Toxin adsorption protein compound, toxin removal method and toxin adsorber prepared from toxin adsorption protein compound

Abstract

The invention discloses a toxin adsorption protein compound, a toxin removal method and a manufactured toxin adsorber, and belongs to the technical field of toxin adsorption, the toxin adsorption protein compound comprises toxin adsorption protein and a positively charged microporous filter membrane medium, and the toxin adsorption protein compound is formed by fixing the toxin adsorption protein on the positively charged microporous filter membrane medium; the microporous filter membrane medium with positive charges is composed of a neutral microporous filter membrane and a polymer treating fluid. The preparation method comprises the following steps: mixing toxin adsorption protein with a positively charged polymer treatment solution to obtain a treatment solution, and then spraying the treatment solution on the surface of a neutral microporous filter membrane serving as a matrix; drying at normal temperature, so that the toxin adsorption protein is fixed on the microporous filter membrane medium with positive charges, and a toxin adsorption protein compound is formed; the toxin adsorber is prepared by filling the toxin adsorption protein compound in an adsorber main body, and bacterial endotoxin and nitric oxide in blood or body fluid can be effectively adsorbed and removed.

Description

Technical Field

[0001] The present invention relates to the technical field of toxin adsorption, and in particular to a toxin adsorption protein complex, a toxin removal method and a prepared toxin adsorber. Background Art

[0002] Sepsis caused by severe bacterial infection is usually accompanied by organ dysfunction, shock and high mortality. The main pathogenic factor of sepsis is bacterial endotoxin, and the median plasma level of patients (515pg / mL) is about 100 times higher than the normal average level (5.1pg / mL) (Opal SM et al, J. Infect. Dis. 1999; 180(5): 1584-9). Endotoxin induces the body to produce excessive nitric oxide, leading to severe hypotension and possibly systemic inflammatory response syndrome. Plasma exchange to remove toxins is a common and high-cost emergency treatment for sepsis. There is an urgent need for safe, efficient and low-cost alternatives in clinical practice.

[0003] Animal-derived hemoglobin is low-cost, and there are also studies on binding to bacterial endotoxins or nitric oxide. For example, endotoxin adsorption research—Bahl N. et al. used surface plasmon resonance technology and found that both the alpha and beta subunits of hemoglobin have high-affinity (KD in the nanomolar range) endotoxin binding sites, which may be related to the evolutionarily conserved surface positive charge region (J. Biol. Chem. 2011; 286 (43): 37793-803). For example, nitric oxide adsorption research—Angelo M. et al. reviewed the interaction between nitric oxide and hemoglobin, and the binding sites include heme and cysteine ​​sulfhydryl groups (Methods Enzymol. 2008; 436: 131-68). When rats were intraperitoneally injected with bacterial endotoxin, the S-nitrosohemoglobin formed after hemoglobin combined with nitric oxide was 25 times higher than that of the control group [Jourd'heuil D et al, Biochem. Biophys. Res. Commun. 2000; 273 (1): 22-6]. The active free cysteine ​​of hemoglobin has important antioxidant functions under physiological or pathological conditions. Mice with knockout gene of cysteine ​​at position 93 of beta subunit had greater hypotension and lung damage after bacterial endotoxin injection (Vitturi DA et al, Free Radic. Biol. Med. 2013; 55: 119-29).

[0004] Based on this, the present invention designs a toxin adsorption protein complex, a toxin removal method and a toxin adsorber to solve the above problems. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention provides a toxin adsorbing protein complex, a toxin removal method and a toxin adsorber prepared therefrom.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] The toxin adsorption protein complex comprises toxin adsorption protein and a positively charged microporous filter membrane medium, wherein the toxin adsorption protein is fixed on the positively charged microporous filter membrane medium to form a toxin adsorption protein complex; the positively charged microporous filter membrane medium is composed of a neutral microporous filter membrane and a polymer treatment liquid.

