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  

Establishment of Pig(Sus scrofa) Small Intestinal Epithelial Cell Line with CD14 Gene Silencing and Its Effect Analysis on the Adhesion Ability to Escherichia coli

作　　者：戴超辉 甘丽娜[1] 钦伟云 孙寿永[1] 朱国强[2,3] 吴圣龙[1,3] 包文斌[1,3] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009 [2]扬州大学兽医学院,扬州225009 [3]江苏省种猪繁育和健康养殖工程技术研究中心,扬州225009

出　　处：《农业生物技术学报》2016年第3期323-331,共9页Journal of Agricultural Biotechnology

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)

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。Cluster of differentiation antigen 14（CD14） plays an important role in both the innate and adaptive immune responses. This study aimed at establishing the short hairpin RNA（sh RNA） interference vectors with pig（Sus scrofa） CD14 gene silencing to package them into lentivirus for transfecting pig small intestinal epithelial cells（IPEC- J2） and conducting function analysis on cell level to provide fundamental basis for function and action mechanism of CD14 gene. Based on the whole coding sequence of pig CD14gene（Gen Bank No. EF626695.1）, 4 interfere sequences were constructed which encoded sh RNAs against pig CD14 gene and cloned into lentivirus expression vectors of p GLV3-CD14-1, p GLV3-CD14-2, p GLV3-CD14-3, p GLV3-CD14-4 and negative control p GLV3-CD14-NC. IPEC-J2 was infected by lentivirus solution after packaging successfully and q RT-PCR was used to detect the interference efficiency of CD14 gene. Lentivirus with the highest interference efficiency continually infected cells through medicinal sieve to obtain pig small intestinal epithelial cell line with stable CD14 gene silencing. Escherichia coli adhesion test was conducted to detect the adhesion ability change of E. coli F18 ab and F18 ac to IPEC-J2. The results showed that 4 sh RNA vectors were constructed successfully and the packaged lentivirus could reduce the m RNA expression level of pig CD14 gene, in which p GLV3-CD14-3 had the best interference efficiency of 94.6%. The adhesion results showed that the E. coli F18ab＇s adhesion ability to IPEC-J2 significantly enhanced after CD14 gene silencing and E. coli F18 ac did not. This result showed that CD14 gene silencing enhanced the susceptibility to E. coli F18 ab in small intestinal epithelial cells, which illustrated that CD14 gene may play an important regulation role in which small intestinal epithelial cells resist infection by E. coli F18 ab. The establishment of pig small intestinal epithelial cell line with CD14 gene silencing stably mediated by lentivirus offered important

关 键 词：猪小肠上皮细胞 白细胞分化抗原14基因(CD14) 基因沉默 大肠杆菌 黏附 

分 类 号：S858.2[农业科学—临床兽医学]