【作者】 柯冬蕾；

【导师】 李和平； 刘伟石；

【作者基本信息】 东北林业大学 ， 特种经济动物饲养， 2008， 硕士

【摘要】 成熟精子的膜表面含有多种膜蛋白,有些是精子本身固有的,有些是在精子成熟过程中粘附到精子表面的。精子膜蛋白是精子发生、成熟过程中的重要标志。随着生命科学的发展,人们发现精子膜蛋白在维持精子形态结构完整、完成精子的新陈代谢和生殖功能中发挥着重要的作用,特别是与精卵识别、精卵结合以及质膜融合等受精活动密切相关。其中部分可作为抗原诱导机体产生相应的抗体,进而影响精子的受精能力,导致免疫性不育。本文以我国重要的经济动物——马鹿的精子作为实验材料,筛选出了马鹿精子膜蛋白提取的适宜方法、建立了其裂解液的理想体系、确定了其SDS-PAGE电泳的最佳条件,同时对其理化性质进行了初步分析。现将研究结果简述如下:1、马鹿精子膜蛋白提取方法的确定采用三种方法对马鹿精子膜蛋白进行提取,根据SDS-PAGE电泳和BCA蛋白含量检测结果确定了NP-40裂解法为马鹿精子膜蛋白提取的适宜方法。2、马鹿精子膜蛋白裂解液体系的建立对马鹿精子膜蛋白裂解液中去垢剂NP-40和蛋白酶抑制剂PMSF各设立四个浓度梯度,对裂解液添加体积设立三个梯度,根据SDS-PAGE电泳和BCA蛋白含量检测结果确定了NP-40浓度为0.5%、PMSF浓度为0.5mM、提取时添加裂解液体积为5倍是膜蛋白裂解液的理想体系。3、马鹿精子膜蛋白SDS-PAGE电泳的最佳条件的确定对马鹿精子膜蛋白SDS-PAGE电泳分离胶设立四个浓度梯度,根据电泳结果确定12%为马鹿精子膜蛋白SDS-PAGE电泳的最佳凝胶条件。4、马鹿精子膜蛋白的理化性质研究发现:(1)共有8种不同分子量的马鹿精子膜蛋白被提取出来;(2)马鹿精子膜蛋白的总含量为468.12μg/ml;(3)马鹿精子膜蛋白有3条较清晰的糖蛋白带,分别与SDS-PAGE电泳图谱上分子量为98.4 kDa、67.6 kDa和47.3 kDa的膜蛋白带相一致。5、马鹿精子膜蛋白的双向电泳实验对不同细胞裂解方法提取获得的马鹿精子膜蛋白的双向电泳结果进行比较,发现采用尿素—盐酸胍两步法裂解精子能获得较好的双向电泳效果。采用软件对图谱进行分析,可检测出约823个蛋白质点,分子量基本分布在10～100 kDa,等电点约为PI 4～9的区域内。更多还原

【Abstract】 Mature sperms had many kinds of Sperm membrane proteins (SMPs) in its membrane. Some of them were sperm-specific, others binded to the sperm surface during the period of sperm maturation. SMPs were important symbols of the mature progress of sperm. By the development of life science, people recognized that these SMPs play an important role in the maintenance of sperm’s shape and structure, the metabolism of sperms and the reproduction, especially in the fertilization. Some of the SMPs as antigen could induce the engine body generate relevant antibody, and caused fertilization failure or immunosterility or to both.With the sperm of wapiti (Cervus elaphus) as the experiment material, we screened out the optimum method of extracting the SMPs of wapiti; established the most ideal system of the extraction solution of the SMPs; determined the best condition of SDS-PAGE of the SMPs, and. analyzed some physicochemical properties of them. The main experimental results of the present work were shown as follows:1. Screened out the optimum method to extract the wapiti SMPsThe SMPs were extracted from the surface of wapiti sperms by three different methods. Base on the result of SDS-PAGE and BCA, the extraction solution with NP-40 was the optimum method of the wapiti SMPs2. Established the ideal system of the extraction solution of the wapiti SMPsThe SMPs were extracted from the surface of wapiti sperms by NP-40 extraction solution under different systems. Base on the result of SDS-PAGE and BCA, the extraction solution with 0.5%NP-40, 0.5%PMSF and 5 times volume was the most ideal system of the extraction solution of the wapiti SMPs.3. Determined the best condition of SDS-PAGE of the wapiti SMPsThe SMPs were separated by SDS-PAGE under 4 different kinds of concentration. Base on the result of SDS-PAGE, the best concentration of the SDS-PAGE for the wapiti SMPs was 12%.4. The analyze of the physicochemical property of the wapiti SMPs(1)8 kinds of SMPs in different Molecule Weight (MW) were extracted from the sperm of wapiti;(2) The concentration of the wapiti SMPs was 468.12μg/ml;(3) There were three kinds of glycoprotein, the MWs of them were 98.4 kDa, 67.6 kDa and 47.3 kDa.5. 2-DE gel electrophoresis of the SMPs of wapitiThe wapiti SMPs was separated by 2-DE technique, the effects of various sample preparation methods were studied .The satisfied result was obtained when two step method of urea-hydrochloride lysed sperm. About 823 protein spots were detected by the imaging analysis system. The molecular weight and isoelectric point (PI) of most protein spots were 10 to 100 kDa and 4.0 to 9.0 respectively.更多还原