应用SELEX技术筛选人类血型IgG适体的实验研究

目的:建立Rh(D)血型的IgG类抗体适体的筛选方法,为后续合成适体并进行新生儿溶血病的防治研究奠定基础。方法:利用SELEX技术,构建含有52个随机序列的单链DNA文库,利用硝酸纤维素膜法筛选与IgG类抗体分子高亲和力结合的核酸分子,同时探索并优化将其扩增为双链DNA核酸库的PCR反应条件;通过膜结合实验检测核酸分子的富集效果并用凝胶阻滞实验初步测定所得核酸适体与Rh(D)血型IgG类抗体的亲和力。结果:随着筛选的进行,核酸分子的富集库向着与靶分子亲和性增强的方向进化,经过了11个循环,筛选出与Rh(D)血型IgG抗体结合力较强的核酸分子,与核酸结合的IgG分子在凝胶阻滞实验中显示出阻滞带。结论:初步建立了利用SELEX技术筛选人类Rh(D)血型IgG抗体适体的方法。

Research on Applicating SELEX Technology Screening Human Blood IgG Aptamer

Objective:To establish the screening methods of human Rh (D) blood IgG aptamers to make foundation for the subsequent synthesis of aptamers and prevention of hemolytic disease of the newborn.Methods:By using the SELEX technology, single-stranded DNA library was constructed containing 52 randomsequences. The nucleic acid molecules of high affinity with IgG class antibody were screened by using the nitrocellulose membrane. Meanwhile, the experimental conditions of amplifying the nucleic acid into double-stranded DNA library were explored and optimized, and the enrichment of nucleic acid molecules was examined by membrane binding assay. The affinity between Rh (D) blood type IgG class antibodies with its aptamer was preliminarily determined by gel shift assay.Results:With the ongoing of screening, nucleic acid molecules enriched library got evolution toward the direction of enhanced affinity with the target molecule. After 11 loops, the nucleic acid molecule of strong affinity with Rh (D) blood IgG antibody was found out, and it shown blocked belt after binding with IgG molecules in gel shift assay.Conclusion:The screening method on aptamer of human Rh (D) blood IgG antibody based on SELEXtechnology was established initially.