Animal blood fibronectin co-production method

Abstract

The invention discloses a method for co-producing animal blood fibronectin, wherein trisodium citrate is added to fresh animal blood for anticoagulation, centrifugation is performed, and plasma and blood cells are collected respectively; in the plasma, a magnetic nanoparticle-polymer composite is added, and the magnetic nanoparticle-polymer composite is combined with a target plasma protein by an external magnetic field to form a precipitate, and filtered to obtain a first filtrate and a magnetic precipitate; the external magnetic field is withdrawn, and the magnetic nanoparticle-polymer composite is separated from the fibronectin to obtain a fibronectin precipitate; the magnetic nanoparticle-polymer composite includes carboxylated iron trioxide magnetic nanoparticles, polyvinyl alcohol and water; the fibronectin precipitate is dissolved, an affinity chromatography column is applied, and the FN main peak liquid is extracted and separated, and then purified by molecular sieve chromatography, ultrafiltration concentration, and finally freeze-dried to obtain an FN product. The quality and stability of the product using the scheme of the present invention are improved.

Description

Technical Field

[0001] The present disclosure relates to a method for co-producing animal fibronectin. Background Art

[0002] The domestic generic name of fibronectin is "FN". FN is a macromolecular glycoprotein discovered by foreign research in 1974, which has multiple biological activities and biological functions. At present, a large number of applied research on FN at home and abroad are mainly manifested in two aspects: 1. As a biochemical reagent, it provides a basis for the industrial application of cell culture and bioengineering. 2. It is used in the diagnosis and treatment of various diseases (such as wound repair and healing, cancer diagnosis and treatment, etc.).

[0003] Patent No. CN200510017717.8 proposes a method for co-producing animal blood fibronectin by separating and extracting several biochemical substances such as FN, SOD, immunoglobulin and hemoglobin in the same process flow. Trisodium citrate is added to fresh animal blood for anticoagulation, and the blood is quickly centrifuged. The plasma and blood cells are collected separately and stored in an environment below 10 degrees. Ammonium sulfate is added to the plasma to a saturation of 20-30%, and the blood is allowed to stand for more than 1 hour. The blood is filtered or suspended to dissolve the precipitate, and the precipitate is applied to an affinity chromatography column to extract and separate the main peak of FN. The FN product is then purified by molecular sieve chromatography, concentrated by ultrafiltration, sterilized, and finally freeze-dried to obtain the FN product. The FN product has the advantages of abundant raw material sources, simple process, and suitability for large-scale production.

[0004] In this scheme, multiple biochemical substances in animal blood are extracted and separated at the same time. However, different biochemical substances may be lost or degraded during the process of centrifugation, addition of ammonium sulfate, freeze drying, etc., resulting in a decrease in the quality and stability of the product. Summary of the invention

[0005] In order to solve the above problems, the present disclosure provides a method for co-producing animal blood fibronectin, comprising:

[0006] Add trisodium citrate to fresh animal blood for anticoagulation, centrifuge, and collect plasma and blood cells separately;

[0007] Adding a magnetic nanoparticle-polymer composite material to the plasma, combining the magnetic nanoparticle-polymer composite material with a target plasma protein by applying an external magnetic field to form a precipitate, and filtering to obtain a first filtrate and a magnetic precipitate;

[0008] The external magnetic field is removed to separate the magnetic nanoparticle-polymer composite material from the fibronectin to obtain a fibronectin precipitate;

[0009] The magnetic nanoparticle-polymer composite material comprises carboxylated iron oxide magnetic nanoparticles, polyvinyl alcohol and water;

[0010] The fibronectin precipitate is dissolved and applied to an affinity chromatography column to extract and separate the FN main peak liquid, which is then purified by molecular sieve chromatography, concentrated by ultrafiltration, and finally freeze-dried to obtain the FN product.

[0011] The inner core diameter of the carboxylated ferric oxide magnetic nanoparticles is 5-30 nm, the hydrodynamic size is 50-150 nm, and the zeta potential of the carboxylated ferric oxide magnetic nanoparticles is -70 mV.

[0012] The molecular weight of the polyvinyl alcohol is in the range of 5000Da to 10000Da.

