中国兽医科学2017，Vol.47Issue(7)：838-842,5.DOI:10.16656/j.issn.1673-4696.2017.07.006
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
摘要
Abstract
According to the whole genomic sequence of PRRSV GSWW strain,using biosynthesis methods,two half-length ofgenomic sequences(7 636 bp and 7 774 bp) were synthesized and cloned into pBR322 vector.And then the full length cDNA clone was constructed by ligation two half part molecules into pBR322 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 Marc 145 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 reach 1 × 1010 copies/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Key words
porcine reproductive and respiratory syndrome virus (PRRSV)/gene synthesis/infectious clone分类
农业科技引用本文复制引用
王治家,张婧,白伟杰,孙普,曹轶梅,李冬,刘在新,卢曾军..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[J].中国兽医科学,2017,47(7):838-842,5.基金项目
甘肃省农业生物技术研究与应用开发项目(GNSW-2009-10) （GNSW-2009-10）
中国农业科学院创新工程经费资助 （）
