Research On Recombinant Vaccine Of Carnivores Parvovirus

Canine parvovirus （CPV）, feline panleukopenia virus（FPV） and mink enteritisvirus （MEV） are of close relationship in antigenicity, belonging to the genusParvovirus of the family Parvoviridae. Animals affected by these viruses could sufferfrom clinical symptoms of severe panleukopenia and acute hemorrhagic enteritisexcept the nascent animals which would develop a series of distinct syndromes.Viruses responsible for these diseases crucially threaten the animal conservation andcultivation because of high morbidity and mortality.Parvovirus genome contains two open reading frames encoding non-structural（NS1,NS2） and structural proteins（VP1,VP2and VP3） respectively. The VP2proteinof parvovirus, accounting for approximately90%of the viral capsid,is the majorcapsid protein, and is responsible for the specific neutralizing antibody induction.In recent years, novel vaccines based on VP2protein become very popular fortheir high safety and significant immunogenicity. In this study, thereplication-defective human adenovirus type5was employed as the virus vector forits safety, convenience and efficacy. By far, recombinant virus vectored vaccine hasalready been used for rabies prevention of wildlife in North America. In this study, the full-length VP2genes were amplified form the three virusesusing primer pair VP2⁽-up and VP2⁽-down and were cloned into pMD18-T vector foridentification via enzyme digestion and sequencing. The identified VP2genes werethen transferred from the T vectors to the shuttle plasmid pacAd5CMV K-NpAthrough a ligation after a double digestion by EcoRI and BamHI. The recombinantplasmids, responsibly named as pacAd5-CVP2, pacAd5-FVP2and pacAd5-MVP2,were subsequently transfected into the HEK293AD cell line respectively with thebackbone plasmid pacAd59.2⁽-100together at a ratio of4:1after PacI linearization.Recombinant adenovirus bearing VP2genes, designated as rAd5v-CVP2,rAd5v-FVP2and rAd5v-MVP2respectively, were packaged and harvested in day7today14post transfection. After10passages, the titers of the viruses reached between10⁸and10^8.25TCID₅₀/ml with the cytopathic effect of the cells remained stable. Theelectron microscope observation of the recombinant viruses presented a typicaladenovirus shape. A381bp band could be directly amplified from the recombinantadenovirus cultures by PCR, indicating that the target genes were integrated into theadenovirus genome. Western blot analysis using canine parvovirus polyclonal serumas the primary antibody confirmed that the target gene was expressed in the infectedHEK293AD cells.To evaluate their immune efficacy,60mice were randomly divided into6groupswhich were respectively injected with500ul （108TCID50/ml） recombinant adenovirus （rAd5v-CVP2, rAd5v-FVP2or rAd5v-MVP2） or500ul (10^3.5TCID₅0/ml) parvovirus（CPV, FPV or MEV） intraperitoneally. The titers of the antibody against theparvovirus were determined via neutralization assay in the serum samples collectedfrom the inoculated mice repeatedly pre-injection,14d and28d post-injection. Thedata showed that the neutralization antibody on pre-injection was0, neutralizationantibody of experimental groups （（rAd5v-CVP2, rAd5v-FVP2or rAd5v-MVP2）） onthe14d post-injection were2^-5,2^-4.9,2^-4.9and the control groups （CPV, FPV or MEV）were2^-4.6,2^-4.6,2^-4.2, experimental groups on the28d post-injection were2^-5.6,2^-5.3,2^-6and the control groups were2^-5.3,2^-5,2^-4.5.In summary, recombinant adenovirus type5expressing VP2protein of CPV,FPV or MEV was successfully constructed and molecular biologically characterized.The subsequent immunogenicity analysis in mice revealed that all the threerecombinant adenoviruses could successfully mediate the specific neutralizingantibody production.