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

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.