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Expression and Purification of Infectious bursal disease virus VP4 protein
傳染性華氏囊炎病毒(Infectious bursal disease virus; IBDV) VP4蛋白是一種絲氨酸蛋白酶，主要功能是將IBDV基因體Segment A的ORF A1所轉譯出的先質多蛋白(polyprotein)切割成pVP2，VP4與VP3。為了解VP4蛋白的結構與功能，必須先能大量表現具有活性的VP4蛋白酶。先前實驗室已將VP4 基因構築於 pET28a載體中，送入大腸桿菌誘導表現VP4蛋白。本研究進一步嘗試利用硫銨沈澱、膠體層析、固定化金屬親和性管柱層析、蔗糖梯度離心等方法純化VP4蛋白。以固定化金屬親和性管柱層析方式純化破菌後之可溶蛋白，所得VP4蛋白純度不高。以20%硫銨沈澱與S-200膠體層析可得到純度約90%的VP4蛋白。S-200膠體層析所收分層構圖可發現兩個peaks, peak I 與 peak II。peak I的分子量大小超過2000 kDa，經由穿透式電子顯微鏡證實 peak I為柱狀蛋白顆粒，peak II則為VP4單體。利用蔗糖梯度離心同樣可得VP4柱狀蛋白顆粒。進一步分析蛋白活性則發現，形成柱狀之VP4蛋白顆粒才具有蛋白酶活性，VP4單體則不具有蛋白酶活性。
Viral protein 4 (VP4) of infectious bursal disease virus (IBDV) is a serine protease, which catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3. In order to characterize the structure and function of VP4, VP4 gene was subcloned and successfully expressed in Escherichia coli system. Several purification methods were employed to obtain VP4 protein, including ammonium sulfate precipitation, gel filtration, immobilized metal-ion affinity chromatography (IMAC) and sucrose gradient ultracentrifugation. When soluble VP4 was purified using 20% ammonium sulfate precipitation following with S-200 gel filtration, the purity is approximately 90%. Two peaks of VP4 were found in the elution profile of S-200 gel filtration. The calculated molecular weight for peak I was more than 2000 kDa. The VP4 in peak I was then subjected to TEM observation and demonstrated that it has a tubule-like structure. Peak II was corresponding to monomeric VP4. The tubular VP4 particle can also be obtained by sucrose gradient ultracentrifugation. Protease activity assay demonstrated that the tubule-like VP4 was a functional protease, but the monomeric VP4 was not.
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