SOLUTION FOR INJECTION, RECOMBINANT PLASMID DNA pSX50 ENCODING SYNTHESIS OF HUMAN RECOMBINANT ALPHA-2b INTERFERON, STRAIN Escherichia coli SX50 AS INDUSTRIAL STRAIN-PRODUCER OF HUMAN RECOMBINANT ALPHA-2b INTERFERON AND METHOD FOR INDUSTRIAL PREPARING INTERFERON ALPHA-2b

摘要

FIELD: biotechnology, pharmacy.;SUBSTANCE: invention relates to creature of a interferon-base solution for injections and to recombinant strains of Escherichia coli (E. coli) and plasmids for its preparing. Invention proposes a novel recombinant multicopy plasmid DNA pSX50 that encodes synthesis of human leukocyte alpha-2b-interferon expression of that is under control of lactose and tryptophan promoters and transcription terminator. The strain E. coli SX50 as a producer of human recombinant leukocyte alpha-2b-interferon with productivity up to 0.901.0 g of alpha-2b interferon is obtained by transformation of the recipient strain E. coli BL21 cells with recombinant plasmid DNA pSX50. Method for preparing recombinant alpha-2b interferon is based on using the created recombinant strain E. coli SX50 and involving its submerged culturing on nutrient medium with decreased content of tryptophan and at continuous addition of nutrient substrates in the process of biosynthesis. The method involves mechanical destruction of microbial cells under high pressure, dissolving precipitated protein in the concentrated solution of guanidine hydrochloride followed by renaturation of interferon in physiological buffer solutions in the presence of chaotropic agents and its purification using three-step chromatography purification of interferon on resins of type Chelating Sepharose Fast Flow immobilized with Cu²⁺ ions, ion-exchange chromatography on ion-exchange resins of type CM Sepharose Fast Flow and gel-filtration chromatography on resins of type Superdex-75. Three-step chromatography purification of interferon provides preparing interferon substance of purity above 98% by data of electrophoresis under reducing and non-reducing conditions, and above 95% by data RF HPLC, and this interferon substance doesn't contain pyrogenes (LAL-test). The yield of the end product is 400 mg from 1 l of cultural medium, not less. Invention provides enhancing yield of interferon and stability of its solution.;EFFECT: improved preparing method of interferon.;12 cl, 5 tbl, 6 dwg, 2 ex