Objective In order to make a foundation for molecular diagnosis and gene engineering studies of Avian encephalomyelitis virus(AEV),the structural protein VP3 gene of AEV and expression of its recombinant protein were cloned and sequenced.Methods We designed a pair of primers to amplify the van-Roekel VP3 gene from AEV infected SPF embryo with specific RT-PCR technique.The amplified product was cloned into PDM18-T vector after purification and withdraw.The VP3 gene was sub-cloned into the eucaryotic expression donor plasmid pFastHTa,which was also digested by EcoR I and Xho I.The recombinant plasmid was transfected into E.coli.DH10Bac competence cells.Then selected by PCR and identified by EcoR I and Xho I degistion,and transposition happened.The transpositional recombinant bacmid was transfected into Sf9 insect cells,and then a kind of recombinant baculovirus was obtained.The antigen activity of the recombinant protein was identified by Western blot.Results The results showed that VP3-c contained 735 bp and 245 amino acids,sharing a 93% and 97% homology with AEV-1143 strain,respectively.It was also compared with other small RNA viruses,and recombinant protein was also achieved by Western-blot.The recombinant protein was about 27 ku.Conclusion Using RT-PCR technique,the van-Roekel VP3 gene has been for first time amplified in vitro.It has been also cloned and sequenced.Using Bac-to-Bac expression system,the recombinant protein has been achieved and proven to exhibit high immune activity.