基于克隆测序技术的人类白细胞抗原基因高分辨率分型方法的建立  

Establishment of Cloning and Sequencing Method for High-Resolution Human Leukocyte Antigen-B Genotype Assay

作　　者：邢晓清 初亚男[2] 项铮 宋沁馨[1,2] 周国华[1,2] 

机构地区：[1]药物质量与安全预警教育部重点实验室、中国药科大学,南京210009 [2]南京军区南京总医院药理科,南京210002

出　　处：《分析化学》2014年第11期1574-1579,共6页Chinese Journal of Analytical Chemistry

基　　金：江苏省科技支撑计划社会发展项目(No.BE2012744);中国博士后科学基金面上项目(No.2012M512179);药物质量与安全预警教育部重点实验室资助项目(No.MKLDP2013MS01);南京军区南京总医院院内课题(No.2013053)资助

摘　　要：运用优化的扩增和克隆测序技术,建立了人类白细胞抗原(HLA-B)基因的高分辨率分型方法。针对HLA-B基因保守区序列设计引物进行等位基因扩增,基于质粒不相容原理将杂合型等位基因有效克隆入质粒DNA中,经细菌培养后进行Sanger测序,根据测序结果经ClustalX2软件分析和IMTG/HLA数据库的BLAST比对即可完成HLA-B基因的高分辨率分型。利用建立的方法对7例临床样本进行了HLA-B基因分型,并且与第三方直接碱基序列分析基因分型技术(PCR-SBT)进行比对,结果完全一致。本方法无需专业分型软件,准确度高,成本低;采用通用引物进行等位基因的扩增和测序,无需传统方法中繁琐的引物设计和过程优化,实现了HLA-B基因的高分辨率分型。A high-resolution method for human leukocyte antigen-B（HLA-B）genotyping was established based on the optimized polymerase chain reaction, cloning and sequencing technology. The exon2 and exon3 of HLA-B gene were amplified with primers based on the HLA-B gene sequence. These produced heterozygous alleles were effectively cloned into plasmid DNA based on the principle of plasmid incompatibility, and were followed by bacterial culture. Then Sanger sequencing was carried out and after analyzing the result by software ClustalX2 and IMTG/HLA database comparison, the HLA-B genotype of the samples was achieved. Seven clinical samples were detected, and the results were consistent with those of PCR-SBT genotyping method. The method was cost-effective, high-resolution and it did not require technical software. The use of universal primers simplified the cumbersome design and optimization process of specific primers.

关 键 词：人类白细胞抗原-B基因 克隆测序技术 Sanger测序 高分辨率基因分型 

分 类 号：Q78[生物学—分子生物学]