长沙人间布鲁菌分离株omp25基因扩增和系统进化分析

目的了解长沙地区人间布鲁菌omp25基因的序列特征并阐明其系统进化关系,为布鲁菌病防控以及omp25基因克隆与表达研究提供参考。方法根据Gen Bank公布的羊种布鲁菌16M的omp25基因序列设计1对PCR引物,采用PCR法从长沙地区布鲁菌分离株基因组中扩增omp25基因,产物用凝胶电泳进行分析后进行双向核苷酸序列测定,测序结果用DNAstar 7.1进行分析,将测序结果提交Gen Bank,并利用Mega 7软件构建布鲁菌长沙分离株系统进化树。结果 41株布鲁菌成功扩增出omp25基因片段,片段长度约640 bp,通过测序后Blast同源性分析,证实41株菌株均为羊种布鲁菌,长沙分离株与10株羊种布鲁菌序列100%相同,进化树显示长沙分离的布鲁菌和国内外羊种布鲁菌位于同一进化分支上,同广西的LA1105参考株亲缘关系最近。结论长沙地区人间布鲁菌分离株omp25基因序列与参考菌株序列同源性高,且与羊种布鲁菌在同一分支,表明omp25基因序列是一个保守序列。该研究结果可为布鲁菌的分子生物学鉴定和诊断试剂的研制提供参考。

omp25 gene amplification and phylogenetic analysis of clinical Brucella isolated from Changsha

Objective To understand the characteristics of the omp25 gene sequence from clinical Brucella isolated from Changsha City and elucidate their phylogenetic relationship in order to provide the references for prevention and control of brucellosis and omp25 gene cloning and expression studies.Methods A pair of primers for the gene sequence of omp25 of Brucella was designed after referring to GenBank released Brucella melitensis strain 16M.Polymerase chain reaction (PCR) was used to amplify the omp25 gene from the Brucella genome,and gene fragments were detected by using agarose gel electrophoresis.The PCR products were sequenced in bidirectional nucleotide and the sequencing results were analyzed by using DNAstar7.1,and then the sequences were submitted to GenBank.The phylogenetic tree of omp25 gene of clinical Brucella isolates from Changsha City was constructed through the Mege7 software.Results The products of omp25 gene of 41 strains were obtained successfully with length about 640 bp.The products were sequenced successfully and the sequences were compared with the relational sequence through online Blast in NCBI,showing the 41 strains of Brucella were B.melitensis.Ten B.melitensis strains of GenBank had similarity of 100％ to the nucleotide sequence of the omp25 gene of clinical Brucella isolates from Changsha City.The phylogenetic tree showed that the Brucella strains of Changsha City were at the same evolutionary branching with most of B.melitensis and had the nearest phylogenetic relationship with B.melitensis strain LA1105 in Guangxi Region.Conclusion The omp25 gene of clinical Brucella isolated from Changsha City is homologous to that of Brucella reference strains and B.rnelitensis is one branch of Brucella's phylogenetic tree.The omp25 gene is a conserved sequence.The current results provide a basis for molecular biological identification of Brucella and preparation of diagnostic reagents to detect Brucella.