本文選題：仔豬腹瀉 + ETEC�。� 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：產(chǎn)腸毒素大腸桿菌(enterotoxigenic Escherichia coli, ETEC) F4ac感染是導(dǎo)致仔豬細(xì)菌性腹瀉最重要的一類病原菌。決定仔豬對(duì)ETEC F4ac抗性與否的關(guān)鍵是小腸上皮細(xì)胞受體蛋白的有無(wú),若有,仔豬表現(xiàn)為易感,若無(wú)則表現(xiàn)為抗性。本研究針對(duì)ETEC F4ac受體基因的篩選、ETEC F4ac感染仔豬上皮細(xì)胞體外模型的建立及候選基因的功能驗(yàn)證展開了研究,深入探討ETEC F4ac型仔豬腹瀉致因基因表達(dá)的分子機(jī)制。試驗(yàn)一,選取4對(duì)ETEC F4ac抗性和易感的全同胞仔豬為試驗(yàn)對(duì)象,采用iTRAQ蛋白質(zhì)組定量技術(shù)鑒定其小腸蛋白表達(dá)差異。共發(fā)現(xiàn)245個(gè)差異表達(dá)蛋白,其中黏附組相對(duì)于非黏附組表達(dá)上調(diào)的有117個(gè),表達(dá)下調(diào)的有128個(gè)。對(duì)差異表達(dá)蛋白進(jìn)行Pathway富集分析,結(jié)果富集到唯一的整合素信號(hào)通路(Integrin signalling pathway:P00034),說(shuō)明該通路對(duì)ETEC F4ac感染仔豬小腸黏膜進(jìn)而引起仔豬腹瀉具有重要作用。從基因位于染色體上的位置、基因的分子功能、基因的蛋白質(zhì)分子量大小角度對(duì)候選基因進(jìn)行分析,發(fā)現(xiàn)TTGB5最有可能為ETEC F4acR的候選基因。試驗(yàn)二,仔豬小腸上皮細(xì)胞系IPEC-J2是唯一一株取自豬空腸上皮細(xì)胞且未轉(zhuǎn)化未癌化的小腸上皮細(xì)胞系,正越來(lái)越多的被應(yīng)用于腸道細(xì)菌與上皮細(xì)胞的互作研究中。通過RT-PCR的方法檢測(cè)ETEC F4ac對(duì)仔豬小腸上皮細(xì)胞IPEC-J2的黏附,結(jié)果表明,當(dāng)大腸桿菌菌液濃度在1×105-1×109CFU/ml范圍之間時(shí),菌液濃度與Ct值之間線性方程為y=-0.3056x+13.418。通過以不同攻菌濃度和攻菌時(shí)間對(duì)IPEC-J2進(jìn)行攻菌,確定了ETEC F4ac進(jìn)行體外攻菌的最佳攻菌濃度為MOI為200：1,最佳攻菌時(shí)間為4h。該方法與平板菌落計(jì)數(shù)法相比較更省時(shí)省力,且定量準(zhǔn)確,重復(fù)性好。試驗(yàn)三,選取ITGB5、MUC4和MUC13作為ETEC F4acR候選基因,同時(shí)選擇無(wú)關(guān)基因SLC12A8作為陰性對(duì)照,利用CRISPR/Cas9基因敲除技術(shù)對(duì)每個(gè)基因分別進(jìn)行缺失突變。對(duì)基因敲除成功的細(xì)胞進(jìn)行體外攻菌試驗(yàn),結(jié)果表明TGB5基因的缺失顯著降低黏附到IPEC-J2細(xì)胞上的ETEC數(shù)目。為更加全面的了解JTGB5基因?qū)TEC F4ac黏附到小腸上皮細(xì)胞的影響,構(gòu)建ITGB5基因真核細(xì)胞表達(dá)載體來(lái)反向驗(yàn)證基因的功能。黏附試驗(yàn)結(jié)果表明ITGB5基因的過表達(dá)會(huì)導(dǎo)致細(xì)菌黏附數(shù)的增加。本研究為深入挖掘ETEC F4acR基因及其所在信號(hào)通路提供了數(shù)據(jù)支持和科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.61
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本文編號(hào)：2005338