通过基因组合成构建美洲型猪繁殖与呼吸综合征病毒感染性克隆  

Construction of infectious clones of porcine reproductive and respiratory syndrome virus via biosynthesis

作　　者：王治家[1] 张婧[1] 白伟杰[1] 孙普[1] 曹轶梅[1] 李冬[1] 刘在新[1] 卢曾军[1] WANG Zhi-jia ZHANG Jing BAI Wei-jie SUN Pu CAO Yi-mei LI Dong LIU Zai-xin LU Zeng-jun(State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute, Chinese A cademy of Agricultural Sciences ,Lanzhou 730046, Chin)

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,甘肃兰州730046

出　　处：《中国兽医科学》2017年第7期838-842,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);中国农业科学院创新工程经费资助

摘　　要：根据已测定的猪繁殖与呼吸综合征病毒(PRRSV)GSWW分离株的全长基因组序列信息,采用合成生物学的方法,将全长基因组序列分为两段(7 636 bp和7 774 bp)分别连接到低拷贝载体p BR-322载体中,然后将2个半长片段相连接得到全长c DNA克隆。将质粒线性化后,体外转录获得全长RNA,经电穿孔转染BHK21细胞拯救获得了初代病毒。将初代病毒接种感染Marc-145细胞进行传代,接毒后第36小时即可观察到完全的致细胞病变效应(CPE)。通过RT-PCR、间接免疫荧光、测序验证,证明成功获得了PRRSV GSWW株的感染性克隆。通过实时荧光定量法检测拯救病毒与原田间分离毒的复制能力,没有显著差异,采用实时荧光定量法测得的拯救病毒复制的最高效价为1×1010copies/m L。本研究表明,采用长片段基因合成的方法进行PRRSV感染性克隆的构建,方法可行且速度快,为PRRSV流行毒感染致病机理、免疫机制和基于反向遗传学的新型疫苗研制提供了重要的分子工具。According to the whole genomic sequence of PRRSV GSWW strain,using biosynthesis methods,two half-length of genomic sequences（7 636 bp and 7 774 bp） were synthesized and cloned into p BR322 vector.And then the full length c DNA clone was constructed by ligation two half part molecules into p BR322 vector.After linearization of the recombinant plasmids,the whole genomic RNA was prepared by in vitro transcription.The first generation of viruses was rescued by electro-transfection of prepared whole genomic RNA into BHK21 cells.The rescued virus werees passaged on Marc145 cells,and the complete cytopathic effect（CPE） was observed within 36 h after inoculation of the first generation of the rescued virus.The rescued PRRSV GSWW virus was further confirmed by RT-PCR,indirect immunofluorescence assay（IFA）and sequencing for the present of virus antigen and ecular marker involved.The growth kinetics of the rescued virus was similar with the parental virus,and the rescued virus titer could reach1×10^10copies/ml.This study showed that field strain PRRSV infectious clone could be effectively established by long fragment DNA synthesis,and would provide a useful tool for study on the molecular mechanisms of PRRSV pathogenesis and development of novel genetically engineered vaccines.

关 键 词：猪繁殖与呼吸综合征病毒 基因合成 感染性克隆 

分 类 号：S852.659.6[农业科学—基础兽医学]