Blood substitute prepared by using hematoglobin-human seralbumin conjugate and its preparation method
A technology of human serum albumin and hemoglobin, applied in the field of blood substitute research, can solve problems such as limitations, short retention time, and large gap in blood function
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Embodiment 1
[0033] Embodiment 1: Preparation of pure matrix-free bovine hemoglobin by electrophoresis
[0034] Take a certain volume of washed fresh bovine blood red blood cells, and use twice the volume of cold swelling solution (20mmol / L KH containing 0.6% NaCl 2 PO 4 / Na 2 HPO 4 (PBS) buffer, pH 7.4) suspension, shake at 4°C for 1 h; pump 20 mmol / L PBS buffer (pH 7.4) twice the volume of red blood cells at a rate of 10% of the total volume of the solution per minute, shake for 1 h and then adjust The concentration of NaCl salt is 0.9%, and the red blood cell swelling and rupture solution is obtained. The rupture fluid was pretreated by a Millipore Pellicon cross-flow membrane filtration system. First, use 0.22μm membrane microfiltration to remove cell debris and macromolecular impurities, and then use membrane ultrafiltration with a molecular weight cut-off of 10KD to remove small molecular impurities. The obtained hemoglobin solution was further purified by DEAE Sepharose Fast Fl...
Embodiment 2
[0035] Embodiment 2: Preparation of chromatographically pure matrix-free porcine hemoglobin
[0036] Take a certain volume of washed fresh porcine red blood cells, and use twice the volume of cold swelling solution (20mmol / L KH containing 0.6% NaCl 2 PO 4 / Na 2 HPO 4 (PBS) buffer, pH 7.4) suspension, shake at 4°C for 1 h; pump 20 mmol / L PBS buffer (pH 7.4) twice the volume of red blood cells at a rate of 10% of the total volume of the solution per minute, shake for 1 h and then adjust The concentration of NaCl salt is 0.9%, and the red blood cell swelling and rupture solution is obtained. The rupture fluid was pretreated by a Millipore Pellicon cross-flow membrane filtration system. First, use 0.45μm membrane microfiltration to remove cell debris and macromolecular impurities, and then use membrane ultrafiltration with a molecular weight cutoff of 30KD to remove small molecular impurities. The resulting hemoglobin solution was further purified by Q Sepharose Big Beads ani...
Embodiment 3
[0037] Example 3: One-step cross-linking of human hemoglobin and human serum albumin by m-maleimide benzoic acid-N-hydroxysuccinimide ester (MBS)
[0038] Since the sulfhydryl group of the HSA molecule will react with MBS, thereby affecting the coupling reaction. Therefore, blocking of remaining sulfhydryl groups is required prior to activation of HSA with MBS. Slowly add 0.2 ml of 30 mM iodoacetamide to 10 ml of 5 mg / ml HSA solution (HEPES buffer at pH 7.8), and react at room temperature for 20 minutes. Subsequently, 0.5 ml of 30 mM MBS solution (dissolved in dimethylformamide) was added and reacted at room temperature for 30 minutes. The reaction mixture was passed through a Sephadex G-25 gel filtration column to remove excess MBS and iodoacetamide, and the protein fraction was collected. Because the hemoglobin molecule has a reactive thiol group (the sulfhydryl group of β-93 cysteine), the activated HSA can directly react with the hemoglobin molecule. The collected prote...
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