布鲁氏菌bp26基因缺失株的构建  

Construction of unmarked bp26 gene-deleted strains of Brucella spp.

作　　者：潘文[1] 王佳莹[1] 赵明秋[1] 琚春梅[1] 易琳[1] 常艳[1] 虞红娇[1] 陈金顶[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医科学》2011年第3期280-286,共7页Chinese Veterinary Science

基　　金：广东省动植物防疫检疫研究专项(粤财农[2009]225);国家自然科学基金-广东省自然科学基金联合项目(U0631006);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0723);广东省自然科学基金创新团队项目(5200638)

摘　　要：选择含有反向筛选基因sacB标记的pRE112质粒为自杀载体,利用等位基因交换的方法成功敲除了布鲁氏菌弱毒疫苗S19株、S2株、M5株的bp26基因,构建了具有非抗性基因标记的缺失株S19-Δbp26、S2-Δbp26、M5-Δbp26。将构建的重组自杀质粒pRE-Δbp26(缺失了bp26基因的681个碱基)电转化入布鲁氏菌后,经过5μg/mL氯霉素筛选单交换子和70g/L蔗糖筛选同源重组双交换子,用菌落PCR及DNA测序的方法进行验证。结果表明,bp26基因的缺失改造成功,连续传20代后菌落PCR及DNA测序的结果显示突变株具有遗传稳定性。以pRE112自杀质粒为基础构建无抗性基因标记的布鲁氏菌缺失株为布鲁氏菌的基因功能研究奠定了基础,同时Δbp26缺失株的构建可为新型布鲁氏菌疫苗的研制奠定基础。Three unmarked gene deletion strains of Brucella spp.,S19-Δbp26,M5-Δbp26 and S2-Δbp26,were constructed by the allelic exchange introduced by the transformation of the pRE112 suicide plasmid containing counter-selection gene sacB.Firstly,the upstream and downstream fragments of bp26 gene were amplified from Brucella spp.genome and then subcloned into suicide plasmid pRE112 to construct the recombinant suicide vector pRE-Δbp26 with 681 bp-deleted bp26 gene fragment.The recombinant suicide vector pREΔbp26 was transformed by electroporation into Brucella spp.and the transformants were selected on TSB-YE plates containing 5 μg/mL chloramphenicol and then 70 g/L sucrose.The successful construction of bp26 gene deletion strains of Brucella spp.were confirmed by the bacterial colony PCR and DNA sequencing.Twenty generations of continuous passage of the S19-Δbp26,M5-Δbp26 and S2-Δbp26 strains showed genetic stability in vitro.The application of pRE112 provided a new suicide plasmid for the construction of unmarked gene deletion strain of Brucella spp.and these strains would be utilized for the study of gene function of Brucella.In addition,these bp26 gene deletion strains could be adapted to develop a live and attenuated Brucella carrier.

关 键 词：布鲁氏菌 bp26基因 自杀质粒 基因缺失株 

分 类 号：S852.614[农业科学—基础兽医学]