畜牧兽医学报 ›› 2010, Vol. 41 ›› Issue (5): 576-580.doi:

• 预防兽医 • 上一篇    下一篇

猪血管内皮细胞中与猪瘟病毒相关microRNA的初步筛选

侯勃,张彦明*,朱晓娟,康恺,代晨,唐青海   

  1. 西北农林科技大学 动物医学院,杨凌 712100
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-05-25 发布日期:2010-05-25

Primary Selection of MicroRNA Acting on Classical Swine FeverVirus in Swine Umbilical Vein Endothelial Cells

HOU Bo, ZHANG Yan-ming*, ZHU Xiao-juan, KANG Kai, DAI Chen, TANG Qing-hai   

  1. College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2010-05-25 Published:2010-05-25

摘要: 本试验旨在筛选猪脐静脉血管内皮细胞(SUVECs)内作用于猪瘟病毒(CSFV)的microRNA。通过构建CSFV 3′和5′非翻译区(Untranslated region, UTR)的双荧光素酶报告基因载体,经生物信息学预测可能与CSFV相互作用的microRNA,然后瞬时共转染重组载体与microRNA的抑制物,最后通过发光检测仪测定荧光素酶活性。结果表明,成功构建了CSFV 5′非翻译区(373 bp)和3′非翻译区(252 bp)的报告基因载体,并合成了4条预测出的microRNA(ssc-miR-let7c、ssc-miR-106a、ssc-miR-18、ssc-miR-139)的抑制物,共转染后发光检测仪检测到荧光素酶的活性被不同程度抑制。本研究发现microRNA作用位点主要在CSFV的3′UTR,而且以上4种microRNA都对CSFV 3′UTR有不同程度的抑制作用,其中ssc-miR-18的抑制作用比较明显。

Abstract: The aim of the study is to find out the microRNAs that acted on classical swine fever virus (CSFV) in swine umbilical vein endothelial cells (SUVECs). CSFV 3′ and 5′untranslated region (UTR) were cloned, and then were inserted into psiCHECHTM-2 plasmid that carrying Firefly and Renilla luciferases reporter genes; the microRNAs that acted with CSFV were forecasted through bioinformatics analysis; then the recombinant plasmids and inhibitors of predicted microRNA were cotransfected into SUVECs. The activity of Firefly and Renilla luciferases was detected by Luminometer. The PCR products of CSFV 5′UTR(373 bp) and 3′UTR (252 bp) were detected by 10 g·L-1 agarose electrophoresis consistent with predicted size, from which four predicted microRNAs (ssc-miR-let7c, ssc-miR-106a, ssc-miR-18, ssc-miR-139) were selected. The CSFV 3′UTR is the important action site of microRNA in SUVECs. The four microRNAs mentioned above had different inhibitory effect on CSFV 3′UTR, of which the ssc-miR-18 play a more important role.