| 李保玉 刘家云 屈园利 董雯 李旭枝 龙铟.SELEX技术筛选纤维蛋白适配子的研究[J].,2015,15(11):2037-2041 |
| SELEX技术筛选纤维蛋白适配子的研究 |
| The Study of Screening Fibrin Aptamer by SELEX Technology |
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| DOI： |
| 中文关键词: 纤维蛋白 适配子 SELEX PCR |
| 英文关键词: Fibrin Aptamer SELEX PCR |
| 基金项目:国家自然科学基金项目（81073095） |
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| 中文摘要: |
| 目的：用纤维蛋白作为靶物质对ssDNA 随机序列文库进行筛选，旨在获得高亲和力的纤维蛋白适配子。方法：在体外人工合
成长度为99 个核苷酸的ssDNA 随机序列文库，文库中间区域为63 个核苷酸的随机序列，两端为18 个核苷酸的固定的引物序
列；然后以羧基磁珠为介质包被纤维蛋白，利用指数级富集的配体系统进化技术（SELEX）对ssDNA 随机序列文库进行反复筛
选，当结合率不再提高时对筛选出的适配子进行连接、转化及测序分析。结果：羧基磁珠成功地包被了纤维蛋白，包被效率为
87.65%，经15 轮逐步递增压力的筛选，获得了纤维蛋白适配子群，经测序分析比对发现适配子有很好的多样性。结论：应用
SELEX技术初步筛选出了亲和力较高的纤维蛋白适配子群，为下一步的鉴定及功能研究奠定了良好基础。 |
| 英文摘要: |
| Objective:In order to obtain the high affinity aptamers of fibrin, fibrin was used as target for screening random
oligonucleotides library.Methods:A single-stranded DNA(ssDNA) random oligonucleotides library of 99 nucleotides was constructed in
vitro, with 63 nucleotides random sequence at middle region and 18 nucleotides immobilized primer sequences at both ends. The
carboxyl magnetic beads coated with fibrin were repeatedly screened by the stematic evolution of ligand by exponential enrichment
(SELEX) technique. When the combination rate was no longer increased, the screened aptamers pool were cloned into T vector and
transformed into Escherichia coli competent cells, and positive clones were sequenced.Results:We successfully coated the carboxyl
magnetic beads with fibrin and the efficiency was 87.65%. The fibrin aptamers were obtained after 15 cycles of gradually increasing
screening pressure by SELEX technique. Sequence alignment results showed a good diversity of aptamers.Conclusion:We successfully
screened the high affinity aptamers of fibrin by SELEX technique, this would lay a good foundation for identification and functional
research of next experiment. |
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