Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36308
標題: 傳染性華氏囊炎病毒VP4蛋白切位與影響柱狀結構形成的序列之研究
Study of Infectious Bursal Disease Virus VP4 Cleavage Sites and the Sequence Affecting the Tubule Formation
作者: 姜威宏
Chiang, Wei-Hung
關鍵字: IBDV;傳染性華氏囊炎病毒;Infectious bursal disease virus;VP4;FRET;螢光共振能量轉移
出版社: 生物科技學研究所
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摘要: 
傳染性華氏囊炎病毒 (Infectious bursal disease virus, IBDV) 感染雞隻造成免疫力下降,且傳染性很高,易引發大規模感染,造成養雞業極大的經濟損失。IBDV VP4蛋白是一種絲胺酸蛋白酶,功能為截切病毒蛋白前驅物polyprotein,產生pVP2、VP4和VP3蛋白,本研究使用大腸桿菌表現VP4蛋白,經硫酸銨沉澱後以膠體層析法純化,純度約為94%。並使用穿透式電子顯微鏡觀察到長度為100 ~ 300 nm,直徑約24 ~ 26 nm的柱狀 (tubule) 結構。以螢光共振能量轉移 (fluorescence resonance energy transfer, FRET) 分析純化後的VP4蛋白活性,測得其 Km 值為10.9 μM,kcat值為0.039 min-1。本研究證實不同的motif序列被VP4蛋白截切的效率有顯著差異,VP4對序列的選擇性為RQLTLA↓AD (72%) > GKARA↓AD (32%) > GKARA↓AS (25%)。另外,本研究發現C端28個胺基酸是柱狀結構形成所需的序列,當此部分刪除後,電顯下無法觀察到VP4柱狀結構。

Infectious Bursal Disease virus (IBDV) is a causative agent for serious immunosuppression in young chickens. VP4 carrying a function of serine protease can cleave viral polyprotein to form pVP2, VP4 and VP3. In this study, the coding sequences of VP4 with various N-terminal cleavage sites and C-terminal deletions were constructed and expressed in E. coli. The recombinant VP4 protein was purified by a two-step purification containing ammonium sulfate precipitation and Sepharose CL-6B gel-filtration to 94% of purity. Electron microcopy demonstrated that native VP4 formed tubule-like structures with 100 ~ 300 nm in length and 24 ~ 26 nm in diameter. Kinetic studies by fluorescence resonance energy transfer (FRET) using a synthetic fluorogenic oligopeptide as a substrate showed that the tubule-like VP4 was a functional protease with Km of 10.9 μM and kcat of 0.039 min-1. The VP4 has preferred selection on cleavage sites by RQLTLA↓AD (72%) > GKARA↓AD (32%) > GKARA↓AS (25%). Furthermore, residues at the C-terminus 28 amino acids are the essential region for tubule assembly.
URI: http://hdl.handle.net/11455/36308
其他識別: U0005-2706201213525700
Appears in Collections:生物科技學研究所

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