Antituberculosis Effect Evaluation Of Mycobacterium Tuberculosis PPK2 Protein Nucleic Acid Aptamers

ObjectiveTo investigate the antibacterial effect of mycobacterium tuberculosis polyphosphate kinase 2?PPK2?aptamer co-cultured with Mycobacterium tuberculosis?MTB?in vitro.Provide new directions for the development of new highly effective and low-toxic anti-tuberculosis drugs.Method1.Bioinformatics analysis was used to analyze the homology of amino acid sequence of PPK2 protein with common pathogenic bacteria in the respiratory tract,and a phylogenetic tree of PPK2 was constructed.2.PPK2 aptamer and MTB standard strain H₃₇Rv,BCG?BCG?,Mycobacterium smegmatis,Pseudomonas aeruginosa,Acinetobacter baumannii,through enzyme-linkedoligonucleotide(enzyme-linked oligonucleotide analysis The binding affinity of PPK2 aptamer to H₃₇Rv was determined by assay,ELONA)method.The binding affinity of PPK2aptamer to normal human and tuberculosis patient's serum of PPK2 was detected by the same method.3.The PPK2 aptamer was incubated with different concentrations of serum for 24 hours,and its stability in different concentrations of serum was analyzed by agarose gel electrophoresis.4.The minimal inhibitory concentration?MIC?of PPK2 aptamer against H₃₇Rv was determined by trace resazurin assay.5.Amplify H₃₇Rv and 1?mol/L PPK2 aptamers into Roche medium and observe the effect of PPK2 aptamer and random sequence on the growth of H₃₇Rv.Co-cultivation of H₃₇Rv with PPK2 aptamer and mutants for 10 days.The D?600nm?value was measured by microplate reader and the effect of PPK2 aptamer on the growth of H₃₇Rv was observed.6.The aptamers of H₃₇Rv and PPK2 were co-cultured for 5 weeks.The effect of PPK2 aptamer on the growth of MTB biofilm was observed by crystal violet staining.7.The PPK2 aptamer was co-cultured with the cells to observe the cytotoxicity of the aptamer.Result1.Bioinformatics analysis and construction of PPK2 phylogenetic tree revealed that the PPK2 protein of H₃₇Rv is closely related to common pathogens in the respiratory tract,and is relatively close to the phylogenetic relationship with Pseudomonas aeruginosa.2.The ELONA assay showed that the PPK2 aptamer selectively binds to the PPK2 protein of H₃₇Rv.In normal human and tuberculosis patients,the PPK2 aptamer shows good affinity in the serum of tuberculosis patients.3.Agarose gel electrophoresis analysis of PPK2 aptamers in the serum at least 8h stable presence.4.The Minimum inhibitory concentration?MIC?of PPK2 aptamer to Mycobacterium tuberculosis standard strain H₃₇Rv was 50nmoL/L.5.Colony growth in Roche medium showed that the PPK2 aptamer had an inhibitory effect on the growth of H₃₇Rv.The growth inhibition assay showed that the D?600nm?value of H₃₇Rv showed a decreasing trend with the increase of aptamer concentration of PPK2,indicating that the PPK2 aptamer inhibited the growth of H₃₇Rv.6.The PPK2 aptamer was co-cultured with MTB for 5 weeks.The crystal violet staining assay showed that the PPK2 aptamer inhibited the formation of MTB biofilm.7.Cytotoxicity tests showed that high and low concentrations of PPK2aptamers were not toxic to cells.Conclusion1.PPK2 protein aptamer showed good antibacterial activity against H₃₇Rv in vitro.2.The PPK2 aptamer binds selectively to the tuberculosis patient serum PPK2 protein.