人卵泡液抑制素B的分离与鉴定

Isolation and identification of human follicular fluid inhibin B

目的探讨人卵泡液抑制素B(INH-B)的分离纯化方法。方法收集25例育龄期女性成熟卵泡液,分别用乙腈、丙酮、乙醇3种试剂处理样品,富集低丰度蛋白,经蛋白浓度测定和SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,比较3种方法对高丰度蛋白去除效果。再通过二乙基氨乙基(DEAE)阴离子交换柱和蛋白质等电点分离法分离目的蛋白。结果采用乙腈沉淀法能去除卵泡液样品中约88.7%的高丰度蛋白。并用乙腈富集、阴离子交换柱分离、等电点分离的"三步分离法"分离人卵泡液后,INH-B蛋白回收率为38.7%,等电点为4.55。结论乙腈沉淀法是一种较为有效的去除样品中高丰度蛋白、富集低丰度蛋白的方法。乙腈富集、阴离子交换柱分离、等电点分离的"三步分离法"能初步纯化获得人体天然INH-B蛋白分子粗制品,纯化回收率达38.70%。

Objective： To explore the method for separating and purificating inhibin B（INH-B）in follicular fluid and identify the molecular form of INH-B in follicular fluid. Methods： The follicular fluids were collected from the women undergone artificial insemination. The follicular fluids were treated with acetonitrile, acetone and ethanol, respectively. The low abundance proteins were concentrated and determined, then analyzed by polyacrylamide gel electrophoresis. The efficiency of removing high abundance proteins with the three reagents was compared. The target protein was isolated by DEAE anion exchange column and the isoelectric point of protein separation method （Rotofor System）. Results： Acetonitrile precipitation can remove 90% high abundance proteins in follicular fluid. The recovery rate of INH-B protein was 38.70% after treated with acetonitrile,DEAE anion exchange column and the isoelectric point method for protein separation. Isoelectric point of INH-B in follicular fluid was 4.55. Conclusions： Acetonitrile precipitation is a more effective method for removal of high abundance proteins and enrichment of low abundance proteins. The recovery rate of INH-B protein was 38. 7 % after treated with acetonitrile, DEAE anion exchange column and the isoelectric point method for protein separation.