Development Of Monoclonal Antibody Against Meq Of Marek's Disease Virus And Its Application

1.Development of monoclonal antibodies specific to the Marek's disease virus Meq geneMeq gene fragment of Marek's disease virus(MDV)was amplified by polymerase chain reaction(PCR)method with specific primers designed according to the published MDV-RBIB-Meq sequence in NCBI.The products of PCR and Pcold-TF vector was digested with two restriction endonucleases EcoRI and XbaI.The digested products were then ligated and transformed into BL21.Positive expressing strains were screened out to obtain recombinant plasmid Pcold-TF-Meq.The results of enzyme digestion,SDS-PAGE and Western blotting showed that the constructed Pcold-TF prokaryotic expression vector was correct and the gene sequence analysis was completely consistent with the expected results.The prokaryotic expression of bacteria transfected with BL21 was induced with ITPG,a large number of expressed MDV-RB1B-Meq recombinant proteins were obtained in the supernatant.SDS-PAGE and Wenstern blot identification showed a specific band about 77KDa molecular weight.The specific protein band was excised ans used as an immunogen.Balb/cc mice were immunized four times.The spleen cells of the immunized mice were fused with SP2/0 cells.Enzyme-linked immunosorbent assay(ELISA)and indirect immunofluorescence(IFA)were used to detect the hybridoma cell supernatant,and a hybridoma cell specific for Meq protein was successfully obtained and named MDV-5F4.After 2 subclonings,the hybridoma cells reached 100%secreting anti-MDV-Meq protein-specific antibodies.MDV-5F4.The subtype of monoclonal antibody was IgM in ELISA kit.The successful development of the monoclonal antibody provided conditions for the differential diagnosis of MDV tumors.2.Establishment of immunohistochemical detection method Poultry tumors mainly include Marek's disease,avian leukemia,reticuloendotheliosis,and other tumors of unknown ongin.In order to rapidly identity tumors in the clinic,an immunohistochemical detection method and indirect immunofluorescence assay(IFA)for three pathogenic tumors including MDV,ALV and REV were established with our monoclonal antibodies to MDV-Meq MDV-5F4,MDV gB protein MDV-BD8.avian leukosis virus ALV-5D3 and reticuloendotheliosis virus REV-6C12.The results showed that this method can be used to rapidly detect the tumor tissues of chickens infected with MDV and ALSV,and has a good application in the future.