阅读说明：本技术 一种人血红蛋白的制备方法 (Preparation method of human hemoglobin ) 是由 徐德红 徐琳 黄红萍 田根根 殷维 于 2019-11-12 设计创作，主要内容包括：本发明公开了一种人血红蛋白的制备方法,具体包括以下步骤：S1、血液的选取：首先从医院或医疗机构收取过期人血,S2、血浆的去除,S3、血红蛋白的释放,S4、基质的除去,S5、血红蛋白的纯化,本发明涉及生物工程技术领域。该人血红蛋白的制备方法,可实现既快速又高效的制备出高纯度人血红蛋白,同时很好的实现了将过期的人血进行回收利用,来提取人血红蛋白,避免了过期人血的直接丢弃造成资源的浪费,大大简化了制备工艺,且提升了制备人血红蛋白的纯度,无需花费工作人员大量的工作时间进行分离和纯化,减轻了工作人员的工作负担,大大提高了工作人员的工作效率,从而大大方便了人们人血红蛋白的制备。(The invention discloses a preparation method of human hemoglobin, which specifically comprises the following steps: s1, selecting blood: the method comprises the steps of collecting expired human blood from a hospital or a medical institution, S2 removing the blood plasma, S3 releasing hemoglobin, S4 removing matrix, and S5 purifying the hemoglobin, and relates to the technical field of biological engineering. The preparation method of the human hemoglobin can realize the rapid and efficient preparation of the high-purity human hemoglobin, simultaneously well realizes the recycling of expired human blood to extract the human hemoglobin, avoids the waste of resources caused by the direct discarding of the expired human blood, greatly simplifies the preparation process, improves the purity of the prepared human hemoglobin, does not need to spend a large amount of working time of workers for separation and purification, reduces the workload of the workers, greatly improves the working efficiency of the workers, and greatly facilitates the preparation of the human hemoglobin.)

1. A preparation method of human hemoglobin is characterized in that: the method specifically comprises the following steps:

s1, selecting blood: firstly, collecting expired human blood from a hospital or a medical institution, and then measuring the expired human blood with the required volume through a weighing device for later use;

s2, removal of plasma: pouring the overdue human blood measured in the step S1 into a centrifugal device, then carrying out rotary centrifugation for 10-20min at the rotation speed of 400-500r/min and the temperature of 25-31 ℃, then precipitating for 30-40min, namely separating the red blood cells in the overdue human blood, and then washing the separated red blood cells for 2-3 times by adopting a buffer system, so that plasma protein and part of membrane phospholipid can be removed;

s3, release of hemoglobin: after the accumulated red blood cells are obtained in the step S2, the red blood cells are placed in a kidney dialyzer, and then poured into hypotonic solution for dialysis for 1-2 hours, so that the red blood cells are gradually expanded until the membrane is broken, and then the mixed solution after dialysis is transferred to 0, l mu m ultrafiltration preparation, so that a hemoglobin solution is filtered;

s4, removal of matrix: transferring the hemoglobin solution obtained in the step S3 into a centrifugal device, centrifuging for 30-120min under the centrifugal force of 10000-36000g to remove erythrocyte membranes and membrane fragment matrixes, then classifying supernate, sequentially passing the supernate through 0.45 pm and 0.2pm filter membranes to remove smaller matrixes and germs, and then adjusting the pH and the salt ion concentration by using a pH regulator;

s5, purification of hemoglobin: filtering with 100kd filter membrane or virus filter to remove phospholipid or virus, and separating with gel exclusion chromatographic column to obtain pure hemoglobin product.

2. The method of claim 1, wherein the method comprises the steps of: the buffer system in the step S2 is 120-130mM NaCl, 10-15mM glucose, 2-3mM MgCl₂52-55mM KH₂PO₄And 18-20mM of K₂HPO₄The buffer solution is capable of preventing rupture of erythrocytes and maintaining stable intracellular environment when washed by using the buffer solution system.

3. The method of claim 1, wherein the method comprises the steps of: the hypotonic solution in the step S3 is MgCh 2-3mM and K5.7-6.3 mM₂HPO₄And 2.3-2.5KH of mM₂PO₄And (3) forming a mixed solution.

4. The method of claim 1, wherein the method comprises the steps of: the supernatant obtained in the step S4 is a hemoglobin solution containing trace amounts of hetero-proteins and lipids.

5. The method of claim 1, wherein the method comprises the steps of: the purification of hemoglobin in step S5 can remove more than 99.7% of phospholipids and more than 99, 9% of blood group antigens.

6. The method of claim 1, wherein the method comprises the steps of: the gel exclusion chromatographic column in the step S5 is a Waters QMA-Acell column which can obtain 20-22g of hemoglobin at one time and only needs 1 h.

