产肠毒素大肠杆菌F4ac型仔猪腹泻候选致因基因的功能验证

Enterotoxigenic Escherichia coli (ETEC) is one of the most important pathogenic bacteria causing diarrhea in neonatal piglets. In pig production, type F4ac of ETEC is the most prevalent pathogen. The pathogenicity of F4ac ETEC is mainly determined by the binding of F4ac to its receptor on intestinal epithelial cells of piglets. The non-adhesive piglets possess the resistibility to F4ac ETEC, however, the casual gene and casual mutation that resist swine diarrhea induced by ETEC F4ac are still not verified. This project first integrates the techniques and methods of protomics, functional genomics, bioinformatics, cytobiology and population genetics to select the F4ac receptors and new candidate casual genes for ETEC F4ac diarrhea by comparing the genome-wide differences of proteins expression between adhesive and non-adhesive Yorkshire piglets from one poplation at the samiliar ages; Then, based on our previous results of genome-wide association study and genome-wide microarray analysis to select the candidate genes of F4ac ETEC type dirrhea for functional verification. To validate the function of the selected candidate casual genes and mutations for F4ac ETEC diarrhea, gene Knock-out, overexpression and causal mutation Knock-in techniques will be performed with cell models of IPEC cell lines. Finally, genotyping the verified casual muations in a Yorkshire population, then, mating the sow and boar with heterozygous genotypes of the casual mutations and validating the function of the casual mutations in their offsprings. The results of the project will deeply reveal the molecular mechanism of piglet resistance to F4ac ETEC diarrhea, and promote the application of anti-disease breeding for diarrhea caused by F4ac ETEC in pig farming worldwide.

产肠毒素大肠杆菌（ETEC）是仔猪腹泻的主要致病菌，其中F4ac型ETEC流行性最强。其致病的关键步骤是菌毛黏附素F4ac与仔猪小肠粘膜组织刷状缘上的受体发生黏附，不黏附的仔猪则具有抗F4ac型ETEC特性。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。本项目首先综合运用蛋白质组学、分子遗传学、细胞生物学及群体遗传学等技术手段，以F4ac黏附及非黏附的同群同龄大白仔猪为试验对象，比较其小肠粘膜组织的蛋白质组表达差异，从功能基因组角度筛选F4ac型仔猪腹泻候选致因基因，结合前期全基因组关联分析获得的仔猪腹泻候选基因；以IPEC为细胞模型，利用基因敲除、过表达及致因突变定点敲入技术验证所选基因的功能，确证仔猪腹泻致因基因；在群体水平，以致因突变杂合基因型公母个体交配，验证致因突变功能，深入探讨ETEC F4ac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）是仔猪腹泻的主要致病菌，其中F4ac型ETEC流行性最强。迄今关于ETEC F4ac型仔猪腹泻的致因基因及突变尚未确定。本项目综合运用蛋白质组学、分子遗传学、细胞生物学及群体遗传学等技术手段，以F4ac黏附及非黏附的同群同龄大白仔猪为试验对象，通过比较其小肠粘膜组织的蛋白质组表达差异，从功能基因组角度筛选F4ac型仔猪腹泻候选致因基因；以IPEC为细胞模型，利用基因敲除、过表达及致因突变定点敲入技术验证候选基因的功能。通过ETEC F4ac抗性和易感仔猪小肠蛋白质组学分析，共发现245个差异表达蛋白，富集到唯一的整合素信号通路；建立了ETEC F4ac感染仔猪小肠上皮细胞的体外模型，确定体外攻菌的最佳攻菌浓度MOI为200:1，最佳攻菌时间为4h；利用CRISPR/Cas9技术对ITGB5基因进行敲除后并进行体外攻菌试验，发现ITGB5基因的缺失显著降低黏附到IPEC-J2细胞上的ETEC数目，而过表达会导致细菌黏附数的增加。本项目结果有助于我们深入探讨ETEC F4ac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。