Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36051
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dc.contributor.advisor王敏盈zh_TW
dc.contributor.advisorMin-Ying Wangen_US
dc.contributor.author陳宜暉zh_TW
dc.contributor.authorChen, Yi-Hueien_US
dc.contributor.other中興大學zh_TW
dc.date2007zh_TW
dc.date.accessioned2014-06-06T07:53:46Z-
dc.date.available2014-06-06T07:53:46Z-
dc.identifier.urihttp://hdl.handle.net/11455/36051-
dc.description.abstract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。zh_TW
dc.description.abstractIn 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.en_US
dc.description.tableofcontents中文摘要.................................................i 英文摘要...............................................ii 第一章、前言.............................................1 一、家禽傳染性華氏囊病病毒之背景介紹.....................1 二、傳染性華氏囊病病毒的基因體及其基因體產物分析.........2 三、傳染性華氏囊病病毒之似病毒顆粒組裝的研究現況.........3 四、傳染性華氏囊病毒與次病毒顆粒的立體結構...............4 五、固定化金屬離子親和層析於純化蛋白上的應用.............6 六、定點突變技術在蛋白質工程上的應用.....................8 七、研究動機及目的.......................................9 第二章、材料與方法.......................................11 一、 大腸桿菌表現系統 1. 質體與細菌菌株......................................11 2. 抗體的製備..........................................12 3. 重組質體之構築及製備................................13 4. 重組質體轉型及其勝任細胞之製備......................14 5. 可溶性重組蛋白之表現及取得..........................15 6. 重組蛋白之分析與鑑定................................15 7. 大腸桿菌表現重組蛋白純化及分析的前處理..............17 二、昆蟲桿狀病毒表現系統 1. 細胞與重組病毒之構築................................18 2. 昆蟲細胞株..........................................22 3. 重組蛋白之表現......................................23 4. 昆蟲細胞所表現重組蛋白之純化及鑑定..................24 三、穿透式電子顯微鏡觀察.................................25 四、 重組蛋白與固定化金屬吸附之分析......................26 第三章、結果.............................................27 1. VP2 441-H249.253A、H253A及H249A重組質體的構築......27 2. VP2 441-H249.253A、H253A及H249A重組蛋白於大腸桿菌表現系統的表現...............................................27 3. 大腸桿菌所表現之VP2 441-H249.253A、H253A及H249A重組點突變蛋白的純化處理.......................................28 4. 氯化銫密度梯度離心純化.............................29 5. 點突變蛋白之穿透式電子顯微鏡觀察...................30 6. 點突變似病毒顆粒與固定化金屬親合管柱吸附特性分析...30 7. H253A點突變蛋白利用昆蟲桿狀病毒系統的蛋白表現......31 8. 昆蟲桿狀病毒系統表現之VP2-441-H253A似病毒顆粒蛋白與固定化金屬親合管柱吸附特性分析.............................32 第四章、討論.............................................34 參考文獻.................................................39 圖與表...................................................45 附錄圖...................................................53zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物科技學研究所zh_TW
dc.subjectsite directed mutagenesisen_US
dc.subject點突變zh_TW
dc.subjectIMACen_US
dc.subjectsubviral particleen_US
dc.subject固定化金屬層析zh_TW
dc.subject似病毒顆粒zh_TW
dc.title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zh_TW
dc.titleInvestigation of the roles of His249&His253 on the affinity of Infectious Bursal Disease Virus Subviral particle and IMAC by Site Directed Mutagenesisen_US
dc.typeThesis and Dissertationzh_TW
item.languageiso639-1en_US-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.grantfulltextnone-
item.fulltextno fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
Appears in Collections:生物科技學研究所
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