基因扫描技术检测人TCR δ2链CDR3多态性的方法

    Methodology for detection of CDR3 polymorphism of human TCRδ2 gene repertoire by gene scan technique

    • 摘要: 目的:建立利用基因扫描技术检测和分析人TCR δ2亚家族CDR3多态性的方法。方法:从健康成人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),或从PBMCs经IL-2或结核杆菌耐热抗原(Mtb-HAg)刺激培养7天的扩增细胞中提取RNA,用RT-PCR方法扩增TCRδ2链基因中CDR3片段,分别用聚丙烯酰胺凝胶电泳和基因扫描观察CDR3长度并分析其多态性。结果:聚丙烯酰胺凝胶电泳结果显示,从三组细胞扩增的RT-PCR产物均显示较宽、模糊的条带,而基因扫描结果显示三组RT-PCR产物均可出现清晰可辨的扫描峰,并发现三组中Mtb-HAg组优势峰CDR3片段,较PBMC组和IL-2组长(P<0.01),后两者之间差异无统计学意义(P>0.05)。结论:基因扫描分析技术能方便、快速检测和分析人外周血γδT细胞TCRδ2链基因CDR3片段长度的多态性。

       

      Abstract: Objective:To establish a method for detecting the CDR3 length polymorphism of TCR δ2 gene repertoire of γδ T cells in human peripheral blood by gene scan technique.Methods:otal RNA was extracted from fresh isolated peripheral blood mononuclear cells (PBMCs) or PBMCs stimulated and expanded with IL-2 cultivated alone (IL-2 group) or with Mtb-HAg plus IL-2 (Mtb-HAg group) cultivated for 7 days.The expression of CDR3 of δ2 gene in the three groups was detected by reverse transcriptase polymerase (RT-PCR),and the CDR3 length and polymorphism of TCR δ2 gene was analyzed by native polyacrylamide gel electrophoresis (native-PAGE) and gene scan technique.Results:The PCR product of the CDR3 of δ2 gene was showed wide and diffused bands by native-PAGE in the three groups,and there were no significant difference.While through gene scan analysis,all PCR products in the three groups were displayed more clear fragments (peaks),and the length of the predominant fragments (peaks) in Mtb-HAg group was significantly increased compared to PBMCs group and IL-2 group (P<0.01),but no difference between PBMCs group and IL2 group.Conclusions:The results suggested that gene scan technique is a convenient and rapid method for detection and analysis of the CDR3 length polymorphism of TCR δ2 gene of γδT cells in human peripheral blood.

       

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