人类白细胞抗原E基因全长序列测定及两个新等位基因的鉴定  

Identification of genomic full-length sequence of human leukocyte antigen-E and its two novel alleles

作　　者：何柳媚[1] 王宋兴[1] 徐筠娉[1] He Liu-mei;Wang Song-xing;Xu Yun-ping(Shenzhen Key Laboratory of Tissue Typing and Immunogenetics, Shenzhen Blood Center, Shenzhen 518035, Guangdong Province, China)

机构地区：[1]深圳市血液中心深圳市组织配型与免疫遗传重点实验室,广东省深圳市518035

出　　处：《中国组织工程研究》2017年第25期4071-4077,共7页Chinese Journal of Tissue Engineering Research

基　　金：广东省医学科研基金(A2016222)资助;深圳市科技研发资金(GJHZ20160229170608241)~~

摘　　要：背景:人类白细胞抗原E是非经典的人类白细胞抗原I类分子,目前的多态性分析研究主要针对其第3外显子人类白细胞抗原E~*01:01及人类白细胞抗原E*01:03进行区分,然而其全基因序列测定方法及新等位基因报道尚不多见。目的:利用中国深圳地区无偿献血正常个体建立人类白细胞抗原E基因全长序列测序分型方法,鉴定人类白细胞抗原E基因全长序列新的等位基因。方法:提取研究对象外周血DNA,根据IMGT/HLA数据库中公布的人类白细胞抗原E基因序列,在保守区设计全基因扩增及测序引物,利用高保真反应体系对人类白细胞抗原E全长序列进行扩增,随后测序、拼接、序列确认及基因定型。结果与结论:(1)成功建立了人类白细胞抗原E的全基因扩增及测序分型方法,发现了两个人类白细胞抗原E新等位基因——人类白细胞抗原E~*01:01:01:06与人类白细胞抗原E~*01:01:01:07,获得了WHO人类白细胞抗原命名因子委员会的命名;(2)人类白细胞抗原E~*01:01:01:06与其最接近的等位基因人类白细胞抗原E~*01:01:01:01相比,突变位点在5’-启动子区的NT-26(G->T),人类白细胞抗原E~*01:01:01:07与其最接近的等位基因人类白细胞抗原E~*01:01:01:01相比,突变位点在3’-非翻译区的NT3345位(T->C);(3)结果提示,中国人群中人类白细胞抗原E全基因序列多态性数据有待填补,试验建立的方法为该项研究提供了关键的技术。BACKGROUND:Human leukocyte antigen-E(HLA-E)is one of non-classical HLA class I genes.Up to now,the polymorphism analysis is mainly aimed at the variation in exon3of HLA-E,which determines HLA-E*01:01or HLA-E*01:03.However,the identification of the full-length HLA-E and its novel alleles is rare reported.OBJECTIVE:To establish the method of identification of HLA-E genomic full-length sequence,and to identify its novel alleles in healthy blood donors in Shenzhen,China.METHODS:Peripheral blood DNA samples were extracted from the subjects,and the amplified primers and sequencing primers in conserved regions were designed according to the sequences of HLA-E published in the IMGT/HLA database.A high-fidelity reaction system was used to amplify the genomic full-length of HLA-E,followed by sequencing,assembling,confirming and typing.RESULTS AND CONCLUSION:Herein,we successfully established the method for amplifying genomic full-length sequence and sequence-based typing.Two novel HLA-E alleles were nominated by WHO HLA Nomenclature committee as HLA-E*01:01:01:06and HLA-E*01:01:01:07.Compared with the most related allele HLA-E*01:01:01:01,HLA-E*01:01:01:06had one nucleotide change at nt-26(G->T)in5’-promoter,and HLA-E*01:01:01:07had one nucleotide change at nt3345(T->C)in3’-UTR.The polymorphism data of genomic full-length HLA-E in Chinese individuals need to be filled,and the method we developed here supplies the key technique for the further studies.

关 键 词：干细胞 移植 人类白细胞抗原E PCR-SBT 全基因序列测定 新等位基因 多态性 

分 类 号：R394.2[医药卫生—医学遗传学]