Objective      To construct a recombinant adenovirus encoding vasoactive intestinal peptide (VIP) for future gene therapy in vivo.        Methods      Full length mouse VIP cDNA linked with an internal ribosome entry site (IRES)-EGFP cassette was subcloned into pAdTrack-CMV shuttle plasmid. The product was linearized to mediate homologous recombination with pAdeasy-1 vector in BJ5183 host bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing. The recombinant adenovirus DNA was transfected into 293 cells for packaging and amplification of pAdeasy-VIP virus

which was purified by SARTOBIND Membrane Adsorbers. The expression of VIP was monitored by EGFP fluorescence in infected cells. VIP peptide expression on 293 cells was detected by PCR

immunofluorescent method and ELISA.       Results      After transfected with adenovirus DNA

infectious virus was only produced to cause cytopathic effect in the permissive cell line 293 but not in the non-permissive cell line HeLa

confirming only replication defective but not wild type virus was generated. The specific expression of mouse pAdeasy-VIP was verified by RT-PCR and immunofluorescent method in 293 cells after infection with pAdeasy-VIP

but not Ad-EGFP

a similarly constructed control virus. pAdeasy-VIP

but not pAd-EGFP

significantly secretes VIP peptide in supernatant.       Conclusion      We have successfully constructed a recombinant adenovirus pAdeasy-VIP that expresses VIP peptide in vitro.