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目的研究HLA-B新的等位基因B*5136的分子机理。方法先证者为浙江省脐带血造血干细胞捐献者。盐析法抽提样本DNA,常规PCR反应扩增先证者HLA-B基因第2~4外显子的编码序列,PCR产物经TOPO试剂盒克隆转染到质粒载体中,分离其两个等位基因,对所得的克隆用第2、3、4外显子引物进行双向测序分析。结果先证者HLA-B基因经TOPO克隆后得到两个等位基因,一个为HLA-B*4601,另一个经BLASTHLA验证为新的等位基因,其序列已递交GenBank(AY601729,AY610730,AY601731)。新的等位基因与最接近的B*5108等位基因比较,在第3外显子上有4个核苷酸的差异:第527位T→A,导致第177位氨基酸Val→Glu;第583位C→T,导致第195位氨基酸His→Tyr;在第559位C→A和第560位T→C,导致第187位氨基酸Leu→Thr。结论该脐血捐献者的HLA-B为新的等位基因,被世界卫生组织HLA命名委员会正式命名为HLA-B*5136。
Objective To study the molecular mechanism of HLA-B novel allele B * 5136. Method of proband for the cord blood hematopoietic stem cell donors in Zhejiang Province. The sample DNA was extracted by salting-out method, and the coding sequence of exon 2 to exon 4 of HLA-B gene was amplified by conventional PCR. The PCR product was cloned by TOPO kit and transfected into plasmid vector, The resulting clone was subjected to bidirectional sequencing using primers 2, 3 and 4. RESULTS: Two alleles of HLA-B gene were obtained by TOPO cloning. One was HLA-B * 4601 and the other was verified as a new allele by BLASTHLA. The sequence was submitted to GenBank (AY601729, AY610730, AY601731 ). The new allele has a 4-nucleotide difference in exon 3 compared with the closest B * 5108 allele: T → A at position 527 resulting in amino acid Val → Glu at position 177; 583 C → T, resulting in amino acid 195 → Tyr at position 195; C → A at position 559 and T → C at position 560, resulting in amino acid Leu → Thr at position 187. Conclusion The cord blood donor HLA-B as a new allele, was officially named HLA HLA-B * 5136 by the WHO HLA Committee.