识别猪SLA的特异性人T细胞系TCR BV基因取用格局和CDR3结构多样性分析

不同T细胞克隆TCR分子的序列不同 ,所识别的抗原特异性也不同。其中第三互补决定区 (CDR3)变异最大 ,是TCR主要的抗原结合部位。本文采用荧光标记半定量PCR技术 ,用DNA测序仪作程序分析 ,了解猪细胞抗原致敏前后的人T细胞群和 5个T细胞系 2 4个TCRBV基因家族取用格局 ,并以TCRα链C区的基因片断作为内参对取用情况作定量估计。发现首次抗原致敏后培养 2周的T细胞除了BV2 4、BV8和BV10未能检测出 ,其它BV基因都有不同程度的取用。然而 ,5个细胞系的TCRBV基因呈现十分有限的取用格局 ,其中两个CD4+ T细胞系都取用BV12和BV14;3个CD8+ T细胞系中都优势取用BV1,有两个还取用BV19。CD4+ T细胞系和CD8+ T细胞系之间TCRBV无交叉取用 ,提示两类细胞识别的抗原表位存在差异。进一步用变性凝胶扫描分析上述T细胞系取用TCRBV中的CDR3的多样性 ,发现未经抗原致敏的T细胞BV的CDR3结构为多峰型且呈正态分布 ,表明涉及多种结构不同的细胞克隆 ;而抗原特异性T细胞系CDR3除了一个CD8+ T细胞系BV1有两个主峰外其它无例外地都显示单峰或者仅一个主峰 ,这从另一个角度证明建系T细胞的单克隆性。

The T Cell Receptor BV Gene Usage Patterns and the Clonal Characterisation of CDR3 in the Xenogeneic Human Anti-swine T Cell Lines

The TCR BV gene repertoire in the human T cell lines specific for swine SLA were analyzed by semi quantitative PCR and Genescan protocol with DNA sequencer The highly restricted TCR Vβ usage was observed from T cell lines in the human anti porcine response system,suggesting that a limited number of xenogeneic epitopes were recognized by human T clones Before priming,the member from all 24 Vβ gene families except BV24,could be identified to be used After multiple repeatedly stimulation with porcine PBMC,however,the usage pattern of Vβ gene segments in the T cell lines changed dramatically Two CD4 + T cell lines shared BV12 and BV14,one of CD4 + T cell lines preferred to express BV12 (usage frequency 79 9%) Moreover,there were common usage of BV1 in three of the CD8 + T cell lines,with a highest usage frequency 52 7 percent in the cell line G TCR Vβ usage patterns within CD8 + T cell lines seemed different from those expressed by CD4 + T cells,a fact consistent with the observation that CD8 + T cells and CD4 + T cells responded to SLA in different manner,probably by a differential recognition of MHC/peptide complexes By scanning with denatured gel,the clonalities characterized for the complementarity determining region 3 (CDR3) of pattern,the used TCR gene segments were further determined Mono or oligoclonal predominance of CDR3 always appeared for all examined T cell lines