| Canine distemper (CD), caused by canine distemper virus (CDV), is acute and highly contagious disease in canine and other carnivores, and is one of the most severe infectious diseases in canine farming, far cultivation and wildlife conservation. Traditional attenuated vaccine has intrinsic shortcomings despite its important role in controlling the occurrence of CD. In order to develop a new kind of safe and efficient CDV genetically engineered vaccine, we cloned and sequenced the nucleocapsid(N) protein gene of CDV Nanjing isolate (CDV-NJ15), and constructed recombinant prokaryotic expression plasmid pET-N and another recombinant eukaryotic expression plasmid pcDNA-N, which express the N gene fragment. We analyzed immunogenicity of expression products in vitro, and subsequently detected immune response in mice induced by DNA immunization.According to the N gene sequence of CDV which was registed in GenBank database, a pair of specific primer was designed and used to amplify the full-length 1572bp N protein gene of CDV-NJ15 with RT-PCR. The PCR product was cloned into pMD 18-T vector routinely, and the positive recombinants were identified by ampicillin screening and endonuclease digestion. The correct positive recombinants were then sequenced and analyzed, The open reading frame length of the N gene of CDV-NJ15 isolate was 1572bp and encoded 523 amino acids. The homology of nucleotide and predicted amino acid of between Nanjing isolate and the vaccine strains(Onderstepoort strain and Convac strain) is 99.7% and 99.4% respectively, while comparison from other CDV isolates shows 94.0-95.1% and 97.5-98.3 identity respectively. It is predicted that CDV-NJ15 isolate was varied from Onderstepoort strain.Using RNA extracted from CDV-NJ15 as template, we designed two pairs of specific primers to amplify the 5' and 3' region of the N protein gene respectively (981bp and... |
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