Method for isolating and purifying immunoglobulin g from bovine colostrum powder

Abstract

This invention discloses a method for isolating and purifying immunoglobulin G (IgG) from bovine colostrum powder, belonging to the field of protein isolation and purification technology. The method includes the step of eluting the immunoglobulin using an 80-120 mM NaCl-8-12 mM PBS solution with a pH of 5.4-6.4. This invention yields immunoglobulin G with a purity greater than 80%, which is beneficial for further research on its functional activity. This helps to understand the true metabolic pathway of IgG in vivo, clarify its immune efficacy mechanism, and provide a theoretical basis and guidance for the application and efficacy enhancement of IgG in infant formula, children's milk powder, and special dairy products.

Description

Technical Field

[0001] This invention relates to the field of protein separation and purification technology, specifically to a method for separating and purifying immunoglobulin G from bovine colostrum powder. Background Technology

[0002] Immunoglobulins (Ig), as a key class of naturally occurring active antibodies in the body, play a central role in regulating immune responses, defending against pathogen invasion, and maintaining immune homeostasis. They are particularly important in the establishment, protection, and maturation of the neonatal immune system, and are highly valuable bioactive protein components in breast milk and animal milk. Among them, immunoglobulin G (IgG) is the most abundant immunoglobulin in human serum. It can cross the placental barrier and be passed to the fetus, providing passive immune protection to newborns and reducing the risk of infectious diseases.

[0003] Immunoglobulin G in bovine colostrum overlaps with human immunoglobulin A (IgA) in terms of antigen-binding activity and immune regulation mechanisms, and can replace or partially replace IgA in human colostrum to perform specific physiological immune functions. Therefore, fortifying infant formula with naturally derived IgG is of significant practical importance and has broad application prospects for compensating for the immune nutritional gap in non-breastfed infants and optimizing the nutritional value and functional characteristics of formula.

[0004] Currently, commercially available immunoglobulin G products are mainly derived from bovine serum, and while their production process is relatively mature, it has significant limitations. Firstly, IgG exists in multiple subtypes (such as IgG1, IgG2, and IgG3). Bovine serum-derived IgG differs significantly from bovine milk-derived IgG in subtype composition ratios and glycosylation modification patterns (such as glycan structure, modification sites, and degree of modification). These differences directly affect the immunomodulatory activity, half-life, tissue targeting, and bioavailability of IgG, making it difficult to meet the specific requirements of infant formula for bovine milk-based IgG. Secondly, bovine serum-derived IgG also faces challenges such as limited raw material sources, high production costs, and potential biosafety risks (such as residual proteins and contaminants in serum), further restricting its widespread application in infant formula.

[0005] Compared to bovine serum, cow's milk, as a natural raw material source, has advantages such as abundant reserves, convenient access, and higher safety. However, there are currently no commercially available cow's milk-based IgG products. The core bottleneck lies in the imperfect technology for the efficient separation and purification of IgG from cow's milk. The cow's milk system has a complex composition, containing various impurities such as casein, whey protein, lactose, and minerals, making the separation of IgG from it quite difficult. At the same time, the IgG content in cow's milk is affected by various factors such as dairy cow breed, lactation stage, and feeding conditions, resulting in a large fluctuation range.

[0006] Bovine colostrum powder, as a deep-processed product of bovine colostrum, has the characteristics of convenient storage and transportation, strong stability, and easy industrial processing. Using it as a raw material to separate and purify IgG can effectively avoid the problems of easy spoilage and short processing window of fresh bovine colostrum, and is more in line with the needs of industrial production.

[0007] In summary, the lack of efficient separation and purification technologies for bovine milk-based (especially bovine colostrum-based) IgG in existing technologies hinders the commercial application of bovine milk-based IgG, thus restricting the optimization and functional upgrading of infant formula. Therefore, developing an efficient, gentle method for separating and purifying IgG from bovine colostrum powder while preserving its bioactivity is a critical technical problem that urgently needs to be solved in the field of dairy product processing and infant formula, and is of great significance for promoting technological progress and product upgrading in related industries. Summary of the Invention

[0008] The purpose of this invention is to provide a method for isolating and purifying immunoglobulin G from bovine colostrum powder.

