Oxygen-carrying protein molecule as well as preparation method and application thereof

Abstract

The invention provides an oxygen-carrying protein molecule and a preparation method and application thereof, and relates to the technical field of functional protein preparation, the oxygen-carrying protein molecule comprises at least one cysteine part and at least one histidine part, the cysteine part comprises acetyl or ethyl maleoyl, and the histidine part comprises histidine. The histidine moiety includes a benzimidazole derivative group. The benzimidazole derivative is adopted as an auxiliary molecule and participates in the modification process of the sulfydryl modified compound on the hemoglobin, the modification site selectivity is improved, the conformational change is reduced, and the modification degree is accurately controlled, so that the oxygen binding force P50 of the modified hemoglobin is effectively improved.

Description

Technical Field

[0001] The present invention provides an oxygen-carrying protein molecule and a preparation method and application thereof, and relates to the technical field of functional protein preparation. Background Art

[0002] Hemoglobin (Hb), as an important functional protein in red blood cells, undertakes key tasks such as transmitting oxygen, removing carbon dioxide, decomposing hydrogen peroxide, and transferring electrons in the body, which is essential for maintaining life activities. With the continuous development of medicine and biotechnology, the research and application of hemoglobin are becoming more and more extensive, especially in the fields of improving the oxygen-carrying function of hemoglobin and developing new blood substitutes, showing great potential and prospects. Hemoglobin is composed of four subunits, each of which contains a heme group, which can reversibly bind to oxygen molecules. The oxygenation curve of hemoglobin is S-shaped, which enables hemoglobin to efficiently absorb oxygen in the lungs and quickly release oxygen in the tissues, thereby meeting the body's demand for oxygen. In addition, hemoglobin also has a certain allosteric ability, that is, when one subunit binds to oxygen, it will promote the binding of other subunits to oxygen. This synergistic effect greatly improves the efficiency of oxygen transportation. In various disease states, such as anemia and shock, the body's demand for oxygen increases sharply, and traditional treatments are often difficult to quickly meet this demand. Therefore, the development of new oxygen-carrying protein molecules, especially enhancing the oxygen-binding capacity of hemoglobin through chemical modification, has become an important research direction. The research and development significance of this technology lies in: Improving the efficiency of treatment: In emergency treatment, such as shock resuscitation, hemoglobin with enhanced oxygen-binding capacity can quickly replenish tissue oxygen supply and improve the success rate of treatment. Reduce dependence on blood transfusions: For patients who need a large amount of blood transfusions, new oxygen-carrying protein molecules can be used as blood substitutes to reduce the problem of blood supply tension. Expanding application areas: In addition to the medical field, this technology can also be applied to oxygen supply in special environments such as plateau operations, diving, aerospace, etc.

[0003] At present, research on the site-specific chemical modification of specific amino acids in hemoglobin molecules to enhance oxygen binding capacity has made some progress. Hemoglobin is composed of two alpha subunits and two beta subunits. In hemoglobin molecules, the active free cysteine ​​contains sulfhydryl groups, which can be easily modified by controlling experimental conditions. Different animals have different numbers of free cysteine ​​that can be modified by site-specific modification. The paper Acharya SA et al. Biochem.J.2007; 405: 503-511 Table 2 shows that when the hemoglobin of different species is bound to oxygen, the human molecule has 2 active free sulfhydryl groups, the dog, chicken, sheep and mouse molecules each have 4, and the cat molecule has 6. The Chinese patent application No. 200710089647.6 of American Aike Co., Ltd. discloses that the cysteine ​​part of hemoglobin contains a sulfhydryl protecting group, the ability of the cysteine ​​site to bind nitric oxide is reduced, and the range of the oxygen binding capacity P50 of the obtained hemoglobin is about 20mmHg to 45mmHg, wherein the reagent used to provide the sulfhydryl protecting group is iodoacetamide. In this patent application, the hemoglobin molecule undergoes additional intramolecular cross-linking, so the oxygen-carrying protein is different in terms of molecule, structure and oxygen binding capacity. The paper GarelM.C.et al, Eur.J.Biochem. 1982;123:513-9 tested the effects of 21 thiol-modified compounds (including iodoacetamide and ethylmaleimide) on human hemoglobin. The data showed that the oxygen binding capacity (P50) was not enhanced after modification with multiple compounds, suggesting that the different spatial structures of the modified proteins may be the reason for the different oxygen binding capacities.

