Construction of Recombinant Adenovirus Vector Containing the Capsid, 3C protease and 3D Polymerase Coding Regions of FMDV by Homologous Recombination

Two recombinant replication-defective human adenovirus serotype 5 vector containing foot-and-mouth disease virus (FMDV) serotype O capsid P1-2A, viral 3C protease and 3D polymerase coding region were constructed using the method of homologous recombination in bacteria. Capsid P1-2A, 3C protease and 3D polymerase coding region were subcloned into pshuttle-CMV vector according its original open-reading frame(orf).The positive recombinant pshuttle-CMV vector was linearized and electroporated into Ad-BJ5183 competent cells which had already been transformed with adenovirus skeletal vector pAdeasy-1.Two recombinant adenovirus vector pAdcmv-p12x3c and pAdcmv-p12x3cd were confirmed by Pac I digestion and sequencing, and then transfected HEK293 cell for packaging of recombinant adenovirus. The cell CPE could be observed within one week after transfected, and complete CPE appeared within 24~48h after four round passages. RT-PCR demon- strated that there were expression of the cloned genes. A PCR method has demonstrated that these two recombinant adenovirus could be stably passaged. These results indicated that these two recombinant adenovirus could be used on animals for FMDV recombinant adenovirus vaccine .