基因扫描分析小鼠T细胞受体CDR3谱型检测T细胞克隆技术的建立

Established genescan technique of diversity gene of T cell antigen receptor complementary-determining region 3 for assaying clonality of mouse TCR α β T cell

目的建立小鼠T细胞受体(TCR)互补决定区3(CDR3)谱型的基因扫描分析术,为分析T细胞克隆提供研究方法。方法应用RT-PCR方法分别扩增近交系小鼠C57BL/6H-2b,Balb/CH-2d和F1代小鼠(Balb/c×C57BL/6F1H-2d/b,CB6F1)脾脏T细胞的21个TCRVα家族和18个TCRVβ家族的CDR3,并用基因扫描分析扩增产物以确定T细胞的克隆性。结果3种小鼠脾脏T细胞均表达所有的TCRVα和VβCDR3基因,基因扫描显示全部TCRCDR3谱型呈高斯(钟型)分布,提示均为多克隆T细胞。各家族CDR3基因表达频率接近,但呈现不同的多态性和长度分布。结论基因扫描分析TCRαβCDR3基因谱型是检测小鼠T细胞克隆性的精确敏感方法。

[Objective] To establish genescan technique of diversity gene of T cell antigen reeeptor（TCR） complementary-determining region 3（CDR3） for assaying elonality of mouse TCR αβ T cell. [Methods] 21 TCR et chain variable gene（TCR Vet） and 18 TCR β chain variable gene （TCR Vβ） of spleen T cell of C57BL/6^H-2b, Balb/ C^H-24 and CB6F1 （Balb/exC57BL/6 F1^H-2d/b） inbreeding line mouse were amplified by RT-PCR. Productions of RT- PCR were assayed by genescan technique to determine clonality of TCR αβ T cell. [Results] All the TCR Vet and TCR Vα genes were expressed in spleen T cell of 3 kinds inbreeding line mouse. All the CDR3 repertoire showed Gaussian distribution, which suggested that T cell were multielonal. The CDR3 of the TCR Vα and Vβ subfamily has the similar frequency of occurrence, but has the different pelymorphism and length profiles. [Conclusion] Genesean technique of diversity gene of TCR CDR3 was stable and precise method for assaying elonality of mouse TCR αβ T cell.