[0004] Modification of the active free cysteine ​​of hemoglobin with thiol-modified compounds such as iodoacetamide or ethylmaleimide can enhance its oxygen binding capacity. However, in some cases, this modification does not achieve the expected effect. The specific reasons may include the following aspects: Selectivity of modification site: Both iodoacetamide and ethylmaleimide can specifically modify cysteine ​​residues, but the selectivity and number of modification sites will affect the conformational changes after modification. If the modification site is inappropriate, the natural conformation of hemoglobin may be destroyed, resulting in no significant increase in oxygen binding capacity. Spatial structural changes after modification: Different modification compounds may cause different degrees of conformational changes in hemoglobin, which will affect the function of hemoglobin. The study of Garel MC et al. showed that the oxygen binding capacity after modification was quite different, which may be caused by the spatial structural changes caused by different compounds. Control of modification degree: Too high or too low modification will affect the function of the protein. Too high a modification degree may cause protein denaturation, while too low a modification degree may not be enough to cause significant functional changes. Therefore, it is necessary to precisely control the conditions of the modification reaction to obtain the best modification effect. Summary of the invention

[0005] To solve the above problems, the present invention is characterized in that a benzimidazole derivative is designed as an auxiliary molecule to participate in the modification process of hemoglobin with acetyl or ethylmaleyl, thereby improving the selectivity of the modification site, reducing conformational changes, and accurately controlling the degree of modification, thereby effectively improving the oxygen carrying capacity of the modified hemoglobin. The specific scheme is as follows:

[0006] The present invention provides an oxygen-carrying protein molecule, comprising at least one cysteine ​​part and at least one histidine part, wherein the cysteine ​​part comprises an acetyl group or an ethylmaleyl group, and the histidine part comprises a benzimidazole derivative group.

[0007] Preferably, the protein molecule is derived from a hemoglobin molecule.

[0008] Preferably, the molecular weight of the oxygen-carrying protein is 64,000 to 66,000 Da.

[0009] Preferably, the molecular oxygen binding capacity P50 of the oxygen-carrying protein ranges from 15.01 mmHg to 19.99 mmHg.

[0010] The present invention provides a method for preparing the above oxygen-carrying protein molecule, comprising the following steps:

[0011] S1. preparing a benzimidazole derivative: adding benzimidazole, a modifying agent and a catalyst into a solvent for reaction, and purifying after the reaction to obtain a benzimidazole derivative;

[0012] S2, preparing a hemoglobin suspension and a benzimidazole derivative solution, mixing the two and incubating them, and purifying them after the end to obtain a hemoglobin suspension modified with a benzimidazole derivative;

[0013] S3. Add iodoacetamide or ethylmaleimide to the obtained hemoglobin suspension modified with the benzimidazole derivative, incubate and purify to obtain the oxygen-carrying protein molecule.

[0014] Preferably, the modifying reagent in step S1 includes one or more of 3-chloropropanol, ethylene glycol, ethanolamine, hydroxylamine hydrochloride, and N-hydroxysuccinimide.

[0015] Preferably, the catalyst in step S1 comprises NaOH or K2CO3.

[0016] Preferably, the solvent in step S1 includes one or more of dimethyl sulfoxide, acetonitrile and ethanol.

[0017] Preferably, the molar ratio of the benzimidazole to the modifying agent in step S1 is 1:(1-3).

[0018] Preferably, the molar ratio of the modifying agent to the catalyst in step S1 is 1:1.

[0019] Preferably, the amount of the solvent used in step S1 is 10-20 mL / 1 g benzimidazole.

[0020] Preferably, the reaction temperature in step S1 is 25-80° C. and the reaction time is 2-24 h.

[0021] Preferably, the purification in step S1 is performed by column chromatography, the solid phase is silica gel, and the mobile phase is n-hexane / ethyl acetate in a ratio range of (5-1):1, or dichloromethane / methanol in a ratio range of (10-1):1.

[0022] Preferably, the hemoglobin suspension in step S2 has a solvent of PBS, a pH range of 7.0 to 7.4, and a hemoglobin content of 0.5 to 0.8 mM; the benzimidazole derivative solution has a solvent of DMSO or acetonitrile, and a concentration of the benzimidazole derivative in the solution is 1 to 2 mM; and the amount of the benzimidazole derivative solution used is 2 to 4 times the volume of the hemoglobin suspension.

[0023] Preferably, the incubation in step S2 is performed at a temperature of 37° C. for 1 to 2 h.

[0024] Preferably, the molar amount of iodoacetamide or ethylmaleimide added in step S3 is 5 to 10 times the molar amount of hemoglobin modified with the benzimidazole derivative in the suspension.

