阪崎克罗诺菌特异性适配子筛选研究

Screening of adaptor specific for Cronobacter sakazakii

目的通过SELEX(指数富集配基系统进化技术)筛选阪崎克罗诺菌特异性DNA适配子,为建立基于适配子技术的阪崎克罗诺菌快速检测方法奠定基础。方法在阪崎克罗诺菌比较基因组分析、蛋白亚细胞定位预测、蛋白跨膜区预测分析、基因mRNA表达量分析、基因克隆表达和蛋白纯化基础上,制备用于适配子筛选的靶蛋白。利用靶蛋白偶联羧基磁珠,筛选ssDNA适配子。并对适配子的靶蛋白结合亲和力和目标菌捕获特异性进行分析。结果适配子S1与靶蛋白结合亲和力较高,对阪崎克罗诺菌有较好捕获能力,捕获率最高为23.1%。对大肠埃希氏菌、沙门氏菌等非目标菌不具有明显的捕获能力,具有较好的阪崎克罗诺菌捕获特异性。结论适配子S1和S2的二级结构以茎环结构和发夹结构为主。

The aim of this study is to screen the specific adaptor of Cronobacter sakazakii for future development of rapid detection technique. Based on comparative genomics analysis of C.sakazakii strains, protein sub cellular localization prediction and protein trans membrane regions prediction, genes mRNA expression level assay, gene clone and protein over expression, certain outer membrane protein was selected and prepared as target protein for adaptor screening. Subsequently, purified target outer membrane protein was bound to carboxyl magnetic beads to screen ssDNA adaptor. The binding affinity of adaptor to target protein and capture specificity for C.sakazakii strain were assayed. It was demonstrated that adaptorS1 presented relatively high affinity with target protein and high capture capability for C.sakazakii strain. Predicted secondary structure of S1 and S2 revealed that loop and hairpin existed generously in these adaptors.