Ikegaki N, Tang X X, Kay B K, Kennett R H
Division of Oncology, Children's Hospital of Philadelphia, Abramson Research Center, PA 19104-4318, USA.
Immunotechnology. 1996 Feb;2(1):37-46. doi: 10.1016/1380-2933(96)00032-2.
During the epitope mapping of monoclonal antibodies specific for myc proteins, two E. coli proteins cross-reactive with an anti-c-myc monoclonal antibody (MYC-X-5/1) were identified. One of the proteins is approximately 90 kDa and the other is over 150 kDa in apparent molecular mass. The molecular masses of these cross-reactive proteins suggested that they may be subunits of E. coli RNA polymerase.
We have investigated whether or not the proteins cross-reactive with MYC-X-5/1 are subunits of E. coli RNA polymerase. In addition, we have attempted to determine the epitope of MYC-X-5/1.
The reactivity of MYC-X-5/1 antibody was tested against highly purified E. coli RNA polymerase holo-enzyme preparations and the cell lysate made from E. coli carrying a multi-copy plasmid with an insert of the rpoD gene, the structural gene for the E. coli sigma subunit. The epitope of MYC-X-5/1 was determined by use of phage display of random peptide libraries.
On immunoblotting assays, MYC-X-5/1 reacted with the 90-kDa protein in the E. coli RNA polymerase preparations and with the 90-kDa protein over-expressed in E. coli carrying the plasmid with the rpoD insert. In addition, we have deduced the epitope of the MYC-X-5/1 antibody to be residues 235-245 of the human c-myc protein. A highly similar sequence to this was also identified in residues 62-72 of the sigma subunit of E. coli RNA polymerase.
These data demonstrated that the 90-kDa protein cross-reactive with MYC-X-5/1 is the sigma subunit of E. coli RNA polymerase. Furthermore, this study shows that random peptide libraries displayed on filamentous phage are useful tools for epitope mapping and defining cross-reactivities of monoclonal antibodies.
在针对 myc 蛋白特异性单克隆抗体的表位作图过程中，鉴定出两种与抗 c-myc 单克隆抗体（MYC-X-5/1）发生交叉反应的大肠杆菌蛋白。其中一种蛋白表观分子量约为 90 kDa，另一种超过 150 kDa。这些交叉反应蛋白的分子量表明它们可能是大肠杆菌 RNA 聚合酶的亚基。
我们研究了与 MYC-X-5/1 发生交叉反应的蛋白是否为大肠杆菌 RNA 聚合酶的亚基。此外，我们还试图确定 MYC-X-5/1 的表位。
针对高度纯化的大肠杆菌 RNA 聚合酶全酶制剂以及由携带含 rpoD 基因（大肠杆菌 σ 亚基的结构基因）插入片段的多拷贝质粒的大肠杆菌制备的细胞裂解物，测试 MYC-X-5/1 抗体的反应性。通过使用随机肽库的噬菌体展示来确定 MYC-X-5/1 的表位。
在免疫印迹分析中，MYC-X-5/1 与大肠杆菌 RNA 聚合酶制剂中的 90-kDa 蛋白以及在携带含 rpoD 插入片段质粒的大肠杆菌中过表达的 90-kDa 蛋白发生反应。此外，我们推断 MYC-X-5/1 抗体的表位为人 c-myc 蛋白的 235 - 245 位残基。在大肠杆菌 RNA 聚合酶 σ 亚基的 62 - 72 位残基中也鉴定出与此高度相似的序列。
这些数据表明与 MYC-X-5/1 发生交叉反应的 90-kDa 蛋白是大肠杆菌 RNA 聚合酶的 σ 亚基。此外，本研究表明丝状噬菌体展示的随机肽库是表位作图和确定单克隆抗体交叉反应性的有用工具。
