人类白细胞抗原-DRB1第3外显子单链测序方法的建立及其多态性分析  

Establishment of allele specific primer polymerase chain reaction sequence-based typing and investigation of the polymorphism in exon 3 of HLA-DRB1

作　　者：朱发明[1] 和艳敏[1] 陶苏丹[1] 章伟[1] 王炜[1] 何俊俊[1] 吕杭军[1] 严力行[1] 

机构地区：[1]浙江省血液中心输血研究所、卫生部血液安全研究重点实验室,杭州310006

出　　处：《中华医学遗传学杂志》2010年第6期639-643,共5页Chinese Journal of Medical Genetics

基　　金：浙江省科学技术研究基金（2009C33164）;浙江省医药卫生科学研究基金（2008A036,2009A047）;浙江省卫生高层次创新人才培养工程

摘　　要：目的 建立人类白细胞抗原（human leukocyte antigen,HLA）-DRB1第3外显子单链测序方法,并分析其多态性.方法 采用组特异性引物扩增HLA-DRB1第2和3外显子序列,扩增产物经双酶切后进行双向测序分析,采用Assign 3.5 SBT分析软件指定等位基因型.结果 PCR产物经直接测序后得到清晰的序列冬,并向IMGT/HLA数据库提交了HLA-DRB1＊08：09和DRB1＊12：02：01第3外显子全部序列.在检测的25个等位基因中,HLA-DRB1第3外显子全长序列中存在27个单核苷酸多态性位点,占第3外显子序列碱基总数的9.56%.建立的方法可有效区分HLA-DRB1＊14∶01∶01/14∶54歧义标本,证实中国人群中存在HLA-DRB1＊14∶01∶01.结论 本实验建立的HLA-DRB1第3外显子测序方法是可行的,HLA-DRB1第3外显子存在多态性.Objective To establish the allele specific primer polymerase chain reaction sequence based typing （PCR-SBT） and investigate the polymorphism of exon 3 of human leukocyte antigen（ HLA）DRB1. Methods The fragment containing exons 2 and 3 of HLA-DRB1 gene was amplified by group specific primers. The amplified products were digested by restriction enzymes and directly sequenced in both directions. The genotype was assigned by using Assign 3.5 SBT software. Results The exon 3 sequences of HLA-DRB1 ＊ 08∶09 and HLA-DRB1 ＊ 12∶02∶01 were identified for the first time. There were 27 polymorphic sites in exon 3 among the twenty-five HLA-DRB1 alleles, which was 9.56% of all nucleotides of exon 3. The method could discriminate the HLA-DRB1 ＊ 14∶01∶01/14∶54 ambiguous samples, and the HLA-DRB1 ＊ 14∶01∶01 was identified in the Chinese population. Conclusion The PCR-SBT method for exon 3 of HLA-DRB1 from the present study was reliable and there were polymorphisms in exon 3 of HLA-DRB1.

关 键 词：人类白细胞抗原-DRB1 第3外显子 测序分析 遗传多态性 

分 类 号：R686[医药卫生—骨科学]