[0009] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: On one hand, the present invention provides a method for isolating and purifying immunoglobulin G from bovine colostrum powder, comprising the steps of: elution of immunoglobulin, wherein the elution is performed using 80-120 mM NaCl - 8-12 mM PBS solution, the pH of which is 5.4-6.4.

[0010] According to some embodiments of the present invention, the concentration of NaCl in the elution solution can be selected from 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, and any intermediate value.

[0011] According to some embodiments of the present invention, the concentration of PBS in the elution solution can be selected from 8mM, 8.5mM, 9mM, 9.5mM, 10mM, 10.5mM, 11mM, 11.5mM, 12mM, and any intermediate value.

[0012] According to some embodiments of the present invention, the pH of the elution solution can be selected from 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and any intermediate value.

[0013] Furthermore, the elution is performed using a 100 mM NaCl - 10 mM PBS solution with a pH of 5.4.

[0014] Furthermore, during elution, the solution is passed through the column at a rate of 3-10 mL / min.

[0015] Specifically, the method for separating and purifying immunoglobulin G from bovine colostrum powder includes: defatting, precipitation of casein, ultrafiltration, microfiltration, water washing and buffer column percolation, sample loading and buffer column percolation, elution of immunoglobulin G, removal of impurities, water washing and ethanol rinsing, dialysis and lyophilization.

[0016] Furthermore, the defatting process involves adding water to bovine colostrum powder, centrifuging, and then filtering.

[0017] According to some embodiments of the present invention, the mass-to-volume ratio of bovine colostrum powder to water is (15-25) g: (200-400) mL; Preferably, the mass-to-volume ratio of bovine colostrum powder to water is 20g:300mL.

[0018] According to some embodiments of the present invention, the centrifugation conditions are 4°C, 3000 r / min for 30 min.

[0019] Furthermore, the precipitated casein is precipitated using an acid precipitation method.

[0020] According to some embodiments of the present invention, the casein is precipitated by adjusting the pH to 4.6 with 1 mol / L HCl, letting it stand at a constant temperature of 37°C for 60 min, centrifuging, sieving, adjusting the pH again, centrifuging again, and filtering.

[0021] According to some embodiments of the present invention, the centrifugation conditions are as follows: centrifugation at 10,000 × g for 15 min at 25°C.

[0022] According to some embodiments of the present invention, the pH is adjusted to 6.8 after sieving.

[0023] According to some embodiments of the present invention, the filtration is to filter whey through a 0.45 μm filter membrane.

[0024] Furthermore, the ultrafiltration involves passing whey through an ultrafiltration centrifuge tube and then concentrating it by centrifugation.

[0025] According to some embodiments of the present invention, the ultrafiltration is performed by passing whey through a 100 kDa ultrafiltration centrifuge tube, and the centrifugation conditions are 20°C, 3000 × g for 16 min.

[0026] Furthermore, the microfiltration involves filtering the ultrafiltered whey through a 0.45 μm filter membrane.

[0027] According to some embodiments of the present invention, the water washing and buffer column percolation are specifically performed as follows: using a constant flow pump to wash the column packed with DEAE-Sep packing material with water for 3 CV, and passing the column through a pH 7.4 10 mM PBS buffer for 3 CV, with the constant flow pump speed being 3-10 mL / min.

[0028] According to some embodiments of the present invention, the sample loading and buffer column permeation are specifically as follows: whey is loaded at a rate of 1 mL / min, and after adsorption saturation, the sample is permeated with pH 7.4 10 mM PBS buffer at a rate of 3-10 mL / min for 2 CV.

[0029] Furthermore, the removal of contaminating proteins specifically involves passing 1 M NaCl-PBS through a column at a rate of 3-10 mL / min.

[0030] Furthermore, the water washing and ethanol rinsing specifically involve rinsing off residual salt with water and sealing with 2 CV 20% ethanol.

[0031] Furthermore, the dialysis and lyophilization are specifically performed as follows: the immunoglobulin G solution is placed in a dialysis bag and dialyzed in a 4°C low-temperature incubator for 24 h, and after dialysis, it is lyophilized in a freeze dryer for 48 h.

