Construction of Synthetic Nanobody Library in Mammalian Cells by dsDNA-Based Strategies**
Construction of Synthetic Nanobody Library in Mammalian Cells by dsDNA-Based Strategies**
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DOI：
10.1002/cbic.202100286
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发表时间：
2021-09-03
期刊：
影响因子：
3.2
通讯作者：
Li, Shuai
中科院分区：
文献类型：
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作者：
Zhao, Yanjie;Wang, Yi;Li, Shuai
纳米体是由单个单体可变的抗原结合域组成的抗体片段。哺乳动物细胞是识别以难以展示的跨膜蛋白为靶标的纳米体和作为细胞表型调节器的纳米体的理想平台。然而，将高多样性纳米体库引入哺乳动物细胞是具有挑战性的。我们开发了两种在哺乳动物细胞中构建纳米体库的新方法。首先通过聚合酶链式反应将互补决定区(CDR)随机序列整合到dsDNA的上游和下游。在第一种方法dsDNA-HR中，含有相同重叠序列的上下游dsDNA共转染培养的哺乳动物细胞，进行细胞内同源重组，形成完整的纳米体库表达盒。在第二种方法中，我们通过连接限制性内切酶的上游和下游的dsDNA，获得了全长的纳米体表达的dsDNA。将获得的全长dsDNA导入哺乳动物细胞进行纳米体库的表达。使用这两种方法，我们产生了超过100万个独特的纳米体序列，高通量测序揭示了这一点。采用单细胞测序的方法分析dsDNA-HR纳米体库的多样性。我们还发现了一个小分子诺可达唑，它可以增强dsDNA-HR的疗效。
A nanobody is an antibody fragment consisting of a single monomeric variable antigen-binding domain. Mammalian cells are ideal platforms for identifying nanobodies targeting hard-to-display transmembrane proteins and nanobodies that function as modulators of cellular phenotypes. However, the introduction of a high-diversity nanobody library into mammalian cells is challenging. We have developed two novel methods for constructing a nanobody library in mammalian cells. Complementarity-determining region (CDR) random sequences were first incorporated into upstream and downstream dsDNAs by PCR. In the first method, named dsDNA-HR, upstream and downstream dsDNAs containing an identical overlapping sequence were co-transfected into cultured mammalian cells for intracellular homologous recombination that resulted in the formation of an intact nanobody library expression cassette. In the second method, named in vitro ligation, we generated full-length nanobody expression dsDNAs via ligation of restriction digested upstream and downstream dsDNAs. The obtained full-length dsDNAs were transfected into mammalian cells for nanobody library expression. Using both methods, we generated over a million unique nanobody sequences, as revealed by high-throughput sequencing. Single-cell sequencing was employed to resolve the diversity of the dsDNA-HR nanobody library. We also identified a small molecule, Nocodazole, which could enhance the efficacy of dsDNA-HR.