RECOMBINANT PLASMIDS pET32a-IFG144, PROVIDING INTERFERON GAMMA SYNTHESIS, BACTERIAL STRAIN ESCHERICHIA COLI JM109/pET32a-IFG144 - INTERFERON GAMMA PROTEIN PRODUCER, METHOD OF OBTAINING INTERFERON GAMMA AND MEDICINAL PRODUCT Russian patent published in 2023 - IPC C12N15/70 C12N15/00 A61K38/21 

FIELD: biotechnology; genetic engineering.

SUBSTANCE: invention relates to a medicinal product, which is a lyophilisate containing mannitol and highly purified interferon gamma (INF-gamma) with the amino acid sequence SEQ ID NO: 1, wherein interferon gamma is obtained by cultivating the producer strain of Escherichia coli JM109/pET32a-IFG144, where after 3–5 hours of cultivation the biosynthesis of the recombinant protein is induced in Escherichia coli cells JM109/pET32a-IFG144 isopropyl-β -D-1-thiogalactopyranoside and incubated for 4–6 hours, obtaining the cell biomass of the producer strain producing the INF-gamma protein, isolating the IFN-gamma protein-producing strain from the resulting cell biomass, subsequent purification of the isolated IFN-gamma protein by cation exchange chromatography under denaturing conditions, renaturation of the INF-gamma protein by sequential fractional addition of denatured protein to a renaturation buffer containing L-arginine, and purification of the renatured INF-gamma protein by cation exchange chromatography in a buffer containing sodium chloride, sodium phosphate disubstituted 12-aqueous and sodium monosubstituted 2-water phosphate, where interferon gamma has a purity of at least 97% and a specific biological activity of at least 1.8×10⁷ IU per 1 mg of protein, and the producer strain was obtained by transforming the parent strain E. coli JM109 with the recombinant plasmid pET32a-IFG144 of 5806 base pairs in size, containing the structural elements: T7 promoter and lacO operator, synthetic nucleotide sequence SEQ ID NO: 2, encoding the INF-gamma protein, the ampicillin antibiotic resistance gene (AmpR) and the bacterial promoter of the ampicillin resistance gene (AmpR promoter) for selection of recombinant cells, the origin of replication of bacteriophage f1 (f1 ori), the lactose repressor gene LacI and the bacterial gene promoter lactose repressor (LacIpromoter), with the following ratio of components (wt.%): interferon gamma with a specific activity of at least 1.8×10⁷ IU/mg and purity of at least 97% 0.03–0.13; sodium chloride 3.2–10; sodium phosphate disubstituted 12-water 6.4–20; sodium phosphate monosubstituted 2-water 0.64–2; the rest is mannitol.

EFFECT: invention makes it possible to obtain a medicinal product containing a purer chemically and biologically stabilized interferon gamma protein, which increased the effectiveness of action and reduced its side effects due to optimally selected composition of the components, as well as their quantity, and also made it possible to increase the shelf life of the medicinal product to 3 years at the temperatures from 2 to 25°C.

2 cl, 4 dwg, 4 tbl, 12 ex