双组分信号转导系统CpxA/CpxR调控副猪嗜血杆菌致病性的分子机制研究

Haemophilus parasuis, the causative agent of swine polyserositis, polyarthritis, and meningitis, is one of the most important bacterial diseases of pigs worldwide. Expounding the molecular mechanism of the two-component signal transduction system CpxA/CpxR regulation HPS pathogenicity is important for prevention and control of the disease. Through bioinformatics for CpxA/CpxR, construction CpxA mutant and pathogenicity in mice, the results showed that the CpxA/CpxR was associated with pathogenicity. Therefore, the CpxA/CpxR is studied as the research object. And then the differential expressed virulence related genes are screened by constructing CpxA, CpxR mutant, RNA-seq and bioinformatic analysis and are validated by fluorescence quantitative PCR. Hence the virulence related genes and binding sites by CpxR directly regulated are identified through electrophoretic mobility shift assay, primer extension, Dnase I footprinting experiments and CHIP-Seq, SPR instrument. Further the directed regulated virulence related genes are carried pathogenicity experiments. Through above study, the molecular mechanism of CpxA/CpxR regulation of pathogenicity of HPS are finally elucidated, which provides the foundation for the development of new vaccines and screening drug target.

副猪嗜血杆菌（HPS），能够引起猪的浆膜炎、关节炎和脑膜炎，是当前危害世界养猪业最重要的细菌性疾病病原之一。阐明双组分信号转导系统CpxA/CpxR调控HPS致病性的分子机制对于预防和控制该病具有重要意义。运用生物信息学对CpxA/CpxR进行分析，构建CpxA突变株并进行小鼠致病性实验，结果表明CpxA/CpxR与致病性相关。因此，本项目拟以CpxA/CpxR为研究对象，通过构建CpxA、CpxR突变株、RNA-seq及生物信息学分析，筛选毒力相关差异表达基因并利用荧光定量PCR进行验证。通过凝胶迁移、引物延伸、DNase I足迹法等实验和CHIP-Seq、SPR仪等筛选CpxR直接调控毒力相关基因及结合位点，并且进一步对直接调控毒力相关基因开展致病性实验。通过以上研究，最终解释CpxA/CpxR调控HPS致病性的分子机制，为HPS新型疫苗开发和药物靶标筛选奠定基础。

副猪嗜血杆菌（HPS），在临床上能够引起猪的浆膜炎、关节炎和脑膜炎，是当前危害世界养猪业最重要的细菌性疾病病原之一。本项目选取了CpxA/CpxR双组分信号转导系统进行了详细的研究，对于阐明HPS致病性的分子机制重要意义。本项目构建了CpxR突变株，证明了CpxR与HPS毒力相关。运用RNA-seq技术筛选了CpxR突变株可能调控的毒力相关差异表达基因并利用荧光定量PCR进行验证。通过实验研究确定了CpxR直接调控的毒力相关基因fis，并且进一步对直接调控毒力相关基因fis开展了致病性实验。通过以上研究结果初步阐明了CpxA/CpxR调控HPS致病性的分子机制，为HPS新型疫苗开发和药物靶标筛选奠定基础。