[0013] The mass ratio of the carboxylated ferric oxide magnetic nanoparticles to polyvinyl alcohol is 1:2-5.

[0014] The co-production method further comprises the following steps:

[0015] Add ammonium sulfate to the first filtrate to a saturation of 50-60%, let it stand for more than 1 hour, filter, collect the precipitate, desalt it through ultrafiltration, and freeze-dry to obtain an immunoglobulin product.

[0016] The blood cells are washed clean, and an equal volume of water is added to hemolyze the blood. The hemolyzed blood is heated at a temperature of 60-75 degrees for 10-20 minutes, and filtered to collect the second filtrate and hemoglobin precipitate.

[0017] The second filtrate was added with 0.25 times of ethanol and 0.15 times of chloroform by volume, and allowed to stand at 4 degrees for more than 1 hour. The supernatant was collected by centrifugation and concentrated by ultrafiltration to obtain a crude SOD solution.

[0018] The hemoglobin precipitate is adjusted into a porridge state with water, and then 3-5 times the volume of a mixed solution of acetone and hydrochloric acid is added, filtered, and 1% sodium acetate or 5% tannic acid is added to the filtrate by volume. The filtrate is allowed to stand for more than 30 minutes, centrifuged or filtered, and the precipitate collected is the crude hemoglobin.

[0019] Beneficial effects of the present invention:

[0020] Superparamagnetic nanoparticles show strong magnetic response under an external magnetic field and can be pulled and manipulated by the magnetic field. They can be precipitated and separated quickly and controllably. Superparamagnetic nanoparticles have high stability and long-term magnetic properties. Polymer coating can increase the interaction between nanoparticles and target biomolecules and improve selectivity. The biocompatibility and stability of magnetic nanoparticles in vivo can be used to achieve biomedical applications.

[0021] The above and other features, aspects and advantages of the present application will be more readily understood with reference to the following detailed description. Specific implementation plan

[0022] Experimental steps:

[0023] Materials preparation:

[0024] Carboxylated iron oxide magnetic nanoparticles were purchased from Beijing Zhongke Leiming Technology Co., Ltd.

[0025] Polyvinyl alcohol, purchased from Aladdin, type 1788.

[0026] Plasma sample: fresh animal blood.

[0027] Magnetic stand or magnetic corner turner.

[0028] Wash buffer (such as phosphate buffered saline or normal saline).

[0029] Preparation of magnetic nanoparticle-polymer composites:

[0030] Carboxylated iron oxide magnetic nanoparticles are synthesized and surface modified, and the magnetic nanoparticles are mixed with polymers to form magnetic nanoparticle-polymer composite materials.

[0031] Sample preparation:

[0032] Collect the plasma samples that require processing and transfer them to centrifuge tubes.

[0033] Addition of magnetic nanoparticles-polymer composites:

[0034] An appropriate amount of magnetic nanoparticle-polymer composite material was added to the plasma sample.

[0035] Stir the sample to ensure uniform mixing.

[0036] External magnetic field treatment:

[0037] The sample is placed in a magnetic stand or magnetic angler to be subjected to an external magnetic field. Within an appropriate time, the magnetic nanoparticle-polymer composite material is allowed to bind to the target plasma protein and form a precipitate.

[0038] Magnetic separation:

[0039] Place the centrifuge tube on a magnetic stand or magnetic corner so that the magnetic nanoparticle-polymer composite material is magnetically adsorbed on the magnetic stand or magnetic corner. Carefully pour out the supernatant or use an aspirator to absorb the supernatant to separate the precipitate from the supernatant.

[0040] Sediment recovery:

[0041] Remove the magnetic stand or magnetic angle from the centrifuge tube to release the precipitate. Wash the precipitate with wash buffer to remove impurities. The washed precipitate can be separated from the supernatant by centrifugation or filtration.

[0042] 1000 ml of fresh animal blood was collected, 142 ml of 3.8% trisodium citrate anticoagulant was added, and the blood was centrifuged at 3000 rpm for 20 minutes to collect blood cells and plasma respectively.

