Method for extracting and separating alpha1 antitrypsin antigen from natural human serum
By using ammonium sulfate precipitation and multi-chromatography in natural human serum, combined with lyophilized protective agent, the purity and stability of α1 antitrypsin antigen were successfully improved, and the low activity and recovery rate in the prior art were solved. The high problem has achieved the preparation of low-cost and high-purity antigens, which is suitable for industrial production and clinical diagnostic applications.
Patent Information
- Application Number
- CN202411487739.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2024-12-17
AI Technical Summary
When extracting and isolating α1 antitrypsin antigen from natural human serum, the prior art has problems such as low activity, low recovery rate and unfavorable to large-scale production of the process, making it difficult to prepare low-cost and active high-purity antigens.
Using human serum as raw material, salting precipitation is carried out by adding ammonium sulfate for the first and second time, followed by hydrophobic chromatography, ion exchange resin and gel chromatography, combined with the use of lyophilized protective agent, high purity is achieved ( ≥95%) of α1 antitrypsin antigen extraction and preservation.
The prepared α1 antitrypsin antigen has high activity, good stability and low cost, is suitable for industrial production, and has wide application value in clinical and in vitro diagnosis.
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Figure CN119143868A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioengineering, and in particular to a method for extracting and separating alpha 1 antitrypsin antigen from natural human serum. Background Art
[0002] α1-antitrypsin (α1-AT) is the most abundant protease inhibitor in human plasma. It is a glycoprotein mainly synthesized by liver cells. It can also be produced in extrahepatic tissues such as mononuclear macrophages, intestinal epithelial cells, renal parenchyma, and some tumor cells. It can inhibit a variety of proteases and serine endopeptidases. The health of the human liver can be reflected by measuring the content of α1-antitrypsin in the blood.
[0003] α1-antitrypsin (α-1-antitrypsin, α-1-AT, AAT), also known as α-1-protease inhibitor, is a single-chain glycoprotein composed of 394 amino acids, containing 10%-20% sugar, and is mainly synthesized by the liver. The isoelectric point is pI4.7-5.0, the molecular weight is 45-56kD, the content of α1-AT in normal human plasma is 2.3-3.3g / L, and the half-life in vivo is 4-6 days. α1-antitrypsin is a member of the serine protease inhibitor family and a protease inhibitor. Its biological function is to inhibit trypsin, hyaluronidase, plasmin, elastase, etc. It is a broad-spectrum protease inhibitor. The inhibitory effect of α1-AT is obviously pH-dependent, with the maximum activity at neutral or weak alkaline, and the activity is basically lost at pH 4.5.
[0004] The clinical significance of α1-antitrypsin in serum can be applied to liver diagnosis. If the α1-antitrypsin level in serum increases, it may reflect tissue damage, inflammation, malignant tumors, viral hepatitis, etc. If the α1-antitrypsin level in serum decreases, it is mainly seen in hereditary α1-AT deficiency, cirrhosis, bronchiectasis, pancreatic fibrosis, etc. caused by α1-AT deficiency. For patients with unexplained cirrhosis, the detection of α1-AT can assist in diagnosis. Therefore, the detection of α1-antitrypsin levels is of great significance.
[0005] Natural α1 antitrypsin protein is mainly found in the liver. Although there are many studies on the preparation process of high-purity α1 antitrypsin antigen, such as ammonium sulfate, polyethylene glycol precipitation and other methods for coarse separation, and then affinity chromatography, gel filtration and other methods for fine separation to obtain high-purity antigen, there are still problems such as low activity and recovery rate, and it is not conducive to large-scale production. However, in order to enable many patients to receive good detection and treatment, the research and development of low-cost, active and high-purity α1 antitrypsin antigen has great economic value and positive practical significance. Summary of the invention
[0006] The purpose of the present invention is to provide a method for extracting and separating α1 antitrypsin antigen from natural human serum, aiming at producing and developing low-cost, active, and highly pure α1 antitrypsin antigen.
