Nanobody is one special type of single-domain antibody fragment with multiple advantages over traditional antibody. Our previous work established linear-double-stranded DNA (ldsDNA, or PCR amplicon) as novel biological parts for building AND gate genetic circuits in mammalian cells. During this AND-gate circuit formation process, the co-transfected up- and down-stream ldsDNAs could be linked together to form intact gene expression cassette. Here, we employed this ldsDNA-based AND-gate (LBAG) strategy to construct nanobody library in mammalian cells. The sequence complexity of complementary determining regions (CDRs) was introduced into ldsDNA by PCR amplification. After being co-transfected into mammalian cells, the up- and down- stream ldsDNAs undergo AND gate linkage and form full nanobody coding regions, containing CDR1-3. High throughput sequencing identified 22,173 unique oligonucleotide sequences in total generated by this strategy. Thus, we developed a novel method to construct nanobody library, which is a start point for building high content nanobody library in mammalian cells.

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基于线性双链DNA和门遗传电路的哺乳动物细胞纳米抗体库的构建

纳米抗体是一种特殊类型的单域抗体片段，具有优于传统抗体的多种优势。我们之前的工作将线性双链DNA（ldsDNA或PCR扩增子）建立为在哺乳动物细胞中构建和门遗传电路的新型生物部分。在此与门电路形成过程中，可以将共转染的上下游ldsDNA连接在一起，以形成完整的基因表达盒。在这里，我们采用该升dsDNA- b ASED甲ND-克特（LBAG）策略在哺乳动物细胞中构建纳米抗体库。通过PCR扩增将互补决定区（CDR）的序列复杂性引入ldsDNA。在共转染哺乳动物细胞后，上游和下游的ldsDNA进行“与”门连接并形成包含CDR1-3的完整纳米抗体编码区。高通量测序鉴定了该策略产生的总共22,173个独特的寡核苷酸序列。因此，我们开发了一种构建纳米抗体库的新方法，这是在哺乳动物细胞中建立高含量纳米抗体库的起点。