Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.
Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.
复制标题
抗人原癌原 c-myc 抗体的一级结构和功能性 scFv 抗体表达。
作者：
P. Fuchs;Frank Breitling;Melvyn Little;Stefan Dübel
从分泌抗人癌基因c-myc的单克隆抗体的Myc 1 - 9 E10杂交瘤细胞中分离免疫球蛋白重链和轻链可变区（Vh和Vl）基因。构建表达载体pOPE 52-c-myc，在大肠杆菌中进行重组表达。杆菌SDS-PAGE和免疫印迹显示，在细胞周质中表达了30 kDa的单链抗体（scFv）。如仅识别经加工的氨基末端的抗血清所示，正确加工了显著部分。证明了scFv片段与母体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。与先前公布的来自该杂交瘤细胞系的部分Vh和Vl序列的序列比较揭示了轻链可变区的遗传异质性。这种单链抗体作为一种新的工具，用于检测和纯化标记的蛋白质，添加共刺激信号的癌细胞的表面，以及用于分析c-myc在活细胞中的功能，通过胞质表达的潜在用途进行了讨论。
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.