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

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 %.