Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3428
標題: 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VP2
作者: 陳建旭
關鍵字: 傳染性華氏囊病毒
出版社: 化學工程學系
摘要: Abstract 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.
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。
URI: http://hdl.handle.net/11455/3428
Appears in Collections:化學工程學系所

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