牛轮状病毒VP6蛋白间接ELISA检测方法的建立

Establishment of iELISA based on the expressed bovine rotavirus VP6 protein for detection of specific antibody in serum

采用反转录-聚合酶链反应扩增了牛轮状病毒NCDV株VP6基因,将其定向插入原核表达载体pProHTa中,构建了重组表达载体pProHTa-NCDV-VP6,重组菌经IPTG诱导表达后,进行SDS-PAGE和Western-blot分析,结果显示,目的蛋白获得表达,表达的蛋白大小约52ku,且有生物活性。以纯化的重组VP6蛋白作为包被抗原,建立了检测牛轮状病毒血清抗体的间接ELISA方法,并与传统的琼脂扩散试验进行了比较。结果显示,建立的间接ELISA的敏感性较琼脂扩散试验高1×104倍。表明,该间接ELISA的建立为牛轮状病毒感染血清抗体的检测提供了一种简单、快速、灵敏的方法。

The VP6gene was amplified from bovine rotavirus（RV）by RT-PCR and sub-cloned into the prokaryotic expression vector pProHTa,then the construction of pProHTa-NCDV-VP6was finished,and the expression of the recombinant protein was carried out under induction of IPTG.SDS-PAGE and Western-blot analyses showed that the recombinant protein was 52ku approximately in size.The expressed protein VP6was used as coating antigen,and an indirect ELISA was developed to detect antibody against bovine RV in serum.The detection system was 1×104 times sensitive than the traditional agar diffusion test, indicating that the developed indirect ELISA was a simple,rapid,sensitive and specific method for the detection of the antibody against bovine RV in practice.