Kretzschmar T, Geiser M
Ciba-Geigy Ltd. (K-681.5.10), Basel, Switzerland.
Gene. 1995 Mar 21;155(1):61-5. doi: 10.1016/0378-1119(94)00897-2.
A gene coding for an anti-(2-phenyl-5-oxazolone) single-chain Fv antibody (Ab) fragment (anti-phOx scFv) was cloned in-frame into phagemid vectors upstream from genes encoding (i) the wild-type (wt) minor coat protein (cp) III of the filamentous bacteriophage M13 of Escherichia coli, (ii) a truncated version of cpIII (amino acid (aa) positions 198-406), (iii) the wt major cpVIII, or (iv) a hybrid of interleukin-1 beta (IL-1 beta; aa 10-152) and wt cpVIII. Recombinant (re-) phage obtained by phagemid rescue were examined for the efficiency of displaying these various anti-phOx scFv::cp hybrids with commercially available anti-M13 enzyme-linked immunosorbent assays (ELISA), by immunoblotting with an anti-c-myc Ab, and by selection experiments. We found that the highest ELISA signals were obtained with the cpIII constructs and also that more immunoreactive material was detected by blotting than with Ab::cpVIII fusions. Consequently, more scFv::cpIII than scFv::cpVIII phage could be recovered in micropanning experiments with the antigen phOx as target.
将编码抗（2-苯基-5-恶唑酮）单链Fv抗体（Ab）片段（抗phOx scFv）的基因读框内克隆到噬菌粒载体中，位于编码以下基因的上游：（i）大肠杆菌丝状噬菌体M13的野生型（wt）次要外壳蛋白（cp）III；（ii）cpIII的截短版本（氨基酸（aa）位置198 - 406）；（iii）wt主要cpVIII；或（iv）白细胞介素-1β（IL-1β；aa 10 - 152）与wt cpVIII的杂交体。通过噬菌粒拯救获得的重组（re-）噬菌体，利用市售的抗M13酶联免疫吸附测定（ELISA）、用抗c-myc抗体进行免疫印迹以及通过筛选实验，检测展示这些不同的抗phOx scFv::cp杂交体的效率。我们发现，cpIII构建体获得的ELISA信号最高，并且通过印迹检测到的免疫反应性物质比Ab::cpVIII融合体更多。因此，在以抗原phOx为靶标的微淘选实验中，回收的scFv::cpIII噬菌体比scFv::cpVIII噬菌体更多。
