【摘要】 根据LAP (leechantihemostaticprotein)理论 ,在分析山蛭素和decorsin结构特征的基础上 ,利用重组PCR方法 ,删除了山蛭素氨基酸序列的 33位至 35位 ,同时分别插入了RGDS和来源于decorsin的一段PRGDADP序列构建成为 2种含RGD序列的山蛭素突变体 ,分别命名为HRGD1和HRGD2 .这 2种突变体在毕赤酵母菌株GS115中得到成功表达 .经过超滤、阳离子交换层析和凝胶过滤层析等纯化步骤之后 ,得到纯度高于 95 %的目的蛋白 .通过以chromozymTH为底物的凝血酶酰胺水解实验和血小板聚集抑制实验证实了其体外生物学活性 ,HRGD1和HRGD2抑制凝血酶的动力学常数达到 10 -13 mol/L水平 ,抑制血小板的IC50 在 10 -6mol/L水平上更多还原

【Abstract】 Two recombinants of haemadin which contain RGD amino acids sequence in its forth loop was cloned, expressed and identificated on the basis of the analysis of structure and function of the haemadin and RGD sequence. The chimera genes coding for mature haemadin with inserted RGD sequence were obtained by overlapping PCR and cloned into the shuttle expression vector pPICZαA. This process was identified by restriction endonuclease digestion and DNA sequence analysis. The recombinant plasmids were electroporated into Pichia pastoris strain GS115. The recombinant proteins, HRGD1 and HRGD2, were expressed and induced by methyl alcohol, secreted into media leading by signal peptides and purificated by combination of ultrafiltration, cation exchange chromatography and gel filtration chromatography. The biological activity assays approved the chimera proteins show activity not only in antithrombin but also in antiplatete aggregation as what have been predicted.更多还原