马鹿精子膜蛋白纯化与理化性质分析
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摘要
成熟精子的膜表面含有多种蛋白质，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。精子表面的膜蛋白是精子发生成熟过程中的重要标志分子，与受精和胚胎的早期发育关系密切，随着生命科学的发展，人们发现精子膜蛋白不仅具有抗原性，而且它们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。其中部分抗原可诱导机体产生相应的自身抗体，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，进行了精子膜蛋白的提取、分离纯化，同时对其理化性质加以分析，并以纯化的马鹿精子膜蛋白作为抗原，制备了多克隆抗血清。利用抗血清进行了精子膜蛋白的定位、对精子运动的影响等的研究，现将研究结果简述如下：
1、马鹿精子膜蛋白的分离纯化及鉴定
采用非离子型去污剂裂解法提取马鹿精子膜蛋白，根据Western-blot和SDS-PAGE电泳实验结果，确定分离纯化的条件。SDS-PAGE电泳结果显示，共有8种马鹿精子膜蛋白被提取出来，其中，有4条膜蛋白带非常清晰。采用胶上纯化法，即利用SDS-PAGE电泳，0.3MKCl进行凝胶染色和反复冻融及高速离心等技术从马鹿精子膜蛋白溶液中分离纯化了该4种膜蛋白，其分子量分别为：98.4kDa，67.6kDa，47.3kDa，29.1kDa。在SDS-PAGE电泳图谱中，纯化的马鹿精子膜蛋白在还原和非还原条件下均为单一条带，无链间二硫键。说明采用上述方法纯化的马鹿精子膜蛋白为单一组分。
糖蛋白的检测中发现，在图谱中有3条很清晰的糖蛋白带，与纯化出来的前3种马鹿精子膜蛋白带吻合，确定为糖蛋白。采用BCA试剂盒对提取的马鹿精子膜总蛋白及纯化的单一马鹿精子膜蛋白进行分析，测得其含量分别为468.12μg／ml，95.67μg／ml，184.23μg／ml，47.68μg/ml，58.32μg／ml。
2、马鹿精子膜蛋白的双向电泳实验
由双向电泳图谱发现，发现马鹿精子膜蛋白有823个蛋白质点；其分子量分布在10kDa—100kDa之间，等电点PI在4-7(pH)之间。
3、马鹿精子膜蛋白多克隆抗血清的制备
用纯化的马鹿精子膜蛋白免疫雄性BALB／C小鼠，制备多克隆抗体。通过ELISA法测得抗血清的效价为1-2×10~3。Western-blot结果表明，纯化的马鹿精子膜蛋白具有免疫反应性，为特异性抗原。
4、抗血清对精子运动的影响
在体外实验中，发现抗马鹿精子膜蛋白抗血清对马鹿精子的直线运动无显著影响；同时还发现抗血清对小鼠精子的直线运动也无明显影响；但对获能的马鹿精子的直线运动却表现为较为显著的影响，并表现为剂量依赖性。
5、马鹿精子膜蛋白在精子上的定位及保守性
通过对马鹿精子和小鼠精子进行免疫细胞化学程序染色并结合激光共聚焦显微镜检测，结果表明，纯化的精子膜蛋白大部分都存在于马鹿精子的顶体区域；而对小鼠精子的定位研究中，也发现膜蛋白存在于顶体区。
6、马鹿精子膜蛋白多抗血清的特异性
通过对马鹿精子膜蛋白的琼脂双向免疫扩散实验，发现马鹿精子膜蛋具有组织特异性。与小鼠的附睾、精巢、输精管有免疫交叉反应，但与重要脏器心、肝、脾、肾无免疫交叉反应。说明马鹿精子膜蛋白具有组织特异性。
It is nearly a century ago that the first document attempts to immunized with male gametephomogenats from heterospecies produced sperm reacting antibodies in animals. Mature spermhave many kinds of proteins in its plasma membrane. Some of them are sperm-spcific, othersbind to sperm surface during the period of sperm maturation. These sperm membrane proteinsplay important role in the maintenance of sperm shape and structure, the metabolism of spermand the reproduction, especially in the fertilization. Since then it has been amply documentedthat deliberated immunization of female animals of numerous species with preparations ofisologous sperm or mature testis results in infertility due to either fertilization failure orpremplantation embryo mortality or to both.
