Prokaryotic Expression and Purification of σB of Novel Duck Reovirus

Objective The soluble σB of novel duck reovirus (NDRV) was obtained with optimized prokaryotic expression for effective disease detection and control.

Method The recombinant plasmid pET-32a-σB identified by double digestion was transformed into Escherichia coli BL21(DE3) competent cells. IPTG was used to induce the expression of recombinant σB (rσB). After ultrasonic crushing, the supernatant and precipitate were analyzed by SDS-PAGE. The rσB in the supernatant was then purified by a His-tag protein purification kit to establish an indirect ELISA for the viral detection.

Result Under the conditions of 0.5mmol·L⁻¹ IPTG and induction at 16℃ for 12h, the rσB was detected in the supernatant with significantly improved expression. The SDS-PAGE analysis showed the purity of rσB to be higher than 90%, and the molecular weight approximately 60 kDa. The purified protein specifically reacted with His-tag monoclonal antibody as shown by western blot. The OD_450nm of the NDRV positive serum tested by the indirect ELISA was 1.75, which was much higher than that of positive sera from Muscovy duck reovirus and Muscovy duck goose parvovirus. The OD_450nm of MDRV and MDGPV positive sera were both less than 0.3. A strong specific reactivity of the purified rσB was confirmed by both western blot and ELISA.

Conclusion The optimized prokaryotic expression conditions of NDRV σB protein established in this study applied low temperature and IPTG concentration to successfully induce the soluble NDRV σB protein that overcame the previously existing bottleneck for the functional and application studies. The secured soluble rσB would lead to the future development of NDRV serum antibody ELISA kit.