人凝血因子7a核酸适配体的筛选与鉴定

Selection and Characterization of Single Stranded DNA Aptamer for Human Coagulation Factor 7a

人凝血因子7a(FVIIa)是外源性凝血途径的核心因子,检测参与凝血过程的FVIIa及针对FVIIa开发安全有效和经济的凝血剂和抗凝血剂是具有重要的意义。本研究基于指数富集配体系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX),采用酸纤维素膜法,通过11轮筛选成功分离出FVIIa的DNA适配体,并模拟了其二级结构。此适配体与FVIIa蛋白结合的表观解离常数为102nmol/L。同时,基于所获得的高亲和力适配体,建立了基于适配体的荧光检测方法。本方法简单高效,对FVIIa蛋白的定量检测范围达到30~80nmol/L,为人凝血因子7a的检测提供了一条新途径。非变性聚丙烯酰胺凝胶电泳和适配体的荧光检测方法结果表明,此适配体具有良好的选择性。

Human coagulation factor 7a (FVIIa) is the core factor for exogenous coagulation pathway. Developing safe, effective and economical coagulants or anticoagulants is still a challenge. In this study, a single-stranded DNA (ssDNA) aptamer for FVIIa was isolated based on systematic evolution of ligands by exponential enrichment (SELEX), which was based on the isolation of the sequences bound to the target protein, which would be absorbed on nitrocellulose membrane. The aptamer was isolated after 11 rounds of selection. The apparent dissociation constant of the aptamer to FVIIa was 102 nmol/L. The aptamer-based fluorescent assay for FVIIa was established, exhibing simplicity and efficiency. The quantitative detection range of FVIIa by this assay was 10-30 nmol/L, providing a new strategy for detection of FVIIa. The non-denaturation polyacrylamide gel electrophoresis and the aptamer-based fluorescent assay results both showed that the aptamer had good selectivity toward FVIIa.