乙型脑炎病毒E蛋白基因的原核表达及ELISA抗体检测方法的建立  

Prokaryotic expression of E protein gene of Japanese encephalitis virus (JEV) and establishment of an indirect ELISA for detection of antibody against JEV

作　　者：刘昊[1,2] 尹革芬[1] 鲁会军[2] 张丹[2,3] 谭磊[2,3] 王开艳[4] 关莎莎[1] 段博芳[1] 金宁一[2] 段纲[1] 

机构地区：[1]云南农业大学动物科技学院,云南昆明650201 [2]中国人民解放军军事医学科学院军事兽医研究所,吉林长春130062 [3]吉林大学畜牧兽医学院,吉林长春130062 [4]延边大学农学院动物医学系,吉林龙井133400

出　　处：《中国兽医科学》2010年第4期390-394,共5页Chinese Veterinary Science

基　　金：国家公益性行业科研专项(200803015);国家高技术研究发展计划(863)项目(2009AA10Z20)

摘　　要：通过PCR方法扩增流行性乙型脑炎病毒(JEV)E基因,全长1 500 bp,将其连入经双酶切的pET-28a(+)载体,构建了重组原核表达质粒pET28a-E。将pET28a-E转化大肠杆菌BL21(DE3)后,经IPTG诱导,进行SDS-PAGE分析。结果显示,E基因在大肠杆菌BL21中获得高效表达,表达的蛋白分子质量约53 ku。Western-blot分析表明,该表达产物具有良好的抗原性。在此基础上,利用该蛋白初步建立了检测猪JEV抗体的间接ELISA方法,并用武汉科前生物制品公司生产的猪JEV ELISA抗体检测试剂盒同时对250份临床采集的猪血清样品进行了检测,结果这两种方法的符合率达到92%,表明建立的ELISA方法具有较高的灵敏性和特异性。According to the E gene sequence of Japanese encephalitis virus（JEV）,a pair of primers was designed.With the primers,the objective gene was amplified with 1500bp in size.Then the amplified E gene was cloned into prokaryotic expression vector pET-28a（＋） to construct recombinant plasmid.The plasmid was transformed into BL21（DE3） competent cell,and the E protein was expressed highly with 53ku approximately in molecular weight.Western-blot analysis showed that E protein could react with positive swine sera.Based on the expressed protein an indirect ELISA was established to detect JEV antibody in swine serum.The coincidence rate of the developed indirect ELISA was 92% with the kit made by Wuhan Keqian Biologicals Company,indicating that the developed indirect ELISA was sensitive and specific.

关 键 词：乙型脑炎病毒 E基因 克隆 表达 抗原性 

分 类 号：S852.659.6[农业科学—基础兽医学]