High-purity chicken plasma fibronectin and a method for preparing the same

Abstract

The application discloses high-purity chicken plasma fibronectin and a preparation method thereof. The method comprises the following steps: taking fresh chicken plasma as raw material, adding ammonium sulfate to a saturation degree of 20% at low temperature to realize targeted extraction and enrichment of fibronectin; then, a gelatin affinity chromatography column is prepared through gelatin-cyanogen bromide (CNBr) activation gel coupling, specific binding of fibronectin and gelatin is utilized to realize efficient capture and preliminary purification, and gradient arginine solution is adopted to realize impurity washing and elution; finally, high-purity and high-activity fibronectin is obtained through desalination of dextran gel G25 chromatography. Through optimization of the combination process of ammonium sulfate precipitation, specific affinity chromatography and gel filtration, the problems of low purity, easy damage of activity, low efficiency and high cost in the traditional method are solved, the purpose of high-value preparation of high-purity fibronectin from by-product chicken blood is realized. The purity can reach 92.93%, and the high-purity chicken plasma fibronectin can be applied to the fields of biomedicine, cosmetics and cell culture.

Description

Technical Field

[0001] This invention belongs to the field of bioactive substance extraction and purification technology, and involves key technologies for fibronectin extraction and purification. Specifically, it provides a method for extracting high-purity fibronectin from chicken plasma. This method can effectively remove impurities such as other proteins to obtain high-purity fibronectin. Background Technology

[0002] Fibronectin (FN) is a multifunctional glycoprotein widely found in animal plasma and extracellular matrix. Its molecular structure contains multiple functional structural segments such as heparin-binding domain, cell-binding domain, and collagen-binding domain. It can participate in various physiological processes such as cell adhesion, migration, proliferation and differentiation, and tissue repair through specific interactions with cell surface receptors, extracellular matrix components, and bioactive molecules. It has extremely high application value in biomedicine, cosmetics, cell culture and other fields.

[0003] Chicken blood is an abundant source, but the large-scale discharge of chicken blood as waste easily leads to resource waste and environmental pollution. Chicken plasma is rich in fibronectin, and using it as a raw material to prepare high-value-added fibronectin products can not only realize the high-value utilization of livestock and poultry by-products and improve the economic benefits of the industry, but also align with the development direction of a green circular economy, resulting in significant economic and social benefits. However, the efficient and economical extraction of high-purity, high-activity fibronectin from complex plasma systems still faces many technical challenges. Existing technologies are mainly divided into two categories: traditional precipitation methods and affinity chromatography methods, but both have significant shortcomings.

[0004] Traditional precipitation methods, such as organic solvent precipitation (ethanol precipitation, acetone precipitation), promote protein precipitation by reducing the dielectric constant of the solution. Although this can improve the separation effect to some extent, organic solvents can easily destroy the spatial conformation of fibronectin and cause it to lose its biological activity. At the same time, this method poses a risk of organic solvent residue, which cannot meet the safety standards of cosmetics and medical-grade products. Furthermore, the cost of organic solvent recovery and treatment is high, which is not conducive to large-scale production.

[0005] Traditional affinity chromatography methods, such as heparin affinity chromatography, are widely used because they specifically bind to the heparin-binding domain of fibronectin. However, single-heparin affinity chromatography has significant limitations: firstly, other proteins in plasma containing heparin binding sites (such as coagulation factors and lipoproteins) compete with fibronectin for binding to the chromatography medium, resulting in a higher residue of these proteins in the elution product and difficulty in achieving a purity exceeding 90%; secondly, some fibronectins bind extremely strongly to heparin, requiring high-concentration salt solutions (such as 1-2 M NaCl) for elution. High-salt environments can easily lead to protein aggregation, affecting the subsequent biological activity and application efficacy of fibronectin. Furthermore, heparin affinity chromatography media are expensive, resulting in high costs per use and a limited lifespan, restricting their application in large-scale production.

[0006] CN201610364078.0 discloses a method for extracting multiple proteins from bovine blood. This technical solution aims to maximize the value of bovine blood, and its process design revolves around "multi-product co-production": first, blood is separated into blood cells and plasma, and then subjected to multi-step pressure filtration and different chromatography steps to sequentially extract more than ten products, including superoxide dismutase, heme, fibrinogen, thrombin, and bovine fibronectin. Within this framework, the extraction of bovine fibronectin is only one step in a long "production line." Its starting material is the "third filtrate," which has undergone multiple separation processes, resulting in diluted fibronectin and a complex composition of impurities. A single purification method, "cation exchange chromatography," is employed: the fifth filter residue is dissolved in 0.008–0.012 M acetate buffer at pH 6.8–7.2, followed by separation via cation exchange chromatography. This method separates target proteins based on the difference in isoelectric points between different proteins, and is a general purification technique. However, it is not very specific. The reason is that other proteins in bovine blood with similar isoelectric points to fibronectin (such as IgM fragments and some plasma enzymes) tend to co-bind with fibronectin, making it difficult to achieve high-purity separation. In addition, the pH and concentration of the buffer solution are both wide-range and not precisely controlled for the binding characteristics of bovine fibronectin, which may lead to weak binding of fibronectin or incomplete elution, affecting purity and recovery rate.

