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研究生:楊函蓁
研究生(外文):Han-Jen Yang
論文名稱:研究傳染性華氏囊病毒次病毒顆粒VP2蛋白表面胺基酸His249與His253對鎳離子之親合性及免疫原性的影響
論文名稱(外文):Investigation of the roles of two surface residues His249&His253 on the affinity to the immobilized Ni2+ ions and immunogenicity of infectious bursal disease virus VP2 subviral particles
指導教授:王敏盈
指導教授(外文):Min-Ying Wang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:58
中文關鍵詞:傳染性華氏囊病毒鎳離子免疫原性
外文關鍵詞:infectious bursal disease virusNi ionsimmunogenicity
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  • 被引用被引用:1
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本實驗室先前研究中證實利用固定化金屬親合性管柱 (Immobilized metal ion affinity chromatography, IMAC) 可以純化未融合His-tag的雞傳染性華氏囊病病毒 (Infectious Bursal Disease Virus, IBDV),以及其結構蛋白VP2所組裝的次病毒顆粒 (Subviral Particle, SVP),且經由結構推測可能是次病毒顆粒表面的胺基酸提供與鎳離子吸附的親和力。此外先前的研究亦指出IBDV VP2胺基酸位置206到350之間包含病毒的中和性抗原決定區,因此任一胺基酸變動都可能導致病毒的抗原變異,並有其他相關研究指出胺基酸253, 279, 284可調控病毒的致病力及進入細胞的機轉。
本實驗首先利用昆蟲細胞表現系統生產點突變VP2-441-H249.253A、 VP2-441-H253A及VP2-441-H249A 蛋白做為研究材料,經由蔗糖超高速離心純化後,藉由穿透式電子顯微鏡觀察次病毒顆粒。並以不同酸鹼值 (power of hydrogen, pH) 的緩衝溶液和不同濃度咪唑 (Imidazole) 進行沖提並藉由分子量篩選的高效能液相層析系統 (High Performance Liquid Chromatography, HPLC) 分析,經由上述實驗證實SVP表面的His253是和Ni-NTA吸附主要的胺基酸。之後進行等溫平衡吸附實驗得到每毫升Ni-NTA可以吸附的蛋白分子莫耳數 (Q),可得到VP2-441與Ni-NTA吸附達飽和之值 (Q) 為7.9 x 10-10 moles/ml Ni-NTA;H249A和H253A之Q值分別為7.3 x 10-10 and 3.5 x 10-10 moles/ml Ni-NTA,但H249.253A因為喪失了與鎳離子的吸附能力並無飽和值,所以取各組之Q值平均可得到2.6 x 10-11 moles/ml Ni-NTA。更進一步研究 H249 和 H253 是否會影響免疫原性,經由單株抗體 MAb SVP-1 和 MAb SVP-4 辨認 wild type VP2-441 SVP 以及突變 His249 與 His253 形成之 SVP,發現突變株均比 wild type VP2-441 SVP 與 mAb1 和 mAb4 結合效率要高,且經由雞隻免疫實驗結果發現VP2-441-H253A產生中和性抗體與VP2-specific IgG 力價均較 wild type VP2-441略為上升,因此推測 His253 可能位於影響IBDV免疫原性之位置。
In previous studies, we have confirmed that both of infectious bursal disease virus (IBDV) virions and the subviral particles (SVPs) composed of IBDV VP2 protein can be purified by immobilized metal ion affinity chromatography (IMAC). This suggests some amino acid residues on the surface of IBDV virions mediate the interaction with Ni2+ metal ions. Earlier reports indicate the region from residue 206 to 350 in VP2 protein contain the major immunogenic determinants. Therefore, any mutations on this region may result in IBDV antigenic variation. Furthermore, other resides include 253, 279, and 284 may determine the viral pathogenicity or play a key role on viral entry.
In this study, we focus on two histidine residues (His249 and His253) and investigate its importance on the interaction with Ni2+ ion and the effect on the immunogenicity. For these purposes, three VP2-441 variants, H249A, H253A, and H249.253A, were constructed and expressed by baculovirus expression system. The particle morphology of these variants examined by transmission electron microscopy (TEM) was similar to the authentic VP2-441 SVPs, exhibiting that the mutation from histidine to alanine doesn’t affect the particle self-assembly. 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 imidazole concentrations. The results demonstrated that the His253 on the particle surface was the major amino acid contributes to the interaction with Ni2+ ion. Afterward, the adsorption of VP2-441 SVP to Ni2+-NTA was increased with the concentration of free VP2. The bound protein per volume of resin (Q) for VP2-441 SVP is 7.9 x 10-10 moles/ml Ni-NTA. The saturated Q of H249A and H253A reached to 7.3 x 10-10 and 3.5 x 10-10 moles/ml Ni-NTA, respectively. The equilibrium adsorption isoterm of H249.253A SVP to Ni-NTA had no accordance to the concentration of SVP and the average Q value was 2.6 x 10-11 moles/ml Ni-NTA, which is the lowest among the four SVPs.
