应用双向电泳技术初步建立人精子头部蛋白质图谱

Establishment of the Two-dimensional Gel Electrophoretic Protein Map of the Human Sperm Head

目的 :利用人类精子蛋白质组分析的双向蛋白电泳 (2 DE)技术建立正常人精子头部蛋白质图谱。 方法 :先用固相pH梯度 SDS电泳技术 (IPG DALT)对全精子和精子头部蛋白质抽提物进行蛋白质分离 ,然后用图像分析软件比较精子头部蛋白质图像与精子蛋白质图像的蛋白质斑点组成差异。其中 ,全精子蛋白质的抽提比较了硫脲 /尿素 /盐酸胍和Kit/盐酸胍两种抽提方法。 结果 :硫脲 /尿素 /盐酸胍法与Kit/盐酸胍法所得的全精子 2 DE图像蛋白质斑点分别为 80 2个和 797个 ,其中相同的有 4 92个 ,将两种方法获得的图像整合而得到的全精子蛋白质图像有1 1 0 7个蛋白质斑点 ;精子头部图像蛋白质斑点有 4 2 8个 ,经匹配 ,全部来源于全精子蛋白质图像。 结论 :综合采用两种蛋白质提取方法初步建立了精子头部蛋白质图谱。

Objective: To construct the two-dimensional electrophoretic (2-DE) protein map of the human sperm head. Methods: Protein extracts of the normal human sperm and sperm head were loaded on an 18 cm immobilized pH gradients (IPG) strip holder and separated with isoelectric focusing electrophoresis as the first dimension, and then with upright SDS-PAGE as the second. Different protein spots between them were compared with Image Master 3.0, and so were the maps of method Ⅰwith Urea/Thiourea/Guanidine HCl and method Ⅱ with Kit/ Guanidine HCl. Results: Of the whole-sperm proteome, 802 protein spots were obtained by method Ⅰ, and 797 by method Ⅱ. Among them, distribution patterns of 492 spots were the same. A sperm protein map was constructed with 1 107 protein spots after the interaction of the spots obtained by both the methods. Of the sperm-head proteome, 428 protein spots were obtained, all found in the whole-sperm protein map after comparison. Conclusion: Both the methods could be complementarily used to construct a sperm protein map, and the map could serve as a model for the establishment of the sperm protein profile.