[0007] In summary, current fibronectin preparation technologies suffer from numerous problems, including low purity, low efficiency, easily compromised activity, high safety risks, and high production costs, failing to meet the demands of biomedicine, cosmetics, and other fields for high-purity, high-activity, and high-safety fibronectin. Therefore, developing a process-optimized, cost-controllable, safe, and efficient technical solution capable of achieving high-purity (high-selectivity), high-efficiency, and high-activity preparation of fibronectin has become a pressing technical challenge in this field. Summary of the Invention

[0008] The purpose of this invention is to innovatively develop extraction and purification technology for chicken plasma fibronectin, and to provide a high-purity, high-activity, and high-efficiency method for extracting and purifying chicken plasma fibronectin.

[0009] This invention addresses the problems of traditional precipitation methods used in plasma fibronectin extraction, which suffer from safety risks (such as residual organic solvents and high operational hazards) and high costs. It provides a method for enriching fibronectin based on ammonium sulfate-based fractional precipitation. This method alters the ionic strength of the extraction system by controlling the concentration of added ammonium sulfate, allowing fibronectin to selectively precipitate at specific salt concentrations, while most other proteins remain dissolved in the solution, achieving highly efficient enrichment of fibronectin. This process requires no complex equipment or expensive reagents, is simple to operate, and low in cost, while avoiding the safety hazards associated with the use of organic solvents in traditional methods.

[0010] This invention addresses the problems of low fibronectin recovery and incomplete removal of impurities leading to insufficient purity and low activity in traditional affinity chromatography methods used for fibronectin purification of plasma. It provides a highly efficient method for the separation and purification of fibronectin. This method employs a combined process of gelatin affinity chromatography and dextran gel G25 chromatography to purify the crude fibronectin extract in stages: firstly, the specific affinity between gelatin and fibronectin is utilized to achieve specific enrichment and preliminary removal of fibronectin through gelatin affinity chromatography; then, desalting is performed using dextran gel G25 chromatography. Analysis shows that the fibronectin purified by this method has high purity (≥90%) and good homogeneity.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing high-purity chicken plasma fibronectin, characterized by comprising the following steps: (1) Plasma pretreatment: Collect fresh chicken blood, add anticoagulant (such as citrate-phosphate-glucose solution, CPD) and protease inhibitor (such as benzyl sulfonyl fluoride, PMSF), separate the plasma by low temperature centrifugation (such as 4℃, 3000 r / min, 20 min), and store at -80℃. (2) Ammonium sulfate extraction: After melting the plasma obtained in step (1), ammonium sulfate was slowly added to the saturation of 20% under ice bath (0-4℃) conditions. After standing and centrifugation, the precipitate was collected and dissolved with PBS buffer to obtain crude extract. (3) Gelatin affinity chromatography purification: The crude extract obtained in step (2) is loaded onto a gelatin affinity chromatography column. After loading, the column is allowed to stand to allow for full adsorption. It is then washed with PBS buffer and 0.2 M arginine buffer in sequence, and finally eluted with 1 M arginine buffer. The eluent containing fibronectin is collected. The packing material of the gelatin affinity chromatography column is prepared by activating the agarose gel matrix with cyanogen bromide, and then covalently coupling gelatin as a functional ligand to the activated matrix. (4) Desalting and concentration: The fibronectin eluent collected in step (3) is desalted by passing it through a dextran gel G25 chromatography column, the eluent is collected, and high-purity chicken plasma fibronectin is obtained after concentration.

[0012] Step (1) established a dedicated system to ensure the activity of fibronectin from the source, preventing coagulation, hemolysis, and protease degradation of fibronectin, and providing highly active and high-quality raw materials for subsequent purification. Storage at -80℃ maintains activity, preventing fibronectin degradation, denaturation, and inactivation throughout the process.

[0013] Step (2) Fibronectin is extracted using the 20% ammonium sulfate precipitation method. Experiments have verified that at this specific saturation level (distinct from the ambiguous precipitation solution range in traditional precipitation methods), fibronectin selectively precipitates, while a large amount of highly soluble contaminating proteins (such as albumin, immunoglobulins, etc.) remain in the supernatant. During the salting process, it is crucial to ensure complete dissolution of the ammonium sulfate to prevent localized supersaturation. After salting, the precipitate is collected after standing and centrifugation. The precipitate is reconstituted with a suitable buffer solution (such as phosphate buffer, PBS). After centrifugation, the supernatant is filtered through a 0.45 μm filter membrane to obtain a crude extract rich in fibronectin. The salting process is carried out at a low temperature of 0-4℃, and the stirring speed should be controlled to approximately 100 r / min to avoid foaming.

