A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells
A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells
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DOI：
10.1016/j.bbrc.2016.05.015
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发表时间：
2016-06-10
影响因子：
3.1
通讯作者：
Muraguchi, Atsushi
中科院分区：
文献类型：
--
作者：
Hamana, Hiroshi;Shitaoka, Kiyomi;Muraguchi, Atsushi
t细胞受体（TCR）基因治疗是治疗感染性疾病和癌症的一种很有前途的方法。然而，配对克隆和抗原特异性TCR α和TCR β的功能分析是费时费力的。在本研究中，我们结合多重一步RT-PCR、转录活性PCR （TAP）和荧光素酶报告基因检测三种技术，建立了一种新颖、快速、高效的抗原特异性tcr克隆系统。从TCR的先导肽序列设计引物的多重一步RT-PCR使我们能够以非常高的效率从单个t细胞中扩增TCR α和β对的cdna。TAP片段和基于hek293的nfat -荧光素酶报告细胞的结合使得无需构建表达载体即可进行快速功能检测。利用该系统，我们在4天内克隆了Epstein-Barr病毒brlf -1衍生肽特异性的人TCRs和黑色素瘤相关抗原酪氨酸酶相关蛋白2 （TRP-2）特异性的小鼠TCRs。这些结果表明，我们的系统提供了快速和有效的克隆功能性抗原特异性的人类和小鼠tcr，并有助于基于tcr的癌症和传染病的免疫治疗。(C) 2016年由Elsevier Inc.出版。
T-cell receptor (TCR) gene therapy is a promising approach for the treatment of infectious diseases and cancers. However, the paired cloning and functional assays of antigen-specific TCR alpha and TCR beta is time-consuming and laborious. In this study, we developed a novel, rapid and efficient antigen-specific TCR-cloning system by combining three technologies: multiplex one-step RT-PCR, transcriptionally active PCR (TAP) and luciferase reporter assays. Multiplex one-step RT-PCR with leader primers designed from leader peptide sequences of TCRs enabled us to amplify cDNAs of TCR alpha and beta pairs from single T-cells with remarkably high efficiency. The combination of TAP fragments and HEK293T-based NFAT-luciferase reporter cells allowed for a rapid functional assay without the need to construct expression vectors. Using this system, we cloned human TCRs specific for Epstein-Barr virus BRLF-1-derived peptide as well as mouse TCRs specific for melanoma-associated antigen tyrosinase-related protein 2 (TRP-2) within four days. These results suggest that our system provides rapid and efficient cloning of functional antigen-specific human and mouse TCRs and contributes to TCR-based immunotherapy for cancers and infectious diseases. (C) 2016 Published by Elsevier Inc.