生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
6期
824-827
,共4页
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
李慧%趙彊%樑超%丁紅梅%李潔%黃皚雪%囌雪婷%張令彊%李少華%邵寧生
리혜%조강%량초%정홍매%리길%황애설%소설정%장령강%리소화%소저생
Western印迹%SELEX%电泳%适配体%未纯化蛋白样品
Western印跡%SELEX%電泳%適配體%未純化蛋白樣品
Western인적%SELEX%전영%괄배체%미순화단백양품
Western blot%SELEX%ssDNA apramer%electrophoresis%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ssDNA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。
目的：建立一種基于Western印跡的指數式富集的配體繫統進化（SELEX）技術，用于未純化蛋白樣品覈痠適配體篩選。方法：將目的蛋白經SDS-PAGE分離後轉移到PVDF膜上，用生物素標記的ssDNA與PVDF膜上的蛋白共同孵育，穫得能與靶蛋白特異結閤的適配體，最後通過生物素-鏈黴親和素-辣根過氧化物酶繫統、基因剋隆測序、MEME在線軟件和RNAstructure軟件分析適配體的一、二級結構，併對篩選得到的適配體進行鑒定。結果：經過4輪篩選，穫得瞭能特異識彆靶蛋白而不識彆無關蛋白的適配體，原庫Gp45則與上述蛋白均沒有結閤。結論：建立瞭Western印跡-SELEX技術，可用于未純化蛋白樣品覈痠適配體篩選。
목적：건립일충기우Western인적적지수식부집적배체계통진화（SELEX）기술，용우미순화단백양품핵산괄배체사선。방법：장목적단백경SDS-PAGE분리후전이도PVDF막상，용생물소표기적ssDNA여PVDF막상적단백공동부육，획득능여파단백특이결합적괄배체，최후통과생물소-련매친화소-랄근과양화물매계통、기인극륭측서、MEME재선연건화RNAstructure연건분석괄배체적일、이급결구，병대사선득도적괄배체진행감정。결과：경과4륜사선，획득료능특이식별파단백이불식별무관단백적괄배체，원고Gp45칙여상술단백균몰유결합。결론：건립료Western인적-SELEX기술，가용우미순화단백양품핵산괄배체사선。
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.
