The Differential Expression Analysis Of Murine IL-1β And MyD88 In Response To Attenuated Brucella Suis Strain S2 Infection And Their Effects On Intracellular Bacterial Colonies

Brucellosis is a bacterial zoonotic disease caused by Brucella spp.which is prevalent worldwide,posing a serious threat to human and animal health,affecting the development of animal husbandry and public health safety.At present,the prevention of brucellosis in animal husbandry mainly relies on attenuated vaccination.However,it is impossible to effectively distinguish the antibody positivity caused by brucellosis vaccine immunization or infected with Brucella,which brings certain difficulties to brucellosis decontamination.Interleukin-1β(IL-1β)as an inflammatory factor plays an important role in host resistance to pathogenic microorganism invasion and defense against infection.Our research group previously constructed the suppression subtractive hybridization(SSH)c DNA library of wild Brucella and B.suis S2 infected sheep leukocytes,and screened out differential expression of IL-1β.It was further found that infection with different virulent strains of Brucella resulted in differential expression of IL-1β at the transcriptional and translational levels.Therefore,in order to reveal the effect of host inflammatory factor IL-1β on intracellular bacterial load of Brucella,the study utilized attenuated B.suis strain S2 to infect mouse macrophages RAW264.7,and analyzed the number of intracellular Brucella by altering IL-1β protein levels.1.Cloning and expression of murine IL-1β coding region DNA and preparation of polyclonal antibodyThe DNA of murine IL-1β coding region was cloned by molecular biology techniques.The IL-1β prokaryotic expression vector p ET-28a-IL-1β was constructed.The IL-1β recombinant protein was expressed and purified and its biological activity was detected by thymocyte proliferation assay.Polyclonal antibody was prepared.Finally,we obtained a biologically active IL-1β recombinant protein and a polyclonal antibody with good specificity.and biologically active IL-1β recombinant protein.2.Analysis of differential expression of IL-1β in Brucella infected mouse macrophagesIn order to analyze the changing trend of IL-1βat transcriptional and translational levels during Brucella infestation,this experiment used attenuated B.suis strain S2 to infect mouse macrophages RAW264.7 at a multiplicity of infection(MOI)of 100 :1(bacteria: cell).Among them,inactivated S2,Listeria monocytogenes and Escherichia coli infection groups were used as comparative analysis groups.RT-q PCR was used to detect IL-1β m RNA during bacterial infection;Western blot was used to analyze proIL-1β at the protein level;ELISA was used to detect extracellular IL-1β secretion.The results showed that bacterial infection,including inactivation S2,could rapidly induce elevated levels of IL-1β transcripts.At 12 h of B.suis S2 infection and 8 h of L.monocytogenes infection,the expression of pro-IL-1β started to increase significantly,while the other groups consistently showed higher levels.The secretion of extracellular IL-1β in the B.suis S2 and L.monocytogenes infection groups was higher than that in the normal control group,while the E.coli and inactivated S2 groups were higher than the normal control group at first and then decreased,with no significant difference from normal controls.3.Effects of changing IL-1β protein content on intracellular bacterial load of BrucellaIn this experiment,macrophages were transfected with interfering RNA or IL-1βoverexpression vector to reduce or increase the level of intracellular IL-1β expression,and by exogenously adding biologically active IL-1β protein to increase extracellular IL-1β content.Finally,the effect of changing IL-1β protein content on the intracellular bacterial load of Brucella was analyzed by colony counting method.The results showed that the changes of IL-1β levels had no significant correlation with the intracellular load of Brucella,which did not affect the intracellular bacterial load of Brucella The RTq PCR results indicated no significant differential expression changes of genes including LAMP1,i NOS,My D88 and TRAF6 genes during Brucella infection.4.Effects of My D88 overexpression on Brucella infectionMy D88 is a key molecule in the intracellular signal transduction of cytokines such as IL-1β and IL-1α.The constructed My D88 overexpression vector was transfected in macrophages.The effect of My D88 overexpression on the intracellular parasitism of Brucella the intracellular viable bacteria count was analyzed by colony counting method to study the effect of My D88 overexpression on the intracellular bacterial load of Brucella.The results demonstrated that overexpression of My D88 reduced the intracellular of Brucella,while the expression of LAMP1,i NOS,Caspase-1 and TRAF6 genes were up-regulated,the expression of Rab7 was significantly down-regulated,the expression of Hex B was no significant difference,and the secretion of IL-1β increased,indicating that My D88 may be closely associated with i intracellular bacterial load of Brucella.In conclusion,the study found no significant correlation between the differential expression of IL-1β in macrophages and intracellular bacterial load of Brucella.My D88,as an important intracellular signaling molecule,its up-regulated expression leads to the reduction of intracellular Brucella,which is helpful for the clearance of intracellular Brucella.However,this effect is apparently not induced through IL-1β binding to IL-1R to activate the My D88-dependent pathway,and the mechanism remains to be further investigated.