Maruyama Kouji, Takigawa Yutaka, Akiyama Yasuto, Hojo Takashi, Nara-Ashizawa Noriko, Cheng Jin-yan, Watanabe Morihiro, Yamaguchi Ken
Growth Factor Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, 104-0045, Tokyo, Japan
Mol Immunol. 2003 Oct;40(6):319-26. doi: 10.1016/s0161-5890(03)00165-2.
Complementary DNAs coding for two subunits of hamster interleukin-12 (IL-12), p35 and p40, were cloned from a hamster dendritic cell (DC) cDNA library. The cloning demonstrated that hamster IL-12 consisted of a p35 subunit with 216 amino acid (aa) residues and a p40 subunit with 327 aa. Structural comparison of hamster p35 and p40 at the protein level showed the highest homologies with each counterpart of sigmodon (hispid cotton rat). The gene expressions of hamster IL-12 p35 and p40 in bone marrow (BM) cells cultured in the presence of mouse granulocyte macrophage-colony-stimulating factor (mGM-CSF) and IL-4 were up-regulated during culture. Immunoblot analysis of 293 cells transfected with hamster p35 and p40 expression vectors suggested the presence of a covalently linked p35/p40 heterodimer. Furthermore, supernatant from the 293 cells transfected with both expression vectors induced the up-regulation of interferon-gamma (IFN-gamma) mRNA in hamster splenocytes, indicating that the p35/p40 heterodimer IL-12 protein present in the supernatant was functional. These results suggest that the vectors containing hamster IL-12 cDNA might be suitable tools for developing an immunotherapeutic approach against experimental cancer in a hamster model.
从仓鼠树突状细胞（DC）cDNA文库中克隆出编码仓鼠白细胞介素12（IL-12）两个亚基p35和p40的互补DNA。克隆结果表明，仓鼠IL-12由一个含216个氨基酸残基的p35亚基和一个含327个氨基酸的p40亚基组成。在蛋白质水平上，仓鼠p35和p40与棉鼠相应亚基的结构比较显示出最高的同源性。在小鼠粒细胞巨噬细胞集落刺激因子（mGM-CSF）和IL-4存在的情况下培养的骨髓（BM）细胞中，仓鼠IL-12 p35和p40的基因表达在培养过程中上调。对转染了仓鼠p35和p40表达载体的293细胞进行免疫印迹分析，提示存在共价连接的p35/p40异二聚体。此外，转染了两种表达载体的293细胞的上清液可诱导仓鼠脾细胞中干扰素-γ（IFN-γ）mRNA上调，这表明上清液中存在的p35/p40异二聚体IL-12蛋白具有功能。这些结果表明，含有仓鼠IL-12 cDNA的载体可能是在仓鼠模型中开发针对实验性癌症的免疫治疗方法的合适工具。
