人白介素18(IL-18)RGD模体抗黑色素瘤细胞B16活性研究

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 chorioallantoic membrane,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。

A study on construction of RGD motif in human interleukin-18 and its inhibitory activity on B16 melanoma

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 B16 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.