Fuchs P, Breitling F, Little M, Dübel S
German Cancer Research Center, Heidelberg, Germany.
Hybridoma. 1997 Jun;16(3):227-33. doi: 10.1089/hyb.1997.16.227.
The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed.
从分泌抗人癌基因c-myc单克隆抗体的Myc1-9E10杂交瘤细胞中分离出免疫球蛋白重链和轻链可变区（Vh和Vl）基因。构建了表达载体pOPE52-c-myc用于在大肠杆菌中进行重组生产。通过SDS-PAGE和免疫印迹在周质空间中发现了一种30 kDa的单链片段（scFv）表达产物。如用仅识别加工后的氨基末端的抗血清所证明，相当一部分产物得到了正确加工。证明了scFv片段与母本单克隆抗体的肽表位的特异性结合，并确定了可变区的一级序列。与该杂交瘤细胞系先前发表的部分Vh和Vl序列进行序列比较，发现轻链可变区存在遗传异质性。讨论了这种scFv作为检测和纯化标记蛋白的新工具、用于向癌细胞表面添加共刺激信号以及通过细胞质表达分析活细胞中c-myc功能的潜在用途。
