ABO基因内含子拼接位点突变影响糖基转移酶活性表达机制的研究

项目基于实验室前期研究在ABO变异型个体中发现的c.28+1-2delGT、c.28+5G>A、c.155+5G>A、c.204-1G>A和c.374 +5G>A内含子拼接位点突变，而内含子拼接位点序列改变常可引起RNA剪接变化；因此提出这些内含子拼接位点突变将通过RNA剪接形成相应外显子序列缺失的mRNA剪接体，进而引起编码蛋白氨基酸和蛋白酶促活性的改变，导致变异型个体抗原表达减弱或不表达的科学假设。为验证该假设，项目通过构建外周血干细胞体外诱导形成红细胞平台，明确这些突变引起的ABO基因剪接体在红系细胞转录情况；综合利用基因重组和体外表达、免疫荧光、酶促活性测定、蛋白结构分析等技术，阐明突变基因对相关糖蛋白合成、分泌、活性表达等功能的影响。项目将明确这些突变点引起ABO变异型抗原差异表达的机制，这对于准确地鉴定和调控ABO变异抗原有重要的意义，有利于提升血液安全。

c.28+1-2delGT、c.28+5G>A、c.155+5G>A、c.204-1G>A和c.374+5G>A mutations were previously found in ABO variant individuals in our laboratory, and the exon-intron splicing site mutations often lead to RNA splicing change. Therefore, a hypothesis is proposed as below: these ABO introns splicing site mutations in the ABO variant individuals will lead to produce different mRNA forms with some exons sequences missing through RNA splicing step, and then it could result in the amino acids and enzyme activity changes in the coding proteins, which will lead to the weakening or non expression antigen in the variant individuals. In order to verify the hypothesis, a erythroid cells generation platform in vitro induced from peripheral blood stem cells was established, and used to test the transcription of ABO gene splices in the erythroid cells caused by these mutations.The effects of the mutated genes on the synthesis, secretion, active expression of the related proteins were determined using the methods including gene recombination, expression in vitro, immunofluorescence, enzyme activity determination, protein structure analysis and so on. The mechanism of the antigen differential expression in the ABO variant caused by these mutations sites will be clarified in the project, which is of great significance for accurate ABO antigen identification and regulation of ABO variants, and will help to improving blood safety.