HLA-Cw基因全长序列分子克隆及测序方法的建立

目的 建立可靠的HLA-Cw基因全长序列的分子克隆和测序技术.方法 设计合成HLA-Cw基因全长序列PCR引物和探索PCR反应体系,采用长距离PCR技术,扩增HLA-Cw基因非翻泽区(untranslated region,5'-UTR)区、8个外显子、7个内含子和3'-UTR区,全长约4.5 kb.PCR产物纯化后进行分子克隆,筛选阳性克隆,提取质粒DNA,采用自行设计的测序引物进行全长双向测序.12份已经AlleleSEQR HLA-Cw测序分型试剂盒进行PCR产物直接测序、基因型已知的样本,分别用TaKaRa LATaq酶和Stratagene Pfu Taq DNA聚合酶进行HLA-Cw基因全长扩增,以及PCR产物分子克隆和序列测定,克隆测序结果分别与PCR产物直接测序结果进行对比分析.结果 PCR扩增获得了特异性目的 片段,测序获得了HLA-Cw基因-962～3576位碱基全长序列.克隆测序结果的对比表明,Pfu酶保真性高于LA Taq酶.比较本文测定的Cw*010201与Cw*07020101等位基因序列,在5'上游-962～-284位碱基区域存在11个单核苷酸多态(single nucleotide polymorphisms,SNPs)和2个插入(或)缺失多态性位点;3'-UTR下游3067～3576位碱基区域存在11个SNPs和1个插入(或)缺失.结论 建立了HLA-Cw基因全长序列分子克隆及测序方法,在HLA-Cw基因全长序列分子多态性及表达调控等研究领域,具有广泛应用前景.     

HLA-DRB1*1218新等位基因克隆测序鉴定及内含子序列分析

Objective To identify a novel human leukocyte antigen (HLA) allele by cloning and sequence-based typing in Chinese population, and analyzing the sequence of the introns 1 and 2. Methods The routine HLA-A, -B, -DRB1 low resolution genotyping for stem cell donor from Guangdong province was performed with polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP). An unknown HLA-DRB1 allele was initially detected by HLA typing. Genomic DNA of the proband was amplified by using HLA-DRB1 locus group-specific primer, the amplified product was cloned, sequenced, and compared to the closest DRB1 * 120201 allele and the closest intron sequence of the DRB1 * 030101 allele. Results The sequencing results showed that a normal DRB1 * 080302 and a novel DRB1 * 1218 variant allele were identified. The sequence of the novel allele has been submitted to GenBank (FJ481086). The novel allele had 1 nucleotide substitution of the closest matching allele HLA-DRB1 * 120201 at nt262(G →C) in exon 2, resulting in an amino acid change from GIu(GAG)→Gln (CAG) at codon 59. The intron 2 sequence is identical between the novel HLA-DRB1 * 1218 and DRB1 * 030101, but there are 12 nucleotides substitution in intron 1. Conclusion A novel HLA allele was confirmed by cloning and sequence-based typing in Chinese. It was officially designated as HLA-DRB1 * 1218 by WHO Nomenclature Committee.     

HLA-DRB1*1218新等位基因克隆测序鉴定及内含子序列分析

Objective To identify a novel human leukocyte antigen (HLA) allele by cloning and sequence-based typing in Chinese population, and analyzing the sequence of the introns 1 and 2. Methods The routine HLA-A, -B, -DRB1 low resolution genotyping for stem cell donor from Guangdong province was performed with polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP). An unknown HLA-DRB1 allele was initially detected by HLA typing. Genomic DNA of the proband was amplified by using HLA-DRB1 locus group-specific primer, the amplified product was cloned, sequenced, and compared to the closest DRB1 * 120201 allele and the closest intron sequence of the DRB1 * 030101 allele. Results The sequencing results showed that a normal DRB1 * 080302 and a novel DRB1 * 1218 variant allele were identified. The sequence of the novel allele has been submitted to GenBank (FJ481086). The novel allele had 1 nucleotide substitution of the closest matching allele HLA-DRB1 * 120201 at nt262(G →C) in exon 2, resulting in an amino acid change from GIu(GAG)→Gln (CAG) at codon 59. The intron 2 sequence is identical between the novel HLA-DRB1 * 1218 and DRB1 * 030101, but there are 12 nucleotides substitution in intron 1. Conclusion A novel HLA allele was confirmed by cloning and sequence-based typing in Chinese. It was officially designated as HLA-DRB1 * 1218 by WHO Nomenclature Committee.     

