Abstract： Objective To screen the repetitive extragenic palindromic sequences with activation of toll-like receptor 9(TLR9) activity from Brucella melitensis DNA,providing new ideas and new targets for prevention and treatment of brucellosis.Methods Bioinformatics methods were used to detect repetitive extragenic palindromic(REP) sequences from Brucella melitensis DNA.The studied REPs were selected and synthesized.RAW264.7 was cultured and transfected with REPs mediated by lipofectamine 3000.Additionally,TLR9-siRNA was used to downregulate TLR9 expression.The content of interferon-α(IFN-α) in the supernatant was then measured by ELISA.Results A total of 2 200 REP sequences in Brucella melitensis DNA were identified.Twelve REP sequences were synthesized for further detecting of the TLR9 agonistic activity.IFN-α expression in RAW264.7 treated with M2,M3,M4,M5,M6,M7,M9,M12 were (26.944 ± 1.868),(46.461 ± 2.562),(34.980 ± 2.055),(43.016 ± 2.162),(62.533 ± 4.031),(67.125 ± 5.069),(18.908 ± 1.633),(39.572 ± 2.465) pg/ml respectively,which significantly increased when compared with the negative control group [(12.594 ± 1.338) pg/ml,t =10.817,20.295,15.812,20.724,20.365,18.016,5.180,16.660,all P ＜ 0.05].Additionally,TLR9-siRNA can significantly decrease the levels of IFN-α in RAW264.7 treated with M6.Conclusion REP sequences presented in Brucella melitensis DNA are able to induce IFN-α expression through TLR9,which can be helpful for the understanding of pathogenesis and immunity of Brucella melitensis.