[0025] Preferably, the incubation in step S3 is performed at a temperature of 37° C. for 30 to 60 minutes.

[0026] Preferably, the purification described in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis fluid, and dialyzing for 12 to 24 hours at 4°C; wherein the dialysis fluid is PBS buffer with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000Da; during the period, the dialysis fluid is replaced multiple times to ensure complete removal of small molecules and solvents.

[0027] Preferably, the method for preparing hemoglobin in step S2 comprises:

[0028] 1) Remove the plasma components by centrifugation or microfiltration, and then wash the cell components with physiological solution;

[0029] 2) Add water or hypotonic solution to the cell components and control the duration of action to ensure selective disruption of red blood cells;

[0030] 3) Add hypertonic solution to restore the overall isotonic environment;

[0031] 4) removing red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collecting the hemoglobin-rich supernatant or ultrafiltration filtrate;

[0032] 5) Remove impurities through methods such as ultrafiltration or column chromatography to further improve the purity of hemoglobin.

[0033] The present invention provides the use of the oxygen-carrying protein molecule in preparing a composition with in vivo and in vitro oxygen supply function.

[0034] Preferably, the application includes the oxygen-carrying protein molecule and other pharmaceutically acceptable carriers or pharmaceutically acceptable components together forming a composition with in vivo and in vitro oxygen supply function.

[0035] Beneficial effects of the present invention:

[0036] The present invention designs a benzimidazole derivative as an auxiliary molecule to participate in the modification process of hemoglobin with acetyl or ethylmaleyl, improve the modification site selectivity, reduce conformational changes, and accurately control the modification degree, thereby effectively improving the oxygen binding capacity P50 of the modified hemoglobin. The oxygen binding capacity P50 of the obtained oxygen-carrying protein molecule ranges from 15.01 mmHg to 19.99 mmHg. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The oxygen dissociation curve is for Example 1;

[0038] Figure 2 The oxygen dissociation curve is for Example 3;

[0039] Figure 3 It is the oxygen dissociation curve of Comparative Example 1;

[0040] Figure 4 It is a comparison diagram of oxygen dissociation curves of samples of Example 1 and Example 3 and a standard bovine hemoglobin (bHb) sample;

[0041] Figure 5 Characteristic absorption spectrum of the sample prepared in Example 1;

[0042] Figure 6 Sample chromatograms were prepared for Example 1. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0044] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0045] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and the experimental materials used in the following examples, unless otherwise specified, are all purchased from commercial channels (Sigma, analytical grade reagents).

[0046] Preparation example: Preparation of hemoglobin:

[0047] 1) Bovine whole blood was obtained from a slaughterhouse, and sodium citrate was added for anticoagulation (final concentration 0.38%). The plasma components were removed by centrifugation (2000 g, 10 min, Beckman JXN-26 centrifuge), and then the cell components were washed with physiological solution (0.9% sodium chloride solution, Shanghai Lingfeng, AR500G);

[0048] 2) adding water to the cell components and controlling the duration of action to ensure selective disruption of red blood cells;

[0049] 3) Add hypertonic sodium chloride (5%, Shanghai Lingfeng, AR500G) solution to restore the overall isotonic environment;

[0050] 4) Centrifuge again to remove red blood cell membrane fragments and unbroken cells and collect the hemoglobin-rich supernatant;

[0051] 5) Remove impurities through dialysis (molecular weight cutoff 7000-8000Da) to further improve the purity of hemoglobin.

[0052] Example 1 Preparation of oxygen-carrying protein molecules:

[0053] S1. preparing a benzimidazole derivative: adding benzimidazole, a modifying agent and a catalyst into a solvent for reaction, and purifying after the reaction to obtain a benzimidazole derivative;

[0054] S2, preparing a hemoglobin suspension and a benzimidazole derivative solution, mixing the two and incubating them, and purifying them after the end to obtain a hemoglobin suspension modified with a benzimidazole derivative;

[0055] S3. Add iodoacetamide to the obtained hemoglobin suspension modified with the benzimidazole derivative, incubate and purify to obtain the oxygen-carrying protein molecule.

[0056] The modification reagent described in step S1 includes 3-chloropropanol.

[0057] The catalyst in step S1 includes NaOH.

[0058] The solvent in step S1 includes acetonitrile.

[0059] The molar ratio of the benzimidazole to the modifying agent in step S1 is 1:1.5.

