含RGD序列水蛭素嵌合抗栓剂的构建与表达

Construction and Expression of Chimeric Antithrombotics with Hirudin and RGD Sequence

通过对水蛭素及一些含有精氨酸 甘氨酸 天冬氨酸 (RGD)序列的多肽类血小板聚集抑制剂结构与功能的分析 ,设计并构建了两个在水蛭素C端融合RGD序列的嵌合分子。嵌合体基因分别重组到表达载体pET 2 1a(+ )中并转化大肠杆菌BL2 1(DE3)。经限制酶消化和DNA序列分析 ,证明两种重组质粒与设计完全一致。由于RGD 水蛭素嵌合基因上游连接了金黄色葡萄球菌蛋白A(SPA)的信号肽序列 ,在IPTG诱导下两种嵌合分子都获得了分泌表达 ,表达产物主要集中在细胞周质空间。通过离子交换层析和凝胶过滤层析分别对两种嵌合体蛋白进行纯化 ,纯化产物在Tricine SDS PAGE中都显示为单一条带。活性分析结果表明两种嵌合体蛋白在保留水蛭素抗凝血酶活力的同时 ,还呈现抗血小板聚集活性。

Based on the structure and function analysis of hirudin,a potent thrombin inhibitor,and some platelet aggregation inhibitors,which contain the recognition sequence Arg Gly Asp(RGD)as their functional motif,two chimeric antithrombotic molecules were designed by introducing RGD sequence to hirudin C terminus.These chimera genes were constructed by PCR and inserted into the expression vector pET 21a(+),the constructs were confirmed by restriction enzyme digestion and DNA sequence analysis.These recombinant plasmids were transformed into E.coli strain BL21(DE3),and gene expression was induced by IPTG.The target proteins were directed into the periplasmic space by the staphylococcal protein A(SPA)signal sequence preceding the RGD hirudin gene.Using ion exchange chromatography and gel filtration chromatography,the chimera proteins were purified,and both of them showed a single band in Tricine SDS PAGE.The results of activity analysis suggested that these two chimera proteins not only have antithrombin activities,but gain platelet aggregation inhibitory activities as well.