[0008] Furthermore, the preparation method of the neutral microporous filter membrane is as follows: polyvinyl chloride, polysulfone, hydroxyethyl cellulose and a porogen are dissolved in an organic solvent to obtain a casting solution; nano-hydrophilic white carbon black and konjac glucomannan microspheres are added to the casting solution, wherein the particle size of the konjac glucomannan microspheres is 200 to 300 μm, and the modified casting solution is obtained after stirring for 12 to 25 minutes under a pressure of 4 to 5.5 MPa, and the modified casting solution is coated on a non-woven fabric to obtain a neutral microporous filter membrane.

[0009] Furthermore, the mass ratio of the polyvinyl chloride, polysulfone, hydroxyethyl cellulose and porogen is 25-40:15-16:6-11:6-8.

[0010] Furthermore, the mass ratio of the nano-hydrophilic white carbon black to the konjac glucomannan microspheres is 3-5:4-7.

[0011] Furthermore, the porogen is polyethylene glycol with a molecular weight of 2000-5000.

[0012] Furthermore, the positively charged polymer treatment solution comprises the following raw materials in parts by weight: 55-65% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch, 10-25% of sodium hydroxide with a pH value of 8.8-9.6, and the remainder is chitosan.

[0013] In order to better achieve the purpose of the present invention, the present invention also provides a method for removing toxins, using the toxin adsorption protein complex, comprising the following steps:

[0014] 1. Preparation of toxin adsorption protein;

[0015] 2. Preparation of neutral microporous filter membrane: dissolving 25-40 parts of polyvinyl chloride, 15-16 parts of polysulfone, 6-11 parts of hydroxyethyl cellulose and 6-8 parts of porogen in 1.3-1.5 times the volume of an organic solvent to obtain a casting solution; adding 3-5 parts of nano-hydrophilic white carbon black and 4-7 parts of konjac glucomannan microspheres to the casting solution, wherein the particle size of the konjac glucomannan microspheres is 200-300 μm, stirring for 12-25 minutes under a pressure of 4-5.5 MPa to obtain a modified casting solution, coating the modified casting solution on a non-woven fabric to obtain a neutral microporous filter membrane;

[0016] 3. Preparation of a positively charged polymer treatment solution: 55-65% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch in a mass ratio of 1:1, 10-25% of sodium hydroxide with a pH value of 8.8-9.6 and the remainder of chitosan are mixed, and stirred at 45-50°C for 1-2h to obtain the solution;

[0017] Fourth, the toxin adsorbing protein and the positively charged polymer treatment liquid are mixed in a weight ratio of 1:70 to 110 to obtain the treatment liquid, and then the neutral microporous filter membrane is used as a matrix and the treatment liquid is sprayed on the surface;

[0018] 5. Drying at room temperature allows the toxin adsorption protein to be fixed on the positively charged microporous filter membrane medium to form a toxin adsorption protein complex;

[0019] 6. Adsorb and remove bacterial endotoxins and nitric oxide in blood or body fluids through toxin adsorption protein complexes.

[0020] Furthermore, in step 4, the spraying amount is controlled at 105-130 g / m 2 .

[0021] Furthermore, step one is specifically as follows:

[0022] 1.1. Made from animal whole blood;

[0023] 1.2. Remove the plasma component from the whole blood by centrifugation or microfiltration, and then wash the red blood cell component with physiological sodium chloride solution;

[0024] 1.3. Add water or hypotonic solution to the red blood cell components for 0.5 to 8 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water and burst, releasing hemoglobin;

[0025] The hypotonic solution can be selected from 5% glucose solution, 5% glucose saline or 10% mannitol injection;

[0026] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0027] The hypertonic solution can be selected from 50% glucose, 10% glucose or 20% mannitol;

[0028] 1.5. Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the hemoglobin-rich supernatant or ultrafiltration filtrate;

[0029] 1.6. Remove impurities through ultrafiltration or chromatography to obtain highly purified toxin adsorbed protein;

[0030] 1.7. Then the system is heated to 80-90°C, treated for 15-30 minutes for inactivation, and cooled to room temperature;

[0031] 1.8. Then, 0.9% sodium chloride was used as the dialysate and the solution was replaced with a 30 kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0032] In order to better achieve the purpose of the present invention, the present invention also provides a toxin adsorber, which is prepared using the toxin adsorption protein complex, and includes an adsorber body and the toxin adsorption protein complex filled inside the adsorber body.

