An improved helper phage system for efficient isolation of specific antibody molecules in phage display
An improved helper phage system for efficient isolation of specific antibody molecules in phage display
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DOI：
10.1093/nar/30.5.e18
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发表时间：
2002-03-01
影响因子：
14.9
通讯作者：
Cha, S
中科院分区：
文献类型：
--
作者：
Baek, H;Suk, KH;Cha, S
噬菌体展示技术已应用于生物和医学科学的许多领域，以研究分子相互作用，特别是用于产生人源性单克隆抗体。然而，噬菌体表面上抗体分子的极低展示水平是基于噬菌粒的展示系统的固有问题，导致分离特异性结合分子的成功率低。我们在这里展示的单链抗体片段（scFv）与pIGT 3噬菌粒产生的展示可以显着增加通过使用遗传修饰的Ex-噬菌体。前噬菌体具有突变的pill基因，其在抑制大肠杆菌菌株中产生功能性野生型pill，但在非抑制大肠杆菌菌株中不产生任何pill。在F+非抑制性大肠杆菌菌株中用Ex-噬菌体包装编码抗体-pill融合的噬菌粒增强了重组噬菌体颗粒表面上抗体片段的展示水平，导致与用M13 K 07辅助噬菌体包装相比抗原结合反应性增加>100倍。因此，Ex-噬菌体和pIGT 3噬菌粒载体提供了从噬菌体展示文库中有效富集特异性结合抗体的系统，从而增加了获得对靶抗原具有特异性的更多样化抗体的机会。
Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pill gene that produces a functional wild-type pill in suppressing Escherichia coli strains but does not make any pill in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pill fusion in F+ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13K07 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.