牛支原体分泌MbovP475进入巨噬细胞核内抑制其增殖的分子机制研究

牛支原体是严重危害养牛业的重要病原体，可引起肺炎、乳腺炎等。牛支原体致病机制不清楚是导致牛支原体病缺少特异高效防治措施的根本原因。申请人前期研究鉴定牛支原体MbovP475为分泌脂蛋白，并发现该蛋白在牛支原体感染牛巨噬细胞（BoMac）后进入细胞核，抑制BoMac增殖。本研究首先通过ChIP-Seq筛选MbovP475蛋白互作基因；然后利用qRT-PCR及Western blot验证MbovP475调控互作基因表达；进一步利用凝胶迁移及Southwestern等技术验证MbovP475蛋白与宿主靶标基因启动子序列结合；并检测靶基因敲降细胞感染牛支原体后的细胞增殖；最终确定MbovP475抑制BoMac细胞增殖的分子机制是通过结合靶基因启动子序列直接调控增殖相关的靶基因表达。鉴于巨噬细胞在先天性和获得性免疫中的重要作用，该研究对阐明牛支原体致病机制、发掘新型药物、疫苗和诊断靶标具有重要意义。

Mycoplasma bovis is the important pathogen which threats the development of cattle industry, and can cause pneumonia and mastitis etc. The unclear pathogenesis makes it is very difficult to control and cure the disease caused by M. bovis. Our previous study identified the secreted MbovP475 which is a nuclear importing protein of M. bovis. It entered into the nuclear of the infected bovine macrophage cell line BoMac cells during M. bovis infection and inhibited the cell proliferation. This research project is aimed to explore the molecular mechanism of MbovP475 in inhibiting BoMac proliferation by investigating on the interaction between this protein and BoMac DNA. ChIP-Seq technique will be applied to identify host genes that MbovP475 bind to. Then the regulation of host gene expression was verified by qRT-PCR and western blot assay. Further the protein-DNA interaction will be verified by southwestern blot assay and EMSA; Finally, the host target genes will be determined to be associated with cell proliferation by RNAi and proliferation assay and MbovP475 will be demonstrated to inhibit BoMac proliferation through binding to the promoter fragments of the host target genes whose expression was regulated. Since macrophages are critical to innate and acquired immunity, the findings will be of significance in elucidating M. bovis pathogenesis and revealing novel targets for developing new drugs, vaccines and diagnostic reagents.