[0060] The molar ratio of the modifying agent to the catalyst in step S1 is 1:1.

[0061] The amount of the solvent used in step S1 is 10 mL / 1 g benzimidazole.

[0062] The reaction temperature in step S1 is 40° C. and the reaction time is 12 h.

[0063] The purification in step S1 is carried out by column chromatography, the solid phase is silica gel, and the mobile phase is dichloromethane / methanol in a ratio range of (10-1):1.

[0064] The hemoglobin suspension in step S2 has a solvent of PBS, a pH range of 7.0 to 7.4, and a hemoglobin content of 0.5 mM. The benzimidazole derivative solution has a solvent of DMSO or acetonitrile, and a concentration of the benzimidazole derivative in the solution of 1 mM. The amount of the benzimidazole derivative solution used is twice the volume of the hemoglobin suspension.

[0065] The incubation in step S2 is performed at 37° C. for 1 h.

[0066] The molar amount of iodoacetamide added in step S3 is 5 times the molar amount of the hemoglobin modified with the benzimidazole derivative in the suspension.

[0067] The incubation in step S3 is performed at 37° C. for 30 min.

[0068] The purification described in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis fluid, and dialyzing for 12 hours at 4°C; wherein the dialysis fluid is PBS buffer with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000Da; during the process, the dialysis fluid is replaced multiple times to ensure complete removal of small molecules and solvents.

[0069] Example 2 Preparation of oxygen-carrying protein molecules:

[0070] S1. preparing a benzimidazole derivative: adding benzimidazole, a modifying agent and a catalyst into a solvent for reaction, and purifying after the reaction to obtain a benzimidazole derivative;

[0071] S2, preparing a hemoglobin suspension and a benzimidazole derivative solution, mixing the two and incubating them, and purifying them after the end to obtain a hemoglobin suspension modified with a benzimidazole derivative;

[0072] S3. Add iodoacetamide to the obtained hemoglobin suspension modified with the benzimidazole derivative, incubate and purify to obtain the oxygen-carrying protein molecule.

[0073] The modification reagent described in step S1 includes ethylene glycol.

[0074] The catalyst in step S1 includes NaOH.

[0075] The solvent in step S1 includes dimethyl sulfoxide.

[0076] The molar ratio of the benzimidazole to the modifying agent in step S1 is 1:1.5.

[0077] The molar ratio of the modifying agent to the catalyst in step S1 is 1:1.

[0078] The amount of the solvent used in step S1 is 20 mL / 1 g benzimidazole.

[0079] The reaction temperature in step S1 is 70° C. and the reaction time is 15 h.

[0080] The purification in step S1 is carried out by column chromatography, the solid phase is silica gel, and the mobile phase is dichloromethane / methanol in a ratio range of (10-1):1.

[0081] The hemoglobin suspension in step S2 has a solvent of PBS, a pH range of 7.0 to 7.4, and a hemoglobin content of 0.8 mM. The benzimidazole derivative solution has a solvent of DMSO or acetonitrile, and a concentration of the benzimidazole derivative in the solution is 2 mM. The amount of the benzimidazole derivative solution used is 4 times the volume of the hemoglobin suspension.

[0082] The incubation in step S2 is performed at 37° C. for 2 h.

[0083] The molar amount of iodoacetamide added in step S3 is 10 times the molar amount of the hemoglobin modified with the benzimidazole derivative in the suspension.

[0084] The incubation in step S3 is performed at 37° C. for 60 min.

[0085] The purification described in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis fluid, and dialyzing for 24 hours at 4°C; wherein the dialysis fluid is PBS buffer with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000Da; during the period, the dialysis fluid is replaced multiple times to ensure complete removal of small molecules and solvents.

[0086] Example 3 Preparation of oxygen-carrying protein molecules:

[0087] S1. preparing a benzimidazole derivative: adding benzimidazole, a modifying agent and a catalyst into a solvent for reaction, and purifying after the reaction to obtain a benzimidazole derivative;

[0088] S2, preparing a hemoglobin suspension and a benzimidazole derivative solution, mixing the two and incubating them, and purifying them after the end to obtain a hemoglobin suspension modified with a benzimidazole derivative;

[0089] S3. Add ethylmaleimide to the obtained hemoglobin suspension modified with the benzimidazole derivative, incubate and purify to obtain the oxygen-carrying protein molecule.

[0090] The modification reagent described in step S1 includes ethylene glycol.

[0091] The catalyst in step S1 includes NaOH.

[0092] The solvent in step S1 includes dimethyl sulfoxide.

