文章摘要
鹅坦布苏病毒E蛋白结构域I的原核表达及鉴定
Prokaryotic expression and identification of goose tembusu virus E protein domain I
投稿时间:2014-06-04  修订日期:2014-07-02
DOI:
中文关键词: 鹅坦布苏病毒  E蛋白  结构域I  基因合成  原核表达
英文关键词: goose tembusu virus  E protein  domain I  gene synthesis  prokaryotic expression
基金项目:江苏省自然科学基金;国家自然科学基金项目(面上项目,重点项目,重大项目)
作者单位E-mail
赵冬敏 江苏省农业科学院兽医研究所 zhaodongmin126@126.com 
黄欣梅 江苏省农业科学院兽医研究所  
刘宇卓 江苏省农业科学院兽医研究所  
韩凯凯 江苏省农业科学院兽医研究所  
杨婧 江苏省农业科学院兽医研究所  
谢星星 江苏省农业科学院兽医研究所  
刘晓燕 江苏省农业科学院兽医研究所  
李银 江苏省农业科学院兽医研究所  
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中文摘要:
      【目的】为了实现鹅坦布苏病毒(GTMUV)E蛋白结构域I在大肠杆菌中的表达并对其进行鉴定。【方法】根据GTMUV JS804株E蛋白基因序列,利用人工合成法获得GTMUV E蛋白结构域I编码基因,构建重组表达质粒pGEX-EI。转化至BL21(DE3)中,经IPTG诱导表达目的蛋白,并对其进行SDS-PAGE分析和Western-blot鉴定。【结果】合成基因片段全长为411 bp并成功构建重组表达载体pGEX-EI。重组均经IPTG诱导5h后达到表达量高峰。SDS-PAGE分析显示重组蛋白以包涵体形式存在。Western-blot鉴定结果显示,重组蛋白与GST单抗和E蛋白阳性血清均可发生特异性反应。【结论】GTMUV E蛋白结构域I在大肠杆菌中成功表达,为进一步研究GTMUV E蛋白结构和功能提供了基础。
英文摘要:
      [Objective] In order to express goose tembusu virus (GTMUV) envelope domain I in Escherichia coli and identify its immunoreactivity. [Method] The encoding gene of GTMUV JS804 strain E protein domain I was artificially synthesised and then was inserted into the prokaryotic vector pGEX-4t-1 for the construction of recombinant expression plasmid pGEX-EI. Then pGEX-EI was transformed into Escherichia coli BL21 (DE3). The recombinant protein was obtained with the induction of IPTG and identified by SDS-PAGE and western-blot. [Result] The length of synthetic gene fragment was 411 bp. The fragment was inserted into pGEX-4t-1 to construct recombinant expression plasmid pGEX-EI successfully. The molecular weight of the recombinant protein was approximately 41kD and the yield of fusion proteins reached peaks after 5h induction by IPTG. The analysis showed that recombinant protein was expressed in Escherichia coli into inclusion bodies. Western-blot demonstrated that the recombinant protein specifically reacted with GST McAb and positive serum for E protein. [Conclusion] E protein domain I of GTMUV was successfully expressed in Escherichia coli. The results provide foundation for further studies on structure and function of GTMUV E protein.
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