7. The method of claim 1, wherein the method comprises the steps of: the pH regulator in the step S4 is sodium carbonate solution with the concentration of 0.6-1.3 mol/L.

8. The method of claim 1, wherein the method comprises the steps of: the kidney dialysis machine in the step S3 is selected from a full-automatic hemocyte dialysis machine with the model number BC 2600.

Technical Field

The invention relates to the technical field of bioengineering, in particular to a preparation method of human hemoglobin.

Background

Hemoglobin is the most predominant protein component in red blood cells and also the major protein component of blood, and red blood cells contain trace amounts of superoxide dismutase (S0D), carbonic anhydrase, glycolytic enzyme system, etc., and plasma fractions contain more than 200 kinds of protein components with different functions, some of which belong to the multienzyme system such as complement system, coagulation system, fibrinolysis system, kininogen system, etc., and some of which belong to the immunoglobulins, protease inhibitors, transportins, lipoproteins, etc.

With the increase of the amount of common blood transfusions and the tension of the supply of safe blood sources (high quality and no virus pollution), the research and development of human blood substitutes are more urgent, so that the research of producing hemoglobin by using animal blood as a raw material and producing hemoglobin by using modern biotechnology has remarkable foresight, bovine hemoglobin has a spatial structure similar to that of human hemoglobin, is less immunogenic, has abundant available blood source supply and great development value, although escherichia coli or yeast can be used for expressing modified human hemoglobin molecules, the difficulty in separating and purifying hemoglobin can be a key point for limiting the path, and the preparation of hemoglobin from microorganisms is more complicated than the preparation of hemoglobin from erythrocytes.

The problem in the hemoglobin preparation process is that almost all 2,3-DPG, namely 2, 3-diphosphoglycerate, an allosteric effector of hemoglobin is lost in the extraction and purification process of hemoglobin, one molecule of 2,3-DPG is arranged in two beta subunits in the molecule of hemoglobin, three negative charge groups in the molecule and three positive charge groups in the beta subunit of hemoglobin form 6 salt bridges, the affinity of hemoglobin to chlorine is adjusted from the surface, so that the affinity of hemoglobin to oxygen is improved, P is half-saturation partial pressure, under normal physiological conditions, the oxygen partial pressure required when the oxygen saturation degree of hemoglobin reaches 50% is reduced to 10 Torr and is greatly reduced compared with that of whole blood (P ═ 26 Torr), the combined oxygen is difficult to release, and because of the membraneless coating, hemoglobin tetramer is easy to be cracked into dimer, is rapidly excreted from kidney, causing renal toxicity, and the half-retention time of hemoglobin in animal body is only 2-4 h.

Most of the existing hemoglobin preparations directly adopt animal cells to express modified human hemoglobin molecules by microorganisms to prepare human hemoglobin, however, the steps of the preparation method are complicated, the purity of the prepared human hemoglobin is low, a large amount of working time of workers is needed to be spent for separation and purification, the working load of the workers is increased, the working efficiency of the workers is greatly reduced, the rapid and efficient preparation of high-purity human hemoglobin cannot be realized, meanwhile, the overdue human blood cannot be recycled to extract the human hemoglobin, the waste of resources caused by the direct discarding of the overdue human blood cannot be avoided, and great inconvenience is brought to the preparation of the human hemoglobin.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a preparation method of human hemoglobin, which solves the problems that the existing preparation method has complicated steps, the prepared human hemoglobin has low purity, a large amount of working time of workers is needed for separation and purification, the working load of the workers is increased, the working efficiency of the workers is greatly reduced, the high-purity human hemoglobin can not be quickly and efficiently prepared, meanwhile, the overdue human blood can not be recycled to extract the human hemoglobin, and the waste of resources caused by the direct discarding of the overdue human blood can not be avoided.

(II) technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme: a preparation method of human hemoglobin specifically comprises the following steps:

s1, selecting blood: firstly, collecting expired human blood from a hospital or a medical institution, and then measuring the expired human blood with the required volume through a weighing device for later use;

s2, removal of plasma: pouring the overdue human blood measured in the step S1 into a centrifugal device, then carrying out rotary centrifugation for 10-20min at the rotation speed of 400-500r/min and the temperature of 25-31 ℃, then precipitating for 30-40min, namely separating the red blood cells in the overdue human blood, then washing the separated red blood cells for 2-3 times by adopting a buffer system, wherein the washing solution is usually isotonic solution with equal volume of the red blood cells, such as normal saline, and the white blood cells and the platelets are removed simultaneously in the washing process, so that plasma proteins and part of membrane phospholipids can be removed;