[0007] To achieve the above object, the present invention provides a method for extracting and separating α1 antitrypsin antigen from natural human serum, comprising the following steps:
[0008] Using human serum as a raw material, adding ammonium sulfate for the first time to obtain a first protein solution, adding ammonium sulfate to the first protein solution for a second time to perform salting out precipitation, and centrifuging to obtain a precipitate;
[0009] The precipitate is subjected to hydrophobic chromatography, ion exchange resin and gel chromatography in sequence to obtain α1-antitrypsin antigen with a purity of ≥95%;
[0010] A lyophilization protective agent is added to the α1-antitrypsin antigen for freeze-drying and storage to obtain a stable α1-antitrypsin antigen.
[0011] The mass concentration of ammonium sulfate added for the first time is 110-120 g / L, the pH of the first protein solution added for the first time is 6.2-6.5, the mass concentration of ammonium sulfate added for the second time is 420-440 g / L, and the pH of the first protein solution added for the second time is 5.4-5.6.
[0012] The specific method of sequentially subjecting the precipitate to hydrophobic chromatography, ion exchange resin and gel chromatography to obtain α1-antitrypsin antigen with a purity of ≥95% is as follows:
[0013] Dialyzing the precipitate using a first buffer to obtain a second protein solution;
[0014] Purifying the second protein solution through hydrophobic chromatography and washing with a second buffer to obtain a third protein solution;
[0015] The third protein solution is subjected to ion exchange chromatography to obtain a fourth protein solution, which is then subjected to gel chromatography to obtain α1-antitrypsin antigen with a purity of ≥95%.
[0016] The components of the first buffer solution include Tris-hcl and Nacl, the concentration of Tris-hcl is 10-15 mmol / L, the pH is 7.8-8.2, and the concentration of Nacl is 260-300 mmol / L.
[0017] The components of the lyoprotectant include sugars, salts, surfactants and polyols, wherein the sugars include any one of lactose, sucrose and trehalose, and the addition amount thereof is 1-2%, the salts include any one of sodium chloride and potassium chloride, and the addition amount thereof is 200-260 mmol / L, the surfactants include any one of glycerol, Tween 20, Tween 80 and Triton X-100, and the addition amount thereof is 0.05-0.1%, and the polyols include any one of sorbitol and polyethylene glycol, and the addition amount thereof is 0.05-0.07%.
[0018] The present invention discloses a method for extracting and separating α1-antitrypsin antigen from natural human serum. The method uses human serum as a raw material, adds ammonium sulfate for the first time to obtain a first protein solution, adds ammonium sulfate to the first protein solution for a second time to perform salting precipitation, and centrifuges to obtain a precipitate; the precipitate is sequentially subjected to hydrophobic chromatography, ion exchange resin and gel chromatography to obtain an α1-antitrypsin antigen with a purity of ≥95%; adds a lyophilization protective agent to the α1-antitrypsin antigen for freeze-drying and storage to obtain a stable α1-antitrypsin antigen. The method uses human serum as a raw material, and obtains a high-purity α1-antitrypsin antigen through three steps of enrichment, crude extraction and refined extraction. The α1-antitrypsin antigen prepared by the method has the characteristics of high activity, good stability, low cost and high process repeatability, can be industrially produced, and has potential and wide application value in clinical diagnosis and in vitro diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0020] Figure 1 The present invention provides a flow chart of a method for extracting and separating α1 antitrypsin antigen from natural human serum.
[0021] Figure 2 The invention is a specific flow chart of a method for obtaining alpha 1-antitrypsin antigen with a purity of ≥ 95% by subjecting the precipitate to hydrophobic chromatography, ion exchange resin and gel chromatography in sequence. DETAILED DESCRIPTION
[0022] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.
[0023] See also Figure 1 to Figure 2 The present invention provides a method for extracting and separating α1 antitrypsin antigen from natural human serum, comprising the following steps:
[0024] S1 uses human serum as a raw material, adds ammonium sulfate for the first time to obtain a first protein solution, adds ammonium sulfate to the first protein solution for a second time to perform salting out precipitation, and centrifuges to obtain a precipitate;
[0025] In an embodiment of the present invention, the mass concentration of ammonium sulfate added for the first time is 110-120 g / L, the pH of the first protein solution (protein solution A) added for the first time is 6.2-6.5, the mass concentration of ammonium sulfate added for the second time is 420-440 g / L, and the pH of the first protein solution added for the second time is 5.4-5.6.
