FIELD: biotechnology.
SUBSTANCE: invention relates to the field of biotechnology, namely to the gene-therapeutic DNA vector GDTT1.8NAS12, Escherichia coli JM110-NAS strain, Escherichia coli JM110-NAS/GDTT1.8NAS12 strain and industrial-scale production of the gene-therapeutic DNA vector GDTT1.8NAS12. Method of producing a genotyping DNA vector GDTT1.8NAS12 involves constructing an intermediate vector with size of 2,408 base pairs, comprising a replication origin of 688 base pairs, 467 bp hGH-TA transcription terminator, a Tn10 RNA-out regulatory region of 137 bp, a kanamycin resistance vector of 1,018 bp and polylinker with size of 68 base pairs. It is then cleaved using Sall and BamHI restriction endonucleases and ligated with a promoter-regulatory site containing a promoter region of the human elongation factor EF1A gene with a proper enhancer of 1,219 bp. Further, the kanamycin resistance gene is cleaved at Spel restriction sites. Method of producing Escherichia coli JM110-NAS strain for producing the gene-therapeutic DNA vector GDTT1.8NAS12 involves constructing a linear DNA fragment containing a regulatory element RNA-in of the transposon Tn10 for selection without antibiotic application with size 64 base pairs, sacB gene of levansucrase, the product of which provides selection on a saccharose-containing medium with size of 1,422 base pairs, a chloramphenicol resistance gene catR required for selection of clones of the strain in which homologous recombination with size of 763 base pairs has passed, and two homologous sequences providing a homologous recombination process in the region of recA gene with its simultaneous inactivation with size of 329 base pairs and 233 base pairs. Said homologous sequences are sequences obtained by PCR amplification of recA gene fragments using genome 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), followed by transformation of Escherichia coli cells by electroporation and selecting clones surviving on medium containing 10 mcg/ml of chloramphenicol. Method of obtaining Escherichia coli JM110-NAS/GDTT1.8NAS12 strain, carrying gene-therapeutic DNA-vector GDTT1.8NAS12, involves production of electrocompetent cells of strain Escherichia coli JM110-NAS and electroporation of these cells with a genetic DNA vector GDTT1.8NAS12. Cells are then sown on petri dishes with agarised selective medium containing yeast extract, peptone, 6 % sucrose, as well as 10 mcg/ml of chloramphenicol.
EFFECT: invention widens the range of equipment.
7 cl, 6 dwg, 10 ex
