Genetic Evolution Analysis Of SYLT-1 Isolated From Brucella Deer And Its Regulation Of Macrophage Apoptosis Through STAT3

Brucellosis is the most common zoonotic disease in the world caused by Brucella,which can infect a variety of animals,among which deer is one of the important hosts.Epidemiological investigations have found that brucellosis is widely prevalent in domesticated sika deer and red deer,and is one of the most important infectious diseases that endanger the development of deer farming.With the expansion of my country’s deer breeding industry and the need to protect wild deer resources,effective prevention of deer brucellosis has become an urgent problem that has important economic and public health significance.In this study,based on the isolation of a strain of Brucella deer origin,the MLVA-16 method was used to analyze its genetic evolution;at the same time,transcriptome sequencing technology and gene silencing technology were used to further explore the effect of Brucella on RAW264.7 The effect of apoptosis in order to provide new clues for revealing the intracellular survival mechanism of Brucella.1.Isolation and identification of Brucella and genetic evolutionary analysis.In this study,a suspected strain of Brucella was isolated from the spleen of a deer fetus,identified by biochemical tests and PCR to identify the isolate as Brucella melitensis type 1,named SYLT-1.The 16 loci information of SYLT-1(1-5-3-12-2-1-3-2-4-40-9-9-4-3-4-6)was determined using the MLVA-16 method,and a genetic evolutionary tree was constructed by comparison and cluster analysis with similar strains in the MLVA database.The results showed that the isolate SYLT-1 differed from 4 to 5 loci from all known Brucella strains in the database suggesting that SYLT-1 might be a new genotype.2.Transcriptome analysis and differential gene validation of Brucella isolate SYLT-1 of deer origin.The isolate SYLT-1 and the vaccine strain M5 were infiltrated with mouse macrophages RAW264.7 at MOI=100,respectively and the optimal time of infiltration was screened by intracellular proliferation assay and CCK8.The results of the intracellular proliferation test showed that the isolate SYLT-1 and the vaccine strain M5 reached their peak proliferation after48 h of infestation;the results of the CCK8 test showed that the isolate SYLT-1 reached its peak proliferation after 12 h of infestation and the vaccine strain M5 reached its peak proliferation at24 h of infestation.In summary,total RNA was extracted from the isolate SYLT-1 and the vaccine M5 at 24 h of infection to construct a c DNA library.The results showed that a total of 1817 genes were differentially expressed between the experimental group of SYLT-1 and the control group of the vaccine strain M5 by RNA-Seq technology,of which 961 genes were down-regulated and 856 genes were up-regulated.GO annotation and KEGG functional annotation of these differential genes revealed that these differential genes reflect differences in immune response and changes in cell proliferation and apoptosis after infection of host cells by different Brucella species.Seven pairs of differential genes were screened and the sequencing results were verified by fluorescence quantitative PCR,and the results were consistent with the sequencing results.3.Effect of interference with STAT3 expression on the regulation of RAW264.7 apoptosis by Brucella.Three si RNAs were designed according to the transcriptome sequencing results,and Stat3-mus-1107 interference was the best by transfection efficiency and silencing efficiency test;the expression of apoptosis-related genes Bax,Bcl-2 and Caspase-3 in transfected cells was detected by fluorescence quantitative PCR,which showed that Bax and Caspase-3 were significantly different between the experimental group and the control group at 24 h and 48 h(P<0.05),showing an increasing trend,while the expression of Bcl-2 gene was extremely different between the experimental group and the control group at 48 h(P<0.01)showing a decreasing trend.The expression changes of apoptosis-related proteins were detected by Western Blot showed that the expression levels of Bax,Caspase-3 and Cleaved Caspase-3 were higher in the experimental group than those in the control group at 24 h and 48 h.The expression levels of Bcl-2 were lower in the experimental group than those in the control group.The results of flow cytometry showed that the apoptosis rate of cells in the experimental group at 24 h and 48 h was higher than that of the control group,and the difference in the apoptosis rate at 24 h was significant(P<0.05)and the difference in the apoptosis rate at 48 h was extremely significant(P<0.01).The experimental results showed that interference with STAT3 expression in the JAK-STAT pathway promoted Brucella induced apoptosis in mouse macrophages.In summary,in this study,a wild strain of Brucella melitensis type 1(SYLT-1)was isolated from the spleen of aborted deer fetuses,and genetic evolutionary analysis was completed;meanwhile,transcriptome analysis of in vitro infected cells from the isolate SYLT-1,based on the sequencing results,found that by interfering with the expression of STAT3 in the JAK-STAT pathway,the apoptosis of Brucella induced macrophages could be promoted.The results of the study not only provide a reference for identifying the main epidemic strains of brucellosis in deer,but also provide new clues for elucidating the mechanism of the interaction between brucella gene regulation and host cells.