【摘要】 在分析鸭瘟病毒gB蛋白抗原性的基础上,设计一对引物克隆gB蛋白N端抗原性较好的抗原域编码基因。将克隆的基因定向插入pET-32a的EcoRⅠ和HindⅢ之间,构建了gB蛋白主要抗原域原核表达载体pET-gB1。将pET-gB1质粒转化BL(21)宿主菌后,对培养和表达条件进行了优化,实现了DPVgB蛋白主要抗原域的高效表达。免疫印迹试验表明获得的表达产物具有良好的反应原性。应用His·Bind亲和层析柱纯化重组DPVgB蛋白,以纯化的重组gB1蛋白作为检测抗原,初步建立了检测鸭瘟病毒抗体的igB1-ELISA。结果表明,抗原的最佳包被浓度为6.5μg/mL,血清的最佳稀释度为1∶80,阳性标准初步定为:待检血清OD490>0.4,且待检血清OD490/阴性血清OD490>2。应用igB1-ELISA对鸭血清样品进行检测,结果表明igB1-ELISA与全病毒包被的iDPV-ELISA符合率达到95.6%。更多还原

【Abstract】 Based on the antigenic analysis of duck plague virus(DPV) gB protein,we designed a pair of primers to amplify the gene fragment encoding high antigenic domain of DPV N-terminal gB protein from the DPV genome.The cloned gene was digested with EcoR Ⅰ and Hind Ⅲ and then inserted into pET32a vector to obtain the recom-binant pET-gB1 plasmid.The recombinant plasmid was transformed into E.coli BL21,and expressed in very high level after induced with IPTG..The expressed product was analyzed by SDS-PAGE and Western blotting.The result indicated that the fusion protein(pET-gB1) existed as inclusion body,which was about 42.4kDa and showed specific immunoreactivity with anti-DPV sera.The recombinant gB1 protein was purifiedwith His·Bind resin pro-tein purification procedure.Then an indirect ELISA was established to detect antibody against DPV with the puri-fied gB1 protein as the coating antigen.The result showed that the optimal concentration of coated antigen was 6.5μg/mL and the optimal dilution of serum was 1∶80.The positive criterion of this ELISA assay was ODthe tested serum>0.4 and ODthe tested serum/ODthe negative serum>2.0.The ELISA was done on 700 sera that were preserved in Shandong,Jiangsu Provinces,and were detected by igB1-ELISA and iDPV-ELISA with duck plague virus as the coating an-tigen respectively.The agreement ratio between the two methods was 95.6%.更多还原