乳腺癌T细胞受体α链全长编码序列多重PCR扩增方法的建立

Establishment of Multi-PCR to amplify the complete DNA sequence of TCR α chains in patients with breast cancer

目的:建立乳腺癌克隆性T细胞TCRα链全长编码序列(CDS)的多重PCR扩增方法。方法:根据Gen Bank现有TCR序列设计扩增TCRα链32个亚家族上游引物35条,下游引物2条。对Mg2+、d NTPs和引物浓度比例以及退火温度进行优化,确定最佳反应体系进行TCRα链多重PCR扩增,构建重组质粒并酶切鉴定。结果:扩增出乳腺癌转移淋巴结中的TCRα链全长编码序列并构建重组质粒,5例患者共获得23个阳性克隆。结论:建立的TCRα链多重PCR扩增方法具有特异、快速、简便的特点,可为研究乳腺癌克隆性T细胞提供帮助。

Objective： To establish a method of Multi-PCR to amplify the complete DNA sequence（ CDS） of TCR α chains of clonal T cells in patients with breast cancer. Methods： The specific 35 upstream primers and 2 downstream primers for 32 subfamilies were designed according to the sequences of TCR α chains provided by Gene Bank. The multiplex PCR reaction system and condition was optimized to find out the best concentration of Mg2 ＋,d NTPs and primers,and the best annealing temperature. The CDS of TCR α chains were amplified and then inserted into cloning vector. The recombinant constructs were identified by the endonucleases. Results： The CDS of TCR α chains were amplified,and constructed the recombinant plasmid. There were 23 positive clones were received in 5 patients with breast cancer. Conclusion： Multi-PCR established in the study is specific,rapid,simple and convenient. It provides the technical support to analyse the Feature of clonal T cell in patients with breast cancer.