Objective: To explore the serological characteristics and molecular biological mechanism of an ael subtype specimen.
Methods: The ABO blood typing was identified by routine blood group serological and absorption/elution methods; PCR-SBT method for ABO genotyping: 7 exons of ABO gene were amplified by PCR, the amplified products were purified, and then sequencing primers were designed and the amplified products were sequenced directly for analysis; 3D molecular model was constructed and the difference of free energy (ΔΔG) was used to predict the GTA mutant stability.
Results: A antigen was not detected on erythrocytes through absorption and elution tests, which was not consistent with the serological characteristics of ael, and the serological typing results were ambiguous. The ABO genotype was ABO*AEL.02/O.01.01, and there were two mutations in exon 7 of the gene, c.467C>T and c.646T>A, which could lead to the replacement of proline with leucine at position 156 (p.Pro156Leu) and phenylalanine with isoleucine at position 216 on the GTA, respectively. The 3D model predicts that the mutations do not introduce new hydrogen bonds to the GTA mutant and do not form a new secondary structure, but can lead to an increase in the ΔΔG value of the GTA mutant, suggesting a decrease in protein stability.
Conclusion: The serological characteristics alone is not reliable to determine the ael subype; the ael phenotype may be due to the GTA mutant that reduces enzyme stability.
题目: AEL.02亚型的血清学特征分析与分子生物学机制研究.
目的: 探讨1例ael亚型标本的血清学特征与分子生物学机理.
方法: 利用血型血清学方法、吸收放散法等技术鉴定ABO血型; PCR-SBT方法进行ABO基因分型: 利用PCR扩增ABO基因的7个外显子，对扩增产物进行纯化，然后设计测序引物，将扩增后的产物直接测序分析；构建3D分子模型，并以自由能的差值(ΔΔG)预测GTA突变体的稳定性.
结果: 吸收放散试验未检出红细胞上的A抗原，与ael的血清学特征不相符，血清学定型结果不明确；ABO基因型为ABO* AEL.02/O.01.01型，基因的外显子7存在c.467C>T与c.646T>A两个突变位点，可分别导致GTA上的156位脯氨酸被替换为亮氨酸(p.Pro156Leu)以及216位苯丙氨酸被替换为异亮氨酸。3D模型预测突变未给GTA突变体引入新的氢键，也未形成新的二级结构，但可导致GTA突变体的ΔΔG值升高，提示蛋白稳定性降低.
结论: 仅仅利用血清学特征来判断是否是ael亚型并不可靠，ael表型可能是由于GTA突变体降低了酶的稳定性所致.
Keywords: ABO blood group; ael subtype; genotyping; molecular model.