结核分枝杆菌多聚磷酸盐激酶2 核酸适配体的抗结核效果评价
Evaluation of anti-tuberculosis effect of Mycobacterium tuberculosis polyphosphate kinase 2 aptamer

目的· 探讨结核分枝杆菌（Mycobacterium tuberculosis，MTB）多聚磷酸盐激酶2（polyphosphate kinase 2，PPK2）核酸适配 体对体外MTB 的抑菌效果。方法· 运用生物信息学方法分析MTB PPK2 与呼吸道部分常见病原菌的同源性，构建PPK2 进化树。通 过酶联寡核苷酸分析（enzyme-linked oligonucleotide assay，ELONA）测定PPK2 核酸适配体与MTB 标准株H37Rv、卡介苗（BCG）、 耻垢分枝杆菌、铜绿假单胞菌、鲍曼不动杆菌的结合亲和力。将PPK2 核酸适配体加入血清中孵育24 h，运用琼脂糖凝胶电泳分析其 在血清中的生物稳定性。采用微量刃天青显色法测定PPK2 核酸适配体对H37Rv 的最低抑菌浓度（minimum inhibitory concentration， MIC）。将H37Rv 与1 μmol/L 的PPK2 核酸适配体在罗氏培养基上培养10 d，观察菌落生长情况。运用酶标仪测定与不同浓度的PPK2 核酸适配体共培养10 d 后H37Rv 菌液的D(600 nm) 值，观察PPK2 核酸适配体对H37Rv 生长的影响。结果· 生物信息学方法构建的 PPK2 进化树显示，H37Rv 的PPK2 蛋白与呼吸道部分常见病原菌亲缘性较远，与铜绿假单胞菌亲缘关系相对较近。ELONA 测定结果 显示PPK2 核酸适配体能与H37Rv 选择性结合。琼脂糖凝胶电泳分析显示PPK2 核酸适配体在血清中至少稳定存在8 h。PPK2 核酸适 配体对H37Rv 的MIC 为50 nmol/L。罗氏培养基菌落生长结果显示PPK2 适配体对H37Rv 生长具有抑制作用。生长抑制试验表明随着 PPK2 核酸适配体浓度增加，H37Rv 的D(600 nm) 呈现下降趋势，说明PPK2 核酸适配体对H37Rv 生长存在抑制作用。结论· PPK2 核 酸适配体对体外H37Rv 表现出良好的抑菌活性。
：Objective · To investigate the bacteriostasis effect of Mycobacterium tuberculosis (MTB) polyphosphate kinase 2 (PPK2) aptamer on MTB in vitro. Methods · The bioinformatics method was used to analyze the homology of MTB PPK2 and common pathogens of respiratory tract, and the PPK2 phylogenetic tree was constructed. The binding affinity of the PPK2 aptamer to H37Rv, BCG, Mycobacterium smegmatis, Pseudomonas aeruginosa and Acinetobacter baumannii was analyzed by enzyme-linked oligonucleotide assay (ELONA). The PPK2 aptamer was incubated for 24 h in serum and its biological stability in serum was analyzed by agarose gel electrophoresis. The minimum inhibitory concentration (MIC) of the PPK2 aptamer to H37Rv was determined by micro-azure method. H37Rv was inoculated with 1 μmol/L PPK2 aptamer or random sequence on Roche culture medium for 10 d and colony growth status was observed. H37Rv was co-cultured with different concentrations of PPK2 aptamer for 10 d, absorbance at 600 nm was measured by microplate reader. The effect of PPK2 aptamer on the growth of H37Rv was observed. Results · PPK2 phylogenetic tree constructed by bioinformatics analysis showed that PPK2 protein of H37Rv was not closely related to the common pathogens of respiratory tract, and it was relatively close to Pseudomonas aeruginosa. The ELONA assay results showed that the PPK2 aptamer binded selectively to H37Rv. Agarose gel electrophoresis analysis showed PPK2 aptamer in serum was at least stable for 8 h. The MIC of the PPK2 aptamer to H37Rv was 50 nmol/L. The colony growth of Roche culture showed that PPK2 aptamer had an inhibitory effect on H37Rv growth. Growth inhibition test showed that the absorbance at 600 nm of H37Rv showed a decreasing