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目的制备人类新的精子结合蛋白HBRP的单克隆抗体,确定其在精子的定位及其与精子功能相关的作用。方法分别利用PCR、Western blot、动物免疫、血清纯化等方法得到HBRP单克隆抗体,用ELISA方法检测抗体滴度;利用间接免疫荧光方法观察HBRP在精子的定位。结果经纯化鉴定获得HBRP融合表达蛋白;首次获得HBRP单克隆抗体;应用制备的单克隆抗体,确定HBRP主要结合在精子尾部的中段和主段。结论 HBRP结合在人成熟精子尾部的中段和主段,提示其参与精子运动功能的调控。
Objective To prepare a monoclonal antibody to human novel sperm-binding protein HBRP and to determine its role in sperm localization and its relevance to sperm function. Methods The monoclonal antibodies against HBRP were obtained by PCR, Western blot, animal immunization and serum purification respectively. The antibody titers were detected by ELISA. The localization of HBRP was observed by indirect immunofluorescence staining. Results HBRP fusion protein was purified and identified. The monoclonal antibody of HBRP was obtained for the first time. The prepared monoclonal antibody was used to confirm the binding of HBRP to the middle and main segments of sperm tail. Conclusion HBRP binds to the middle and main segments of human mature spermatozoa, suggesting that it is involved in the regulation of sperm motility.