Cloning, expression, and characterization of single-chain variable fragment antibody against mycotoxin deoxynivalenol in recombinant Escherichia coli
Cloning, expression, and characterization of single-chain variable fragment antibody against mycotoxin deoxynivalenol in recombinant Escherichia coli
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DOI：
10.1016/j.pep.2003.12.008
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发表时间：
2004-05-01
影响因子：
1.6
通讯作者：
Seo, JH
中科院分区：
文献类型：
--
作者：
Choi, GH;Lee, DH;Seo, JH
脱氧雪腐镰刀菌烯醇（DON）是一种由镰刀菌属真菌产生的真菌毒素，是一种世界性的食品和饲料污染物。在重组大肠杆菌中产生DON特异性单链可变片段（scFv）抗体。使用重叠延伸聚合酶链反应，将从杂交瘤3G 7克隆的重链（V-H）和轻链（V-L）的可变区与柔性接头连接。核苷酸序列分析表明，抗DON VH属于V-H Ⅲ基因家族IA亚群，VL属于V-H Ⅲ基因家族II亚群。在大肠杆菌中表达了功能性的单链抗体。大肠杆菌中进行基因融合和伴侣蛋白共表达。分子伴侣（DnaK-DnaJ-GrpE）的共表达允许scFv的可溶性表达。通过固定化金属亲和层析（IMAC）纯化在C-末端与六组氨酸残基融合的scFv抗体。以这种方式产生的可溶性scFv抗体的抗原结合特征进行了表征。通过表面等离子体共振（SPR）分析测定其作为抗体的生物学亲和力，证明其具有显著的生物学亲和力，但弱于完整的抗DON mAb。(C)2004年爱思唯尔公司All rights reserved.
Deoxynivalenol (DON), a mycotoxin produced by several Fusarium species, is a worldwide contaminant of food and feedstuffs. The DON-specific single-chain variable fragment (scFv) antibody was produced in recombinant Escherichia coli. The variable regions of the heavy chain (V-H) and light chain (V-L) cloned from the hybridoma 3G7 were connected with a flexible linker using an overlap extension polymerase chain reaction. Nucleotide sequence analysis revealed that the anti-DON VH was a member of the V-H III gene family IA subgroup and the VL gene belonged to the V, gene family II subgroup. Extensive efforts to express the functional scFv antibody in E. coli have been made by using gene fusion and chaperone coexpression. Coexpression of the molecular chaperones (DnaK-DnaJ-GrpE) allowed soluble expression of the scFv. The scFv antibody fused with hexahistidine residues at the C-terminus was purified by immobilized metal affinity chromatography (IMAC). Soluble scFv antibody produced in this manner was characterized for its antigen-binding characteristics. Its biological affinity as antibody was measured by surface plasmon resonance (SPR) analysis and proved to be significant but weaker than that of the whole anti-DON mAb. (C) 2004 Elsevier Inc. All rights reserved.