Preliminary Study On The Molecular Mechanism Of Brucella Infection In Host Cells

Brucellosis is a bacterial infectious disease caused by Brucella that is widely prevalent among humans and animals.It is widely spread throughout the world,from land to sea.So far,vaccines for the treatment of brucellosis have not been very effective,and the preventive effect is not good.The diagnosis and treatment of diseased organisms are mostly treated with antibiotics or combined with multiple antibiotics,but the overall treatment is not effective,patients The probability of recurrence is large.Although the control of brucellosis has achieved certain effects in recent years,it still has a significant harm to animal husbandry and human safety.Therefore,it is of great significance to study the invasion and pathogenic process of Brucella.In this study,the linear regression of B.melitensis M5-90 concentration and OD600nm was obtained,and the linear regression equation was y=1E×10x+4E+06（R2=0.9997,0≤OD600nm≤1）.Determine the optimal multi-incubation time of goat fibroblasts with MOI=100,and determine the optimal co-incubation time of the two for 3 h.Observe the morphological changes of goat fibroblasts during infection,from the time of infection to the time of cell disintegration.For 72 h,a model of B.melitensis M5-90 infected goat fibroblasts was successfully constructed.Based on this,RNA-seq sequencing and small RNA were extracted from goat fibroblasts（negative control）and B.melitensis M5-90 4 h,24 h,48 h,and 72 h goat fibroblasts.Sequencing was performed to analyze the dynamic changes of mRNA and miRNA during infection.Through bioinformatics analysis,the number of differentially expressed mRNA genes increased significantly with the increase of infection time,and the differentially expressed miRNAs were significantly more up-regulated than the down-regulated genes.After GO and KEGG analysis,it was found that differentially expressed mRNA was clustered into GO term,such as nucleus,extracellular and nucleoli,and 3922 genes were annotated into 307 KEGG pathways.After STEM analysis,7298 differentially expressed mRNAs were aggregated.From the dynamic expression trend of 8 genes,613 differentially expressed miRNA genes clustered 20 genes dynamic expression trends.qRT-PCR verification of mRNA with continuous up-regulation and down-regulation and enrichment in two signaling pathways of ko04060 and ko04062,and selection of the correct nuclear transcription factor NFKBI,interferon receptor IFNAR2,interleukin-10 receptor IL10RB,trend The factor CCL25 was selected as the research object.The miRNAs with continuous up-regulation and sustained down-regulation were selected for the joint analysis.A total of 19 miRNAs were found to have a corresponding relationship.The expression of 10 miRNAs was consistent with the results of small RNA sequencing by qRT-PCR.Consistent.Transfection of RNA mimics further verified the regulatory relationship between the two,and found that the corresponding relationship was bta-miR-744,chi-miR-25-5p,pc-5p-11241₄10,pc-5p-40217₇2 and NFKB1;miR-29a-5p_R+4,bta-miR-2285j_R+1 and IFNAR2;cfa-mir-8903-p3,chi-miR-193b-5p and IL10RB.This experiment provides a theoretical basis for further exploring the invasion and infection mechanism of Brucella infecting natural host goat fibroblasts and provides new ideas for the diagnosis and vaccine development of brucellosis.