FIELD: biotechnology.
SUBSTANCE: invention refers to biotechnology, namely to gene-therapeutic DNA vectors for targeted therapy, Escherichia coli JM110-NAS strain and selection for targeted gene therapy of a gene-therapeutic DNA vector. Method of a gene-therapeutic DNA vector for targeted gene therapy involves constructing 2408 bps vector comprising a replication origin of 688 bps, 467 bps hGH-TA transcription terminator, a Tn10 RNA-out regulatory region of 137 bps, a kanamycin resistance vector of 1018 bps and polylinker with size of 68 bps. It is then cleaved using XhoI and BamHI restriction endonucleases and ligated to a promoter-regulatory region, wherein to obtain a gene-therapy gene of interest for the targeted gene therapy, a site containing the promoter region of the person of interest is used, and then the kanamycin resistance gene is cleaved at the SpeI restriction sites, after which the remaining fragment is ligated by itself. Method of producing Escherichia coli JM110-NAS strain for producing a gene-therapeutic DNA vector involves constructing a linear DNA fragment containing a regulatory element RNA-in of a transposon of Tn11 for selection without applying antibiotics with size of 64 bps, sacB gene of levansucrase, the product of which provides selection on a saccharose-containing medium with size of 1422 bps, a gene of resistance to chloramphenicol catR required for selection of clones of the strain in which homologous recombination with size of 763 bps has passed. and two homologous sequences providing a homologous recombination process in the recA gene region with simultaneous inactivation thereof with size of 329 bps and 233 bps, wherein said homologous sequences are sequences obtained by PCR amplification of recA gene fragments using genomic DNA Eshcerichia coli JM110-NAS as a matrix and a pair of primers LHA-F (5'-GCTGACGCTGCAGGTGATC) and LHA-R (5-GACAAGATGTGTGTCTACCGCTTCAGGTTACCCGCCAG), and a pair of primers RHA-F (5'-TGGCAGGGCGGGGCGTAACTACGCCTCTGTTCGTCTCGA) and RHA-R (5'-CTCAGCAGCAACTCACGTAC). That is followed by transformation of Escherichia coli cells by electroporation, and clones surviving on medium containing 10 mcg/ml of chloramphenicol are selected.
EFFECT: invention widens the range of equipment.
28 cl, 1 tbl, 38 dwg, 39 ex
