鸭瘟病毒gB蛋白N端主要抗原域的表达及间接ELISA检测

Prokaryotic expression of N-terminal antigenic domain of duck plague vi-rus gB protein and the establishment of putative indirect ELISA assay

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Hind Ⅲ 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DPV gB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DPV gB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%。

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 EcoRⅠand Hind Ⅲ and then inserted into pET32a vector to obtain the recombinant 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 %.