CAJ | 학술논문

目的对中国部分汉族人群人类白细胞抗原(human leukocyte antigen,HLA)A座位常见等位基因的内含子进行全长序列研究.方法从“中国造血干细胞捐献者资料库广东分库”中抽取165份样本,用美国Gentra公司的试剂盒快速提取全血DNA.自行设计引物,特异性扩增HLA-A座位的目的3 kb片段,共涵盖8个外显子和7个内含子.凝胶电泳回收目标条带,连接于T载体,E.coli DH5α转化,挑选阳性克隆正反向测序.采用MEGA 4.0软件进行分析,并用ClustalW比对序列,相邻连接法构建进化树.其中距离矩阵通过p-distance计算,采用自举法评价这些树的准确性,重复取样部分数据1000次.结果共获得33个HLA-A等位基因全长序列,长度为2902～2918 bp.7个内含子共发现138个点突变以及9个插入或缺失,具有明显的组特异性.第1内含子的多态性、GC含量和平均遗传距离D值最高.根据全长及第1～7内含子序列分别构建进化树.其中,以全长序列构建的进化树将HLA-A基因分为5组:Ⅰ组包括A* 01/03/11/30；Ⅱ组包括A*23/24(A9)；Ⅲ组包括A*02/68/69(A2/28)；Ⅳ组包括A*26/34(A10)；Ⅴ组包括A*29/31/32/33/74(A19).其中Ⅰ～Ⅱ组属于同一世系,Ⅲ～Ⅴ组属于另一世系.用7个内含子序列构建的进化树在结构上有小的差异,较为特殊的是Ⅱ组和A*30.Ⅱ组的第2～5内含子与Ⅰ组处于同一世系,而第1和7内含子却处于相对立的另一世系.A*30与A* 03家族的关系最近,而与A19家族较远.结论 18个等位基因全长序列填补了GenBank数据库的空白,并得到了免疫基因标记数据库的确认.HLA-A等位基因的内含子多态性具有显著的组特异性,并对基因的重组具有重要意义.A*30可能是一个具有高突变率和高重组率的特殊组系.
목적 대중국부분한족인군인류백세포항원(human leukocyte antigen,HLA)A좌위상견등위기인적내함자진행전장서렬연구.방법 종“중국조혈간세포연헌자자료고엄동분고”중추취165빈양본,용미국Gentra공사적시제합쾌속제취전혈DNA.자행설계인물,특이성확증HLA-A좌위적목적3 kb편단,공함개8개외현자화7개내함자.응효전영회수목표조대,련접우T재체,E.coli DH5α전화,도선양성극륭정반향측서.채용MEGA 4.0연건진행분석,병용ClustalW비대서렬,상린련접법구건진화수.기중거리구진통과p-distance계산,채용자거법평개저사수적준학성,중복취양부분수거1000차.결과 공획득33개HLA-A등위기인전장서렬,장도위2902～2918 bp.7개내함자공발현138개점돌변이급9개삽입혹결실,구유명현적조특이성.제1내함자적다태성、GC함량화평균유전거리D치최고.근거전장급제1～7내함자서렬분별구건진화수.기중,이전장서렬구건적진화수장HLA-A기인분위5조:Ⅰ조포괄A* 01/03/11/30；Ⅱ조포괄A*23/24(A9)；Ⅲ조포괄A*02/68/69(A2/28)；Ⅳ조포괄A*26/34(A10)；Ⅴ조포괄A*29/31/32/33/74(A19).기중Ⅰ～Ⅱ조속우동일세계,Ⅲ～Ⅴ조속우령일세계.용7개내함자서렬구건적진화수재결구상유소적차이,교위특수적시Ⅱ조화A*30.Ⅱ조적제2～5내함자여Ⅰ조처우동일세계,이제1화7내함자각처우상대립적령일세계.A*30여A* 03가족적관계최근,이여A19가족교원.결론 18개등위기인전장서렬전보료GenBank수거고적공백,병득도료면역기인표기수거고적학인.HLA-A등위기인적내함자다태성구유현저적조특이성,병대기인적중조구유중요의의.A*30가능시일개구유고돌변솔화고중조솔적특수조계.
Objective To analyze the full intronic sequences of human leukocyte antigen (HLA)-A alleles in Han Chinese.Methods The full-length HLA-A alleles,including 8 exons and 7 introns,were amplified with a long-template PCR system from 165 donors from the Chinese Marrow Donor Program (CMDP).The products were cloned into a PGEM-T Vector System and sequenced from both directions.Genetic analysis was performed using a MEGA4.0 software.All sequences were aligned with a ClustalW algorithm.Phylogenetic trees were constructed with a neighbor-joining method.Genetic distances were estimated based on p-distance,and a bootstrap analysis was applied for assessing the confidence limits of the trees.Results A total of thirty-three full-length sequences of HLA-A alleles,containing 2902-2918 nucleotides,were derived.A total of 138 point mutations and 9 insertions or deletions were found among the 7 introns,which showed remarkable group specificity.Intron 1 appeared to be most polymorphic with the highest average GC content and evolutionary distances. Eight phylogenetic trees were constructed respectively with the derived full-length sequences as well as each of the 7 introns sequences.Based on fulllength sequences,sequences of the HLA-A locus were classified into five groups:group Ⅰ consisted of A *01/03/11/30; group Ⅱ consisted of A* 23/24(A9); group Ⅲ consisted of A * 02/68/69(A2/28); group Ⅳ consisted of A * 26/34(A10); and group Ⅴ consisted of A * 29/31/32/33/74(A19).The five groups were derived from two ancient lineages,one including groups Ⅰ and Ⅱ,and another including groups Ⅲ,Ⅳand Ⅴ.No substantial difference was detected between the trees constructed with the 7 intronic sequences,except that group Ⅱ belonged to different lineages based on introns 2-5 and introns 1 and 7.The A * 30 variant cluster was close to group Ⅰ (A * 01/03/11/30) and differed from group Ⅴ (A19).Conclusion The full-length sequences of 18 alleles have been submitted to GenBank and accepted by the international ImMunoGeneTics database (IMGT).Polymorphisms identified within the introns of HLA-A alleles showed remarkable group specificity.Such sequences seem to have substantially contributed to the recombination of the HLA-A alleles.The A * 30 may represent an atypical group in which the rates of gene conversion and mutation have been unusually high.