[0032] In another aspect, the present invention provides immunoglobulin G obtained by the above-described method for separation and purification.

[0033] The beneficial effects of this invention are as follows: This invention uses bovine colostrum powder as raw material. After processes such as reconstitution, defatting, casein removal, and ultrafiltration concentration, immunoglobulin G (IgG) in bovine colostrum is separated and purified using anion exchange chromatography. Finally, desalting and drying are performed to obtain immunoglobulin G with a purity greater than 80%. The extraction of IgG from bovine colostrum powder by this invention facilitates further research on its functional activity, thereby helping to understand the true metabolic pathway of IgG in vivo, clarify its immune efficacy mechanism, and provide a theoretical basis and guidance for the application and efficacy enhancement of IgG in infant formula, children's milk powder, and special dairy products. Attached Figure Description

[0034] Figure 1 The SDS-PAGE results (12.5% ​​electrophoresis) are for the optimized separation and purification ion strength parameters in Example 1.

[0035] Figure 2The results of SDS-PAGE after optimizing the pH parameters of the separation and purification buffer in Example 1 are shown (12.5% ​​electrophoresis; sample ① was obtained by elution with pH 7.4 100 mM NaCl - 10 mM PBS solution, and sample ② was obtained by elution with pH 5.4 100 mM NaCl - 10 mM PBS solution).

[0036] Figure 3 The results of SDS-PAGE (8% electrophoresis) after optimizing the pH parameters of the separation and purification buffer in Example 1 are shown. Sample ① was obtained by elution with pH 7.4 100 mM NaCl - 10 mM PBS solution, and sample ② was obtained by elution with pH 5.4 100 mM NaCl - 10 mM PBS solution.

[0037] Figure 4 SDS-PAGE results (8% electrophoresis) for the purification of immunoglobulin G.

[0038] Figure 5 MALDI-TOF MS / MS results for determining the purity of purified immunoglobulin G (pH 7.4, 100 mM NaCl - 10 mM PBS solution elution, 150 kDa).

[0039] Figure 6 MALDI-TOF MS / MS results for determining the purity of purified immunoglobulin G (pH 5.4, 100 mM NaCl - 10 mM PBS solution elution, 150 kDa).

[0040] Figure 7 MALDI-TOF MS / MS results for determining the purity of purified immunoglobulin G (pH 5.4, 100 mM NaCl - 10 mM PBS solution elution, 150 kDa). Figure 7 for Figure 6 The results of parallel samples.

[0041] Figure 8 MALDI-TOF MS / MS results for determining the purity of purified immunoglobulin G (pH 5.4, 100 mM NaCl - 10 mM PBS solution elution, 250 kDa).

[0042] Figure 9 HPLC results (solvent) for determining the purity of purified immunoglobulin G.

[0043] Figure 10 HPLC results for the purity determination of purified immunoglobulin G (standard).

[0044] Figure 11The HPLC results for the purity determination of purified immunoglobulin G (sample before method optimization, eluted with 100 mM NaCl - 10 mM PBS solution (pH 7.4) in Example 1).

[0045] Figure 12 The results are HPLC results of the purity determination of purified immunoglobulin G after the method optimization in Example 2. Detailed Implementation

[0046] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0047] Example 1 1.1 Preprocessing 1.1.1 Reagent Preparation Prepare 10 mM PBS buffer (pH 7.4). 50, 100, 150, 200, 250 mM NaCl - 10 mM PBS solution (pH 7.4); 100 mM NaCl - 10 mM PBS solution (pH 5.4); 1 M NaCl - 10 mM PBS solution (pH 7.4); The solution was filtered through a microporous membrane (0.45 μm) and air bubbles were removed using ultrasound. A 1 mol / L solution of hydrochloric acid and a 1 mol / L solution of sodium hydroxide were prepared to adjust the pH.

[0048] 1.1.2 Degreasing Bovine colostrum powder (PRODIET F520 C20, purchased from INGREDIA, product number U23029) was dissolved in deionized water at a specific ratio (20g bovine colostrum powder: 300mL deionized water). Then, it was centrifuged in a vertical centrifuge at 4℃ and 3000 r / min for 30 min to remove the separated fat layer. The resulting product was then filtered through a sieve to obtain defatted bovine colostrum.

