Isolation and characterization of nucleic acid-binding antibody fragments from autoimmune mice-derived bacteriophage display libraries.
Isolation and characterization of nucleic acid-binding antibody fragments from autoimmune mice-derived bacteriophage display libraries.
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从自身免疫小鼠衍生的噬菌体展示文库中分离和表征核酸结合抗体片段。
DOI：
10.1016/0378-1119(93)90254-z
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发表时间：
1993
期刊：
影响因子：
3.5
通讯作者：
Deutscher,SL
中科院分区：
文献类型：
--
作者：
Calcutt,MJ;Kremer,MT;Giblin,MF;Quinn,TP;Deutscher,SL
抗体片段（Fab）在丝状噬菌体表面上的展示和结合特定抗原的噬菌体的选择使得能够分离具有许多特异性的Fab，包括半抗原、蛋白质和病毒颗粒。我们已经研究了通过构建来自自身免疫（MRL/1 pr）小鼠的噬菌体展示Fab的组合文库来分离核酸结合Fab的可能性。选择自身免疫小鼠是因为它们含有对核组分（包括DNA、RNA和蛋白质复合物）具有反应性的抗体（Ab）。针对单链（ss）小牛胸腺（CT）DNA淘选文库，并进一步分析选择的Fab。核酸结合噬菌体的表征导致两种性质完全不同的Fab的鉴定。一个Fab以高亲和力结合多种ssDNA分子以及几种模型RNA底物。该Fab已被亲和纯化至大于95%，并且竞争研究揭示了对与聚（dT）结合的显著偏好。第二个Fab显示与RNA配体的结合减少，ssDNA分子的数量有限。对Fab可变（V）区的推导的氨基酸（aa）序列的分析揭示，来自强核酸结合Fab的重（H）链V区衍生自在自身抗体中反复使用的vh基因。该vh结构域与抗ssDNA自身免疫单克隆抗体（mAb）最相似，表明存在于自身免疫库中的抗原结合特异性可以通过该方法直接获得。进行轻（L）链改组实验以研究来自核酸结合Fab的H链片段如何可以是无差别的并且仍然保留抗原结合。与以前使用半抗原或蛋白质的报道相反，仅分离出少数能够结合DNA和RNA的L链。此外，推导的氨基酸序列的这些L链是几乎相同的原始Fab。几个氨基酸的变化导致抗原特异性的转变，这表明底物识别的微调是可能的。本报告表明，噬菌体Fab展示库将是一个强大的方法，用于分离的核酸结合抗体在体外，并可能适用于免疫识别的研究，特别是当它发生在自身免疫性疾病。
The display of antibody fragments (Fab) on the surface of filamentous bacteriophage and selection of phage that bind to a particular antigen has enabled the isolation of Fab with numerous specificities, including haptens, proteins and viral particles. We have examined the possibility of isolating nucleic acid-binding Fab by constructing a combinatorial library of phage displaying Fab derived from autoimmune (MRL/1pr) mice. Autoimmune mice were chosen because they contain antibodies (Ab) reactive against nuclear components, including DNA, RNA and protein complexes. The library was panned against single-stranded (ss) calf thymus (CT) DNA and the selected Fabs were analyzed further. Characterization of the nucleic acid-binding phage led to the identification of two kinds of Fab with quite different properties. One Fab bound with high affinity a variety of ssDNA molecules, as well as several model RNA substrates. This Fab has been affinity purified to greater than 95% and competition studies revealed a marked preference for binding to poly(dT). The second Fab showed a reduced binding to RNA ligands and a restricted number of ssDNA molecules. Analysis of the deduced amino acid (aa) sequences of the Fab variable (V) regions revealed that the heavy (H) chain V region from the strong nucleic acid-binding Fab was derived from a vh gene that is used recurrently in autoantibodies. This vhdomain was most similar to an anti-ssDNA autoimmune monoclonal antibody (mAb) suggesting that antigen-binding specificities present in an autoimmune repertoire may be directly accessed by this approach. A light (L) chain shuffling experiment was performed to investigate how indiscriminate a H chain fragment from a nucleic acid-binding Fab could be and still retain antigen binding. In contrast to previous reports employing haptens or proteins, only a few L chains were isolated that enabled DNA and RNA binding. Furthermore, the deduced aa sequences of these L chains were almost identical to that of the original Fab. The few aa changes present resulted in a shift in antigen specificity, suggesting that fine tuning of substrate recognition may be possible. This report demonstrates that phage Fab display libraries will be a powerful method for the isolation of nucleic acid-binding Ab in vitro and may be applicable to the study of immune recognition, especially as it occurs in autoimmune disease.