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標題: 利用點突變探討傳染性華氏囊病病毒VP2蛋白中His249與His253對於IBDV次病毒顆粒與固定化金屬親和性管柱親和力之重要性
Investigation of the roles of His249&His253 on the affinity of Infectious Bursal Disease Virus Subviral particle and IMAC by Site Directed Mutagenesis
作者: 陳宜暉
Chen, Yi-Huei
關鍵字: site directed mutagenesis;點突變;IMAC;subviral particle;固定化金屬層析;似病毒顆粒
出版社: 生物科技學研究所
本實驗室先前的研究中証實利用固定化金屬親合性管柱(Immobilized Metal ion Affinity Chormatography column, IMAC)不但能純化融合His-tag的雞傳染性華氏囊病(Infectious Bursal Disease Virus , IBDV)VP2-452H蛋白所自行組裝的次病毒顆粒 (Sub-Viral Particle , SVP),亦能純化未融合His-tag的IBDV病毒與VP2-441次病毒顆粒。因此推論提供純化的吸附力並非是VP2蛋白所融合的His-tag,而是因為SVP本身表面的胺基酸提供與固定化鎳離子吸附的親和力,但其確切胺基酸位置尚未得知。本研究利用基因工程的方式置換SVP表面可能跟IMAC產生吸附的特定胺基酸His-249或His-253,並以大腸桿菌表現此VP2-441-H249.253A 、VP2-441-H253A及VP2-441-H249A重組蛋白,以及利用昆蟲細胞-桿狀病毒系統表現VP2-441-H253A重組蛋白,並經由破菌、酸沉澱、硫酸銨沉澱、透析及超高速離心純化後,並藉由穿透式電子顯微鏡觀察有粒徑為20~23 nm的次病毒顆粒。而上述兩系統表現後可自行組裝為似病毒顆粒之H249.253A、 H253A及H249A重組蛋白,經IMAC之純化,結果顯示蛋白主要於flow through與pH 7.8 binding buffer緩衝液下沖提出,證實具有His-249與His-253突變之次病毒顆粒喪失與IMAC間的吸附能力。因此大腸桿菌與昆蟲細胞-桿狀病毒系統表現的VP2-441蛋白,於蛋白組裝成似病毒顆粒後,蛋白胺基酸序列中的His-249與His-253控制了次病毒顆粒是否能與固定化鎳離子親和性鍵結的獨特吸附現象。

In previous research, it demonstrated that Immobilized Metal ion Affinity Chormatography column(IMAC)not only can purify the His-tag fusion IBDV VP2-452H subviral particle (SVP), but also can purify the IBDV VP2-441 subviral particle without his-tag fusion. Therefore, the VP2 protein absorption with IMAC is not depending on the His-tag. It was suggested that SVP purified by IMAC was due to its surface amino acid residues of VP2. Two histidine residues which were located on the most exposed loops (Loop DE) may engage directly in contact with the immobilized Ni2+ ions.
In this work, we engineered SVP surface amino acid, His-249 or His-253, which is related with IMAC absorption, and produced three mutants, VP2-441-H249.253A, VP2-441-H253A and VP2-441-H249A. Mutants VP2-441-H249.253A and VP2-441-H249A were expressed in E.coli and VP2-441-H253A was expressed in both E.coli and baculovirus expression system. After purification by ultracentrifugation, the VP2 SVP assembled as 20~23 nm particles which can be visualized under Transmission electromicroscopy (TEM). The results show that histidine mutation will not affect the self assembly of SVP. However, the three SVP mutants are present in the flow-through and pH 7.8 binding buffer eluent after IMAC purification. It indicates that these three mutants had lost the absorption ability with IMAC and provides evidence that the His 249 and His253 of VP2 play an important role in the binding affinity of SVP with Ni2+ ion of IMAC.
Appears in Collections:生物科技學研究所

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