犬瘟热病毒重组H蛋白间接ELISA检测方法的建立

Development of an indirect ELISA for detection of antibody against the recombinant H protein of canine distemper virus

为建立一种检测水貂犬瘟热病毒(CDV)血清抗体的间接ELISA方法,将CDV的血凝素蛋白(H)基因的主要抗原表位片段克隆至p ET-30a-c(+)载体中,转化至感受态细胞Rosetta(DE3)中进行诱导表达,并对表达产物进行SDS-PAGE分析和Western-blot鉴定。以纯化的重组H蛋白作为包被抗原,建立了检测CDV抗体的间接ELISA方法,并证实该方法具有良好的特异性、重复性和敏感性。采用建立的方法与国外商品化的犬瘟热抗体检测试剂盒对80份血清进行检测,两者符合率为92.5%。本研究建立的间接ELISA方法为水貂抗体水平检测和评价犬瘟热疫苗免疫效力提供了简便快速的方法。

To establish an indirect ELISA method for detection of serum antibody to canine distemper virus（CDV）,the main antigen fragment of HEMAGGLUTININ（H） gene was amplified from CDV genome and cloned into the p ET-30 a-c（＋） vector.Then the recombinant H protein could be expressed in Rosetta（DE3） by IPTG induction.The results of SDS-PAGE and Western-blot analysis revealed that the recombinant H protein was successfully expressed and had good reactivity with the antiserum to CDV.Using the purified recombinant H protein as coating antigen,an indirect ELISA was developed for detection of antibody against CDV,and the ELISA method showed good results in specificity,sensitivity and reproducibility.The agreement rate of the indirect ELISA relative to commercial CDV antibody kit was above92.5% in detecting 80 mink sera.The indirect ELISA established in this study will provide a simple and rapid means for monitoring the antibody levels and evaluating the vaccine potency.