Construction And Experimental Immune Of Gene Vaccines And Recombinant Canine Adenovirus Of CDV

Canine distemper (CD) caused by canine distemper virus (CDV) is an acute infectious disease which is characterized by high pathogenicity and infectivity. It is imminent to prevent CD for its high mortality to canine, fur animal and wildlife, etc. Among structural proteins of CDV, nucleocapsid protein (N) is an immunogenicity protein with strong conservatism and can cause CTL response to CDV. Fusion protein (F) is a protective antigen to induce neutralization antibody against CDV. Therefore, it is of great significance to construct gene vaccines and recombinant canine adenovirus containing N and F protein gene of CDV to prevent the infection of CDV. We report the engineering and the characterization of two gene vaccines and three CDV recombinant canine adenovirus type 2 expressing the N protein and F protein, respectively.According to the genome sequence of CDV reported in GenBank, two pairs of specific primers were designed and synthesized. Both N protein gene and F protein gene of CDV LP strain were respectively amplified by RT-PCR, cloned, sequenced, and phylogenetic analysis. The results demonstrated that the full length of N protein gene of CDV LP strain was 1571bp, which encoded 523 amino acids. The full length of F protein gene of CDV LP strain was 1989bp, which encoded 662 amino acids. There were five potential N-glycosylation sites in deduced amino acids sequence of F protein. The analysis of hydrophobicity, antigenic epitopes and surfaceprobability plot of N and F proteins with DNAStar Program indicated that there were distinct differences between CDV LP strain and Onderstepoort vaccine strain. Contrast to Onderstepoort vaccine strain, there were higher antigen surface indices in N and F proteins of CDV LP strain. Based on the above, two expressing plasmid vectors pCIN and pCIF were successfully constructed with pCI vector. After pCIF and pCIN plasmid vectors were transfected into MDCK cells with Lipofectamine, transcriptions of targeted genes were detected by RT-PCR and the expressing objective proteins were detected by ELISA method. The results showed that the transcription of objective genes and expression of interesting proteins could be detected at 72h post-transfection, respectively. Both the two expressed proteins were biological active.It is well known that foreign protein expressed by adenovirus vector possesses activity of the natural protein.Recombinant plasmid pCAV-2-pVAXLPN containing N protein gene expressing cassette and recombinant plasmid pCAV-2-pVAXLPF containing F protein gene expressing cassette were successfully constructed on the basis of pPoly2-CAV-2 vector containing the whole genome of CAV-2 SY strain. The linearized genomes of CAV-2-pVAXLPN and CAV-2-pVAXLPF were gained after recombinant plasmids pCAV-2-pVAXLPN and pCAV-2-pVAXLPF were digested with Asc I, respectively. These linearization genomes were mixed with the accessorial segments of CAV-2 SY genome digested with Sal I and Nru I together. Then the mixtures were co-transfected into MDCK cells by Lipofectamine, respectively. After three times of transfection, typical CAV-2 cytopathic effect (CPE) was observed under microscope. Recombinant canine adenovirus as expressing respectively N protein and F protein of CDV were successfully constructed and identified by electronmicroscopy, enzyme digestion, PCR and sequencing, respectively. The molecular weight of N protein and F protein was 58 KDa and 72 KDa, respectively. The genetic characteristics of recombinant viruses were all stable in serial passage experiment. To improve the expressing amount of F protein in recombinant virus, recombinant piasmid pCAV-2-pCILPF containing F protein gene cassette was successfully constructed on the basis of the expressing elements CMVI.E, intron and SV40 Late poly (A) of pCI vector as well as the backbone vetor pPoly2-CAV-2. Then this recombinant piasmid was transfected into MDCK cells with three kinds of methods by Lipofectamine. The recombinant virus CAV-2-pCILPF was gained through 3 passages in MDCK, and the typical cytopathic effects of CAV-2 could be observed. A recombinant CAV-2 expressing fusion protein of CDV was successfully constructed, which was demonstrated by observation in electron microscopy, enzyme digestion, PCR and sequencing. F protein with immunological activity expressed by recombinant virus could be specifically recognized by monoclonal antibody against CDV by western blot analysis and the molecular weight of fusion protein was 72kDa. Moreover, the expressing level of F protein in recombinant virus CAV-2-pCILPF was higher than that in recombinant virus CAV-2-pVAXLP.The genetic characteristics of recombinant virus CAV-2-pCILPF was also good in stability proved by serial passage experiment.The mice were immunized by intramuscular injection with single gene piasmid or two gene plasmids of pCIF, pCIN, pVAXF, and pVAXN as CDV gene vaccine. The puppies which were free of CDV antibody were immunized with individual or different combinations of the three strain recombinant viruses and the four gene vaccines by intramuscular injection. Indirect ELISA, serum neutralization test and lymphocyte transformationassays were conducted to determine the immunological response after three times of immunization. The results revealed that the specific cellular and humoral immunoresponse were induced by the recombinant plasmids in mice. Of all the experimental groups, the antibody titer against CDV in the group immunized by pCIF+pCIN together was the highest, but it was lower than that of positive control group which immunized by CDV attenuated strain.The specific immunoresponse could be induced in dogs immunized by single recombinant virus or recombinant virus combining gene vaccines.The group immunized by gene vaccines and recombinant virus together could effectively induce the higher antibody titer than the group immunized by sole recombinant virus. CAV-2-pCILPF could induce higher antibody titer than CAV-2-pVAXLPF when they were used to immune canines, respectively. But antibody titer against CDV in immunized canines was lower than that in canines inoculated by attenuated CDV. The recombinant virus could induce not only the specific antibody against CDV, but also the specific against CAV-2.The results indicated that immune protection could be induced not only in groups immunized with single recombinant viruses but also in groups immunized with recombinant viruses and gene vaccines together after virulent CDV was injected into the immunized canines.Several key technical problems were resolved by this work in developing safe and effective gene vaccine as well as recombinant canine adenovirus vaccine, which give a new way to prevent CD.