SELEX技术筛选纤维蛋白适配子的研究

目的:用纤维蛋白作为靶物质对ss DNA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ss DNA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ss DNA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。

The Study of Screening Fibrin Aptamer by SELEX Technology

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.