摘要
目的进一步纯化人精浆中与精子前向运动相关的蛋白并进行活性鉴定.方法运用ConA包被珠的蛋白质亲和层析技术及超滤技术对热处理后的人精浆进行蛋白分离,随后对获得的层析洗脱液经浓缩后进行聚丙酰胺凝胶电泳及Commassie Blue R-250染色,根据染色部位将电泳凝胶切割成5部分并获得各部分的蛋白浸出液.结果含第2和第3部分蛋白浸出液的实验组与公牛附睾头部精子及茶碱共同孵育后,与仅用茶碱的对照组相比,前者的精子前向运动值显著高于后者(P＜0.01).聚丙酰胺凝胶电泳及银染色分析显示,第2和第3部分蛋白的分子量分别介于85.0～135.0kDa及44.1～85.0 kDa之间;而SDS-聚丙酰胺凝胶电泳分析却发现:第2部分蛋白解离成分子量分别为89,74,18,16 kDa的条带痕迹,第3部分蛋白则解离成46.8和57.5k Da的亚基.结论人精浆中与精子前向运动相关蛋白的纯化及活性分析可能对了解该蛋白的结构和功能具有重要的意义.
Objective To purify and analyze the proteins that related to the sperm forward motility from human seminal plasma. Methods We Isolated and concentrated some proteins from the heated human seminal plasma by the techniques of affinity chromatography covered with ConA beads and protein ultrafiltration. We did separate the mixed proteins eluted from the affinity chromatography into five portions according to the results of PAGE and Coomassie blue R-250 staining and finally, got the proteins from the portions gel. Results The proteins’ concentration was mainly focused on the portion II and III. Furthermore, these portions could stimulate the bovine spermatozoa from epididymal caput to gain forward motility compared with the control group incubated with theophylline only. The differences revealed significant (P <0.01), especially for the portion III. The molecular weight for portion II was between the 85 kDa and 135k Da under the PAGE condition, whereas it becomes cleft into about 89 kDa, 74 kDa, 18 kDa and 16 kDa trance or weak bands under the SDS-PAGE condition. On the other hand, the portion III was an obvious band whose molecular weight was between 44.1 kDa and 85 kDa under the PAGE condition. It also cleft into about 46.8 kDa and 57.5 kDa main bands under the SDS-PAGE condition however. Conclusion The purification and analysis of proteins related to the sperm forward motility from human seminal plasma was one of the important procedures for the mechanism elucidation of molecular interactions between the proteins and spermatozoa.
出处
《中国男科学杂志》
CAS
CSCD
2005年第3期6-8,15,共4页
Chinese Journal of Andrology
基金
上海市教育委员会重点学科基金项目编号为S970203
上海市高等学校科学技术发展基金资助
项目编号03BK17
