白喉毒素/IL-6的高效表达及细胞毒作用的研究  

HighLevel Expression and Characterization of the Fusion Protein Consisting of Diphtheria Toxin and Human Interleukin 6

作　　者：张新建[1] 李晶[1] 张宝云[1] 洪涛[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《生物化学与生物物理学报》1998年第2期169-173,共5页

摘　　要：白喉毒素是由携带β噬菌体基因组的白喉杆菌产生的由５３５个氨基酸组成的单链外毒素，毒性极强，１～２个分子即可灭活１个真核细胞，肿瘤细胞对它尤为敏感。在骨髓瘤、原发性肝癌等肿瘤细胞表面，ＩＬ－６受体可过度表达，利用受体与配体的特异性结合，可将细胞毒性药物定向导入肿瘤细胞。基于上述理论，用ＩＬ－６ｃＤＮＡ取代白喉毒素的受体结合区，构建了白喉毒素／ＩＬ－６融合蛋白表达载体ｐΔＤＴ／ＩＬ－６。ＩＰＴＧ诱导后，ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ印迹结果显示，在６４ｋＤ处，有一明显产物带，该产物即能与白喉毒素单抗结合而显色，又能与ＩＬ－６单抗结合。表达量在菌体蛋白的２０％以上。经初步纯化后，ＩＬ－６受体竞争性结合实验及细胞毒性实验表明，该融合蛋白对表面有大量ＩＬ－６受体的骨髓瘤细胞Ｕ２６６有较强的细胞毒作用；而ＩＬ－６受体较少的正常细胞对其有一定的耐受作用。Diphtheria toxin is a single chain exotoxin of 535 amino acids, secreted from β Corynebacteriophage diphtheriae. Eukaryotic cells, especially tumor cells are very sensitive to DT. Just one or two molecules of DT can kill a cell. On the surface of some tumor cells, such as human myeloma, hepatoma, etc, IL6 receptor has been demonstrated to be expressed at a veryhigh level. Selective cytotoxicity mediated by IL6 receptor could be useful in the targeting therapy of these tumors. Based on this strategy, a hybrid protein consisting of DT and IL6 was constructed, expressed and characterized. IL6 cDNA was first modified for constructing the fusion protein of DT/IL6 and the receptor binding domain of DTDNA was replaced by IL6 cDNA to obtain the expression vector pΔDT/IL6. After induction by IPTG, the fusion protein was expressed successfully, accounting for 20% of the total bacterial protein. The results of SDSPAGE and Westernbloting showed that there was a band of about 64 kD. After preliminary purification, the IL6 receptor competitive binding test and the cytotoxic activity assay of the ΔDT/IL6 showed that the fusion protein possessed significant cytotoxic activity to U266 cells; while the cells expressing IL6 receptors at a mediumlevel was resistant it to a certain degree.

关 键 词：白喉毒素 白细胞介素6 表达 细胞毒 肿瘤治疗 

分 类 号：R392.11[医药卫生—免疫学] R730－56[医药卫生—基础医学]