克隆测序确认HLA新等位基因B~*3712^(★○)  

A new allele HLA-B*3712 defined by cloning and sequencing

作　　者：张伯伟[1] 邢培清[1] 赵磊[1] Ana Lazaro Jennifer Ng 

机构地区：[1]河南省红十字血液中心,河南省郑州市450012 [2]美国海军骨髓库,洛克维尔马里兰州美国20852

出　　处：《中国组织工程研究与临床康复》2007年第50期10131-10133,共3页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:由于技术原理的限制,目前尚不能对所有的HLA等位基因进行严格的区分,特别是以往没有发现的新基因序列只能通过测序的方法解决,然而,当遇到等位基因杂合时,测序给出的结果仍然无法确认新的序列改变发生在等位基因的哪一侧,这时需要用分子生物学方法分离杂合子然后进行测序才能确定新的基因序列。采用基因克隆方法确认HLA新等位基因。方法:实验于2006-01/05在河南省红十字血液中心HLA实验室,美国海军骨髓库HLA实验室完。成造血干细胞血样由中华骨髓库提供。采用荧光微珠HLA分型方法对中华骨髓库捐献者血样进行HLA分型检测,无法给出确切结果的摸棱两可结果标本用基因克隆(TOPO TA Cloning)、DNA测序的方法确认新的HLA基因序列。结果:通过克隆分离杂台等位基因,再进行测序确认发现新的序列与B~*3709相比,出现4个核苷酸改变:1.355nt C>A,2.363nt C>G,3.412ntG>A,4 477nt C>G,而且均发生在HLA-B基因外显子3(exon 3)。4处改变引起氨基酸编码改变:①编码95 CTC>ATC,氨基酸改变L>I(亮氨酸>异亮氨酸)。②97 AGC>AGG,S>R(丝氨酸>精氨酸)。③114 GAC>AAC D>N(天门冬氨酸>天冬酰胺)。④135 GCC>GCG A=A无氨基酸改变。结论:①新的基因序列已经在GenBnak注册,被WHO的HLA因子命名委员会得到正式命名为HLA-B~*3712基因。②基因克隆是确认HLA新基因的根本方法。AIM： We cannot strictly distinguish HLA allele at present, because of limited technique. A new gene sequencing should be distinguish by sequencing. However, when allele heterozygosis appeared, where the new sequencing changes occurred on allele. Thus, heterozygote is isolated by molecular biology method, and then sequencing was performed to define the new gene sequencing. This article defines a new HLA allele by gene cloning. METHODS： Experiments ware performed from January to May 2006 at HLA Lab of Henan Red Cross Blood Center and the HLA Lab of C.W.Bill Young Marrow Donor Program of Naval Medical Research Center. Blood samples of haemopoietic stem cells ware provided by Chinese Bone Marrow Bank. HLA typing was determined by fluorescent superbead HLA typing. An HLA-B ambiguous result was found to define a new HLA gene sequencing by gene clone （TOPO TA Cloning） and DNA sequencing. RESULTS： Heterozygous allele was separated by cloning. An novel HLA-B^＊3712 was identified with sequencing, 4 nucleotide substitution was found in exon 3： 1.355nt C 〉 A ,2.363nt C 〉 G,3.412nt G 〉 A,4.477nt C 〉 G, resulting in 4 amino acid changes： ①coding 95 CTC 〉 ATC,amino acids change L 〉 I （aminoisocaproic acid〉isoleucine）; ②97 AGC 〉 AGG, S 〉 R（serine〉arginine）; ③114 GAC 〉 AAC D 〉 N（aspartic acid〉asparagine）; ④135 GCC 〉 GCG A=A no changes in amino acids. CONCLUSION： ①The new sequencing is registered in GenBank and officially assigned as novel HLA-B^＊3712 by WHO Nomenclatrue Committee. ②DNA sequencing should be the golden stander for HLA typing to distinguish the ambiguity.

关 键 词：HLA新基因 基因克隆 DNA测序 

分 类 号：R596[医药卫生—内科学]