中文摘要：

目的利用同源重组技术构建金黄色葡萄球菌RN4220附属基因调节系统（agr）的敲除株，探讨agr基因缺失对金黄色葡萄球菌分泌膜泡毒力的影响。方法 分别扩增agr基因的上下游同源臂，利用重叠PCR获得agr基因上下游同源臂的融合片段，经酶切后连接敲除载体PYT3，构建同源重组质粒pYT3：：Δagr，电转化至金黄色葡萄球菌RN4220，利用pYT3质粒对温度敏感的特点筛选agr缺失的突变株。分别制备野生菌RN4220和突变株RN4220：：Δagr的膜泡，小鼠毒力实验观察agr基因缺失后对金黄色葡萄球菌膜泡毒力的影响。 结果同源重组质粒pYT3：：Δagr通过酶切鉴定证明构建成功；经PCR及DNA测序证实获得金黄色葡萄球菌RN4220 agr缺失突变株，重组效率为5.6%；agr突变株与野生株相比，其分泌膜泡的毒力大大降低，72 h内小鼠存活率为100%。 结论 agr基因缺失后能够显著降低膜泡的毒力。

英文摘要：

Objective To construct the agr gene knockout mutant of Staphylococcus aureus （S. aureus） and evaluate the effect of agr on the virulence of secreting membrane vesicles. Methods A plasmid pYT3：：Δagr was constructed for homologous recombination of S. aureus and transferred into S. aureus strain RN4220. S. aureus RN4220 with the recombination vector was incubated at 42 ℃ and 25 ℃ alternatively to select the agr deletion mutant. Membrane vesicles were prepared from both RN4220：：Δagr mutant and the wild type strain, and the virulence of the membrane vesicles was evaluated after challenging BALB/c mice. ResultsRestriction endonucleases analysis indicated that the homologous recombination vector was correctly constructed and the agr deletion mutant was characterized by PCR and direct sequencing of the chromosomal DNA. The virulence of membrane vesicles prepared from the RN4220：：Δagr mutant （72 h mouse survival rate 100%） was significantly decreased as compared to that from the wild type RN4220. Conclusion The agr gene of S. aureus has great effect on the virulence of membrane vesicles. The construction of the RN4220：：Δagr mutant will lay down a solid foundation for preparation of safe staphylococcal membrane vesicle vaccine.