人类精子蛋白质组分析的双向蛋白电泳技术研究

Establishment of two-dimensional gel electrophoretic protein map of human spermatozoa

目的 :利用人类精子蛋白质组分析的双向蛋白电泳技术 (2 DE)建立正常精子的蛋白质图谱。方法 :比较不同蛋白质提取方法和上样量、不同的第一向等点聚焦方法所得图谱的差别 ,并初步建立人类正常精子的蛋白质图谱。结果 :用蛋白质提取方法一制得的样本电泳后有 6 70～ 70 3个蛋白质斑点 ;方法二所得样本电泳后仅有 194～ 2 10个蛋白质斑点。蛋白质提取方法一所制样本进行载体两性电解质pH梯度 SDS电泳技术 (ISO DALT)得到约 2 80～ 30 0个蛋白质斑点 ,用固相pH梯度 SDS电泳技术 (IPG DALT)得到多达 70 0个蛋白质斑点。结论 :采用蛋白质提取方法一制备精子蛋白质进行IPG DALT电泳的方法适用于建立精子全细胞蛋白质谱。

Objective To establish the method of 2 dimensional electrophoresis(2 DE) for proteins of human spermatozoa and to construct a protein map of human spermatozoa. Methods The sperm pellet was prepared with simple Percoll layer protocol. We studied the effects of various sample preparation methods, loading quantities and isoelectric focusing protocols on the quality of silver stained 2 DE map, and constructed a primary protein map of human spermatozoa. Result Up to 703 protein spots were acquired with sample preparation Method Ⅰwhile only 194～210 spots with Method Ⅱ.With immobilized pH gradients and sodium dodecyl sulfate polyacrylamide gel electrophoresis(IPG DALT) we could acquire over 700 spots while only 280～300 with isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis(ISO DALT). Conclusion It is satisfactory to lyse sperm with sample preparation Method Ⅰ and to separate sperm proteins by IPG DALT for establishing 2 D map of human sperm.