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.        Results       Restriction 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.