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

摘要

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.%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。