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標題: 番茄斑點萎凋病毒及鳳仙花壞疽斑點病毒非結構 性 NSs 蛋白多元和單元抗體之製備及其應用
Preparation and Application of Polyclonal and Monoclonal Antibodies against the Nonstructural NSs Proteins of Tomato spotted wilt virus and Impatiens necrotic spot virus
作者: 蔡偉婷
Tsai, Wei-Ting
關鍵字: 番茄斑點萎凋病毒
Tomato spotted wilt virus (TSWV)
Impatiens necrotic spot virus (INSV)
nonstructural NSs protein
polyclonal antibody (PAb)
monoclonal antibody (MAb)
出版社: 植物病理學系所
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摘要: 依照病毒核鞘蛋白 (nucleocapsid protein, NP) 及地緣分佈關係,可將番茄斑點萎凋病毒 (Tomato spotted wilt virus, TSWV) 血清群病毒和鳳仙花壞疽斑點病毒 (Impatiens necrotic spot virus, INSV) 獨立血清型歸類為歐美型番茄斑萎病毒屬病毒 (Euro-America type tospoviruses)。及時有效又正確的檢測系統有助台灣防堵歐美型病毒的入侵或進一步擴散。因此,本研究擬發展精確且快速偵測歐美型病毒之多元抗體 (抗血清) 及單元抗體,其中又以歐美型病毒中兩種會造成嚴重經濟損害的典型病毒 INSV 和 TSWV 主要為目標。由於此屬病毒的非結構性 NSs 蛋白 (Nonstructural protein, NSs) 為基因沉寂抑制子 (gene silencing suppressor) ,幫助病毒抵抗寄主抗病反應,並在病毒感染植物內會大量聚集成內含體。因此本研究以細菌表現系統來表現 INSV 和 TSWV 的 NSs 蛋白,蛋白經純化後用以生產抗體。 INSV NSs (INSs) 老鼠抗血清中 (antiserum) 的多元抗體 (polyclonal antibody) 不僅偵測到INSV 病毒,更可同時檢測到TSWV 血清群中的 TSWV 、花生輪斑病毒 (Groundnut ringspot virus, GRSV) 和番茄黃化斑點病毒 (Tomato chlorotic spot virus, TCSV) 病毒。另外 TSWV NSs (TNSs) 老鼠抗血清中的多元抗體也可同時偵測到此三種 TSWV 血清群病毒及 INSV 病毒。經融合瘤細胞篩選過程,挑 選能產生高度辨識 INSV 或 TSWV 抗體之融合瘤細胞株系,用以生產腹水。細胞株 3E12D4 產生之腹水MAb-INSs-3E12D4 只專一性辨認 INSV,力價達 2.05 × 106 倍;而細胞株 81D9E2 產生之腹水 MAb-TNSs-81D9E2 只專一性辨識TSWV ,力價為 5.12 × 105 倍。另外,細胞株 55G4E7 所產生的單元抗體 MAb-INSs-55G4E7 ,可同時與 INSV、 GRSV 和 TCSV 反應,其力價為 5.12 × 105 倍。上述結果顯示,利用 INSs 及 TNSs 蛋白作為抗原所生產出來的多元和單 元抗體,可有效作為廣泛偵測歐美型病毒及單獨檢測 INSV 或 TSWV 的工具,將有助於番茄斑點萎凋病毒屬檢測工作及 NSs 蛋白功能之研究。結合 MAb-INSs-55G4E7 和 MAb-TNSs-81D9E2,做成二合一抗體套組,可專一性辨認歐美型番茄斑萎病毒屬病毒,區別亞洲型番茄斑萎病毒屬病毒 (Asia type tospoviruses) 及其他病毒,為防檢疫上防止歐美型番茄斑萎病毒屬病毒入侵及監控歐美型番茄斑萎病毒屬病毒在台灣分佈情形之利器。此外,大部分歐美型和亞洲型番茄斑萎病毒屬病毒可被三合一單元抗體套組 (MAb-INSs-55G4E7、 MAb-TNSs-81D9E2 及本研究室先前生產的專一性辨識亞洲型番茄斑萎病毒屬病毒的單元抗體MAb-WNSs-231E6D12) 所辨識,此組合加速田間檢測效率及精確度。本研究生產之血清學診斷鑑定利器,配合本研究團隊針對番茄斑萎病毒屬病毒設計的簡併式引子對,可建立起及時有效又正確的番茄斑萎病毒屬病毒檢測系統。
Members of the Tomato spotted wilt virus (TSWV) serogroup and Impatiens necrotic spot virus (INSV) serotype are regarded as Euro-America type tospoviruses according to their nucleocapsid protein (NP) and geographical distribution. Accurate and prompt detection for members of Euro-America type tospoviruses is crucial for effective quarantine to prevent possible invasion or limit further spreading of these viruses in Taiwan. In this investigation, we attempted to develop polyclonal antibody (antisera) and monoclonal antibodies (MAbs) to INSV and TSWV, two representative and economically important Euro-America type tospoviruses, as effective and accurate detection tools. The tospovirus-encoded nonstructural NSs protein, which accumulates abundantly in infected cells, is the gene silencing suppressor to protect the virus from host gene silencing in plants. In this investigation, the NSs proteins of INSV and TSWV were individually expressed in the bacterial system and the purified proteins were used for producing of antibodies from mouse. The mouse antisera produced against the purified recombinant INSV NSs (INSs) proteins reacted strongly with the crude antigen of INSV and also cross-reacted with three members of the TSWV serogroup tospoviruses, TSWV, Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV), in indirect ELISA and western blot analysis. Similarly, the anti-TSWV NSs (TNSs) antisera reacted with the crude antigens from the three analysed members of TSWV serogroup and INSV serotype. In addition, MAbs against purified NSs proteins were generated by hybridoma technique. The anti-INSs hybridoma line 3E12D4 generated MAb-INSs-3E12D4 that reacted specifically with INSV NSs protein, with a titer (ascitic fluid) up to 2.05 106 X. The anti-TNSs hybridoma line 81D9E2 generated MAb-TNSs-81D9E2 that reacted specifically with the NSs protein of TSWV, with titers (ascitic fluids) up to 5.12 105 X. Moreover, the MAb-INSs-55G4E7 of anti-INSs hybridoma line 55G4E7 reacted with INSV NSs protein and with the NSs proteins of other two viruses from TSWV serogroup, GRSV and TCSV , with a titer (ascitic fluid) up to 5.12 105 X. Hence, the presently produced antisera and MAbs to INSV NSs and TSWV NSs proteins can be used to study the roles of muti-funtion NSs proteins and also as effective tools not only for the specific detection of INSV and TSWV but also for the detection of Euro-America type tospoviruses. In addition, our resilts indicated that the combination of MAb-INSs-55G4E7 and MAb-TNSs-81D9E2 can be used as a two-in-one kit to distinguish the Euro-America type tospoviruses from Asia type tospoviruses, and the three-in-one kit (e.g. MAb-INSs-55G4E7, MAb-TNSs-81D9E2 and the previously developed MAb-WNSs-231E6D12) can be used as a accurate and prompt detection tool for the detection of both Euro-America type and Asia type tospoviruses. A fast and accurate detection method can be established by the cooperation of the specific degenerate primers privously developed in our group with the MAb kits generated in this study.
其他識別: U0005-2806201009584100
Appears in Collections:植物病理學系



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