猪轮状病毒VP4蛋白基因在大肠杆菌中的表达Expression of Porcine Rotavirus VP4 in Escherichia coli
肖长峰;刘成倩;卢永红;陈斌;吴双林;张天庆;易建中;
摘要(Abstract):
[目的]研究猪的轮状病毒VP4蛋白基因在BL21大肠杆菌中的表达。[方法]以猪肺脏组织中分离的总RNA为模板,采用反转录聚合酶链式反应(RT-PCR)技术扩增VP4蛋白的部分基因,将PCR产物克隆到表达载体pGEX-4T-1中,构建原核表达质粒PGEX-4T-1-VP4并对重组质粒进行酶切和测序鉴定。将重组载体PGEX-4T-1-VP4转化至大肠杆菌BL21(DE3)中进行诱导表达和SDS-PAGE分析,并对重组大肠杆菌进行可溶性分析。[结果]对猪肺脏组织中分离的总RNA进行RT-PCR扩增,获得875 bp的VP4部分基因,重组质粒PGEX-4T-1-VP4经BamHⅠ和XhoⅠ双酶切和测序鉴定后,插入的猪轮状病毒VP4的cDNA编码区序列与已公布的猪GenBank核酸序列比对结果为99.5%;将重组质粒pGEX-4T-1-VP4-转化至大肠杆菌BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约58 kDa融合蛋白带。[结论]该研究为进一步研究轮状病毒的疫苗研制奠定了基础。
关键词(KeyWords): 轮状病毒;VP4基因;克隆;原核表达
基金项目(Foundation): 上海市浦江人才计划(PJ[20])
作者(Author): 肖长峰;刘成倩;卢永红;陈斌;吴双林;张天庆;易建中;
Email:
DOI: 10.13989/j.cnki.0517-6611.2009.22.139
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