Preparation And Identification Of Anti-sheep Early Pregnancy Factor Monoclonal Antibodies

Early pregnancy factor is a pregnancy associated protein in serum after fertilization,and the protein has immune-suppressive. Early pregnancy factor can confirm the one of the earliest pregnancy biochemical indicators. In graziery production, using the specificity of EPF can confirm the sheep pregnancy diagnosis in time, so development and application in detection of EPF is important for sheep production.Objective: To Preparate anti-sheep early pregnancy factor( EPF) monoclonal antibodies by molecular biological and cell fusion techniques.Methods: The expression fungus was induced by IPTG to express the intersted protein. Spleen cells from Balb/c mice immunized four times with purified EPF were fused with homology myeloma cells(SP2/0). The cell strains of monoclonal antibodies were detected by the indirect ELISA method, and ascitic fluid were obtained by injecting cell strains to enterocoelia of mice. Monoclonal antibodies were purified by G-sepharose column affinity chromatography. Purity and specificity of monoclonal antibodies after purification were detected by SDS-PAGE with Western-blot.Results: When the gene engineering E.coli strain p GEX4t-EPF-BL21(DE) was grown into LB medium,shaking incubated at 37 for OD= 0.6℃-0.8,and induced for 4 hours by 0.5 m M IPTG,a high yield of EPF protein was obtained.A Cell strain of anti- sheep EPF monoclonal antibodies was obtained after the cell fused(B4D5), Anti- sheep EPF monoclonal antibodies were obtained with a high purity and specificity after purification.Conclusion: The recombinant protein EPF-GST was obtained, the immunized Balb/c mice and antibody effectiveness was analyzed. The monoclonal antibodies of EPF were obtained.