Ohnuma Katsuhiko, Ito Kazuei, Takahashi Jutaro, Nambo Yasuo, Miyake Yoh-Ichi
Department of Bioscience and Technology, Faculty of Agriculture, Iwate University, Ueda, Morioka-shi, Iwate, Japan.
Am J Reprod Immunol. 2004 Feb;51(2):95-101. doi: 10.1046/j.8755-8920.2003.00124.x.
Early pregnancy factor (EPF) is an immunosuppressive protein detected in the serum in early pregnancy. We have already reported the development of the rosette inhibition test for mare EPF and have detected EPF in thoroughbreds and ponies. Here, we attempted to purify equine EPF from pregnant mare serum.
Mare EPF was purified by ultrafiltration and ion-exchange chromatography. Purified EPF was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and a neutralization test. EPF activity was estimated as the rosette inhibition titer (RIT) by the rosette inhibition test.
Purified EPF bound to carboxymethyl (CM) sepharose and did not adsorb to diethylaminoethyl (DEAE) sepharose. SDS-PAGE revealed that in the final purified fraction there were many proteins. In the immunoblotting analysis, a protein band of 25.8 kDa was detected as the pregnancy-specific band. Further, antibody gained from the 20 to 30 kDa protein band of the final purified fraction neutralized the RIT activity of pregnant mare serum.
Mare EPF was detected in the final purified fraction and had a molecular weight of 25.8 kDa. EPF in the mare is similar to that obtained from the serum of pregnant cows.
早期妊娠因子（EPF）是一种在妊娠早期血清中检测到的免疫抑制蛋白。我们已经报道了马EPF玫瑰花环抑制试验的开发，并在纯种马和小马中检测到了EPF。在此，我们试图从孕马血清中纯化马EPF。
通过超滤和离子交换色谱法纯化马EPF。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、免疫印迹和中和试验对纯化的EPF进行分析。通过玫瑰花环抑制试验将EPF活性估计为玫瑰花环抑制效价（RIT）。
纯化的EPF与羧甲基（CM）琼脂糖结合，不吸附于二乙氨基乙基（DEAE）琼脂糖。SDS-PAGE显示，在最终纯化的组分中有许多蛋白质。在免疫印迹分析中，检测到一条25.8 kDa的蛋白带作为妊娠特异性条带。此外，从最终纯化组分的20至30 kDa蛋白带获得的抗体中和了孕马血清的RIT活性。
在最终纯化的组分中检测到马EPF，其分子量为25.8 kDa。母马中的EPF与从妊娠母牛血清中获得的EPF相似。
