牛疱疹病毒Ⅰ型gB基因的原核表达及间接ELISA检测方法的建立

Prokaryotic expression of gB gene of bovine herpesvirus-1 and establishment of an indirect ELISA based on the recombinant fusion protein

采用PCR方法从牛疱疹病毒Ⅰ型(BHV-1)Bartha Nu/67株扩增出完整的gB基因编码区,将其克隆到pGEM-T Easy载体。根据抗原性将gB基因分成3段,分别克隆到原核表达载体pET-32a中,构建了重组质粒,将其转化大肠杆菌BL21(DE3),经IPTG诱导表达,SDS-PAGE分析。结果显示,目的蛋白均获得了高效表达,均以包涵体形式存在,纯化后目的蛋白的纯度在90%以上。Western-blot和ELISA分析表明,重组蛋白pET-gBⅢ的反应原性最好。以纯化的pET-gBⅢ为诊断抗原包被酶标板,建立了检测BHV-1抗体的间接ELISA方法。用该间接ELISA与法国IDEXX公司的IBR抗体检测试剂盒对临床样品进行平行检测,结果二者的总符合率为93.8%。表明建立的gBⅢELISA检测方法具有很好的特异性和敏感性。

A full gB gene was amplified from bovine herpesvirus-1（BHV-1） Bartha Nu/67 strain by PCR,and the gB PCR product was cloned into pGEM-T Easy vector to construct plasmid pGEM-T-gB.Three different fragments of the gB gene were amplified from pGEM-T-gB by 3 different pairs of primers according to the antigenicity of the proteins encoded by the 3 different fragments of the gB gene,respectively.Three different fragments were inserted into prokaryotic expression vector pET-32a to construct recombinant plasmids,respectively,and the recombinant plasmids were transformed into Escherichia coli BL21（DE3） for expression.SDS-PAGE analysis showed that the proteins pET-gBⅠ,pET-gBⅡ and pET-gBⅢ were expressed in inclusion bodies and the purity of the fusion proteins were over 90%.Western-blotting and ELISA showed that pET-gBⅢ had the best antigenicity among the 3 recombinant proteins.An indirect ELISA for the detection of BHV-1 antibody was established with the purified fusion protein pET-gB Ⅲ after the optimal working circumstance was tried out with chessboard titration.Comparison of the established ELISA with the abroad kit IDEXX ELISA showed the two methods were 93.8% in agreement for the detection of the same serum samples,indicating that the established indirect gB Ⅲ ELISA was specific and sensitive.