Bai Xuelian, Shim Hyunbo
Department of Life Science, Ewha Womans University, 52 Ewhayeodae-gail, Seodaemun-gu, Seoul, Korea.
Department of Bioinspired Science, Ewha Womans University, Seoul, Korea.
Methods Mol Biol. 2017;1575:15-29. doi: 10.1007/978-1-4939-6857-2_2.
Many large synthetic antibody libraries have been designed, constructed, and successfully generated high-quality antibodies suitable for various demanding applications. While synthetic antibody libraries have many advantages such as optimized framework sequences and a broader sequence landscape than natural antibodies, their sequence diversities typically are generated by random combinatorial synthetic processes which cause the incorporation of many undesired CDR sequences. Here, we describe the construction of a synthetic scFv library using oligonucleotide mixtures that contain predefined, non-combinatorially synthesized CDR sequences. Each CDR is first inserted to a master scFv framework sequence and the resulting single-CDR libraries are subjected to a round of proofread panning. The proofread CDR sequences are assembled to produce the final scFv library with six diversified CDRs.
许多大型合成抗体库已经被设计、构建，并成功产生了适用于各种苛刻应用的高质量抗体。虽然合成抗体库具有许多优点，如优化的框架序列和比天然抗体更广泛的序列范围，但它们的序列多样性通常是通过随机组合合成过程产生的，这会导致许多不需要的互补决定区（CDR）序列的掺入。在这里，我们描述了一种使用包含预定义的、非组合合成的CDR序列的寡核苷酸混合物构建合成单链抗体（scFv）库的方法。每个CDR首先被插入到一个主scFv框架序列中，然后对所得的单CDR库进行一轮校正淘选。将校正后的CDR序列组装起来，以产生具有六个多样化CDR的最终scFv库。
