RECOMBINANT GENE ENCODING HBD-EPO PROTEIN, RECOMBINANT PLASMID DNA PL610, METHOD FOR PRODUCING A RECOMBINANT HBD-EPO PROTEIN, A RECOMBINANT HBD-EPO PROTEIN, A COMPOSITION FOR SPECIFICALLY INDUCING BONE REGENERATION, METHOD OF SPECIFIC INDUCTION OF BONE TISSUE REGENERATION Russian patent published in 2018 - IPC C12N15/70 C12N15/03 C12N15/09 C12N15/12 C12N15/18 C12N15/62 A61P19/10 A61K39/00 

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, genetic engineering, biochemistry, medicine, veterinary medicine. Synthetic DNA sequence corresponding to gene encoding the protein sequence of the heparin-binding domain (HBD) from Danio rerio fused with human erythropoietin (Epo) sequence (HBD-Epo protein) that nucleotide composition of the codons is optimized for heterologous expression in the non-pathogenic laboratory strain of E. coli. This synthetic HBD-Epo gene is flanked by 5'-end of the NcoI site, and 3'-end site of Kpn2I and inserted into plasmid pQE6 at the NcoI and Kpn2I sites. Based on this, a recombinant plasmid pL610 is obtained containing an artificial bacterial operon of the recombinant HBD-Epo protein comprising the promoter region of the early promoter of bacteriophage T5, the recombinant HBD-Epo protein gene, the transcription terminator; bacterial operon beta-lactamase; a bacterial region of the initiation of replication of the ColE1 type encoding the recombinant HBD-Epo protein and providing its synthesis in E. coli strains. Invention also includes a method for purifying the HBD-Epo protein by ion exchange chromatography. Invention relates to HBD-Epo recombinant protein itself, the expression vector pL610 and the composition (demineralized bone matrix (DCM) containing the HBD-Epo protein, as well as a DCM containing the protein HBD-Epo and BMP-2), aimed at stimulating osteogenesis. Invention allows to obtain a stable purified active protein HBD-Epo, as well as HBD-Epo, immobilized on DCM, including DCM with BMP-2 bone morphogenetic protein.

EFFECT: invention further includes a method for specifically inducing bone regeneration, comprising filling the defects of bone tissue with a composition consisting of HBD-Epo immobilized on DCM, including on the DCM with bone morphogenetic protein BMP-2.

7 cl, 4 dwg, 5 ex