PA0833是课题组前期通过反向疫苗学技术筛选得到的一个铜绿假单胞菌疫苗候选抗原，在动物实验中表现出良好的免疫原性和免疫保护效果，优于已进入临床试验的疫苗抗原OprF。被动免疫和B细胞敲除小鼠实验结果提示其主要通过抗体介导的免疫应答发挥免疫保护效果，但具体机制并不清楚。进一步研究发现PA0833滴鼻免疫时能够诱发肺组织明显的炎症反应，其潜在的致病性严重限制了其应用。本项目拟在前期研究基础上，构建PA0833基因敲除株和回复突变株，通过与野生株的比较研究，进一步对PA0833的致病性进行验证。同时，解析得到PA0833蛋白的晶体结构，通过结构比对和分析明确其结构表面的关键氨基酸残基等信息，设计制备相应的突变体并对突变体的致病性和免疫保护效果进行评价，得到安全有效的突变体蛋白，在此基础上深入探讨PA0833通过抗体介导的免疫保护的具体机制，为铜绿假单胞菌疫苗抗原的筛选和疫苗的成功制备奠定基础。

PA0833 is a candidate antigen of Pseudomonas aeruginosa vaccine identified by our group previously based on reverse vaccine technology. It exhibits stronger immunogenicity and protective efficacy than OprF, a candidate antigen that has already been evaluated by clinical trials. The results from passive immunization and B cell knockout mice suggested that PA0833 specific antibody plays a key role in PA0833 mediated protection, but the mechanism remains unclear. Further study revealed that nasal immunization in mice with PA0833 resulted in obvious inflammatory response in lung tissue, and the potential pathogenicity limited its application for vaccine development. In this study, we will construct the PA0833 gene knockout strain and rescue strain of Pseudomonas aeruginosa strain PAO1 firstly, and to verify the pathogenicity of PA0833 by comparative studies with the wild type strain. At the same time, we will determine the crystal structure of PA0833, which will be used as a tool to analyze and identify the key amino acid residues that may be involved in the pathogenicity of the protein. The corresponding mutants will be designed and prepared, and the pathogenicity and protective efficacy of these mutants will be evaluated, in this way to obtain a safe and effective mutant of PA0833 for vaccine development. Finally, we will systematically investigate the mechanism of PA0833 mediated protection. This study will lay a solid foundation for the development of vaccine against Pseudomonas aeruginosa infection.