新HLA-B-4061等位基因核苷酸序列分析.docVIP

新HLA-B*4061等位基因核苷酸序列分析 　 作者：章伟　吕沁风　王炜　韩浙东　朱发明　严力行 【摘要】 　　本研究探讨HLA 新的等位基因HLA-B*4061的分子基础。采用盐析法抽提样本DNA，利用PCR方法扩增先证者HLA-B基因的第1-8外显子，PCR产物直接经TOPO克隆到质粒载体中得到单链，对所得克隆进行HLA-B基因第2、3、4外显子双向测序分析。应用PCR-SSP方法证实测序所发现的突变。结果表明：先证者样本克隆测序得到2个等位基因，其中1个等位基因为B*4601，另1个经blast验证其为新的等位基因，新的等位基因序列已递交GenBank（DQ089628，DQ089629，DQ089630）。与最接近的B*400101等位基因序列相比，新的等位基因仅在第2外显子上有1个核苷酸不同，即第272位C→A，导致第67位氨基酸Ser→Tyr。结论：HLA-B*4061等位基因为新的HLA-B等位基因，已荣获世界卫生组织HLA因子命名委员会正式命名。 【关键词】 HLA-B；HLA-B*4061； 等位基因；序列分析 　　Sequence Analysis of HLA-B*4061 Allele Newly Found 　　AbstractThe aim of this study was aimed to investigate the molecular genetic basis for a novel HLA allele, HLA-B*4061, in Chinese population. DNA was extracted from whole blood by salting-out method. The HLA-B exons 1-8 of the proband was amplified and the amplified product was cloned using TOPO TA cloning sequencing kit to split the two alleles apart. Both strands of exons 2, 3 and 4 of chosen colonies were sequencing. The PCR-SSP was performed to confirm the mutations detected by sequencing. The sequencing results showed HLA-B alleles of the proband as B*4601 and the novel allele. The sequences of the novel allele have been submitted to GenBank (DQ089628，DQ089629，DQ089630). After HLA blast analysis, the novel allele showed a single nucleotide mismatch with B*400101 in exon 2 at position 272 C→A, as the results, changing amino acid from Ser to Tyr at codon 67. It is concluded that this allele is a novel one and has been officially named B*4061 by the WHO Nomenclature Committee. 　　Key wordsHLA-B；HLA-B*4061; allele; sequence analysis 　　人类白细胞抗原（HLA）在器官移植和造血干细胞移植中起重要的作用，HLA抗原相容能显著改善移植物的存活，目前HLA的DNA分型已广泛应用于造血干细胞移植、肾移植等。HLA系统是目前所知最具多态性的系统，现已命名的HLA等位基因超过2 000个，并且仍不断在发现新的等位基因［1-3］。本实验室在检测的造血干细胞捐献者样本中发现1例新的HLA-B等位基因，被世界卫生组织（WHO）HLA命名委员会正式命名为HLA-B*4061，现将结果报告如下。 　　材料和方法 　　样本来源 　　先证者为男性，浙江籍汉族，为造血干细胞捐献者，在本实验室HLA常规分型中因HLA-B位点出现罕见的反应格局而进一步确认。 　　样本DNA提取 　　按快速盐析法［4］。 　　HLA分型 　　HLA-A、-B、-DRB1基因分型采用PCR-SSO方法（tepnel lifecodes, stamford,CT），严格按试剂说明书进行操作，结果采用自动软件判读。H