Preparation Of Monoclonal Antibodies Against P27 And Gp85 Proteins Of Avian Leukemia And Establishment Of Detection Methods

Avian leukemia virus(ALV)is a retrovirus that causes a variety of neoplastic diseases in birds.ALV is divided into endogenous and exogenous viruses with 11 subgroups.ALV is primarily vertically transmitted by exogenous genes.Infection with ALV can cause gradual growth and weight loss in broilers and reduced egg production in laying hens,and can even cause death in chickens.Of these,subpopulation ALV-J is the most infected subpopulation,with horizontal and vertical transmission,rapid and infectious,and ALV transmission can only be isolated by purifying the population.In order to improve the decontamination rate of poultry farms,it is particularly essential to develop a rapid detection method for ALV.In this experiment,the p27 and gp85 genes were amplified by PCR using the cDNA of ALV-J as template,the p ET-32a-p27 and p ET-28a-Mysb-gp85 vectors were constructed using a prokaryotic expression system and induced to be expressed in BL21 recipient cells,and the recombinant proteins were purified by Ni chromatography column.The ALV-J-p27 and ALV-J-gp85 proteins were used as antigens to immunize BABL/c mice,whose spleen cells were fused with SP2/0 cells to screen for hybridoma cells capable of secreting stable antibodies by a limited dilution method.The results showed that p27 and gp85 recombinant proteins were successfully expressed with sizes of 45 k Da and 65 k Da,respectively.Four hybrid cells,named 3C9,1H15,1H6,7G2,were screened from mice immunized with the p27 protein,and the heavy chains were identified as Ig G2 b,Ig G2 b,Ig G1,Ig G1,and the light chains were identified as Kappa by antibody isotype identification.The gp85 protein-immunized mice were screened for a hybridoma cell line,named 2G9,identified by antibody isotypes Ig G1 for the heavy chain and Kappa for the light chain.A precipitation method using octanoic acidammonium sulfate and protein G agar was used.The monoclonal antibodies were purified by gel affinity chromatography column method.Western blotting identified the ability of the monoclonal antibody to react with ALV-J virus,demonstrating its excellent specificity.The monoclonal antibody 7G2 was labeled with HRP and the best capture antibody was 3C9,the best coating concentration was 2 μg/m L,the best dilution of the enzyme-labeled antibody was 1:2 000,and the best color development time was15 min.The samples were detected by using the established double antibody sandwich ELISA method,and the results showed that this method has great sensitivity for the detection of ALV The results showed that the method was sensitive and provided a theoretical basis for the detection of ALV.The eukaryotic p27 and gp85 genes were also amplified by PCR using the cDNA of ALV-J as a template,and the p YBDM-IM-p27 and p YBDM-IM-gp85 vectors were constructed from the baculovirus system and transfected into Am receptor cells to obtain Bacmid-IM-p27 and Bacmid-IM-gp85 strains,which were then transfected into Sf9 cells.The appearance of red fluorescence indicates the successful transfection of the baculovirus.The p27 and gp85 proteins were identified by western blotting,which revealed specific bands at 27 and 36 k Da.Sf9 cells were infected with a transfected baculovirus,and 3C9 and 2G9 antibodies were incubated as primary antibodies.The IFA analysis showed that the monoclonal antibody was able to respond to the baculovirus,demonstrating the excellent specificity of the screened monoclonal antibody.This sets the stage for subsequent refinement of ALV assays.In summary,this experiment used a prokaryotic expression system to prepare the p27 and gp85 proteins and prepared monoclonal antibodies to initially establish a double antibody sandwich ELISA with excellent specificity.We also expressed the proteins via baculovirus to lay the foundation for subsequent improvements in the assay.