关键词: 水蛭素, RGD序列, 嵌合抗栓剂

Abstract: 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.

Key words: hirudin, RGD sequence, chimeric antithrombotic