SELECTION AND RAPID PURIFICATION OF MURINE ANTIBODY FRAGMENTS THAT BIND A TRANSITION-STATE ANALOG BY PHAGE DISPLAY
SELECTION AND RAPID PURIFICATION OF MURINE ANTIBODY FRAGMENTS THAT BIND A TRANSITION-STATE ANALOG BY PHAGE DISPLAY
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DOI：
10.1007/bf02787932
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发表时间：
1994-05-01
影响因子：
3
通讯作者：
KENTEN, J
中科院分区：
文献类型：
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作者：
MCCAFFERTY, J;FITZGERALD, KJ;KENTEN, J
通过在编码病毒外壳蛋白的基因处将可变区基因引入病毒基因组中，可以在丝状噬菌体的表面上展示功能性抗体片段。“噬菌体展示”使得能够根据抗体基因编码的结合特异性从大型文库中分离抗体基因。我们构建了一个新的噬菌体展示载体，编码一个多组氨酸标签，已用于快速纯化可溶性抗体片段。将来自免疫小鼠的抗体文库克隆到该载体中。该文库针对过渡态类似物RT3淘选，并且在两轮淘选后分离出高比例的结合物。PCR分析显示，有24个不同的模式组。对主要模式组内的15个克隆进行测序，发现10个具有一系列点突变的相关克隆。因此，基于TSA选择和筛选，噬菌体展示可以提供大量多样的候选催化抗体库。
Functional antibody fragments may be displayed on the surface of filamentous bacteriophage by introducing variable region genes into the viral genome at a gene encoding a viral coat protein. ''Phage display'' enables the isolation of antibody genes from large libraries according to the binding specificities they encode. We have constructed a new phage-display vector encoding a polyhistidine tag that has been used for rapid purification of soluble antibody fragments. An antibody library derived from immunized mice was cloned into this vector. This library was panned against the transition state analog RT3, and a high proportion of binders isolated after two rounds of panning. PCR analysis revealed that there were 24 different pattern groups. Sequencing of 15 clones within the major pattern group revealed 10 related clones with a range of point mutations. Thus, phage display can provide a large diverse repertoire of candidate catalytic antibodies based on TSA selection and screening.