[0049] 1.1.3 Precipitating Casein Acid precipitation was used to remove casein from skim milk.

[0050] The pH of skim milk was adjusted to 4.6 with 1 mol / L HCl, and the mixture was kept at 37°C in a thermostatic water bath for 60 min to allow casein to precipitate completely. Then, it was centrifuged at 10,000 × g for 15 min at 25°C. After sieving, the whey pH was adjusted to 6.8, and it was centrifuged again at 10,000 × g for 15 min at 25°C and then sieved. The whey was then filtered through a 0.45 μm microporous membrane for ultrafiltration.

[0051] 1.1.4 Ultrafiltration The whey was ultrafiltered through a 100 kDa ultrafiltration centrifuge tube, and the ultrafiltration centrifuge tube was centrifuged in a vertical centrifuge at 20°C and 3000 × g for 16 min to concentrate the volume by one-fold.

[0052] 1.1.5 Microfiltration The ultrafiltered whey was filtered through a microporous membrane (0.45 μm) and then placed in a 4°C low-temperature incubator for loading.

[0053] 1.2 Through the column 1.2.1 Resin treatment and column packing Stir the swollen strong anion exchange resin in 20% ethanol solution until homogeneous. Take out the excess amount according to the volume of the chromatography column to be used. After standing, replace the 20% ethanol solution with ultrapure water. Let the removed resin stand for a period of time, then pour out the solution and add clean ultrapure water, and let it stand again. Repeat the above steps 3-5 times. Pack the treated resin into a chromatography column (Φ16 mm × 10 cm) to obtain a chromatography column with a volume of approximately 20 mL. First, equilibrate the chromatography column with ultrapure water, and then continue to equilibrate the chromatography column to neutral pH with 10 mM PBS buffer (pH 7.4).

[0054] 1.2.2 Washing with water and column pass through PBS buffer Use a constant flow pump to wash the column packed with DEAE-Sep packing material with water for about 3 CV (CV represents column volume / mL) to remove ethanol, and then pass the column through the column with pH 7.4 10 mM PBS buffer for about 3 CV to equilibrate the column. The constant flow pump speed is 3 mL / min.

[0055] 1.2.3 Sample loading and PBS buffer column permeation Whey was loaded at a rate of 1 mL / min. After adsorption saturation, the unadsorbed protein was removed by passing the column through a 10 mM PBS buffer at a rate of 3 mL / min for about 2 CV.

[0056] 1.2.4 Elution of Immunoglobulin G The protein concentrations were collected and measured by sequentially eluting with 50, 100, 150, 200, and 250 mM NaCl - 10 mM PBS solution (pH 7.4) in a gradient. SDS-PAGE was used for validation, and the content and purity of immunoglobulin G were determined by enzyme-linked immunosorbent assay (ELISA). The ion exchange experimental conditions were then optimized based on the experimental results.

[0057] Elution was performed using 100 mM NaCl - 10 mM PBS solution (pH 7.4) and 100 mM NaCl - 10 mM PBS solution (pH 5.4), with the elution collected separately (the pH 5.4 elution was adjusted back to neutral). Protein concentrations were determined, and the results were validated by SDS-PAGE. Immunoglobulin G content and purity were determined using enzyme-linked immunosorbent assay (ELISA). Ion exchange experimental conditions were optimized based on the results.

[0058] The solution was passed through the column at a rate of 3 mL / min to elute immunoglobulin G, and the protein concentration was detected using an ultra-micro biodetector until the protein concentration dropped below 0.5 mg / mL.

[0059] 1.2.5 Wash away any remaining proteins that have not been eluted. Elute impurity proteins by passing 1 M NaCl-PBS through the column at a rate of 3 mL / min.

[0060] 1.2.6 Wash with water and rinse with 20% ethanol Residual salts were rinsed with ultrapure water after membrane treatment, and finally sealed with 2 CV 20% ethanol.

[0061] 1.2.7 Regeneration of the ion column When the chromatography column has been used for an extended period, it should first be rinsed with 3 CV ultrapure water, then with 2 CV 0.1 mol / L NaOH solution, followed by washing with 10 mM PBS buffer (pH 7.4) until neutral, and finally washed with ultrapure water and sealed with 2 CV 20% ethanol for future use. When the chromatography column is not used for a long time, the packing material should be removed and soaked in 20% ethanol for storage.

