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目的研究HLA新的等位基因HLAB5614的分子基础。方法样本DNA抽提采用盐析法,利用PCR方法扩增先证者HLAB基因的第2～4外显子,PCR产物直接经TOPO转染克隆到质粒载体中分离其等位基因,对所得克隆进行第2～4外显子双向测序分析。应用序列特异性引物PCR方法证实测序所发现的突变。结果先证者样本克隆测序得到两个等位基因,其中1个等位基因为B1502,另一个经BLAST验证为新的等位基因,新的等位基因序列已递交GenBank(AY601726,AY601727,AY601728)。与最接近的B5608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277位G→C,导致第93位氨基酸Gly→Arg。结论该等位基因为新的HLAB等位基因,被世界卫生组织HLA因子命名委员会正式命名为HLAB5614。
Objective To study the molecular basis of HLA new allele HLAB5614. Methods DNA extraction was carried out by salting-out method. Exon2 and exon4 of HLAB gene of proband were amplified by PCR. The PCR products were directly cloned into plasmid vector by TOPO transfection and the alleles were separated. The obtained clones Two to four exons bi-directional sequencing analysis. Sequence-specific primers were used to confirm the mutations found in sequencing. RESULTS: Two alleles were obtained by cloning and sequencing of proband samples. One allele was B1502 and the other was BLAST-verified as a new allele. The new allele sequences were submitted to GenBank (AY601726, AY601727, AY601728 ). Compared to the closest B5608 allele, the new allele has only one nucleotide difference on exon 2, ie, G → C at position 277, resulting in amino acid Gly → Arg at position 93. Conclusion This allele is a new HLAB allele and was officially named as HLAB5614 by the WHO Nomenclature Committee of HLA Factors.