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.
Abstract
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.
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