鸡传染性支气管炎病毒S1蛋白抗原表位的串联表达及间接ELISA方法的建立  

Development of an Indirect ELISA of Infectious Bronchitis Virus by Using Tandem Epitopes of S1

作　　者：孙罗美[1] 易林[1] 邹年莉[1] 柳萍[1,2] 黄勇[1,2] 

机构地区：[1]四川农业大学动物医学院,雅安625014 [2]四川农业大学动物疫病与人类健康四川省重点实验室,雅安625014

出　　处：《畜牧兽医学报》2012年第11期1780-1787,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：教育部<长江学者和创新团队发展计划>创新团队项目(IRTO848)

摘　　要：本研究旨在建立一种快速、简便的鸡传染性支气管炎抗体的检测方法。通过生物信息学软件对传染性支气管炎病毒(IBV)ZY3株的S1蛋白进行分析后,筛选出4段S1蛋白抗原表位优势区域(F1～F4),将4段表位串联成1条新基因F,对F基因编码的蛋白质进行二级结构预测,结果表明该蛋白抗原指数性高且具有良好的柔韧性。构建重组表达载体pET-32a(+)-F,并在原核表达系统中表达,获得大小为42ku的融合蛋白,经Western blot分析表明表达的串联蛋白具有良好的反应原性。以纯化的串联蛋白作为包被抗原,建立了一种检测IBV抗体的间接ELISA方法。利用建立的ELISA方法对采集来175份血清样品进行检测,并与商品化试剂盒进行对比,显示其阳性符合率为90.2%,阴性符合率为85.7%,总体符合率为89.7%,表明建立的ELISA方法具有良好的特异性、重复性和敏感性。The aim of this study was to establish an indirect ELISA for detection of antibodies against avian infectious bronchitis （IB）. The published amino acid sequences of S1 gene of avian Infectious bronchitis virus （IBV） strain ZY3 were analyzed by bioinformatics software, and four dominant epitopes named as F1, F2, F3 and F4 were selected and ligated together as a chimeric gene F. Bioinformatic analysis showed that this protein if highly antigenic and flexible. Then the chimeric gene was then inserted into expression vector pET-32a（＋）for the expression of target gene and a 42 kD recombinant protein was obtained. The result of westernblot showed that the chimeric protein could react specifically with anti-IBV positive serum. An indirect ELISA was then developed using purified protein as coating antigen.175 sera samples were examined by this ELISA and commercial kit, the results showed that the positive coincidence rate could reached 90.2%, negative coincidence rate could reached 85.7%, and the total coincidence rate reached 89.7%. The results indicated that the indirect ELISA was sensitive and specific, and no cross-reaction with positive sera of other chicken diseases. The indirect ELISA for detection of chicken antibodies against Infectious bronchitis were successfully developed.

关 键 词：传染性支气管炎病毒 S1蛋白 表位串联 间接ELISA 建立 

分 类 号：S852.659.6[农业科学—基础兽医学]