Isolation And Identification Of A New Myxococcus And Studies On Its Antitumor Activity Substance

Myxobacteria is a kind of aerobic gram-negative bacteria,belongs to the delta branches of Proteobacteria.Myxobacteria are famous for their complicated multicellular behavior and excellent ability of producing secondary metabolites,which have important application in medicine,agriculture and ecological environment.Myxobacteria has been attracted more and more researchers.This paper isolated myxoccocus new strain from the soil,and obtained the antitumor activity substance from the fermentation supernatant of the myxoccocus,and study of its mechanism of antitumor.The study isolated myxobacteria from the soil sample collected from parts of the country through inactivated E.coli inducing method,studied of the antitumor activity of the isolated myxobacteria,and screen out strain S54 which has important antitumor activity.identified and catalogued strain S54 as Myxococcus macrosporus STXZ54 according to morphological observation,physiological and biochemical characteristics and the homologous analysis of 16S rRNA sequences of nucleotides.16S rRNA gene sequences of strain S54 were submitted to the GenBank database under accession number:KF482866.This study enrich the resources of myxobacteria strains,which provided an important basis for excavating the secondary metabolite of myxobacteria.The antitumor activity of S54 was studied by cytotoxicity test.The growth curve of strain S54 and its corresponding period antitumor activity were measured.The fermentation supernatant has antitumor activity against B16,Hela,4T1,Hep-3B with IC50 values of 3?L biomass of the fermentation broth,and HCT-116 cell with IC50 values of 10 ?L.S54 was different from most myxobacteria which need more time in adjustment period,S54 can turn into logarithmic phase rapidly after inoculating for 8 h.The changes in the anti-tumor activity of the fermentation broth after teated with different methods,which contains temperature gradient,organic solvent treatment,different pH treatment,ammonium sulfate precipitation,acetone precipitation.The antitumor activity of fermentation broth was not detected when put in 80? for half a hour.and the antitumor substances was not stable when teated with methanol,acetonitrile,ethanol.The precipitate but not the supernatant had antitumor anctivity after teated by 75%ammonium sulfate precipitation.The precipitate of acetone precipitation had antitumor activity.so,the purpose antitumor substance may be a kind of protein.The antitumor substances were separated and purified.Firstly,crude extract was obtained through ammonium sulfate precipitation and acetone precipitation.Then the crude extract was purified by cation exchange chromatography,gel chromatography,and obtained pure substance named as SGF5 which showed a single band on SDS-PAGE strip.The active component SGF5 has antitumor activity against B16,Hela,4T1,Hep-3B.With the increase of drug concentration and response time,the inhibition rate of tumor cells corresponding increased.The active component SGF5 has antitumor activity against B16,Hela,4T1 with IC50 values of 2.20?g/mL?2.767?g/mL?3.758?g/mL,which indicate that SGF5 can inhabit kinds of tumor cells.When tumor cells were treated with DAPI,tubulin Alexa Fluor 488 and MitoTracker immunofluorescence,Subcellular structure of B16 cells were unnormal observed by confocal laser scanning microscope after treated with SGF5 for 24h:Cells from the spindle to a circle,pleomorphic nuclei showing of,nuclei shows pleomorphic,tubulin cytoskeleton degradation lead to collapse.All above mentioned gave us a preliminary presumption that SGF5 can induce apoptosis of B16 cells.The isolated Myxococcus macrosporus STXZ54 has a significant effect on tumor cells,which was worth exploiting as potential antitumor drugs.