结核分枝杆菌MPT64蛋白适配子的筛选与鉴定

Selecting and affinity analysis of DNA aptamers to MPT64 protein from Mycobacterium tuberculosis

目的利用指数富集配体系统进化（SELEX）技术筛选能与结核分枝杆菌分泌蛋白MPT64特异性结合的寡核苷酸适配子，寻找早期诊断结核病的方法。方法体外合成随机单链DNA（ssDNA）文库，以MPT64蛋白为靶物质，采取SELEX技术进行12轮筛选，将适配子库克隆、测序后，用DNAMAN软件对其结构进行分析，经生物素-链亲和素-辣根过氧化物酶显色系统测定亲和力，并利用捕获适配子与检测适配子组成的“三明治”夹心法对获得的适配子进行初步验证。将13个非结核分枝杆菌菌株和BCG菌株作为阴性组计为0，将结核分枝杆菌和牛结核分枝杆菌组作为阳性组计为1，采用MedCalc软件分析受试者操作特征曲线，确定最佳阳性判定值，并构建散点图。结果经12轮筛选后，随机挑选15个适配子与MPT64蛋白的亲和性进行分析，吸光度值为0．492—1．243，73．3％的适配子的吸光度值在1．0以上；二级结构分析显示，适配子与MPT64蛋白亲和性的基础主要是大口袋茎环结构，口袋与环之间的茎桥含有不同数量的GC碱基对；“三明治”夹心法对阴性组[非结核分枝杆菌标准菌株及卡介苗（BCG）株]和阳性组（结核分枝杆菌实验室H37Rv株及临床株、牛结核分枝杆菌标准株）共47个菌株培养上清的检测结果显示，在临界（cut-off）值为0．61时，H37Rv、牛结核分枝杆菌组为阳性，BCG株为阴性；阴性组标本的阴性检出率为85．7％，阳性组标本的阳性检出率为87．9％，表现出一定的检测价值。结论已初步筛选到与MPT64蛋白具有高亲和性的DNA适配子。

Objective To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX technology. Methods 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 biotion 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 H37 Rv, Mycobacterium bovis reference strain, and clinical strains from Mycobacterium tuberculosis. Results 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 H37 Rv and Mycobacterium bovis, and negative BCG, when the cut-off value for a positive response was 0. 61 OD. Conclusion A set of aptamers with considerable binding affinity to MPT64 protein were successfully selected from the initial random DNA library.