供、患者HLA-A、B、DRB1高分辨分型的无关供者外周血造血干细胞移植32例回顾分析

目的探讨供、患者的人类白细胞抗原HLA-A、B、DRB1基因高分辨相合及亚型相合,无关供者外周血造血干细胞移植后效果的情况,为临床造血干细胞移植治疗选择适配供者提供理论依据。方法采用PCR-SSP或SBT技术对供、患者HLA-A、B、DRB1的基因进行高分辨分型,定期随访患者在造血干细胞移植后状况,统计分析移植后效果。结果32例患者与供者的HLA低分辨分型结果完全相合,HLA-A、B、DRB1基因高分辨分型全相合的患者与HLA-A、B、DRB1基因高分辨分型不相合的患者,造血干细胞植活和两年半内的移植存活率比较,无统计学差异。结论HLA低分辨分型结果完全相合是临床医生选择造血干细胞移植的重要条件之一,供、患者HLA-A、B、DRB1高分辨分型是外周血造血干细胞移植治疗前组织配型不可少的实验之一,但HLA-A、B、DRB1基因高分辨分型错配对无关供者外周血造血干细胞移植的造血干细胞植活和两年半内的移植存活率影响较小,HLA基因高分辨分型可以帮助临床医生选择合适的供者。     

HLA-DRB1*1218新等位基因克隆测序鉴定及内含子序列分析

Objective To identify a novel human leukocyte antigen (HLA) allele by cloning and sequence-based typing in Chinese population, and analyzing the sequence of the introns 1 and 2. Methods The routine HLA-A, -B, -DRB1 low resolution genotyping for stem cell donor from Guangdong province was performed with polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP). An unknown HLA-DRB1 allele was initially detected by HLA typing. Genomic DNA of the proband was amplified by using HLA-DRB1 locus group-specific primer, the amplified product was cloned, sequenced, and compared to the closest DRB1 * 120201 allele and the closest intron sequence of the DRB1 * 030101 allele. Results The sequencing results showed that a normal DRB1 * 080302 and a novel DRB1 * 1218 variant allele were identified. The sequence of the novel allele has been submitted to GenBank (FJ481086). The novel allele had 1 nucleotide substitution of the closest matching allele HLA-DRB1 * 120201 at nt262(G →C) in exon 2, resulting in an amino acid change from GIu(GAG)→Gln (CAG) at codon 59. The intron 2 sequence is identical between the novel HLA-DRB1 * 1218 and DRB1 * 030101, but there are 12 nucleotides substitution in intron 1. Conclusion A novel HLA allele was confirmed by cloning and sequence-based typing in Chinese. It was officially designated as HLA-DRB1 * 1218 by WHO Nomenclature Committee.     

HLA-A和HLA-B与HLA-DRB1基因低分辨相合的无关供/受者对HLA高分辨水平匹配性分析

位基因6/6相合的样本中,等位基因8/8相合及10/10相合的比例分别为63.2%(48/76)和57.9%(44/76);但其中1～2个HLA-Cw等位基因不相合的比例为36.8%(28/76),且主要为抗原水平不相合.结论 实现HLA-A、HLA-B、HLA-DRB1的高分辨分型数据入库虽有助于提高无关供/受者对与HLA高分辨配型检索的成功率,但HLA-Cw抗原水平的不相合不能被忽视,建议将HLA-Cw基因分型纳入骨髓库骨髓志愿捐献者HLA入库数据的常规检测.     

人类白细胞抗原及单倍型与中国北方汉族急性非淋巴细胞白血病相关性研究

急性非淋巴细胞白血病(AML)是我国白血病分类构成中最常见的一类,其疾病的发生、发展机制目前尚不十分清晰,可能与遗传因素、外界环境因素和自身免疫因素等有关.人类白细胞抗原(HLA)与肿瘤的免疫监视作用密切相关,HLA与白血病相关性研究国内外文献均有报道[1-3],但结论不一.     

