Lederle Strains Of Canine Distemper Virus H And N Gene Cloning, Sequence Analysis And Expression

Canine distemper caused by canine distemper virus (CDV) is a highly infectious, frequently lethal disease with high mortality in dogs and other carnivores. CDV belongs to the genus morbillivirus, family Paramyxoviridae and has a single-stranded, negative sense RNA genome with a size of approximately 16 kb. The CDV genome contains six non-overlapping gene regions, organized 3'-N-P-M-F-H-L-5. Hemagglutinin protein and Nucleocapsid protein play an important role in immuno-prevention of canine distemper.Epidemiology survey of canine distemper showed that CDV infection is ubiquitous in dogs. Based on this situation, the H and N genes of CDV were cloned and sequenced from the genome of CDV Lederle strain. The partial fragments of the two genes were expressed in prokaryotic vector and the recombinant proteins showed positive reaction with anti-CDV serum. These results would be helpful on diagnosis and immuno-prevention of CDV.In epidemiology survey of canine distemper RT-PCR was used directly to detect the CDV antigen from the stool swabs of sick dogs. The positive ratio was 18.7% in all-sick dogs. The result may give a hint on the relationship between age, immunity situation of dogs and the occurrence risk of canine distemper. The young dogs are easier to infect CD than the elder and the dogs that have immunized still have high infected ratio.CDV growth was in Vero, DK and CEF in this study. Multiple blind cell-passages were in the three cell lines before obvious cytopathic effect (CPE) appeared. The two former cell lines had no CPE and the infected CEF cells showed obvious CPE including cell rounding, detachment of cells and destruction of the cell monolayer. The cell adaptation strain was obtained, which would offer a substantial basis for monoclone of entire virus.Pairs of primers were designed and synthesized based on the sequence of the haemagglutinin (H) and nucleocapsid (N)protein genes of CDV Onderstepoort strain. Full-length fragments of the H and N genes were amplified by RT-PCR from the RNA genome of CDV Lederle strain. The two fragments were cloned into pGM-T easy vector for sequencing. Sequence analysis showed that the nucleotic sequence of the H and N genes had identity of 97.0% and 97.4% with CDV Onderstepoort strain respectively. And the deduced amino acid sequence showed identity of 99.1% and 97.9%. The sequences of H and N registered in Genbankand the Genbank accession were EF418782 and EF418783.The partial fragments of the H and N genes of CDV were amplified from the recombinant plasmids containing the full-length of the H and N genes by PCR respectively. The amplified fragments were also cloned into pGM-T easy vector and sequenced. Then the cloned fragments were subcloned into prokaryotic expression vector pET-32a, and the recombinant expression plasmids were expressed in E.coli BL21 (DE3) with inducement. The size of the recombinant fusion proteins was 57 kDa (H) and 37 kDa (N), respectively.The recombinant fusion proteins showed positive reaction with anti-CDV positive serum by Western blotting. Purification and immunogenicity analysis of the two recombinant proteins will offer a substantial basis for preparing an ELISA detection kit of CDV.