RECOMBINANT PLASMID DNA, CODING SYNTHESIS; METHOD OF PRODUCTION AND MEDICINAL PREPARATION OF THE RECOMBINANT PROTEIN OF LYSOSTAFIN

摘要

1. The recombinant plasmid DNA coding for synthesis is characterized by the creation of a recombinant plasmid pBBSLis2, using the expression vector pNCMO2 which contains the recombinant mature lysostaphin gene for synthesis and secretion in Bacillus brevis cells, further contains a P2 promoter, the restriction analysis of Pstl and Kpnl, reprecipitation with ethanol, Amp- and Nm-resistant clones were selected, cells of the transformed strain were cultured. The method for producing a recombinant plasmid DNA according to claim 1, comprising culturing the cells of the transformed strain, ultrafiltration and chromatographic purification of the culture liquid for the elution of the desired product - mature lysostaphin, characterized in that two primers are synthesized for cloning the mature lysostaphin in the vector plasmid pNCMO2, secrete mature lysostaphin in Bacillus brevis, determine the NH2-terminal formyl-methionine residue of pre-prolysolistophine as residue -143 and NH2-terminal alanine of the mature lysostaphin is defined as the residue with +1, the 1.5-bp fragment. plasmid DNA that contains a lysostaphin gene from Staphylococcus simulans and its adherence sequences is completely sequenced, the gene initially encodes the preprofile Mr = 42205 bp, the NH2 terminal sequences are identified, the plasmid vector pNCMO2 containing the P2 promoter is transformed into E. coli HB101 is selected Amp-resistant clones are cultured with E. coli HB101, plasmid vector pNCMO2 is isolated, PstI and KpnI are digested, ethanol is digested, and transformed Baccillus brevis cells are cultured in a liquid medium of MT with a pH of 7.0 at 30-37 ° C of 120 rpm in for 48-64 hours,