The Effects Of Repetitive Extragenic Palindromic Sequences From Brucella Melitensis DNA On The TLR9-Mediated IFN-? Production

Objective To screen the repetitive extragenic palindromic sequences with activation of TLR9 activity from Brucella melitensis DNA,providing new ideas and new targets for prevention and treatment of brucellosis.Method Bioinformatics methods were used to detect 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 IFN-? in the supernatant was then measured by ELISA.The selected sequences which can make IFN-alpha secretion significantly increased was transfected into Murine macrophage RAW264.7.Results(1)A total of2200 REP sequences in Brucella melitensis DNA were identified.Twelve REP sequences were synthesized for further detecting the TLR9 agonistic activity.(2)IFN-? 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 could significantly decrease the levels of IFN-? in RAW264.7 treated with M6.Conclusions REP sequences present in Brucella melitensis DNA are able to induce IFN-? express through TLR9,which can be useful for the understanding of pathogenesis and immunity of Brucella melitensis.