Shingarova L N, Sagaidak L N, Berkova N, Korobko V G
Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
FEBS Lett. 1996 May 13;386(1):72-4. doi: 10.1016/0014-5793(96)00396-1.
In order to map the immunogenic epitope for the monoclonal antibody E7H2 on the human tumour necrosis factor (hTNF-alpha) molecule, a number of chimeric proteins were developed by in-frame joining segments of the human genes encoding TNF-alpha and lymphotoxin (TNF-beta) as well as by coupling appropriate coding regions for human and mouse TNF-alpha. High level expression of these chimeric genes was achieved in Escherichia coli by placing the coding sequences under control of either E. coli trp-promoter or a tandem of bacteriophage T7 constitutive promoters A2 and A3. As revealed by Western blot analysis with monoclonal antibody E7H2 directed against human TNF-alpha, the region involved in the binding of this antibody includes sequence ValGluLeuArg in the N-terminal part of the TNF-alpha molecule.
为了在人肿瘤坏死因子（hTNF-α）分子上定位单克隆抗体E7H2的免疫原性表位，通过将编码TNF-α和淋巴毒素（TNF-β）的人类基因片段进行读框内连接，以及通过连接人和小鼠TNF-α的适当编码区，构建了一些嵌合蛋白。通过将编码序列置于大肠杆菌trp启动子或噬菌体T7组成型启动子A2和A3串联的控制之下，这些嵌合基因在大肠杆菌中实现了高水平表达。用针对人TNF-α的单克隆抗体E7H2进行的蛋白质印迹分析表明，该抗体结合所涉及的区域包括TNF-α分子N端部分的序列ValGluLeuArg。
