Primery Research Of B.melitensis FliC Gene Affecting Gene Expression Of RAW264.7 Cell

Brucellosis is a highly contagious zoonosis that is widespread throughout the world.Brucella is the pathogenic bacterium with Gram negative and belongs to facultative intracellular bacteria.The flagellin FliC is a major component of the Brucella flagellum and is involved in the pathogenesis of bacteria on the host.It is an important pathogen-associated molecular pattern(PAMPs).Previous study has investigated the infection of mice with intraperitoneal injection of the Brucella strain lacking the fliC gene and found that its virulence was enhanced.However,the gene expression changes of host cells infected by the fliC deletion strain remains to be studied.In this study,the homologous recombination plasmid pGEM-7Zf(+)-?fliC was successfully constructed by overlap extension PCR reaction and T4 DNA ligase ligation.Then the recombinant plasmid was transformed into Brucella strain M5-90 by electroporation.The genetic stability was confirmed by PCR and serial passage.Finally,the M5-90 ?fliC gene deletion strain was successfully obtained.Mice macrophages RAW264.7 were infected the B.melitensis M5-90 and B.melitensis M5-90 AfliC deletion strain respectively and the total RNA was extracted by TRIzol.The expression profiles of miRNAs were detected by miRNA Microarray-Single chip,and the mRNA expression profiles were detected by Agilent gene expression microarray.The results of the microarray experiment indicated that 15 miRNAs with a difference of ?2 and 388 mRNAs with a difference of>2 were found.After qRT-PCR verification,confirmed that the expression of miRNAs was 10 down-regulated,which are mmu-miR-6965-3p,mmu-miR-328-5p,mmu-miR-7683-3p,mmu-miR-7072-5p,mmu-miR-691,mmu-miR-3070-2-3p,mmu-miR-7056-5p,mmu-miR-1907,mmu-miR-486a-3p,and mmu-miR-409-3p.After the conjoint analysis of differential miRNAs and differential mRNA expression profiles using TargetScan and miRDB databases,39 target genes were identified.After qRT-PCR verification,the expression of 38 target genes was confirmed to be consistent with the chip results,and finally 80 pairs of miRNAs-Target gene were predicted.After that,the pro-apoptotic protein Bmf,interleukin-17 receptor Il17ra and Frizzled Fzd7 gene were selected for immunity and apoptosis.According to the predicted miRNA-mRNA relationship,miRNA mimics were used to transfect RAW264.7,and targets were identified by qRT-PCR validation.The confirmed miRNA-predicated target gene were obtained,which is mmu-miR-7056-5p and Bmf,mmu-miR-328-5p and Bmf,mmu-miR-691 and Il17ra,mmu-miR-328-5p and Fzd7.This study provides theoretical support for the elucidating the function of fliC in the process of Brucella infects RAW264.7.