Construction and expression of chimeric antithrombotics with hirudin and rgd sequence 含rgd序列水蛭素嵌合抗栓剂的构建与表达
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Protective effect of hirudin against apoptosis on cultured rat brain microvascular endothelial cells 水蛭素对凝血酶所致脑微血管内皮细胞损伤的保护作用
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Abstract : hirudin is the potent antithrombotic factor found in nature . hirulog is a dind of peptides designed by imitating hirudin 文摘:水蛭素是迄今发现的最强的天然凝血酶抑制剂，水蛭肽是模仿水蛭素与凝血酶的相互作用机制而合成的肽类化合物。
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This paper introduces their mechanism , function and clinical research , illustrates their advantages compated with heparin , believing that both hirudin and hirulog are new promising antithrombotic agents 本文介绍了他们的作用机理及其临床研究，叙述了他们与肝素相比的优越之外，认为水蛭素与水蛭肽是两类非常有前途的抗栓新药。
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Based on the structure and function analysis of hirudin , a potent thrombin inhibitor , and some platelet aggregation inhibitors , which contain the recognition sequence argglyasp as their functional motif , two chimeric antithrombotic molecules were designed by introducing rgd sequence to hirudin cterminus . these chimera genes were constructed by pcr and inserted into the expression vector pet21a , the constructs were confirmed by restriction enzyme digestion and dna sequence analysis . these recombinant plasmids were transformed into 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。
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Strain bl21 , and gene expression was induced by iptg . the target proteins were directed into the periplasmic space by the staphylococcal protein a 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 通过离子交换层析和凝胶过滤层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。
