摘要
旨在细胞水平上进一步探讨FUT2基因的功能及其在猪小肠上皮细胞受到F18大肠杆菌(Escherichia coli,E.coli)侵染时的调控机制,为提高仔猪对F18大肠杆菌病抵抗力的分子选育提供理论基础。运用Real-time PCR方法检测分析FUT2基因在大肠杆菌F18ab、F18ac感染和脂多糖(LPS)诱导小肠上皮细胞前后的mRNA表达差异,并采用Western blotting方法检测大肠杆菌F18ab、F18ac感染小肠上皮细胞前后FUT2的蛋白表达差异;同时设计并构建猪FUT2基因慢病毒干扰载体,筛选并获得FUT2基因稳定沉默的小肠上皮细胞系,检测FUT2基因沉默对其所在球系列鞘糖脂生物合成通路其他关键基因(FUT1、ST3GAL1、HEXA、HEXB、B3GALNT1、NAGA)表达以及小肠上皮细胞E.coli F18黏附能力的影响。结果表明,在F18大肠杆菌感染和LPS诱导小肠上皮细胞后,FUT2基因的mRNA相对表达水平均极显著升高(P<0.01),同时大肠杆菌感染后小肠上皮细胞的FUT2蛋白表达上升。此外,本研究成功构建FUT2基因慢病毒干扰载体,并获得FUT2基因稳定沉默的小肠上皮细胞系;FUT2基因沉默后,其所在的球系列鞘糖脂生物合成通路其他关键基因的表达都存在不同程度的下降,其中FUT1基因表达水平显著下调(P<0.05),ST3GAL1、HEXA和HEXB基因表达水平极显著下调(P<0.01),而B3GALNT1和NAGA基因表达水平未发生显著变化,同时FUT2基因沉默后F18大肠杆菌对小肠上皮细胞的黏附能力显著降低。结果显示,FUT2基因低表达可能不利于仔猪大肠杆菌受体的形成,以增强猪小肠上皮细胞抵抗E.coli F18感染的能力,本试验结果同时为球系列鞘糖脂生物合成通路在仔猪抗大肠杆菌感染过程中的作用机制研究提供一定的理论基础和依据。
The purpose of this study was to further explore the function of FUT2 and its regulation mechanisms in swine epithelial cells infected by E. coli F18 at the cellular level, which provide theoretical basis for molecular breeding of improving the resistance of piglets to E. coli F18. The expression differences of FUT2 gene in intestinal epithelial cells before and after E. coli F18ab, F18ac infection and lipopolysaccharide（LPS） induction were detected by Real-time PCR. Then, the Western blotting was performed to identify the protein expression differences of FUT2 in intestinal epithelial cells before and after E. coli F18ab and F18ac infection. Meanwhile the lenti- viral interference vectors of porcine FUT2 were designed and constructed, and the intestinal epi- thelial cells line with FUT2 gene silencing was obtained. The effects of FUT2 gene silencing on the mRNA expression of key genes （FUT1, ST3GAL1, HEXA, HEXB, B3GALNT1, NAGA） involved in glycosphingolipid biosynthesis-globo series pathway and on E. coli F18 adhesion to intestinal epithelial cells were further analyzed. The results showed that the FUT2 expression levels in intestinal epithelial cells were significantly increased after E. coli infection and LPS in- duction （P;0.01）, and the FUT2 expression level in intestinal epithelial cells was also increased after E. coli infection. Moreover, the lentiviral interference vectors of porcine FUT2 were suc- cessfully constructed, and the intestinal epithelial cells line with FUT2 gene silencing were ob- tained. After FUT2 gene silencing, the expression level of key genes in glycosphingolipid biosyn- thesis-globo series pathway were decreased at different degrees. The expression level of FUT1 af- ter FUT2 silencing was significantly reduced （P%0.05）, and the expression levels of ST3GAL1, HEXA, HEXB were extremely significantly reduced （P%0.01）, while the expression levels of B3GALNT1 and NAGA genes showed no significant changes. In addition, the adhesion ability of E. coli F18 to swine epithelial cells was decreased significantly （P%0.05）. These results show that the lower expression level of FUT2 may hinder the formation of E. coli F18 receptor, and thus elevate the resistance capability of porcine small intestine epithelial cells to E. coli F18 infection. Our findings can also provide theoretical foundations and evidence for further investigating on the mechanisms of glycosphingolipid biosynthesis-globo series pathway in resistance of pigs to E. coli infection.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2017年第11期2034-2045,共12页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31572360
31372285)
江苏省重点研发计划(现代农业)(BE2015329
BE2016315)
江苏高校优势学科建设工程资助项目(PAPD)
