Kato Mieko, Hanyu Yoshiro
Bio-Peak Co., Ltd., 584-70 Shimonojo, Takasaki, 370-0854, Japan.
Structure Physiology Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566, Japan.
Methods Mol Biol. 2017;1575:31-44. doi: 10.1007/978-1-4939-6857-2_3.
Recombinant monoclonal antibodies can be established by displaying single-chain variable fragment (scFv) antibody libraries on phages and then biopanning against the target. For constructing superior scFv libraries, antibody light-chain variable region (V) and heavy-chain variable region (V) fragments must be assembled into scFvs without loss of diversity. A high-quality scFv library is a prerequisite for obtaining strong binders from the scFv library. However, the technical challenges associated with the construction of a diverse library have been the bottleneck in the establishment of recombinant antibodies through biopanning. Here, we describe a simple and efficient method for assembling V and V fragments through the concerted action of λ-exonuclease and Bst DNA polymerase. We successfully used this method to construct a diverse chicken scFv library.
重组单克隆抗体可以通过在噬菌体上展示单链可变片段（scFv）抗体文库，然后针对靶标进行生物淘选来建立。为构建优质的scFv文库，抗体轻链可变区（V）和重链可变区（V）片段必须组装成scFv，同时不损失多样性。高质量的scFv文库是从scFv文库中获得强结合剂的先决条件。然而，与构建多样化文库相关的技术挑战一直是通过生物淘选建立重组抗体的瓶颈。在此，我们描述了一种通过λ核酸外切酶和Bst DNA聚合酶的协同作用来组装V和V片段的简单有效方法。我们成功地使用该方法构建了一个多样化的鸡scFv文库。