[0043] The FN finished product was prepared by the above method. (The core diameter of the carboxylated iron oxide magnetic nanoparticles is about 10 nm, the hydrodynamic size is about 80 nm, and the zeta potential of the carboxylated iron oxide magnetic nanoparticles is -70 mV. The molecular weight range of polyvinyl alcohol is 8000 Da. The mass ratio of carboxylated iron oxide magnetic nanoparticles to polyvinyl alcohol is 1:2.)

[0044] The filtrate was added with ammonium sulfate for the second time according to volume until the homogeneity reached 60%, filtered, and the precipitate was collected, desalted by ultrafiltration, and freeze-dried to obtain immunoglobulin.

[0045] Wash the blood cells with physiological saline for 3 times, add an equal amount of distilled water to the clean red blood cells by volume, stir and hemolyze. Heat the hemolyzed blood to 75℃ and keep it warm for 20 minutes. Then filter or centrifuge to collect the filtrate and hemoglobin precipitate respectively.

[0046] The filtrate is added with 0.25 times ethanol (95%) and 0.15 times chloroform by volume, and allowed to stand at 10°C for more than 1 hour. Centrifuge for 20 minutes, collect the supernatant, and concentrate by ultrafiltration to obtain a crude SOD solution. The crude SOD solution is subjected to ion exchange chromatography, wherein the ion exchange chromatography is diethylaminoethyl-dextran gel A50, and molecular sieve chromatography, wherein the molecular sieve chromatography is dextran gel G200, to separate and purify the SOD finished solution.

[0047] The hemoglobin precipitate is adjusted into a porridge state with a small amount of water, and then 5 times acetone hydrochloric acid solution (Ph3) is added and stirred for hydrolysis for 30 minutes, filtered or centrifuged, and the red clear liquid (filtrate) is collected. 5% tannic acid (or 1% sodium acetate) is added by volume, stirred thoroughly, and allowed to stand for more than 30 minutes, centrifuged or filtered, and the precipitate is collected. After dehydration, the crude hemoglobin is obtained by drying.

[0048] Experimental evaluation

[0049] Equipment and reagents required for the experiment:

[0050] Dynamic Light Scattering (DLS) Equipment

[0051] FN Product Samples

[0052] Appropriate solvent or buffer

[0053] Sterile filter (such as 0.22μm filter membrane)

[0054] Completely transparent sample container;

[0055] Experimental steps:

[0056] Prepare samples: Take an appropriate amount of FN product sample and dissolve it in an appropriate solvent or buffer as needed. Ensure that the sample solution is evenly mixed and avoid obvious particles or bubbles.

[0057] Adjust sample concentration: According to the experimental requirements and instrument requirements, adjust the concentration of FN product samples, usually in the range of 0.1mg / mL to 10mg / mL.

[0058] Filter the sample: To remove large particles or impurities in the sample, filter the sample using a sterile filter.

[0059] Filling sample: Transfer the filtered sample into a completely transparent sample container, making sure the sample container is clean, dust-free and bubble-free.

[0060] Start the DLS instrument: According to the instrument's operating manual, turn on and start the dynamic light scattering (DLS) instrument and perform necessary warm-up and system calibration. According to the instrument's requirements, set appropriate experimental parameters, such as measurement temperature, detection angle, etc. Make sure to select the correct wavelength and detector configuration to obtain the best results.

[0061] Start measurement: Place the sample container in the DLS instrument and start the measurement.

[0062] Example 1

[0063] As mentioned above, the core diameter of carboxylated iron oxide magnetic nanoparticles is about 10nm, the hydrodynamic size is about 80nm, and the zeta potential of carboxylated iron oxide magnetic nanoparticles is -70mV. The molecular weight range of polyvinyl alcohol is 8000Da. The mass ratio of carboxylated iron oxide magnetic nanoparticles and polyvinyl alcohol is 1:2. The particle size distribution analysis of the FN product was performed using the above method, and the average particle size was 180nm and the distribution width was ±45nm.

