CN111848807A - Method for protecting fibronectin stability and biological activity - Google Patents
Method for protecting fibronectin stability and biological activity Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a method for protecting the stability and biological activity of fibronectin, which relates to the field of protein activity research and comprises the steps of titanium powder loading, storage solution preservation, solid-liquid separation and extraction of fibronectin and the like. When the fibronectin purification kit is used, the fibronectin can be extracted by a solid-liquid separation method to remove harmful protein protection components such as toxic dose PMSF or EDTA or other protection components interfering experiments, so that the extraction and use of the fibronectin are simply and efficiently completed, and the activity application and research of the fibronectin in cell experiments are not influenced.
Description
Technical Field
The invention relates to the field of protein activity research, in particular to a method for protecting the stability and biological activity of fibronectin.
Background
Fibronectin (Fn) is an extracellular matrix (ECM) protein that bridges cells to cells and between cells and basement membranes, maintains the normal morphology and function of cells, and binds to heparin, actin, fibrin, collagen, DNA, and cells. The important functions of the cell growth promoter are promoting cell adhesion and adhesion, improving the cell adherence rate and confluence rate, shortening the cell confluence time, ensuring good cell morphological structure, enhancing the metabolic rate and obviously improving the protein synthesis speed.
For the fibronectin which is frequently used, the fibronectin is generally preserved at-20 ℃, and in order to avoid repeated freezing and thawing of the fibronectin, the common method is to add a certain amount of glycerol into the fibronectin, but only the glycerol is added, so that the repeated freezing and thawing of the fibronectin can be avoided, but the degradation of the fibronectin cannot be effectively prevented. And because the components of the protective agent are more, the effect on fibronectin is different due to different adding amounts, so a method for protecting the stability and the biological activity of fibronectin is needed.
Commonly used protein protective agents include glycerol, mannitol, polyethylene glycol, glycine, glutamic acid, arginine, bovine serum albumin, and the like. In practice, it is difficult to achieve effective protection of fibronectin with a single protectant, and thus, in order to enhance the protection effect, it is usually necessary to use a combination of protectants. There is a need to develop better fibronectin protectors.
The existing protease inhibitor can prevent protein degradation, but has cytotoxic effect, and influences the application of fibronectin stock solution in vitro cell experiments.
The LBL technology generally comprises the steps of firstly immersing a substrate with charges on the surface into a polyelectrolyte solution with opposite charges, standing for a period of time, taking out the substrate, cleaning and removing the polyelectrolyte which is not firmly adsorbed, and completing the adsorption process of surface substances. The method can be used for preparing various adsorption film layers at normal temperature, has good stability, and is an effective method for constructing the composite organic/inorganic coating. The LBL self-assembled membrane can be loaded with a variety of biomolecules such as polysaccharides, proteins, and enzymes. Commercially pure titanium (Ti) and its alloys have excellent corrosion resistance and good biocompatibility, and have been widely used in the fields of plastic materials, dental prosthetic materials, and cardiovascular implant materials, such as artificial joints, dental implants, and heart valve annuluses, without cytotoxicity.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the method of storing and preventing fibronectin from degrading prevents the protein activity research application of fibronectin in cell experiments, and particularly influences the biological activity application of fibronectin in cell experiments due to the toxic effect of toxic doses of protease inhibitors on cells in protein stock solutions.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for protecting the stability and biological activity of fibronectin comprises the following steps:
(1) fixing fibronectin on the surface of the activated pure titanium powder;
(2) adding the fibronectin-immobilized solid powder into a storage solution, fully and uniformly mixing, and storing at-20 ℃, wherein the storage solution contains a protease inhibitor and a protein protective agent;
(3) and centrifuging to remove the stock solution before use, washing the precipitate with PBS or TBS, and then resuspending to a working concentration to obtain the fibronectin working solution.
