CAJ | 학술논문

目的分析HLA-B新等位基因HLA-B*9534的核苷酸序列,并建立HLA-B * 9534单链扩增技术.方法采用商品化快速抽提试剂盒抽提标本基因组DNA,采用PCR技术扩增先证者HLA-B基因的第1～8外显子序列,PCR产物经双酶切后直接测序分析第2、3、4外显子.应用序列特异性引物PCR建立HLA-B*9534单链扩增技术,获得HLA-B*9534等位基因的单链产物,并对单链产物进行第2、3、4外显子测序分析.结果先证者标本存在2个HLA-B等位基因,直接测序结果经软件分析显示与最接近的HLA-B*1518和B*4601组合存在1个碱基不匹配,即第593位A/G杂合.单链扩增技术将先证者等位基因分离后,测序得到两个等位基因为HLA-B*4601和HLA-B*9534.与最接近的HLA-B*1518的第2～4外显子序列相比,HLA-B*9534仅在第3外显子存在一个碱基的不同,即第593位A→G的改变,导致第174位氨基酸天冬酰胺改变为丝氨酸,该等位基因序列已递交GenBank(EU046491),并经世界卫生组织HLA命名委员会正式命名为HLA-B*9534.结论发现一个新的HLA-B*9534等位基因,建立的HLA-B*9534单链扩增技术是可行的.
목적 분석HLA-B신등위기인HLA-B*9534적핵감산서렬,병건립HLA-B * 9534단련확증기술.방법 채용상품화쾌속추제시제합추제표본기인조DNA,채용PCR기술확증선증자HLA-B기인적제1～8외현자서렬,PCR산물경쌍매절후직접측서분석제2、3、4외현자.응용서렬특이성인물PCR건립HLA-B*9534단련확증기술,획득HLA-B*9534등위기인적단련산물,병대단련산물진행제2、3、4외현자측서분석.결과 선증자표본존재2개HLA-B등위기인,직접측서결과경연건분석현시여최접근적HLA-B*1518화B*4601조합존재1개감기불필배,즉제593위A/G잡합.단련확증기술장선증자등위기인분리후,측서득도량개등위기인위HLA-B*4601화HLA-B*9534.여최접근적HLA-B*1518적제2～4외현자서렬상비,HLA-B*9534부재제3외현자존재일개감기적불동,즉제593위A→G적개변,도치제174위안기산천동선알개변위사안산,해등위기인서렬이체교GenBank(EU046491),병경세계위생조직HLA명명위원회정식명명위HLA-B*9534.결론 발현일개신적HLA-B*9534등위기인,건립적HLA-B*9534단련확증기술시가행적.
Objective To analyze the molecular genetic basis of novel allele HLA-B * 9534 and establish the allele group specific primer PCR method. Methods Genomic DNA was extracted from whole blood by commercial DNA extraction kit. The HLA-B exons 1 to 8 coding sequences of the proband were am-plified by PCR and the amplification product was purified with double enzymes digestion and both strands of exons 2, 3 and 4 were sequenced. The exon 2-4 amplification of the HLA-B * 9534 was performed with al-lele group specific primers PCR and the PCR product was directly sequenced for exon 2 to 4. Results The proband has two HLA-B alleles. The result was assigned for HLA-B * 1518 and B * 4601 combination with a mismatch in 593A/G heterozygote by DNA sequencing of exon 2 to 4 with loci primers. After separating the two alleles of the proband with allele group specific primers polymerase chain reaction method, HLA-B * 4601 and HLA-B * 9534 alleles were identified after sequencing. The HLA-B * 9534 is identical to HLA-B * 1518 except for one nucleotide substitutions in exon 3 at position 593 A→G, this results in amino acid substitution at cedon 174 from Asn to Ser. The sequences of the novel allele have been submitted to GenBank (EU046491) and the allele has been officially nominated by the WHO Nomenclature Committee. Conclusion Identification of a novel HLA-B * 9534 allele and allele group specific primer PCR for HLA-B * 9534 was re-liable.