Construction of recombinant adenoviral shuttle vectors carrying the rabies virus single G and double G genes.

The viral RNA was isolated from the rabies virus (RV) HEP-Flury strain,and then the glycoprotein (G) gene were amplified by RT-PCR and cloned into pMD18-T plasmid.The G gene was then double digested and subcloned into adenoviral shuttle vector pShuttle-IRES-hrGFP-1.The recombinant shuttle vector pShuttle-G carrying G gene was identified by kanamycin resistance,PCR,restriction endonuclease and sequencing.Moreover,the 2A self-shearing peptide gene of foot-and-mouth disease virus (FMDV) was added to the end of G gene by PCR strategy and cloned into T vector.The G+2A sequence and G gene were respectively cleaved from the pMD18-T plasmids by double restriction endonuclease,and then subcloned directedly into the shuttle vector pShuttle-IRES-hrGFP-1.The recombinant shuttle vector pShuttle-dG carrying double G genes was obtained after being selected by kanamycin resistance,and identitied by restriction endonuclease and sequencing.After transfecting into Ad-293 cells with the above two expression plasmid respectively,GFP expression was observed using fluorescence microscopy,Dot-blot results showed the success of glycoprotein expression.