利用Red重组系统敲除APEC毒力岛irp2基因

应用质粒pKD46介导的Red同源重组系统,以质粒pKD3为模板,设计irp2基因序列敲除引物,引物5'端有51 bp的拟敲除基因的同源臂,3 '端为扩增引物,扩增两侧含FRT位点的氯霉素抗性基因,通过第1次同源重组将拟敲除的irp2基因替换为氯霉素抗性基因,再通过重组酶质粒pCP20在FRT位点发生第2次同源重组,消除抗性基因.结果表明,利用该重组系统成功敲除了禽致病性大肠杆菌HPI毒力岛irp2基因.为深入研究irp2基因在禽致病性大肠杆菌致病过程中所发挥的作用打下基础.

Deletion of irp2 gene of avian pathogenic E.coli strain with HPI by red recombination

The aim of this research was to construct irp2 gene deletion mutant of avian pathogenic E.coli (APEC) which harbored the HPI by using Red recombination system. PCR products were obtained by using primers with 51 bp extension homologous to irp2 using template plasmid pKD3 which carrying chloramphenicol resistance gene flanked by FRT sites. With the plasmid pKD46 mediated Red recombinant system, the PCR products were transformed into APEC (CE02) by electroporation to recombine irp2 gene, and the recombinant was selected on chloramphenicol agar plate. Then the plasmid pCP20 which encoded the Flp recombinase was introduced into the recombinant to eliminate chloramphenicol resistance gene. The results showed that gene of irp2 deletion mutant of APEC was successfully constructed. The recombinant strains would be the foundation for the further research on the role of irp2 gene in pathogenic mechanism of APEC.