猪脑心肌炎病毒感染性克隆构建及病毒拯救OA北大核心

Three cDNA fragments covering the complete genome of encephalomyocarditis virus(EMCV)XX1-19 were amplified using RT-PCR.The eukaryotic expression vector pcDNA3.1 was linearized by EcoR Ⅰdigestion,and the three cDNA fragments were simultaneously ligated into the vector to construct a re-combinant plasmid containing the complete genome of EMCV XX1-19.The recombinant plasmid was identi-fied by restriction enzymes BamH Ⅰ and Not Ⅰ digestion,combined with bacterial liquid PCR amplifica-tion,and sequencing verification.The results showed that the recombinant plasmid was successfully constructed and named pcDNA/XX1-19.The recombinant plasmid was transfected into BHK21 cells using li-posomes to rescue virus.The rescued virus was identified through cytopathic effect observation,nu-cleic acid integrity detection,transmission electron microscopy observation,TCID50 detection,and other methods.The results showed that the constructed full-length cDNA clone of EMCV was infectious and could rescue the virus.The one-step growth curve results indicate that the rescued virus has simi-lar in vitro proliferation characteristics to the wild-type virus.The method established in this study eliminates the tedious steps of gradually cloning and splicing gene fragments,as well as modify-ing vectors,avoiding the drawbacks of transcription of viral RNA in vitro.By using the CMV promoter of pcDNA3.1 vector,homologous recombination and transcription of viral RNA in vivo,infectious clones were rapidly and efficiently constructed and the virus was successfully rescued.