伤寒沙门菌rpoE基因缺陷变异株的制备及其生存能力

Construction of a rpoE Gene-Deleted Mutant in Salmonella Enterica Serovar Typhi and Its Survival Ability

目的为深入研究rpoE基因在伤寒沙门菌侵袭致病中的作用,制备rpoE基因缺陷变异株;观察rpoE基因缺陷变异株在应激条件下的生存能力。方法根据伤寒沙门菌rpoE基因序列,设计PCR特异性引物,制备rpoE基因缺陷性同源性核苷酸片段导入自杀质粒PGMB151后再导入伤寒沙门菌野生株,进行同源重组,用PCR观察重组现象;绘制生长曲线,对比rpoE基因缺陷变异株与野生株在应激条件下的生存情况。结果PCR及序列分析证实,缺陷变异株的rpoE基因缺失288个碱基;生长曲线提示rpoE基因缺陷变异株在氧、酸、高渗应激条件下生存能力明显低于野生株,差异有统计学意义(P<0.05)。结论成功构建了伤寒沙门菌rpoE基因缺陷株,并发现在氧、酸、高渗应激条件下其生存能力明显降低,为进一步研究其在伤寒沙门菌中的功能奠定了基础。

Objective To investigate the invasion and pathogenicity of rpoE gene, the deletion mutant of the rpoE gene was constructed in Salmonella enterica serovar Typhi; To observe the survival a- bility of rpoE mutant in stress. Methods As the genomic information, two pair＇s primers were designed, upper-and down-stream of the rpoE gene to amplify two homologous DNA fragments. The homologous recombinant DNA fragment of the defective target gene was cloned into the suicide plasmid pGMB151 which was then transferred into the target cell of S. enterica serovar Typhi GIFU 10007. The recombination was visualized by PCR;Under the stress conditions the rpoE mutant and parent the survival ability was prepared by drawing growth curve. Results A deletion of 288 bp of the rpoE gene was confirmed by PCR and sequencing analysis. Under the conditions such as oxidative stress,acid,high osmolarity,the rpoE mutant was significantly （P〈0.05） compromised compared to the parent. Conclusion The rpoE gene-deleted mutant of S. enterica serovar Typhi was generated successfully and it was found that the rpoE mutant was significantly （P〈0.05） compromised compared to the parent under the stress which was the foundation to study the function of the rpoE gene in S. enterica serovar Typhi.