RECOMBINANT PLASMID DNA PKEP-9 ENCODING HUMAN ERYTHROPOIETIN, STRAIN OF CULTURED CHINESE HAMSTER OVARY CELLS CHOPE-9 - A PRODUCER OF HUMAN ERYTHROPOIETIN

摘要

FIELD: molecular biology, biotechnology, genetic engineering. SUBSTANCE: invention relates to preparing recombinant plasmid vector pKEP-9 containing 8 116 nucleotide pairs (molecular mass is 5 mDa) that provides biosynthesis of human erythropoietin in cultured Chinese hamster ovary cells. Method of preparing involves fusion of Bst EII-Bgl II-fragment (2 383 nucleotide pairs) of plasmid pSY-Ep-gpt containing human erythropoietin structural gene into Smaq I-site of vector plasmid pNut (5 733 nucleotide pairs) containing activated murine metallothionein promoter MT-1 and dihydrofolate reductase gene under control of early promoter SV-40. Recombinant strain CHOpE-9 is obtained after transformation of cells CHO-KIdhfr with constructed recombinant plasmid pKEP-9 followed by culturing on growth medium and selection of colonies showing resistance to methotrexate and hybridizing with pKEP-9 DNA. Cells of new strain under conditions of stationary culturing stably produce human erythropoietin in cultural fluid at the yield 400 UA/ml medium. EFFECT: plasmid and cells indicated above, increased yield of erythropoietin. 2 cl, 3 ex, 2 dwg