早孕因子分离纯化方法的优化  

Optimization of a method for isolation and purification of early pregnancy factor

作　　者：温志锋[1] 练玉银[1] 王家骥[1] 聂湘辉[1] 林燕棉[1] 

机构地区：[1]广州医学院公共卫生学院,510182

出　　处：《免疫学杂志》2012年第12期1073-1076,共4页Immunological Journal

基　　金：广东省科技计划重点项目(2008A030201022)

摘　　要：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。This study aimed to optimize the method for isolation and purification of early pregnancy factor（EPF） in serum from the normal early pregnancy women so as to improve the access rate of EPF.EPF was purified from the mixed serum of normal early pregnancy women with 12 gestational weeks by using the DEAE Fast Flow ion exchange chromatography and Heparin Fast Flow affinity chromatography.The biological activity of each extraction was detected by erythrocty active rosette inhibition test（Ea-RIT） and the purified product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） to determinate its molecular weight.And its specificity was analyzed by Western blot.The Ea-RIT showed that the DE-Ⅰ peak of the two emerged peaks in DEAE Fast Flow ion exchange chromatography,and the H-Ⅱ peak of the two arisen peaks in Heparin Fast Flow affinity chromatography were the EPF activity peak.The SDS-PAGE profile further revealed there was only one band with a molecular weight of 38 100 kD for the extraction of the H-II peak,and Western blot analysis showed that the matching of antibody and antigen was good.The quantity of the protein was 0.615 mg with a recovery rate was 0.034%,which was much higher than the traditional four-step method.The result indicated that the optimized method for the isolation and purification of EPF from the early pregnancy women serum is feasible,which can greatly improve the purification efficiency.

关 键 词：早孕因子 分离纯化 优化 

分 类 号：R392.2[医药卫生—免疫学]