Sequence Analysis Of F Gene And The Immune Response In Mice Co-immunized By H,F And N Gene Of CDV Isolated From Fox And Raccoon Dog

Canine distemper is acute and highly contagious in many species of carnivores caused by canine distemper virus(CDV).It is one of the most important viral infectious diseases commonly occur among farmed fur animals.Once prevalence,it will be followed by high mortality of 30%-100%.A great number of deaths can not only lead to sever economic loss for the owners,but also badly influence and restrict the development of the whole industry. Although the extensive use of traditional attenuated vaccine has provided the CD well prevented and better controlled than before,it still has intrinsic shortcomings.Such as attenuated vaccines for CDV are easily interference from maternal antibodies;they are likely to return virulence and are not safe for use for some wild animals need to be protected from CDV. Especially,as the genome of CDV evolves continually and the emergence of field strains in recent years,traditional attenuated vaccine can not offer full protection.As a result,it is very important to study genetic variation characters and develop a new kind of safe and efficient CDV genetically engineered vaccine for the disease of canine distemper that we could prevent and control them more effectively.Six canine distemper virus(CDV)isolated strains which were named MS01,SCO1,ZD01,NM01,GN01,HL01,were obtained from foxes and raccoon dogs in some areas of Heilongjiang province.A pair of primers was designed according to the F gene sequences of CDV published in GenBank.The F genes of six isolated strains were amplified by RT-PCR, cloned and sequenced.Then the amino acid sequcences of fusion protein were deduced and the phylogenetic tree was constructed.Sequence analysis showed the F genes of six isolated strains were all contained a 1989bp open reading frame which coded for a polypeptide of 663 amino acid.All of the 13 Cysteine residues and 4 potential asparagines-linked glycosylation sites in F glycoprotein were competely conserved among these strains.Cleavage site amino acid sequence was²²⁰R-R-Q-R-²²⁴R.The phylogenetic tree showed the six Chinese isolated strains were all belongs to the virulent group which the representative strain is A75/17.The HL01 strain has a high homology with phocid distemper virus-2(PDV-2),and far distance with other five isolated strains.The F gene of CDV MS01 isolate were cloned into the eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid pcDNA-F was identified by PCR and digestion with restriction enzymes.Subsequently the mice were immuned with pcDNA-H and pcDNA-N which constructed by our lab and pcDNA-N recombinant plasmids respectively or mixed by intramuscular injection,attenuated CDV vaccine,pcDNA empty vector and negative control were also on trial.As a result,the result of CD4+/CD8+of the three genes mixed immunization group is apparently higher than sigle gene or both genes immunization group and is close to the group of attenuated CDV vaccine.The immunization of DNA vaccine of H,F,N can been detected CDV antibody.All in all,the mice inoculated with these plasmids developed specific humoral and cellular immune responses against CDV.The experiments are benefitial to the following dog trial and the fundamental study of more researches of DNA vaccine for fur animals.