MDV Meq-clustered miRNAs基因缺失BAC克隆株的构建及其体外增殖特性

Construction and in vitro proliferation characteristics of MDV Meq-clustered miRNAs deletion of BAC clone

为构建缺失马立克氏病病毒(Mareks disease virus,MDV)Meq基因簇microRNAs(Meq-clustered miRNAs的突变株,本研究在vv MDV GX0101细菌人工染色体(bacterial artificial chromosome,BAC)克隆的基础上,采用Red/ET同源重组技术将Meq-clustered miRNAs的编码基因进行缺失突变,经PCR鉴定及序列分析证明Meq-cluster miRNAs序列成功缺失后,提取Δmeq-miRNAs BAC DNA,转染鸡胚成纤维细胞(CEF)进行病毒拯救,用SYBR GreenⅠqRT-PCR检测病毒体外增殖特性。结果表明成功拯救出缺失MDV Meq-clustered miRNAs基因的感染性BAC克隆株GX0101Δmeq-miRNAs,且该缺失株与亲本株GX0101BAC具有相似的增殖曲线,Meq-clustered miRNAs是MDV体外复制的非必需基因。MDV Meq-clustered miRNAs基因缺失感染性BAC克隆株的成功构建为进一步研究MDV Meq-clustered miRNAs在MDV致病和致肿瘤方面的作用奠定了基础。

To construct the Meq-clustered miRNAs-deleted mutant of Marek's disease virus(MDV),the Meq-clustered miRNAs were deleted from the viral genome of the bacterial artificial chromosome(BAC)clone of very virulent(vv)MDV strain GX0101 by the technique of Red/ET homologous recombination.The deletion was verified by PCR and DNA sequencing.Then,the mutant GX0101Δmeq-miRNAs was successfully rescued by the transfection of the BAC DNA containing the deletion of the Meq-clustered miRNAs into primary chicken embryo fibroblast(CEF)cells,and the in vitro replication kinetics of the mutant virus was determined utilizing a SYBR Green real-time qPCR.In conclusion,a full-length infectious BAC clone of MDV-GX0101 strain with the deletion of the Meq-clustered miRNAs was successfully constructed,a similar replication kinetics was observed between the parent GX0101 BAC and the mutant GX0101ΔMeq-miRNAs,indicating that the Meq-clustered miRNAs are not essential for MDV replication.The infectious BAC clone provides a useful tool for further studies on the roles of the Meq-clustered miRNAs in MDV pathogenesis.