Preferably, the method of immobilizing fibronectin to a Ti surface is as follows:
(a) ultrasonically cleaning pure titanium powder with acetone, absolute ethyl alcohol and distilled water twice for 5min each time, and drying for later use;
(b) preparing 0.5M NaOH solution, immersing the dried pure titanium powder in the solution, reacting for 24 hours at 70 ℃, fully washing with distilled water, and drying to obtain activated titanium powder;
(c) preparing 2% aminopropyl triethoxysilane APTE solution, wherein the solvent is absolute ethyl alcohol, and placing activated titanium powder into the APTE solution for oscillation reaction for 10 hours at 37 ℃; cleaning the titanium powder by absolute ethyl alcohol, and then carrying out curing reaction for 6h at 120 ℃ to enhance the combination of APTE and the surface of the activated titanium powder, so as to obtain silanized titanium powder A;
(d) preparing a fibronectin solution with the concentration of 150 mu g/mL by using an EDC/NHS/PBS system, wherein the pH value of the fibronectin solution is 7.0;
(e) resuspending and soaking the silanized titanium powder A in a fibronectin solution for reaction for 15min, washing away unbound fibronectin molecules by 0.01M PBS, centrifuging and removing a supernatant.
Preferably, the protease inhibitor is 40-100 mM PMSF and 1mM Pepstatin A.
Preferably, the protein protectant consists of the following raw materials: arginine with a final concentration of 50-200 mM, bovine serum albumin with a total weight of 0.2-2 wt% of a protein protective agent, EDTA with a final concentration of 0.3mg/ml, and glycerol with a total weight of 40 wt% of the protein protective agent, wherein a solvent is Tris-NaCl buffer solution, the Tris-NaCl buffer solution is Tris with a final concentration of 50mM, NaCl with a final concentration of 100mM, and the pH value is 8.0.
Preferably, the method further comprises the following steps:
soaking the silanized titanium powder B in a leupeptin solution of 5-10 mu g/mL for adsorption and fixation for 15min, then washing away unbound leupeptin with 0.01M PBS, adding the titanium powder fixedly adsorbed with the leupeptin into a storage solution, and fully mixing with the solid powder fixed with fibronectin.
Preferably, the particle size of the titanium silanized powder B is 100 times of that of the titanium silanized powder A.
The invention has the following beneficial effects:
the surface of the pure titanium powder can be effectively loaded with fibronectin after being activated, the bioactivity of the fibronectin is kept, and the high-concentration protease inhibitor and the protein protective agent in the storage solution can effectively prevent protein degradation and better maintain the bioactivity of the fibronectin in the storage process. When the fibronectin purification kit is used, the fibronectin can be extracted by a solid-liquid separation method to remove harmful protein protection components such as toxic dose PMSF or EDTA or other protection components interfering experiments, so that the extraction and use of the fibronectin are simply and efficiently completed, and the activity application and research of the fibronectin in cell experiments are not influenced.
Detailed Description
The following examples are included to provide further detailed description of the present invention and to provide those skilled in the art with a more complete, concise, and exact understanding of the principles and spirit of the invention.
Example 1: fibronectin was stored and used as follows:
(1) fixing fibronectin on the surface of the activated pure titanium powder by the following specific method:
(a) ultrasonically cleaning 10g of pure titanium powder with the particle size of 0.2mm with acetone, absolute ethyl alcohol and distilled water for two times, 5min each time, and drying for later use;
(b) preparing 0.5M NaOH solution, immersing the dried pure titanium powder in the solution, reacting for 24 hours at 70 ℃, fully washing with distilled water, and drying to obtain activated titanium powder;
(c) preparing 2 wt% Aminopropyltriethoxysilane (APTE) solution, wherein the solvent is absolute ethyl alcohol, and placing activated titanium powder into the APTE solution for oscillation reaction at 37 ℃ for 10 hours; cleaning the titanium powder by absolute ethyl alcohol, and then carrying out curing reaction for 6h at 120 ℃ to enhance the combination of APTE and the surface of the activated titanium powder, so as to obtain silanized titanium powder A;
(d) preparing a fibronectin solution with the concentration of 150 mu g/mL by using an EDC/NHS/PBS system, wherein the pH value of the fibronectin solution is 7.0;
(e) resuspending and soaking 1g of silanized titanium powder A in 5mL of fibronectin solution for reaction for 15min, washing away unbound fibronectin molecules by 0.01M PBS, centrifuging, and removing the supernatant.
