- 學位論文
利用點突變探討傳染性雞華氏囊病毒 VP2蛋白之Cys99與Cys197對於形成次病毒顆粒之影響
Study of the Role of Cys99 and Cys197 on the Formation of VP2 Subviral Particle of Infectious Bursal Disease Virus by Site-directed Mutagenesis
摘要
摘要 傳染性雞華氏囊病病毒 (Infectious bursal disease virus, IBDV)結構蛋白 VP2的胺基酸序列僅含有兩個Cysteine基團，分別是位於SD domain的Cys-99與 PA’ domain的 Cys-197。本研究以大腸桿菌表現系統與昆蟲細胞桿狀病毒表現系統生產重組點突變 Cysteine的 VP2蛋白，探討 Cysteine在VP2蛋白中的重要性。大腸桿菌所表現的蛋白有VP2-C99A、VP2-C197A、VP2-C197T、VP2-C197S、VP2-C99.197A；昆蟲細胞表現系統則表現 VP2-C99A及VP2-C197A蛋白。利用純化次病毒顆粒系統-固定化金屬離子親和性層析法 (Immobilized metal-ion affinity chromatography, IMAC)進行蛋白質純化，結果顯示大腸桿菌或昆蟲細胞桿狀病毒所表現的 VP2-C99A點突變蛋白仍會形成次病毒顆粒並可被 IMAC純化，電子顯微鏡下觀察出粒徑約 20- 25 nm。除了VP2-C99A蛋白，其它由大腸桿菌所生產之點突變蛋白皆喪失與 IMAC親和性吸附力。昆蟲細胞所表現之 VP2-C197A突變蛋白，表現量下降，大部份蛋白於 Flow-through及 pH 7.8 binding buffer緩衝溶液被沖堤下來，此突變蛋白對 Ni-NTA的親和鍵結能力變弱，電子顯微鏡下觀察具有少量似膨脹且不完整的次病毒顆粒構型。實驗結果證實 VP2蛋白胺基酸序列中的 Cys-197為 VP2蛋白組裝成次病毒顆粒的重要胺基酸。
並列摘要
Abstract VP2 is a major structural protein of Infectious bursal disease virus (IBDV), which contains only two Cysteine residues, Cys-99 and Cys-197, located at SD domain and PA’ domain, respectively. In this study, VP2 was engineered by site-directed mutagenesis to investigate the role of Cysteines on the formation of VP2 subviral particle (SVP). Mutants VP2-C99A, VP2-C197A, VP2-C197T, VP2-C197S and VP2-C99.197A were expressed in E. coli and VP2-C99A and VP2-C197A were also expressed in insect cell/ baculovirus system. VP2-C99A expressed in both systems remains its ability of SVP formation and can be purified by immobilized metal-ion affinity chromatography (IMAC), with a particle size of 20-25 nm. The other mutants expressed in E. coli lost their affinity to Ni-NTA resin, indicating that they do not exist in SVP form. Most of the insect cell-derived VP2-C197A was present in the flow-through and pH 7.8 binding buffer eluant, suggesting that they have lost the absorption ability with Ni-NTA. The small amount of VP2-C197A purified by IMAC exhibits swelling and incomplete particle form observed under electron microscope (EM). Our results indicate that Cys-197 is an essential and important site for the self-assembly of VP2 into subviral particle.