Ziegler A, Macintosh S M, Torrance L, Simon W, Slabas A R
Scottish Crop Research Institute, Invergowrie, Dundee, United Kingdom.
Lipids. 1997 Aug;32(8):805-9. doi: 10.1007/s11745-997-0103-3.
Purified Brassica napus enoyl acyl carrier protein reductase (ENR) was used to select specific antibodies from a library of antibody fragments, single-chain Fv (scFv), displayed on filamentous phage. Analysis of the selected clones by BstNI fingerprinting and nucleotide sequencing showed that the scFv were derived from three different human VH germline genes. The binding specificities were confirmed by Western blots and ELISA. The scFv preparations reacted with B. napus ENR, but not with beta-keto reductase, nor enoyl reductase from Escherichia coli. Analysis of fragments generated by CNBr treatment indicates that the scFv 3.13 recognized an epitope located within the N-terminal 80 amino acids of the enzyme molecule. The scFv were used to detect ENR directly in extracts of B. napus seeds.
使用纯化的甘蓝型油菜烯酰基载体蛋白还原酶（ENR）从展示在丝状噬菌体上的抗体片段文库——单链Fv（scFv）中筛选特异性抗体。通过BstNI指纹图谱分析和核苷酸测序对所选克隆进行分析，结果表明scFv源自三种不同的人类VH种系基因。通过蛋白质免疫印迹法和酶联免疫吸附测定法确认了结合特异性。scFv制剂与甘蓝型油菜ENR发生反应，但不与β-酮还原酶反应，也不与大肠杆菌的烯酰还原酶反应。对溴化氰处理产生的片段进行分析表明，scFv 3.13识别位于酶分子N端80个氨基酸内的一个表位。scFv被用于直接检测甘蓝型油菜种子提取物中的ENR。
