Bading H, Hansen J, Moelling K
Max-Planck-Institut für Molekulare Genetik, Berlin, FRG.
Oncogene. 1987;1(4):395-401.
The bacterially expressed v-myb protein served as antigen for the isolation of several monoclonal antibodies, one of which recognized the human cellular myb protein (p75hu-c-myb) indicating a conserved epitope. The epitope was mapped to amino acid positions 208-232 by the use of several bacterially expressed v-myb proteins with various deletions. Furthermore, a synthetic oligopeptide which had been selected on the basis of its hydrophilicity by computer analysis of the v-myb oncogene (amino acids 213-231) blocked the action of this monoclonal antibody, indicating the immunological significance of this region. The monoclonal antibody allowed efficient purification of the p75hu-c-myb protein by immunoaffinity chromatography. The purified protein binds to double-stranded DNA in vitro in a filter-binding assay. Since the monoclonal antibody does not interfere with DNA binding it allowed analysis of DNA-protein interaction in a modified McKay assay using the purified p75hu-c-myb protein. Specific binding was observed predominantly to one of 12 lambda DNA fragments in vitro in the presence of high molar excess of competing co-polymer poly [d(I:C)]. Enhancer/promoter-like sequences of SV40 were not preferentially recognized.
细菌表达的v-myb蛋白用作分离几种单克隆抗体的抗原，其中一种识别人类细胞myb蛋白（p75hu-c-myb），表明存在保守表位。通过使用几种具有不同缺失的细菌表达的v-myb蛋白，将该表位定位到氨基酸位置208 - 232。此外，通过对v-myb癌基因（氨基酸213 - 231）进行计算机亲水性分析而选择的合成寡肽可阻断该单克隆抗体的作用，表明该区域具有免疫意义。该单克隆抗体可通过免疫亲和层析有效纯化p75hu-c-myb蛋白。纯化后的蛋白在体外滤膜结合试验中与双链DNA结合。由于该单克隆抗体不干扰DNA结合，因此可使用纯化的p75hu-c-myb蛋白在改良的麦凯试验中分析DNA - 蛋白质相互作用。在存在高摩尔过量的竞争性共聚体聚[d（I:C）]的情况下，体外观察到特异性结合主要发生在12个λDNA片段中的一个上。SV40的增强子/启动子样序列未被优先识别。
