Preparationan Of Monoclonal Antibodies Against Recombinant NS1 Of GPV And Its Identification Of Antigenic Epitopes Domain

Goose parvovirus (GPV) infection known as goose plaque, belongs to Parvovirinae Dependovirus, and causes high mortality in domestic goslings and Muscovy dueklings. It caused 90% mortality in susceptible goslings under the age of 4 days to 20 days. It's can cause acute enteritis and also liver, kidney, heart and other organs septic lesions, particularly the fibrous of the small intestine. GPV infection spread every year in different degree that hinder the healthy development of breeding industry, requires an accurate and convenient diagnostic method for diagnosis. Owing to the hinder the breeding industry development because of this desease, there needs accurate,convenient diagnostic method for early diagnosis avoiding the interference of maternal antibodies. GPV-NS1 is the main non-structural protein. The monoclonal antibodies (McAbs) against GPV-NS1 were producted. It not only can help us comprehend the relations between its antigen constitution and its function by studying the epitides of GPV- NS1 with its monoclonal antibodies, but also has a significant meaning for early diagnosis and designing on the basis of the molecule level of epitopes.In this study, we constructed plasmid pcDNA3.1-GPV-NS1 and a recombinant protein GPV- NS1, and expressed recombinant protein GPV- NS1 in the system of E.coli Rosetta. The secreted McAbs were obtained by fusing mouse myeloma cells and spleen cells of Balb/c mice which were immunized with the plasmid pcDNA3.1-GPV-NS1 and recombinant protein of GPV-NS1. Six hybridoma cell lines against GPV-NS1 were selected by screening with indirect ELISA. The subtypes of the two McAbs were IgG2a, the others were IgM. The light chain areκ. Six hybridoma cell lines showed strong reactivity in indirect immunofluorescence test on the GEF with transient expressed pcDNA3.1-GPV-NS1, indicating that six hybridoma cell lines were GPV-NS1 specific monoclonal antibodies.Western blot analysis showed that the six McAbs could react with recombinant GPV-NS1. GPV-NS1 was dissected into 15 overlapping epitopes, which were used to react with McAbs by Western blot. The results showed that 1B2,3F12 and 4C6 reacted with fusion peptide GPV-NS1 498～532aa; 3D9 reacted with fusion peptide GPV-NS1 485～532aa; 2D4 reacted with fusion peptide GPV-NS1 485～514aa; 3H8 reacted with fusion peptide GPV-NS1 543～573aa. It indicated sthat the non-structural protein linear B-cell epitopes located at the C-terminus 485～514aa, 498~532aa and 543～573aa.This study has prepared McABs against protein GPV-NS1, and detected the epitopes of them. It can help us to better comprehend the characteristics of its antigen, and has a significant meaning for early diagnosis and designing on the basis of the molecule level of epitopes.