[0014] Step (3) Gelatin affinity chromatography was used to purify fibronectin. Gelatin can specifically bind to the collagen-binding domain of fibronectin under mild binding conditions. Arginine, as a mild eluent, can effectively competitively dissociate fibronectin while maintaining its biological activity.

[0015] Step (4) utilizes the molecular sieve principle of gel filtration. Fibronectin, with its large molecular weight, is eluted first because it cannot enter the internal pores of the gel; while salt ions with very small molecular weights (such as Na+) are eluted. + Cl - Arg + (etc.) are then eluted after entering the long-path gel channels. Through this step, buffer replacement and desalting are efficiently achieved, and trace small molecule impurities that may be co-eluted are further removed, resulting in a high-purity fibronectin solution.

[0016] This protocol employs a combined strategy of "specific affinity chromatography + targeted desalting" to achieve "precise purification + activity assurance": The first step uses gelatin affinity chromatography, where gelatin specifically binds to chicken plasma fibronectin via its collagen-binding domain. After loading, the sample is allowed to stand to ensure sufficient binding. A gradient elution process of "PBS removal – 0.2M Arg washing – 1M Arg elution" is then used to gradually remove non-specifically bound proteins, resulting in highly targeted elution. The second step uses dextran gel G25 chromatography, which removes salt ions introduced in the previous purification process based on the molecular sieve effect, while further removing small molecule proteins, achieving a final product purity of 92.93% ± 1.4%.

[0017] Preferably, in step (1), 15 mL of CPD and PMSF with a final concentration of 1 mM are added to every 100 mL of blood sample.

[0018] Preferably, in step (1), the centrifugation conditions are 4℃, 3000 r / min for 20 min.

[0019] Preferably, in step (2), the plasma is melted at a strictly controlled and uniform temperature (such as a 37°C constant temperature water bath) to avoid protein aggregation caused by temperature fluctuations, and the plasma melting temperature is not higher than 37°C to ensure the structural integrity and biological activity of fibronectin.

[0020] Preferably, in step (2), the ammonium sulfate addition and settling process are carried out under ice bath conditions of 0-4℃, and the centrifugation conditions are 4℃, 4000 r / min for 40 min. Using these centrifugation parameters can ensure sufficient precipitation of fibronectin. Operating at room temperature increases the risk of fibronectin denaturation. More preferably, in step (2), the precipitate obtained by centrifuging every 40 ml of plasma is dissolved in 20 ml of 1 mM PBS buffer, and then centrifuged at 4℃, 4000 r / min for 40 min. Subsequently, the supernatant is filtered through a 0.45 μm filter membrane to obtain the filtrate, which is the crude extract.

[0021] Preferably, in step (2), the plasma is melted at a strictly controlled and uniform temperature, not exceeding 37°C. The ammonium sulfate addition and settling process are carried out under ice bath conditions of 0-4°C, and the centrifugation conditions are 4°C, 4000 r / min for 40 min.

[0022] Preferably, in step (3), the gelatin affinity chromatography column is prepared by coupling the gelatin solution with cyanogen bromide activated gel at room temperature for 2 h, and then packing the column after sealing and cleaning.

[0023] Preferably, in step (3), the crude extract obtained in step (2) is loaded onto a gelatin affinity chromatography column that has been equilibrated with PBS at a low flow rate (e.g., 1 mL / min). After loading, the flow path is paused and the column is allowed to stand for a period of time (e.g., 15 min) to ensure that fibronectin can fully and specifically bind to the gelatin on the packing material.

[0024] Preferably, in step (3), washing and elution are performed at a low flow rate (e.g., 2 mL / min), with the washing buffer being PBS buffer and 0.2 M arginine buffer, and the elution buffer being 1 M arginine buffer.

[0025] Preferably, in step (3), the sample loading flow rate is 1 mL / min, the standing time after sample loading is 15 min, and the washing flow rate and elution flow rate are 2 mL / min.

[0026] Preferably, in step (4), the dextran gel G25 chromatography column is pre-equilibrated with PBS before loading the sample.

[0027] Preferably, in steps (3) and (4), the chromatography column is pre-equilibrated with PBS before loading the sample.

[0028] Preferably, in step (4), the loading flow rate of the dextran gel G25 chromatography column is 5 mL / min, the elution flow rate is 10 mL / min, and the elution buffer is PBS buffer.

[0029] Preferably, this method includes the following steps: Step 1: Preparation of Plasma Samples. Collect fresh chicken blood samples and add the anticoagulant citrate-phosphate-glucose (CPD) solution and the protease inhibitor PMSF. Add 15 mL of CPD and 1 mM PMSF to every 100 mL of blood sample. After mixing, centrifuge at 3000 r / min for 20 min at 4℃ to obtain the supernatant plasma. Store at -80℃ for later use.

[0030] Step 2: Extraction of Plasma Fibronectin. Melt the plasma at 37°C, then slowly add ammonium sulfate to 20% saturation while in an ice bath (0-4°C), stir, and allow to stand. Centrifuge the plasma at 4000 rpm for 40 min in a refrigerated centrifuge (4°C). Discard the supernatant after centrifugation and dissolve the precipitate in PBS buffer. Centrifuge the solution again (4°C, 4000 rpm, 40 min). After centrifugation, filter the supernatant through a 0.45 μm filter membrane to obtain the filtrate, which is then used.

[0031] Step 3: Gelatin affinity chromatography ① Coupling of gelatin with cyanogen bromide (CNBr) activated gel: First, calculate the amount of packing material needed based on the volume of the chromatography column. The sedimentation volume of the gel used is 1.15 times the column volume. Accurately weigh 500 mg of gelatin and add it to 50 mL of coupling solution B (containing 0.5 mol / L NaCl and 0.1 mol / L NaHCO3, pH 8.3). Dissolve the gelatin in a 50℃ water bath and incubate overnight at 4℃. Take 50 mL of CNBr activated gelatin in a clean sintered glass funnel and wash it with coupling solution A (1 mM HCl). Then dilute the washed activated gelatin with 25 mL of coupling solution A. After dilution, add 50 mL of the gelatin solution incubated overnight at 4℃ and stir at room temperature for 2 h until well mixed. After the coupling of CNBr activated gelatin and gelatin is completed, the affinity chromatography packing material is sequentially blocked and washed. First, add blocking solution (0.1 M Tris-HCl, pH 8.3) to the packing material and allow it to stand at room temperature for 3 hours. Then, wash the packing material 3 times its volume with alternating solutions of 0.1 M Tris-HCl (pH 8.3) and 0.2 M acetic acid (pH 4.2). Finally, wash the packing material with PBS to complete the solution replacement. When preparing the gel suspension, add 0.5 to 1 times the volume of the settled gel to PBS, mix thoroughly, and set aside.

[0032] ② Column Packing: Take a clean chromatography column, remove any air bubbles from the bottom membrane, and leave a water column about 1 cm high at the bottom of the column. Pour the gel suspension into the chromatography column, avoiding the introduction of air bubbles during the process, and gently stir with a plastic rod until homogeneous. Then connect the upper column head to the protein purifier, remove any air bubbles below the screen on the upper column head, and then pack the column head into the chromatography column and seal it. Set the packing flow rate to 30 mL / min, open the lower end cap of the chromatography column, and start the protein purifier at the set flow rate to pack the column. After the gel suspension has settled, continue rinsing for 3-5 column volumes. Press the column head vertically downwards to about 0.5 cm below the gel surface, continue pressing at the set flow rate, and mark the gel surface position. Press the gel surface down to 0.5 cm below the marked position, tighten the sealing ring on the column head, and close the bottom valve to complete the column packing.

[0033] ③ Equilibrate the column: Rinse the chromatography column with PBS buffer at a flow rate of 2 mL / min. The column is equilibrated when the pH and conductivity of the buffer at the outlet are the same as those of the buffer before entering the chromatography column.

[0034] ④ Sample loading: Load the processed plasma sample into the pre-equilibrated gelatin affinity chromatography column at a flow rate of 1 mL / min. After loading, pause the instrument for 15 min to ensure that fibronectin can be adsorbed to the maximum extent.

[0035] ⑤ Washing and elution: Wash the affinity chromatography column with PBS buffer at a flow rate of 2 mL / min. Collect unadsorbed proteins at 280 nm until A280 drops to baseline. Wash away other plasma contaminants with 0.2 M arginine (Arg) buffer at the same flow rate. Collect unadsorbed proteins at 280 nm and elute with buffer containing 1 M Arg at a constant flow rate. Collect fibronectin. Once A280 drops to baseline, wash two column volumes sequentially with PBS buffer, deionized water, and 20% ethanol.

[0036] ⑥ Concentration: Add the collected fibronectin to an ultrafiltration centrifuge tube and centrifuge at 4000 r / min for 15 min to obtain concentrated fibronectin.

