Expression Of Sheep Early Pregnancy Factor And Preparation Of Polyclonal Antibody Against Early Pregnancy Factor

Early pregnancy factor (EPF) is a pregnancy-associated protein that hasimmune-suppressive and growth-regulatory activities. It is regarded as a biochemical indexby which super-early pregnancy can be determined even than HCG. EPF has a highspecificity for gravida, so development and application in detection of EPF is important forlivestock production.Objective: Applying prokaryotic expression system to prepare EPF，Rabbits wereimmunized by Freund’s adjuvant immunization method, and then obtain EPF polyclonalantibodies, which is laying a foundation of pregnancy factor detection kit for furtherresearch.Methods: The total RNA was extracted from pregnancy sheep ovarian tissue. Theobjective gene of EPF obtained by RT-PCR and cloned into the expression vectorpGEX-4T-1and pET20b, and transform into the BL21(DE3). The recombinant protein wasanalyzed through SDS-PAGE and Western-blot after induced expression by IPTG. Mixingrecombinant protein and freund’s adjuvant as1:1, and obtaining polyclonal antibodythrough immunized rarebit. The recombinant protein with His as coating antigen wasanalyzed by polyclonal antibody through Western Blot. The twenty sheep’s pregnanciessituations were detected by polyclonal antibody.Results: Through GST Resin or Ni-NTA Resin，we gained two recombinant protein.The One containing GST Tab is about36kDa, the other containing His Tab is about12kDa. The antibody titre at57d after immunization reached1:12000and the accuracy ofantibody is95%in the practical application. The antibody can identify EPF protein.Overall, EPF recombination got a high expression in e.coli and the polyclonal antibodywas prepared successfully with good biological activity and specificity.