Recombinant plasmid DNA pPicZαA / BChE, containing the gene of human butyrylcholinesterase and butyrylcholinesterase METHOD FOR PRODUCING HUMAN methylotrophic yeast Pichia pastoris FOR TREATMENT OF POISONING organophosphorus toxins

摘要

1. Recombinant DNA plasmid pPicZαA / BChE, coding for human BuChE consisting of pPicZαA plasmid and the nucleotide sequence of the synthetic gene of human BuChE comprising inducible alcohol oxidase gene promoter AOX1 yeast P. pastoris wild type native terminator and polyadenylation signal AOX1 gene Sh ble gene sequence the product of which provides resistance to the antibiotic Zeocin transformants, and the nucleotide sequence of the signal peptide of the alpha-factor from S.cerevisiae, required for secretion Expro essiruemyh products into the culture sredu.2. Producing strain P. pastoris GS115 / BChE / a2, obtained by transforming P. pastoris cells GS115 line recombinant plasmid DNA pPicZαA / BChE.3. A method for producing human butyrylcholinesterase based producer strain GS115 / BChE / a2 under conditions of acidity of the culture medium pH of 7.6, the starting optical density of 3.0 pu mode and adding methanol: the starting concentration of 0.5% methanol, 1 day without making methanol (1 day) after 48 hours - 0.1% (day 2), after 72 hours, and further at 0.2% (3-7 days).