Preparationan Of Monoclonal Antibodies Against VP3 Protein Of Goose Parvovirus And Location Of Epitope

Gosling plague also known as Goose Parvovirus (GPV) infection, causes domestic goslings and Muscovy ducklings with chordapsus and liver, kidney, the heart substance organ inflamm violent contagious disease. GPV has high pathogenicity and lethality, makes severity effect to goose-producing countries, and causes huge economic loss. Owing to the hindrance to the development of goose cultural industry because of the disease, there needs Monoclonal Antibodies (McAb) and epitope for diagnosis and prevention. VP3 is the main structural protein of GPV, and also the main capsid protein, exposing the superficies of the nucleocapsid, contains GPV main antigen determinant, and it is the immune protective antigen of GPV, 80% of total protein. Because it can excite neutralization antibody, it is the preferred protein to develop genetic engineering recombinate vaccine. Preparation of McAb against VP3 protein and the location of linear B-cell epitope with McAbs will conduce to understand the relation of antigenic structure and function. It plays an important role in locating linear B-cell epitope of VP3, setting up gold colloid and ELISA clinical detect methods of gosling plague. It is also important for vaccine development, disease management and so on.The recombinant protein GPV VP3 was used as immunogen after purification by Ni-NTA. The 4 to 6 weeks-old BALB/c mice were intraperitoneally immunized with the VP3 protein for three times, then myeloma cells SP2/0 were fused with the spleen cells of the immunized BALB/c mice on day 3 post-last-immunization. Four hybridoma cell lines against the VP3 protein were obtained by screening with the indirect ELISA, named 1F1, 2A9, 3B11, 4A2, respectively. The first McAbs were identified to be IgG2b, and the others to be IgG1 withκlight chain. The ELISA titer of the 4 McAbs ascites were 1:819 200, 1:1 638 400, 1:409 600, 1:819 200, respectively. Western-blot analysis showed that the four McAbs could react with GPV VP3 recombinant protein and GPV native protein VP3 specifically, and the four McAbs could combine with GPV certified by IFA and immunoelectron microscopy.On the basis of the epitopes results of GPV VP3 protein which have already been identified in previous study, oligomeric nucleic acid fragments of ten amino acid fragments mutually coinciding five amino acids were contrived, after annealing, connected with pET-32a, derivation expressed, small fragment fusion proteins were gained. The antigenicity were identified by Western blot. With the same method, amino acids were deleted one by one, and two epitopes were located at 430-435aa and 643-647aa. This study has prepared McAbs against protein GPV VP3 and located the epitope of them. It has a significant meaning for early diagnosis and designing the safe and effective genetic engineering epitide vaccine against GPV on the basis of the molecule level of epitopes. McAbs and epitope will be used as material foundation for the further experiment of GPV, and will also offer far-reaching prospect for their association.