高通量 T 细胞受体克隆和表达策略。
A High-Throughput Strategy for T-Cell Receptor Cloning and Expression.
机构信息
Institute of Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA.
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
出版信息
Methods Mol Biol. 2022;2574:251-264. doi: 10.1007/978-1-0716-2712-9_12.
Expression of T-cell receptor (TCR) genes is a critical step for TCR characterization and epitope identification. The recent interest in using specific TCRs for cancer immunotherapy has further increased the demand for practical and robust methods to rapidly clone and express TCRs. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TCR transgene into different expression systems. In this protocol, we first constructed all the human TRAV and TRBV genes into individual plasmid. To clone any TCR, we only need to ligate a short CDR3 fragment to its corresponding V gene plasmid using Golden Gate cloning. This strategy significantly improves the efficiency of individual TCR cloning and mutagenesis, providing a flexible high-throughput method for TCR analysis and TCR-mediated therapeutics.
T 细胞受体 (TCR) 基因的表达是 TCR 鉴定和表位识别的关键步骤。最近，人们对使用特定 TCR 进行癌症免疫治疗的兴趣日益浓厚，这进一步增加了对实用且强大的方法的需求，以便快速克隆和表达 TCR。我们展示了一种基于重组的克隆方案，该方案促进了 TCR 转基因快速简单地转移到不同的表达系统中。在本方案中，我们首先将所有人类 TRAV 和 TRBV 基因构建到单个质粒中。要克隆任何 TCR，我们只需要使用 Golden Gate 克隆将短的 CDR3 片段连接到其对应的 V 基因质粒上。这种策略显著提高了单个 TCR 克隆和突变的效率，为 TCR 分析和 TCR 介导的治疗提供了一种灵活的高通量方法。
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