肠聚集性大肠杆菌ybtE基因缺失突变株的构建

Construction of the ybtE gene-deleting mutant of enteroaggrerative E.coli 17-2 strain

目的构建肠聚集性大肠杆菌ybtE缺失突变株,为下一步ybtE基因功能的研究奠定基础。方法应用PCR扩增肠聚集性大肠杆菌的ybtE基因,将其克隆至pUC18载体,酶切缺失988bp ybtE片段,并插入998bp的卡那霉素抗性基因,利用定向克隆技术将突变的ybtE片段克隆至自杀质粒pKTN701,形成重组自杀质粒。将携带重组自杀质粒的SM10λpir与EAEC17-2进行接合转移,利用同源重组,根据卡那霉素抗性筛选并鉴定接合子。结果经PCR、酶切和探针杂交鉴定,得到2株肠聚集性大肠杆菌ybtE缺失突变株。结论成功构建肠聚集性大肠杆菌ybtE基因缺失突变株。

In order to construct the ybtE gene-deleting mutant of enteroaggregrative E. coli 17-2 strain EAEC17-2 for the further studies on the function of this gene, the ybtE gene was firstly amplified by PCR technique from EAEC 17-2 and then cloned into vector pUC18, thus to construct a recombinant plasmid pUC18-E. After the reeombinant plasmid pUC18-E was digested by Hpa I, the 988 bp fragment was missing from the ybtE gene and then a 988 bp kanamyein-resistant gene（kan） was introduced into ybtE through ligase reaction. The mutative gene was cloned to suicide plasmid pKTN701, resulting in the recombinant suicide pKTN-KE, which was then introduced into EAEC 17-2 strain by conjugation transfer. By identification with PCR, restriction analysis and dot hybridization, two ybtE-deleeting mutants of enteroaggrerative E. col i were thus obtained and these strains can be used for the further studies.