Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3428
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dc.contributor.advisor孫幸宜zh_TW
dc.contributor.author陳建旭zh_TW
dc.date2004zh_TW
dc.date.accessioned2014-06-06T05:31:54Z-
dc.date.available2014-06-06T05:31:54Z-
dc.identifier.urihttp://hdl.handle.net/11455/3428-
dc.description.abstractAbstract VP2, matured from the polyprotein encoded by the genome of infectious bursal disease virus (IBDV), is the primary host-protective immunogen of this virus. The matured VP2 protein (mVP2) and two of mVP2 N-terminal truncated mutants were cloned and expressed in E. coli in this study. To obtain pure recombinant proteins for the development of an efficient subunit vaccine against IBDV infection, they were fused with six additional histidine residues at its C-terminus as a His-purification-tag. Following purification employing immobilized metal-ion (Ni2+) affinity chromatography (IMAC), all the E. coli-derived three proteins have the morphology of icosahedral particles of approximately 25 nm in diameter. To reduce the cost of resins used for IMAC, self-prepared immobilized metal-ion affinity membranes (IMAMs), which were commercial regenerated cellulose membranes that modified with IDA and immobilized with nickel ions, were also applied to directly purify mVP2H and two mutant proteins. Results indicated that particles formed by mVP2H and its truncated mutants can also be efficiently purified, a purification fold of 104 was reached. The abundant quantities of pure rVP2H particles coupled with their uniform dimensions facilitates an understanding of higher order structure of the immunogenic particles and can therefore result in improved vaccines against the virus.en_US
dc.description.abstract中文摘要 本實驗由大腸桿菌表現系統生產傳染性雞華氏囊病毒之VP2結構蛋白,藉由在此蛋白上融合His-Tag,得以固定化金屬親和層析方法加以純化。由於大腸桿菌表現系統所生產的VP2蛋白可溶部分表現量並不多,直接以固定化金屬親和層析方法純化並無法得到理想效果,所以針對VP2蛋白對於酸的耐受性良好的特性,本實驗以調整pH值至4.0並且離心的方式作初純化,即可幫助去除部份非目標蛋白,再加上使用硫酸銨沉澱法,更進一步去除雜蛋白,之後再進行固定化金屬親和層析加以純化,即能得到相當純度的VP2蛋白,在固定化金屬管柱層析純化的回收率為6.728%,固定化金屬親和薄膜純化的回收率為5.278%,兩種純化方式的純化倍數為104.167。 另外再將VP2蛋白分別在N端截切5個及10個胺基酸,再使用固定化金屬管柱層析純化並且在TEM下觀察,發現有似病毒顆粒的形成,表示VP2蛋白即使在N端截切5個及10個胺基酸,仍可經由固定化金屬親和方法加以純化,且最終仍保有形成似病毒顆粒的能力。zh_TW
dc.description.tableofcontents目錄 中文摘要.......................................................1 英文摘要.......................................................2 目錄...........................................................3 一.前言........................................................7 二.文獻回顧....................................................9 2.1固定化金屬親和層析技術原理..................................9 2.2影響蛋白質吸附的因素.......................................10 2.2.1固體相的組成及對吸附的影響...............................10 2.2.1.1固體載體的種類.........................................11 2.2.1.2延伸臂.................................................11 2.2.1.3螯合劑.................................................12 2.2.1.4金屬離子...............................................13 2.2.2 蛋白質結構..............................................14 2.2.3 鹽類及濃度..............................................14 2.2.4溫度.....................................................15 2.2.5pH值.....................................................16 2.3影響脫附蛋白質的因素.......................................16 2.3.1 質子化..................................................17 2.3.2 配位基交換..............................................17 2.3.3 螯合消除................................................17 2.4傳染性華氏囊病毒...........................................18 三.材料與方法.................................................19 3.1實驗材料...................................................19 3.2實驗藥品...................................................19 3.3以IDA為螯合劑之固定化金屬薄膜製備..........................19 3.3.1鎳離子鍵結量的量測.......................................20 3.4大腸桿菌表現系統...........................................20 3.4.1菌種與質體...............................................20 3.4.2 蛋白質之表現及取得......................................21 3.4.3 發酵槽操作培養..........................................21 3.5蛋白質純化及分析的前處理...................................22 3.5.1 酸處理..................................................22 3.5.2 硫酸銨沈澱..............................................22 3.6固定化金屬親和層析管柱.....................................23 3.7固定化金屬親和性薄膜.......................................23 3.8蛋白質的分析...............................................24 3.8.1 SDS-聚丙烯醯胺凝膠電泳(SDS-PAGE)........................24 3.8.2三氯醋酸沉澱(Trichloroacetic acid preciptipation)........24 3.8.3 西方墨點法(Western blot)................................25 3.8.4 硝酸銀染色法 (silver staining)..........................25 3.9蛋白質定量.................................................26 3.9.1總蛋白定量方法...........................................26 3.9.2純化前目標蛋白質的定量...................................26 3.9.3純化後目標蛋白質定量.....................................27 四.結果與討論.................................................28 4.1 E.coli表現蛋白之最佳化條件................................28 4.2 VP2H,VP2HN5,VP2HN10蛋白的表現與純化.................29 4.2.1 純化蛋白的前處理........................................29 4.2.1.1調整pH值做酸處理.......................................29 4.2.1.2 以硫酸銨沉澱作初純化..................................30 4.2.1.3以固定化金屬親合層析管柱純化不同截切長度的VP2蛋白......30 4.2.2VP2H蛋白表現與純化.......................................31 4.2.3 VP2HN5蛋白表現與純化..................................32 4.2.4 VP2HN10蛋白表現與純化.................................33 4.3以固定化金屬薄膜純化不同截切長度的VP2蛋白..................34 4.3.1 VP2H蛋白表現與純化......................................34 4.3.2 VP2HN5蛋白表現與純化..................................35 4.4回收率與純化倍數...........................................36 4.5穿透式電子顯微鏡(Transmission Electron Microscope)下觀察似病毒顆粒........................................................37 五.結論.......................................................38 六.參考文獻...................................................39 七.圖表.......................................................44zh_TW
dc.language.isozh_TWzh_TW
dc.publisher化學工程學系zh_TW
dc.subject傳染性華氏囊病毒zh_TW
dc.title以固定化金屬親和方法純化在E.coli表現傳染性華氏囊病毒之結構蛋白VP2zh_TW
dc.typeThesis and Dissertationzh_TW
item.languageiso639-1zh_TW-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.grantfulltextnone-
item.fulltextno fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
Appears in Collections:化學工程學系所
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