重组多功能抗凝多肽的表达及活性测定

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／Ⅲα受体与纤维蛋白原的结合;(2)纤维蛋白单肽A的7-16残基(FBA^7-16),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(100μmol／L时其活性为对照组的20．7％);随浓度增加而显著延长部分凝血活酶时间(80μmol／L时凝血活酶时间为74．7s)和凝血酶时间(80μmol／L时为102．3s)并抑制凝血酶的蛋白酶分解活性(100μmol／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。结论 通过基因重组的方法设计一具复合抗凝功能的多肽是可行的。

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

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 31 amino 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 DH5 alpha cells and the fusion protein was purified using affinity chromatography. RESULTS: The purified fusion protein significantly lengthened the activated partial thromboplastin time (74.7 s tested in 80 micromol/L) and thrombin time (102.3 s tested in 80 micromol/L) and inhibited the amidolytic activity of thrombin 11% activity compared with the control tested in 100 micromol/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 micromol/L. CONCLUSION: Our results confirm that it is feasible to design a hybrid multifunctional protein that targets various components of the haemostatic process.