多重PCR扩增乳腺癌克隆性T细胞TCRβ链全长序列

Establish a method of Multi-PCR to amplify complete DNA sequence of clonal TCRβ chains in patients with breast cancer

目的建立乳腺癌克隆性T细胞TCRβ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TCRβ链24个亚家族上游引物24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TCRβ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TCRβ链全长编码序列并成功构建重组质粒,5例患者共获得24个阳性克隆。结论建立的TCRβ链多重PCR扩增方法具有特异、快速、简便的特点,为研究乳腺癌克隆性T细胞的特点提供了帮助。

Objective To establish a method of Multi - PCR to amplify the comPlete DNA sequence （CDS） of TCRβ chains of clonal T cell in patients with breast cancer. Methods The specific 24 upstream primers and 2 downstream primers for 24 subfamilies were designed according to the sequences of TCRβ chains provided by GeneBank. The multi- plex PCR reaction system and condition was optimized to find out the best concentration of Mg^2＋~ , dNTPs, and primers, and the best annealing temperature. The CDS of TCRβchains were amplified and then inserted into cloning vector. The recombinant constructs were identified by the endonucleases. Results The CDS of TCRβchains were amplified, and constructed the recombinant plasmid. There were 24 positive clones were received in 5 patients with breast cancer. Conclusion Multiplex 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.