全基因组测序用于宁夏羊种3型布鲁氏菌毒力基因筛选的研究

Application of Whole-genome Sequencing to Screening Virulence Genes of B. Melitensis Biovar 3 in Ningxia

目的从3株布鲁氏菌分离株的全基因组序列中筛选出可能导致脑损害的关键毒力基因,为后续筛选布鲁氏菌致脑损害毒力基因的研究奠定基础。方法通过BCSP31PCR、传统生化实验对3株分离于布鲁氏菌病患者外周血中的布鲁氏菌进行鉴定分型。利用Illumina Hiseq2000平台对3株布鲁氏菌进行全基因组测序与比较基因组学分析,根据文献报道分析筛选出布鲁氏菌的经典毒力基因,通过检索NCBI核酸数据库查找出所选毒力基因的参考序列,利用所选参考序列分别与3株布鲁氏菌全基因组序列做BLAT比对分析。结果3株布鲁氏菌经BCSP31PCR、传统生化实验鉴定为羊种3型;比较基因组分析显示3株布鲁氏菌与标准株参考序列的同源性比例均达到99.02%。BLAT比对分析发现所选毒力基因wbkA、pgm、hemolysin、hemolysin III和Omp25在这3株布鲁氏菌全基因组序列中的比对分数均达到99%以上。结论本研究所选取的3株羊种3型布鲁氏菌的全基因组中存在经典的毒力基因序列。

Objective To screen key virulence genes which probably induced brain damage from whole -ge- nome sequence of three field Brucella strains in Ningxia and to establish foundation for following study about screening virulence genes which induced brain damage of Brucella. Methods Three field Brucella strains which isolated from human peripheral blood were identified using BCSP31PCR and traditional biochemical assays. Whole genomic DNA of these strains was sequenced by an Illumina High Seq 2000 sequencer. Comparative genomic analyses were also performed. Typical viruLence genes of Brucella were analyzed and selected according to literature while reference sequence of selected viruLence genes were searched through NCBI database. BLAT analysis was made between these reference sequence and whole - genome sequence of three Brucella melitensis biovar 3 strains, Respectively. Results Three strains were identified as Brucella melitensis biovar 3. Comparative genomic analyses showed that homology ratio between the three Brucella melitensis bio- var 3 strains and standard reference sequence all reached 99.02%. BLAT comparative analysis demonstrated that matching proportion of selected virulence genes found in these Brucella genome sequences reached 99% or more. Conclusion This study proved that whole - genome sequences of these three Brucella strains contain typical virulence genes.