胞内劳森菌效应蛋白及靶标蛋白鉴定及功能研究

proliferative enteropathy is an important disease of commercial pig production worldwide. Lawsonia intracellularis, a fastidious anaerobic gram-negative intracellular bacterium, is the generally accepted pathogenesis. The infection is hypothesized to infect the mitotically active cells, induces cells proliferation of crypt epithelial and inhibits their differentiation into mature enterocytes, while the initial pathogenesis of L. intracellularis infection is to a large extent unexplored. Analysis of the genome sequence of the L. intracellularis strain PHE/MN1-00 has revealed the presence of a T3SS secretion system in this bacterium，while none effectors has been reported. In this work, we try to select potential L. intracellularis effectors through bioinformatics methods and sequence analysis, screen the effectors using the budding yeast Saccharomyces cerevisiae as the alternative host; verify the phenotype in the porcine intestinal epithelial cells, screen and identify the target proteins of effectors through GST-pull down method, use RT-PCR to detect transcription levels of the effectors and the target proteins in the intestinal epithelial tissue samples of proliferative enteropathyof ,and finally analysis the correlationship. The completion of the project help to reveal the biological function of L. intracellularis effectors and its target proteins, to develop the understanding of the pathogenic mechanism of L. intracellularis, and provide guidance for prevention and control of porcine proliferative enteritis.

猪增生性回肠炎是一种世界各地广泛流行、给养猪业造成严重经济损失的常见猪病，其病原为胞内劳森菌。劳森菌侵染猪肠上皮细胞，使其持续进行有丝分裂、高度增生但不能成熟，形成猪增生性肠病，但作用的分子机制不明。现有研究对劳森菌进行序列分析发现其存在保守的Ⅲ型分泌系统，但分泌底物-作用于宿主细胞的效应蛋白还未有报道。本研究尝试用生物信息学方法挑选可能的效应蛋白；用酿酒酵母作为替代宿主初步筛选促进酵母生长的劳森菌基因；用猪肠上皮细胞进行表型验证和定位观察；用GST-Pull down方法来筛选和鉴定与效应蛋白相互作用的猪肠上皮细胞靶标蛋白；最后用RT-PCR检测临床样品中，病程不同的增生性回肠炎患猪肠上皮组织、同一患猪小肠病灶上皮组织及周围正常组织的劳森菌效应蛋白与靶标蛋白的转录水平，并进行相关性分析。本项目的完成，有助于揭示劳森菌效应蛋白和靶标蛋白在增生性回肠炎中的生物学功能，为它的防治提供新的思路。