白眉蝮蛇去整合素Adinbitor定点突变体的构建及其功能检测

Construction and Function of Site-directed Mutant of Adinbitor, A Disintegrin from Gloydius blomhoffi brevicaudus

目的构建白眉蝮蛇去整合素Adinbitor的RIARGD→RPTRGD突变体,研究其主要功能及其作用的分子机制。方法重叠延伸PCR法获得Adinbior定点突变cDNA序列,表达并纯化突变体蛋白。血小板聚集试验检测其对血小板聚集的影响;鸡绒毛尿囊膜模型体内血管新生试验检测其对血管新生的抑制功能;流式细胞仪检测其对血小板膜受体去整合素αⅡbβ3与CD41结合的影响。结果双酶切、PCR及测序证实突变体构建正确,纯化的突变体蛋白纯度在90%以上。野生型Adinbitor可识别αⅡbβ3,竞争性抑制纤维蛋白原与血小板结合,从而发挥抑制血小板聚集的功能;在相同剂量下,突变型Adinbitor几乎不具备此功能,而保留了抑制血管新生的功能。结论已成功构建了白眉蝮蛇去整合素Adinbitor定点突变体,与野生型Adinbitor比较,突变体抑制血小板聚集的功能得到了有效抑制,抑制血管新生功能保持不变,为其今后的功能开发奠定了理论基础。

Objective To construct a site-directed mutant of Adinbitor, a disintegrin from Gloydius blomhoffi brevicaudus and investigate its primary function and molecular mechanism. Methods A site-directed mutant of cDNA sequence of Adinbitor, in which sequence RIARGD was substituted with RPTRGD, was amplified by splicing by overlap extension PCR, based on which the mutant protein was expressed and purified. The mutant was determined for effect on platelet aggregation by platelet aggregation test,for inhibitory effect on angiogenesis by in vivo angiogenesis test in chick chorioallantoic membrane model, and for effect on binding of disintegrin αⅡβ3 of platelet membrane receptor to CD41 by flow cytometry, Results Restriction analysis, PCR and sequencing proved that the mutant was constructed correctly. The expressed mutant protein reached a purity of more than 90% after purification. Wild Adinbitor recognized αⅡβ3 and competitively inhibited the binding of fibrinogen to platelet, thereby inhibited platelet aggregation. However, the Adinbitor mutant at the same dosage showed little inhibitory effect on platelet aggregation, while remained the flmction of inhibiting angiogenesis. Conclusion A site-directed mutant of Adinbitor was successfully constructed. Compared with those of wild Adinbitor. the inhibitory effect of Adinbitor mutant on platelet aggregation was significantly inhibited, while the inhibitory effect on angiogenesis showed no significant change, which provided a theoretical basis for development of function of Adinbitor.