Bio-Peak Co., Ltd., 584-70 Shimonojo, Takasaki, 370-0854 Japan; Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566 Japan.
J Immunol Methods. 2013 Oct 31;396(1-2):15-22. doi: 10.1016/j.jim.2013.07.003. Epub 2013 Jul 31.
The single-chain Fv fragment (scFv) is the most frequently used form of recombinant antibody. It is possible to establish clones specific to a certain target by displaying the scFv library on phages followed by biopanning against the target. For the construction of superior scFv libraries, the light-chain variable region (VL) and the heavy-chain variable region (VH) fragments should be assembled into the scFv without loss of diversity. We have provided an efficient method for constructing scFvs by enzymatic assembly of the VL and VH domains using the concerted action of λ-exonuclease and Bst DNA polymerase. First, we amplified the chicken VL and VH fragments using a phosphorylated primer with a 21-nucleotide overlap in the linker region. Then we recessed the overlapping parts of the VL and VH fragments with λ-exonuclease, which yielded single-stranded overhangs that specifically annealed between the VL and VH fragments; the complete double-stranded scFv was formed using Bst DNA polymerase. Complete scFvs were obtained using this method, whereby a library of scFvs was constructed from the immune library of chicken IgG. The diversity of this scFv library was analyzed by DNA fingerprinting method. The scFvs specific to the antigen could be isolated from this library after 5 rounds of panning.
单链 Fv 片段（scFv）是最常使用的重组抗体形式。通过将 scFv 文库展示在噬菌体上，然后针对目标进行生物淘选，可以建立针对特定目标的克隆。为了构建优越的 scFv 文库，应该在不损失多样性的情况下将轻链可变区（VL）和重链可变区（VH）片段组装成 scFv。我们提供了一种通过使用 λ-核酸外切酶和 Bst DNA 聚合酶的协同作用酶促组装 VL 和 VH 结构域来构建 scFv 的有效方法。首先，我们使用带有在连接区有 21 个核苷酸重叠的磷酸化引物扩增鸡的 VL 和 VH 片段。然后，我们用 λ-核酸外切酶凹入 VL 和 VH 片段的重叠部分，产生特异性退火在 VL 和 VH 片段之间的单链突出端；使用 Bst DNA 聚合酶形成完整的双链 scFv。使用这种方法获得了完整的 scFv，从而从鸡 IgG 的免疫文库中构建了 scFv 文库。通过 DNA 指纹图谱分析方法分析了该 scFv 文库的多样性。可以从该文库中分离出针对抗原的特异性 scFv。