(2) According to the weight ratio of 1 g: adding 100mL of fibronectin-immobilized solid powder into a stock solution, fully mixing uniformly, and storing at-20 ℃, wherein the stock solution comprises PMSF (permanent magnet phosphate) with a final concentration of 40mM, Pepstatin A with a final concentration of 1mM and a protein protective agent; the protein protective agent is composed of the following raw materials: arginine with a final concentration of 50mM, bovine serum albumin with a total weight of 0.2 wt% of a protein protective agent, EDTA with a final concentration of 0.3mg/ml, glycerol with a total weight of 40 wt% of the protein protective agent, and adopting a Tris-NaCl buffer solution as a solvent, wherein the Tris-NaCl buffer solution is Tris with a final concentration of 50mM, NaCl with a final concentration of 100mM, and the pH value is 8.0. Because PMSF and Pepstatin A are volatile in low-concentration aqueous solution, the high-concentration protein close to or equal to the storage concentration is adopted for long-term protection to prevent protease degradation, and the storage solution is removed subsequently, so that the high-concentration protein inhibitor can not produce toxic effect on cell experiments.
(3) And centrifuging to remove the stock solution before use, washing the precipitate with PBS or TBS, and then resuspending to a working concentration to obtain the fibronectin working solution.
Example 2: fibronectin was stored and used as follows:
(1) fixing fibronectin on the surface of the activated pure titanium powder by the following specific method:
(a) ultrasonically cleaning 10g of pure titanium powder with the particle size of 0.8mm with acetone, absolute ethyl alcohol and distilled water for two times, 5min each time, and drying for later use;
(b) preparing 0.5M NaOH solution, immersing the dried pure titanium powder in the solution, reacting for 24 hours at 70 ℃, fully washing with distilled water, and drying to obtain activated titanium powder;
(c) preparing 2 wt% aminopropyltriethoxysilane solution, wherein the solvent is absolute ethyl alcohol, and placing activated titanium powder into APTE solution for oscillation reaction for 10 hours at 37 ℃; cleaning the titanium powder by absolute ethyl alcohol, and then carrying out curing reaction for 6h at 120 ℃ to enhance the combination of APTE and the surface of the activated titanium powder, so as to obtain silanized titanium powder A;
(d) preparing a fibronectin solution with the concentration of 150 mu g/mL by using an EDC/NHS/PBS system, wherein the pH value of the fibronectin solution is 7.0;
(e) resuspending and soaking 1g of silanized titanium powder A in 5mL of fibronectin solution for reaction for 15min, washing away unbound fibronectin molecules by 0.01M PBS, centrifuging, and removing the supernatant.
(2) Soaking 1g of silanized titanium powder B in 2mL of leupeptin solution of 10 mu g/mL for adsorption and fixation for 15min, then washing away unbound leupeptin by 0.01M PBS, and adding the titanium powder fixedly adsorbed with leupeptin into the storage solution in a ratio of 1 g: 500 mL.
(3) Then, 1 g: adding 50mL of fibronectin-immobilized solid powder into the stock solution, fully mixing uniformly, and storing at-20 ℃, wherein the stock solution contains PMSF with a final concentration of 100mM, Pepstatin A with a final concentration of 1mM and a protein protective agent; the protein protective agent is composed of the following raw materials: arginine with a final concentration of 200mM, bovine serum albumin with a total weight of 2 wt% of a protein protective agent, EDTA with a final concentration of 0.3mg/ml, glycerol with a total weight of 40 wt% of the protein protective agent, and adopting a Tris-NaCl buffer solution as a solvent, wherein the Tris-NaCl buffer solution is Tris with a final concentration of 50mM, NaCl with a final concentration of 100mM and pH is 8.0.
