RECOMBINANT PLASMID DNA PSS5 ENCODING SYNTHESIS OF RECOMBINANT HUMAN ALPHA-2B INTERFERON, STRAIN OF ESCHERICHIA COLI SS5 AS PRODUCER OF RECOMBINANT HUMAN ALPHA-2B INTERFERON AND METHOD OF ALPHA-2B INTERFERON PREPARING

摘要

FIELD: biotechnology, molecular biology, genetic engineering. SUBSTANCE: cells of recipient strain E. coli BL 21 (DE3) is transformed by recombinant multicopy plasmid DNA pSS5 encoding synthesis of leukocyte human alpha-2b interferon. Expression of the latter is under control of lactose gene, phage T7 and tryptophan promoters and terminator of translation rrn B-T₁T₂ containing gene determining the resistance to kanamycin and containing DNA fragment from plasmid pUC19 that is responsible for replication of recombinant plasmid. Obtained strain E. coli SS5 has productivity 800 mg of alpha-2b interferon, not less, with total activity 1-2 x 10¹¹ IU/1 l of cultural medium or 15-20 g of wet biomass of strain cells. Method involves culturing on nutrient medium, freezing cultural liquid at -70 C, not above, its defrosting by temperature increase to 4 C followed by addition of lysozyme to cultural liquid, removal of DNA and RNA by treatment of lyzate with DNase, concentration of obtained product by protein washing off with solutions of nonionic detergents, centrifugation and dissolving formed precipitate in solution of guanidine hydrochloride followed by renaturation of interferon and its purification using ion-exchange chromatography on ion-exchange resins of type Whatman CM-52-cellulose. Invention can be used for preparing recombinant human leukocyte alpha-2b interferon for medicinal aims. EFFECT: increased yield of interferon producing. 7 cl, 4 dwg, 3 ex