PADI2基因在绵羊肺炎支原体感染巨噬细胞过程中的作用机制研究

绵羊肺炎支原体是引起绵羊患传染性胸膜肺炎的病原微生物，其传播速度日趋严重是当前我国养羊业面临重大现实问题。PADI2基因编码一个催化蛋白瓜氨酸化的蛋白酶，已在人和小鼠巨噬细胞抗病菌感染过程中被证实发挥重要促炎作用。申请人在之前研究中通过全基因组测序数据发现PADI2基因可能在绵羊抗支原体肺炎过程中发挥重要调控作用，但目前其作用机制尚不清楚。本项目拟在绵羊肺炎支原体感染绵羊原代肺泡巨噬细胞中检测PADI2基因表达变化，明确该基因与绵羊肺炎支原体感染过程相关性；利用CRISPR/Cas9系统制备敲除PADI2基因肺泡巨噬细胞，采用绵羊肺炎支原体感染细胞，从三个方面研究：细胞凋亡、粘附性、促炎因子和瓜氨酸化蛋白表达水平变化；细胞吞噬和自噬功能变化；细胞转录组动态变化。本项目不仅有助于深入了解绵羊肺炎支原体致病机理，也为PADI2基因作为分子标记在绵羊抗病育种中利用奠定理论基础。

Mycoplasma ovipneumoniae (MO) has been identified as a pathogenic agent of sheep contagious pleuropneumonia, and the continuing epidemic of its has currently become a serious problem in Chinese sheep industry. PADI2 gene encodes a protease that causes protein citrullination. It had been reported that the gene play a crucial role in regulating the proinflammatory process of anti-bacterial infection in human and mouse macrophages. In the previous study, the applicant found that PADI2 gene might play an important role in regulating the process of resistance to MO in sheep based on whole-genome sequencing data. To data, the underlying mechanism of PADI2 gene in sheep infected with MO remains unclear. In the current project, we will detect the change in PADI2 gene expression in sheep primary alveolar macrophages infected with MO to confirm the correlation between the gene and the infection process of MO. Then, CRISPR/Cas9 system will be used to knockout PADI2 gene in alveolar macrophages. The cells will be infected with MO, and the studies from three aspects will be performed: observe the change of cell apoptosis and adhesion, cytokines secretion and citrullinated protein expression; detect the phagocytosis and autophagy function of the macrophages; investigate the dynamic changes in cell transcriptomes. This project will not only provide deeper insight into the pathogenesis of MO, but also lay theoretical basis for PADI2 gene used as molecular marker in disease resistance breeding in sheep.