中文摘要：

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TCRVα32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＆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CD8T细胞TCRVα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CD8T细胞中扩增到了所有TCRVα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。TCRVα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TCRVα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。

英文摘要：

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.