南方汉族强直性脊柱炎患者及健康人群HLA-B27基因的分子多态性及分布

目的研究南方汉族强直性脊柱炎(AS)患者及健康人群HLA-B27亚型等位基因的多态性和分布特点。方法采用HLA测序分型方法,对收集到的46份B*27阳性的南方汉族AS患者血样以及随机选择的80份经rS-SOHLA流式磁珠低分辨检测为B*27阳性的造血干细胞南方汉族捐献者血样,做HLA-B基因的第2—4外显子测序分型,测序反应产物用ABI PrismTM3730测序仪检测,Assign3.5分析软件分析结果。对检出的模棱两可结果及"罕见型"等位基因,辅以PCR-SSP法HLA-B27高分辨基因分型。结果46名南方汉族AS患者中,共检出4个HLA-B*27相关的等位基因:B*2704的检出比例最高,为82.98%(39/47);其次是B*2705,为12.77%(6/47);B*2707和B*2724各为2.13%(1/47)。80名南方汉族造血干细胞捐献者中,共检出7种B*27相关等位基因:以B*2704的检出比例最高,为57.32%(47/82);其次为是B*2705,为26.83%(22/82);B*2706和B*2707各为6.10%(5/82);B*2703、B*2715和B*2724各为1.22%(1/82)。结论南方汉族AS患者HLA-B*27基因中以B*2704最为常见,健康人群以B*2704和B*2705等位基因为主要亚型。     

广东汉族人群HLA-Cw基因座的等位基因多态性分析

目的调查广东汉族人群人类白细胞抗原（HLA）-Cw基因座的等位基因分布,并比较分析中国人群HLA-Cw基因的分布特征。方法采用结合聚合酶链式反应（PCR）-测序分型（sequence based typing,SBT）方法对160例广东汉族骨髓供者的HLA-Cw基因的第2,3外显子进行序列分析。结果广东汉族群体中共检出20种HLA-Cw等位基因,频率分布为0．0031～0．2094,其中Cw＊070201（0．2063）、Cw＊0102（0．1937）、Cw＊0304（0．1563）、Cw＊0302（0．0906）和Cw＊080101（0．0781）比较常见。70检验表明其基因型分布符合Hardy-Weinberg平衡定律。结论建立广东汉族人群HLA-Cw基因座的等位基因频率数据库,为法医学、人类学、医学研究和应用提供重要的群体遗传学资料;同时发现HLA-Cw等位基因在中国人群中呈现规律性分布。     

HLA-DRB1*1218新等位基因克隆测序鉴定及内含子序列分析

目的 鉴定中国人群人类白细胞抗原(human leukocyte antigen,HLA)DRB1基因,分析新等位基因第1和2内含子序列信息.方法 采用聚合酶链反应-序列特异寡核苷酸探针反向杂交法(polymerase chain reaction-sequence specific oligonucleotide probes,PCR-SSOP)对广东地区随机正常人群进行HLA常规基因分型,发现1个与HLA-DRB1*120201相近的未知基因,对先证者DNA应用组特异性引物扩增HLA-DRB1位点第2外显子,PCR产物经克隆到质粒载体中以获得单链,对克隆所得产物进行HLA-DRB1基因的第2外显子及第1和第2内含子双向测序分析.并与DRB1*120201基因序列的第2外显子和DRB1*03010101等位基因内含子相比较.结果 发现该个体的一个HLA-DRB1*080302基因被确认,而另一个HLA-DRB1基因为新等位基因,其序列被GenBank接受(编号为FJ481086).新等位基因与最相近的DRB1*120201相比,在第2外显子上有1个核苷酸的不同,即第262位G→C(密码子59 GAG→CAG,氨基酸59 Glu→Gln).DRB1*1218与DRB1*03010101等位基因第2内含子序列完全相同,而与DRB1*03010101等位基因第1内含子序列相比较有12个碱基不同.结论 发现并鉴定一个新的HLA等位基因,经世界卫生组织HLA因子命名委员会正式命名为HLA-DRB1*1218.