关键词: 犬瘟热病毒小熊猫株, 反向遗传系统, 构建

Abstract: In this study,the CDV originated from lesser pandas was isolated and served as the template for the infectious cDNA clone of CDV. After genome sequencing,seven cDNA fragments containing full-length CDV genome sequence were obtained by RT-PCR. The seven different fragments were digested and spliced together,and then inserted into eukaryotic expression vector pCI. Thus,we obtained the full-length cDNA of CDV lesser panda named pCI-CDV-LP. Furthermore, three helper plasmids were constructed by cloning the N,P,L protein ORF of lesser panda strain CDV in pCI vectors,respectively. The sequences of nuclease and CDV cDNA in pCI vector were verified by restriction enzyme digestion and sequencing. The full-length plasmid and three helper plasmids were then co-transfected into BSR cells using transfection reagent Lipofectamine TM2000. The results of RT-PCR,indirect immunofluorescence assay and viral infection assay showed that the lesser panda CDV was rescued successfully,and the reverse genetics of lesser panda CDV was built successfully. The results of the current investigation provide an important insight into the pathogenesis of canine distemper virus.