一种精子表面GPI锚定蛋白初步鉴定方法的建立

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菌培养、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45 kD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15~130 kD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.

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

Glycosyphosphatidylinositol anchored proteins (GPI-Aps) on spermatozoon surface play crucial roles in spermatogenesis. In the present study, aerolysin was found to recognizespermatozoa 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.