炭疽芽孢杆菌芽孢适配子的结构与亲和性分析

STRUCTURES AND AFFINITY ANALYSIS OF APTAMERS TO BACILLUS ANTHRACIS SPORES

为了对炭疽芽孢杆菌疫苗株A .16R芽孢的适配子进行亲和性分析 ,体外构建了 78个碱基的随机DNA库 ,以炭疽芽孢杆菌疫苗株A .16R芽孢为靶标 ,用SELEX技术进行了 15轮的筛选 ,将筛选到的适配子库进行克隆、测序 ,用Macaw 2 .0 5和DNAsisv2 .5软件对每一适配子的保守序列和二级结构进行分析 ,并用生物素 链酶亲和素 辣根酶系统检测 ,根据OD值的高低判定亲和力的大小 ,对每一适配子进行亲和力测定。结果表明 ,适配子的亲和力高低不一 ,OD值最高为 1.2 ,最低为 0 .2 5 ,二级结构分析结果显示 ,茎环和茎等二级结构可能是适配子与芽孢结合的基础 ;其一级结构提示有AGGGG、CCCCG、GGGTT、ACACT等保守序列 。

In order to investigate the affinity of aptamers to Bacillus anthracis spore, a custom synthesized 78 mer random DNA library was subjected to 15 rounds of selection against spores of vaccine strain A.16R by using SELEX method. The selected aptamers were cloned and sequenced. Macaw 2.05 and DNAsis 2.5 package were employed to analyze the conserved sequences and second structure of the aptamers, respectively. Affinities of aptamers to the spores were visualized by biotin streptavidin horseradish peroxidase system. The results showed that affinities of the aptamers were different. The highest OD at 450nm was 1.2, and the lowest was 0.25. The second structure analysis revealed possible stem loops for binding to the spores. The conserved sequences, AGGGG, CCCCG, GGGTT and ACACT, were found and the aptamers having same conserved sequence demonstrated similar affinity to the spores.