Primary Screening Of Virulence Genes Of B.melitensis Base On Whole-genome Sequencing

Objective In order to establish foundation for further study on pathogenesis of the braindamage caused by Brucella, This study have screening key virulence genes which fromwhole-genome sequence of three wild Brucella strains by whole-genome sequencingtechnology.Methods Three wild Brucella strains which isolated from Brucellosis patients peripheralblood were cultured, and identified used BCSP31-PCR and traditional biochemical assays.Whole genomic DNA of these strains were extracted and sequenced by an Illumina HighSeq2000sequencer. Comparative genomic analyses were also performed. We analyzed andselected key virulence genes which probably induce brain damage of Brucella according tobioinformatics analysis and literature, searched for reference sequence of selected virulencegenes through NCBI database, and made BLAT analysis between these reference sequencesand whole-genome sequence of three Brucella strains, respectively.Results Three strains were identified as Brucella melitensis biovar3by BCSP31-PCRand traditional biochemical assays. Whole-genome sequencing data showed that each genomesize of Brucella strains was approximately3.29M; the bias of GC is not serious, GC contentis about57.24%. By means of comparative genomic analysis, statistical results of SNP andIn-del revealed that some variation exist in these wild strains; Homology ratio of sequencebetween the three wild strains and standard strains all reached99.02%. Bioinformaticsanalysis about three Brucella strains indicated that gene regulation function on evading hostphagocytic cells killing is powerful. These strains have developed function on replication and metabolism, can take advantage of amino acids, some of the inorganic ions and carbohydratesas the main source of nutrients and energy. BLAT comparative analysis demonstrated thatmatching proportion of selected virulence genes (wbkA, pgm, hemolysin, hemolysin III,Omp25) found in these Brucella genome sequences reached99%or more.Conclusions Illumina Hiseq2000whole-genome sequencing platforms can providetechnical support for study on screening virulence genes of Brucella. The three Brucellastrains show a relatively high conserved over a long period of evolution. Bioinformaticsanalysis about three Brucella strains indicated that gene regulation function on replication,metabolism and evading host phagocytic cells killing is developed. The study have primarilyscreening these key virulence genes of Brucella melitensis biovar3arewbkA,pgm,hemolysin,hemolysin III and Omp25through whole-genome sequencing and BLATcomparative analysis. According to literature, we infer that these genes are the key genes,which are likely to regulate in the process of causing brain damage by Brucella. But its exactpathogenesis requires further study to verify.