| 龙宇鹏 谭琳琳 刘利 石小平 任君.应用SELEX技术筛选人类血型IgG适体的实验研究[J].,2014,14(29):5658-5661 |
| 应用SELEX技术筛选人类血型IgG适体的实验研究 |
| Research on Applicating SELEX Technology Screening HumanBlood IgG Aptamer |
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| DOI： |
| 中文关键词: Rh（D）血型 IgG 抗体 适体 配体系统进化的指数富集技术 |
| 英文关键词: Rh（D）blood type IgG antibody aptamer Systematic Evolution of Ligands by Exponential Enrichment |
| 基金项目:重庆市卫生局医学科研计划项目（2011-2-590） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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