Shen, Tze-Huang
關鍵字: Watermelon silver mottle virus
single-chain variable fragment
Turnip mosaic virus vector
出版社: 植物病理學系所
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摘要: 利用在轉基因植物或植物病毒載體於植物體中表現抗體或抗體的片段來直接對抗抗原的方式,能夠有效達到抵抗病原或調節植物代謝之目的。西瓜銀斑病毒(Watermelon silver mottle virus, WSMoV)屬於Bunyaviridae科、Tospovirus屬,在台灣造成西瓜、冬瓜、甜瓜和其他葫蘆科植物栽培之危害。番茄斑點萎凋病毒群(tospoviruses)的非結構性蛋白NSs與病徵嚴重性有關,為一個基因靜默(gene silencing)抑制子。本實驗室已於先前的研究中製備了對抗西瓜銀斑病毒NSs蛋白的單株抗體,並且證實這些單株抗體可辨識NSs蛋白N端第98至120個胺基酸的位置。此單株抗體辨識區域同時也存在於西瓜銀斑病毒血清群病毒的NSs蛋白中,這些病毒包括花生頂芽壞疽病毒(Peanut bud necrosis virus, PBNV)、甜椒黃化病毒(Capsicum chlorosis virus, CaCV)和海芋黃化斑點病毒(Calla lily chlorotic spot virus, CCSV)。這個高保留性區域可能在NSs蛋白功能上扮演重要角色。本研究乃自一個可分泌西瓜銀斑病毒NSs蛋白抗體的融合瘤細胞株239F1B9中選殖出單鏈抗體變異區(single-chain variable fragment, scFv)基因片段,並將此scFv基因片段構築在強、弱系蕪菁嵌紋病毒(Turnip mosaic virus, TuMV)病毒載體和二元載體pBI121上用以於植物體內表現之。利用對六個histidine胺基酸標誌的單株抗體可於感染強系重組病毒TYC5-WNSssf或弱系重組病毒THCIG5-WNSssf的奎藜和煙草上偵測到scFv蛋白的表現。此外,利用聚合酶連鎖反應(polymerase chain reaction, PCR)亦證實scFv基因已存在於轉殖菸草染色體中。而轉入有WNSs蛋白的轉基因植物在溫室下挑戰接種西瓜銀斑病毒後有些植株呈現延緩7到20天的病徵發展。
Antibodies or antibody fragments expressed by transgenes or viral vectors in plants can directly target crucial antigens and accumulate in the right cell compartments, they are able to effectively trigger pathogen resistance and to modulate plant metabolism. Watermelon silver mottle virus (WSMoV) is a member of the genus Tospovirus in the Family Bunyaviridae, causing severe damages for the production of watermelon, wax gourd, melon and other cucurbits in Taiwan. The NSs protein of tospoviruses is a gene-silencing suppressor and is a pathogencity determinant for the severity of symptoms. In our previous report, the monoclonal antibodies (MAbs) against the WSMoV NSs protein were produced and the recognition site of the MAbs was identified at the amino acid 98-120 of the N-terminal region of the WSMoV NSs protein. The MAbs-recognized region is also conserved among the NSs proteins of the WSMoV-serogroup tospoviruses, including WSMoV, Peanut bud necrosis virus (PBNV), Capsicum chlorosis virus (CaCV) and Calla lily chlorotic spot virus (CCSV), and it is considered to play an important role in functions of NSs protein. In this investigation, we cloned a single-chain variable fragment (scFv reading frame) of the MAb, produced from a hybridoma cell line 239F1B9, against the common epitope of WSMoV NSs protein. Subsequently, the scFv reading frame was constructed in the severe and mild strains of Turnip mosaic virus (TuMV) vectors and binary vector pBI121 of Agrobacterium for expression of scFv in Nicotiana benthamiana plants. Expression of the free-form scFv protein in the N. benthamiana and Chenopodium quinoa plants infected with the severe TuMV recombinant TYC5-WNSssf or the mild TuMV recombinant THCIG5-WNSssf was detected by western blotting using the monoclonal antibody against the hexa histidine tag. Moreover, the transgenic N. benthamiana plants carrying the scFv transgene were also generated and the presence of the scFv reading frame was confirmed by polymerase chain reaction (PCR). The WNSs scFv transgenic tobacco plants were challenged with WSMoV under greenhouse conditions and symptom development was significantly delayed for 7 to 20 days.
其他識別: U0005-2808200616093300
Appears in Collections:植物病理學系



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