[0062] 1.3 Dialysis and Lyophilization The received immunoglobulin G solution was placed in a dialysis bag and then placed in a large beaker filled with deionized water. Dialysis was performed at 4°C for 24 h, with the deionized water changed periodically. After dialysis, the sample was freeze-dried for 48 h and then stored at -80°C for subsequent studies.

[0063] 2. Experimental methods for index detection 2.1 Determination of protein content in samples The protein content of the samples was determined according to the method in GB 5009.5-2016. Accurately weigh 0.1 g of each sample and transfer it to a dry nitrogen determination flask. Add 0.5 g of anhydrous copper sulfate, 4.5 g of potassium sulfate, and 20 mL of sulfuric acid. Gently shake and place a small funnel at the mouth of the flask. Place the nitrogen determination flask on a digestion furnace and digest according to the procedure until the liquid turns pale blue and becomes clear and transparent. Remove the digestion tube and place it in a Kjeldahl nitrogen determination apparatus. Place an Erlenmeyer flask at the receiving end and distill according to the procedure. After the procedure begins, add two drops of indicator solution to the Erlenmeyer flask. During distillation, keep the receiving tube below the liquid level in the Erlenmeyer flask. After distillation, rinse the receiving tube with deionized water, remove the Erlenmeyer flask, and titrate with 0.1 mol / L hydrochloric acid standard solution to the endpoint as soon as possible.

[0064] The formula for calculating the protein content of a sample is as follows: Nitrogen content (%) = 1.4 * c * (V - V0) / m; Protein content (%) = N (%) * X; Where c is the standard acid concentration (mol / L); V is the volume of acid solution titrated with the sample (mL); V0 is the volume of acid solution titrated with the blank sample (mL); m is the sample mass; and X is the conversion factor.

[0065] 2.2 Determination of protein content and immunoglobulin G content and purity Protein content was determined according to the method described on the BCA Protein Assay Kit (The Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, catalog number 23225). Immunoglobulin G content and purity were determined according to the method described on the ELISA kit (Bovine IgG ELISA Kit, Bethyl Laboratories, catalog number E11-118).

[0066] 2.3 SDS-PAGE Validation of Purified Immunoglobulin G The sample was prepared into a 2 mg / mL solution using deionized water, mixed 1:1 with 2× sample buffer, and loaded with 10 μL. The separating gels were prepared at concentrations of 12.5% ​​and 8% (m / v), and the stacking gel was 5% (m / v). Specific formulations were provided in the electrophoresis kit instructions (PAGE Gel Quick Preparation Kit (12.5%), Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 20326ES62; PAGE Gel Quick Preparation Kit (8%), purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 20324ES62). Electrophoresis was performed under constant voltage: 60 V for the stacking gel and 120 V for the separating gel. The staining agent was 0.1% (m / v) Coomassie Brilliant Blue R-250 solution, and the destaining agent was a mixture of ethanol and acetic acid. After the gel is run, gently remove the gel, rinse off the electrophoresis buffer, stain with Coomassie Brilliant Blue, and place on a horizontal shaker for 2 hours. Then destain with destaining solution to make the electrophoretic bands clearly visible. Finally, take pictures using the Bio-Rad gel imaging system.

[0067] 3. Experimental Results 3.1 Parameters 3.1.1 Parameter results of ionic strength The protein concentrations were collected and measured by eluting with 50, 100, 150, 200, and 250 mM NaCl - 10 mM PBS solutions (pH 7.4) in a gradient, and then verified by SDS-PAGE.

[0068] like Figure 1 The image shows the optimized SDS-PAGE for separation and purification using ionic strength parameters. The results show that elution with 50 mM and 150 mM NaCl - 10 mM PBS solution (pH 7.4) yielded a large amount of BSA contaminants, while elution with 200 mM and 250 mM NaCl - 10 mM PBS solution (pH 7.4) yielded a large amount of β-lactoglobulin. Elution with 100 mM NaCl - 10 mM PBS solution (pH 7.4) yielded less IgG contaminants, with only a small amount of α-lactalbumin present. Therefore, the elution solution was initially determined to be 100 mM NaCl - 10 mM PBS solution (pH 7.4).