s3, release of hemoglobin: after the packed red blood cells are obtained in the step S2, the red blood cells are placed in a kidney dialyzer, and then poured into hypotonic solution for dialysis for 1-2 hours, so that the red blood cells are gradually expanded until the membrane is broken, and then the mixed solution after dialysis is transferred to 0, l mu m ultrafiltration preparation, so that a hemoglobin solution is filtered out, wherein the hemoglobin solution does not contain any lipid basically, the red blood cells are swelled through the hypotonic solution, part of membrane pores are opened to release the hemoglobin, and the hemoglobin is released to the maximum extent, and the structure of the cell membrane is completely preserved;

s4, removal of matrix: transferring the hemoglobin solution obtained in the step S3 into a centrifugal device, centrifuging for 30-120min under the centrifugal force of 10000-36000g to remove erythrocyte membranes and membrane fragment matrixes, then classifying supernate, sequentially passing the supernate through 0.45 pm and 0.2pm filter membranes to further remove smaller matrixes and germs, and then adjusting the pH and the salt ion concentration by using a pH regulator;

s5, purification of hemoglobin: filtering with 100kd filter membrane or virus filter to remove phospholipid or virus, and separating with gel exclusion chromatographic column to obtain pure hemoglobin product.

Preferably, the buffer system in the step S2 is 120-130mM NaCl, 10-15mM glucose, 2-3mM MgCl₂52-55mM KH₂PO₄And 18-20mM of K₂HPO₄The buffer solution is capable of preventing rupture of erythrocytes and maintaining stable intracellular environment when washed by using the buffer solution system.

Preferably, the hypotonic solution in step S3 is 2-3mM MgCh and 5.7-6.3mM K₂HPO₄And KH of 2.3-2.5mM₂PO₄And (3) forming a mixed solution.

Preferably, the supernatant obtained in step S4 is a hemoglobin solution containing trace amounts of hetero-proteins and lipids.

Preferably, the purification of hemoglobin in step S5 can remove more than 99.7% of phospholipids and more than 99, 9% of blood group antigens.

Preferably, the gel exclusion chromatography column in step S5 is a Waters QMA-Acell column, which can obtain 20-22g hemoglobin at a time, and only 1h is needed.

Preferably, the pH regulator in the step S4 is 0.6-1.3mol/L sodium carbonate solution.

Preferably, the kidney dialyzer in the step S3 is selected from a full-automatic hemocyte dialyzer with the model number BC 2600.

(III) advantageous effects

The invention provides a preparation method of human hemoglobin. Compared with the prior art, the method has the following beneficial effects: the preparation method of the human hemoglobin specifically comprises the following steps: s1, selecting blood: first collecting expired human blood from hospital or medical institution, then measuring the required volume of expired human blood by weighing device for standby, S2, removing plasma: pouring the expired human blood measured in the step S1 into a centrifugal device, then carrying out rotary centrifugation for 10-20min at the rotation speed of 400-500r/min and the temperature of 25-31 ℃, then precipitating for 30-40min, namely separating the red blood cells in the expired human blood, then washing the separated red blood cells for 2-3 times by adopting a buffer system, and removing plasma protein and part of membrane phospholipid, S3, and releasing hemoglobin: after the stacked red blood cells are obtained in the step S2, the red blood cells are placed in a kidney dialyzer, and then poured into hypotonic solution for dialysis for 1-2 hours, so that the red blood cells gradually expand until the membrane is ruptured, and then the mixed solution after dialysis is transferred to 0, l μm ultrafiltration preparation, so as to filter out the hemoglobin solution, S4, removal of stroma: transferring the hemoglobin solution obtained in the step S3 into a centrifugal device, centrifuging for 30-120min under the centrifugal force of 10000-36000g to remove erythrocyte membranes and membrane fragment matrixes, then classifying the supernatant, sequentially passing the obtained supernatant through 0.45 and 0.2pm filter membranes to further remove smaller matrixes and germs, then adjusting the pH and the salt ion concentration by using a pH regulator, S5, purifying the hemoglobin: the method has the advantages that the 100kd filter membrane or virus filter is used for filtering to play a role in removing phospholipid or virus, and then the hemoglobin solution is separated through a gel exclusion chromatographic column, so that a pure hemoglobin product can be obtained, high-purity human hemoglobin can be prepared quickly and efficiently, expired human blood can be recycled well, human hemoglobin is extracted, waste of resources caused by direct discarding of expired human blood is avoided, the preparation process is greatly simplified, the purity of the prepared human hemoglobin is improved, a large amount of working time of workers is not needed for separation and purification, the work burden of the workers is reduced, the working efficiency of the workers is greatly improved, and great convenience is brought to the preparation of human hemoglobin of people.

Drawings

FIG. 1 is a flow chart of the present invention;

FIG. 2 is a data statistics chart of the control experiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-2, the embodiment of the present invention provides three technical solutions: a method for preparing human hemoglobin specifically comprises the following embodiments: