猪血管内皮细胞中与猪瘟病毒相关microRNA的初步筛选

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

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CSFV3′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CSFV5′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-mi R-let7c、ssc-mi R-106a、ssc-mi R-18、ssc-mi R-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CSFV3′-UTR有不同程度的抑制作用,其中ssc-mi R-18的抑制作用比较明显。

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.