Hemoglobin conjugate and its preparing method and use

A technology for hemoglobin and conjugates, applied in the field of hemoglobin conjugates, can solve the problems of short retention time, large gap in blood function, limitations, etc., and achieve the effects of enhancing stability, prolonging circulatory half-life, and avoiding aggregation.

Inactive Publication Date: 2006-05-03
苏志国 +3
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some side effects of blood substitutes (such as vasoconstriction and high blood pressure) limit their use, especially in the elderly with various medical conditions
Secondly, the product has a short retention time in the internal circulat

Method used

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  • Hemoglobin conjugate and its preparing method and use
  • Hemoglobin conjugate and its preparing method and use
  • Hemoglobin conjugate and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Preparation of pure matrix-free bovine hemoglobin by electrophoresis

[0036] 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, pH7.4) suspension, shake at 4°C for 1h; pump 20mmol / LPBS buffer (pH7.4) twice the volume of red blood cells at a rate of 10% of the total volume of the solution per minute, adjust NaCl after shaking for 1h The salt concentration 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 Flow anion e...

Embodiment 2

[0037] Embodiment 2: Preparation of chromatographically pure matrix-free porcine hemoglobin

[0038] 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, pH7.4) suspension, shake at 4°C for 1h; pump 20mmol / LPBS buffer (pH7.4) twice the volume of red blood cells at a rate of 10% of the total volume of the solution per minute, adjust NaCl after shaking for 1h The salt concentration 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 molecule impurities. The obtained hemoglobin solution was further purified by Q Sepharose BigBeads anion exchange c...

Embodiment 3

[0039] Example 3: One-step cross-linking of human hemoglobin and human serum albumin by m-maleimide benzoic acid-N-hydroxysuccinimide ester (MBS)

[0040] 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. 0.2 ml of 30 mM iodoacetamide was slowly added to 10 ml of 5 mg / ml HSA solution (HEPES buffer at pH 7.8), and reacted 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 collect...

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Abstract

The invention provides a hemoglobin-human serum albumin conjugate and a preparation method thereof. By preparing stroma-free hemoglobin, then coupling hemoglobin with human serum albumin, and the coupling product is purified by ion exchange chromatography, ultrafiltration or gel filtration chromatography, and the molecular weight distribution of the coupling product is between 100KD and 300KD Among them, there are 1 to 3 hemoglobin molecules that have been intramolecularly cross-linked or not intramolecularly cross-linked and 1 to 3 human serum albumin molecules. The prepared protein conjugates have good properties of blood substitutes.

Description

technical field [0001] The invention relates to a conjugate of hemoglobin, in particular to a conjugate of hemoglobin and human serum albumin used as a blood substitute, and a preparation method of the conjugate. Background technique [0002] Blood transfusion is an indispensable medical method for clinical operations, disaster relief and battlefield rescue. Relying on people to donate blood not only faces the problem of shortage of blood sources, but also due to the complexity of blood types, blood transfusion must be strictly matched. Security is threatened. In recent years, the demand for blood has been increasing, but safe and effective blood sources have become increasingly scarce. Therefore, in recent decades, human blood substitutes have always been a research and development focus in the international scientific and business circles. [0003] Human blood substitutes, that is, human red cell substitutes, are artificial preparations that have the function of oxygen ...

Claims

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Application Information

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IPC IPC(8): A61K38/42C07K14/765C07K14/805
Inventor 苏志国路秀玲郑春扬徐宇红
Owner 苏志国
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