“荧光定量PCR溶解曲线法”监测HIV感染者TCR α链CDR3谱系漂移的研究

Analysis of variations in the CDR3 region of the TCR α chain in individuals with HIV using real-time fluorescence quantitative RT-PCR with DNA melting curve analysis

目的利用"荧光定量PCR溶解曲线分析技术"监测HIV感染者外周血T细胞TCRα链CDR3谱系漂移(单/寡/多克隆增生)。方法提取6例HIV感染者、4例健康者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的总RNA,逆转录成cDNA,设计人32个TRAV基因家族上、下游引物,荧光定量PCR(FQ-PCR)扩增32个TRAV基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的单/寡/多克隆增生。结果健康者外周血T细胞TCRα链32个家族CDR3表达频率不一致,各家族PCR产物的"溶解曲线谱型图"显示多克隆增生的高斯分布,呈现溶点不同的CDR3多态性,6例HIV感染者的外周血TCRα链CDR3谱系的32个家族CDR3表达频率不一致,患者各家族PCR产物的"溶解曲线谱型图"显示多数家族为多克隆增生的高斯分布,但每个患者均出现数量不等的单克隆和寡克隆增生家族。结论荧光定量PCR溶解曲线法监测人TCRα链CDR3谱系漂移的方法稳定、简便,可以用来监测健康者和HIV感染者外周血T细胞TCRα链CDR3谱系漂移。

Objective To analyze variations in the CDR3 region of the TCR α chain in the peripheral blood of individuals with HIV using real-time fluorescence quantitative RT-PCR （FQ RT-PCR） with DNA melting curve analysis.MethodsThe total RNA of peripheral blood mononuclear cells （PBMCs） from 4 healthy donors and individuals with HIV were reverse-transcribed into cDNA.CDR3 cDNA of 32 TRAV gene families was amplified by FQ RT-PCR,and monoclonal,oligoclonal,and polyclonal CDR3 fragments were used in DNA melting curve analysis.Results CDR3 expression by the 32 TRAV families differed for healthy donors and patients.CDR3 spectratyping of 26 TRAV families produced a polyclonal peak （Gaussian distribution） for healthy donors but produced different monoclonal/oligoclonal/polyclonal peaks for individuals with HIV according to DNA melting curve analysis.Conclusion FQ RT-PCR with DNA melting curve analysis can be used to analyze variations in the CDR3 region of the TCR α chain in individuals with HIV.