[0037] Step 4: G25 Gel Chromatography. First, calculate the required amount of G25 powder based on the column volume: Powder amount (g) = (column volume × 1.15) ÷ 4.5. Accurately weigh 40.684 g of G25 powder and pour it into 5 times its weight of 0.1 M NaCl solution (80 ~ 100℃). Stir slightly and allow it to swell for at least 1 hour until fully swollen. Swelling is complete when the gel volume shows no change. After cooling, stir evenly and then pack the gel. Equilibrate with PBS until A280 drops to baseline. Set the loading rate of the fibronectin collection buffer to 5 mL / min. After loading, use PBS as the elution buffer and elute fibronectin at a flow rate of 10 mL / min. Collect the fibronectin eluent at the elution peak using an automatic collector. Add the collected sample to an ultrafiltration centrifuge tube and centrifuge at 4000 r / min for 15 min to obtain desalted concentrated fibronectin. Store at -80℃.

[0038] The present invention also provides a high-purity chicken plasma fibronectin, which is prepared by the preparation method described above.

[0039] The high-purity chicken plasma fibronectin has a purity of ≥90%, preferably 92.93%±1.4%.

[0040] Due to the adoption of the above technical solution, the beneficial effects of the present invention are: (1) This scheme uses ammonium sulfate precipitation to extract fibronectin. The ammonium sulfate saturation is controlled at 20%. This concentration can specifically promote the precipitation of fibronectin, while minimizing the co-precipitation of albumin and other impurities. This achieves the initial targeted enrichment of fibronectin and solves the problems of low fibronectin recovery rate and many impurities in traditional extraction methods.

[0041] (2) This invention employs gelatin affinity chromatography to purify fibronectin. Gelatin, as a specific ligand for fibronectin, can achieve highly efficient and specific binding to fibronectin through its collagen-binding domain. Compared to traditional methods such as ion exchange chromatography, this method effectively reduces the non-specific adsorption of other proteins. Furthermore, the price of gelatin raw material is far lower than that of heparin, significantly reducing the preparation cost of the affinity chromatography medium. The entire process requires no expensive equipment and is suitable for large-scale production.

[0042] (3) This invention utilizes dextran gel G25 chromatography after fibronectin purification to remove salt ions from fibronectin while preserving its biological activity during elution. Specifically, dextran gel G25 is a gel filtration chromatography medium whose core mechanism is based on the "molecular sieve effect"—the gel particles form a three-dimensional network structure with a specific pore size range. When a mixed solution containing fibronectin and salt ions flows through the chromatography column, smaller salt ions (such as Na+ that may remain in the affinity chromatography eluent) are removed. + Cl - Arg + (e.g., salt ions) can enter the micropores inside the gel particles, resulting in a long flow path and slow elution rate; while fibronectin (relative molecular mass of about 500 kDa), whose molecular mass is much larger than that of salt ions, cannot enter the gel micropores and can only flow along the gaps between gel particles, resulting in a short flow path and fast elution rate, thus achieving efficient separation of the two.

[0043] (4) The entire process of this invention avoids prolonged exposure to strong denaturants, organic solvents, and high-concentration salt solutions. The core process steps balance mildness and effectiveness: ammonium sulfate precipitation is performed at low temperatures; an arginine gradient elution strategy is used during gel affinity chromatography; and subsequent gel filtration provides rapid and efficient desalting. The synergistic effect of these multiple process measures fully ensures the integrity of fibronectin's complex biological structure and functional conformation, maintaining its excellent biological activity. Attached Figure Description

[0044] Figure 1Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of crude fibronectin extracts from broiler chicken plasma dissolved in different volumes of PBS: Marker lane: pre-stained standard protein; Lane 1: broiler chicken plasma (5-fold dilution in PBS); Lane 2: crude fibronectin extract dissolved in 5 mL PBS buffer; Lane 3: crude fibronectin extract dissolved in 10 mL PBS buffer; Lane 4: crude fibronectin extract dissolved in 15 mL PBS buffer; Lane 5: crude fibronectin extract dissolved in 20 mL PBS buffer; Lane 6: fibronectin purified by gelatin affinity chromatography (reduced, molecular weight approximately 250 kDa); Lane 7: fibronectin purified by gelatin affinity chromatography (non-reduced, molecular weight approximately 500 kDa).

[0045] Figure 2 Elution curves of fibronectin purified by gelatin affinity chromatography: Peak I (breakthrough peak): unadsorbed protein collected with PBS buffer; Peak II (elution peak): unadsorbed protein collected with 0.2 M arginine buffer; Peak III (elution peak): fibronectin collected with 1 M arginine buffer.

[0046] Figure 3 SDS-PAGE patterns of fibronectin detection during gelatin affinity chromatography purification: Lane 1: pre-stained standard protein; Lane 2: crude fibronectin extract; Lane 3: PBS buffer elution peak collected from impurities; Lane 4: 0.2 M arginine buffer elution peak collected from impurities; Lane 5: 1 M arginine buffer elution peak collected from fibronectin.

