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目的对正常早孕妇女血清中早孕因子(EPF)的分离纯化过程进行优化,以提高EPF的回收率。方法依次采用DEAEFast Flow离子交换色谱和Heparin Fast Flow亲和色谱,从妊娠12周内的正常早孕妇女混合血清中提纯EPF,采用活性玫瑰花结抑制试验(Ea-RIT)检测各阶段产物的EPF活性,SDS-聚丙烯酰胺凝胶(SDS-PAGE)电泳鉴定纯化物并测定其相对分子质量,用Western blot鉴定其特异性。结果经Ea-RIT检测后,DEAE Fast Flow离子交换色谱所现2峰中DE-Ⅰ峰为EPF活性峰,HeparinFast Flow亲和色谱所现2峰中H-Ⅱ峰为EPF活性峰。H-Ⅱ峰收集液经SDS-PAGE电泳,结果显示为1条相对分子质量为38 100的蛋白带,Western blot分析表明抗体与抗原匹配性良好。经检测其蛋白含量为0.615 mg,回收率为0.034%,与传统分离纯化四步法的回收率相比明显提高。结论本优化方法对于提纯孕血清中的EPF是可行的,大大提高了纯化效率。
OBJECTIVE: To optimize the isolation and purification of early pregnancy factor (EPF) in normal pregnant women to improve the recovery rate of EPF. Methods EPF was purified from mixed serum of normal early pregnant women within 12 weeks of gestation by DEAEFast Flow ion exchange chromatography and Heparin Fast Flow affinity chromatography, and the activity of EPF was detected by the active rosette inhibition assay (Ea-RIT) , SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to identify the purified product and determine its relative molecular mass, and its specificity was identified by Western blot. Results The results of Ea-RIT showed that the DE-Ⅰ peak in the two peaks of DEAE Fast Flow ion exchange chromatography was the peak of EPF activity and the peak of the peak of EPF in the peak of Heparin Fast Flow affinity chromatography was H-Ⅱ peak. H-Ⅱ peak collection by SDS-PAGE electrophoresis, the results showed that a relative molecular mass of 38 100 protein bands, Western blot analysis showed that the antibody and antigen match well. After testing the protein content of 0.615 mg, the recovery rate of 0.034%, compared with the traditional separation and purification of four-step recovery was significantly improved. Conclusion The optimization method is feasible for the purification of EPF in serum and greatly improves the purification efficiency.