Prokaryotic Expression And Genetic Evolution Analysis Of Phosphoprotein Gene Of Canine Distemper Virus

【Objective】Two strains of Canine distemper virus (CDV) were as the experimental materials of this study. The genetic evolution analysis was done by comparing the phosphoprotein gene sequences of two strains of CDV with 10 different strains in GenBank to provide a scientific basis for molecular epidemiological investigation and control measures of Canine distemper (CD). To establish a fast, simple and specific method of diagnosis and lay experimental basis for genetic engineering vaccine development, an prokaryotic expression of phosphoprotein gene of Canine distemper virus from dog was done as well.【Method】Phosphoprotein gene of Canine distemper virus amplied by RT-PCR was cloned and sequenced. The nucleotide and amino acid sequences of the gene of two strain CDVs and 10 different strain CDVs in GenBank was analyzed and the phylogenetic tree with DNA Star and Mega(version3.1) was drawed. The prokaryotic expression system of CDV phosphoprotein gene was established by using of recombinant DNA technology, and identified immune activity of the expression products though SDS-PAGE, indirect ELISA and western-blot method..【Results】The results showed that the homology of Canine distemper virus phosphoprotein gene from dog and tiger with sequences AY466011, AY964113, AF181446, AY386315, AF259549, AJ582385, AB212727, AB212962, AY130857 and U53712 in GenBank were as follows: 93% and 96%, 95% and 95%, 93% and 99%, 91% and 94%, 91% and 94%, 95% and 96%, 94% and 96%, 96% and 98%, 98% and 97%, 93% and 95%. Six nucleotide mutations were identified in phosphoprotein gene's conservation region of twelve strain CDVs, in which 2243C and 2465A were only found in the tigger's sequence, AF466011 and AY130857. The result of tigger strain related to AF466011 and AY130857 showed that the site 2243 and 2465 nucleotide mutations may have some kind of relationship with Canine distemper virus spreading between species. In addition, the recombinant bacterium of dog's CDV phosphoprotein gene had been obtained successfully. The recombinant bacterium could express the fusion protein about 92ku at the condition of 1.0 mmol/L IPTG-induced and 37℃for 6 hours. The purified recombinant protein identified through Western-blot and indirect ELISA test showed that there was a specific reaction with the polyclonal antibody against CDV, and that it was the recombinant phosphoprotein of CDV.【Conclusion】Six nucleotide mutations were identified in phosphoprotein gene's conservation region of twelve strain CDVs. The genetic evolution analysis of the region showed the closest kinship between strains from the tigger, coon (AF466011) and mink (AY130857). The prokaryotic expression system of CDV phosphoprotein gene was established sussessfully, and the recombinant phosphoprotein was obtained.