鸡肠炎沙门氏菌转座突变体库的构建及减毒菌株的筛选  

Construction of a transposon-mediated mutagenesis library in Salmonella enterica Serovar Enteritidis and screening for the attenuated strains

作　　者：吕爽[1] 朱婷[1] 王秀梅[1] 刘慧芳[1] 司微[1] 于申业[1] 陈利苹[1] 刘思国[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物细菌病研究室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2013年第3期210-213,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家高技术研究发展计划(863)项目(2011AA10A210)

摘　　要：为筛选减毒肠炎沙门氏菌(S.enteritidis)菌株,本研究以含有Mini-Tn5转座子的重组自杀质粒pUT对S.enteritidis SM6株进行转座诱变,利用双亲本滤膜杂交方法与供体菌E.coliβ2155进行接合转移,构建并优化接合转移体系,利用卡那抗性和DAP营养缺陷培养平板筛选法构建了SM6的随机转座突变体库。其中包含5个子库,共计获得364株突变体,经PCR鉴定表明转座子均插入SM6株的基因组中。通过Caco-2细胞侵袭试验筛选S.enteritidis侵袭力减弱并在细胞内增殖受到抑制的减毒突变株,经初筛及验证最终获得3株稳定减毒菌株,为进一步研究减毒菌株突变毒力基因的定位及SM6株的侵袭及感染机制奠定了基础。For screening the attenuated strains of Salmonella enteritidis, a mutagenesis library was constructed by conjugal transfer method. Under the optimized conjugation conditions, the filter hybridization was proceeded between recipient bacteria SM6 and donor bacteria E.coli 132155 carrying a recombinant suicide plasmid pUT-MiniTn5-Km2. The mutants were preliminarily screened by kanamycin resistance and DAP heterotrophia and further identified by PCR. A total of 364 mutants with Tn5 transposon inserting at different loci were obtained from the S.enteritidis mutagenesis library. They were investigated in epithelial cell Caco-2 to test the invasion ability. With primary screening and repeated verification, eventually 3 stable attenuated strains showed poorly invasion and proliferation in cells. These results provided essential basis to further study on the virulence gene location in the mutants and the invasion mechanism of SM6 strain.

关 键 词：肠炎沙门氏菌 转座子 突变体库 减毒菌株 

分 类 号：S852.61[农业科学—基础兽医学]