Löwenadler B, Jansson B, Paleus S, Holmgren E, Nilsson B, Moks T, Palm G, Josephson S, Philipson L, Uhlén M
Kabigen AB, Stockholm, Sweden.
Gene. 1987;58(1):87-97. doi: 10.1016/0378-1119(87)90032-1.
A novel method to obtain specific antibodies against short peptides is described, involving synthesis of the corresponding oligodeoxynucleotides followed by cloning into a new set of fusion vectors, pEZZ8 and pEZZ18, based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus protein A. The soluble gene fusion product thus obtained, can be collected from the culture medium of Escherichia coli and rapidly recovered in a one-step procedure by IgG affinity chromatography. The system was used to express a fusion protein consisting of the two Z fragments and the C-terminal part [amino acids (aa) 57-70] of human insulin-like growth factor I (IGF-I). This 16-kDa protein was purified by affinity chromatography on IgG Sepharose and antibodies were raised in rabbits. The fusion protein elicited peptide-specific antibodies, as measured by solid-phase radioimmuno assay and Western blotting, reactive with both synthetic C-terminal peptide and the native human IGF-I protein. The results suggests that the gene fusion system can be used for efficient antibody production against short peptides encoded by synthetic oligodeoxynucleotides.
本文描述了一种获得针对短肽的特异性抗体的新方法，该方法包括合成相应的寡脱氧核苷酸，然后克隆到基于金黄色葡萄球菌蛋白A的两个合成IgG结合结构域（ZZ）的一组新的融合载体pEZZ8和pEZZ18中。由此获得的可溶性基因融合产物，可以从大肠杆菌的培养基中收集，并通过IgG亲和层析在一步操作中快速回收。该系统用于表达由两个Z片段和人胰岛素样生长因子I（IGF-I）的C末端部分[氨基酸（aa）57 - 70]组成的融合蛋白。这种16 kDa的蛋白通过IgG琼脂糖亲和层析纯化，并在兔中产生抗体。通过固相放射免疫测定和蛋白质印迹法测定，融合蛋白引发了肽特异性抗体，该抗体与合成的C末端肽和天然人IGF-I蛋白都有反应。结果表明，该基因融合系统可用于高效生产针对由合成寡脱氧核苷酸编码的短肽的抗体。