(4) And centrifuging to remove the stock solution before use, washing the precipitate with PBS or TBS, and then resuspending to a working concentration to obtain the fibronectin working solution.
Example 3: fibronectin was stored and used as follows:
(1) fixing fibronectin on the surface of the activated pure titanium powder by the following specific method:
(a) ultrasonically cleaning 10g of pure titanium powder with the particle size of 0.5mm with acetone, absolute ethyl alcohol and distilled water for two times, 5min each time, and drying for later use;
(b) preparing 0.5M NaOH solution, immersing the dried pure titanium powder in the solution, reacting for 24 hours at 70 ℃, fully washing with distilled water, and drying to obtain activated titanium powder;
(c) preparing 2 wt% of APTE solution, wherein the solvent is absolute ethyl alcohol, and placing activated titanium powder into the APTE solution for oscillation reaction for 10 hours at 37 ℃; cleaning the titanium powder by absolute ethyl alcohol, and then carrying out curing reaction for 6h at 120 ℃ to enhance the combination of APTE and the surface of the activated titanium powder, so as to obtain silanized titanium powder A;
(d) preparing a fibronectin solution with the concentration of 150 mu g/mL by using an EDC/NHS/PBS system, wherein the pH value of the fibronectin solution is 7.0;
(e) resuspending and soaking 1g of silanized titanium powder A in 5mL of fibronectin solution for reaction for 15min, washing away unbound fibronectin molecules by 0.01M PBS, centrifuging, and removing the supernatant.
(2) Soaking 1g of silanized titanium powder B in 2mL of leupeptin solution of 7.5 mu g/mL for adsorption and fixation for 15min, then washing away unbound leupeptin solution with 0.01M PBS, adding the titanium powder on which leupeptin is fixedly adsorbed into the storage solution, and adding the titanium powder into the storage solution according to the proportion of 1 g: 1L of the compound. The grain diameter of the titanium silanization powder B is 100 times of that of the titanium silanization powder A.
(3) Then, 1 g: adding 25mL of fibronectin-immobilized solid powder into the stock solution, fully mixing uniformly, and storing at-20 ℃, wherein the stock solution contains PMSF with a final concentration of 70mM, Pepstatin A with a final concentration of 1mM and a protein protective agent; the protein protective agent is composed of the following raw materials: arginine with a final concentration of 125mM, bovine serum albumin with a total weight of 1.0 wt% of a protein protective agent, EDTA with a final concentration of 0.3mg/ml, glycerol with a total weight of 40 wt% of the protein protective agent, and adopting a Tris-NaCl buffer solution as a solvent, wherein the Tris-NaCl buffer solution is Tris with a final concentration of 50mM, NaCl with a final concentration of 100mM, and the pH value is 8.0.
(4) Centrifuging to remove the storage solution before use, screening out the large-particle-size titanium silanization powder B by using a screen after resuspending the precipitate, centrifuging the small-particle-size titanium silanization powder A to obtain the precipitate, washing the precipitate by PBS or TBS, and then resuspending to working concentration to obtain the fibronectin working solution. The titanium silanized powder B adsorbed with leupeptin can be repeatedly applied to the storage of fibronectin.
Comparative example 1: the procedure of example 1 was repeated, except that the solid-liquid mixed stock of fibronectin was diluted with 2 wt% glycerol-containing Tris-NaCl buffer to a fibronectin concentration of 0.1mg/mL and used in the cell assay without removing the stock solution.
Fibronectin-loaded solid powder was isolated and extracted from the stock prepared in examples 1 to 3, and subjected to quantitative fibronectin determination and activity detection by indirect ELISA, comprising the following steps: firstly, blocking a sample by 1% BSA for 30min, washing the sample by PBS for 3 times, adding a mouse anti-human fibronectin monoclonal antibody (primary antibody), culturing the sample at 37 ℃ for 1h, washing the sample, adding an HRP-labeled goat anti-mouse IgG polyclonal antibody (secondary antibody: 1: 100dilution in PBS), culturing the sample at 37 ℃ for 1h, fully washing the sample by PBS for 5 times,adding TMB substrate, developing in dark for 10min, and finally using 1M H2SO4The development was stopped and the absorbance value was read at 450 nm. The loading concentration of fibronectin on the surface of the titanium powder is obtained according to a standard curve.
TABLE 1 determination of the results of the surface loading of fibronectin on titanium powder
The results in table 1 show that pure titanium powder efficiently adsorbs fibronectin after activation and alkylation.
Fibronectin stability and biological activity assay methods:
the detection operation steps are as follows: the storage mixtures prepared in example 1 and comparative example 1 were placed in a 37 ℃ incubator for accelerated disruption, the pellets were centrifuged at 0, 1, 3, 5 and 7 days, respectively, and their biological activity was measured by a prokaryote assay. The titanium silanized powder A loaded with fibronectin was weighed according to the loading concentration of fibronectin on the surface of the titanium powder, and resuspended in a fibronectin concentration of 0.1mg/mL using a Tris-NaCl buffer containing 2 wt% glycerol as a sample for the subsequent cell experiments, and a 0.1mg/mL fibronectin solution and control example 1 were used as controls. The prepared samples were subjected to an accelerated destruction test at 37 ℃ for 7 days.
The specific steps of the 'cell adherence promotion experiment determination' are as follows: the fibronectin has the important functions of promoting the adhesion and the adhesion of cells, improving the adherence rate and the confluence rate of the cells, shortening the cell confluence time, ensuring that the morphological structure of the cells is good, enhancing the metabolic rate and obviously improving the protein synthesis speed. Cell adhesion and cell adhesion are essential conditions for cell repair and cell growth, so that fibronectin has the effect of promoting cell repair. The experiment counts the number of cells in each hole under different fibronectin concentrations through an adhesion test of fibronectin MDBK cells, and obtains the half Effective Dose (ED) of fibronectin through four-parameter fitting50). a. The main apparatus is as follows: superclean bench, cell culture case.
b. Reagent preparation
Complete cell culture solution: 10ml of fetal bovine serum and 1ml of double antibody are measured and added into 90ml of DMEM culture solution for storage at 4 ℃.
Serum-free medium: 1ml of double antibody is measured, 99ml of 1640 culture solution is added, and the mixture is stored at 4 ℃.
Digestion solution: 0.25% trypsin.
PBS buffer: weighing 8.0g of sodium chloride, 0.20g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate, adding water to dissolve, fixing the volume to 1000ml, and sterilizing at 121 ℃ for 15 minutes under high pressure.
BSA blocking solution: 3g of bovine serum albumin was weighed, dissolved in PBS and made to volume of 100 ml.
Cell: MDBK cells (bovine kidney cells) grow in a complete cell culture solution in a single layer and attached to the wall, are passaged once every 4-5 days at a ratio of 1: 2, and grow and propagate in the complete cell culture solution.
c. Test procedure
Sample dilution and incubation: fibronectin was diluted in a 2-fold gradient in 96-well plates for a total of 10 dilutions, 50. mu.l of fibronectin samples at different dilutions per well, and a negative control (no fibronectin) was set up, 50. mu.l of PBS was added as a control, and incubated overnight at 4 ℃.
Cell adherence-promoting assay: after incubation, the liquid in the plate is discarded, 100 mul BSA is added into each hole for blocking, and the plate is placed in an incubator at 37 ℃ for incubation for 1 h; the plate was removed and the liquid was discarded and MDBK cell suspension (resuspended in serum-free medium) was added at a cell seeding density of 1.0X 105Each well was inoculated with 100. mu.l of each, and incubated in an incubator for 5 hours.
