鸭瘟病毒gB蛋白N端主要抗原域的表达及间接ELISA检测

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EcoR Ⅰ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%.

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

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 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 purified with His-Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against DPV with the purified gB1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 6.5 microg/mL and the optimal dilution of serum was 1 : 80. The positive criterion of this ELISA assay was OD(the tested serum) > 0.4 and OD(the tested serum)/OD(the 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 antigen respectively. The agreement ratio between the two methods was 95.6%.