| 摘要 |
FIELD: molecular pharmacology, in particular human endostatin preparation and method for production thereof. ^ SUBSTANCE: claimed preparation is isolated after cell incubation E.coli BL21 (DE3) in cultural medium up to dense level of 0.4-0.6 OD6-00. Cell contains plasmide vector pBSH-ED15 or pBSH-ED16 having size of 6675 n.p. and gene of canamicine resistance gene; vector contains replicative sites pUS ori and M13 and gene encoding lac-repressor controlled by T7-promoter, and finished product has purity of 99 % and apyretic properties. Claimed preparation is obtained by expression of endostatin gene using plasmide DNA containing in E.coli cells; product refolding and purification, wherein expression is carried out in E.coli BL21 (DE3) cells, transformed by recombinant plasmide DNA pBSH-ED15 or pBSH-ED16. Cells are cultivated in cultural medium up to dense level of 0.4-0.6 OD6-00, then 0.1-0.3 mM of isopropyltiogalactoside is added into medium and cultivation is carried out for 2-3 h up to product accumulation. Further cells is exposed with ultrasound for 50-70 s at 0°C in presence of 0.1 % sodium deoxycholate. Bodies are dissolved in presence of 1 % sodium N-lauryl sarcosine. Suspension is multiply washed and centrifuged at 5000 g. In refolding process oxidation with air oxygen is carried out for 35 h, and target product is purified using chromatography depending on used incorporated plasmide. ^ EFFECT: apyretic product of high purity. ^ 6 cl, 9 ex, 7 dwg |
