Song Z, Cai Y, Song D, Xu J, Yuan H, Wang L, Zhu X, Lin H, Breitling F, Dübel S
Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China.
Hybridoma. 1997 Jun;16(3):235-41. doi: 10.1089/hyb.1997.16.235.
The immunoglobulin heavy- and light-chain variable region (VH and VK) genes were isolated from 8E5 hybridoma cells, which secreted monoclonal antibody against human fibrin by RT-PCR. An expression vector pOPE51-8E5 was constructed for the recombinant VH-VK scFv expression. The primary sequence of the variable regions was determined. Expression product was found in the periplasmic space and inclusion bodies by SDS-PAGE and immunoblotting. It was a 30 KDa single chain fragment (scFv) with the antigen-binding specificity of the parental monoclonal antibody. A light chain shuffling with an unspecific VL did not result in a loss of fibrin binding specificity.
通过逆转录聚合酶链反应（RT-PCR）从分泌抗人纤维蛋白单克隆抗体的8E5杂交瘤细胞中分离出免疫球蛋白重链和轻链可变区（VH和VK）基因。构建了用于重组VH-VK单链抗体片段（scFv）表达的表达载体pOPE51-8E5。测定了可变区的一级序列。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹法在周质空间和包涵体中发现了表达产物。它是一个30千道尔顿的单链片段（scFv），具有亲本单克隆抗体的抗原结合特异性。与非特异性VL进行轻链改组不会导致纤维蛋白结合特异性丧失。
