医学论文-前列腺精子结合蛋白的二维凝胶电泳及质谱分析.docVIP

医学论文-前列腺精子结合蛋白的二维凝胶电泳及质谱分析 【摘要】? 【目的】 应用精子膜蛋白亲和层析柱分离金黄地鼠前列腺精子结合蛋白，并用二维电泳和质谱对金黄地鼠前列腺精子膜结合蛋白进行初步分析。【方法】 应用附睾精子膜亲和层析柱纯化前列腺精子结合蛋白,采用二维凝胶电泳对纯化的前列腺精子膜结合蛋白进行蛋白分离和图像分析，并结合基质辅助激光解吸附电离飞行时间质谱分析及数据库搜索从而鉴定部分蛋白质斑点; RT-PCR检测已鉴定蛋白在前列腺及未受精卵子中的mRNA的表达。 【结果】 附睾精子膜蛋白亲和层析柱可纯化出前列腺结合蛋白，前列腺附睾精子膜蛋白结合蛋白的二维凝胶电泳蛋白图谱。分离的蛋白质斑点为30个，质谱鉴定出2个蛋白点，一为热休克蛋白105，另一羰基还原酶（NADPH）-3。前列腺和未受精卵中均表达热休克蛋白105和羰基还原酶-3的mRNA。 【结论】 自前列腺分泌物中???离出精子膜结合蛋白，金黄地鼠前列腺中精子结合蛋白经双向电泳及飞行质谱分析鉴定出蛋白热休克蛋白和羰基还原酶。 【关键词】? 前列腺分泌蛋白; 附睾精子膜蛋白; 二维凝胶电泳; 质谱 ??? Abstract: 【Objective】 The aims of this study was to identify and purify the ventral prostate secretory sperm binding proteins and analyze the ventral prostate sperm binding proteins by 2D- electrophoresis and MOTIF. 【Methods】 The ventral prostate secretory sperm binding proteins were identified and purified by epdidymal sperm membrane affinity chromatography.? The protein spots excised from 2D gels were digested with trypsin and then the resulting peptides were identified by using peptide mass fingerprinting feature by database searching. The mRNA of heat shock protein-105 and carbonyl reductase（NADPH）-3 were detected in ventral prostate gland and unfertilized oocyte by RT-PCR. 【Results】 It was purified sperm membrane proteins from ventral prostate secretory proteins. About 30 spots were separated by 2D-electrophoresis. Heat shock protein-105 and carbonyl reductase-3 are identified by MOTIF from ventral prostate sperm binding proteins. Both mRNA of heat shock protein-105 and carbonyl reductase （NADPH）-3 were expressed in ventral prostate glands and unfertilized oocyte.? 【Conclusions】About 30 of ventral prostate secretory proteins bind to sperm. Heat shock protein-105 and carbonyl reductase （NADPH）-3 are identified from the ventral prostate secretory sperm binding proteins. ??? Key words: ventral prostate secretory protein; epdidymal sperm membrane protein; 2D-electrophoresis; mass spectometry ????? 前列腺为雄性附属性腺之一，而精浆主要由雄性附属性腺分泌。精浆对精子生殖力的影响有许多矛盾报道；体外获得的牛附睾尾部的精子的生殖力低于射出的精子的生殖力，但在猪猫和大鼠则相反[1]。