Activity Detection And Purification Of Early Pregnancy Factor In Beef Cattle

EPF (early pregnancy factor, EPF) is a protein with immune suppression and growth regulation in maternal serum during early pregnancy, which was found for the first time by an Australia scholar named Morton in 1974, because EPF can be detected after mating 6h in mice,it was named as early pregnancy factor. Early pregnancy factor is the earliest appeared immunosuppressive peptide substance in the blood associated with pregnancy after mammal fertilization. It is generally believed that early pregnancy factor is homologues of heat shock chaperonin 10 family members. E Rosette inhibition test(E-RIT)can be used to identify EPF biologically. EPF prevents maternal immune system from eliminating the fetus as foreign substance during early pregnancy. The secretion of early pregnancy factor depends on an appropriate hormonal environment, which associate with the ovaries, fallopian tubes, eggs, placenta, pituitary and so on. Early pregnancy factor can be detected in maternal blood several hours after fertilization and throughout the whole gestation. EPF in the mother disappears after childbirth. EPF can be used for early pregnancy diagnosis and prediction of early embryonic death and abortion, embryo transplantation identification, monitoring fetal status, then, EPF as a gentle and natural immune inhibitors can be used in treatment of autoimmune diseases, prevention of organ transplant rejection and so on. So, the study on this protein is valuable for livestock development.Beef cattle blood samples and vaginal mucus was collected after fertilization 20h, 25h and 30h respectively. This study tried to establish a simple and effective E rosette inhibition test to detect activity of early pregnancy factor quickly and accurately, and to determinate the earliest time of the early pregnancy factor was detected in blood and vaginal mucus, Serum was collected from healthy pregnant bovine in 1-9 month gestation, serum was isolated by DEAE-52 ion exchange chromatography, Con A-Sepharose 4B affinity chromatography and Heparin Sepharose Cl 6B affinity chromatography purification, in order to get purified EPF. Rosette inhibition test was used to detect activity of the various stages of product. SDS-polyacrylamide gel electrophoresis ( SDS-PAGE ) and isoelectric focusing polyacrylamide gel electrophoresis was used to analyzed purified EPF.The results showed that EPF cloud be detected in 20h after the insemination feom beef cattle serum and EPF can be detected in 25h from the vaginal mucus. Pregnant bovine and non-pregnant bovine serum rosette inhibition titer significantly different (P < 0.05), the purified EPF has shown activity by E rosette inhibition test, the molecular mass is 53.6 ku, and isoelectric point of 6.8. Conclusion is that EPF activity was detected by rosette inhibition test for identification of early pregnancy factor activity in pregnant beef cattle is possible, and the first 25h after insemination can be detected in the blood. The purified EPF was detected by rosette inhibition test shows activity. The molecular weight was detected by SDS-PAGE appears 53.6Ku. The isoelectric point was detected by isoelectric focusing polyacrylamide gel electrophoresis appears 6.8.