表达FMDVB7多表位基因的重组PRRSV的拯救与复制水平的测定  

Recombinant porcine reproductive and respiratory syndrome virus expressing B7 multi-epitope gene of foot-and-mouth disease virus and its replication capacity

作　　者：白伟杰[1] 曹轶梅[1] 包慧芳[1] 孙普[1] 付元芳[1] 李平花[1] 白兴文[1] 陈应理[1] 李坤[1] 马雪青[1] 刘在新[1] 李冬[1] 卢曾军[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《中国兽医科学》2016年第4期449-453,共5页Chinese Veterinary Science

基　　金：甘肃省农业生物技术研究与应用开发项目(GNSW-2009-10)

摘　　要：利用北美PRRSV Fl12毒株感染性c DNA克隆,在病毒基因组ORF1b和ORF2a之间插入绿色荧光蛋白（EGFP）基因,构建了表达EGFP的重组PRRSV（v Fl12-EGFP）。拯救病毒v Fl12-EGFP感染Marc-145细胞后,可观察到明显的细胞病变和绿色荧光,表明EGFP基因正确表达。随后,采用相同的策略构建了表达口蹄疫病毒（FMDV）B7多表位基因的重组PRRSV（v Fl12-B7）,拯救病毒v Fl12-B7感染Marc-145细胞后,可观察到典型的CPE;对拯救病毒进行RT-PCR扩增和序列测定,表明多表位基因正确插入;荧光定量PCR检测,第1代和第4代拯救病毒的拷贝数分别为3.76×10^5 copies/L与1.58×10^8 copies/L,说明重组病毒具有较好的复制能力,为以PRRSV为载体表达口蹄疫多表位基因活载体疫苗研究奠定了基础。Using an infectious c DNA clone of North American PRRSV isolate Fl12,a recombinant PRRSV（v Fl12-EGFP） was generated for expression of enhanced green fluorescent protein（EGFP） gene by insertion between ORF1 b and ORF2 a. When the rescued virus v Fl12-EGFP infected Marc-145 cells,obvious cytopathic effect（CPE） and green fluorescent were observed,indicating that the inserted EGFP gene was correctly expressed. Subsequently,a recombinant PRRS（v Fl12-B7） was constructed and rescued by the same strategy,expressing B7 multi-epitope gene of foot-and-mouth disease virus（FMDV）. Marc-145 cells infected with v Fl12-B7 showed the typical CPE after PRRSV infection. The results of RT-PCR and sequence determination showed that FMDV multi-epitope gene was correctly inserted in PRRSV genome. Fluorescent quantitative PCR was used to detect rescued virus, and viral copies of the first and fourth passage v Fl12-B7 reached 3.76×10^5 copies/ L and 1.58×10^8 copies/ L, respectively, indicating that the recombinant virus possessed good vitality and replicated well. This work Laid a good foundation for development of a recombinant PRRSV vector vaccine against FMD.

关 键 词：PRRSV 感染性克隆 FMDV B细胞表位 病毒载体疫苗 

分 类 号：S852.659.6[农业科学—基础兽医学]