一种高效扩增人T细胞受体α链可变区基因方法的建立

A high-performance method for amplifying human T-cell receptor Vα-encoding genes

目的建立一种高效扩增人T细胞受体(TCR)α链可变区(Vα)基因的方法。方法根据TCR Vα32个亚家族的基因序列特点,设计扩增Vα基因的上游内、外引物各42条,并将上游内、外引物各分为5组简并引物。在恒定区(C区)设计下游内、外引物,测序引物以及扩增Cα基因的上游引物各1条。提取细胞RNA,用PolyA介导反转录后,采用巢式PCR扩增正常人外周血单个核细胞(PBMC)中CD8 T细胞TCR Vα的32个亚家族基因,以Jurkat T淋巴瘤细胞作为对照。用T-easy载体克隆RT-PCR产物,对克隆基因进行测序分析。结果从正常人CD8 T细胞中扩增到了所有TCR Vα亚家族基因,以10个Jurkat细胞所提取的RNA作为模板即可获得成功扩增。TCR Vα同一亚家族基因的同源性均大于75%,序列差异主要在CDR3高变区。结论建立了一种高效灵敏的TCR Vα编码基因扩增方法,为抗原特异性细胞毒T淋巴细胞(CTL)的TCR克隆和功能研究打下了良好基础。

Objective To develop an effective method for amplifying human Tcell receptor （TCR） variable region of a chain （Vα）- encoding genes. Methods Based on the property of 32 subfamilies of human TCR Vα-encoding gene sequence, 42 sets of outer and inner sense primers which were divided into 5 degenerate primer groups, and a set of outer and inner antisense primers located in conserved region chain （Cα） were designed for the amplification of the TCR Vα-encoding genes. In addition, a sequencing primer and a sense primer for amplifying Cα-encoding gene were also designed. CD8 Tcell RNA was extracted and subjected to reverse transcription mediated by poly A, followed by a nested polymerase chain reaction （PCR） for the amplification of 32 subfamilies of human TCR V^-encoding genes. Jurkat T lymphoma cells were used as control. Target genes were cloned into T-easy vector and sequenced. Results All subfamilies of the Vα-encoding gene were obtained from CD8 T cells of a healthy donor, while the Val was the only subfamily obtained from Jurkat cells as anticipa- ted, and it could be amplified successfully from RNA extracted from only 10 cells. The results were confirmed by DNA sequencing. The homology of one TCR subfamily was beyond 75%, while CD3 high variable region showed great sequence differences. Conclusion The nested RTPCR assay developed in present study is capable of amplifying human TCR Vc^encoding genes with high effectiveness with broad spectrum, which will be helpful for cloning and functional study of the TCR expressed by antigen-specific cytotoxic T lymphocytes.