[0033] Compared with the prior art, the present invention has the following beneficial effects: the present invention prepares toxin adsorption protein, neutral microporous filter membrane, and positively charged polymer treatment liquid, mixes the toxin adsorption protein with the positively charged polymer treatment liquid to obtain the treatment liquid, and then uses the neutral microporous filter membrane as a matrix, sprays the treatment liquid on the surface, and controls the spraying amount to 120g / m 2 ; Dry at room temperature to fix the toxin adsorption protein on the positively charged microporous filter medium to form a toxin adsorption protein complex; Use the toxin adsorption protein complex to fill the toxin adsorption protein complex inside the adsorber body to prepare a toxin adsorber, which can effectively adsorb and remove bacterial endotoxins and nitric oxide in blood or body fluids. It can be used as a safe, efficient and low-cost alternative to plasma exchange. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the characteristic absorption spectrum of hemoglobin prepared in step 1 of Example 1. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1: In some embodiments, the toxin adsorption protein complex is prepared as follows:

[0037] 1. Preparation of toxin adsorption protein

[0038] 1.1. Made from animal whole blood;

[0039] 1.2. Remove the plasma component from the whole blood by centrifugation or microfiltration, and then wash the red blood cell component with physiological sodium chloride solution;

[0040] 1.3. Add water or hypotonic solution to the red blood cell component for 5 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water and burst, releasing hemoglobin;

[0041] The hypotonic solution can be selected from 5% glucose solution;

[0042] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0043] The hypertonic solution may be selected from 50% glucose;

[0044] 1.5. Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the hemoglobin-rich supernatant or ultrafiltration filtrate;

[0045] 1.6. Remove impurities by ultrafiltration or chromatography to obtain highly purified toxin adsorbing protein (hemoglobin);

[0046] 1.7. Then the system is heated to 80°C, treated for 30 minutes for inactivation, and cooled to room temperature;

[0047] 1.8. Then, 0.9% sodium chloride was used as the dialysate and the solution was replaced with a 30 kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0048] 2. Preparation of neutral microporous membrane

[0049] 25 parts of polyvinyl chloride, 16 parts of polysulfone, 6 parts of hydroxyethyl cellulose and 8 parts of a porogen (polyethylene glycol with a molecular weight of 4000-5000) are dissolved in 1.3 times the volume of an organic solvent to obtain a casting solution; 5 parts of nano-hydrophilic white carbon black and 4 parts of konjac glucomannan microspheres are added to the casting solution, wherein the particle size of the konjac glucomannan microspheres is 200 μm, and the modified casting solution is obtained after stirring for 12 minutes under a pressure of 5.5 MPa, and the modified casting solution is coated on a non-woven fabric to obtain a neutral microporous filter membrane;

[0050] 3. Preparation of positively charged polymer treatment solution

[0051] 65% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch in a mass ratio of 1:1, 10% of sodium hydroxide with a pH value of 9.6 and the remainder of chitosan are mixed, and the mixture is stirred at 50°C for 1 hour to obtain;

[0052] 4. Mix the toxin adsorbing protein and the positively charged polymer treatment liquid in a weight ratio of 1:70 to obtain the treatment liquid. Then, use the neutral microporous filter membrane as the matrix and spray the treatment liquid on the surface. The spraying amount is controlled at 130g / m 2 ;

[0053] 5. Drying at room temperature allows the toxin adsorption protein to be fixed on the positively charged microporous filter membrane medium to form a toxin adsorption protein complex;

[0054] Bacterial endotoxins and nitric oxide in blood or body fluids can be adsorbed and removed by toxin adsorption protein complexes.

[0055] The toxin adsorber is prepared by using the toxin adsorption protein complex filled in the adsorber body.

[0056] Hemoglobin is the most abundant protein molecule in the blood and can bind certain substances that are harmful to the body, such as bacterial endotoxins and excessive nitric oxide. The present invention fixes animal-derived hemoglobin on a positively charged microporous filter medium to form a toxin-adsorbing protein complex, which can effectively adsorb and remove harmful components such as bacterial endotoxins and nitric oxide in blood / body fluids. The toxin-adsorbing protein complex can be filled inside the adsorber body to form a toxin adsorber.

