禽致病性大肠埃希菌强毒力岛(HPI)全岛缺失株的构建

Deletion of high pathogenicity island in chromosome of APEC by red recombination

目的构建大肠埃希菌强毒力岛(HPI)全岛缺失突变株,为进一步评价大肠埃希菌HPI的功能打下基础。方法根据已知大肠埃希菌HPI基因序列设计PCR敲除引物,引物5′端有50 bp的拟敲除基因的同源臂,3′端为扩增引物,以pKD3为模板,扩增两侧含FRT位点的氯霉素抗性基因,利用pKD46的λ重组系统替换E.coli ZL基因组上的毒力岛全岛基因,再利用表达Flp重组酶的质粒pCP20,可将FRT位点之间的氯霉素抗性基因删除,用鉴定引物进行鉴定并测序。结果构建的全岛缺失株与预期一致。结论成功构建了禽致病性大肠埃希菌强毒力岛(HPI)全岛缺失突变株。

Objective To construct E.coli mutants knockouted the gene encoding high pathogenicity island（HPI） in order to evaluate the function of HPI.Method First,PCR products were obtained by using primers with 50 bp extension which were homologous to HPI and by using template plasmid named pKD3 carrying chloramphenicol resistance gene flanked by FRT sites.Then the products were reformed by the program with the pKD46 recombination system replacing high pathogenicity island in the chromosome of E.coli ZL.Secondy,the strains expressing chloramphenicol resistance gene were selected by chloramphenicol agar.The chloramphenicol resistance gene was then eliminated by using a helper plasmid pCP20.Finally,the purpose gene was identificated and sequenced.Result The constructed mutants were consistent with the expectations.Conclusion The construction of the mutants deleted HPI in chromosome of Escherichia coli was completed successfully.