全细胞SELEX法筛选单增李斯特菌的ssDNA适配体

Selection of ssDNA Aptamers of Listeria monocytogenes by Whole SELEX

摘要:

以单增李斯特菌活细胞为靶标，采用指数富集配基的系统进化技术（Systematic Evolution of Ligands by Exponential Enrichment，SELEX）从含40个随机碱基序列的单链DNA（single- stranded DNA，ssDNA）文库中筛选与之特异性结合的适配体。酶联配体吸附法（ELASA）测定筛选过程中次级文库与单增李斯特菌结合力的变化。对筛选得到的适配体进行序列测定，RNA structure软件预测二级结构，ELASA测定适配体与靶标的结合能力和特异性。结果显示，每轮筛选后次级文库与靶标的亲和力增强，特异性适配体逐步得到富集。测序获得23条适配体，其序列同源性不高，但预测的二级结构有些相似，根据二级结构将其分为3个群，分析结果表明二级结构与适配体的亲和力有关。挑选亲和力较强的21号(Lma-21)和35号(Lma-35)适配体能特异性地识别单增李斯特菌。本研究为开发食源性病原菌单增李斯特菌的新型检测试剂提供了基础材料。

Abstract:

In this paper, aptamers against Listeria monocytogenes live cells were screened by systematic evolution of ligands by exponential enrichment (SELEX) from single-tranded DNA (ssDNA) library which contained 40 random bases. During the SELEX selection, affinity ability between ssDNA sub-library and L. monocytogenes was evaluated by enzyme linked aptamer absorbent assay (ELASA). The characteristics of aptamers including sequence, secondary structure, affinity and specificity to L. monocytogenes were studied. The results revealed that aptamers were enriched after 15 SELEX rounds and the affinity against target enhannced round by round. The aptamers of 23 different sequences obtained via sequencing were divided into 3 groups, according to the similarity of secondary structure predicted by RNA structure program. Also, it indicated that the secondary structure was related with the binding ability against the target. Two aptamers named Lma-21 and Lma-35 with high affinity could specifically recognize L. monocytogenes. This work provided necessary materials for the development of new type of detection reagents against L. monocytogenes.