模式寄生蠕虫乳酸脱氢酶的结构与功能研究

项目名称： 模式寄生蠕虫乳酸脱氢酶的结构与功能研究

项目编号： No.30860070

项目类型： 地区科学基金项目

立项/批准年度： 2009

项目学科： 医药、卫生

项目作者： 吕刚

作者单位： 海南医学院

项目金额： 26万元

中文摘要： 糖酵解是寄生虫能量代谢的主要途径，乳酸脱氢酶（LDH）为其末端酶。通常认为LDH是胞内蛋白，但根据前期对日本血吸虫LDH（SjLDH）及华支睾吸虫LDH（CsLDH）的研究我们认为寄生蠕虫LDH具有膜蛋白的特性，是理想的药物及疫苗靶标。本课题拟对三种模式寄生蠕虫（日本血吸虫、细粒棘球绦虫、欧猬迭宫绦虫）LDH进行研究，以揭示寄生蠕虫LDH作为膜蛋白的普遍意义，并从分子水平探讨其结构与功能的关系及作为药物和疫苗靶点的应用价值。本课题获得了日本血吸虫LDH（SjLDHA、SjLDHB）、细粒棘球绦虫LDH（EgLDH）及欧猬迭宫绦虫LDH（SeLDH）全长cDNA序列，通过对其编码蛋白质序列的生物信息学分析、基因克隆表达及功能研究，证实了寄生蠕虫LDH具有跨膜蛋白的特性，位于蠕虫表皮表面；对SjLDHB、EgLDH的酶学特性进行了分析，结果表明在生理条件下其主要催化丙酮酸还原为乳酸的反应；通过重组蛋白抗体与重组蛋白体外酶活性抑制试验，证实重组蛋白及主要表位抗体可有效抑制重组蛋白酶活性，提示LDH是潜在的药物及疫苗作用分子，课题基本达到了预期目的，具有重要的理论意义及应用价值。

中文关键词： 寄生虫；乳酸脱氢酶；膜蛋白；结构；功能

英文摘要： Glycolysis process is the mian energy metabolic way of parasites, lactate dehydrogenase (LDH) is the terminal enzyme in glycolysis which is known as an intracellular protein.Previous studies on LDHs from Schistosoma japonicum and Clonorchis sinensis implied that the LDHs from helminth has the characterization of transmembrane protein and are ideal targets of drugs or vaccine. This project aim to study the structure and function of LDHs from 3 model parasitic helminth (Schistosoma japonicum, Echinococcus granulosus, Spirometra erinaceieuropaei), prove they are transmembrane protein and evaluate these LDHs as the targets of drugs and vaccine. We obtained the full-length cDNA sequences of SjLDHA, SjLDHB, EgLDH, SeLDH. By the bioinformatics analysis,molecular cloning, prokaryotic expression and functional studies of the recombinant protein of these LDHs, we proved that these protein are transmembrane proteins and located on the surface of tegument of parasites. Enzymatic properties showed that LDHs are responsible for catalyzing the reduction of pyruvate acid to lactic acid in physiological conditions.We also proved that the enzymatic activity of the recombinant proteins of these LDHs were inhibited by antibodies directed against the intact protein or against epitopes that contain key residues in the catalytic center or substrate binding sites, which implied the LDHs of parasites were potential target for drugs and vaccines against parasites. This project basically reached the intended purpose and have important theoretical significance and application value.

英文关键词： parasite；lactate dehydrogenase;membrane protein;structure;function