Evaluation of anti-tuberculosis effect of Mycobacterium tuberculosis polyphosphate kinase 2 aptamer / 上海交通大学学报（医学版）

ABSTRACT

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 trend with the increase of PPK2 aptamer concentration,which indicated that PPK2 aptamer had an inhibitory effect on H37Rv growth.Conclusion·PPK2 aptamer has good antibacterial activity against H37Rv in vitro.