For investigating the effect of such mutation on its immunogenicity, the corresponding SVPs were recognized by two neutralizing monoclonal antibodies (MAb SVP-1 and MAb SVP-4) against IBDV in an ELISA test. The data have shown such mutations offer the SVP a better binding affinity to the two neutralizing monoclonal antibodies. The protections afforded by three VP2-441 SVP variants were also compared by immunizing the specific-pathogen-free chickens and following with a homologous virus challenge. The sera collected from the chickens immunized with H253A SVPs have a higher virus neutralizing (VN) titer than authentic VP2-441 SVPs did. These suggested that the His253 may locate on the center of immunogenic determinants and mutation to alanine has fine-tuned the local structure to fit the binding pocket of neutralization antibody much more tightly.
目錄

摘要.................................................................i
Abstract..............................................................ii
目錄...............................................................iv
第一章 文獻回顧......................................................1
一、 傳染性華氏囊病病毒之簡介.......................................1
二、 傳染性華氏囊病之病徵與防範.....................................1
三、 傳染性華氏囊病病毒之基因體 (Genome) 及其產物分析...............2
四、 傳染性華氏囊病病毒之結構組成...................................3
五、 傳染性華氏囊病病毒之宿主細胞...................................3
六、 傳染性華氏囊病病毒之病毒接受器 (receptor)........................4
七、 固定化金屬離子親和層析法於純化蛋白上的應用.....................4
八、 定點突變技術在蛋白質工程上的應用...............................5
九、 點突變對於固定化金屬離子層析法純化次病毒顆粒之影響.............6
十、 研究動機與目的.................................................7
第二章 材料與方法....................................................8
一、 大腸桿菌表現系統...............................................8
(一) 重組質體之構築 (Construction of recombinant plasmid) ...............8
二、 細胞與病毒以及抗體之製備.......................................8
(一) 昆蟲細胞株Sf-9 (Spodoptera frugiperda) ............................8
(二) 昆蟲細胞株Hi-5 (Trichoplusia ni, BTI-TN-5B1-4).....................8
(三) 雞胚胎纖維母細胞 (Gallus gallus, chicken embryo fibroblast cell line) ....9
(四) 實驗所選用之病毒.............................................9
(五) 病毒力價測定 (virus titer).......................................9
(六) 雞隻免疫試驗之攻毒用病毒.....................................10
(七) 多價抗體 (polyclonal antibody)...................................10
(八) 單株抗體 (monoclonal antibody)..................................10
三、 昆蟲細胞桿狀病毒表現系統......................................10
(一) 重組轉移質體之構築...........................................10
(二) 桿狀病毒轉移質體 (Baculovirus transfer vector) pBlueBac4............11
(三) 重組病毒之取得 (co-transfection).................................11
(四) 純化重組桿狀病毒之病毒溶斑試驗 (plaque assay)...................11
(五) 以聚合酶連鎖反應鑑定純化後之重組病毒.........................12
(六) 高效價重組病毒之製備 (High-titer viral stock)......................12
(七) 病毒力價之測定 (Viral plaque assay)..............................13
四、 重組蛋白之生產、純化與鑑定....................................13
(一) 重組蛋白之表現...............................................13
(二) 蔗糖梯度離心分離法 (Sucrose gradient centrifugation)................13
(三) 酵素連結免疫分析法 (Enzyme-linked immunosorbent assay, ELISA)....14
(四) SDS-聚丙烯胺凝膠電泳 (SDS-PAGE).............................14
(五) 西方墨點法 (Western blotting)...................................15
(六) 穿透式電子顯微鏡觀察 (Transmission Electron Microscopy) ..........15
(七) 高效液相層析儀 (High Performance Liquid Chromatography, HPLC)....15
(八) 利用PH值進行重組蛋白與固定化金屬離子之脫附分析.............16
(九) 利用咪唑進行重組蛋白與固定化金屬離子之脫附分析...............16
(十) 重組蛋白對Ni-NTA樹脂之等溫平衡吸附分析......................17
(十一) 重組蛋白與單株抗體吸附分析.................................17
五、 雞隻之免疫試驗 (chicken immunization)............................17
(一) 實驗動物.....................................................17
(二) 動物免疫.....................................................17
(三) 攻毒試驗.....................................................18
(四) 華氏囊之均質化...............................................18
六、 免疫試驗之分析................................................18
(一) VP2持異性抗體力價之測試 (VP2-specific IgG titer).................18
(二) 病毒中和性試驗 (virus neutralization assay, VN assay)................18
(三) 華氏囊中病毒之檢測...........................................19
第三章 結果.........................................................20
一、 VP2-441之點突變蛋白於昆蟲桿狀病毒系統表現....................20
二、 利用PH值進行重組蛋白與固定化金屬離子之脫附分析...............21
三、 利用咪唑進行重組蛋白與固定化金屬離子之脫附分析..............21
四、 重組蛋白對Ni-NTA樹脂之等溫平衡吸附分析......................22
五、 重組蛋白與單株抗體之吸附分析..................................22
六、 次病毒顆粒之免疫原性分析......................................23
第四章 討論.........................................................25
參考文獻............................................................30
結果圖表............................................................38
附錄................................................................55
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