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

作　　者：张凯丽 王怡 李怡凡 赵雁杰 李帅 Zhang Kaili;Wang Yi;Li Yifan;Zhao Yanjie;Li Shuai(Department of Breast Cancer Pathology and Research Laboratory,Tianjin Medical University Cancer Institute and Hospital,National Clinical Research Center for Cancer,Key Laboratory of Cancer Prevention and Therapy,Tianjin,Tianjin’s Clinical Research Center for Cancer,Key Laboratory of Breast Cancer Prevention and Therapy,Ministry of Education,Tianjin 300060,China)

机构地区：[1]天津医科大学肿瘤医院乳腺病理研究室、国家恶性肿瘤临床医学研究中心、天津市"肿瘤防治"重点实验室、天津市恶性肿瘤临床医学研究中心、乳腺癌防治教育部重点实验室,天津300060

出　　处：《中华生物医学工程杂志》2023年第5期488-494,共7页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(31870860);天津市医学重点学科(专科)建设项目(TJYXZDXK-012A)。

摘　　要：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.

关 键 词：纳米抗体 互补决定区 与门 线性双链DNA 

分 类 号：R392[医药卫生—免疫学]