[0069] Table 1 shows the ELISA results of the optimized ion strength parameters for separation and purification in the preliminary experiment. The results show that the recovery rate of the column chromatography step is relatively low.

[0070] Table 1. ELISA results of immunoglobulin G content during isolation and purification (elution with pH 7.4, 100 mM NaCl, and 10 mM PBS solution).

[0071] 3.1.2 Experimental results of buffer pH Elution was performed using 100 mM NaCl - 10 mM PBS solution (pH 7.4) and 100 mM NaCl - 10 mM PBS solution (pH 5.4), with the elution collected separately (the pH 5.4 elution was adjusted back to neutral). Protein concentrations were determined, and the results were validated by SDS-PAGE. Immunoglobulin G content and purity were determined using enzyme-linked immunosorbent assay (ELISA). Ion exchange experimental conditions were optimized based on the results.

[0072] like Figures 2-3 The results show the SDS-PAGE and ELISA results after optimizing the pH parameters of the separation and purification buffer. The results show that the purity of IgG obtained by elution at different pH values ​​is similar, but the recovery rate of the column step increased from 50.63% to 70.00%. Therefore, the final elution solution was determined to be 100 mM NaCl - 10 mM PBS solution (pH 5.4).

[0073] Table 2. ELISA results of immunoglobulin G content during preliminary isolation and purification (pH 7.4 / 5.4, 100 mM NaCl - 10 mM PBS solution elution)

[0074] Example 2 2.1 Preprocessing 2.1.1 Reagent Preparation Prepare 10 mM PBS buffer (pH 7.4). 100 mM NaCl - 10 mM PBS solution (pH 5.4); 1 M NaCl - 10 mM PBS solution (pH 7.4); The solution was filtered through a microporous membrane (0.45 μm) and air bubbles were removed using ultrasound. A 1 mol / L solution of hydrochloric acid and a 1 mol / L solution of sodium hydroxide were prepared to adjust the pH.

[0075] 2.1.2 Degreasing Bovine colostrum powder (purchased from INGREDIA, product number U23029) was dissolved in deionized water at a specific ratio (20g bovine colostrum powder: 300mL deionized water). Then, it was centrifuged in a vertical centrifuge at 4℃ and 3000 r / min for 30 min to remove the separated fat layer. The resulting product was then filtered through a sieve to obtain defatted bovine colostrum.

[0076] 2.1.3 Precipitating Casein Acid precipitation was used to remove casein from skim milk.

[0077] The pH of skim milk was adjusted to 4.6 with 1 mol / L HCl, and the mixture was kept at 37°C in a thermostatic water bath for 60 min to allow casein to precipitate completely. Then, it was centrifuged at 10,000 × g for 15 min at 25°C. After sieving, the whey pH was adjusted to 6.8, and it was centrifuged again at 10,000 × g for 15 min at 25°C and then sieved. The whey was then filtered through a 0.45 μm microporous membrane for ultrafiltration.

[0078] 2.1.4 Ultrafiltration The whey was ultrafiltered through a 100 kDa ultrafiltration membrane to concentrate it by one-fold.

[0079] 2.1.5 Microfiltration The ultrafiltered whey was filtered through a microporous membrane (0.45 μm) and then placed in a 4°C low-temperature incubator for loading.

[0080] 2.2 Through the column 2.2.1 Resin treatment and column packing The treated packing material was packed into a chromatography column (Φ26 mm × 60 cm) to obtain a chromatography column with a volume of approximately 260 mL. The rest is the same as in 1.2.1.

[0081] 2.2.2 Washing with water and column pass through PBS buffer The constant flow pump speed was 10 mL / min during water washing and PBS buffer column permeation. The rest is the same as in 1.2.2.

[0082] 2.2.3 Sample loading and PBS buffer column permeation The constant flow pump speed was 10 mL / min when passing PBS buffer through the column. The rest is the same as in 1.2.3.

