将RGD模体改变为RGDNM可减弱Echistatin对血小板聚集的抑制但增强血管新生的抑制作用

Replacement of RGD Motif with RGDNM Attenuates Echistatin's Anti-platelet Aggregation Effect,but Enhances Its Inhibition on Angiogenesis

为了研究去整合素echistatin RGD(Arg-Gly-Asp)模体周边氨基酸突变后对其生物学功能的影响,根据echistatin序列设计合成6个片段,利用重叠延伸PCR法合成cDNA,使echistatin RGD模体周边序列变为ARGDNM(D27→N),与载体pTXB1连接后转化E.coliBL21(DE3),建立echistatin(ARGDNM)的表达体系.工程菌经IPTG诱导后融合蛋白表达量占菌体总蛋白约30%,几丁质亲和纯化后,DTT裂解释放目的蛋白分子量约5.4kD.体外血小板聚集和体内鸡胚绒毛尿囊膜(chick chorioallantoic membrane,CAM)血管新生实验结果表明,echistatin(ARGDNM)抑制血小板聚集的作用减弱,而其抑制血管新生的作用增强.RGD模体周围氨基酸的改变影响了去整合素的生物学功能,echistatin(ARGDNM)增强了与αⅤβ3结合的特异性,本工作为研究特异性更强的去整合素药物奠定了基础.

In order to study the influences of amino acid mutation surrounding echistatin RGD（Arg-Gly-Asp） motif on its biological activity,six fragments were synthesized and used to obtain echistatin mutant cDNA by overlaping extension polymerase chain reaction.The amino acid residues flanking RGD loop was changed into ARGDNM（D27→N）.The target gene was ligated with pTXB1 and transformed into E.coli BL21（DE3）.The mutant was efficiently expressed as a fusion protein and accounted for about 30% of the total protein under IPTG induction. The target protein was cleaved from the fusion protein by DTT treatment on the chitin column with a molecular weight 5.4 kD. The biological activity was determined by platelet aggregation in vitro and CAM （chick chofioallantoic membrane） angiogenesis in vivo. The platelet aggregation inhibition activity of purified echistatin （ARGDNM） was less efficient than echistatin, but the mutant was more effective in inhibiting CAM angiogenesis. These results indicated that the binding specificity of echistatin （ARGDNM） to av 133 was improved. Disintegrin with RGDNM motif is more inclined to bind with αvβ3. This work laid the foundation for further research of specific disintegrin agents.