[0047] Figure 4 SDS-PAGE spectra of fibronectin detection during G25 desalting process: Lane 1: fibronectin obtained during the rising phase of the elution peak; Lane 2: fibronectin obtained during the peak phase of the elution peak. Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0049] Example 1: This embodiment provides a method for preparing high-purity fibronectin from broiler chicken plasma.

[0050] (1) Preparation of plasma samples: Healthy 39-day-old white-feathered chickens raised in Luanfeng Township, Guangze County, Fujian Province were selected and slaughtered on-site. Anticoagulant citrate-phosphate-glucose (CPD) solution and protease inhibitor (PMSF) were added to blood samples. 15 mL of CPD and 1 mM PMSF were added per 100 mL of blood sample. The samples were centrifuged at 3000 r / min for 20 min at 4℃ to obtain the supernatant plasma. The plasma was transferred to sterile blood bags using a syringe and stored at -80℃ for later use.

[0051] (2) Pretreatment of broiler chicken plasma samples: a) Plasma thawing: Place the frozen plasma bag inside a sealed bag and thaw it in a 37°C water bath.

[0052] b) Plasma fibronectin extraction and treatment: The thawed plasma was placed in a beaker in an ice bath, and ammonium sulfate was added while stirring until saturation reached 20%. After the ammonium sulfate was dissolved, it was incubated at 4°C for 1 h. The incubated plasma was then centrifuged at 4000 r / min for 40 min in a refrigerated centrifuge (4°C). After centrifugation, the crude fibronectin extract precipitate was dissolved in 1 mM PBS buffer, and centrifuged again under the same conditions. The supernatant was then filtered through a 0.45 μm filter membrane to obtain the filtrate.

[0053] Screening for the optimal volume of PBS for dissolving crude fibronectin extract precipitate: The centrifuged crude fibronectin extract precipitate (40 mL plasma / tube) was dissolved in different volumes of 1 mM PBS buffer (5 mL, 10 mL, 15 mL, 20 mL), and the presence of flocculent precipitate was observed. After dissolution, the solution was centrifuged again, and the supernatant was filtered through a 0.45 μm filter membrane to obtain the filtrate. Dissolving the crude fibronectin extract precipitate with 5 mL PBS buffer resulted in slow dissolution, a small amount of flocculent material formation, a yellow solution, and easy clogging of the filter membrane, leading to PBS spillage and waste. Dissolving the crude fibronectin extract precipitate with 10 mL and 15 mL PBS buffer resulted in moderate dissolution, a small amount of flocculent material formation, a slightly yellow solution, slight clogging of the filter membrane, frequent filter membrane replacements, and slow filtration. Dissolving the crude fibronectin extract precipitate with 20 mL PBS buffer resulted in fast dissolution, no flocculent material formation, a slightly yellow and clear solution, and easy clogging of the filter membrane, with a fast filtration speed. SDS-PAGE analysis was performed on crude fibronectin extract precipitates dissolved in different volumes of PBS buffer. Figure 1 Lanes 1-5 were used, and 20 mL of PBS solution was loaded onto a gelatin affinity chromatography column for further detection of the target band of fibronectin. Figure 1 (lanes 6-7). Based on this, the volume ratio of PBS used to dissolve the crude fibronectin extract to the amount of broiler chicken plasma was calculated to be 1:2.

[0054] (3) Coupling of gelatin with CNBr activated gel: a) Accurately weigh 500 mg of gelatin and add it to 50 mL of coupling solution B (containing 0.5 mol / L NaCl and 0.1 mol / L NaHCO3, pH 8.3). Dissolve the gelatin by stirring in a 50°C water bath and incubate overnight at 4°C. Take 50 mL of the CNBr activated gelatin into a clean sintered glass funnel and wash it with pre-cooled coupling solution A (4°C, 1 mM HCl) for at least 30 min (using approximately 3000 mL of coupling solution A). Dilute the washed activated gelatin with 25 mL of coupling solution A, and then add 50 mL of the gelatin solution incubated overnight at 4°C. Stir at room temperature for 2 h until well mixed. Collect the supernatant for concentration analysis of the uncoupled gelatin (using the BCA method). After the CNBr activated gelatin and gelatin are coupled, the affinity chromatography packing material is sequentially blocked and washed. First, add blocking solution (0.1 M Tris-HCl, pH 8.3) to the packing material and allow it to stand at room temperature for 3 hours. Then, wash the packing material 3 times its volume with alternating solutions of 0.1 M Tris-HCl (pH 8.3) and 0.2 M acetic acid (pH 4.2). Finally, wash the packing material with PBS to complete the solution replacement. When preparing the gel suspension, add 0.5 to 1 times the volume of the settled gel to PBS, mix thoroughly, and set aside.

