OBJECTIVE To prepare and characterize rat polyclonal antibodies highly specific for Drosophila regeneration (rgn). METHODS Gene fragments rgn were cloned from Drosophila W(1118) cDNAs by PCR and then subcloned into vector pRSETA. Its expression was induced in E.coli JM109(DE3). Then the fusion protein was purified by Ni-NTA superflow chromatography. After the products was identified by Western blotting, SD male rats were immunized with the rgn fusion protein. The polyclonal antibodies were obtained from the rat serum. The specificity and titer of the polyclonal antibodies were detected by Western blotting and immunohistochemistry. RESULTS Through the recombinant plasmid pRSETA-rgn, 6×His-rgn fusion protein was expressed abundantly in E.coli JM109(DE3). After purification, it was used to immunize SD rats as antigen to generate the polyclonal antibodies against rgn. Immunohistochemistry showed that rgn is located in the cytoplasm of hemocytes of the third instar larvae of Drosophila. CONCLUSION The experiment has prepared high-specificity rat polyclonal antibodies against rgn.