Cloning and Sequence Analysis of CDV H,F,N Gene

The cDNAs of H,F and N genes of the CDVLP strain were amplified by RT-PCR,cloned and sequenced.Molecular and phylogenetic analyses of H gene revealed that the CDVLP strain genotype belongs to the lineage of the field isolate strains.The H gene has more potential glycosylation sites. The F and N genes are more conserved than the H gene.By molecular epidemiology assay,it implied that the CDVLP strain has higher immunogenicity than the vaccine strains.From a practical point of view,CDV DNA vaccine and recombinant vaccine based on canine adenovirus type-2 can be constructed with CDVLP H,F and N genes.The H,F and N genes of CDVLP strain were sub-cloned into the eukaryotic expression plasmid pVAX1,which is driven by the CMV promoter,named pVAXLPH,pVAXLPF and pVAXLPN,respectively.Purified plasmids DNA were transfected into BHK-21 cells by lipofectamine.The CDV H,F and N proteins were expressed in BHK-21,which do specifically react to the CDV antisera.The mice inoculated with these plasmids induced the immune response against CDV.