靶向TLR2基因的siRNA对牛疱疹病毒Ⅰ型增殖规律的影响

Effect of si RNA targeting TLR2 on the proliferation profile of BHV-1

为研究牛胚胎气管（EBTr）细胞Toll样受体2（TLR2）在牛疱疹病毒Ⅰ型（BHV-1）感染的天然免疫反应中的作用,本研究利用小分子干扰RNA（si RNA）技术,以TLR2为靶向设计并合成了3条si RNA干扰序列,用SYBR GreenⅠ定量PCR方法筛选出最佳的si RNA-TLR2干扰片段,继而转染EBTr细胞使其TLR2沉默后感染BHV-1,用Taq Man定量PCR方法检测BHV-1不同时间点的增殖变化。结果显示,与对照组相比转染后第48小时si TLR2A、si TLR2B和si TLR2C在EBTr细胞中分别对TLR2 m RNA表达量下调了35.05%、69.20%和84.85%,筛选出的si TLR2C片段在第12~72小时可以显著抑制TLR2 m RNA的表达（P〈0.05）,用该片段转染EBTr细胞再感染BHV-1后第6~72小时,si RNA-TLR2组BHV-1 DNA拷贝数显著低于对照组BHV-1 DNA拷贝数（P〈0.05）。本试验成功筛选出了特异性抑制EBTr细胞TLR2基因的si RNA片段,并证明抑制TLR2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 receptors2（TLR2）of the bovine embryonic tracheal（EBTr）cells in the innate immune response mediated by bovine herpesvirus typeⅠ（BHV-1）.Using small interfering RNA（si RNA）technology,three si RNA interference sequences target for TLR2 were designed and synthesized in this study.After si RNA-TLR2 interference,the expression levels of TLR2 gene were detected by SYBR GreenⅠreal-time PCR. The EBTr cells were transfected with the best si RNA interference sequences,and then infected with BHV-1.The changes of BHV-1 DNA were detected by Taq Man real-time PCR at different time points. After 48 h transfecting si TLR2 A,si TLR2 B,and si TLR2 C in EBTr cells,the results showed that the expression levels of TLR2 m RNA were reduced 35.05%,69.20% and 84.85%,respectively.Comparing with the control group,the expression levels of TLR2 m RNA were significantly inhibited by si TLR2 C fragments in 12-72 h（P〈0.05）,and after transfecting the best fragments and infecting with BHV-1,the si RNA-TLR2 group BHV-1 DNA copy numbers were significantly lower than the control group BHV-1 DNA in 6-72 h（P〈0.05）.The specific si RNA fragments target for TLR2 were successfully screened out in this test,and demonstrated that inhibition of TLR2 expressions can re-duce the BHV-1 proliferation in EBTr cells.