And (4) observing and calculating results: washing the incubated cell plate with PBS for 3 times, observing the cell adherence condition under a mirror, selecting five points at the edge under the mirror with the magnification of 200 times to count the number of adherence cells, and fitting a curve according to the counting result to obtain the half effective amount (ED) of fibronectin50)。
Calculating the formula: y ═ A-D)/(1+ (X/C) } B) + D
ED50Sample concentration/(2 ^ C)
The test results are shown in the following table:
TABLE 2 fibronectin degradation disruption test results
The results in table 2 show that at 37 ℃, fibronectin is gradually degraded and inactivated with the passage of time, the cell adhesion promoting activity of fibronectin is gradually reduced with the passage of time, the degradation of fibronectin is remarkably inhibited by a high-concentration protease inhibitor in the storage process after the method is adopted, the activity is slowly reduced in the process of 0-7 d, and the titanium powder loaded with fibronectin after the storage solution is removed is nontoxic to cells and does not influence the exertion of the bioactivity of fibronectin, and at 0d, the bioactivity is the same as that of a pure Fn solution. Whereas the high concentration of protease inhibitor in control example 1 was cytotoxic, cell survival was not guaranteed at the working concentration of fibronectin, and the ED of fibronectin could not be determined50. The simple Fn solution is easy to degrade at high temperature, the activity is rapidly reduced in the process of 0-7 d, the biological activity is almost lost after 7 days, and the Fn solution cannot be used for biological research or experiments. Therefore, the protein protection technology can effectively prevent protein from being denatured and decomposed, maintain the biological activity of the protein, and is separated from a storage system when being applied, is not influenced by the cytotoxicity of the storage system, and is beneficial to the exertion of the fibronectin activity.
In conclusion, the surface of the pure titanium powder can be effectively loaded with fibronectin after being activated, the bioactivity of the fibronectin is kept, and the high-concentration protease inhibitor and the protein protective agent in the storage solution can effectively prevent protein degradation and better maintain the bioactivity of the fibronectin in the storage process. When the fibronectin purification kit is used, the fibronectin can be extracted by a solid-liquid separation method to remove harmful protein protection components such as toxic dose PMSF or EDTA or other protection components interfering experiments, so that the extraction and use of the fibronectin are simply and efficiently completed, and the activity application and research of the fibronectin in cell experiments are not influenced.
The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby, and any modification made on the basis of the technical scheme according to the technical idea proposed by the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.
Claims (6)
1. A method for protecting the stability and biological activity of fibronectin is characterized by comprising the following steps:
(1) fixing fibronectin on the surface of the activated pure titanium powder;
(2) adding the fibronectin-immobilized solid powder into a storage solution, fully and uniformly mixing, and storing at-20 ℃, wherein the storage solution contains a protease inhibitor and a protein protective agent;
(3) and centrifuging to remove the stock solution before use, washing the precipitate with PBS or TBS, and then resuspending to a working concentration to obtain the fibronectin working solution.
2. A method of preserving the stability and biological activity of fibronectin as claimed in claim 1 wherein: the method for fixing the fibronectin on the surface of the titanium powder comprises the following steps:
(a) ultrasonically cleaning pure titanium powder with acetone, absolute ethyl alcohol and distilled water twice for 5min each time, and drying for later use;
(b) preparing 0.5M NaOH solution, immersing the dried pure titanium powder in the solution, reacting for 24 hours at 70 ℃, fully washing with distilled water, and drying to obtain activated titanium powder;
(c) preparing 2% aminopropyl triethoxysilane APTE solution, wherein the solvent is absolute ethyl alcohol, and placing activated titanium powder into the APTE solution for oscillation reaction for 10 hours at 37 ℃; cleaning the titanium powder by absolute ethyl alcohol, and then carrying out curing reaction for 6h at 120 ℃ to enhance the combination of APTE and the surface of the activated titanium powder, so as to obtain silanized titanium powder A;
(d) preparing a fibronectin solution with the concentration of 150 mu g/mL by using an EDC/NHS/PBS system, wherein the pH value of the fibronectin solution is 7.0;
(e) resuspending and soaking the silanized titanium powder A in a fibronectin solution for reaction for 15min, washing away unbound fibronectin molecules by 0.01M PBS, centrifuging and removing a supernatant.
