目的：建立Native-PAGE结合Western blot检测水蛭活性多肽与凝血酶相互作用产物的方法，探究水蛭有效抗凝成分。方法：本研究分别提取宽体金线蛭、日本医蛭两种水蛭虫体总蛋白及日本医蛭唾液蛋白，用凝血酶滴定法测定其抗凝血酶活性，Western blot检测凝血酶-活性多肽复合体。结果：研究发现两种水蛭Native-PAGE-WB均检测到凝血酶-活性多肽复合体，且SDS-PAGE-WB检测到复合体变性后产生凝血酶条带。结论：建立的Native-PAGE-WB法可有效检出水蛭活性多肽和凝血酶相互作用产物，且发现同水蛭素类似，宽体金线蛭活性抗凝多肽与凝血酶在体外也能形成稳定的蛋白复合体。

Native-PAGE can conserve the bioactivity of protein after the separation of electrophoresis.Western blot is a protein detection technique used to detect specific antigens (such as thrombin) with specific antibodies. In this researchNative-PAGE-WB methods was established to detect Protein-Protein Interactions between thrombin and anticoagulantcomponents and to explore the active anticoagulants from two leech species. In this method NP-40 lysis buffer was usedto extract the total proteins from Whitmania pigra Whitman and Hirudo nipponica Whitman, then Antithrombin activity oftwo leech species was determined by thrombin titration. It was found that both leech species the thrombin - activepeptides was detected through Native- PAGE -WB, and the SDS- PAGE -WB found that the strips produced after thedegeneration of the complex were the same as that of standard thrombin.It comes to the conclusion that the anticoagulants from Whitmania pigra Whitman can form stable protein complexes with thrombin in vitro like hirudin.

湖北省科技厅自然科学基金项目（2016CFB404）：水蛭体外抗凝血机制及活性评价的研究，负责人：肖凌；湖北省食品药监督管理局科研项目（2016010+29）：以水蛭为示范的动物药材DNA分子鉴定系统构建，负责人：汪波；中医药行业科研专项（201507002）：中药饮片质量保障系统研究（一），负责人：郑健。