CD14沉默的猪小肠上皮细胞系的建立及其对大肠杆菌黏附能力的变化分析

发布时间：2018-06-11 23:00

本文选题：猪小肠上皮细胞 + 白细胞分化抗原基因(CD)　；参考：《农业生物技术学报》2016年03期

【摘要】：白细胞分化抗原14(cluster of differentiation antigen 14,CD14)在先天性免疫和适应性免疫反应中都发挥重要调控作用,本研究旨在构建猪(Sus scrofa)CD14基因的短发夹RNA(short hairpin RNA,sh RNA)干扰载体,包装成慢病毒,转染猪小肠上皮细胞,在细胞水平上进行相关功能探讨。本研究参照CD14基因(Gen Bank登录号:EF626695.1)全长编码区序列,设计4个靶向猪CD14基因的sh RNA干扰序列,将其克隆至慢病毒表达载体质粒中,分别命名为p GLV3-CD14-1、p GLV3-CD14-2、p GLV3-CD14-3和p GLV3-CD14-4,以及阴性对照p GLV3-CD14-NC。包装成功后转染猪小肠上皮细胞系IPEC-J2,利用q RTPCR检测其干扰效率。选择干扰效率最高的慢病毒进行药筛,获得CD14基因稳定沉默的猪小肠上皮细胞系。大肠杆菌(Escherichia coli)黏附实验检测大肠杆菌F18ab和F18ac对CD14基因沉默小肠上皮细胞黏附能力的变化。结果表明,本研究成功构建4个sh RNA表达载体,包装成的慢病毒均能降低猪CD14m RNA表达水平,其中p GLV3-CD14-3慢病毒的干扰效果最好,其干扰效率达到94.6%;CD14基因沉默后F18ab对小肠上皮细胞的黏附能力显著增强,而F18ac虽然也有上调,但并未产生显著性差异。研究结果表明,CD14基因沉默后的小肠上皮细胞对大肠杆菌F18ab的黏附能力显著增强,CD14基因可能在小肠上皮细胞抵御大肠杆菌F18ab感染过程中发挥了重要的调控作用。慢病毒介导的CD14沉默的猪小肠上皮细胞系的建立,不仅为在细胞水平研究CD14基因功能提供了有效模型,也为进一步探究其所在的Toll样受体/白介素-1受体(toll-like receptors/interleukin-1 receptor,TLRs/IL-1R)信号通路在猪肠道病原微生物引起的免疫反应和抵御病原入侵的调控机制奠定了基础。
[Abstract]:Leukocyte differentiation antigen 14 (cluster of differentiation antigen 14, CD14) plays an important role in both congenital and adaptive immune responses. The aim of this study is to construct the short hairpin RNA (short hairpin RNA) of the swine (Sus scrofa) CD14 gene (short hairpin RNA), which is packaged into lentivirus, transfected to pig small intestinal epithelial cells, and in cell water. On the basis of the CD14 gene (Gen Bank login number: EF626695.1) full length coding region, 4 sh RNA interference sequences targeting the CD14 gene of swine were designed and cloned into the lentivirus vector plasmid, named P GLV3-CD14-1, P GLV3-CD14-2, P, and negative controls, respectively. After the LV3-CD14-NC. package was successfully transfected to the pig small intestinal epithelial cell line IPEC-J2, the interference efficiency was detected by Q RTPCR. The sieves with the highest interference efficiency were selected to obtain the stable and silent pig small intestinal epithelial cell line of the CD14 gene. The adhesion test of Escherichia coli (Escherichia coli) was used to detect the silencing of the CD14 gene of the Escherichia coli F18ab and F18ac. The changes in the adhesion ability of small intestinal epithelial cells showed that 4 sh RNA expression vectors were successfully constructed in this study. The packaged lentivirus could reduce the expression level of CD14m RNA in pigs. The interference effect of P GLV3-CD14-3 lentivirus was the best and the interference efficiency reached 94.6%, and the adhesion ability of F18ab to small intestinal epithelial cells after the CD14 gene was silent was significant. The results showed that the adhesion ability of small intestinal epithelial cells to Escherichia coli F18ab was significantly enhanced after the CD14 gene silencing, and the CD14 gene may play an important role in preventing the F18ab infection of Escherichia coli in the intestinal epithelial cells. The CD14 gene may play an important role in the F18ab infection of the Escherichia coli. The slow virus mediated CD1. The establishment of the 4 silent pig intestinal epithelial cell line not only provides an effective model for the study of CD14 gene function at the cellular level, but also further explores the immune response and counterpart of the Toll like receptor / interleukin -1 receptor (Toll-like receptors/interleukin-1 receptor, TLRs/IL-1R) signaling pathway in the pig intestinal pathogenic microorganism. The regulatory mechanism of the invasion of the pathogen has laid the foundation.
【作者单位】：扬州大学动物科学与技术学院;扬州大学兽医学院;江苏省种猪繁育和健康养殖工程技术研究中心;
【基金】：国家自然科学基金(No.31572360和No.31372285) 转基因生物新品种培育科技重大专项(No.2014ZX08006-001B) 江苏省科技支撑计划(No.BE2015329和No.BE2014357) 江苏高校优势学科建设工程资助项目(PAPD)
【分类号】：S852.6;Q78

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