磁珠-流式细胞术SELEX技术的建立

Establishment of magnetic beads-flow cytometry SELEX

目的:建立磁珠—流式细胞术SELEX技术方法。方法:不对称PCR扩增并制备ssDNA文库,选用商品化的、表面包被链霉亲合素的磁珠作为分离介质进行SELEX筛选,流式细胞术检测筛选得到的配基与链霉亲合素结合的特异性,及配基竞争生物素与链霉亲合素的结合情况。结果:筛选获得了特异识别链霉亲合素的ssDNA配基,流式细胞术检测确定:筛选得到的配基与链霉亲合素特异结合,并能竞争生物素与链霉亲合素的结合。结论:成功建立了磁珠—流式细胞术SELEX技术方法。

Objective： To establish the magnetic beads-flow cytometry SELEX method. Methods： Asymmetric PCR was used to amplify random single strangded DNA （ssDNA） pool, streptavidin-coated magnetic beads were used as the target in the SELEX selection process. The specificity of aptamers obtained were detected by flow cytometry using FITC（fluorescein isothiocyanat）-labeled aptamers, and the inhibiter activity of aptamer to combine streptavidin and biotin were measured by flow cytometry. Results： After 12 round SELEX selections, ssDNA pool was enriched, and streptavidin-specific ssDNA aptamers were obtained. Aptamers obtained could specifically bind to streptavidin-coated magnetic beads, and inhibit the combination of streptavidin and biotin. Conclusion： The feasible magnetic beads-flow cytometry SELEX method was established successfully.