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 siRNA-TLR9 interference,the expression levels of TLR9 gene were detected by Real-time PCR.After 48 h,the expression levels of TLR9 mRNA induced by siRNA-TLR9A,siRNA-TLR9B and siRNA-TLR9C were 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 siRNA-TLR9A fragments at 12 to 72 h (P＜0.05),and after transfecting the best fragments and infecting with BHV-1,the BHV-1 DNA copy numbers of siRNA-TLR9A group were lower than BHV-1 DNA of the control group at 6 to 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-1 proliferation in EBTr cells.%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 Green Ⅰ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48 h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12～72 h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,6～72 h siRNA-TLR9A组BHV-1 DNA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.
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