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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 effect of amino acid mutations around the motif of integrin echistatin RGD (Arg-Gly-Asp) on its biological function, six fragments were designed and synthesized based on the echistatin sequence. The cDNA was synthesized by overlap extension PCR. The echistatin RGD motif The expression of echistatin (ARGDNM) in E.coli BL21 (DE3) was induced by ligating with the vector pTXB1.After induced by IPTG, the fusion protein expressed in E.coli reached about 30 %, Chitin affinity purification, DTT cleavage release target protein molecular weight of about 5.4kD.In vitro platelet aggregation and in vivo chick chorioallantoic membrane (chick chorioallantoic membrane, CAM) angiogenesis experiment results show that echistatin (ARGDNM) inhibit platelet Aggregation effect weakened, and its role in inhibiting angiogenesis.RGD motifs around the amino acid changes affect the biological function of disintegrin, echistatin (ARGDNM) enhanced the specificity of αVβ3 binding, the work for the study of specificity Stronger to integrin drugs laid the foundation.