刺参蛋白酶及其内源性抑制剂与基质降解关系的研究

项目名称： 刺参蛋白酶及其内源性抑制剂与基质降解关系的研究

项目编号： No.31201300

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 水产食品

项目作者： 孙黎明

作者单位： 大连工业大学

项目金额： 20万元

中文摘要： 海参在捕捞、运输及加工过程中常发生强烈的自体降解，导致"化皮、溶解"，是海参养殖及加工中的一个困扰。研究表明，这种自溶现象是以刺参体内蛋白酶为主导，对自身基质的降解过程。正常情况下，蛋白酶的活性受到多种因素，尤其是内源性蛋白酶抑制剂的严格调控，使蛋白酶的活性限制在一定范围内，不至于降解自身的结构蛋白。海参自溶时，蛋白水解酶为何能够肆意水解海参基质，内源性蛋白酶抑制剂自身及其对蛋白酶的抑制作用发生哪些改变，上述问题均无报道。本项目拟从体内、体外两个层面入手，首先，采用基因克隆及异源表达技术获得刺参自溶关键蛋白酶及其内源性抑制剂纯品；接着，以其为抗原制备特异性单克隆抗体，建立免疫组织化学技术，用于自溶关键蛋白酶及其抑制剂的定位、定量研究；最后，在体外研究蛋白酶与内源性抑制剂相互作用方式（亲和力、复合体形成及蛋白酶对抑制剂的降解等）。从蛋白酶-内源性抑制剂相互关系的角度，揭示自溶机理。

中文关键词： 海参；自溶；蛋白酶；内源性抑制剂；基质降解

英文摘要： Self-degradation (autolysis) of sea cucumber often occurs during fishing, transportation and processing. It is a problem perplexing the aquaculture and processing industries. Researches demonstrated that autolysis is a matrix-degrading process caused mainly by endogenous proteases. At normal condition, the proteases are strictly controlled by many factors, especially the endogenous inhibitors, which limited the activity of proteases within a normal range to avoid degrading self-structuring proteins. When autolysis occurs, why could the proteases be out of control in degrading the matrix of sea cucumber? How the endogenous inhibitors and their effect on proteases change? There is no report on them until now. This project is to resolve the above problesms from two levels of in vivo and in vitro. First, the key proteases and endogenous inhibitors are obtained and purified by gene cloning and heterologous expression system. Second, monoclonal antibodies against the above protains are produced to establish immunohistochemical methods for locating and quantifying the key proteases and endogenous inhibitors during autolysis in vivo. Third, the interaction of the key proteases and endogenous inhibitors (affinity, complex forming and the degradation effect of the protease on the inhibitors) is analysed in vitro. At last,

英文关键词： sea cucumber；autolysis；protease；endogenous inhibitor；matrix degradation