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通过片段的一步酶促 DNA 组装实现高通量的计算衍生合成抗体的生成。

High-Throughput Generation of In Silico Derived Synthetic Antibodies via One-step Enzymatic DNA Assembly of Fragments.

机构信息

Department of Molecular Genetics, Charles Best Institute, University of Toronto, 112 College Street, 112 College Street, Room 70, Toronto, ON, M5G 1L6, Canada.

出版信息

Mol Biotechnol. 2020 Feb;62(2):142-150. doi: 10.1007/s12033-019-00232-z.

Abstract

Phage-display technology offers robust methods for isolating antibody (Ab) molecules with specificity for different target antigens. Recent advancements couple Ab selections with in silico strategies, such as predictive computational models or next-generation sequencing metadata analysis of Ab selections. These advancements result in enhanced Ab clonal diversities with potential for enlarged epitope coverage of the target antigen. A current limitation however, is that de novo Ab sequences must undergo DNA gene synthesis, and subsequent expression as Ab proteins for downstream validations. Due to the high costs and time for commercially generating large sets of DNA genes, we report a high-throughput platform for the synthesis of in silico derived Ab clones. As a proof of concept we demonstrate the simultaneous synthesis of 96 unique Abs with varied lengths and complementary determining region compositions. Each of the 96 Ab clones undergoes a one-step enzymatic assembly of distinct DNA fragments that combine into a circularized Fab expression plasmid. This strategy allows for the rapid and efficient synthesis of 96 DNA constructs in a 3 day window, and exhibits high percentage fidelity-greater than 93%. Accordingly, the synthesis of Ab DNA constructs as Fab expression plasmids allow for rapid execution of downstream Ab protein validations, with potential for implementation into high-throughput Ab protein characterization pipelines. Altogether, the platform presented here proves rapid and also cost-effective, which is important for labs with limited resources, since it utilizes standard laboratory equipment and molecular reagents.

摘要

噬菌体展示技术为分离针对不同靶抗原的特异性抗体 (Ab) 分子提供了强大的方法。最近的进展将 Ab 选择与计算策略相结合,例如预测计算模型或 Ab 选择的下一代测序元数据分析。这些进展导致 Ab 克隆多样性增强,有可能扩大靶抗原的表位覆盖范围。然而,目前的一个限制是,从头 Ab 序列必须经过 DNA 基因合成,然后作为 Ab 蛋白进行表达,以进行下游验证。由于商业生成大量 DNA 基因的成本高且耗时,我们报告了一种用于合成计算衍生 Ab 克隆的高通量平台。作为概念验证,我们展示了同时合成 96 种具有不同长度和互补决定区组成的独特 Ab。这 96 种 Ab 克隆中的每一种都经过独特 DNA 片段的一步酶组装,这些片段组合成一个环形 Fab 表达质粒。该策略允许在 3 天的窗口内快速高效地合成 96 个 DNA 构建体,并且具有高的保真度(大于 93%)。因此,Ab DNA 构建体作为 Fab 表达质粒的合成允许快速执行下游 Ab 蛋白验证,并且有可能将其纳入高通量 Ab 蛋白表征管道中。总之,这里提出的平台证明了快速且具有成本效益,这对于资源有限的实验室很重要,因为它利用了标准的实验室设备和分子试剂。

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