Study On The Function Of Fth1 In The Course Of Brucella Ovis 019 Infecting Raw264.7 Macrophages

Object:Brucellosis is a zoontic disease caused by members of the genus Brucella. Brucella may parasite in macrophage and inhibit cell apoptosis at long-term, which causes many organs damage and chronic infection for human and livestock. It is very important to explore the relation between brucella's intracellullar mechanism and chronic infection. The results of current etiologic study show that outer membrane proteins (OMPs) and type IV secretion system (TFSS) proteins are putative virulent factors because these elements play an important role in brucella's invasion, survival and multiplication. The function study of macrophage target proteins interacting with brucella virulent factors becomes molecular basis of brucella intracellullar parasite mechanism. This study is useful to screen specific candidate drugs to brucellosis.Methods:(1)To verificate ferritin heavy chain 1(FTH1) interacted with brucella TFSS VirB5 protein by co-immunoprecipitation (Co-IP). (2)The target genes including FTH1,16S rRNA, GAPDH and apoptosis-related genes were cloned, whose Std and melt curve were made. (3)Three positive and one negative pSIREN-siRNAs targeted to FTH1 were transfected into mouse RAW264.7 macrophages with TurboFectTM in vitro Transfection Reagent. The optimal pSIREN-siRNA were screened by detecting the expression of FTH1 by Real-time PCR. (4)The optimal pSIREN-siRNA was transfected into mouse RAW264.7 macrophages. After 48h the transfected macrophages were infected with B.ovis 019 strain for 4h and the expressions of 16S rRNA and apoptosis-related genes were quantitated and compared by Real-time PCR. (5) The morphologic change of RAW264.7 macrophages infected with B.ovis 019 strain before RNAi and after RNAi were observed by electron microscope. (6) The cell culture fluid supernatant of Mouse RAW264.7 macrophages transfected with optimal pSIREN-siRNA and infected with B.ovis 019 strain for 15min,30min, 1h and 4h was detected by ELISA.Results:(1) The results of Co-IP showed that VirB5 interacted with FTH1. (2) The eleven target genes including FTH1,16S rRNA,GAPDH and apoptosis-related genes were cloned and their Std value were made. (3)Three positive siRNA plasmids were successfully transfected into mouse RAW264.7 macrophages. The optimal pSIREN-C was screened and its inhibition rate reached to 98%. (4) The results of Real-time PCR showed that the capability of Brucella's invasion was inhibited by 84% after RNAi while two apoptosis suppressor gene (Mkll and Birclb) expression were inhibited by 76.3% and 88.8%, respectively. (5) After RNA interfering RAW264.7 macrophages were infected with B.ovis 019 strain, the observation of transmission electron microscope showed that macrophages appeared apoptosis symptom. (6) During the difference stage of infection, the results of ELISA showed that the expression of IL-2, IL-4, IL-6, IL-10 and TNF-a had different changes compared with that in negative control.Conclusion:(1) VirB5 can interact with FTH1. (2) RAW264.7 macrophages transfected the pSIREN-C can inhibit Brucella infection and promote apoptosis. (3) The secretory volume change of cytokine IL-2, IL-4, IL-6 and TNF-a were disadvantage for brucella's endoparasitism.