在哺乳动物细胞内基于线性双链DNA"与门"基因线路构建纳米抗体文库

Construction of nanobody library in mammalian cells by linear-double-stranded DNA based and gate genetic circuit

目的在哺乳动物细胞内基于线性双链DNA"与门"基因线路构建纳米抗体文库。方法应用基于线性双链DNA的"与门"逻辑基因线路构建纳米抗体文库,首先通过PCR扩增将互补决定区(CDR)随机序列引入上、下游线性双链DNA,将其共转染至HEK293T细胞,转染48 h后,经RNA提取、cDNA合成、PCR扩增、高通量测序和数据处理步骤,鉴定"与门"形成的纳米抗体文库序列,对本策略进行了分析与评价。结果深度测序共测得4 173 356条序列,其中约88.18%的序列为纳米抗体文库序列。文库核酸序列最丰富的序列长度为264 bp,为理论设计长度。后续在264 bp序列中鉴定出22 172种纳米抗体序列,且CDR1~3序列中的核苷酸频率符合NNK模式。结论本研究开发了一种基于线性双链DNA"与门"逻辑基因线路在哺乳动物细胞中构建纳米抗体文库的新方法。

Objective A linear-double-stranded DNA(ldsDNA)based AND-gate strategy was developed to construct nanobody library in mammalian cells.Measures We employed the ldsDNA-based AND-gate genetic circuit to introduce nanobody library into cultured mammalian cells.The sequence complexity of the complementary determining regions(CDRs)was introduced into the up-and down-stream ldsDNA by PCR amplification,respectively.After input ldsDNAs being co-transfected into HEK293T cells for 48h,RNA was extracted then cDNA was synthesized.PCR was employed to amplify the library nanobody sequences.High-throughput sequencing(HTS)was employed to analyze the library nanobody sequences.Results We combined the clean merged paired-end reads from three biological repeats and got 4,173,356 reads.About 88.18%of the merged reads contain both upstream-and downstream-ldsDNA sequences.The most abundant read length is 264-bp,which corresponds to the intact sequence length.A total of 22,172 unique nanobody sequences were identified by high-throughput sequencing.Moreover,the library CDR sequences followed the NNK degeneracy.Conclusion We developed a novel ldsDNA-based AND gate genetic circuit to construct nanobody library in mammalian cells.