抗体修饰DNA测序酶的开发及其应用

doi:10.13560/j.cnki.biotech.bull.1985.2023-1103

摘要/Abstract

摘要：

【目的】开发一种新型DNA测序酶，解决一代测序技术应对复杂DNA结构模板所面临的主要挑战，如测序信号中断或测序信号快速衰减。【方法】从NCBI数据库中挖掘到了Taq DNA聚合酶和单链结合蛋白SSB基因信息，利用遗传融合、定点突变及基因设计技术获得了一种新型的测序酶Sso-Sequenase。利用亲和层析和离子交换层析，获得了纯化的测序酶。利用抗体修饰技术改良了Sso-Sequenase的性能，并且利用STR技术对其热启动性能进行了表征。选取多组不同类型的复杂模板，采用一代技术对比分析了Sso-Sequenase测序酶试剂盒和传统BigDye测序试剂盒的测序表现。【结果】Sso-Sequenase在大肠杆菌中稳定表达，纯度达到95%以上，产量高达10.5 mg/L。当温度低于35℃时，Sso-Sequenase表现出热启动活性。在复杂模板的一代测序反应中，如重复序列、高GC和发夹结构等样本，Sso-Sequenase测序酶成功完成了所有样本的测序，序列的平均碱基质量QV大于20。相比而言，BigDye测序试剂盒在处理这些复杂样本时，多数样本测序信号出现了显著衰减或中断。【结论】开发了一种纯度好、产量高，兼具热启动活性的DNA测序酶Sso-Sequenase及测序试剂盒，显著提高了复杂DNA结构模板（重复序列、高GC和发夹结构）测序成功率。

关键词: DNA测序酶, 抗体修饰, 复杂模板, 测序

Abstract:

【Objective】To develop a novel DNA sequencing enzyme that addresses the main challenges faced by first-generation sequencing technology when dealing with complex DNA structure templates, such as interruption of sequencing signals or rapid signal decay. 【Method】Taq DNA polymerase and single-strand binding protein SSB gene sequences were mined from the NCBI database, and a novel sequencing enzyme, Sso-Sequenase, was obtained using genetic fusion, site-directed mutagenesis, and gene design techniques. The purified sequencing enzyme was obtained through affinity chromatography and ion exchange chromatography. The performance of Sso-Sequenase was improved using antibody modification technology, and its hot start performance was characterized using STR technology. A variety of complex templates were selected, and the sequencing performance of the Sso-Sequenase sequencing enzyme kit was compared with the traditional BigDye sequencing kit using Sanger sequencing. 【Result】Sso-Sequenase was stably expressed in Escherichia coli, with a purity of over 95% and a yield of up to 10.5 mg/L. When the temperature was below 35℃, Sso-Sequenase demonstrated hot-start activity. In first-generation sequencing reactions with complex templates, such as those with repetitive sequences, high GC content, and hairpin structures, Sso-Sequenase successfully completed sequencing for all samples, with an average base quality value QV > 20. In contrast, the BigDye sequencing kit experienced significant signal decay or interruption when processing these complex samples. 【Conclusion】A DNA sequencing enzyme, Sso-Sequenase, has been developed to possess exceptional purity, remarkable yield, and hot-start activity. These advancements have significantly bolstered the sequencing success rate for intricate DNA templates, including those characterized by repetitive sequences, high GC content, and hairpin structures.

Key words: DNA sequencing enzyme, antibody modification, complex templates, sequencing

宋辉, 曹文刚, 肖晓文, 杜军. 抗体修饰DNA测序酶的开发及其应用[J]. 生物技术通报, 2024, 40(5): 84-93.

SONG Hui, CAO Wen-gang, XIAO Xiao-wen, DU Jun. Development and Application of Antibody Modified DNA Sequencing Enzymes[J]. Biotechnology Bulletin, 2024, 40(5): 84-93.

图/表 6

图1 DNA测序酶Sso-Sequenase的基因结构模式图

Fig. 1 Schematic diagram of gene structure of DNA sequencing enzyme Sso-Sequenase

图2 Sso-Sequenase的蛋白纯化

Fig. 2 Purification of Sso-Sequenase protein

图3 Sso-Sequenase的STR检测

Fig. 3 STR detection of Sso-Sequenase

图4 DNA 测序酶Sso-Sequenase在重复序列模板上的表现 Raw：原始视图；Sequence：序列质量视图

Fig. 4 Performance of DNA sequencing enzyme Sso-Sequenase on repeat sequence templates

图5 DNA 测序酶Sso-Sequenase在高GC模板上的表现

Fig. 5 Performance of DNA sequencing enzyme Sso-Sequenase on high GC templates

图6 DNA 测序酶Sso-Sequenase在发夹结构模板上的表现

Fig. 6 Performance of DNA sequencing enzyme Sso-Sequenase on hairpin structure templates

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