Study On Constructing The DNA Vaccine Of Canine Distemper And Vaccination Experiments

The Canine distemper virus which naturally infected Canis,Mustela,Ursidae and cat and belonged to the number of genus Morbillivirus in Paramyxoviridae is minor strand RNA virus having capsular,is one of the most acute and highly contagious disease in canine farming,fur cultivation and wildlife conservation.The useful way of preventing Canine distemper is vaccine inoculation,there are lots of defects in inactivated vaccine and live vaccine now.Traditional attenuated vaccine has intrinsic shortcomings despite its important role in controlling the occurrence of CD:it can cause immunosuppression and lesion of CNS,it may be lethal some susceptible animal and possess a wild host spectrum.Moreover,mutation of CDV can also cause epidemic of this disease,so safer,more effective and more pragmatic vaccine is being studied.Gene vaccine and genetically engineering vaccine are studied by more and more people.Many studies have shown that two glycoproteins(Hemagglutinin H and Fusion F protein) of CDV are major target antigens for the host immune system.Nucleocapsid(N) protein is highly conserved immunogenicity protein.In addition,N protein contains T cell epitope.Hemagglutinin protein,Fusion protein and Nucleocapsid protein play an important role in immuno-prevention of canine distemper.To develop a new kind of safe CDV genetically engineered vaccine,four pairs of primers were designed according to theH,F,N genes of Canine Distemper virus strains.Then the H,F,N genesof CDV strain were amplified by reverse transcription polymerase chain reaction(RT-PCR).The amplified fragments were cloned into pMD-18T simple vector.Four objective genes were obtained and four eukaryon recombinant expression plasmids were constructed by re-cloning the objective gene into pIRES1neo vector.The eukaryon recombinant expression plasmid pIRES H,pIRES F,pIRES N1 and pIRES N2 could express respectively objective protein by Western-blot.Then vaccination experiments were done by mouses and raccoon dogs,antibody was detected by ELISA,conteracting toxic substances experiment was done by raccoon dogs at same time.The results indicated that the conducting gene vaccine could induce specific immune response of mouses and raccoon dogs,but the protection ratio of gene vaccine was inconsistent(33%-100%).