[0083] 2.2.4 Elution of Immunoglobulin G Immunoglobulin G was eluted by passing a pH 5.4 100 mM NaCl - 10 mM PBS solution through a column at a rate of 10 mL / min. The pH of the elution was then adjusted back to neutral, and the protein concentration was detected using an ultra-micro biodetector until the protein concentration dropped below 0.5 mg / mL.

[0084] 2.2.5 Wash away any remaining contaminating proteins. When passing PBS buffer and 1 M NaCl-PBS solution through the column, the constant flow pump speed is 10 mL / min. The rest is the same as in 1.2.5.

[0085] 2.2.6 Wash with water and rinse with 20% ethanol Same as 1.2.6.

[0086] 2.2.7 Regeneration of the ion column Same as 1.2.7.

[0087] 2.3 Dialysis and Lyophilization Same as 1.3.

[0088] 2.4 MALDI-TOF MS / MS Identification of Purified Immunoglobulin G Carefully cut the target band from the gel using a gel plate and place it in an EP tube, cutting the gel block into 1 mm³ pieces. After washing with water, transfer to a new EP tube, add 200 μL-400 μL of destaining solution, destain on a shaker for 30 min, wash until clear, remove the supernatant, add 100 μL of acetonitrile, aspirate after 5 min, and allow to evaporate until the gel particles are dry and white. Add 90 μL of 100 mmol / L NH₄HCO₃ and 10 μL of 100 mmol / L DTT to each tube, incubate at 56℃ for 30 min to reduce the protein. Next, remove the supernatant, add 100 μL of acetonitrile to each tube, aspirate after 5 min. Add 70 μL of 100 mmol / L NH₄HCO₃ and 30 μL of 200 mmol / L IAA iodoacetamide to each tube, and incubate in the dark for 20 min. Remove the supernatant. Add 100 μL of 100 mmol / L NH4HCO3 to each tube and incubate at room temperature for 15 min. Discard the supernatant, add 100 μL of acetonitrile to each tube, aspirate after 5 min, add 50 μL of acetonitrile, aspirate after 5 min, and repeat once. Evaporate until the gel particles are dry and white. After the gel particles are completely dry, add 5 μL-10 μL of enzyme digestion solution to each tube and incubate at 4℃ for 30-60 min to allow the gel particles to fully swell. Then add 20-30 μL of 25 mmol / L NH4HCO3 buffer and react overnight at 37℃. Spot 0.7 μL of the enzyme digestion solution onto a target plate, air dry, cover with 0.7 μL of matrix, air dry again, and then analyze. Aspirate the enzyme hydrolysate, transfer it to a new EP tube, centrifuge and concentrate to the remaining 3 μL. Take 0.7 μL and spot it onto the target plate. After drying, cover it with 0.7 μL of matrix. After drying, cover it with 2 μL of ice-cold 0.1% (m / m) TFA trifluoroacetic acid. Immediately aspirate after 10 s to remove salt. After drying, analyze it on the instrument.

[0089] 2.5 Purity determination of purified immunoglobulin G—HPLC method The reagents were prepared as follows: Buffer 1: 0.1 mol / L 1,3-bis((tris(hydroxymethyl)methylamino)propane (Bis-Tris-Propane), pH 7.0, 8 mol / L urea, 20 mmol / L DTT, 1.3% (m / v) NaCA·2H2O (trisodium citrate dihydrate); Buffer 2: acetonitrile-water-trifluoroacetic acid (100:900:1 v / v / v), 6 mol / L urea.

[0090] First, dissolve the purified bovine milk immunoglobulin lyophilized powder and mix it with a sample buffer (Sample:Buffer 1) ratio of 1:1 (v / v). Vortex for 30 s, then mix on a shaker at room temperature for 1 h. Centrifuge at 10000×g for 3 min to remove residual fat, and collect the intermediate layer sample as the mixture. Next, mix the mixture with buffer (Buffer 2) at a ratio of 1:3 (v / v), vortex for 30 s, and then pass the mixture through a 0.45 μm organic or nylon membrane.

