重组多功能抗凝多肽的表达及活性测定

Cloning expression and characterization of a multifunctional anticoagulated peptide gene in E.Coli

目的 探讨多功能抗凝多肽基因的克隆、表达的可行性及其活性的测定。方法 设计一个具有双重抗凝功能的重组多肽分子结构 ,它由谷胱甘肽 S 转移酶 (GST)作为载体蛋白与重组抗凝多肽融合而成。编码重组抗凝多肽的DNA序列是根据氨基酸序列逆向翻译后经人工合成 ,并插入表达质粒pGEX 5X 3中 ,与其中的编码GST序列的 3′端融合。采用大肠杆菌DH5α进行原核表达。应用亲和层析的方法得到纯化的融合蛋白。重组抗凝多肽含有 31个氨基酸 ,由 3个功能部分组成 :(1)Lys Gly Asp(KGD)氨基酸序列 ,可抑制血小板GpⅡb/Ⅲa受体与纤维蛋白原的结合 ;(2 )纤维蛋白单肽A的 7 16残基 (FBA7 16) ,是凝血酶的抑制剂。 (3)水蛭素C末端的 12肽 ,可直接抑制凝血酶。结果 纯化的融合蛋白能抑制二磷酸腺苷诱导的血小板聚集 (10 0 μmol/L时其活性为对照组的 2 0 7% ) ;随浓度增加而显著延长部分凝血活酶时间 (80 μmol/L时凝血活酶时间为 74 7s)和凝血酶时间 (80 μmol/L时为 10 2 3s)并抑制凝血酶的蛋白酶分解活性 (10 0 μmol/L时活性为对照的 11% )。同时 ,也发现GST也有抗血小板作用 ,但其作用要明显弱于融合蛋白。

Objective To investigate the feasibility about the cloning?expression and characterization of multifunctional anticoagulated peptide. Methods We designed a construct using glutathione S transferase (GST) as a protein vector, fused via a cleavable linker to an antithrombotic peptide of 31 amino acids. The peptide was designed to include three inhibitory regions:(1)the Lys Gly Asp(KGD) amino acid sequence to prevent fibrinogen binding to platelets; (2)a part of fibrinopeptide A, an inhibitor of thrombin; and (3)the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 31amino acid peptide was reverse translated, and the gene was chemically synthesized and cloned into an expression vector pGEX 5X 3 as a 3′fusion to the GST gene. Gene expression was induced in E. coli DH5α cells and the fusion protein was purified using affinity chromatohraphy. Results The purified fusion protein significantly lengthened the activated partial thromboplastin time (74.7 s tested in 80 μmol/L) and thrombin time (102.3 s tested in 80 μmol/L) and inhibited the amidolytic activity of thrombin 11% activity compared with the control tested in 100 μmol/L. The ADP induced platelet aggregation was markedly inhibited by the purified fusion protein. The study has also shown that GST exhibits relevant activity of antiplatelet weaker than the purified fusion protein. 20.7% activity compared with the control tested in 100 μmol/L. Conclusion Our results confirm that it is feasible to design a hybrid multifunctional protein that targets various components of the haemostatic process.