多系统萎缩患者TCR α链CDR3谱系的荧光定量PCR溶解曲线

An Analysis on FQ-PCR Melting Curve of CDR3 Gene Spectratype on TCR alpha Chain of A Patient with Multiple System Atrophy

目的:利用荧光定量PCR溶解曲线分析技术检测1例多系统萎缩患者TCRα链CDR3谱系。方法:提取1例多系统萎缩患者和1例正常人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的总RNA,逆转录成cDNA,以32个人TCRα链胚系可变区基因家族(TRAV)设计上游引物,共同的TCRα链恒定区基因家族(TRAC)设计下游引物,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,挑选单克隆进行PCR扩增,扩增产物行普通琼脂糖凝胶回收后测序分析。结果:多系统萎缩患者和正常人PBMC的32个TCRα链TRAV家族CDR3谱型的FQ-PCR产物的各家族相对定量表达不一;正常人的32个TCRα链TRAV家族CDR3谱型的溶解曲线图表现为多个峰型的高斯分布,多系统萎缩患者的TRAV10、TRAV15、TRAV29家族CDR3谱型的溶解曲线表现为单峰的克隆性增生,其他家族为多峰、寡峰的多克隆及寡克隆增生,未出现缺失的家族,对单峰的TRAV15家族的测序得到的CDR3区的氨基酸组成为:SAIYFCAEDRDSTLTFG。结论:该多系统萎缩患者的PMBC中,存在TCRα链CDR3谱系漂移。

Objective： To analyze CDR3 spectratype of TCR alpha gene in peripheral blood monouclear cells （PBMC） of one patient with multiple system atrophy （MSA） by using DNA melting curve technique with real-time fluorescence quantitative polymerase chain reaction. Methods： Total RNA from PBMC of a healthy donor and a MSA patient were transcripted reversely into cDNA respectively. The forward primers were designed according to the 32 human TRAV gene families, and the reverse primers designed according to TRAC. FQ-PCR was carried out to amplify CDR3 spectratyping of the 32 human TRAV gene families （HTGF） , and the monoclonal/oligoclonal/polyclonal CDR3 were analyzed with DNA melting curves. Monoclonal T cells of TRAV families were amplified and sequenced. Resuits： The FQ-PCR products of HTGF of the MSA patient were different from those of normal people. The 32 HTGF melting curves of normal people showed Gaussian distribution with several peaks, while those of the MSA patient showed mono-peak Cloning increase in TRAV10, TRAV15, and TRAV29 gene families, and multipeak/oligpeak-multclone/oligclone increase in other gene families. No deletion family was found. The CDR3 amino acid sequence of TRAV15 which showed monopeak was inferred as SAIYFCAEDRDSTLTFG according to the DNA sequence obtained. Conclusion： There might＇ be spectratyping drift in CDR3 on TCR alpha chain in PBMC of MSA patient.