【摘要】 目的:建立多重PCR方法扩增α/βTCR全长序列,分析活动性肺结核患者病变局部T淋巴细胞α/βTCR基因重排特点及CDR3谱型。方法:分离结核患者肺泡灌洗液中的淋巴细胞,提取RNA后用SMART方法逆转录,运用根据现有文献设计的α和βTCR扩增引物,进行多重PCR扩增以获得其全长序列,构建重组质粒并测序,利用DNAstar及网上TCR资源分析序列。结果:3例患者共获得24个α链序列,13个β链序列。α链以AV1S2(54%)、AV12S3(41%)、AV12S2(5%)为主;β链以BV2(38%)、BV29S1(46%)、BV14(3%)、BV4S2(3%)为主。同一病例及不同病例之间CDR3区呈现多样性,但是标本1、标本3各有一条β链的CDR3氨基酸序列相同:SVGTGTLHQETQY;标本2和标本3的各有2条α链CDR3序列相同:AVRDWAGNMLT;标本2的一个α链和标本3的一个α链的CDR3均有:AV…DNN…RLM序列。结论:建立了TCRα和β链全长序列的多重PCR扩增方法。结核患者病变局部克隆性增殖的TCRα和β链谱型呈限制性取用,克隆增生的T淋巴细胞来自不同的亚群,但是相同的CDR3序列对于识别MTB多肽可能具有特异性。更多还原

【Abstract】 Objective:To establish a method of multi-PCR to amplify the complete DNA sequence (CDS) of TCR α and β chain of the antigen-specific T lymphocytes in local pathologic specimen of active pulmonary tuberculosis patients, and to analyze α/β T cell receptor gene rearrangement and CDR3 repertoire.Methods:The lymphocytes in bronchoalveolar lavage (BAL) of active pulmonary tuberculosis patients were separated. Following total RNA extraction, cDNA synthesis, Multi-PCR, recombinant clones construction, and sequencing, the CDS of TCR α and β chains from these lymphocytes were analyzed by using software of DNAstar and internet TCR resources.Results:24 of α chain CDS and 13 of β chain CDS from 3 samples of BAL were obtained. As for TCR α chain, AV1S2 (54%), AV12S3 (41%), and AV12S2(5%) appeared frequently. BV2(38%), BV29S1(46%), BV14(3%), and BV4S2(3%) in TCR β chain appeared more often. There were CDR3 diversities between samples and even in the same sample by amino acid sequence analysis, but there were a few identical or similar amino acid sequences. There was the same amino acid sequence of SVGTGTLHQETQY in CDR3 region of β chain of BAL sample No.1 and No.2; The sequence of AVRDWAGNMLT appeared in two α chains of BAL sample No.2 and No.3; Moreover, the sequence of AV…DNN…RLM appeared in α chains of BAL sample No.2 and No.3.Conclusion:A method of Multi-PCR is used to amplify TCR α and β chain CDS of tuberculosis patients. There are characteristic T cell clones to proliferate,with TCR α and β chain repertiore skewing in local infective focus. The sequences of CDR3 in different TCR clones are mostly different but there are a few identical or similar sequences in the same patient or even between different patients. The identical amino acid sequences of CDR3 are possibly specific for recognizing MTB polypeptide.更多还原