Construction of recombinant adenoviral vector co-expressing G gene of RV and IFN-a gene

To construct adenoviral vector co-expressing G gene of RV and IFN-a gene by utilizing an internal ribosome entry site(IRES) sequences to link the two genes,the G gene of RV was obtained by reverse transcriptase-polymerase chain reaction(RT-PCR),and the IFN-a gene was amplified by PCR from a T-vector containing IFN-a cDNAs.The DNA fragments were inserted into T vector and sequenced,then subcloned IFN-a gene into pIRES.The amplification product of G gene and the plasmid of pIRES-a were digested with double restriction endonuclease,and then simultaneously cloned into shuttle vector pAdTrack-CMV.Then the recombinant adenoviral plasmid was obtained by homologous recombinant of the linearized shuttle plasmid and pAdEasy-1 backbone vector in E.coli.Finally the recombinant adenoviral plasmid was digested with Pac I and transfected into cell 293 to pack the adenovirus.Strong green fluorescence were observed by fluorescence microscope.The results showed that the recombinant adenovirus containing G gene and IFN-a gene were successfully constructed,thus provided a basis for the research of recombinant replica-tion-defective adenovirus based RV vaccine.