VTN基因介导整合素信号通路调控产肠毒素大肠杆菌F4ac型仔猪腹泻的分子机制

Enterotoxigenic Escherichia coli (ETEC) F4ac is one of the most important pathogenic bacteria causing diarrhea in neonatal piglets. The pathogenicity of ETEC F4ac is mainly determined by its binding capacity of adhesin to the receptor on intestinal epithelial cells, and piglets with non-adhesive trait possess the resistibility to ETEC F4ac. However, the host receptor gene and its mutation sites giving rise to the resistance of ETEC F4ac-induced diarrhea are still obscure. Integrin protein encoded by ITGB5 gene have been verified involved in the adherence of ETEC F4ac to host cell in our previous study, and vitronectin have a significant expression difference with resistant and susceptible piglets. This results suggesting that ETEC F4ac may interact with integrin by using vitronectin as bridging molecule. In this project, ETEC F4ac receptors on porcine intestinal epithelial cells will be identified by Y2H and BiFC experiments. Then, gene function will be verified in IPEC-J2 cell lines as in vitro by gene knockout and overexpression techniques. Finally, association studies will be performed to determine the key mutation sites associated with non-adhesive trait, and CRISPR/Cas9 site directed mutagenesis will be used to validate the function and mutations of the ETEC F4ac receptor gene. This project will illuminate the molecular mechanism of piglet resistance to F4ac ETEC induced diarrhea, and provide scientific reference to the anti-disease breeding in pig farming worldwide.

产肠毒素大肠杆菌（ ETEC）F4ac是导致仔猪细菌性腹泻最重要的一类病原菌，其致病的关键步骤是菌毛黏附素与仔猪小肠粘膜组织刷状缘上的受体发生黏附。迄今关于ETEC F4ac型仔猪腹泻的致因基因及致因突变尚未确定。我们前期研究发现整合素参与ETEC F4ac的黏附和定植，且玻连蛋白在抗性和易感的仔猪小肠组织表达存在显著性差异，提示ETEC F4ac可能是通过玻连蛋白的桥梁作用结合于小肠上皮细胞整合素受体蛋白上。本项目拟首先运用酵母双杂交和双分子荧光互补分析技术确定ETEC F4ac黏附于猪小肠上皮细胞的受体基因；然后以IPEC-J2为细胞模型，利用基因敲除、过表达等手段对受体基因进行效应分析；最后关联分析确定关键突变位点，并利用CRISPR/Cas9基因定点突变技术对致因突变进行体外功能验证。研究结果将明确ETEC F4ac感染引起仔猪腹泻的分子机制，为仔猪腹泻的分子抗病育种提供参考依据。

产肠毒素大肠杆菌（ ETEC）F4ac是导致仔猪细菌性腹泻最重要的一类病原菌，其致病的关键步骤是菌毛黏附素与仔猪小肠粘膜组织刷状缘上的受体发生黏附。迄今关于ETEC F4ac型仔猪腹泻的致因基因及致因突变尚未确定。本研究利用GST-Pull-down、基因敲除等技术，探究ETEC F4ac感染引起仔猪腹泻的分子机制。主要研究结果包括：1）明确了ITGB5为ETEC F4ac黏附小肠上皮细胞的受体基因；2）利用RACE技术克隆了猪的完整的ITGB5基因；3）利用CRISPR/Cas9技术构建了ITGB5基因小肠条件性敲除flox小鼠；4）鉴定了可能影响ETEC F4ac黏附小肠上皮细胞的甲基化位点。