[0093] The molar ratio of the benzimidazole to the modifying agent in step S1 is 1:1.5.

[0094] The molar ratio of the modifying agent to the catalyst in step S1 is 1:1.

[0095] The amount of the solvent used in step S1 is 20 mL / 1 g benzimidazole.

[0096] The reaction temperature in step S1 is 70° C. and the reaction time is 15 h.

[0097] The purification in step S1 is carried out by column chromatography, the solid phase is silica gel, and the mobile phase is dichloromethane / methanol in a ratio range of (10-1):1.

[0098] The hemoglobin suspension in step S2 has a solvent of PBS, a pH range of 7.0 to 7.4, and a hemoglobin content of 0.8 mM. The benzimidazole derivative solution has a solvent of DMSO or acetonitrile, and a concentration of the benzimidazole derivative in the solution is 2 mM. The amount of the benzimidazole derivative solution used is 4 times the volume of the hemoglobin suspension.

[0099] The incubation in step S2 is performed at 37° C. for 2 h.

[0100] The molar amount of ethylmaleimide added in step S3 is 10 times the molar amount of the hemoglobin modified with the benzimidazole derivative in the suspension.

[0101] The incubation in step S3 is performed at 37° C. for 60 min.

[0102] The purification described in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis fluid, and dialyzing for 24 hours at 4°C; wherein the dialysis fluid is PBS buffer with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000Da; during the period, the dialysis fluid is replaced multiple times to ensure complete removal of small molecules and solvents.

[0103] Comparative Example 1 Preparation of modified hemoglobin molecules (the difference from Example 3 is that no benzimidazole derivative modification is performed):

[0104] A hemoglobin suspension is prepared, ethylmaleimide is added, and the oxygen-carrying protein molecule is purified after incubation.

[0105] The molar amount of the added ethylmaleimide is 10 times the molar amount of hemoglobin in the suspension.

[0106] The incubation temperature is 37° C. and the time is 60 min.

[0107] The purification includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis fluid, and dialyzing for 24 hours at 4°C; wherein the dialysis fluid is a PBS buffer solution with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000Da; during the process, the dialysis fluid is replaced multiple times to ensure complete removal of small molecules and solvents.

[0108] Comparative Example 2 Preparation of modified hemoglobin molecules (the difference from Example 2 is that no benzimidazole derivative modification is performed):

[0109] A hemoglobin suspension is prepared, iodoacetamide is added, and the oxygen-carrying protein molecule is purified after incubation.

[0110] The molar amount of the added iodoacetamide is 10 times the molar amount of hemoglobin in the suspension.

[0111] The incubation temperature is 37° C. and the time is 60 min.

[0112] The purification includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis fluid, and dialyzing for 24 hours at 4°C; wherein the dialysis fluid is a PBS buffer solution with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000Da; during the process, the dialysis fluid is replaced multiple times to ensure complete removal of small molecules and solvents.

[0113] Characterization and performance detection:

[0114] Oxygen binding capacity test (P50): Use an oxygen dissociation curve meter to measure the oxygen dissociation curve of the modified hemoglobin solution. Calculate its P50 value, which is the oxygen partial pressure when the hemoglobin reaches 50% oxygen saturation.

[0115] The characteristic absorption spectrum and chromatogram of the sample in Example 1 are shown in Figure 5 , Figure 6 , the oxygen dissociation curve is shown in Figure 1, sample P50 = 18.19 mmHg, Example 2 sample P50 = 18.05 mmHg, Example 3 sample P50 = 18.54 mmHg, the Example P50 value is below 20.0 mmHg, and the modified hemoglobin has enhanced oxygen binding capacity. Comparative Example 1 sample P50 = 27.29 mmHg, Comparative Example 2 sample P50 = 28.62 mmHg, the comparative example P50 value is equivalent to the level of unmodified hemoglobin in previous studies (26.0-30.0 mmHg); Blank Example: Standard bHb sample was tested for oxygen binding capacity (P50), sample P50 = 27.47 mmHg, and the P50 value is equivalent to the level of unmodified hemoglobin in previous studies (26.0-30.0 mmHg).

