穿孔素适配体的筛选及电化学传感器的制备

Screening of perforin aptamers and preparation of electrochemical aptasensor

目的研发能特异性结合穿孔素的DNA适配体并构建适配体电化学传感器,为发展穿孔素的新型检测技术提供基础。方法体外合成全长为81个碱基并含有35个随机寡核苷酸的单链DNA文库,库容量大约为1×1010,以穿孔素蛋白为靶标,利用指数富集配体系统进化技术(SELEX),从单链DNA文库中筛选能够结合穿孔素蛋白的DNA适配体;采用M-fold程序(version 3.2)预测二级结构;设计并构建适配体电化学传感器,进行穿孔素电化学特异性检测,绘制标准曲线。结果经多轮筛选,获得6条穿孔素蛋白的DNA适配体,分别为序列SEQ1~SEQ6,适配体SEQ6能够特异性结合穿孔素,并可通过电化学方法用于检测穿孔素,而其与牛血清清蛋白、γ-干扰素、肿瘤坏死因子α结合较弱。该电化学传感器检测穿孔素的线性水平范围为1~20nmol,检出限为0.7nmol。结论成功利用SELEX筛选获得特异结合穿孔素的DNA适配体,其在研发针对穿孔素的新型检测技术方面具有应用潜能。

ObjectiveTo research and develop DNA aptamers specifically combining with perforin and to construct an aptamers electrochemical aptasensor to provide a basis for developing perforin new type detection technique.MethodsThe total length of 81 basic groups and single stranded DND library containing 35 random oligonucleotides were synthesized in vitro.The DNA library capacity was approximately 1×10^10.The DNA aptamers capable binding with perforin were screened by using the systematic evolution of ligands by exponential enrichment（SELEX）technique from single stranded DNA library with perforin protein as target.The secondary structures of aptamers were predicted by MFold program（version 3.2）.The electrochemical aptasensor was design and constructed for conducting the perforin electrochemical specificity detection and drawing the standard curve.ResultsSix perforin protein DNA aptamers were obtained by multiple screening,which were sequences SEQ1-SEQ6 respectively.The aptamer SEQ6 could specifically bind with perforin and could be used to detect perforin by electrochemical method,while its binding with BSA,TNF α and IFN γ were all weaker.The aptasensor exhibited a linear range of 1-20 nmol for detecting perforin with a detection limit of 0.7 nmol.ConclusionThe DNA aptamers specifically combining with perforin is successfully obtained by using the SELEX technique,which may have application potentials in the aspects of researching and developing new type detection technique aiming at perforin.