Studies On Production Technology And Quality Of Recombinant Staphylokinase Variants

Thrombus disease prejudices the health of human kind seriously.SK,UK andits mutant are used by thrombolytics commonly,but so many defects existed.(recanalizate mis-classification,thrombus re-formation,bleeding and allergicresponse etc).SAK(Staphylokinase) to have thrombolysis activitycorresponding totissue-type plasminogen activator.SAK have higher FIB specificity.It is one kindof thrombolytics with outlook application.Recombination to change constructionof staphylokinase mutation plasmid was done for degrade the immunogenicity ofwild-typeSAK,andexpressedbyBacilluscoli.We investigated the produce technology of constructed DH5α/pBV220/rSAKengineering bacteria strain,the M9 medium was selected from several kinds ofbasic culture media,and adapted to fermentation and expression of rSAK.Mon-factorial experiment was practiced to select the carbon source and nitrogensource of M9 medium.Carbon source is glucose and nitrogen source is PGYE andTGY powder.We designed the orthogonal experiment;optimized the compoundingof culture media,and selected the adapted culture media.Mg2+ can accelerate thegrowthofbacteria.We studied the effect corresponding to the growth and expression of rSAK bythe fermentation conditions with shaking-flask(Culture Temp;Induce opportunity;Induce maintenance time).And incipient pH value of broth,dissolved oxygen(DO),seed age,inoculation amount were study. The adapted fermentation conditions withshaking-flask:culture temperature is 30 degree;incipient pH value of broth is7.2-7.4;rotation speed of rocking bed is 180r/min;rise the temperature to 42 degreeto induce the expression for 3 hours in logarithmic growth period,passage on 35degree,this method will raise the production of bacteria and the expression ofrSAK. The expression of rSAK is 24.6%,production is 0.276g/L.The character ofsubmerged fermentation showed that dissolved oxygen deficiency mainly limited...