Prelimenary Research Of The Effects Of B.Melitensis OMP25 On Gene Expression Profile Of RAW264.7 Cells

Brucellosis is a worldwide zoonosis caused by the Brucella, and threats to the security of humans and animals. OMP (outer membrane protein) is a kind of important virulence factors of Brucella. OMP25 is one of the Brucella OMP, which plays an important role in the pathogenic process of Brucella. Constructing B.melitensis M5-90-? OMP25 strain, To provide a theoretical basis for the development of efficient vaccines for Brucella and Brucella pathogenesis explanation.According to the B.melitensis M5-90 genome sequence and OMP25 gene sequence in GenBank, the up primers and down primers of the left(C) and right (N) homology arm of OMP25 gene were designed, the B.melitensis M5-90 genome were used as the template to amplify C and N homologous arms fragments by PCR. According to the pEGFP-N1 vector sequence, the up primers and down primers of Kanr gene were designed, the pEGFP-N1 plasmid were used as the template to amplify Kanr gene fragments by PCR. The C homology arms fragments, Kanr fragments and N homology arms fragments, were digested by double enzyme of Sac I+BamH I; BamH I+Kpn I; Kpn I+Apa I, respectively, suicide plasmid, pGEM-7Zf-?OMP25, was constructed successfully. The suicide plasmids were electroporated into B.melitensis M5-90 competent cells. After screening double homologous recombination B.melitensis M5-90-?OMP25 by kanamycin and ampicillin selection medium, B.melitensis M5-90-?OMP25 strain were successfully constructed.RAW264.7 cells were infected with B.melitensis M5-90 strain and B.melitensis M5-90-?OMP25 strain, respectively, Total RNA was extracted for LC microRNA Microarray-single microarray and Agilent microarray analysis. The miRNAs microarray results indicated that mmu-miR-146-a-5p and mmu-miR-155-5p were up-regulated, mmu-miR-149-3p and mmu-miR-5126 were down-regulated. The mRNAs microarray results indicated that 3897 genes were significantly different, in which there are 967 genes whose fold changes were ?2. The results of miRNAs-mRNAs analysis and qRT-PCR verification of the predicted target genes suggested that Bc16b?Nos2?Ikbke? Slc31a2?Dusp16?Ifit1?Rhoc?Slc7a11?Illr11?Olr1 were the potential targets of mmu-miR-149-3p.