[0116] Through the above experiments and analysis, we have obtained a technical solution that uses benzimidazole derivatives to participate in the modification process of hemoglobin with acetyl or ethylmaleyl, thereby effectively improving the oxygen binding capacity of hemoglobin molecules. The binding of benzimidazole derivatives to hemoglobin may cause local conformational changes on the protein surface, making the overall structure of hemoglobin more open or flexible. In this open or flexible state, the use of IAA or NEM modification may more effectively expose and modify key residues near the oxygen binding site, resulting in significant changes in oxygen affinity. The synergistic effect of the two modifications may significantly change the quaternary structure of hemoglobin or the microenvironment of the oxygen binding site, thereby greatly improving the oxygen binding capacity and significantly reducing the P50 value. In addition, we also verified the solution of replacing the benzimidazole derivatives we prepared with benzimidazole, but the experimental results showed that benzimidazole could not form a stable bond with hemoglobin.

[0117] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0118] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. An oxygen-carrying protein molecule, characterized in that: It comprises at least one cysteine ​​moiety and at least one histidine moiety, wherein the cysteine ​​moiety comprises an acetyl group or an ethylmaleyl group and the histidine moiety comprises a benzimidazole derivative group.

2. The oxygen-carrying protein molecule according to claim 1, characterized in that: The protein molecule is derived from the hemoglobin molecule.

3. The oxygen-carrying protein molecule according to claim 1, characterized in that: The molecular weight of the oxygen-carrying protein is 64,000 to 66,000 Da.

4. The oxygen-carrying protein molecule according to claim 1, characterized in that: The molecular oxygen binding capacity P50 of the oxygen-carrying protein ranges from 15.01 mmHg to 19.99 mmHg.

5. A method for preparing the oxygen-carrying protein molecule according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. preparing a benzimidazole derivative: adding benzimidazole, a modifying agent and a catalyst into a solvent for reaction, and purifying after the reaction to obtain a benzimidazole derivative; S2, preparing a hemoglobin suspension and a benzimidazole derivative solution, mixing the two and incubating them, and purifying them after the end to obtain a hemoglobin suspension modified with a benzimidazole derivative; S3. Add iodoacetamide or ethylmaleimide to the obtained hemoglobin suspension modified with the benzimidazole derivative, incubate and purify to obtain the oxygen-carrying protein molecule.

6. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The modifying reagent in step S1 includes one or more of 3-chloropropanol, ethylene glycol, ethanolamine, hydroxylamine hydrochloride, and N-hydroxysuccinimide.

7. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The catalyst in step S1 includes NaOH or K2CO3.

8. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The solvent in step S1 includes one or more of dimethyl sulfoxide, acetonitrile and ethanol.

9. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The molar ratio of the benzimidazole to the modifying agent in step S1 is 1:(1-3).

10. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The molar ratio of the modifying agent to the catalyst in step S1 is 1:

1.

11. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The amount of the solvent used in step S1 is 10-20 mL / 1 g benzimidazole.

12. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The reaction temperature in step S1 is 25-80° C. and the reaction time is 2-24 h.

13. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The purification in step S1 is carried out by column chromatography, the solid phase is silica gel, the mobile phase is n-hexane / ethyl acetate, the ratio range is (5-1):1, or dichloromethane / methanol, the ratio range is (10-1):

1.

14. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The hemoglobin suspension in step S2 has a solvent of PBS, a pH range of 7.0 to 7.4, and a hemoglobin content of 0.5 to 0.8 mM. The benzimidazole derivative solution has a solvent of DMSO or acetonitrile, and a concentration of the benzimidazole derivative in the solution of 1 to 2 mM. The amount of the benzimidazole derivative solution used is 2 to 4 times the volume of the hemoglobin suspension.

15. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The incubation in step S2 is performed at a temperature of 37° C. for 1 to 2 hours.

16. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The molar amount of iodoacetamide or ethylmaleimide added in step S3 is 5 to 10 times the molar amount of the hemoglobin modified with the benzimidazole derivative in the suspension.

17. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The incubation in step S3 is performed at a temperature of 37° C. for 30 to 60 minutes.

18. The method for preparing the oxygen-carrying protein molecule according to claim 5, characterized in that: The purification described in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis fluid, and dialyzing for 12 to 24 hours at 4°C; wherein the dialysis fluid is PBS buffer with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000Da; during the period, the dialysis fluid is replaced multiple times to ensure complete removal of small molecules and solvents.

19. Use of the oxygen-carrying protein molecule according to any one of claims 1 to 4 in the preparation of a composition having the functions of supplying oxygen and removing carbon dioxide in vivo and in vitro.

20. The use according to claim 19, comprising the oxygen-carrying protein molecule and other pharmaceutically acceptable carriers or pharmaceutically acceptable components together forming a composition having the functions of supplying oxygen and removing carbon dioxide in vivo and in vitro.

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