With sperm of wapiti (Cervus elaphus) as the experiment material, The sperm membraneproteins were studied in such respects as extraction, isolation, purification and physiochemicalcharacteristics analysis. To raised polyclonal antibodies preparation with purified spermmembrane proteins of wapiti as antigens, and study on localization of the sperm membraneproteins of wapiti on sperm, the effect on the ability of sperm. The main experimental result ofthe present work were shown as follows:
1. Isolation, purification and identification of the sperm membrane proteins of wapiti:The sperm membrane proteins were extracted from the surface of wapiti sperms by the use.ofdecontaminant, base on the result of western-blot and SDS-PAGE, the condition of isolationand purification were estabilished. 4 sperm membrane proteins were purification by SDS-PAGE, staining 0.3mol/L KCl, repeated freeze-thaw and centrifugation. The purified spermmembrane proteins migrated as a single homogeneous band when analyzed by SDS-PAGEunder reducing and nonreducing conditions, respectively. This result reveals that the spermmembrane proteins has no inter-chain disulfide bond. The purity was attested to thehomogeneity.
Physicochemical properties of the sperm membrane proteins were determined. Theestimated molecular weight of the sperm membrane proteins were 98.4kDa, 67.6kDa,47.3kDa, 29.1kDa, and the PI were 4-7(pH). The concentration of sperm membrane proteinsof wapiti were 95.67μg/ml, 184.23μg/ml, 47.68μg/ml, 58.32μg/ml; the concentration ofthe total protein was 468.12μg/ml by BCA. 3 sperm membrane proteins of wapiti wereglycoprotein in 4 purified proteins.
2.2-D gel electrophoresis: Our results have been shown, there were 823 point obtainde inthe map of 2-DE of the sperm membrane proteins of wapiti. The purity molecular weight was10-100 kDa and the PI was 4-7(pH).
3. Polyclonal antibodies prepareation: In order to study the function of the spermmembrane proteins, polyclonal antibodies were raised in BALB/C mouse. The titer of theantisera was about 1-2×10~3 assayed by ELISA. The result of Western-blot has been indiciatedthat the immunity reactive of the sperm membrane proteins of wapiti, and it was a kind ofspecific antigen.
4. The effct of polyclonal antibodies on sperm activity: The effct of the polyclonalantibodies was not significant.on the activity of wapiti sperm before capacitation in vitro. Itwas not effct on mouse; but the effct on the activity of wapiti s.perm after capacitationmarkedly, The effects mentioned above were dose-dependent
5. Localization of the sperm membrane proteins of wapiti on sperm: The result ofconfocal laser scaning microscope showed that the purified sperm membrane proteins werelocated in the acrosomal of wapiti sperm, Location of sperm of mouse was the same as.
6. identification of polyclonal antibodies of sperm membrane proteins: an agar geldiffusion method showed that the sperm membrane proteins were specific antigen, Theantibodies antiserum was heart. Liver. Kidney.and lung, the purity was reacted with testist andvasdeferens. It was a kind of tissue specificity.
本文以我国重要的经济动物—马鹿的精子作为实验材料，进行了精子膜蛋白的提取、分离纯化，同时对其理化性质加以分析，并以纯化的马鹿精子膜蛋白作为抗原，制备了多克隆抗血清。利用抗血清进行了精子膜蛋白的定位、对精子运动的影响等的研究，现将研究结果简述如下：
1、马鹿精子膜蛋白的分离纯化及鉴定
采用非离子型去污剂裂解法提取马鹿精子膜蛋白，根据Western-blot和SDS-PAGE电泳实验结果，确定分离纯化的条件。SDS-PAGE电泳结果显示，共有8种马鹿精子膜蛋白被提取出来，其中，有4条膜蛋白带非常清晰。采用胶上纯化法，即利用SDS-PAGE电泳，0.3MKCl进行凝胶染色和反复冻融及高速离心等技术从马鹿精子膜蛋白溶液中分离纯化了该4种膜蛋白，其分子量分别为：98.4kDa，67.6kDa，47.3kDa，29.1kDa。在SDS-PAGE电泳图谱中，纯化的马鹿精子膜蛋白在还原和非还原条件下均为单一条带，无链间二硫键。说明采用上述方法纯化的马鹿精子膜蛋白为单一组分。
糖蛋白的检测中发现，在图谱中有3条很清晰的糖蛋白带，与纯化出来的前3种马鹿精子膜蛋白带吻合，确定为糖蛋白。采用BCA试剂盒对提取的马鹿精子膜总蛋白及纯化的单一马鹿精子膜蛋白进行分析，测得其含量分别为468.12μg／ml，95.67μg／ml，184.23μg／ml，47.68μg/ml，58.32μg／ml。
2、马鹿精子膜蛋白的双向电泳实验
由双向电泳图谱发现，发现马鹿精子膜蛋白有823个蛋白质点；其分子量分布在10kDa—100kDa之间，等电点PI在4-7(pH)之间。
3、马鹿精子膜蛋白多克隆抗血清的制备
用纯化的马鹿精子膜蛋白免疫雄性BALB／C小鼠，制备多克隆抗体。通过ELISA法测得抗血清的效价为1-2×10~3。Western-blot结果表明，纯化的马鹿精子膜蛋白具有免疫反应性，为特异性抗原。
4、抗血清对精子运动的影响
在体外实验中，发现抗马鹿精子膜蛋白抗血清对马鹿精子的直线运动无显著影响；同时还发现抗血清对小鼠精子的直线运动也无明显影响；但对获能的马鹿精子的直线运动却表现为较为显著的影响，并表现为剂量依赖性。
5、马鹿精子膜蛋白在精子上的定位及保守性
通过对马鹿精子和小鼠精子进行免疫细胞化学程序染色并结合激光共聚焦显微镜检测，结果表明，纯化的精子膜蛋白大部分都存在于马鹿精子的顶体区域；而对小鼠精子的定位研究中，也发现膜蛋白存在于顶体区。