[0057] Example 2: In some embodiments, the toxin adsorption protein complex is prepared as follows:

[0058] 1. Preparation of toxin adsorption protein

[0059] 1.1. Made from animal whole blood;

[0060] 1.2. Remove the plasma component from the whole blood by centrifugation or microfiltration, and then wash the red blood cell component with physiological sodium chloride solution;

[0061] 1.3. Add water or hypotonic solution to the red blood cell component for 8 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water and burst, releasing hemoglobin;

[0062] The hypotonic solution can be selected from 5% glucose saline;

[0063] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0064] The hypertonic solution may be selected from 10% glucose;

[0065] 1.5. Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the hemoglobin-rich supernatant or ultrafiltration filtrate;

[0066] 1.6. Remove impurities by ultrafiltration or chromatography to obtain highly purified toxin adsorbing protein (hemoglobin);

[0067] 1.7. Then the system is heated to 90°C, treated for 15 minutes for inactivation, and cooled to room temperature;

[0068] 1.8. Then, 0.9% sodium chloride was used as the dialysate and the solution was replaced with a 30 kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0069] 2. Preparation of neutral microporous membrane

[0070] 40 parts of polyvinyl chloride, 15 parts of polysulfone, 11 parts of hydroxyethyl cellulose and 6 parts of a porogen (polyethylene glycol with a molecular weight of 2000-3000) are dissolved in 1.5 times the volume of an organic solvent to obtain a casting solution; 3 parts of nano-hydrophilic white carbon black and 7 parts of konjac glucomannan microspheres are added to the casting solution, wherein the particle size of the konjac glucomannan microspheres is 300 μm, and the modified casting solution is obtained after stirring for 25 minutes under a pressure of 4 MPa, and the modified casting solution is coated on a non-woven fabric to obtain a neutral microporous filter membrane;

[0071] 3. Preparation of positively charged polymer treatment solution

[0072] 55% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch in a mass ratio of 1:1, 25% of sodium hydroxide with a pH value of 8.8 and the remainder of chitosan are mixed, and the mixture is stirred and reacted at 45°C for 2 hours to obtain the product;

[0073] 4. Mix the toxin adsorbing protein and the positively charged polymer treatment liquid in a weight ratio of 1:110 to obtain the treatment liquid. Then, use the neutral microporous filter membrane as the matrix and spray the treatment liquid on the surface. The spraying amount is controlled at 105g / m 2 ;

[0074] 5. Drying at room temperature allows the toxin adsorption protein to be fixed on the positively charged microporous filter membrane medium to form a toxin adsorption protein complex;

[0075] Bacterial endotoxins and nitric oxide in blood or body fluids can be adsorbed and removed by toxin adsorption protein complexes.

[0076] The toxin adsorber is prepared by using the toxin adsorption protein complex filled in the adsorber body.

[0077] Example 3: In some embodiments, the toxin adsorption protein complex is prepared as follows:

[0078] 1. Preparation of toxin adsorption protein

[0079] 1.1. Made from animal whole blood;

[0080] 1.2. Remove the plasma component from the whole blood by centrifugation or microfiltration, and then wash the red blood cell component with physiological sodium chloride solution;

[0081] 1.3. Add water or hypotonic solution to the red blood cell component for 7 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water and burst, releasing hemoglobin;

[0082] The hypotonic solution can be selected from 10% mannitol injection;

[0083] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0084] The hypertonic solution can be selected from 20% mannitol;

[0085] 1.5. Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the hemoglobin-rich supernatant or ultrafiltration filtrate;

[0086] 1.6. Remove impurities by ultrafiltration or chromatography to obtain highly purified toxin adsorbing protein (hemoglobin);

[0087] 1.7. Then the system is heated to 85°C, treated for 20 minutes for inactivation, and cooled to room temperature;

[0088] 1.8. Then, 0.9% sodium chloride was used as the dialysate and the solution was replaced with a 30 kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0089] 2. Preparation of neutral microporous membrane

