一种精子表面GPI锚定蛋白初步鉴定方法的建立  

Development of a Novel Method for Identification of GPI-anchored Surface Proteins on Spermatozoon

作　　者：辛玲[1] 王慧萍[1] 王小强[1] 于和鸣[1] 

机构地区：[1]国家人口计生委科学技术研究所,北京100081

出　　处：《中国生物化学与分子生物学报》2014年第11期1155-1159,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：中央级公益科研院所基本科研业务费专项(No.2010GJSSJKA0I)~~

摘　　要：精子表面糖基磷脂酰肌醇（GPI）锚定蛋白（GPI-Aps）在精子不同阶段都发挥着关键作用．本研究介绍了嗜水气单胞菌毒素（aerolysin）作为生物探针来鉴定精子表面的GPI锚定蛋白．实验依次进行了嗜水气单胞茵培养、毒素提取纯化、多克隆抗体制备以及三明治蛋白印迹验证实验．凝胶蛋白分离实验显示，硫酸铵沉淀加Sephadex G-100层析柱纯化得到了分子质量为45kD高纯度细菌毒素；蛋白印迹实验验证了制备的多克隆抗体的特异性；三明治蛋白印迹结果显示，嗜水气单胞菌毒素能识别出12条精子脂筏提取蛋白条带，其分子质量主要分布在15～130kD之间；Alex488标记的免疫荧光实验显示，嗜水气单胞菌毒素识别的GPI锚定蛋白主要分布在精子头部和尾部．研究结果提示，嗜水气单胞菌毒素能够有效地识别精子表面的GPI锚定蛋白．Glycosyphosphatidylinositol anchored proteins （GPI-Aps） on spermatozoon surface play crucial roles in spermatogenesis. In the present study, aerolysin was found to recognize spermatozoa GPI-APs. Aerolysin was extracted and purified from the culture of aeromonas hydrophila. Polyclonal antibody against aerolysin was prepared and used for detecting GPI-Aps by Sandwich Western blot. Aerolysin purifed by Sephadex G-100 chromatography was determined as a 45 kD molecule in SDS- PAGE. Western blot showed that the prepared anti-aerolysin polyclone antibody recognized the target with high specificity. Sandwich Western blot indicated that aerolysin interacted with 12 GPI-Aps ranging from 15 to 130 kD. Immunofluorescence showed that Alex488 labeled aerolysin was distributed at the head and tail of spermatozoa. The results demonstrated that aerolysin could be a probe for detecting GPI-anchored proteins on spermatozoon.

关 键 词：糖基磷脂酰肌醇(GPI)锚定蛋白(GPI-Aps) 嗜水气单胞菌毒素 精子 

分 类 号：Q51[生物学—生物化学]