METHOD FOR PRODUCING A BIOLOGICALLY ACTIVE COMPLEX. BIOLOGICALLY ACTIVE PROTEIN/POLYPEPTIDE COMPLEX

摘要

The claimed Invention relates to the method of production of the biologically active protein-polypeptide complex, wherein the quick-frozen mid first third to mid last third gestational age hoof livestock embryonic brain is gradually unfrozen, and, step by step, within the temperature ranges 2°C to 28°C: - is homogenized in buffer solution, simultaneously extracting in presence of the buffer solution proteolysis reversible inhibitors and nonionic detergents at pH min. 5.2 and max. 8.5, at the solution / tissue volume ratios max. 1:0.5; - the homogenate is separated from the undissolved tissular and cellular components by centrifuging at 10,000 g to 30,000 g during 90 to 30 minutes, respectively; - the supernatant is stepwise filtered down to the max. 0.45 µm mesh filters; - the filtrate undergoes the anion-exchange chromatography using a saline buffer solution as mobile phase and separating the linked to sorbent proteins and polypeptides in stepwise gradient, with the help of a 0.08 to 0.26 mol/I ionic strength and 5.2 to 8.5 pH eluent, increasing the mobile phase ionic strength in the 0.02 mol/I steps, and beginning to collect the target fractions with the min. 0.1 ionic strength solution; and - the collected target fractions are desalinized in dialysis or gel filtration, and, having added the bacteriostatic and fungicidal preservatives, 0.06 mg/ml max. total concentration, and solubilizer, 0.01 mg/ml max. total concentration, proceed to the ultra filtration at max. 0.22 µm mesh, and then sterile packed. In addition, the claimed Invention relates to the biologically active protein-polypeptide complex itself, obtained as defined above, having the tissue-specific reparative effect on the nervous tissue, obtained from the brain tissue of the gestation mid first third to mid last third hoof livestock embryos, including the negatively charged faintly acid neutral proteins and polypeptides relating to the growth factor, differentiation factor, signaling molecules, that determine its biologic and pharmacologic activity, with the 5 to 200 kDa molecular masses, wherein at least 80% of the total protein mass has the 10 to 120 kDa molecular mass, and characterized by a peak at the 274-284 nm wave length in the UV-visible spectrum and bands in the pI 4.2 to 8.4 range at isoelectric focusing in a 5% polyacrylamide gel.