A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells

Hiroshi Hamana, Kiyomi Shitaoka, Hiroyuki Kishi*, Tatsuhiko Ozawa, Atsushi Muraguchi

*この論文の責任著者

研究成果: ジャーナルへの寄稿学術論文査読

32 被引用数 (Scopus)

抄録

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 TCRα and TCRβ 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 TCRα and β 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.

本文言語英語
ページ(範囲)709-714
ページ数6
ジャーナルBiochemical and Biophysical Research Communications
474
4
DOI
出版ステータス出版済み - 2016/06/10

ASJC Scopus 主題領域

  • 生物理学
  • 生化学
  • 分子生物学
  • 細胞生物学

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