摘要
为研究牛胚胎气管(bovine embryonic tracheal,EBTr)细胞Toll样受体9(Toll-like receptors 9,TLR9)在牛疱疹病毒Ⅰ型(bovine herpesvirus type 1,BHV-1)感染的天然免疫反应中的作用,本试验利用小分子干扰RNA(siRNA)技术,以TLR9为靶向分别设计并合成3条siRNA干扰序列,用SYBR GreenⅠ实时荧光定量PCR技术检测应用siRNA-TLR9干扰后TLR9基因的表达变化,筛选出最佳的siRNA-TLR9干扰序列,继而转染EBTr细胞使其TLR9基因沉默后感染BHV-1,用TaqMan实时荧光定量PCR方法检测BHV-1不同时间点的增殖变化。结果显示,转染48h后,与阴性对照组相比,siRNA-TLR9A、siRNA-TLR9B和siRNA-TLR9C分别对TLR9mRNA表达量下调了60.90%、24.05%和40.75%。筛选出siRNA-TLR9A片段在12~72h可以显著抑制TLR9mRNA表达(P<0.05)。用该片段转染EBTr细胞再感染BHV-1后,6~72hsiRNA-TLR9A组BHV-1DNA拷贝数低于对照组。本试验成功筛选出了特异性的抑制EBTr细胞TLR9基因的siRNA序列,并证明抑制TLR9的表达可降低BHV-1的增殖能力。
The aim of this study was to investigate the role of Toll-like receptors 9（TLR9）of the bovine embryonic tracheal（EBTr）cells in the innate immune response mediated by bovine herpesvirus type 1（BHV-1）.Using small interfering RNA（siRNA）technology,three siRNA interference sequences target for TLR9 were designed and synthesized in this study.After siRNATLR9 interference,the expression levels of TLR9 gene were detected by Real-time PCR.After48 h,the expression levels of TLR9 mRNA induced by siRNA-TLR9 A,siRNA-TLR9 Band siRNA-TLR9 Cwere reduced to 60.90%,24.05% and 40.75%,respectively.Comparing with the control group,the expression levels of TLR9 mRNA were significantly inhibited by siRNATLR9 Afragments at 12 to 72h（P〈0.05）,and after transfecting the best fragments and infecting with BHV-1,the BHV-1 DNA copy numbers of siRNA-TLR9 A group were lower than BHV-1DNA of the control group at 6to 72 h.The specific siRNA fragments target for TLR9 were successfully screened out in this test,and demonstrated that inhibition of TLR9 expressions could reduce the BHV-1proliferation in EBTr cells.
出处
《中国畜牧兽医》
CAS
北大核心
2017年第2期303-310,共8页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金项目(31260597)
