用于未纯化蛋白样品核酸适配体筛选的Western印迹-SELEX筛选技术的建立

Establishment of Western Blot-SELEX Used for Selection of Aptamers of Unpurified Protein Samples

目的:建立一种基于Western印迹的指数式富集的配体系统进化(SELEX)技术,用于未纯化蛋白样品核酸适配体筛选。方法:将目的蛋白经SDS-PAGE分离后转移到PVDF膜上,用生物素标记的ss DNA与PVDF膜上的蛋白共同孵育,获得能与靶蛋白特异结合的适配体,最后通过生物素-链霉亲和素-辣根过氧化物酶系统、基因克隆测序、MEME在线软件和RNAstructure软件分析适配体的一、二级结构,并对筛选得到的适配体进行鉴定。结果:经过4轮筛选,获得了能特异识别靶蛋白而不识别无关蛋白的适配体,原库Gp45则与上述蛋白均没有结合。结论:建立了Western印迹-SELEX技术,可用于未纯化蛋白样品核酸适配体筛选。

Objective： To develop a rapid and efficient SELEX technology based on Western blot used for selec？tion of aptamers of unpurified protein samples. Methods： Firstly, the target protein was transferred to PVDF mem？brane after SDS-PAGE. Secondly, the PVDF membrane which contained protein was used as the target in the SELEX selection process. The aptamers to specific bind target protein were obtained by Bio-labeled ssDNA pool incubated with PVDF membrane. Finaly, the aptamers were selected and identified by stabilized streptavidin-horse？radish peroxidase, gene cloning and sequencing. MEME online software and RNAstructure analysis software were employed to predict the secondary structures of the aptamers. Results： After 4 round SELEX selections, ssDNA pool was enriched, and target protein-specific ssDNA aptamers were obtained. The specific aptamers could only bind to target protein but not bind to negative protein .The Bio-Gp45 couldn’t bind to both target protein and negative protein. Conclusion： The Western blot- SELEX technology which could be used for selection of aptamers of unpurified protein samples was established successfully.