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

作　　者：余祖华[1,2] 丁轲[1] 滕蔓[2] 迟佳琦[2] 宿靖伟[2] 程相朝[1] 

机构地区：[1]河南科技大学动物疫病与公共卫生重点实验室河南省动物疫病与公共安全院士工作站,河南洛阳471003 [2]河南省农业科学院农业部动物免疫学重点实验室河南省动物免疫学重点实验室,河南郑州450002

出　　处：《中国兽医学报》2015年第6期851-857,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金项目(31072145;31372445)

摘　　要：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.

关 键 词：马立克氏病病毒 细菌人工染色体 Red/ET 克隆 

分 类 号：S852.65[农业科学—基础兽医学]