3. A method of preserving the stability and biological activity of fibronectin as claimed in claim 1 wherein: the protease inhibitor is PMSF of 40-100 mM and Pepsstatin A of 1 mM.
4. A method of preserving the stability and biological activity of fibronectin as claimed in claim 1 wherein: the protein protective agent is composed of the following raw materials: arginine with a final concentration of 50-200 mM, bovine serum albumin with a total weight of 0.2-2 wt% of a protein protective agent, EDTA with a final concentration of 0.3mg/ml, and glycerol with a total weight of 40 wt% of the protein protective agent, wherein a solvent is Tris-NaCl buffer solution, the Tris-NaCl buffer solution is Tris with a final concentration of 50mM, NaCl with a final concentration of 100mM, and the pH value is 8.0.
5. The method of claim 2, wherein the method further comprises the steps of:
soaking the silanized titanium powder B in a leupeptin solution of 5-10 mu g/mL for adsorption and fixation for 15min, then washing away unbound leupeptin with 0.01M PBS, adding the titanium powder fixedly adsorbed with the leupeptin into a storage solution, and fully mixing with the solid powder fixed with fibronectin.
6. The method of claim 5, wherein the size of the titanium silanized powder B is 100 times the size of the titanium silanized powder A, and the titanium silanized powder B is screened off during use.
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Cited By (2)
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|---|---|---|---|---|
| CN114085292A (en) * | 2021-11-01 | 2022-02-25 | 上海淳誉健康科技有限公司 | Method for protecting fibronectin stability and biological activity |
| CN114395595A (en) * | 2021-12-20 | 2022-04-26 | 广州栋方生物科技股份有限公司 | Preparation method of recombinant human collagen, product and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101357240A (en) * | 2008-09-12 | 2009-02-04 | 西南交通大学 | Method for fixing laminar adhesive protein on Ti-O film surface |
| CN109925540A (en) * | 2019-03-14 | 2019-06-25 | 西南交通大学 | Albumin is mediated to form method, biomaterial and its application of albumin layer on the surface of the material |
-
2020
- 2020-07-28 CN CN202010737835.0A patent/CN111848807A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101357240A (en) * | 2008-09-12 | 2009-02-04 | 西南交通大学 | Method for fixing laminar adhesive protein on Ti-O film surface |
| CN109925540A (en) * | 2019-03-14 | 2019-06-25 | 西南交通大学 | Albumin is mediated to form method, biomaterial and its application of albumin layer on the surface of the material |
Non-Patent Citations (2)
| Title |
|---|
| 季士委等: "钛表面蛋白质微图形的构建及其对人体成骨细胞粘附行为的影响", 《材料导报》 * |
| 张慧婷等: "利用MicroBCA和QCM对钛薄膜表面固定纤连蛋白的量化研究与对比", 《功能材料》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114085292A (en) * | 2021-11-01 | 2022-02-25 | 上海淳誉健康科技有限公司 | Method for protecting fibronectin stability and biological activity |
| CN114395595A (en) * | 2021-12-20 | 2022-04-26 | 广州栋方生物科技股份有限公司 | Preparation method of recombinant human collagen, product and application thereof |
| CN114395595B (en) * | 2021-12-20 | 2024-05-10 | 广州栋方生物科技股份有限公司 | Preparation method of recombinant human collagen, product and application thereof |
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