Immunogenicity Analysis Of H Protein Of Asia-1 Serotype Canine Distemper Virus And Construction Of Recombinant Canine Adenovirus Type-2

Canine distemper (CD) is an acute or subacute, highly contagious febrile disease in dogs and other camivores caused by infection with canine distemper virus (CDV), and is one of the most severe diseases in canine farming, far cultivation and wildlife conservation. Although conventional live modified vaccines are commercially available and play an important role in controlling the occurrence of CD have several severe drawbacks. And with the changes of environment and CDV adaptation to prevailing factors, new CDV variants, the number of typical CD cases has increased throughout the world, and several episodes of CDV disease in vaccinated animals have been reported. Recently, it is very important to to develop new kinds of safe and efficient CDV genetically engineered vaccines.In the study, we cloned and sequenced the haemagglutinin (H) protein gene of Asia-1 serotype CDV field isolate (HLJ2-07 strain), and constructed recombinant eukaryotic expression plasmid pcDNA3.1-H,which express the H gene fragment. We analyzed immunogenicity of expression products in vitro, and subsequently detected immune response in mice induced by DNA immunization. A transfer vector named as pUC-2â–³E3-H was congstructed based on the generated vector pUC-â–³E3-EGFP with insertion of H gene. Recombinant virus rCAV-2â–³E3-EGFP successfully constructed by our lab infected MDCK cells, and obtained after several cycles of plaque purification and PCR identification, then we analysised its bionomics. Based on these, The transfer vector pUC-2â–³E3-H was transfected into MDCK cells infected with rCAV-â–³E3-EGFP by calcium phosphate-DNA coprecipitation method. The recombinant virus was selected by virus plaque and identified by PCR, named rCAV-2â–³E3-H.The results showed that the full length of H protein gene of HLJ2-07 strain was 1824 bp, which encoded 607 amino acids. There were nine potential N-glycosylation sites, and twelve cysteine residues. Homology analysis showed that the genetic relationship between HLJ2-07 and Onderstepoort (91.4 %), Convac (91.3 %)was the farthest of all. Phylogenetic analysis showed that HLJ2-07 belonged to Asia-1 type and its comparison with 32 strains CDV isolated in china indicated that most of them belonged to Asia-1 type. Alteration of gene type may be the important reason of changing antigenicity of H protein. So it is necessary to find a proper stains in domestic to construct virus vaccine. The result showed that the sera of immunized goups could react with MDCK-SLAM infected by virus and had specifically bright red fluorescence. The titers of antibody of ELISA and SN could relatively reach1:28-1:79 and 1:11-1:32. Three tests indicated that H protein had the Immunogenicity. PCR, IFA, Western blot confirmed that recombinant virus rCAV-2â–³E3-H could exress H protein whose molecular weight was about 84 kD. After the recombinant virus rCAV-â–³E3-EGFP infected MDCK cells, when cytopathic effect was observed the cells were frozen and thawed three times, sonicated and centrifuged. The supernatant was generated after several cycles of plaque purification and PCR identification. Compared to its parental virus CAV-2, the recombinant virus rCAV-â–³E3-EGFP showed no obvious differences in virus multiplication and growth kinetics in MDCK cells.After 25 passages in MDCK cells , the EGFP gene was expressed stably in the recombinant virus rCAV-â–³E3-EGFP, which was shown a front view of application. The transfer vector pUC-2â–³E3-H was transfected into MDCK cells infected with rCAV-â–³E3-EGFP by calcium phosphate-DNA coprecipitation method. The recombinant virus was selected by virus plaque and identified by PCR, named rCAV-2â–³E3-H. PCR, IFA, confirmed that recombinant virus rCAV-2â–³E3-H could exress H protein.In this study, based on clone and sequence analysis of H gene of CDV HLJ2-07 strain, the Immunogenicity of H protein expressed in eukaryotic system was analyzed. A recombinant virus rCAV-â–³E3-EGFP expressing EGFP gene was generated after plaque purification and PCR identification. The recombinant adenovirus vector expressing H protein was successfully constructed. All of these lay a foundation for the next studies of genetic stability, growth kinetics and immunology.