靶向TLR9基因siRNA的筛选及其对BHV-1增殖规律的影响

doi:10.16431/j.cnki.1671-7236.2017.02.001

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (2): 303-310.doi: 10.16431/j.cnki.1671-7236.2017.02.001

• 生物技术 • 下一篇

靶向TLR9基因siRNA的筛选及其对BHV-1增殖规律的影响

呼凤丽, 徐丽媛, 张志飞, 周伟光

内蒙古农业大学兽医学院, 农业部动物疾病临床诊疗技术重点实验室, 呼和浩特 010018

收稿日期:2016-08-19 出版日期:2017-02-20 发布日期:2017-02-25
通讯作者: 周伟光 E-mail:zhouwg2001@aliyun.com
作者简介:呼凤丽(1989-), 女, 陕西榆林人, 硕士生, 研究方向:兽医免疫学, E-mail:hufengli@aliyun.com
基金资助:
国家自然科学基金项目（31260597）

Screening of siRNA Targeted for TLR9 Gene and Its Effect on the Proliferation Profile of BHV-1

HU Feng-li, XU Li-yuan, ZHANG Zhi-fei, ZHOU Wei-guang

Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China

Received:2016-08-19 Online:2017-02-20 Published:2017-02-25

摘要/Abstract

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48 h后，与阴性对照组相比，siRNA-TLR9A、siRNA-TLR9B和siRNA-TLR9C分别对TLR9 mRNA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TLR9 mRNA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。

关键词: 小分子干扰RNA; 牛胚胎气管细胞; Toll样受体9; 牛疱疹病毒Ⅰ型; 实时荧光定量PCR

Abstract:

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.

Key words: siRNA; EBTr cells; TLR9; BHV-1; Real-time PCR

中图分类号:

S852.65⁺9.1

呼凤丽, 徐丽媛, 张志飞, 周伟光. 靶向TLR9基因siRNA的筛选及其对BHV-1增殖规律的影响[J]. 《中国畜牧兽医》, 2017, 44(2): 303-310.

HU Feng-li, XU Li-yuan, ZHANG Zhi-fei, ZHOU Wei-guang. Screening of siRNA Targeted for TLR9 Gene and Its Effect on the Proliferation Profile of BHV-1[J]. , 2017, 44(2): 303-310.

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靶向TLR9基因siRNA的筛选及其对BHV-1增殖规律的影响

Screening of siRNA Targeted for TLR9 Gene and Its Effect on the Proliferation Profile of BHV-1

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