Qin Lian-Hua, Ma Zhan-Zhong, Zheng Rui-Juan, Liu Zhong-Hua, Lu Jun-Mei, Hu Zhong-Yi
Shanghai Tuberculosis Key Laboratory, Shanghai Pulmonary Hospital Affiliated to Tongji University, Shanghai 200433, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2008 Jun;31(6):453-8.
To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX technology.
An in vitro synthesized 78 per random DNA library was subjected to 12 rounds of selection by SELEX (Systematic evolution of ligands by exponential enrichment) method against MPT64 protein. Binding of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. DNAMAN package was employed to analyze the sequences and the second structures of the aptamers. Moreover, target protein was bound to one aptamer and another aptamer modified with biotin together forming a sandwich-like complex, which was captured in microwell, to be tested in negative group including BCG and reference strains from nontuberculous mycobacteria, and positive group including H37Rv, Mycobacterium bovis reference strain, and clinical strains from Mycobacterium tuberculosis.
After 12 rounds of selection, high-affinity aptamers to MPT64 was obtained. The OD value at 450 nm of affinity of aptamers to MPT64 protein was from 0. 492 to 1.243, in which 73.3% was over 1.0. Pocket and stem-loops was the basis of aptamers binding to MPT64 protein by the analysis of structures,with several GC pairs among bridges between pocket and stem-loops. The analysis of the sandwich-like complex system based on two aptamers and protein showed that the positive percentage was 87. 9% in the positive group while the negative percentage was 85.7% in the negative group, with positive H37Rv and Mycobacterium bovis, and negative BCG, when the cut-off value for a positive response was 0.61 OD.
A set of aptamers with considerable binding affinity to MPT64 protein were successfully selected from the initial random DNA library.
利用指数富集的配体系统进化技术（SELEX）获得能特异性结合结核分枝杆菌MPT64蛋白的DNA寡核苷酸适配体。
体外合成78个随机DNA文库，采用SELEX方法针对MPT64蛋白进行12轮筛选。通过生物素 - 链霉亲和素 - 辣根过氧化物酶系统检测适配体与蛋白的结合情况。使用DNAMAN软件包分析适配体的序列和二级结构。此外，将靶蛋白与一个适配体结合，另一个用生物素修饰的适配体一起形成夹心样复合物，捕获于微孔板中，在包括卡介苗和非结核分枝杆菌参考菌株的阴性组以及包括H37Rv、牛分枝杆菌参考菌株和结核分枝杆菌临床菌株的阳性组中进行检测。
经过12轮筛选，获得了对MPT64具有高亲和力适配体。适配体与MPT64蛋白亲和力在450nm处的OD值为0.492至1.243，其中73.3%超过1.0。通过结构分析，口袋和茎环是适配体与MPT64蛋白结合的基础，口袋和茎环之间的桥中有几个GC对。基于两个适配体和蛋白的夹心样复合物系统分析表明，当阳性反应的临界值为0.61OD时，阳性组的阳性率为87.9%，阴性组的阴性率为85.7%，H37Rv和牛分枝杆菌为阳性，卡介苗为阴性。
从初始随机DNA文库中成功筛选出一组对MPT64蛋白具有相当结合亲和力的适配体。