[0064] Example 2

[0065] As mentioned above, the core diameter of carboxylated iron oxide magnetic nanoparticles is about 10nm, the hydrodynamic size is about 80nm, and the zeta potential of carboxylated iron oxide magnetic nanoparticles is -70mV. The molecular weight range of polyvinyl alcohol is 8000Da. The mass ratio of carboxylated iron oxide magnetic nanoparticles and polyvinyl alcohol is 1:1. The particle size distribution analysis of the FN product was performed using the above method, and the average particle size was 240nm and the distribution width was ±90nm.

[0066] Example 3

[0067] As mentioned above, the core diameter of the carboxylated iron oxide magnetic nanoparticles is about 10nm, the hydrodynamic size is about 80nm, and the zeta potential of the carboxylated iron oxide magnetic nanoparticles is -70mV. The molecular weight range of polyvinyl alcohol is 8000Da. The mass ratio of carboxylated iron oxide magnetic nanoparticles to polyvinyl alcohol is 1:6. The particle size distribution analysis of the FN product was performed using the above method, and the average particle size was 210nm and the distribution width was ±70nm.

[0068] Example 4

[0069] Add ammonium sulfate by volume to a saturation of 20%, stir and dissolve, and then let stand for more than 1 hour. Filter. Use the above method to analyze the particle size distribution of the FN product, and the average particle size is 320nm and the distribution width is ±105nm.

[0070] As described above, 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 thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for co-producing animal fibronectin, characterized in that: The following steps are involved: Add trisodium citrate to fresh animal blood for anticoagulation, centrifuge, and collect plasma and blood cells separately; Adding a magnetic nanoparticle-polymer composite material to the plasma, combining the magnetic nanoparticle-polymer composite material with a target plasma protein by applying an external magnetic field to form a precipitate, and filtering to obtain a first filtrate and a magnetic precipitate; The external magnetic field is removed to separate the magnetic nanoparticle-polymer composite material from the fibronectin to obtain a fibronectin precipitate; The magnetic nanoparticle-polymer composite material comprises carboxylated iron oxide magnetic nanoparticles, polyvinyl alcohol and water; The fibronectin precipitate is dissolved, applied to an affinity chromatography column, the FN main peak solution is extracted and separated, then purified by molecular sieve chromatography, concentrated by ultrafiltration, and finally freeze-dried to obtain the FN product; The core diameter of the carboxylated ferric oxide magnetic nanoparticles is about 10 nm, the hydrodynamic size is about 80 nm, and the zeta potential of the carboxylated ferric oxide magnetic nanoparticles is -70 mV; the molecular weight range of polyvinyl alcohol is 8000 Da; the mass ratio of the carboxylated ferric oxide magnetic nanoparticles to the polyvinyl alcohol is 1:2; The average particle size of the animal blood fibronectin is 180 nm, and the distribution width is ±45 nm.

2. The method for co-producing animal fibronectin according to claim 1, characterized in that: The co-production method further comprises the following steps: Add ammonium sulfate to the first filtrate to a saturation of 50%-60%, let it stand for more than 1 hour, filter, collect the precipitate, desalt by ultrafiltration, and freeze-dry to obtain an immunoglobulin product.

3. The method for co-producing animal fibronectin according to claim 1, characterized in that: The co-production method further comprises the following steps: The blood cells are washed clean, and an equal volume of water is added to hemolyze the blood. The hemolyzed blood is heated at a temperature of 60-75 degrees for 10-20 minutes, and filtered to collect the second filtrate and hemoglobin precipitate.

4. The method for co-producing animal fibronectin according to claim 3, characterized in that: The co-production method further comprises the following steps: The second filtrate was added with 0.25 times of ethanol and 0.15 times of chloroform by volume, and allowed to stand at 4 degrees for more than 1 hour. The supernatant was collected by centrifugation and concentrated by ultrafiltration to obtain a crude SOD solution.

5. The method for co-producing animal fibronectin according to claim 3, characterized in that: The co-production method further comprises the following steps: The hemoglobin precipitate is adjusted into a porridge state with water, and then 3-5 times the volume of a mixed solution of acetone and hydrochloric acid is added, filtered, and 1% sodium acetate or 5% tannic acid is added to the filtrate by volume. The filtrate is allowed to stand for more than 30 minutes, centrifuged or filtered, and the precipitate collected is the crude hemoglobin.