6、马鹿精子膜蛋白多抗血清的特异性
通过对马鹿精子膜蛋白的琼脂双向免疫扩散实验，发现马鹿精子膜蛋具有组织特异性。与小鼠的附睾、精巢、输精管有免疫交叉反应，但与重要脏器心、肝、脾、肾无免疫交叉反应。说明马鹿精子膜蛋白具有组织特异性。
It is nearly a century ago that the first document attempts to immunized with male gametephomogenats from heterospecies produced sperm reacting antibodies in animals. Mature spermhave many kinds of proteins in its plasma membrane. Some of them are sperm-spcific, othersbind to sperm surface during the period of sperm maturation. These sperm membrane proteinsplay important role in the maintenance of sperm shape and structure, the metabolism of spermand the reproduction, especially in the fertilization. Since then it has been amply documentedthat deliberated immunization of female animals of numerous species with preparations ofisologous sperm or mature testis results in infertility due to either fertilization failure orpremplantation embryo mortality or to both.
With sperm of wapiti (Cervus elaphus) as the experiment material, The sperm membraneproteins were studied in such respects as extraction, isolation, purification and physiochemicalcharacteristics analysis. To raised polyclonal antibodies preparation with purified spermmembrane proteins of wapiti as antigens, and study on localization of the sperm membraneproteins of wapiti on sperm, the effect on the ability of sperm. The main experimental result ofthe present work were shown as follows:
1. Isolation, purification and identification of the sperm membrane proteins of wapiti:The sperm membrane proteins were extracted from the surface of wapiti sperms by the use.ofdecontaminant, base on the result of western-blot and SDS-PAGE, the condition of isolationand purification were estabilished. 4 sperm membrane proteins were purification by SDS-PAGE, staining 0.3mol/L KCl, repeated freeze-thaw and centrifugation. The purified spermmembrane proteins migrated as a single homogeneous band when analyzed by SDS-PAGEunder reducing and nonreducing conditions, respectively. This result reveals that the spermmembrane proteins has no inter-chain disulfide bond. The purity was attested to thehomogeneity.
Physicochemical properties of the sperm membrane proteins were determined. Theestimated molecular weight of the sperm membrane proteins were 98.4kDa, 67.6kDa,47.3kDa, 29.1kDa, and the PI were 4-7(pH). The concentration of sperm membrane proteinsof wapiti were 95.67μg/ml, 184.23μg/ml, 47.68μg/ml, 58.32μg/ml; the concentration ofthe total protein was 468.12μg/ml by BCA. 3 sperm membrane proteins of wapiti wereglycoprotein in 4 purified proteins.
2.2-D gel electrophoresis: Our results have been shown, there were 823 point obtainde inthe map of 2-DE of the sperm membrane proteins of wapiti. The purity molecular weight was10-100 kDa and the PI was 4-7(pH).
3. Polyclonal antibodies prepareation: In order to study the function of the spermmembrane proteins, polyclonal antibodies were raised in BALB/C mouse. The titer of theantisera was about 1-2×10~3 assayed by ELISA. The result of Western-blot has been indiciatedthat the immunity reactive of the sperm membrane proteins of wapiti, and it was a kind ofspecific antigen.
4. The effct of polyclonal antibodies on sperm activity: The effct of the polyclonalantibodies was not significant.on the activity of wapiti sperm before capacitation in vitro. Itwas not effct on mouse; but the effct on the activity of wapiti s.perm after capacitationmarkedly, The effects mentioned above were dose-dependent
5. Localization of the sperm membrane proteins of wapiti on sperm: The result ofconfocal laser scaning microscope showed that the purified sperm membrane proteins werelocated in the acrosomal of wapiti sperm, Location of sperm of mouse was the same as.
6. identification of polyclonal antibodies of sperm membrane proteins: an agar geldiffusion method showed that the sperm membrane proteins were specific antigen, Theantibodies antiserum was heart. Liver. Kidney.and lung, the purity was reacted with testist andvasdeferens. It was a kind of tissue specificity.
引文
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