口蹄疫病毒2C3AB基因的原核表达及ELISA方法的建立

口蹄疫病毒(FMDV)的2C蛋白中存在对自然感染和灭活疫苗免疫动物具有鉴别诊断意义的抗原表位。为建立一种敏感的血清学鉴别诊断方法,本研究截取FMDV 2C蛋白的C端B细胞表位较为富集的区域与全长3AB基因组合,经原核表达获得分子量约为44 ku的目的蛋白。Western blot分析表明,表达产物2C3AB与FMDV感染动物的阳性血清呈特异性反应。以纯化的目的蛋白作为包被抗原建立间接ELISA方法,敏感性检测表明该方法比3ABC-ELISA具有更高的敏感性;同时检测猪圆环病毒、猪繁殖和呼吸障碍综合征病毒、猪瘟病毒标准阳性血清均无交叉反应。批内和批间重复性试验显示,OD450nm值的变异系数小于9%。采用该方法检测不同背景的临床样品,并与3ABC-ELISA及进口试剂盒比较,总符合率分别为98.5%和90%。结果表明,该ELISA检测方法具有更高的敏感性和良好的特异性、重复性。

Establishment of indirect ELISA for detection of antibodies against FMDV with prokaryotic expressed 2C3AB as coating antigen

To establish a differential detection method for antibodies from foot-and-mouth disease virus(FMDV) naturally infected and inactivated vaccine immunized animals,an indirect ELISA was developed with the nonstructural proteins of FMDV as coating antigen,which expressed from partial 2C gene and 3AB gene of FMDV as a fusion protein in E.coli.Western blot analysis indicated that the expressed protein 2C3AB reacted specifically with the sera from FMDV-infected animal and no cross-reaction with the positive sera of other swine diseases.The coefficient of variability(C.V) of intra-assay and inter-assay reproducibility was less than 9%.Sera derived from swine with different immune and infectious status were detected for antibodies against nonstructural proteins(NSP) of FMDV by the 2C3AB-ELISA,3ABC-ELISA and prioCHECKR NSP ELISA,the coincidence rate was 98.5% and 90%.These results demonstrated that the indirect ELISA method was higher sensitive and specific.