MICA基因第2～4外显子大规模双向测序及分子克隆检测平台的建立

摘要

Objective To establish a stable and large-scale bi-directional sequencing platform for genotyping MICA gene exons 2 to 4,and to analyze single nucleotide polymorphisms(SNP) of the region.Methods Primers for particular alleles of MICA gene exons 2 to 5 were designed.Optimal conditions for PCR amplification and sequencing reaction were explored.A commercialized one-way sequencing kit for MICA allele was used as a parallel control.Four samples carrying a MICA * 010 allele were subjected to cloning and haplotype sequencing.Results Results of MICA allele typing of 100 samples for a parallel control group were confirmed by the establish method.Twenty-two SNP in MICA gene exons 2 to 4 were detected in Chinese population.Two novel allelic sequences were accepted by GenBank and IMGT/HLA database and officially named as MICA * 065 and MICA * 066 by the WHO Nomenclature Committee.A novel SNP in MICA gene intron 3 was discovered,with allelic sequence submitted to GenBank and IMGT/HLA database.Conclusion The bi-directional sequencing genotyping platform may be applied for largescale study of MICA allelic polymorphisms,tissue typing,organ transplantation and disease research.%目的 建立稳定的、大规模的MICA基因第2～4外显子双向测序分型检测技术平台,并分析其单核苷酸多态性(single nucleotide polymorphism,SNP).方法 自行设计MICA基因第2～5外显子扩增引物及测序引物,探索PCR扩增及测序的最佳反应条件.用商品化的MICA基因单向测序分型试剂盒作为平行对照,对4个包含MICA* 010等位基因的样本采用自行设计引物扩增,并进行分子克隆和单倍体测序.结果 采用自行建立的MICA基因双向测序分型方法验证了100人份平行对照组单向测序分型结果.应用本研究建立的方法获得了中国人群MICA基因第2～4外显子22个SNP位点.两个新等位基因MICA* 065、MICA* 066获得了世界卫生组织的正式命名.首次发现了MICA等位基因第3内含子新的SNP位点,序列已提交IMGT/H LA数据库.结论 建立的MICA基因双向测序方法可大规模应用于中国人群的MICA基因多态性、组织器官移植配型和疾病研究.