In the present study, we designed and constructed a chimeric multi-epitope gene of ALV-J to develop a potential multi-epitope vaccine using a reverse vaccinology approach. The chimeric gene includes 4 multi-epitope concentrated fragments (Gag (278-376aa), Pol (784-855aa), Env (Gp85:145-156aa and Gp37:412-538aa) screened from major structural proteins of ALV-J using epitope prediction software. The recombinant chimeric multi-epitope protein (rCMEPX) encoded by the cloned chimeric gene was successfully expressed using an Escherichia coli expression system. The rCMEPX was induced optimally at 37 degrees C for 4.0 h with 0.5 mM IPTG. The identity and purity of the expressed rCMEPX was analyzed on a SDS-PAGE. The specific recognition of the purified rCMEPX by the chicken anti-ALV-J serum on a western analysis demonstrated a good immunoreactivity of the expressed rCMEPX, which indicates that the construction and expression of the multi-epitope based chimeric gene for ALV-J vaccine development is successful. The antigenicity and reactionogenicity of the rCMEPX were evaluated by western blot and indirect ELISA. Our results showed good reactionogenicity, specificity, and sensitivity for the expressed rCMEPX, suggesting that it may be a promising vaccine candidate against ALV-J infections. (C) 2014 Elsevier Inc. All rights reserved.