臺灣博碩士論文加值系統

固定化金屬離子親和性層析法(Immobilized-Metal ion Affinity Chromatography,IMAC)，因具有單一步驟純化、再生容易優點，可做為大量純化蛋白質之方式。先前研究，以IMAC可直接純化未融合His-tag的VP2蛋白，發現位於P domain DE loop結構上的His 253胺基酸可與IMAC的鎳離子形成配位鍵結，做為病毒顆粒與IMAC管柱結合的重要位置。
本研究為尋找可取代His 253位置與固定化金屬離子結合的胺基酸位置，先利用軟體分析蛋白的暴露面積篩選，在His 253為丙胺酸的情形下，將其個別突變為組胺酸(Histidine)，經篩選後可Gln221、Ser222、Ser251、Ser317、Gln320及Ala321六個位置。分別以大腸桿菌表現系統與昆蟲桿狀病毒表現系統表現各突變株重組蛋白，並進行不同酸鹼值的緩衝溶液及不同濃度之咪唑沖提，結果顯示VP2-P domain 的Loop結構上人工His能取代His 253與鎳離子結合，且透過多種實驗觀察證實經突變後蛋白並不影響其構形、密度等物理特性。

The advantage of Immobilized-Metal ion Affinity Chromatography (IMAC) – one- step purification, recycling is easy, etc.- are decisive when developing large-scale purification procedures for industrial applications. Previous results indicated that SVPs can be purified directly by IMAC and the His 253 on DE loop of P domain plays an important role binding Ni2+ ions. Besides His 253, outward superficial residues on P domain of SVPs provide higher chances for Ni2+ ions binding.

In this study, to search the residues this can be substituted for the binding of immobilized Ni2+ ions. First, the calculation of exposure areas of residues on the loop was performed by Surface Racer 5.0 software. Six resides - Gln221, Ser222, Ser251, Ser317, Gln320 and Ala321-have been chosen, than been substituted with histidine. Recombinant proteins was expressed in Escherichia coli and Bac-to-Bac baculovirus expression system, and then purified by IMAC. The binding strength of these variants with immobilized Ni2+ ion was measured by quantitating the unbound VP2 protein after a series of wash with various pH and immidazole concentrations. The results have shown some residues on the loop structure of P domains of SVPs substituted and complement the mutation of H253A to bind Ni2+ ion, and don’t affect density and other physical characteristics.

目錄
摘要......................................................i
Abstract..................................................ii
目錄.....................................................iii
第一章 文獻回顧............................................1
一、固定化金屬離子親和性層析法...........................1
(一) 固定相基質........................................1
(二) 金屬螯合劑........................................1
(三) 金屬離子..........................................2
二、病毒與次病毒顆粒之純化...............................2
三、固定化金屬離子親和性層析法優點與應用.................2
四、傳染性華氏囊炎病毒...................................3
五、傳染性華氏囊炎病毒之基因組及其產物分析...............3
六、VP2次病毒顆粒與固定化金屬離子親和性層析結合特性分析..4
七、VP2次病毒顆粒之原核與真核表現系統....................5
八、巨分子與溶劑接觸面積計算概念及其應用.................5
九、定點突變技術在蛋白質工程上之應用.....................6
第二章 研究動機與目的......................................7
第三章 材料與方法..........................................8
一、利用蛋白結構分析軟體分析各胺基酸殘基在P3009 VP2蛋白的
暴露程度.............................................8
二、以大腸桿菌(Escherichia coli)表現系統生產重組蛋白.....8
(一) 重組質體與細菌菌株製備與保存......................8
1.大腸桿菌表現載體...................................8
2.大腸桿菌保存質體菌株TOP10F’.......................8
3.大腸桿菌表現菌株BL21(DE3)codon plus R.P............8
4.以PCR構築VP2突變蛋白的重組質體.....................9
5.勝任細胞的製備.....................................9
6.重組質體轉型入宿主細胞.............................9
7.抽取質體...........................................9
8.限制酶切割鑑定重組質體與DNA定序...................10
(二) 重組蛋白表現.....................................10
三、以昆蟲桿狀病毒表現系統(Insect cell/ Baculovirus
Expression System)生產蛋白..........................10
(一) 重組桿狀病毒之構築...............................10
1.昆蟲細胞 Sf-9.....................................10
2.桿狀病毒轉移質體(Baculovirus transfer vector)
pFastBacTM1.......................................11
3.重組病毒轉移質體之構築............................11
4.轉型DH10BacTME.coli以生產重組bacmid...............11
(二) 重組桿狀病毒 (Recombinant Baculovirus)之生產.....12
1.轉染(transfection)昆蟲細胞產生重組病毒............12
2.放大(amplify)重組病毒.............................12
3.以PCR方式鑑定重組病毒DNA..........................12
(三) 重組蛋白之表現...................................13
1.昆蟲細胞Hi-5......................................13
2.重組蛋白之表現....................................13
四、重組蛋白純化及分析前處理............................13
(一) 以固定化金屬離子親和性管柱純化重組蛋白...........13
(二) 蔗糖梯度離心分離法...............................14
(三) 重組蛋白分析前處理...............................14
1.以100 kDa 濃縮膜置換緩衝液及濃縮目標蛋白..........14
2.墊糖離心..........................................15
五、VP2蛋白多價抗體製備.................................15
六、重組蛋白分析與鑑定..................................15
(一) SDS-聚丙醯胺凝膠電泳(SDS-PAGE)及考馬斯亮藍
(Coomassie Blue)染色法...........................15
(二) 西方墨漬法(Western blotting).....................16
(三) 酵素連結免疫分析 (Enzyme-Linked Immunosorbent
Assay, ELISA)....................................16
(四) 高效能液相層析系統分析 ( High Performance Liquid
Chromatography,HPLC).............................17
七、以穿透式電子顯微鏡觀察(Transmission Electron
Microscopy).........................................17
八、利用咪唑進行重組蛋白與固定化金屬離子之脫附分析......17
第四章 結果...............................................19
一、P3009 VP2蛋白胺基酸側鏈之暴露面積計算...............19
二、大腸桿菌表現系統生產重組蛋白及純化分析..............19
(一) 點突變重組載體之構築.............................19
(二) 重組蛋白表現.....................................19
(三) 點突變蛋白與固定化金屬親和管柱吸附特性分析.......19
(四) 重組蛋白之高效能液相層析系統分析及穿透式電子顯微鏡
觀察.............................................20
(五) 重組蛋白之定量...................................20
三、昆蟲桿狀病毒表現系統生產突變蛋白及純化分析..........21
(一) 重組基因及重組桿狀病毒之構築.....................21
(二) 重組蛋白表現.....................................21
(三) 次病毒顆粒之固定化金屬親和管柱吸附分析...........21
(四) 重組蛋白之高效能液相層析系統分析及穿透式電子顯微鏡
觀察.............................................22
(五) 重組蛋白之定量...................................22
(六) 蔗糖梯度超高速離心分離次病毒顆粒.................22
(七) 酵素連結免疫分析超高速離心後之次病毒顆粒.........22
(八) 利用咪唑進行重組蛋白與固定化金屬離子之脫附分析...23
第五章 討論...............................................24
一、胺基酸突變位置與固定化鎳離子吸附特性關係............24
二、抗原決定位與固定化鎳離子吸附位置探討................24
圖表結果..................................................26
參考文獻..................................................48
附錄圖表..................................................54

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