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目的构建HPI毒力岛缺失的EAggEC172突变株,初步研究EAggEC菌株携带的耶尔森菌HPI毒力岛合成铁载体Ybt的功能。方法以EAggEC172为出发菌株,irp8基因部分序列作为同源重组的一侧序列,irp5基因序列作为同源重组的另一侧序列,中间插入有氯霉素(Cm)抗性基因(cat基因)标记。通过接合转移和同源重组,构建了缺失约24kb的HPI毒力岛功能核心区区域EAggEC172的全岛缺失株EA85。应用流式细胞技术(FACS)检测指示菌株WACSirp1::KN(pCJG3.3N)荧光强度的变化情况,对EA85缺失株和出发菌株进行了合成Ybt的功能比较研究。结果成功构建了EAggEC172HPI全岛缺失株EA85。EAggEC172菌株具有表达Ybt的功能,而缺失株EA85丧失了合成Ybt的能力。结论EAggEC172HPI毒力岛的缺失,使Ybt的合成彻底阻断。EAggEC172具有的合成Ybt的功能是由其染色体携带的HPI毒力岛所决定的。
Objective To construct the EAggEC172 mutant with deletion of HPI virulence island and study the function of Ybt of Yersinia pestis HPI virulence island carried by EAggEC strain. Methods EAggEC172 was used as a starting strain, partial sequence of irp8 gene was homologous recombination side sequence, irp5 gene sequence was homologous recombination sequence of the other side, with chloramphenicol resistance gene (cat gene) . The whole island deletion strain EA85 of EAggEC172, a core region of HPI virulence domain lacking approximately 24 kb, was constructed by junctional transfer and homologous recombination. The fluorescence intensity of the indicator strain WACSirp1 :: KN (pCJG3.3N) was detected by flow cytometry (FACS), and the function of Ybt was compared between the EA85 deletion strain and the starting strain. Results EAggEC172HPI deletion mutant EA85 was successfully constructed. The EAggEC172 strain has the function of expressing Ybt, whereas the deletion strain EA85 loses its ability to synthesize Ybt. Conclusion The deletion of EAggEC172HPI virulence island completely blocked the synthesis of Ybt. The function of EAggEC172 to synthesize Ybt is determined by the HPI virulence island carried by its chromosome.