Objective:To investigate the physiological and pathological function of HSP27 by construct and identify the recombinant adenoviruses containing green fluorescence protein(GFP)and mouse HSP27 gene with PAdEasy system. Methods:The coding region of HSP27 was subcloned into the shuttle vector pAdTrack-CMV to form pAdTrack-CMV-HSP27. The identification was performed by PCR,sequencing and restriction digest. Chemical transformation of the plasmid pAdEasy-1 into E. coli BJ5183 strain was performed to prepare BJ5183-pAdEasy-1 as the competent bacterium,in which pAdEasy-1 and pAdTrack-CMV-HSP27 were cotransformed. Finally,the recombinant adenovirus containing the coding region of HSP27 gene was constructed by transfecting 293 cells with linearized adenoviral genomes of Ad-CMV-HSP27,and produce was used to infect mice spermatocyte,the expression of HSP27 protein was detected by Western blot. Results:The recombinant plasmids identified by PCR and directly sequencing were positive. GFP was observed after construction,high concentration of spermatocytes infected by recombinant adenortrus was harvested,and HSP27 protein expressed correctly. Conclusion:The recombinant adenoviruses expressing HSP27 and GFP using AdEasy system,which will facilitate further functional studies of HSP27,are successfully performed in this study.