[0090] 30 parts of polyvinyl chloride, 15.5 parts of polysulfone, 8 parts of hydroxyethyl cellulose and 7 parts of a porogen (polyethylene glycol with a molecular weight of 3000-5000) are dissolved in 1.4 times the volume of an organic solvent to obtain a casting solution; 4 parts of nano-hydrophilic white carbon black and 5 parts of konjac glucomannan microspheres are added to the casting solution, wherein the particle size of the konjac glucomannan microspheres is 250 μm, and the modified casting solution is obtained after stirring for 18 minutes under a pressure of 4.5 MPa, and the modified casting solution is coated on a non-woven fabric to obtain a neutral microporous filter membrane;

[0091] 3. Preparation of positively charged polymer treatment solution

[0092] 60% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch in a mass ratio of 1:1, 20% of sodium hydroxide with a pH value of 9 and the remainder of chitosan are mixed, and the mixture is stirred at 48°C for 1.7 hours to obtain the product;

[0093] 4. Mix the toxin adsorbing protein and the positively charged polymer treatment liquid in a weight ratio of 1:90 to obtain the treatment liquid. Then, use the neutral microporous filter membrane as the matrix and spray the treatment liquid on the surface. The spraying amount is controlled at 120g / m 2 ;

[0094] 5. Drying at room temperature allows the toxin adsorption protein to be fixed on the positively charged microporous filter membrane medium to form a toxin adsorption protein complex;

[0095] Bacterial endotoxins and nitric oxide in blood or body fluids can be adsorbed and removed by toxin adsorption protein complexes.

[0096] The toxin adsorber is prepared by using the toxin adsorption protein complex filled in the adsorber body.

[0097] Comparative Example 1

[0098] The toxin adsorption protein complex is prepared as follows:

[0099] 1. Preparation of toxin adsorption protein

[0100] 1.1. Made from animal whole blood;

[0101] 1.2. Remove the plasma component from the whole blood by centrifugation or microfiltration, and then wash the red blood cell component with physiological sodium chloride solution;

[0102] 1.3. Add water or hypotonic solution to the red blood cell component for 7 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water and burst, releasing hemoglobin;

[0103] The hypotonic solution can be selected from 10% mannitol injection;

[0104] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0105] The hypertonic solution can be selected from 20% mannitol;

[0106] 1.5. Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the hemoglobin-rich supernatant or ultrafiltration filtrate;

[0107] 1.6. Remove impurities by ultrafiltration or chromatography to obtain highly purified toxin adsorbing protein (hemoglobin);

[0108] 1.7. Then the system is heated to 85°C, treated for 20 minutes for inactivation, and cooled to room temperature;

[0109] 1.8. Then, 0.9% sodium chloride was used as the dialysate and the solution was replaced with a 30 kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0110] 2. Preparation of neutral microporous membrane

[0111] 30 parts of polyvinyl chloride, 15.5 parts of polysulfone, 8 parts of hydroxyethyl cellulose and 7 parts of a porogen (polyethylene glycol with a molecular weight of 3000-5000) are dissolved in 1.4 times the volume of an organic solvent to obtain a casting solution; 4 parts of nano-hydrophilic white carbon black and 5 parts of konjac glucomannan microspheres are added to the casting solution, wherein the particle size of the konjac glucomannan microspheres is 250 μm, and the modified casting solution is obtained after stirring for 18 minutes under a pressure of 4.5 MPa, and the modified casting solution is coated on a non-woven fabric to obtain a neutral microporous filter membrane;

[0112] 3. Mix the toxin adsorbing protein and water in a weight ratio of 1:90 to obtain the treatment liquid. Then use the neutral microporous filter membrane as the matrix and spray the treatment liquid on the surface. The spraying amount is controlled at 120g / m 2 ;

[0113] 4. Dry at room temperature to fix the toxin adsorption protein on the neutral microporous filter membrane medium to form a toxin adsorption protein complex.

[0114] The toxin adsorber is prepared by using the toxin adsorption protein complex filled in the adsorber body.

