Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31031
標題: 利用與基因沉寂抑制子共同抗原決定基結合之單鏈抗體建構對蕃茄斑萎病毒群之轉基因抗性
Transgenic resistance against tospoviruses conferred by a single-chain antibody against the common epitope of the gene silencing suppressor
作者: 莫雅莉
Maulidiyah, Alik
關鍵字: 轉基因植物
Transgenic plant
單鏈抗體變異區
西瓜銀斑病毒
非結構性蛋白
single-chain variable fragment (scFv)
Watermelon silver mottle virus (WSMoV)
non-structural protein
出版社: 植物病理學系所
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摘要: 利用轉基因植物表現抗體使植物避免病毒感染是一種抗病毒之可行策略。本研究室先前研發出西瓜銀斑病毒 (Watermelon silver mottle virus, WSMoV) 基因沉寂抑制子 (gene silencing suppressor, NSs protein) 之單株抗體 (monoclonal antibodies, MAbs),此抗體可以辨認WSMoV 血清群 NSs 之高保留區抗原決定基 (common epitope, WNScon)。本研究將此單株抗體融合瘤細胞株239F1B9之單鏈抗體變異區 (single-chain variable fragment, scfv) 構築於載體中,並以農桿菌為媒介進行菸草轉殖。首先利用 scFv 專一性引子確認30個擬轉基因菸草中可增幅出0.74 kb的專一性片段。經 WSMoV 接種後,結果有一個品系不發病被歸類為免疫抗性,四個為品系高度抗性,兩個為中度抗性及一個為微度抗性。以 WSMoV 血清群病毒進行廣泛抗病毒能力分析,結果顯示轉基因菸草對彩色海芋黃化斑點病毒 (Calla lily chlorotic spot virus)、番椒黃化病毒 (Capsicum chlorosis virus)、花生頂芽壞疽病毒 (Peanut bud necrosis virus)、西瓜頂芽壞疽病毒 (Watermelon bud necrosis virus) 具有延遲性抗性,但對同一血清群關係較疏遠的甜瓜黃斑病毒 (Melon yallow spot virus) 則不具抗性。北方及西方轉漬法結果顯示一個免疫品系、三個高抗及一個中抗品系具有較高量的scFv 轉錄體及蛋白累積,此表示 scFv 轉基因植物之抗性與其轉錄體及蛋白表現呈正相關性。利用純化的NSs-HA蛋白當作探針,以蛋白-蛋白套疊法分析顯示 scFv 與 NSs 具結合能力,因此scFv轉基因植物對WSMoV具有抗性應該是藉由scFv 與 NSs之結合產生的蛋白主導的抗病性狀。
Expression of antibodies in transgenic plants is a possible strategy to prevent plant viral infection. The monoclonal antibodies (MAbs) against a common epitope WNSscon (amino acid 98-120) of the gene silencing suppressors (NSs proteins) of Watermelon silver mottle virus (WSMoV) serogroup tospoviruses were previously produced from our laboratory. In this study, a clone encoding a single-chain variable fragment (scFv) against a common epitope WNSscon was generated from a hybridoma cell line 239F1B9 by polymerase chain reaction (PCR) and used for Agrobacterium-mediated transformation of Nicotiana benthamiana plants. Using scFv-specific primers, a DNA fragment of 0.74 kb was amplified from 30 putative transgenic tobacco lines by PCR. The R0 scFv transgenic lines challenged inoculated with WSMoV showed various degrees of resistance: one line immune, four lines resistant, two lines moderately resistant, and one line weakly resistant to WSMoV. Evaluation of their broad-spectrum resistance against other WSMoV serogroup tospoviruses showed delayed-type resistance to Calla lily chlorotic spot virus (CCSV), Capsicum chlorosis virus (CaCV), Peanut bud necrosis virus (PBNV), and Watermelon bud necrosis virus (WBNV), but not obvious resistance to Melon yellow spot virus (MYSV). Northern hybridization revealed that one immune line, three resistant lines and one moderately resistant line accumulated detectable levels of scFv transcript. Moreover, immunoblot analysis demonstrated that the one immune line, three resistant lines and one moderately resistant line expressed high levels of scFv protein. Our results indicated that the expression levels of scFv transcript and protein are correlated to the levels of transgenic resistance. The protein-protein overlay assay using purified NSs-HA protein as a probe indicated the binding activity of expressed scFv and NSs protein.We conclude that the resistance to the WSMoV infection is resulted from the binding activity of the scFv and NSs protein.
URI: http://hdl.handle.net/11455/31031
其他識別: U0005-2107201118394600
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2107201118394600
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