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 |
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引用本文: | 李慧,;赵强,;梁超,;丁红梅,;李洁,;黄皑雪,;苏雪婷,;张令强,;李少华,;邵宁生.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[J].生物技术通讯,2014(6):824-827. |
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作者姓名: | 李慧 ;赵强 ;梁超 ;丁红梅 ;李洁 ;黄皑雪 ;苏雪婷 ;张令强 ;李少华 ;邵宁生 |
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作者单位: | [1]军事医学科学院基础医学研究所,北京100850; [2]军事医学科学院放射与辐射医学研究所,北京100850 |
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基金项目: | 国家高技术研究发展计划(2012AA022501) |
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摘 要: | 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。
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关 键 词: | Western印迹 电泳 适配体 未纯化蛋白样品 |
Establishment of Western Blot-SELEX Used for Selection of Aptam-ers of Unpurified Protein Samples |
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Institution: | LI Hui, ZHAO Qiang, LIANG Chao, DING Hong-Mei, LI Jie, HUANG Ai-Xue, SU Xue-Ting, ZHANG Ling-Qiang, LI Shao-Hua, SHAO Ning-Sheng (a. Institute of Basic Medical Sciences; b. Institute of Radiation Medicine; Academy of Military Medical Sciences, Beijing 100850, China) |
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Abstract: | 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. |
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Keywords: | SELEX Western blot SELEX ssDNA apramer electrophoresis unpurified protein samples |
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