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研究生(外文):Sheng-che Ding
論文名稱(外文):The contribution of non-native structure with recombinant cobrotoxin to its immunoreactivity toward anti-cobrotoxin antibodies
指導教授(外文):Long-Sen Chang
外文關鍵詞:Anti-cobrotoxin antibodiesELISAConformation-dependentCobrotoxinbinding affinity
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在生物體內,外生性抗原被降解並帶到抗原呈現細胞表面以誘導產生特異性抗體,在這過程中,抗原可能因結構變化而導致抗原決定部位受到遮蔽使抗體無法與原生性蛋白有良好反應。 先前關於重組臺灣眼鏡蛇毒蛋白Cobrotoxin與原生性Cobrotoxin之抗原性比較已有深入報導,本論文目標在於探討是否存在某些非原生性Cobrotoxin之結構,暴露原生性毒蛋白所隱蔽之抗原決定部位而提升對抗體反應性。 首先以圓二色偏極光譜及Acrylamide淬熄Trp螢光分析得知重組與原生性Cobrotoxin二級結構之差異。 在ELISA及dot blotting實驗中發現重組Cobrotoxin對抗體之反應性明顯優於原生性毒蛋白,以特異性認氮端及碳端之抗體分析也是同樣結果,推測因結構差異導致,而以Ammonium thiocyanate elution分析發現重組Cobrotoxin對抗體親和力也有所提升。 在Trypsin及Chymotrypsin digestion實驗中得知重組Cobrotoxin較原生性毒蛋白易受到酵素水解,推測其對抗體之表達位向有所差異。 當Cobrotoxin碳端兩對雙硫鍵Cys43-54及Cys55-60突變後發現其抗原性有顯著衰退現象,且當重組及原生性毒蛋白回到線性狀態時抗原性則沒有差異,顯示抗體主要辨認部位具結構依賴性。 綜合上述結果得知原生態Cobrotoxin之結構可能隱蔽了某些抗原決定部位而使其對抗體反應性並非最佳狀態。
To induce the production of antibodies, exogenous antigens are taken up and degraded in antigen presenting cells in vivo. Since this process inevitably lead to distort antigen’s structure, it is likely that some arising antibodies following immunization may not react appropriately with native protein. In the present study, comparative studies on the reactivity of cobrotoxin and recombinant cobrotoxin toward anti-cobrotoxin antibodies were carried out. CD spectra and acrylamide quenching of Trp fluorescence showed that global structure of recombinant cobrotoxin was different from that of native toxin. Results of ELISA and dot blotting assay revealed that recombinant cobrotoxin had a superior reactivity toward anti-cobrotoxin antibodies than native toxin did. Reactivity with antibody fractions specifically against N-terminal region or C-terminal region of cobrotoxin also showed the same results. The binding of recombinant cobrotoxin with antibodies was stronger than that of cobrotoxin as revealed by ammonium thiocyanate elution assay. Recombinant protein was susceptible to reduce its antigenicity after tryptic digestion compared to cobrotoxin. Distorting disulfide linkages at C-terminus caused a marked decrease in immunoreactivity of recombinant cobrotoxin, indicating that anti-cobrotoxin antibodies mostly recognized conformation-dependent epitopes. Moreover, cobrotoxin and recombinant cobrotoxin showed a similar immunoreactivity under denaturing condition. Taken together, these results suggest that native conformation with cobrotoxin may unfavorably impede the interaction of some epitope(s) with anti-cobrotoxin antibodies.
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