仔猪产肠毒素大肠杆菌（ETEC）F4ac受体基因的鉴定及功能验证

发布时间：2018-06-11 13:02

  本文选题：仔猪腹泻 + ETEC　； 参考：《中国农业大学》2016年博士论文

【摘要】：产肠毒素大肠杆菌(enterotoxigenic Escherichia coli, ETEC) F4ac感染是导致仔猪细菌性腹泻最重要的一类病原菌。决定仔猪对ETEC F4ac抗性与否的关键是小肠上皮细胞受体蛋白的有无,若有,仔猪表现为易感,若无则表现为抗性。本研究针对ETEC F4ac受体基因的筛选、ETEC F4ac感染仔猪上皮细胞体外模型的建立及候选基因的功能验证展开了研究,深入探讨ETEC F4ac型仔猪腹泻致因基因表达的分子机制。试验一,选取4对ETEC F4ac抗性和易感的全同胞仔猪为试验对象,采用iTRAQ蛋白质组定量技术鉴定其小肠蛋白表达差异。共发现245个差异表达蛋白,其中黏附组相对于非黏附组表达上调的有117个,表达下调的有128个。对差异表达蛋白进行Pathway富集分析,结果富集到唯一的整合素信号通路(Integrin signalling pathway:P00034),说明该通路对ETEC F4ac感染仔猪小肠黏膜进而引起仔猪腹泻具有重要作用。从基因位于染色体上的位置、基因的分子功能、基因的蛋白质分子量大小角度对候选基因进行分析,发现TTGB5最有可能为ETEC F4acR的候选基因。试验二,仔猪小肠上皮细胞系IPEC-J2是唯一一株取自猪空肠上皮细胞且未转化未癌化的小肠上皮细胞系,正越来越多的被应用于肠道细菌与上皮细胞的互作研究中。通过RT-PCR的方法检测ETEC F4ac对仔猪小肠上皮细胞IPEC-J2的黏附,结果表明,当大肠杆菌菌液浓度在1×105-1×109CFU/ml范围之间时,菌液浓度与Ct值之间线性方程为y=-0.3056x+13.418。通过以不同攻菌浓度和攻菌时间对IPEC-J2进行攻菌,确定了ETEC F4ac进行体外攻菌的最佳攻菌浓度为MOI为200：1,最佳攻菌时间为4h。该方法与平板菌落计数法相比较更省时省力,且定量准确,重复性好。试验三,选取ITGB5、MUC4和MUC13作为ETEC F4acR候选基因,同时选择无关基因SLC12A8作为阴性对照,利用CRISPR/Cas9基因敲除技术对每个基因分别进行缺失突变。对基因敲除成功的细胞进行体外攻菌试验,结果表明TGB5基因的缺失显著降低黏附到IPEC-J2细胞上的ETEC数目。为更加全面的了解JTGB5基因对ETEC F4ac黏附到小肠上皮细胞的影响,构建ITGB5基因真核细胞表达载体来反向验证基因的功能。黏附试验结果表明ITGB5基因的过表达会导致细菌黏附数的增加。本研究为深入挖掘ETEC F4acR基因及其所在信号通路提供了数据支持和科学依据。
[Abstract]:Enterotoxigenic Escherichia coli (ETEC-F4ac) infection is the most important pathogen causing bacterial diarrhea in piglets. The key to determine the resistance of piglets to ETEC F4ac is the presence or absence of small intestinal epithelial cell receptor proteins. If there are, piglets are susceptible to ETEC F4ac, and if not, they are resistant. In this study, the screening of ETEC F4ac receptor gene and the establishment of in vitro model of ETEC F4ac infected piglet epithelial cells and the functional verification of candidate genes were studied, and the molecular mechanism of ETEC F4ac type diarrhea induced gene expression in piglets was studied. In the first experiment, four whole sibling piglets with ETEC F4ac resistance and susceptibility were selected as experimental objects, and their small intestine protein expression was identified by using the technique of iTRAQ proteome quantification. A total of 245 differentially expressed proteins were found, of which 117 were up-regulated and 128 down-regulated in adhesion group compared with non-adhesion group. The differentially expressed proteins were enriched by Pathway. The results showed that integrin signalling pathway: P00034 was the only integrin signaling pathway, which indicated that this pathway played an important role in infecting the intestinal mucosa of piglets with ETEC F4ac and causing diarrhea in piglets. According to the position of the gene on the chromosome, the molecular function of the gene and the molecular weight of the protein, the candidate gene was found to be the most likely candidate gene for ETEC F4acR. In experiment 2, IPEC-J2, a porcine intestinal epithelial cell line, is the only cell line derived from porcine jejunal epithelium and has not been transformed into non-cancerous intestinal epithelial cell line. It is being used more and more in the study of the interaction between intestinal bacteria and epithelial cells. The adhesion of ETEC F4ac to IPEC-J2 was detected by RT-PCR. The results showed that when the concentration of Escherichia coli was in the range of 1 脳 105-1 脳 109CFU / ml, the linear equation between the concentration of Escherichia coli and the Ct value was yang-0.3056x 13.418. The optimum attack concentration of ETEC F4ac on IPEC-J2 was determined to be 200: 1, and the optimal attack time was 4 h. Compared with the plate colony counting method, this method saves more time and effort, and is accurate and reproducible. In experiment 3, ITGB5, MUC4 and MUC13 were selected as candidate genes for ETEC F4acR and SLC12A8 as negative control. CRISPRR / Cas9 gene knockout technique was used to carry out deletion mutation of each gene. The results showed that the deletion of TGB5 gene significantly reduced the number of ETEC adhered to IPEC-J2 cells. In order to fully understand the effect of JTGB5 gene on the adhesion of ETEC F4ac to intestinal epithelial cells, the eukaryotic expression vector of ITGB5 gene was constructed to reverse verify the function of the gene. The results of adhesion test showed that the overexpression of ITGB5 gene resulted in the increase of bacterial adhesion. This study provides data support and scientific basis for further mining ETEC F4acR gene and its signaling pathway.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61
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本文编号：2005338