人白介素18(IL-18)RGD模体抗黑色素瘤细胞B16活性研究  

A Study on Construction of RGD Motif in Human Interleukin-18 and Its Inhibitory Activity on B16 Melanoma

作　　者：卢忠民[1] 赵宝昌[1] 

机构地区：[1]大连医科大学生物化学和分子生物学教研室,大连116027

出　　处：《中国生物工程杂志》2006年第2期13-19,共7页China Biotechnology

摘　　要：利用定点突变及DNA重组技术,在人白细胞介素18(IL-18)的cDNA序列中插入了GGC序列,使IL-18第39精氨酸残基和第40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Pichia Pastoris酵母GS115,利用表达系统进行了高效表达。用Sephadex G-100凝胶过滤纯化表达产物,获得初步纯化的蛋白。研究表明,IL-18在体外对黑色素瘤细胞株B16没有作用,而IL-18-RGD抑制作用明显,IC50=8·10μmol/L;应用小鼠动物模型研究结果显示,IL-18及IL-18-RGD均对黑色素瘤细胞B16有抑制作用,且IL-18-RGD比IL-18的作用强;同时,对鸡胚绒毛尿囊膜(chicken chorioallantoicmembrane,CAM)血管生成的抑制实验发现,IL-18-RGD对CAM血管生成的抑制作用也比IL-18强。但IL-18-RGD仍保存对PBMC诱导产生IFN-γ能力。结果提示,IL-18-RGD在具有抗炎,抗感染作用的同时增添了抑制肿瘤血管新的功能。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, then 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. The cell culture of melanoma B16 showed that IL-18-RGD efficiently inhibited B16 tumor growth, IC50 = 8. 10μmol/L, but the inhibitory effect of IL-18 was not detected. Both IL-18-RGD and IL-18 showed inhibitory activities on mice loaded BI6 in vivo, the inhibitory efffct of IL-18-RGD was stronger than that of IL-18. The inhibitory activities of IL-18-RGD and IL-18 on bFGF induced angiogenesis on chorioallantoic membranes were detected. There was no differences between IL-18-RGD and wild IL-18 in the activity of inducing IFN-γ in human PBMC.

关 键 词：IL-18 RGD模体 毕赤酵母 B16细胞 

分 类 号：Q518.4[生物学—生物化学]