【作者】 王琦；

【导师】 康现江；

【作者基本信息】 河北大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 中华绒螯蟹(Eriocheir sinensis)是我国特有的水产珍品，也是我国一种重要的经济蟹类，广泛存在于我国南北各地淡水水域中。中华绒螯蟹是我国名优淡水养殖业的主要品种。近年来，其养殖业在沿海各地蓬勃兴起，它以优良的生物学品质，极高的营养价值和独特的风味蜚声海内外。养殖业的发展，进一步促进了基础科学研究的发展，而基础科学的发展，又反过来加速了养殖业的蓬勃发展。 本文就中华绒螯蟹精子膜蛋白提取、抗体制备及其免疫定位进行了研究，为其受精生物学、发育生物学方面的深入研究提供参考资料。主要从以下几个方面进行了研究： 1．对中华绒螯蟹雄性生殖系统进行解剖，得到贮精囊，进而分离到精荚，利用玻璃匀浆器匀浆的方法，对中华绒螯蟹精荚进行破碎，得到精荚基质和精子；使用两种去污剂对中华绒螯蟹精子膜蛋白进行了提取，在变性聚丙烯酰胺凝胶电泳(SDS-PAGE)条件下，对其精子膜蛋白组分进行了分析，实验结果表明，在双去污剂作用下，提取得到的精子膜蛋白与脱膜精子部分蛋白谱带差异明显；并对提取的精子膜蛋白进行了分子量、等电点和糖蛋白检测，结果显示，所提取的12种精子膜蛋白，其分子量均在100ku以下，等电点范围在4.4-6.2之间，并均为糖蛋白，说明所提取的中华绒螯蟹精子膜蛋白是低分子量的酸性糖蛋白，这与其他一些哺乳动物精子膜蛋白的研究结果类似； 2．乳酸脱氢酶(LDH)尤其是精子特异性乳酸脱氢酶，定位于膜上，也为精子膜蛋白的一种。利用天然聚丙烯酰胺凝胶电泳(Native-PAGE)和特异性染色的方法，对中华绒螯蟹进行了乳酸脱氢酶同工酶电泳分析，实验结果表明，以两种不同底物(乳酸钠和α-羟基丁酸钠)分别作用于血清、鳃、心脏、肌肉、肝胰腺、成熟蟹精巢、未成熟蟹精巢和精子，实验结果显示，乳酸脱氢酶同工酶具有组织特异性，并且首次在中华绒螯蟹体内发现了精子特异性乳酸脱氢酶，这种酶在中华绒螯蟹精子成熟过程中均存在，并随着精子的成熟，其含量和酶活性逐渐增强，可能在精子成熟过程中起作用； 3．利用抽提的精子膜蛋白免疫雌性新西兰白兔，制备多克隆抗血清；利用琼脂糖双向免疫扩散分别对多克隆抗血清活性、效价进行了分析，实验结果表明，制备的多克隆抗血清发生抗原抗体沉淀反应，具有免疫活性，且其效价为1：16；在精子膜蛋白雌雄组织特异性检测中，其实验结果表明，制备的多克隆抗血清与精巢、输精管和贮精囊粗提更多还原

【Abstract】 Eriocheir sinensis is an unique aquatic treasure as well as important economic crab, which extensively exists throughout South and North of freshwater in China. As one of the famous freshwater species for aquaculture, in recent years, its aquaculture in coastal areas is prosperous. It is having great renown for its excellent biological character, the extremely high nutritional value and the unique flavor all the world. The progress of aquaculture further promotes that of fundamental research, or otherwise, fundamental research accelerates aquaculture, too.This paper is about extraction, antisera preparation and immunolocalization of the sperm membrane proteins in E. sinensis. It provides some references for the further researches on the Fertilization Biology and Developmental Biology. Our study is mainly from the following aspects:1. Male reproductive system of E. sinensis was dissected, spermatophore was obtained from seminal vesicle and then matrix of spermatophores as well as sperms was separated from the spermatophores by using glass homogenizer. Next, sperm membrane proteins of the crab were extracted by two detergent solutions and subjected to SDS Polyacrylamide Gel Electrophoresis(SDS-PAGE). Our results indicated that under the function of double-detergent, the proteins we got had significant differences from those of the demembranated sperms. We found that the molecular weight of 12 extracted proteins was less than 100ku, isoelectric point(pI) ranged from 4.4-6.2, and all of them were glycoproteins. Thus demonstrate that sperm membrane proteins of E. sinensis are lower molecular acidic glycoproteins, which is similar with those in mammalian sperms.2. Lactate dehydrogenase(LDH), is one of the membrane proteins, which is also found on sperm membrane, so we speculated that sperm membrane of E. sinensis may contain this protein. By Native-PAGE and specific staining, we researched on the sperm-specific LDH of E. sinensis, the experiments showed that in the presence of two substrates(sodium lactate and α-hyd roxybutyric acid sodium), we analyzed its serum, gill, heart, muscle, hepatopancreas,更多还原