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標題: 傳染性華氏囊炎病毒次病毒顆粒表面胺基酸的替換對其與鎳離子間吸附作用之研究
The Correlation between the Substitution of Superficial Residues on Subviral Particles of the Infectious Bursal Disease Virus and their Interactions with Nickel Ions
作者: 余仁豪
Yu, Ren-Hao
關鍵字: Infectious Bursal Disease Virus;傳染性華氏囊炎病毒;Homology Modeling;IMAC;同源模擬法;固定化金屬親和性層析分離法
出版社: 生物科技學研究所
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傳染性華氏囊炎病毒(Infectious Bursal Disease Virus, IBDV) 的VP2蛋白為病毒主要外鞘結構蛋白,其單獨表現可自行組裝成T=1正二十面體之次病毒顆粒(Subviral Particle, SVP)。VP2 單體(Monomer) 蛋白結構主要區分為三個domains,分別為Protrusion(P) 、Shell(S) 和Base (B) domain。其中P domain 位於次病毒顆粒的表面,由胺基酸202~341序列範圍組成loop以及β-sheet 結構。
本研究利用同源模擬法(Homology Modeling) 以台灣傳統株P3009 VP2 模擬出伊朗株IR01 VP2 蛋白結構,以Surfacr Racer 5.0及Discovery Studio 2.5 結構分析軟體計算P3009 VP2 及IR01 VP2 P domain 胺基酸暴露面積及暴露程度。由本實驗室先前研究得知,VP2 P domain上DE loop的His253位於SVP結構的表面與鎳離子接觸而達到吸附作用,P3009 VP2 、IR01 VP2以點突變技術將特定位置胺基酸置換成組胺酸,經蔗糖梯度離心分離結果與野生株之次病毒顆粒相同,接續以穿透式電子顯微鏡分析確定與野生株有相似粒徑之次病毒顆粒,利用固定化金屬親和性管柱測試突變組是否同樣產生吸附作用,結果顯示在P3009 VP2 P domain之β-sheet 結構的D279H、Q324H 位置可提供與鎳離子進行吸附作用;而IR01 VP2的S251H、G285H 及Q324H位置皆與鎳離子進行吸附作用。總結本研究,P3009 VP2位於β-sheet 結構的D279H、Q324H 能與IMAC間具有吸附力,以及IR01 VP2純化結果證明利用同源模擬法所得之IR01次病毒顆粒model之可信度。

VP2 is the major viral capsid protein of infectious bursal disease virus (IBDV), a causative agent of a highly contagious immunosuppressive disease in young chickens. In vivo expression of VP2 can cause the formation of icosahedral (T=1) subviral particles (SVPs). Monomeric VP2 protein contains three domains, ie., protrusion (P), shell(S) and base (B) domain. Outward P domain (aa.202~341) is to compose the loop and β-sheet.
In this study, to simulate the protein structure of iran strain IR01 VP2 base on local strain P3009 VP2 by using homology modeling, then employed Surface Racer 5.0 and Discovery Studio 2.5 to calculate the exposure area and percent solvent accessibility of side chains of P3009 VP2 and IR01 VP2, respectively. Previous results demonstrated that SVPs can bind to Ni ion by His253 on the DE loop of VP2 P domain of SVP, the specific amino acid were substituted with histidine via site-directed mutagenesis to generate few variants. P3009 VP2, IR01 VP2 mutants were separated by sucrose gradient centrifugation. In the results, themutants have same distribution campare with the WT SVP, the follow-up transmission electron microscopy identified P3009 and IR01 VP2 particles structures have similar diameter with wild type SVP . To test the adsorption of each mutants by immobilized metal affinity chromatography, the results showed the D279H, Q324H on the β-sheet structure of P domain of P3009 VP2 provide the adsorptive to Ni ion and S251H, G285H and Q324H of IR01 VP2 were carried out with the nickel ion adsorption, respectively. Conclusion of this study, the D279H, Q324H in the β-sheet structure of P3009 VP2 provide the adsorption with IMAC and adsorption results of IR01 VP2 showed that credibility of IR01 subvirus particle model from homology modeling.
其他識別: U0005-2008201012461300
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