【作者】 李张伟；

【导师】 韩雅莉；

【作者基本信息】 汕头大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 土鳖虫为传统活血化淤类中药，首载于《神农本草经》，被广泛用于治疗血滞经闭、淤血腹痛和因跌打损伤引起的血淤不通等病症，治疗效果显著。然而迄今，未见到对土鳖虫抗血栓机理的深入研究，土鳖虫体内的纤溶活性成分也未被完全分离纯化出来，这对充分开发祖国医学这一宝贵动物药材，研制高效低副作用抗血栓药物造成了极大困难。本文通过生化分离手段，从土鳖虫体内提取纯化出三种具有纤溶活性的物质，并对其中一种进行了生化性质的研究，并在此基础上深入至分子水平，对土鳖纤溶活性成分进行了基因克隆和分析。 本研究中，首先通过硫酸铵分段沉淀、DEAE—纤维素柱和Sephadex G-75柱层析从活雌性地鳖体内分离纯化得到一种纤溶酶原激活物(EFF-1)，该激活物经SDS-PAGE电泳检测，显示为一条带，其分子量为41.3KD，含糖量为10.5％。其水解纤维蛋白的比活力为547.9U／mg，进一步研究表明，该成分属于丝氨酸蛋白酶类。此外，通过制备电泳法，从活雌性土鳖体内分离纯化到另外两种纤溶成分：EFF-2、EFF-3，分子量分别为32.9KD和30.6KD，比活力为234.0U／mg和148.5U／mg，EFF-2和EFF-3均具有直接纤溶酶活性，提示它们为新的具有纤溶酶活性的蛋白酶。 其次根据已登陆NCBI网站的蚯蚓纤溶酶、蜈蚣纤溶激酶和人的激肽释放酶的氨基酸保守序列设计出一对简并引物，用RT-PCR的方法从土鳖体内克隆到一段长度为434bp基因序列，共编码144个氨基酸，经蛋白质Blastp比对，证实获得的氨基酸序列为胰蛋白酶家族成员，与多种动物的胰蛋白酶有较高的同源性，与蚯蚓纤溶酶、蜈蚣纤溶酶激酶及人类激肽释放酶的蛋白质序列有一定的同源性，保守序列也一致，证明所克隆出来的cDNA序列是土鳖胰蛋白酶的一段核苷酸序列，为首次发现的基因，记为序列一，命名为ET-Ⅰ；再跟据上述蚯蚓纤溶酶、蜈蚣纤溶激酶的成熟肽N端氨基酸保守序列和序列一设计出一对简并引物，从土鳖虫体内克隆到一段长度为535bp基因，其氨基酸序列与序列一有81％的一致性，为另一种胰蛋白酶家族成员，同样与多种动物的胰蛋白酶有较高的同源性。根据序列二，运用3′末端快速扩增法(3′RACE)法成功克隆出序列二的3′端序列，记为序列三。将序列二和序列三进行拼接，得出完整成熟肽，记为序列四，命名更多还原

【Abstract】 Nowadays thrombi has became the most serious sick in the world.It is important for medicinals expert to invent some usefull medicine to cure the thrombi. Eupolyphaga sinensis Walker is a kind of tradional Chinese herbal medicine. It proves to be an useful medicine for deal with thrombi. But for a long time, people have known a little about it. According to this situaion, this experiment research the useful parts for cure thrombi in Eupolyphaga sinensis Walker by modern biology technology.At first, A kind of the plasminogen activator of Eupolyphaga sinensis Walker has been separated and purified by ammonium sulphate precipitation, DEAE-cellulose and SephadexG-75 Column chromatography. The activator which named EFE1 is appeared to be a single band on SDS-PAGE. It is showed that the MW of the activator is 41300. In addition, it includes ten point five percent sugar. Its specific activity is 547.9U/mg. Three kind of fibrinolytic factors of Eupolyphaga sinensis Walker which are named as EFF-1 EFF-2 and EFF-3 have been separated and purified by ammonium sulphate precipitation, DEAE-cellulose and preparative PAGE electropheresis. Three purified components were proved to be homogeneous in SDS-PAGE electropheresis with moleculare weights 41000,32900 and 30600 respectively. Their Activity as plasminogen activator were 171.3U/mg 234.0U/mg和 148.5U/mg. In addition, EFF-2 and EFF-3 showed fibrinolytic activities on fibrinous plate lacked of Plasminogen. So we could consider EFF-2 and EFF-3 as two new kind of fibrinolytic enzymes.A section of trypsin(ET-I)cDNA was amplified by RT-PCR from female Eupolyphaga sinensis Walker. Sequence analysis revealed that the cDNA was 434 base pair, and that encoded a protein of 144 amino acid residues. With the help of protein Blast, it was proved that the gene belongs to the serine protease trypsin family. In addition, it shows homology in protein sequence compared with Earthworm fribrinolytic enzyme, Scolopendra subspinipes mutilans scolonase and更多还原