猪脑心肌炎病毒HB10株cDNA感染性克隆的构建  

Construction of infectious cDNA clone of encephalomyocarditis virus HB10 strain

作　　者：于会彬[1] 黄丽[1] 张元峰 李江南[1] 蔡雪辉[1] 崔尚金[1] 翁长江[1] 

机构地区：[1]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/动物病原监测与流行病学研究创新团队,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2015年第9期665-668,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31300139;31172333)

摘　　要：为建立猪脑心肌炎病毒(EMCV)HB10株cDNA感染性克隆,本研究利用反向遗传操作技术,采用RT-PCR一次性扩增EMCV-HB10株全长基因组(包括5'和3'UTR)序列,并直接克隆于低拷贝载体pOK12中,构建了EMCV感染性重组质粒pOK12-EMCV-HB10。将重组质粒体外转录后转染BHK-21细胞进行病毒拯救。结果显示,转染细胞36 h后,出现典型的细胞病变。拯救病毒rEMCV-HB10全基因组经测序鉴定显示,与亲本病毒开放阅读框比较,其仅在1D基因出现两个核苷酸突变:即A37G(R13K)和G187C(M63I),该突变可以作为区别亲本病毒的标志。间接免疫荧光和生长曲线试验结果显示,这两个突变并不影响VP1蛋白的表达及病毒拯救,并且拯救病毒与亲本病毒具有相似的生长特性。本研究构建了感染性EMCV-HB10 cDNA克隆,为深入研究EMCV的致病分子机理提供了有效的反向遗传操作平台。The aim of this study is to construct reverse genetic system of encephalomyocarditis virus （EMCV）. The full length cDNA of EMCV HB10 strain was amplified by RT-PCR, and inserted into a low-copy plasmid pOK12 to construct the infectious cDNA clone of pOK-EMCV-HB10. Then, the pOK-EMCV-HB10 was linearized with BamH I and the transcript RNA was synthesized using the RiboMAXTM Large Scale RNA Production Systems in vitro. The transcripts were transfected into BHK-21 cells and typical cell pathogenic effect （CPE） apeared after 36 hrs. In addition, the full length genome was amplified by RT-PCR to identify the rescued EMCV virus. Compared with the ORF of parental virus, only two nucleotide mutations of AaTG and G187C were found in 1D gene of the rescued virus contains, The expression of VP1 was detected by indirect immunofluorescence assay and the rescued virus had similar growth characteristics compared with the parental virus, suggesting the two existed nucleotide mutations did not affect the expression of VP1 and viral rescue, whereas, the mutations could be used to distinguish the rescued virus from its parental virus. Here, we have successfully constructed the EMCV-HB10 cDNA clone, which provided an essential tool for investigating the molecular basis of pathogenicity of EMCV.

关 键 词：猪脑心肌炎病毒 cDNA感染性克隆 转录转染 病毒拯救 

分 类 号：S852.65[农业科学—基础兽医学]