gga-miR-21过表达细胞株对传染性法氏囊病病毒复制的影响  

gga-miR-21 regulates infectious bursal disease virus(IBDV)replication by targeting VP1 gene of IBDV

作　　者：欧阳伟[1] 朱向东[1,2] 王永山[1] 王永强[3] 潘群兴[1] 毕振威[1] 王笑梅[3] 

机构地区：[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095 [3]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2012年第11期847-853,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31272537);江苏省自然科学基金(BK2010471;BK2012787);中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室开放基金(SKLVBF201105)

摘　　要：为研究microRNA(gga-miR-21)对传染性法氏囊病病毒(IBDV)复制的影响,本研究利用PCR方法从鸡的肝细胞基因组中扩增细胞pri-gga-miR-21,克隆于慢病毒表达质粒pL-EGFP中构建重组质粒pL-miR-21。将pL-miR-21与3个包装质粒pLP1、pLP2和pLP/VSVG共转染293FT细胞,制备含gga-miR-21基因的重组慢病毒VL+miR-21。将VL+miR-21感染DF-1细胞,经杀稻瘟菌素筛选获得过表达gga-miR-21的细胞株DF-miR-21,采用定量RT-PCR(qRT-PCR)检测表明,gga-miR-21的表达量比正常DF-1细胞提高60%。将IBDV接种于DF-miR-21细胞,在96 h后,其病毒滴度(104.75TCID50/0.1 mL)显著低于正常DF-1细胞的病毒滴度(108.75TCID50/0.1 mL)。采用生物信息学方法和双荧光素酶报告系统分析验证,在IBDV基因组VP1基因中存在gga-miR-21的结合靶点。将IBDV感染DF-miR-21细胞,采用western blot分析,VP1蛋白的表达量比正常DF-1细胞显著降低;用qRT-PCR分析,VP1 mRNA水平与正常DF-1细胞没有明显差异。本实验结果表明:细胞gga-miR-21可以抑制IBDV的复制,其分子作用机理是在VP1基因翻译水平上实现的。MicroRNAs （miRNAs）, small non-coding RNAs, contribute to the repertoire of host-pathogen interactions during virus infection. To explore the function of the miRNA of gga-miR-21 in regulating replication of infectious bursal disease virus （IBDV）, the sequence of pri-gga-miR-21 was amplified from genomic DNA of chicken hepatocytes and inserted into the lentiviral vector pL-EGFP to construct the recombinant plasmid of pL-miR-21. The lentiviral particles of VL＋miR-2, were generated by co-transfecting the 293FT cell with the pL-miR-21 and three packaging vectors of pLP1, pLP2 and pLP/VSVG, and the DF-miR-21 ceils stably overexpressing gga-miR-21 were prepared from DF-1 cell line by infecting VL＋miR-21 and selecting in the presence of blasticidin. Furthermore, IBDV inhibition test indicated that virus titer was dramatically reduced in the DF-miR-21 cells （104.75 TCID50/0.1 mL） than that in DF-1 cells （108.75 TCID50/0.1 mL）. Bioinformatics analysis indicated the VP1 gene in IBDV B segment had a putative binding site for gga-miR-21, which was confirmed by the Luciferase assays. Moreover, western blot analysis showed that the viral VP1 protein level was significantly lower in the IBDV infected DF-miR-21 cells than that in DF-1 cells, but the mRNA level of VP1 remained the same in both cells detected by real-time RT-PCR. The results demonstrated that gga-miR-21 down-regulated IBDV VP1 expression at the translational levels, but not the mRNA levels. These findings suggest that gga-miR-21 plays a role in protecting host ceils from the virus in addition to its known cellular functions.

关 键 词：传染性法氏囊病病毒 miRNA gga-miR-21 慢病毒表达系统 病毒复制 

分 类 号：S852.65[农业科学—基础兽医学]