To explore the possibility of stable expression of cationic peptide beta-defensin-2 in eukaryocytes with adenovirus vector. The rat beta-defensin-2 (rBD2) gene was cloned at the downstream of CMV promoter of the adenoviral shuttle plasmid pShuttle-CMV. Then pShuttle-CMV-rBD2 was transformed into E. coli BJ5183-AD-1, in which recombination occurred between plasmids and pAdEasy-1 to construct pAdEasy-rBD2. After confirmation by endonuclease, linear pAdEasy-rBD2 was transformed into 292 cells to obtain packaged adenoviral expression vector, which was used to infect cos-7 cells and to establish respiratory adenovirus infection model of rat. The in vivo and in vitro expression activity of recombinant adenovirus was detected by Western blot and immunohistochemistry. The inserted DNA of pShuttle-CMV-rBD2 consisted of rat beta-defensin-2 gene. The pathological effect of infected cells, electronic microscopic observation and PCR showed that the recombinant adenovirus vector was constructed successfully. The concentration of the adenovirus was 10(9) PFU/ml. The vector expressed rat beta-defensin-2 efficiently in vivo and in vitro. The recombinant adenovirus vector can express cationic peptide beta-defensin-2 in eukaryocytes.