含RGD模体的人白细胞介素18(IL-18)突变体的酵母表达及其抗血小板聚集活性

利用定点突变及DNA重组技术,在人白细胞介素18(IL18)cDNA序列中插入GGC序列,使IL18第39位精氨酸残基和第40位天冬氨酸残基之间插入一个甘氨酸残基,从而构建了RGD模体.此重组的cDNA序列构建入表达质粒pPIC9K,并转化Pichiapastoris酵母GS115,利用表达系统进行了高效表达.用SephadexG100凝胶过滤纯化表达产物,获得初步纯化的蛋白.对该蛋白进行了血小板聚集抑制实验和对GPⅡbⅢa与Fn结合的抑制实验.含RGD模体的重组IL18(IL18RGD)显示了较强的体外抑制血小板聚集活性,IC50=8.8μmolL;并具有与GPⅡbⅢa的竞争结合活性,IC50=8.0μmolL.该含有RGD模体的重组IL18仍保存对PBMC诱导产生IFNγ能力.结果表明,此IL18RGD嵌合体在具有抗炎,抗感染的同时增添了新的抑制血小板聚集功能.

Expression of Human Interleukin-18 Mutant Containing RGD Motif in Yeast and Its Inhibitory Effect on Platelet Aggregation

Using site-directed mutagenesis and DNA recombinant technology, GGC fragment was inserted into human IL-18 cDNA. The mutated IL-18 cDNA was constructed into plasmid pPIC9K, thereafter transformed into Pichia pastoris GS115 and efficiently expressed. A Glycine residue was inserted into the recombinant IL-18 between Arg39 and Asp40 to form a RGD motif. The mutated IL-18 was termed IL-18-RGD. The protein was purified with Sephadex G-100 showing a single band on SDS-PAGE. Inhibitory activity of IL-18-RGD on ADP induced platelet aggregation and on the binding of GPⅡb-Ⅲa(platelet fibrinogen receptor) to fibronectin (Fn) were dose-dependent with IC 50=8.8 μmol/L and IC 50=8.0 μmol/L, respectively. The results showed that IL-18-RGD had an obvious inhibitory potency on ADP induced platelet aggregation,but the effect on the binding of GPⅡb-Ⅲa to Fn was the same as the wild IL-18. There were no differences between IL-18-RGD and wild IL-18 in inducing IFN-γ production from human PBMC in vitro.