[0091] The specific chromatographic conditions were as follows: Mobile phase A: acetonitrile-water-trifluoroacetic acid (100:900:1 v / v / v); Mobile phase B: acetonitrile-water-trifluoroacetic acid (900:100:0.7 v / v / v). Column: Waters Xbridge BEH C4, 300A, 3.5 μm, 4.6 × 250 mm; Column temperature: 25℃; Detection wavelength: 280 nm; Injection volume: 30 μL; Flow rate: 1.0 mL / min.

[0092] Protein purity (%) = Sample protein peak area / Total peak area × 100%.

[0093] 3. Experimental Results 3.1 Determination of Immunoglobulin G Content and Purity Table 3 shows the results of the determination of the purity of immunoglobulin G content in bovine colostrum powder raw materials. The results show that the immunoglobulin G content in bovine colostrum powder is 20.66g / 100g milk powder.

[0094] Table 3. Results of the determination of immunoglobulin G content in raw bovine colostrum powder

[0095] 3.2 SDS-PAGE Validation of Purified Immunoglobulin G like Figure 4The image shows the SDS-PAGE verification results for the purity determination of purified immunoglobulin G. The 8% electrophoresis comparison standard and sample are basically consistent, indicating high purity from a qualitative perspective.

[0096] 3.3 MALDI-TOF MS / MS Identification of Purified Immunoglobulin G like Figures 5-8 The image shows the MALDI-TOF MS / MS results for determining the purity of purified immunoglobulin G. Before method optimization, the sequence match rate of the 150 kDa sample was 6%, with a score of 115. After method optimization, the sequence match rate of the 150 kDa sample was 10%, with a score of 245, and the sequence match rate of the 250 kDa sample was 6%, with a score of 117. This indicates that the band with a molecular weight of approximately 250 kDa is also immunoglobulin G, although its score is slightly lower than that of the 150 kDa band, possibly indicating a multimer. The presence of this band in the standard further confirms this. The slight difference between the standard and sample bands in 12.5% ​​electrophoresis may be due to their different sources (serum and bovine colostrum).

[0097] 3.4 Purity determination of purified immunoglobulin G—HPLC method like Figures 9-12 The figure shows the HPLC results of the purity determination of purified immunoglobulin G. The sample purity after method optimization is as high as 95% or more.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for isolating and purifying immunoglobulin G from bovine colostrum powder, characterized in that, The procedure includes the following steps: elution of immunoglobulins, wherein the elution is performed using an 80-120 mM NaCl-8-12 mM PBS solution with a pH of 5.4-6.

4.

2. The method according to claim 1, characterized in that, The elution was performed using a 100 mM NaCl - 10 mM PBS solution with a pH of 5.

4.

3. The method according to any one of claims 1-2, characterized in that, During elution, the solution is passed through the column at a rate of 3-10 mL / min.

4. The method according to claim 1, characterized in that, The method for separating and purifying immunoglobulin G from bovine colostrum powder includes the following steps: defatting, casein precipitation, ultrafiltration, microfiltration, water washing and buffer column permeation, sample loading and buffer column permeation, elution of immunoglobulin G, removal of impurity proteins, water washing and ethanol rinsing, dialysis and lyophilization.

5. The method according to claim 4, characterized in that, The defatting process involves adding water to bovine colostrum powder, centrifuging, and filtering; the mass-to-volume ratio of bovine colostrum powder to water is (15-25) g: (200-400) mL.

6. The method according to claim 4, characterized in that, The precipitated casein was precipitated using an acid precipitation method.

7. The method according to claim 4, characterized in that, The ultrafiltration process involves passing the whey through an ultrafiltration centrifuge tube and then concentrating it by centrifugation; the microfiltration process involves passing the ultrafiltered whey through a 0.45 μm filter membrane.

8. The method according to claim 4, characterized in that, The washing and buffering column pass-through were performed by using a constant flow pump to wash the column packed with DEAE-Sep packing material with water, followed by passing the column through a pH 7.4 10 mM PBS buffer at a constant flow rate of 3-10 mL / min.

9. The method according to claim 4, characterized in that, The sample loading and buffer column permeation were performed as follows: whey was loaded at a rate of 1 mL / min, and after adsorption saturation, the sample was permeated with pH 7.4 10 mM PBS buffer at a rate of 3-10 mL / min.

10. Immunoglobulin G isolated and purified by the method of any one of claims 1-9.

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