Analysis and expression of nucleocapsid protein gene of atypical canine distemper virus in Escherichia coli

To investigate the canine distemper virus(CDV) isolated from the lung and liver samples of an atypical clinical cases of canine distemper,the nucleocapsid protein(N) gene was amplified by RT-PCR with a pair of primers designed based on the N gene sequence of CDV reference strains in GenBank.The sequence analysis demonstrated that the homology of the atypical CDV N gene with other 12 virulent reference strains in GenBank were from 96.6% to 99.2% in nucleotide and 97.9% to 99.4% in amino acid sequence,and shared 93.2% to 93.6% nucleotide sequence homology and 96.4% to 97.5% amino acid sequence with that of 4 attenuated vaccine stains.The phylogenetic tree based on the sequences of the N gene showed that the atypical CDV was in the same subgroup with the virulent strains.Furthermore,the N gene from the atypical CDV was sub-cloned into pET-28a(+) and expressed in E.coli.SDS-PAGE analysis indicated that the recombinant N protein was 62 ku and mainly existed as inclusion bodies in E.coli.Western blot showed that the recombinant N protein could be recognized by CDV positive serum.An indirect ELISA coated with the purified recombinant N protein showed specificity for the detection of antibody against CDV.