[0115] Comparative Example 2

[0116] The toxin adsorption protein complex is prepared as follows:

[0117] 1. Preparation of toxin adsorption protein

[0118] 1.1. Made from animal whole blood;

[0119] 1.2. Remove the plasma component from the whole blood by centrifugation or microfiltration, and then wash the red blood cell component with physiological sodium chloride solution;

[0120] 1.3. Add water or hypotonic solution to the red blood cell component for 7 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water and burst, releasing hemoglobin;

[0121] The hypotonic solution can be selected from 10% mannitol injection;

[0122] 1.4. Add hypertonic solution to restore the overall isotonic environment;

[0123] The hypertonic solution can be selected from 20% mannitol;

[0124] 1.5. Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the hemoglobin-rich supernatant or ultrafiltration filtrate;

[0125] 1.6. Remove impurities by ultrafiltration or chromatography to obtain highly purified toxin adsorbing protein (hemoglobin);

[0126] 1.7. Then the system is heated to 85°C, treated for 20 minutes for inactivation, and cooled to room temperature;

[0127] 1.8. Then, 0.9% sodium chloride was used as the dialysate and the solution was replaced with a 30 kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

[0128] 2. Preparation of positively charged polymer treatment solution

[0129] 60% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch in a mass ratio of 1:1, 20% of sodium hydroxide with a pH value of 9 and the remainder of chitosan are mixed, and the mixture is stirred at 48°C for 1.7 hours to obtain the product;

[0130] 3. Mix the toxin adsorbing protein and the positively charged polymer treatment liquid in a weight ratio of 1:90 to obtain the treatment liquid. Then use the commercially available nylon microporous filter membrane as the matrix and spray the treatment liquid on the surface. The spraying amount is controlled at 120g / m 2 ;

[0131] 4. Dry at room temperature to fix the toxin adsorption protein on the positively charged microporous filter membrane medium to form a toxin adsorption protein complex;

[0132] Bacterial endotoxins and nitric oxide in blood or body fluids can be adsorbed and removed by toxin adsorption protein complexes.

[0133] The toxin adsorber is prepared by using the toxin adsorption protein complex filled in the adsorber body.

[0134] In the experimental example, the toxin adsorbers prepared in Examples 1 to 3 of the present invention and Comparative Examples 1 to 2 were used to detect the adsorption effects of bacterial endotoxins and nitric oxide.

[0135] Test method: (1) Prepare a culture medium containing bacterial endotoxin (2EU / mL) and excess nitric oxide (70 μg / L (nitrate ion NO 2 - / NO 3 -)) of plasma; (2) using a toxin adsorber to adsorb the plasma, and detecting the endotoxin, colony count and nitric oxide content at the outlet of the toxin adsorber. The test results are shown in Table 1.

[0136] Table 1 Endotoxin, colony count and nitric oxide content test results

[0137]

[0138] The present invention prepares toxin adsorption protein, neutral microporous filter membrane, and positively charged polymer treatment liquid, mixes the toxin adsorption protein and the positively charged polymer treatment liquid to obtain the treatment liquid, and then uses the neutral microporous filter membrane as a matrix, sprays the treatment liquid on the surface, and controls the spraying amount to 120g / m 2 ; Dry at room temperature to fix the toxin adsorption protein on the positively charged microporous filter medium to form a toxin adsorption protein complex; Use the toxin adsorption protein complex to fill the toxin adsorption protein complex inside the adsorber body to prepare a toxin adsorber, which can effectively adsorb and remove bacterial endotoxins and nitric oxide in blood or body fluids. It can be used as a safe, efficient and low-cost alternative to plasma exchange.

[0139] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A toxin adsorption protein complex, characterized in that It includes toxin adsorption protein and positively charged microporous filter membrane medium, which fixes the toxin adsorption protein on the positively charged microporous filter membrane medium to form a toxin adsorption protein complex; the positively charged microporous filter membrane medium is composed of a neutral microporous filter membrane and a polymer treatment liquid.

2. The toxin adsorption protein complex according to claim 1, characterized in that The preparation method of the neutral microporous filter membrane comprises the following steps: dissolving polyvinyl chloride, polysulfone, hydroxyethyl cellulose and a porogen in an organic solvent to obtain a casting solution; adding nano hydrophilic white carbon black and konjac glucomannan microspheres into the casting solution, wherein the particle size of the konjac glucomannan microspheres is 200 to 300 μm; stirring the solution for 12 to 25 minutes under a pressure of 4 to 5.5 MPa to obtain a modified casting solution; coating the modified casting solution on a non-woven fabric to obtain a neutral microporous filter membrane.

