抗凝、溶栓双功能水蛭素12肽-瑞替普酶融合蛋白的模拟、构建与表达

Simulation,Construction and Expression of A Fusion Protein Made of 12 Peptides of Hirudin and Reteplase

将水蛭素12肽通过柔性肽(Gly)3与r-PA连接,以期望获得既具有抗凝活性,又具有溶栓活性的新型基因工程蛋白质分子。采用计算机辅助分子设计手段模拟了该融合蛋白的分子结构与其活性区可以正常发挥功能。构建并表达了兼有溶栓和抗凝活性的瑞替普酶(r-PA)与水蛭素12肽双功能融合基因。通过两轮加端PCR获得水蛭素12肽与r-PA通过柔性肽(Gly)3连接得到的融合基因,再克隆到pET-21a载体上,经测序正确后转化大肠杆菌BL21(DE3)中诱导表达,融合蛋白约占菌体总蛋白的12%,以包涵体形式存在,经过体外复性后,融合蛋白的纤溶比活达到8 400.5 IU/mg,抗凝比活达到930.2 ATU/mg,具有较高的溶栓抗凝双功能活性。该双功能融合蛋白有望成为治疗血栓疾病的新型药物。

To combine the fibrinolytic and anticoagulant activities in therapy agains thrombotic disease,a fusion protein made of 12 peptides of hirudin and reteplase was constructed and expressed.The structure of the designed protein was predicted and simulated by computer-assisted molecular design.The objective gene from rPA gene was amplified with two cycles PCR,adding the(Gly)3link and 12 peptides of hirudin to the original gene.Then it is cloned into pET-21a vector and the sequence is identical with that predicted.The recombinant protein was produced in E.coli BL21(DE3) after IPTG induction,it constituted about 12% of total bacterial protein,and existed from inclusion body after sonication.After renaturation by dilution and molecular-exclusion chromatography in vitro,the fusion protein has showed effective biofuntional activities.The fibrinolytic activity and anticoagulant activity was 8400.5IU/mg and 930.2ATU/mg respectively.So the fusion protein is probably a new effective therapeutic agent against thromobotic disease.