猪巨细胞病毒gB基因优势抗原表位区的原核表达及间接ELISA方法的建立  

Prokaryotic expression of immunodominant region of PCMV gB gene and development of an indirect ELISA for detecting antibodies against porcine cytomegalovirus

作　　者：刘骁[1] 廖珊[1] 朱玲[1,2] 周远成[1] 周璐[1] 

机构地区：[1]四川农业大学动物生物技术中心,四川雅安625014 [2]功物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国兽医科学》2013年第2期152-158,共7页Chinese Veterinary Science

基　　金：四川省青年科技基金项目(200930421)

摘　　要：扩增了猪巨细胞病毒(PCMV)gB基因的优势抗原表位区,将构建成功的gB基因优势抗原表位区原核表达重组质粒(pET32a-gB)转化入表达菌Rosetta(DE3)中进行表达,以表达的重组蛋白作为包被抗原建立了一种检测PCMV抗体的间接ELISA方法,并对该间接ELISA检测方法进行了优化。结果显示,最佳抗原包被浓度为1.9μg/mL,最佳血清稀释度为1∶160,二抗最佳稀释度为1∶10 000;当样品D450nm≥0.26时判为阳性,当样品D450nm<0.215时判为阴性,介于两者之间时判为可疑。用该间接ELISA方法对138份PCMV阴性血清与240份PCMV阳性血清进行检测的结果显示,它的特异性为97.83%,敏感性为97.9%,变异系数均小于10%。用该ELISA方法和Western-blot对184份临床样品进行检测,结果二者的符合率为92.4%。结果表明,该间接ELISA方法简便快捷,具有良好的特异性、敏感性及重复性,适用于对PCMV抗体的大规模检测。In this study,the major epitope region of porcine cytomegalovirus（PCMV） gB gene was amplified.The prokaryotic expression recombinant plasmid with the gB major epitope region（pET32a-gB） was constructed and expressed with Rosetta（DE3） prokaryotic expression system.The purified recombinant protein was used as coating antigen in the development of an indirect ELISA method for detecting antibodies against PCMV.The ELISA detection method was optimized with the optimal antigen coating concentration of 1.9 μg/mL,the optimal serum dilution of 1∶160,and the optimal HRP-SPA dilution of 1∶10 000.It was judged as positive when the sample D450 nm≥0.26,as negative when the sample D450 nm0.215,and as suspicious when the D450 nm value was in between 0.215 and 0.26.Total of 138 PCMV negative serum and 240 positive serum were detected with the ELISA method,the results showed the specificity of the method was 97.83%,and the sensitivity was 97.9%.The results showed that the indirect ELISA was simple and practical with good specificity,sensitivity and reproducibility,and it was suitable for large-scale detection of PCMV antibodies.

关 键 词：猪巨细胞病毒 原核表达 gB基因优势抗原表位区蛋白 间接ELISA 

分 类 号：S852.659.1[农业科学—基础兽医学]