3. The toxin adsorption protein complex according to claim 2, characterized in that: The mass ratio of the polyvinyl chloride, polysulfone, hydroxyethyl cellulose and porogen is 25-40:15-16:6-11:6-8.

4. The toxin adsorption protein complex according to claim 3, characterized in that The mass ratio of nano hydrophilic white carbon black to konjac glucomannan microspheres is 3-5:4-7.

5. The toxin adsorption protein complex according to claim 4, characterized in that: The porogen is polyethylene glycol with a molecular weight of 2000-5000.

6. The toxin adsorption protein complex according to claim 5, characterized in that The positively charged polymer treatment liquid comprises the following raw materials in parts by weight: 55-65% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch, 10-25% of sodium hydroxide with a pH value of 8.8-9.6, and the remainder is chitosan.

7. A method for removing toxins, using the toxin adsorbing protein complex according to claim 6, characterized in that: The following steps are involved:

1. Preparation of toxin adsorption protein; 2. Preparation of neutral microporous filter membrane: dissolving 25-40 parts of polyvinyl chloride, 15-16 parts of polysulfone, 6-11 parts of hydroxyethyl cellulose and 6-8 parts of porogen in 1.3-1.5 times the volume of an organic solvent to obtain a casting solution; adding 3-5 parts of nano-hydrophilic white carbon black and 4-7 parts of konjac glucomannan microspheres to the casting solution, wherein the particle size of the konjac glucomannan microspheres is 200-300 μm, stirring for 12-25 minutes under a pressure of 4-5.5 MPa to obtain a modified casting solution, coating the modified casting solution on a non-woven fabric to obtain a neutral microporous filter membrane; 3. Preparation of a positively charged polymer treatment solution: 55-65% of a mixture of polydiallyldimethylammonium chloride and cationic etherified starch in a mass ratio of 1:1, 10-25% of sodium hydroxide with a pH value of 8.8-9.6 and the remainder of chitosan are mixed, and stirred at 45-50°C for 1-2h to obtain the solution; Fourth, the toxin adsorbing protein and the positively charged polymer treatment liquid are mixed in a weight ratio of 1:70 to 110 to obtain the treatment liquid, and then the neutral microporous filter membrane is used as a matrix and the treatment liquid is sprayed on the surface; 5. Drying at room temperature allows the toxin adsorption protein to be fixed on the positively charged microporous filter membrane medium to form a toxin adsorption protein complex; 6. Adsorb and remove bacterial endotoxins and nitric oxide in blood or body fluids through toxin adsorption protein complexes.

8. The toxin removal method according to claim 7, characterized in that: In step 4, the spraying amount is controlled at 105-130 g / m 2 .

9. The toxin removal method according to claim 7, characterized in that: Step 1 is as follows: 1.

1. Made from animal whole blood; 1.

2. Remove the plasma component from the whole blood by centrifugation or microfiltration, and then wash the red blood cell component with physiological sodium chloride solution; 1.

3. Add water or hypotonic solution to the red blood cell components for 0.5 to 8 minutes. Due to the action of water or hypotonic solution, water molecules enter the red blood cells, causing the red blood cells to absorb water and burst, releasing hemoglobin; The hypotonic solution can be selected from 5% glucose solution, 5% glucose saline or 10% mannitol injection; 1.

4. Add hypertonic solution to restore the overall isotonic environment; The hypertonic solution can be selected from 50% glucose, 10% glucose or 20% mannitol; 1.

5. Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the hemoglobin-rich supernatant or ultrafiltration filtrate; 1.

6. Remove impurities through ultrafiltration or chromatography to obtain highly purified toxin adsorbed protein; 1.

7. Then the system is heated to 80-90°C, treated for 15-30 minutes for inactivation, and cooled to room temperature; 1.

8. Then, 0.9% sodium chloride was used as the dialysate and the solution was replaced with a 30 kDa ultrafiltration membrane to convert the solution into a 0.9% sodium chloride solution.

10. A toxin adsorber, prepared using the toxin adsorption protein complex according to claim 6, characterized in that: The invention comprises an adsorber body and a toxin adsorption protein complex filled in the adsorber body.

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