[0055] b) Coupling ligand density of gelatin and CNBr activated gel: The concentration of uncoupled gelatin in the collected coupling supernatant was calculated using the BCA method, and then the coupling ligand density of CNBr was calculated using the formula: .

[0056] M1: Determined total amount of ligand added before coupling (unit: mg); M2: Measured amount of free protein in the solution after coupling (unit: mg); V: Packing volume (unit: mL).

[0057] The final calculated coupling ligand density of CNBr in this example is 4.38 mg / mL.

[0058] c) Packing of gelatin-CNBr affinity packing material: Take a clean chromatography column, remove air bubbles from the bottom membrane, and leave a water column about 1 cm high at the bottom of the column. Pour the gel suspension into the chromatography column, avoiding air bubbles during the process, and gently stir with a plastic rod until homogeneous. Then connect the upper column head to the protein purifier, remove air bubbles below the screen of the upper column head, and then pack the column head into the chromatography column and seal it. Set the packing flow rate to 30 mL / min, open the lower end cap of the chromatography column, and start the protein purifier at the set flow rate to pack the column. After the gel suspension has settled, continue rinsing for 3-5 column volumes, press the column head vertically downwards to about 0.5 cm below the gel surface, continue pressing at the set flow rate, and mark the gel surface position. Press the gel surface down to 0.5 cm below the marked position, tighten the sealing ring of the column head, close the bottom valve, and the column packing is complete.

[0059] (4) Swelling of Sephadex G25: Accurately weigh 40.684 g of G25 dry powder, pour it into 0.1 M NaCl at 80 ~100℃ with 5 times the weight of dry powder and stir slightly. Swell for at least 1 h until fully swollen. When the volume of the gel does not change, the swelling is complete. After cooling, stir evenly before use.

[0060] Packing of Sephadex G25: Set the flow rate to 40 mL / min and follow the same procedure as packing gelatin CNBr affinity filler.

[0061] (5) Equilibration, loading, elution, and sample detection of gelatin affinity chromatography: The chromatography column was rinsed with PBS buffer at a flow rate of 2 mL / min until the pH and conductivity of the buffer at the outlet were consistent with those before entering the column, indicating that the column equilibration was complete. The processed plasma sample was loaded into the equilibrated gelatin affinity chromatography column at a flow rate of 1 mL / min. After loading, the instrument was paused for 15 min to ensure that fibronectin was adsorbed to the maximum extent. Then, the affinity chromatography column was rinsed with PBS buffer at a flow rate of 2 mL / min. Unadsorbed proteins (breakthrough peak, peak I) were collected at a wavelength of 280 nm until A280 dropped to baseline. Next, other plasma contaminants were washed away with 0.2 M Arg buffer at a flow rate of 2 mL / min. Unadsorbed proteins were collected at 280 nm (0.2 M Arg elution peak, peak II). Finally, elution was performed with buffer containing 1 M Arg at a constant flow rate, and fibronectin was collected (1 M Arg elution peak, peak III). After A280 dropped to baseline, the column was washed sequentially with PBS buffer, deionized water, and 20% ethanol for two column volumes. The elution curve for fibronectin purification by gelatin affinity chromatography is shown below. Figure 2 As shown, the reducing SDS-PAGE assay detected the target band of fibronectin as follows: Figure 3 As shown.

[0062] (6) Equilibration, loading, elution, and sample detection of the Sephadex G25 column: The G25 column was equilibrated with PBS until the A280 level dropped to baseline. The fibronectin collection buffer was then loaded at a flow rate of 5 mL / min. After loading, fibronectin was eluted with PBS at a flow rate of 10 mL / min, and an automatic collector was set up to collect the eluent at the elution peak. The collected sample was concentrated and stored at -80℃. The target band of fibronectin was detected by reducing SDS-PAGE. The results are shown below. Figure 4 As shown.

[0063] The results of the implementation case diagram analysis are shown below: SDS-PAGE spectra of gelatin affinity chromatography eluents ( Figure 1 The results showed that dissolving the crude fibronectin extract in 5 mL, 10 mL, and 15 mL PBS buffer (lanes 2-4) yielded a high amount of other proteins and a low amount of fibronectin. The solution obtained by dissolving the crude fibronectin extract precipitate in 20 mL PBS buffer not only showed a clear target band at 250 kDa but also had fewer other protein bands (lane 5). Fibronectin purified by gelatin affinity chromatography using this solution showed a clear target band at 250 kDa in a reducing SDS-PAGE experiment (lane 6), consistent with the molecular weight characteristics of fibronectin monomer subunits. Under non-reducing conditions, the target band migrated to approximately 500 kDa (lane 7), consistent with the molecular weight of the fibronectin dimer, further confirming the extracted protein as fibronectin; however, the presence of a band at 250 kDa (lane 7) may be due to the partial breakdown of fibronectin into monomer subunits. The above results indicate that dissolving the crude fibronectin extract in 20 mL of PBS buffer is more effective for further purification of fibronectin in broiler chicken plasma.

