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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CommassieBlueR-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44.1~85.0kDa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89,74,18,16kDa的条带痕迹,第3部分蛋白则解离成46.8和57.5kDa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 further purify and identify the activity of sperm forward-movement-related proteins in human seminal plasma. Methods ConA coated beads affinity chromatography and ultrafiltration technology heat-treated human seminal plasma protein separation, followed by the chromatographic eluate was concentrated after polyacrylamide gel electrophoresis and CommassieBlueR-250 Dyeing, the electrophoresis gel was cut into 5 parts according to the stained part and the protein leachate of each part was obtained. Results Compared with theophylline-only control group, the experimental group with the second and third partial protein leaching solutions incubated with spermatozoa and theophylline in the head of the epididymis of the bull showed significantly higher forward sperm motility P <0.01). Polyacrylamide gel electrophoresis and silver staining analysis showed that the molecular weights of the second and third partial proteins ranged from 85.0 to 135.0 kDa and 44.1 to 85.0 kDa, respectively. However, SDS-polyacrylamide gel electrophoresis analysis revealed that the second part Proteins were dissociated into bands with molecular weights of 89, 74, 18 and 16 kDa, respectively. The third part of the protein was dissociated into subunits of 46.8 and 57.5 kDa. Conclusion Purification and activity analysis of proteins related to the forward movement of sperm in human seminal plasma may be of great significance for understanding the structure and function of this protein.