RECOMBINANT PLASMIDS pET32a-TNF-Thy, PROVIDING SYNTHESIS OF HYBRID PROTEIN α-TUMOR NECROSIS FACTOR - THYMOSIN ALPHA 1, BACTERIAL STRAIN ESCHERICHIA COLI BL21(DE3)/PET32a-TNF-Thy - PRODUCER OF HYBRID PROTEIN TNF-Thy, METHOD OF OBTAINING TNF HYBRID PROTEIN-Thy AND MEDICINAL PRODUCT Russian patent published in 2023 - IPC C12N15/70 C12N15/00 C12N15/62 C12N15/28 C07K19/00 C07K14/525 C07K14/77 

FIELD: biotechnology; genetic engineering.

SUBSTANCE: invention relates to a medicinal product, which is a lyophilisate containing sodium chloride, sodium phosphate disubstituted 12-water, sodium phosphate monosubstituted 2-water, mannitol and highly purified hybrid protein TNF-Thy with amino acid sequence SEQ ID NO 1, wherein the hybrid protein TNF-Thy was obtained by cultivating the producer strain of Escherichia coli, BL21(DE3)/pET32a-TNF-Thy, where after 3–5 hours of cultivation, induction of biosynthesis is carried out in Escherichia coli BL21(DE3)/pET32a cells TNF-Thy isopropyl-β-D-1-thiogalactopyranoside and incubation is performed for 4–6 hours to obtain cell biomass of the TNF-Thy protein-producing strain, isolation from the resulting cell biomass of the TNF-Thy protein-producing strain, subsequent purification of the isolated TNF-Thy hybrid protein by anion-exchange and gel permeation chromatography under denaturing conditions, renaturation of the TNF-Thy hybrid protein by diluting a urea solution and purification of the renatured TNF-Thy hybrid protein by anion exchange chromatography to obtain a hybrid protein with a purity of at least 97% and Escherichia coli strain BL21(DE3)/pET32a-TNF-Thy — producer of the hybrid protein TNF-Thy — was obtained by transforming the parent strain E. coli BL21(DE3) with the recombinant plasmid pET32a-TNF-Thy with a size of 5929 base pairs (bp), providing the synthesis of a protein having the amino acid sequence SEQ ID NO 1 is 20.5 kDa in size and contains the following structural elements: the T7 promoter and the lacO operator, the synthetic nucleotide sequence SEQ ID NO 2 encoding the TNF-Thy hybrid protein, the ampicillin 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 promoter of the lactose repressor gene (Laclpromoter) with the following ratio of components (wt.%): 0.18–0.22 of tumor necrosis factor — thymosin alpha 1 recombinant with specific activity not less than 1.7×10⁶ U/mg; 27–35 of sodium chloride; 8–12 of sodium phosphate disubstituted 12-water; 0.3–1.8 sodium phosphate monosubstituted 2-water; the rest is mannitol.

EFFECT: invention makes it possible to obtain a product containing a purer chemically and biologically stabilized hybrid protein TNF-Thy, which increases the effectiveness of action and reduces its side effects due to optimally selected composition of components and their quantity, and also allows to increase the shelf life of the medicinal product to 3 years at the temperature from 2 to 8°C.

3 cl, 4 dwg, 2 tbl, 12 ex