检测人TCR BD基因重排时RSS断裂末端的LM-PCR方法的建立

Established the ligation mediated PCR for detecting the ends of recombinant signal sequences of human TCRβ chain diversity gene

目的:建立检测人T细胞TCRBD基因(Diversity Gene)经历重排的连接介导PCR方法(Ligation Mediate dPCR,LMPCR),为T细胞重排和疾病的关系研究提供基础。方法:在人TCRβ链BD1与BD25′端重组信号序列(RSS)前,BD1与BD23′端RSS后各设计两套用于巢式PCR引物;设计并合成和双链RSS平断裂末端相接的特殊接头(BWLinker),提取胸腺组织、正常人和急性T淋巴细胞白血病(TALL)外周血单个核细胞(PBMC)样本总DNA,和BWlinker连接,进行巢式PCR,PCR产物琼脂糖电泳分析,阳性产物做胶回收,并克隆测序鉴定。结果:在1例胸腺组织提取的总DNA中证实存在BD1和BD25′和3′的RSS断裂末端,在2例TALLs的PBMC中检测到BD25′和3′的RSS断裂末端,2例正常人PBMC中未能检测到RSS断裂末端,阳性的LMPCR产物通过测序鉴定,和基因组中序列完全相符合。结论:1例胸腺组织和2例TALL的PBMC中检测到RSS断裂末端,提示TCRBD基因正经历重排,测序结果表明建立的监测TCR重排的LMPCR方法可靠,可用于T细胞重排模型和相关疾病的机制研究。

Objective：To established a special ligation-mediated PCR for analyzing the breaks end of recombination signal sequence of human TCR BD gene. Methods： Designing four primers in front of nanomer-spacer（ 12 ）-heptamer（ RSS 9-12-7 ） and four primers behind of heptamer-spacer（23）-nanomer（ RSS 7-23-9） from human TCR diversity gene and synthesizing A partially doublestranded oligonucleotide linker（BW-hnker）. Isolating total DNA from thymus ,and from the PBMC samples of T-hneage acute lymphoblastic leukemia（T-ALLs） and healthy controls ,linking BW-linker to the end of DNA RSS and identified the signal ends by PCR and sequencing analysis. Results：In the thymus（ one samples） could find RSS breaks end of TCR BD1 and TCR BD2, and in PBMC of two T-ALL patients could find RSS breaks end of TCR BD2, but in two controls, it is negative. The products of LM-PCR has exactly nucleotide sequence corresponding to the human TCR BD gene. Conclusion：The positive LM-PCR results demonstrated ongoing TCR gene recombination in thymus and T-ALL patients PBMC ,the LM-PCR represents a simple and reliable tool for detecting human TCRβ chain gene rearrangement.