肉牛早孕因子(EPF)的活性检测及分离纯化

Purification and characterization of early pregnancy factor(EPF) from beef cattle serum

【目的】检测怀孕肉牛早孕因子（Early pregnancy factor,EPF）的活性,并对其进行分离纯化,以期为EPF快速检测方法的建立提供依据。【方法】采集妊娠1~9个月的肉牛血样,并进行妊娠诊断,通过玫瑰花环抑制试验（Rosette inhibition test,RIT）检测其早孕因子的活性。对有活性的血样依次进行DEAE-52离子交换层析、ConASepharose 4B亲和层析和Heparin Sepharose Cl 6B亲和层析,分离纯化EPF,对纯化产物进行SDS-PAGE检测,并测定其等电点。【结果】怀孕牛和未怀孕牛血清的玫瑰花环抑制滴度差异显著（P〈0.05）。玫瑰花环抑制试验显示,纯化出的EPF具有活性,其分子质量为53.6 ku,等电点为6.8。【结论】利用玫瑰花环抑制试验检测早孕因子活性,可以鉴定肉牛的妊娠状态;利用层析的方法可以分离出早孕因子的活性蛋白。

【Objective】 The study was done to detect the activity of EPF to and purify EPF from pregnant beef cattle serum using rosette inhibition test.【Method】 Serum was obtained from pregnant beef cattle of 1-9 months.Rosette inhibition test was used to detect the activity of EPF in pregnant beef cattle,and then serum was purified by DEAE-Sepharose,ConA Sepharose 4B affinity chromatography and Heparin Sepharose Cl 6B affinity chromatography.The purified products were detected by SDS-PAGE and isoelectric points were detected.【Result】 The results indicated that RIT significantly deviated（P0.05） between pregnant and non-pregnant beef cattle.The EPF＇s activity of beef cattle was detected in the final purified products and its molecular weight was 53.6 ku,the isoelectric point 6.8.【Conclusion】 The study indicates that RIT has the potential to distinguish pregnant status of beef cattle,and chromatography method can be used to isolate protein of early pregnancy factor.