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目的 研究红细胞ABO血型系统Bw亚型的分子基础。方法 通过标准血型血清学方法明确鉴定2个家庭3例Bw亚型,PCR扩增Bw 亚型ABO糖基转移酶基因的增强子、启动子和第1～7外显子及侧翼内含子序列,PCR产物经割胶纯化后直接测序。同时将第6和7外显子克隆到pc DNA3.1(- )质粒,转化DH5α后进行序列分析。采用序列特异性引物-聚合酶链反应方法证实测序所发现的突变。结果 直接测序发现3例Bw亚型的基因型为B/ O杂合,其中糖基转移酶基因的第2 6 1位G杂合缺失,第72 1位C/ T杂合。克隆证实一条染色体上为正常的O等位基因,另一条染色体上B等位基因(α1,3半乳糖基转移酶基因)存在第72 1位C>T突变,导致多肽链Arg2 4 1Trp替换。序列特异性引物-聚合酶链反应检测14 0份随机样本未发现此突变。结论 α1,3半乳糖基转移酶基因第7外显子72 1C>T突变可能是Bw亚型分子遗传基础之一。
Objective To study the molecular basis of the Bw subtype of erythrocyte ABO blood group system. Methods Three Bw subtypes of two families were identified by standard blood group serological methods. The enhancer, promoter, exon 1 to exon 7 and flanking intron of Bw subtype ABO glycosyltransferase gene were amplified by PCR The PCR products were directly sequenced after being gel-purified. The exon 6 and 7 were cloned into pcDNA3.1 (-) plasmid and transformed into DH5α for sequence analysis. Sequence-specific primer-polymerase chain reaction method was used to confirm the mutations found by sequencing. Results The direct genotypes of three Bw isotypes were found to be B / O heterozygous. Among them, the 661th G gene of glycosyltransferase gene was heterozygous and the 721th C / T was heterozygous. The clone confirmed a normal O allele on one chromosome and the C allele of the B allele on the other chromosome (α1,3 galactosyltransferase gene) on the other chromosome led to the substitution of Arg2 4 1Trp polypeptide chain. Sequence-specific primers - polymerase chain reaction detected 14 random samples of this mutation was not found. Conclusion The mutation 72C> T of exon 7 of α1,3 galactosyltransferase gene may be one of the molecular basis of Bw subtypes.