一种核酸结合抗体片段的分析：重链互补决定区切换变体的构建与表征

Analysis of a nucleic-acid-binding antibody fragment: Construction and characterization of heavy-chain complementarity-determining region switch variants.

作者信息

Calcutt M J, Komissarov A A, Marchbank M T, Deutscher S L

机构信息

Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, 65212, USA.

出版信息

Gene. 1996 Feb 2;168(1):9-14. doi: 10.1016/0378-1119(95)00717-2.

DOI:10.1016/0378-1119(95)00717-2

PMID:8626072

Abstract

The display of antibody (AB) fragments (Fab) on the surface of filamentous bacteriophage (phage) and selection of phage that interact with a particular antigen (Ag) has enabled the isolation of Fab that bind nucleic acids. Nucleic acid (NA) binding Ab occur in vivo in connective tissue disease patients and certain inbred strains of mice and are thought to be pathogenic. Although there is ample data concerning the amino acid (aa) sequence of murine monoclonal Ab (mAb) reactive with DNA, significantly less is known about how autoAb interact with NA. The complementarity-determining regions (CDR) contained in the Fab contribute to most Ag binding, especially through heavy (H)-chain CDR 3. We have examined the role of individual H-chain CDR of a previously isolated recombinant single-stranded DNA-binding Fab (DNA-1) in nucleic acid interaction using a combination of H-chain CDR switching and solution-binding experiments. The three H-chain CDR of DNA-1 Fab were independently switched with the H-chain CDR of a Fab (D5) with very similar sequence and framework (FR) that binds DNA poorly in order to create all possible H-chain CDR combinations. The chimeric Fab genes were bacterially expressed, and their products were purified and analyzed. Results indicated that the H-chain CDR 3 of DNA-1 Fab, in the context of the remainder of the H-chain of D5 Fab, restored binding to oligo(dT)15 to 60% of DNA-1 levels, whereas H-chain CDR 1 and 3 of DNA-1 with CDR 2 of D5 Fab restored binding to 100% A combination of H-chain CDR 2 and 3 of DNA-1 Fab with H-chain CDR 1 of D5, unexpectedly resulted in the ability of the chimeric Fab to bind RNA preferentially over DNA. These studies demonstrate the importance of both H-chain CDR 1 and 3 in DNA recognition and further suggest that the specificity of the type of NA recognized by a particular Fab can be drastically altered by exchanging CDR.

摘要

丝状噬菌体（噬菌体）表面抗体（AB）片段（Fab）的展示以及与特定抗原（Ag）相互作用的噬菌体的筛选，使得能够分离出与核酸结合的Fab。核酸（NA）结合抗体在结缔组织病患者和某些近交系小鼠体内存在，被认为具有致病性。尽管有大量关于与DNA反应的鼠单克隆抗体（mAb）氨基酸（aa）序列的数据，但对于自身抗体如何与NA相互作用的了解却少得多。Fab中包含的互补决定区（CDR）对大多数Ag结合起作用，尤其是通过重链（H）-链CDR 3。我们使用H链CDR交换和溶液结合实验相结合的方法，研究了先前分离的重组单链DNA结合Fab（DNA-1）的单个H链CDR在核酸相互作用中的作用。DNA-1 Fab的三个H链CDR分别与序列和框架（FR）非常相似但结合DNA能力很差的Fab（D5）的H链CDR独立交换，以产生所有可能的H链CDR组合。嵌合Fab基因在细菌中表达，其产物被纯化并分析。结果表明，在D5 Fab H链其余部分的背景下，DNA-1 Fab的H链CDR 3将与寡聚（dT）15的结合恢复到DNA-1水平的60%，而DNA-1的H链CDR 1和3与D5 Fab的CDR 2结合可将结合恢复到100%。DNA-1 Fab的H链CDR 2和3与D5的H链CDR 1的组合意外地导致嵌合Fab优先结合RNA而非DNA。这些研究证明了H链CDR 1和3在DNA识别中的重要性，并进一步表明，通过交换CDR，特定Fab识别的NA类型的特异性可能会发生巨大改变。