[0064] In the purification process analysis, the reducing SDS-PAGE spectrum of the gelatin affinity chromatography eluted fraction ( Figure 3 The results showed that the fibronectin band was not obvious in the crude extract precipitated with ammonium sulfate (lane 2), while the fraction eluted with 1 M Arg buffer showed a significant single band of fibronectin at 250 kDa, with high purity (lane 5), indicating that fibronectin was effectively eluted, purified, and enriched. The fractions eluted with PBS buffer and 0.2 M Arg buffer were mainly impurities (lanes 3 and 4), indicating that the chromatography process effectively removed most of the impurities.

[0065] Further desalting of fibronectin was performed using G25 gel chromatography, and samples from the rising and peak segments of the elution peak were collected for reducing SDS-PAGE analysis. Figure 4Both lanes showed clear and single target bands at 250 kDa, indicating that the desalting process did not cause protein degradation or significant loss, and a high-purity fibronectin solution was successfully obtained.

[0066] The purity of fibronectin in broiler chicken plasma was further quantitatively evaluated using an ELISA kit. The purified fibronectin had a high purity of 92.93% ± 1.4%, indicating good purification effect.

Claims

1. A method for preparing high-purity chicken plasma fibronectin, characterized in that, Includes the following steps: (1) Plasma pretreatment: Fresh chicken blood was collected, anticoagulant and protease inhibitor were added, and plasma was obtained by low-temperature centrifugation and stored at -80℃; (2) Ammonium sulfate extraction: After melting the plasma obtained in step (1), ammonium sulfate was slowly added under ice bath conditions until the saturation was 20%. After standing and centrifugation, the precipitate was collected and dissolved with PBS buffer to obtain crude extract. (3) Gelatin affinity chromatography purification: The crude extract obtained in step (2) is loaded onto a gelatin affinity chromatography column. After loading, the column is allowed to stand to allow for full adsorption. It is then washed with PBS buffer and 0.2 M arginine buffer in sequence, and finally eluted with 1 M arginine buffer. The eluent containing fibronectin is collected. The packing material of the gelatin affinity chromatography column is prepared by activating the agarose gel matrix with cyanogen bromide, and then covalently coupling gelatin as a functional ligand to the activated matrix. (4) Desalting and concentration: The fibronectin eluent collected in step (3) is desalted by passing it through a dextran gel G25 chromatography column, the eluent is collected, and high-purity chicken plasma fibronectin is obtained after concentration.

2. The preparation method according to claim 1, characterized in that, In step (1), the centrifugation conditions are 4℃, 3000 r / min for 20 min; the anticoagulant is a citric acid-phosphate-glucose solution, and 15 mL of anticoagulant is added for every 100 mL of blood sample; the protease inhibitor is benzyl sulfonyl fluoride, and its final concentration is 1 mM.

3. The preparation method according to claim 1, characterized in that, In step (2), the plasma is melted at a strictly controlled and uniform temperature, not exceeding 37°C. The ammonium sulfate addition and settling process are carried out under ice bath conditions of 0-4°C. The centrifugation conditions are 4°C, 4000 r / min for 40 min.

4. The preparation method according to claim 3, characterized in that, In step (2), the precipitate obtained by centrifuging 40 mL of plasma is dissolved in 20 mL of 1 mM PBS buffer, and then centrifuged at 4℃ and 4000 r / min for 40 min. The supernatant is then filtered through a 0.45 μm filter membrane to obtain the filtrate, which is the crude extract.

5. The preparation method according to claim 1, characterized in that, In step (3), the gelatin affinity chromatography column is prepared by coupling the gelatin solution with cyanogen bromide activated gel at room temperature for 2 h, and then packing the column after sealing and cleaning.

6. The preparation method according to claim 1 or 5, characterized in that, In step (3), the sample loading flow rate is 1 mL / min, the standing time after sample loading is 15 min, and the washing flow rate and elution flow rate are 2 mL / min.

7. The preparation method according to claim 1, characterized in that, In step (4), the loading flow rate of the dextran gel G25 chromatography is 5 mL / min, the elution flow rate is 10 mL / min, and the elution buffer is PBS buffer.

8. The preparation method according to claim 1, characterized in that, In steps (3) and (4), the chromatography column is pre-equilibrated with PBS before loading the sample.

9. A high-purity chicken plasma fibronectin, characterized in that, It is prepared by any one of claims 1 to 8.

10. The high-purity chicken plasma fibronectin according to claim 9, characterized in that, Its purity is ≥90%.

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