荧光定量PCR溶解曲线监测HIV感染者TCRα、β链CDR3谱系漂移技术的建立  

Detection of CDR3 shewing of TCR α,β gene repertoire in HIV infected individuals by using real-time fluorescence quantitative reverse transcription polymerase chain reaction with DNA melting curve analysis

作　　者：李志峰[1,2] 宋方洲[2] 冯连贵[1] 周全华[1] 凌华[1] 张敏[1] 

机构地区：[1]重庆市疾病预防控制中心,400042 [2]重庆医科大学生物化学与分子生物学教研室,400016

出　　处：《国际检验医学杂志》2011年第14期1535-1537,共3页International Journal of Laboratory Medicine

基　　金：卫生部2007年艾滋病防治应用性研究项目(WA-2007-03)

摘　　要：目的建立荧光定量PCR(FQ-PCR)溶解曲线分析技术监测人类免疫缺陷病毒(HIV)感染者外周血T细胞受体(TCR)α、β链CDR3谱系漂移。方法提取HIV感染者外周血单个核细胞中的总RNA,逆转录成cDNA,设计人32个TCRAV、26个TCRBV基因家族上、下游引物,FQ-PCR扩增HIV感染者TCRAV和TCRBV基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的寡克隆增生,并与传统的CDR3检测技术基因扫描检测结果比较分析。结果 FQ-PCR溶解曲线分析技术与基因扫描检测结果一致,HIV感染者TCRα链和β链家族CDR3表达频率不一致,多数家族为多克隆增生的高斯分布,但出现数量不等的寡克隆增生家族。结论 FQ-PCR溶解曲线法监测HIV感染者TCRα、β链CDR3谱系漂移技术操作简单,检测成本低,可以用来监测HIV感染者外周血T细胞TCRα、β链CDR3谱系漂移。Objective To detect the CDR3 shewing of TCR α,β gene repertoire in peripheral blood of HIV infected individuals by using real-time fluorescence quantitative reverse transcription polymerase chain reaction（FQ-PCR） with DNA melting curve analysis. Methods The total RNA of peripheral blood mononuclear cell （PBMC） from HIV infected individuals were transcripted reversely into cDNA. The cDNA of 32 TCRAV and 26 TCRBV gene family CDR3 were amplified by FQ-PCR. Polyclonal CDR3 spectratyping was analyzed with DNA melting curve and compared with the results of Genescan method. Results The two methods showed the same results that the TCRAV and TCRBV family CDR3 expressed with different frequencies and the most CDR3 spectratyping of TCRα,β families showed polyclonal peak （Gaussian distribution） ,but some showed different polyelonal peaks in HIV infected individuals. Conclusion The technique of FQ-PCR with DNA melting curve analysis could be used for detecting the CDR3 shewing of TCRα,βgene repertoire in HIV infected individuals.

关 键 词：互补决定区 聚合酶链反应 溶解曲线 

分 类 号：R512.91[医药卫生—内科学]