Jobim M I M, Oberst E R, Salbego C G, Wald V B, Horn A P, Mattos R C
Department of Veterinary Clinical Pathology, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
Theriogenology. 2005 Apr 15;63(7):2053-62. doi: 10.1016/j.theriogenology.2004.10.002. Epub 2004 Nov 18.
The objective of this study was to assess the protein profile of ovine seminal plasma using 2D-PAGE and verify if BSP A1/A2 are present in ovine seminal plasma. Seminal plasma was collected from three mature rams and pooled to eliminate individual differences. Seminal plasma samples were submitted to 2D-PAGE using 12% acrylamide gels. The image analysis software identified 21 protein spots on the air-dried gel, with molecular weight ranging from 15 to 115 kDa and pI 3.2 to 8.7. The most prominent spots were those <30 kDa. The most intensely stained spots were: 3 (18-19 kDa, pI 4.8-5.0), 5 (17-18 kDa, pI 5.0-5.2), 7 (15-16 kDa, pI 6.2-6.4), and 23 (105-108 kDa, pI 6.8-7.0). Three of these spots (spots 3, 5 and 7, respectively) accounted for 41.1% of the relative intensity of the spots of the gels, based on the intensity of the Comassie blue staining. Western blot analysis indicated that spots 3 and 5 were similar to BSP A1/A2 (16.5, pI 4.7-5.0 and 16 kDa, pI 4.9-5.2) identified in Manjunath's studies [Manjunath P, Sairam MR. Purification and biochimical characterization of three major acid proteins (BSP A1, BSP A2 and BSP A3) from bovine seminal plasma. Biochem J 7 (1987) 685-92.], based on the specific reaction of the polyclonal antibody to those spots.
本研究的目的是使用二维聚丙烯酰胺凝胶电泳（2D-PAGE）评估绵羊精浆的蛋白质谱，并验证BSP A1/A2是否存在于绵羊精浆中。从三只成年公羊采集精浆并混合以消除个体差异。精浆样品使用12%丙烯酰胺凝胶进行二维聚丙烯酰胺凝胶电泳。图像分析软件在空气干燥的凝胶上鉴定出21个蛋白质斑点，分子量范围为15至115 kDa，等电点为3.2至8.7。最突出的斑点是分子量小于30 kDa的那些。染色最深的斑点是：3（18 - 19 kDa，等电点4.8 - 5.0）、5（17 - 18 kDa，等电点5.0 - 5.2）、7（15 - 16 kDa，等电点6.2 - 6.4）和23（105 - 108 kDa，等电点6.8 - 7.0）。基于考马斯亮蓝染色的强度，这些斑点中的三个（分别为斑点3、5和7）占凝胶斑点相对强度的41.1%。蛋白质免疫印迹分析表明，基于多克隆抗体对这些斑点的特异性反应，斑点3和5与Manjunath研究中鉴定的BSP A1/A2（16.5，等电点4.7 - 5.0和16 kDa，等电点4.9 - 5.2）相似[Manjunath P, Sairam MR. Purification and biochimical characterization of three major acid proteins (BSP A1, BSP A2 and BSP A3) from bovine seminal plasma. Biochem J 7 (1987) 685 - 92.]。
