结核分枝杆菌ESAT-6抗原适体的筛选与亲和性分析

Screening and affinity analysis of aptamers to ESAT-6 protein from Mycobacterium tuberculosis

目的通过建立指数级富集配体的系统进化技术(SELEX),筛选结核分枝杆菌ESAT-6抗原适体的方法,获得ESAT-6的高亲和性适体,为结核病诊断和治疗试剂的开发奠定基础。方法体外构建一个长度为78个碱基的随机单链DNA(ssDNA)文库,以微孔板为筛选介质,SELEX技术筛选获得ESAT-6的适体库,将适体库克隆、测序,用DNAMAN软件对其结构进行分析,利用生物素-链亲和素-辣根过氧化物酶显色系统测定亲和力,找出高亲和性适体。结果经过13轮筛选,ssDNA文库与ESAT-6亲和力从0.257提高到1.163,且12轮后A值趋于平衡,克隆、测序获得ESAT-6的高亲和性适体,其中适体A10亲和力最高,达到1.515,且与CFP10-ESAT6融合蛋白无交叉反应。二级结构显示,口袋和茎环结构可能是适体与ESAT-6结合的结构基础。结论成功建立了SELEX筛选技术,并初步获得了ESAT-6的高亲和性适体。

Objective To set up a method of systematic ew）lution of ligands by exponential enrichment （SELEX） for＇ screening and obtain the high-affinity aptamers to ESAT-6 protein from Mycobac＇terium tubercuh）sis, lay a foundation of developing a diagnostic and therapeutic reagent of TB. Methods A 78 nt random single-strand DNA （ssDNA） library was designed and synthesized in vitro. Selected by 96 well plate,obtained aptamers to ESAT-6 protein by SELEX method, the aptamers was cloned and sequenced. DNAMAN package was employed to analyze the conserved sequences and the second structure of the aptamers, affinity of aptamers to ESAT-6 was visualized by biotin-streptavidin-horseradish peroxidase system. Results After 13 rounds of selection,high-affinity aptamers to ESAT-6 was accessed. The affinity of aptamer A3 to ESAT-6 protein was highest,with A 1. 515. The second structure analysis revealed possible pocket and stem-loops for binding to ESAT-6. Conclusion The high-affinity aptarners binding to ESAT-6 was successfully selecting from the initial random ssDNA library.