人类精子蛋白质组分析的双向蛋白电泳技术研究

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670-703个蛋白质斑点；方法二所得样本电泳后仅有194-210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280-300个蛋白质斑点，用固相pH梯度-SDS电泳技术（IPG-DALT）得到多达700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电泳的方法租用于建立精子全细胞蛋白质谱。

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

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 I while 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.