猪乙脑病毒重组E基因的克隆表达及间接ELISA检测方法研究

Research on cloning,expression of recombinant E gene and indirect ELISA for detection of antibody of swine JEV

参照已发表的猪乙脑病毒(JEV)基因组序列,设计引物,扩增出E基因,克隆到pMD19-T载体中。序列分析表明:E基因与NCBI登录的JEV毒株E基因序列同源性达到96.6%—99.7%;遗传进化分析表明:该JEV属于GIII病毒。将E基因片段亚克隆到pET32a-E,IPTG诱导表达E蛋白。Western Blot检测表明:该重组蛋白能与JEV高免血清发生非特异性反应,证明该表达产物为JEV E蛋白。以重组E蛋白为包被抗原,建立检测猪JEV抗体的间接ELISA方法,检测160份猪血清样品,与猪JEV抗体检测试剂盒进行平行性对比,结果显示二者的吻合率为92.2%,表明建立的ELISA方法具有较好的特异性,有望为临床提供一种JEV血清抗体检测方法。

Primers were designed according to the sequences of the JEV E gene published in GenBank.E gene was amplified and cloned into pMD19-T vector.The sequence homology of E gene and JEV E gene from GenBank were up to 96.6%—99.7%.Phylogenetic analysis indicated that the JEV belonged to the genotype III.E gene was subcloned into pET32a vector and the recombinant E protein was expressed induced by IPTG.It was proved by Western Blot that the expression product was recombinant E protein which had nonspecific reac-tion with JEV hyper immune serum.Indirect ELISA was established to detect JEV antibody with E protein as coa-ted antigen.Compare to porcine JEV ELISA diagnostic kit,the coincidence rate of indirect ELISA in the study was 92.2% by detecting 160 clinical pig serum samples.The established ELISA method was specific and could provide a method for the clinical detection of JEV serum antibodies.