Epitope Determination And Construction Of Recombinant Adenovirus Expressing VP3Gene Of Waterfowl Parvovirus

Goose parvovirus and Muscovy duck parvovirus are one of the smallest single-stranded DNAin animal virus, which both belong to Dependovirus genus of Parvovirdae family. In infectedgoslings and ducklings, clinical signs show diarrhoea, chordapsus and so on. Parvovirus ischaracterized by high mortality and morbidity, causing huge economic losses in waterfowl-farming.To provide a new inspiration for the study of vaccine and therapeutics, we mapped the epitopes ofthe VP3protein by phage display and construction of recombinant adenobirus expressing VP3gene of waterfowl.Precise epitope mapping may help design more diagnostic reagents and reasonable vaccines.A consensus sequence, DYRFHH, was screened from a random12-peptide library by biopanningusing a monoclonal antibody （McAb）4A2against GPV VP3protein. The motif showed highhomology with the amino acid sequence⁶⁵DYRFHH⁸⁸in GPV VP3protein. Selected8phages andidentified by ELISA, the sequence of DYRFHH was shown to be reactive with4A2. We concludethat⁶⁵DYRFHH⁸⁸represents a structure epitope of GPV VP3protein. The result was helpful forfurther structural and functional research on VP3protein and development of vaccine of GPV andMDPV.The VP3protein is the most abundant of three core proteins and can induce neutralizingantibodies in GPV-or MDPV-infected waterfowl. The primers specific for VP3-encoding genes ofGPV and MDPV were designed, respectively. The VP3gene was amplified from GPV and MDPVby PCR technique and cloned into pShuttle-CMV vector. The recombinant plasmid which wasidentified correctly was linearized by PmeⅠ and transformed into E.coil BJ5183（AdEasy-1-BJ5183） competent cells, which containing adenovirus backbone vector, to producerecombinant adenovirus plasmid by homologous recombination. The positive recombinantplasmids could be digested by PacⅠinto30kb and3kb/4.5kb fragments. The positive plasmidswere linearized by PacⅠ and transfected into HEK-293cells. After10days, obvious cytopathicappeared, identified the recombinant adenovirus by PCR and indirect immunofluorescence assay,the results showed that the recombinant adenovirus rAd-GPV-VP3and rAd-MDPV-VP3couldefficiently express VP3protein, which laid the foundation for new GPV and MDPV vaccine.