基于T7RNA聚合酶的犬瘟热病毒微型基因组构建与拯救OA北大核心CSTPCD

To develop a reverse genetic system for canine distemper virus(CDV)and recover the minigenome of CDV3 strain,the genome 3'-leader sequence,5'-tailer sequence and enhanced green fluorescent protein(EGFP)gene sequence were synthesized based on the genomic sequence of the commercial attenuated live vaccine CDV3 strain and reversely cloned into the plasmid PCR script regulated by the T7 promoter.Hammerhead ribozyme and hepatitis D ribozyme sequences were respectively introduced at the 5'and 3'ends of the minigenome to construct a minigenome plasmid pCR-mCDV3-EGFP.The pCR-mCDV3-EGFP plasmid and three helper plasmids expressing measles virus(MeV)N,P and L proteins were co-transfected into BSR cells and EGFP fluorescence was observed by fluorescence microscope.After 48 hours of co-transfection with plasmids,specific green fluorescence was observed in BSR cells.The successful development of a reverse genetics system of CDV minigenome was expected to be used for recovering CDV in the later period This study laid the foundation for the development of a new CDV vaccine and study of pathogenesis of infection.