| Avian leukosis is the variety of tumor diseases of chickens which was induced by avian leukos is virus(ALV). It can directly induce tumors and death in chickens and can also result in immunosuppression, mixed infection and lower immune response ability. Since 2008, avian leukosis, especially the J subgroup avian leukosis, has become widespread throughout most regions of China and has resulted in severe economic losses to the poultry industry. Effective vaccines and medicines against ALV are not available. The control of ALV infections is to establish exogenous ALV free poultry flocks through adopting eradication as the strategy of choice. Therefore, the development of ALV diagnostic method using group and subgroup specific monoclonal antibody(MAb) will be of great help for the diagnosis and control of ALV.P27 protein was the group specific antigen of ALV. For p27 protein had many virus antigen sites easy to detect and its gene is highly conserved, it is the first choice to prepare detection antibody. In this study, we successfully expressed and purified p27 protein through prokaryotic expression system. Four hybridoma cells, named 3A9、2E5、3D5、3H12, stably secreting MAb against p27 protein was obtained by hybridoma technology. One linear B-cell epitope 181PPSAR185 specific for p27 protein was identif ied by pepscan technology. The epitope was highly conserved among different ALV subgroups by sequence alignment analysis, indicating that it is a group specific epitope. Avian leukosis virus group specific antigen ELISA method was developed using MAb 2E5 as capture antibody and MAb 3D5 as detection antibody. This diagnostic method exhibits high specificity, for it can react with the common A, B and J subgroup of ALV, but cannot react with other avian pathogens, s uch as NDV, IBDV, MDV and AI V. The detection limit of recombinant p27 protein was as low as 1.25ng/m L. The sensitivity of the diagnostic method developed in this study was a little higher than the IDEXX Avian Leukosis Virus Antigen Test Kit( ALV Ag). The coefficients of variation intra-assay and inter-assay were less than 10%. The ELISA kit stored at 4℃ could be maintained for 12 months. The coincidence rate of detection result of 2555 cloacal swab samples between the diagnostic method developed in this study and the IDEXX ALV Ag was 96.32%. And the rate of 1141 albumen samples was 97.20%. In animal experiments, the first positive result of the PCR detection method was 11 days after infection, while the ELISA method developed in this study was 12 days.Gp85 protein exhibits high variability among different ALV subgroups. The ALV-J gp85 gene exhibited only 30% homology with other subgroups of ALV. In this study, we successfully expressed and purified gp85 protein through prokaryotic expression system. One hybridoma cell stably secreting MAb against gp85 protein was obtained by hybridoma technology. The MAb cannot react with ALV- A and ALV- B, but it can react with variety of ALV-J strains isolated from different contries. One linear B-cell epitope 134AEAELRDFI142 specific for gp85 protein was identif ied by pepscan technology. T he epitope was highly conserved among ALV-J strains by sequence alignment analysis. The MAbs and the identified epitope might provide valuable tools for the development of immunodiagnos tic approaches for ALV-J.In this study, four hybridoma cells stably secreting MAb against p27 protein and one hybridoma cell stably secreting MAb against gp85 protein were obtained. Through pepscan technology, one p27 linear B-cell epitope 181PPSAR185 and one gp85 linear B-cell epitope 134AEAELRDFI142 were identif ied. Avian leukos is virus group specific antigen ELIS A method was developed using MAb 2E5 and MAb 3D5. This diagnostic method exhibits high specificity, sensitivity and coincidence rate with other diagnostic methods, indicating that it will be useful for the diagnosis of ALV and establishing exogenous ALV free poultry flocks through adopting eradication. |
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