Early Pregnancy Factor Separation, Purification And Identification From Pregnant Cows' Milk

The early pregnancy factor(EPF)was separated and purified by cellulose ion exchange chromatography, sephadex gel chromatography and affinity chromatograph from milk of pregnant cows, to gain the highly purified EPF, and try to make anti-EPF monoclonal antibody at first stage.Experimental Methods: Milk of four-month pregnant cows was gathered and degreased by hypothermy freezing centrifugation.The degreased milk was separated and purified by ammonium sulfate fractionation salting-out, DEAE-52 cellulose ion exchange chromatography,G-100 dextran gel chromatography,ConA-Sepharose FF 6B affinity chromatography technology in turn to obtain EPFof high-purity.The EPF activities of various stages were detected by rosette suppression test. Molecular weight of EPF was examined by SDS-polyacrylamide gel electrophoresis.Isoelectric point of EPF was examined by polyacrylamide gel isoelectric focusing electrophoresis.Experimental Results: After ion-exchange chromatography and rosette suppression test,the EPF activity of every received peak was detected,with the result that peak of EPF activity is DE-â… . After dextran gel chromatography and rosette suppression test,the EPF activity of every received peak was detected,with the result that peak of EPF activity is G-â…¢.C-â… component of 0.6800mg was obtained by affinity chromatography technology from 400 ml milk of pregnant cows.Two protein bands can be seen after SDS-polyacrylamide gel electrophoresis with the relative olecular weight of 21.0 kDa,67.0kDa and isoelectric point of 6.80.Conclusion: This research indicated that the separation and purification technical route of early pregnancy factor is feasible,early pregnancy factors of high-purity were obtained.