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 C07K19/00 C07K14/525 C07K14/47 

FIELD: biotechnology; genetic engineering.

SUBSTANCE: subject of the invention is a medicinal product which is a solution for parenteral administration, containing osmotic pressure regulators, a buffer system maintaining a pH of 7.0–7.8, and a highly purified TNF-Thy hybrid protein with the amino acid sequence SEQ ID NO 1, wherein the TNF-Thy hybrid protein was obtained by cultivating the Escherichia coli producer strain, BL21(DE3)/pET32a-TNF-Thy, where, after 3–5 hours of cultivation, biosynthesis induction in Escherichia coli cells BL21(DE3)/pET32a-TNF-Thy isopropyl-β -D-1-thiogalactopyranoside and incubation for 4–6 hours are performed 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 fusion protein by diluting a urea solution and purification of the renatured TNF-Thy fusion protein by anion exchange chromatography to obtain a fusion protein with a purity of at least 97%, and Escherichia coli strain BL 21(DE3)/pET32a -TNF-Thy — producer of the hybrid protein TNF-Thy — obtained by transforming the parent strain E. coli BL21(DE3) with the recombinant plasmid pET32a-TNF-Thy 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 containing 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 antibiotic resistance gene (AmpR) and the bacterial ampicillin resistance gene promoter (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) in the following ratio of components (wt.%): 0.0052–0.0064 of TNF-Thy with a specific activity of at least 1.7×10⁶ U/mg and purity of at least 97%; 1.8–2.9 of osmotic pressure regulators; 2.2–3.6 of buffer system maintaining pH 7.0–7.8; the rest is water.

EFFECT: invention makes it possible to obtain an effective agent for parenteral administration, which is a solution containing a purer chemically and biologically stabilized hybrid protein TNF-Thy, which increases the efficiency of action and reduces the side effects of the hybrid protein due to optimally selected composition of components and their quantity, and also allows to increase the shelf life of the medicinal product up to 3 years at the temperatures from 2 to 8°C.

3 cl, 4 dwg, 1 tbl, 12 ex