膜泡是细菌生长过程中自然释放的纳米级脂质结构，可携带细菌DNA和蛋白质等成分，在疫苗或疫苗载体研发中具有广阔应用前景；然而膜泡形成的调控机制复杂，如何提高产量是膜泡开发利用的关键瓶颈。我们前期研究发现，SigB(Q225P)突变可显著促进金葡菌膜泡产生，并经筛选发现耐热核酸酶nuc基因表达下调，生物信息学分析发现nuc基因调控区存在SigB可能调控位点，故推测金葡菌SigB(Q225P)突变通过直接下调Nuc表达，导致细菌DNA累积，促进细菌膜泡的形成。为证实该假设，本项目拟构建nuc基因敲除和回补菌株，通过膜泡分离和定量分析，探讨SigB(Q225P)突变通过Nuc调控金葡菌膜泡形成的作用；再通过基因报告系统、EMSA及蛋白-核酸相互作用检测，阐明SigB(Q225P)突变下调Nuc表达，进而促进膜泡形成的分子机制；并验证突变菌的高产膜泡疫苗质量，为高效制备细菌膜泡疫苗提供新的途径。

Release of membrane vesicles (MVs) is a ubiquitous process that occurs during normal bacterial growth, and MVs are spherical onreplicating nanoparticles with the diameter of 30−250 nm and contain phospholipid bilayers that are incorporated with various bacterial proteins and lumens carrying periplasmic constituents, such as DNA. MVs secreted by both Gram-positive and Gram-negative bacteria have since emerged as attractive and eective vaccines or delivery systems. The hypervesiculation is the key for MVs development and application, however, the mechanism underlying bacterial vesiculogenesis is complicated. In our previous study, we demonstrated a substitution mutation in the alternative sigma factor B, SigB(Q225P), promotes the production of Staphylococcus aureus MVs. The hypervesiculation was associated with downregulation of nuc gene that encodes thermonuclease. Bioinformatic analysis revealed a putative binding site of the SigB exits in the nuc promoter region. Therefore, we suppose that SigB(Q225P) promotes the production of Staphylococcus aureus MVs through downregulation of the nuc expression. To text this hypothesis, the nuc deletion mutant and complement S.aureus strains will be constructed to verify the role of SigB(Q225P) in promotion of the MVs production through controlling nuc expression; then, the underlying mechanism will be explored by a gene reporter system, EMSA, and DNA-protein interaction analysis. The results will not only be important for understanding the hypervesiculation in S. aureus, but also lay down a great foundation in the preparation and screen of high-level MVs production S. aureus engineering strains.