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

Abstract

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

CLC Number:

S852.65⁺9.1

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.

References

[1] Woodbine K A, Medley G F, Moore S J, et al. A four year longitudinal sero-epidemiological study of bovine herpesvirus type-1 (BHV-1) in adult cattle in 107 unvaccinated herds in Southwest England[J].BMC Veterinary Research,2009,5:1-12.
[2] Verma A K, Kumar K, Sahzad, et al. Sero-prevalence of infectious bovine rhinotracheitis in dairy animals with reproductive disorders in Uttar Pradesh, India [J]. Pakistan Journal of Biological Sciences,2014,17(5):720-724.
[3] Jacevicius E, Salomskas A, Milius J, et al. Five year serological study of bovine herpesvirus type-1 in cattle in Lithuania[J]. Bulletin of the Veterinary Institute in Pulawy, 2010, 54:289-292.
[4] 李程,陈颖钰,索朗斯珠,等. 牛传染性鼻气管炎的流行与防控进展[J]. 中国奶牛,2015,17:40-43.
[5] 王洪梅,侯佩莉,宋玲玲,等. 国内外奶牛主要病毒病的流行现状及防控技术研究进展[J]. 中国畜牧杂志,2014,10:42-49.
[6] 徐晓琴,冷雪,李真光,等. 牛传染性鼻气管炎诊断方法研究进展[J]. 动物医学进展,2010,1:81-86.
[7] Fulton R W,d'Offay J M,Dubovi E J,et al.Bovine herpesvirus-1: Genetic diversity of field strains from cattle with respiratory disease, genital, fetal disease and systemic neonatal disease and their relationship to vaccine strains[J].Virus Research,2016, 223(2):115-121.
[8] 马思慧,杨欢,吴天成,等. Toll样受体信号传导通路的研究进展[J].中国畜牧兽医,2014, 41(8):160-164.
[9] 常晓彤,辇晓峰,王振辉. Toll样受体信号转导途径研究进展[J]. 生理科学进展,2011,5:340-346.
[10] Duffy L, O'Reilly S C.Toll-like receptors in the pathogenesis of autoimmune diseases: Recent and emerging translational developments[J].Immunotargets Ther, 2016, 22(5):69-80.
[11] 同戈,孙瑶. Toll样受体9介导的免疫激活研究进展[J].中国畜牧兽医,2010,37(9):183-187.
[12] Lester N, Li K. Toll-like receptors in antiviral innate immunity[J]. Journal of Molecular Biology,2014, 426(6):1246-1264.
[13] Li Z,Rana T M. Molecular mechanisms of RNA-triggered gene silencing machineries[J]. Accounts of Chemical Research,2012,45(7): 1122-1131.
[14] Chery J,Näär A.RNA therapeutics:RNAi and antisense mechanisms and clinical applications[J].Postdoc J, 2016,4(7):35-50.
[15] Wang J, O'Keefe J, Orr D,et al.An international inter-laboratory ring trial to evaluate a Real-time PCR assay for the detection of bovine herpesvirus 1 in extended bovine semen[J].Veterinary Microbiology, 2008, 126(1-3):11-19.
[16] 徐丽华,刘春雷,常玉梅,等. 双标准曲线相对定量PCR试验原理与方法[J]. 生物技术通报,2011,1:70-75.
[17] 张宇飞,刘月,张亚坤,等. 靶向enJSRV env基因shRNA真核表达质粒的构建及其干扰效率的测定[J]. 中国兽医科学,2013,1:53-59.
[18] 张赟,龚敏,毕杨,等. 大鼠Toll样受体2基因siRNA重组腺病毒载体的构建及鉴定[J]. 细胞与分子免疫学杂志,2012,2:144-147.
[19] 汤建萍. Toll样受体2和9在单纯疱疹病毒感染发病中的作用研究[D].长沙:中南大学,2008.
[20] 罗兵,李涛,徐元宏. Toll样受体的信号转导及抗感染免疫研究进展[J]. 免疫学杂志,2011,2:165-169.
[21] Ayuko S, Melissa S M, Akiko I. Dual recognition of herpes simplex viruses by TLR2 and TLR9 in dendritic cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006,13(46) :17343-17348.

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

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