CHARACTERISTICS OF EXTRACELLULAR DNA CONTAINING UNMETHYLATED CpG MOTIFS ISOLATED FROM BACILLUS SUBTILIS CULTURE MEDIUM FILTRATE

Olishevsky Sergiy1, Kozak Vyatcheslava1, Yanish Yuriy1, Potebnya Grygoriy1, Lisovenko Galyna1, Olexienko Ivan1, Mazur Olga1, Rybalko Svitlana1, Shlyakhovenko V.A.2

Summary. The application of CpG DNA is one of the most modern and sufficiently perspective trends in immunotherapy of cancer. The aim of the investigation was to characterize the DNA as a part of nucleoprotein fraction isolated from Bacillus subtilis culture medium filtrate (CMF) and to evaluate the dynamic of accumulation of such DNA and level of its unmethylated CpG motifs enrichment in the process of cultivation of bacterium. Methods: Two bacterial strains — B. subtilis 7025 and B. subtilis GP1-807-03 were used. Polyacrylamide and agarose gel electrophoresis, restriction analysis and transmission electron microscopy were used. The results of the investigations showed that DNA was accumulated in culture medium irregularly. DNA-containing fractions isolated on the 7th–9th day of cultivation have the greatest sensitivity to restriction endonuclease HpaII. The presence of phages in CMF concentrates has been shown. Conclusion: The present findings suggest that 7th–9th day of cultivation are the most optimal to obtain the bacterial extracellular DNA for the following immunotherapy application.

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