鸭疫里氏杆菌CH3株ompA基因缺失株的构建及其部分生物学特性的研究  

Construction of ompAgene deletion mutant of Riemerella anatipestifer CH3 strain and study on the biological characteristics of the mutant

作　　者：崔俊生[1,2] 姜盼[2] 孙冰清[1,2] 陶敏捷 薛亚飞[2] 丁云竹 吴金节[1] 胡青海[2] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医科学》2015年第11期1171-1176,共6页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31072156;31272590;31472224)

摘　　要：为了分析OmpA缺失对鸭疫里氏杆菌生物学特性的影响,采用自杀质粒同源重组的方法构建了CH3株的ompA基因缺失株。用PCR方法扩增ompA基因左右两侧的同源臂和壮观霉素抗性基因表达盒后连入自杀性质粒pDS132,构建得到重组自杀质粒pDS132-ompA-LSR。再通过双亲本接合转导的方法将此重组质粒导入到CH3株,用含卡那霉素和壮观霉素的TSA筛选可能的基因缺失株,并用PCR和Western-blot进行鉴定,获得基因缺失株CH3ΔompA。生物学特性分析表明,OmpA缺失后鸭疫里氏杆菌对盐离子浓度的耐受能力降低、对抗生素的敏感性增强,而对去污剂SDS、EDTA和酸碱(pH)耐受性没有明显的影响。To analyze the effect of ompA gene deletion on the biological characteristics of Riemerella anatipestifer,ompAgene of strain CH3 was deleted using homologous recombination with a suicide plasmid.The left and right flanking sequences of ompAgene,and a spectinomycin resistance cassette were amplified by PCR,respectively.These three fragments were ligated into the suicide vector pDS132 to produce a recombinant suicide plasmid pDS132-ompA-LSR.A bi-parental conjugation using Escherichia coli S 17-1（pDS132-ompA-LSR）as the donor strain into R.anatipestifer CH3 as the recipient strain CH3.Transconjugants were selected on TSA agar in the presence of kanamycin and spectinomycin.The deletion of the ompAgene was confirmed by PCR amplification and Western-blot,and the mutant strain was designated CH3ΔompA.Analysis of the biological characteristics showed that the reduced tolerance to high concentration of NaCl and increased sensitivity to some antibiotics of the mutant CH3ΔompA was observed,as compared to those of the wild-type CH3,but there was no obvious effect on their tolerance to SDS,EDTA and pH in TSB medium.

关 键 词：鸭疫里氏杆菌 ompA基因 缺失突变株 自杀质粒 

分 类 号：S852.612[农业科学—基础兽医学]