重组白眉蝮蛇去整合素定点突变对其生物活性的影响(英文)  

Influences of Site-directed Mutagenesis of a Recombinant Didintegrin from Gloydius blomhoffi brevicaudus on Its Biological Activities

作　　者：陈洪岩[1] 崔秀云[1] 张春鹏[1] 王继红[1] 赵霆[1] 吕莉[1] 韩国柱[1] 赵宝昌[1] 

机构地区：[1]大连医科大学生物化学与分子生物学教研室,大连116027

出　　处：《中国生物化学与分子生物学报》2006年第8期621-626,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.30572141)~~

摘　　要：RGD为存在于许多糖蛋白配体中的氨基酸序列,对整合素具有识别作用.此序列也发现于许多蛇毒去整合素分子中.采用基因克隆技术从大连产白眉蝮蛇的毒腺中克隆出的去整合素adinbitor是含73个氨基酸残基的去整合素,分子中含有12个半胱氨酸和RGD模体.实验证明,adinbitor作为去整合素的新成员,具有典型的抗ADP诱导的人血小板聚集作用和抗肿瘤血管新生作用.为了将adinbitor的这2种功能分开,采用PCR基因定点突变的方法,将其cDNA序列中RGD模体改变成KGD.重组adinbitor(KGD)在E.coliBL21得到表达,并通过His·Bind亲和层析予以纯化.实验发现,adinbitor对ADP诱导的人血小板聚集具有明显抑制作用,其IC50=85nmol/L,明显优于adinbitor(RGD)(IC50=150nmol/L).然而,与adinbitor(KGD)相比,adinbitor(KGD)则丧失了对血管生成的抑制作用.结果说明,adinbitor(KGD)可作为专一的抗人血小板聚集药具有潜在的开发前景.Arg-Gly-Asp （RGD） is a unique minimal integrin-binding sequence found within several glycoprotein ligands and also in snake-venom disintegrins. Adinbitor, a protein with 73 amino acid residues including 12 cysteins and an RGD motif, was cloned from Gloydius blomhoffi brevicaudus in my laboratory. As a new member of disintegrin family, adinbitor can inhibit both human platelet aggregation induced by ADP and angiogenensis in vivo and in vitro, the typical characters of disintegrin family. To separate the effect of inhibiting platelet aggregation from that of inhibiting angiogenensis, the motif KGD was introduced into adinbitor cDNA to replace RGD by site-directed and PCR-based mutagenesis. The recombinant protein （recombinant adinbitor （KGD）） was expressed in E. coli BL21 and purified through the His· Bind affinity chromatography. Recombinant adinbitor （KGD） could inhibit ADP-induced platelet aggregation with IC50 value of 85 nmol/L. Considerably, it was a more effective inhibitor on platelet aggregation than recombinant adinbitor （RGD）, which has an IC50 of 150 nmol/L. Interestingly, recombinant adinbitor （KGD） has no potency in inhibiting angiogenesis in vivo compared with recombinant adinbitor （RGD）. These findings showed that KGD containing adinbitor was more suitable for inhibiting ADP-induced human platelet aggregation as a potential and specific inhibitor of human platelet aggregation, which might have promising therapeutic potential as an antithrombotic agent.

关 键 词：去整合素 定点突变 血管生成作用 血小板聚集 白眉蝮蛇 

分 类 号：Q71[生物学—分子生物学] Q78