导　　师： 王群

学科专业： 071002

授予学位： 硕士

作　　者： ;

机构地区： 华东师范大学

摘　　要： 组织蛋白酶A,是一种重要的酸性丝氨酸羧肽酶,在中性pH条件下,具有脱酰胺酶和酯酶活性。其在高等脊椎动物中的研究比较多,但是在无脊椎动物中的研究较少。早期研究表明,组织蛋白酶A在酸性条件下能够优先水解芳香族和大多数疏水性氨基酸/(羧肽酶活性/)；在中性条件下水解N-端氨基酸/(脱酰胺酶和酯酶活性/),这些都表明了其在蛋白水解过程中的重要功能。 副性腺是中华绒螯蟹雄性生殖系统中重要的组成部分之一,在8月-10月开始发育,明显晚于精巢的发育时间。副性腺在11月份发育到高峰期,这一时期同时也是中华绒螯蟹开始进行交配的时间。因此推测副性腺与中华绒螯蟹雄性生殖发育之间存在着重要的关系。而早期研究也证明,中华绒螯蟹副性腺发育后期的分泌物能快速有效地破裂精荚壁。但是到目前为止,对于副性腺内容物内目标蛋白的相关研究还未见报道。 本研究旨在鉴定参与到精荚消化的功能蛋白。通过硫酸铵沉淀、DEAE-Sephacel离子层析柱和Sephacryl S-200凝胶层析柱从副性腺发育后期内容物蛋白中分离纯化出一种50-kDa左右的蛋白。纯化得到的蛋白能够对精荚破裂起有效作用,随后的HPLC-ESI-MS//MS shotgun分析鉴定这种蛋白为组织蛋白酶A。此外,Western blot分析结果验证了组织蛋白酶A存在于能够消化精荚的蛋白组分里,而它的存在进一步证明了其与精荚消化之间的重要联系。通过ELISA分析可得,副性腺分泌物当中组织蛋白酶A的活性随着分离纯化地进行而逐步增加。并且在中华绒螯蟹副性腺发育后期/(11月份/),同时也是河蟹进行交配的期间,组织蛋白酶A的活性显著高于其他时期。 基于以上研究结果,首次研究证明了存在于中华绒螯蟹副性腺内容物中的组织蛋白酶A,在精荚消化中可能起着很重要的作用。通过对副性腺内容物消化精荚机制的初步探究,必将有利于对中华绒螯蟹的生殖相关作用机制的研究,同时也将进一步为副性腺在雄性的生殖发育中的作用提供相关的依据。 Cathepsin A is an important acidic serine carboxypeptidase with deamidase and esterase activities at a neutral pH. Although there were many studies about cathepsin A in higher vertebrates, fewer reports on it in invertebrates. It appears to prefer the hydrolysis of aromatic and large hydrophobic amino acids residues at acidic pH /(carboxypeptidase activity/) and neutral pH /(amidase//esterase activity/) in the N-terminal amino acids as shown by previous reports. Accessory sex gland is one of the most important male reproductive systems, it begins to develop between Augest and October, significantly delayed than testis's developing period. However, the accessory sex gland reaches the peak of development in Novermber, which is the time of mating in E. sinensis. Therefor, we speculate the important connection between accessory sex gland and reproductive development in E. sinensis. Previous studies have showed the late period of accessory sex gland /(ASG/) secretory proteins in the Chinese mitten crab /(E. sinensis/) can effectively digest the spermatophore wall. But so far, the target secretory proteins have not been reported. Here, we aimed to identify proteins participating in spermatophore digestion. About50-kDa protein secreted from the late period of accessory sex gland was purified to homogeneity by a series of isolation steps including ammonium sulfate fractionation, ion exchange chromatography on a DEAE-Sephacel column and Sephacryl S-200gel-filtration. The purified protein is effective in the spermatophore wall rupture, and the following HPLC-ESI-MS//MS shotgun analysis allowed to identified such protein as cathepsin A. Furthermore, the identification of such protein as cathepsin A was also confirmed by Western blot analysis. It appeared in the proteins which can digest the spermatophore walls.The presence of cathepsin A further confirmed its important connection to spermatophore digestion. By ELISA analysis, cathepsin A enzymatic activity from ASG secretions was shown to increase during its purification process. Moreover, in the late development of accessory sex gland /(Novermber/), with the same time in the mating period of the crab, enzymatic activity was significantly higher than other period. Based on the above results, we first report the characterization of cathepsin A from the accessory sex gland, which might play a key role in digesting the spermatophore wall of E. sinensis. According to the primary study of accessory sex gland secretory proteins on mechanism of spermatophore digestion, it will contribute to clarify the reproductive mechanism of E. sinensis, and provide theory basis on the role of accessory sex gland in male reproduction.

关 键 词： 副性腺分泌蛋白 蛋白纯化 组织蛋白酶 中华绒螯蟹

分 类 号： [S917.4]

领　　域： [农业科学]