大肠杆菌-钩端螺旋体重组穿梭质粒pGKINVA的构建及重组钩体的筛选  

Construction of Recombinant E. coli-L. interrogans Shuttle Plasmid pGKINVA and Screening Recombinant Leptospira Strains

作　　者：朱庆平[1] 鲍朗[1] 张会东[1] 赵明才[1] 

机构地区：[1]四川大学华西基础医学与法医学院感染免疫研究室,成都610041

出　　处：《四川大学学报（医学版）》2007年第1期81-83,共3页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30471546)资助

摘　　要：目的构建大肠杆菌-钩体穿梭质粒pGK INVA,并重组于钩体PatocⅠ株,为进一步确定钩体invA基因的功能奠定基础。方法将invA基因克隆于pGK b le24载体,PCR、限制性内切酶双酶切及测序鉴定重组穿梭质粒pGK INVA。质粒pGK INVA经电穿孔法转化无毒钩体PatocⅠ株,并筛选重组钩体菌株。结果从钩体基因组中克隆出长558 bp的invA基因,定向插入pGK b le24构建重组穿梭质粒pGK INVA,抗生素及蓝/白斑筛选和测序鉴定并获得正确的重组质粒后,电穿孔法转化钩体PatocⅠ株,在K orthof固体培养基生长并抗生素筛选、PCR鉴定出重组钩体菌株。结论成功构建重组大肠杆菌-钩端螺旋体穿梭质粒pGK INVA,并筛选出2个重组钩体菌株,将有助于进一步阐明invA基因在钩体体内中的功能分析。Objective To further investigate pathogenic function of invA in Leptospira, recombinant L. biflexa-Escherichia coli shuttle vector pGKINVA was constructed, and electropored into L. biflexa strain Patoc Ⅰ . Methods The invA gene was cloned into shuttle vector pGKble24 to construct a recombinant L. biflexa- Escherichia coli shuttle vector pGKINVA. The recombinant vectors were electropored into L. biflexa serovar Patoc strain Patoc Ⅰ . Results A 558 bp invA was amplified from L. interrogans serovars Lai strain 017 and ligated with Aat Ⅱ and Xma Ⅰ digested pGKble24. After screening by kanamycin and blue/white color, the recombinant pGKINVA was obtained and sequenced. Subsequently, the recombinant pGKINVA was electropored into L. bijQexa strain Patoc Ⅰ. The selected mutants were identified by PCR. Conclusion Recombinant L. biflexa- Escherichia coil shuttle vector with invA insert was constructed and two strains of pGKINVA transformed Leptospira were obtained. These pGKINVA transformed Leptospira strains would provid an experimental model to investigate potential pathogenic functions of invA in vivo.

关 键 词：invA基因 大肠杆菌-钩端螺旋体重组穿梭载体 钩端螺旋体 

分 类 号：R346[医药卫生—基础医学]