肠出血性大肠杆菌O157:H7 z0986-z1444双缺失突变株构建

摘要

Objective To construct the double deletion mutant of enterohemorrhagic Escherichia coli( EHEC) O157: H7 by knocking out z0986 gene and z1444 gene with Red recombinant system. Methods Firstly, the target frag-ments of z0986 and z1444 were established. Secondly, competent cells were struck by lightning, then the bacteria were identified through PCR. The growth curve of the ultimate positive monoclone was assayed. Results The solo deletion mutant(Δz1444/933) and the double deletion mutant(Δz0986/Δz1444/933) were successfully construc-ted and their growth curves had no significant difference compared to the wild type EHEC. Conclusion The dele-tion mutant of z0986 and z1444 has no effect on growth of EHEC. The construction of Δz1444/933 as well asΔz0986/Δz1444/933 would be helpful for further study of functions and mechanisms of z1444 gene.%目的 采用Red重组方法敲除肠出血性大肠杆菌(EHEC)O157:H7 z0986和z1444基因,构建双缺失突变株.方法 首先构建z0986、z1444打靶片段;其次以电击法转化感受态细胞,之后通过PCR鉴定所得菌株;最终所得阳性单克隆测定生长曲线.结果 z1444单基因缺失突变株(Δz1444/933)及z0986-z1444双基因缺失突变株(Δz0986/Δz1444/933)构建成功,其生长曲线与野生型EHEC O157:H7差异均无统计学意义.结论 z0986与z1444基因缺失对EHEC的生长无影响.构建Δz1444/933及Δz0986/Δz1444/933为深入研究z1444基因的功能及作用机制奠定了基础.