[0026] S2 sequentially subjecting the precipitate to hydrophobic chromatography, ion exchange resin and gel chromatography to obtain α1-antitrypsin antigen with a purity of ≥95%;
[0027] Specific method:
[0028] S21 dialyzes the precipitate using a first buffer to obtain a second protein solution;
[0029] In an embodiment of the present invention, the components of the first buffer solution (buffer A) include Tris-hcl and Nacl, the concentration of Tris-hcl is 10-15 mmol / L, the pH is 7.8-8.2, and the concentration of Nacl is 20-30 mmol / L.
[0030] S22 purifying the second protein solution by hydrophobic chromatography and washing with a second buffer to obtain a third protein solution;
[0031] In an embodiment of the present invention, the components of the second buffer (buffer B) include Tris-hcl and NaCl, which are purified by hydrophobic chromatography and dialyzed with the second buffer to obtain a third protein solution (protein solution C).
[0032] S23: subjecting the third protein solution to ion exchange chromatography to obtain a fourth protein solution, and further subjecting the fourth protein solution to gel chromatography to obtain α1-antitrypsin antigen with a purity of ≥95%.
[0033] In an embodiment of the present invention, the third protein solution (protein solution C) is subjected to ion exchange chromatography, and the α1-antitrypsin antigen with a purity of 75%-85% is eluted with buffer C, i.e., the fourth protein solution. The components of buffer C are sodium acetate, Nacl and EDTA, and the concentrations of sodium acetate, Nacl and EDTA are 10-15mmol / L, 17-20mmol / L and 0.8-1.2mmol / L, respectively. The gel chromatography uses buffer D to elute the α1-antitrypsin antigen with a purity of 75%-85%, to obtain the α1-antitrypsin antigen with a purity ≥95%, and the components of buffer D are 20mmol / LTris-Hcl+500mmol / LNacl+0.3%NaN3 (pH=8.0-8.2).
[0034] S3: adding a lyophilization protective agent to the α1-antitrypsin antigen for freeze-drying and storage to obtain a stable α1-antitrypsin antigen.
[0035] In an embodiment of the present invention, the components of the lyoprotectant include sugars, salts, surfactants and polyols, the sugars include any one of lactose, sucrose and trehalose, and the addition amount thereof is 1-2%, the salts include any one of sodium chloride and potassium chloride, and the addition amount thereof is 200-260 mmol / L, the surfactants include any one of glycerol, Tween 20, Tween 80 and Triton X-100, and the addition amount thereof is 0.05-0.1%, and the polyols include any one of sorbitol and polyethylene glycol, and the addition amount thereof is 0.05-0.07%.
[0036] In order to better understand the present technical solution, the following embodiments are provided for further explanation:
[0037] Example 1
[0038] Crude Extraction and Enrichment of α1-Antitrypsin Antigen in Human Serum
[0039] Take the human serum out of the -20℃ refrigerator. After the human serum is completely dissolved, measure 100mL of human serum into a beaker, weigh a certain amount of solid ammonium sulfate, slowly add it to the human serum, stir while adding, stir for 30-60min after adding, and place it in a 2-8℃ refrigerator for 4-8h. Centrifuge the sample at 4℃, 7000-9000rpm for 20-30min after standing to remove immunoglobulin and other macromolecular impurities in the serum, and collect the supernatant to obtain protein solution A. Weigh a certain amount of solid ammonium sulfate, slowly add it to protein solution A, stir while adding, stir for 30-40min after adding, centrifuge at 4℃, 7000-9000rpm for 20-30min after completely dissolving, take the precipitate, use 30ml buffer A to re-dissolve, and dialyze in buffer A for 48h, changing the dialysate 3 times to obtain the enriched protein solution B.
[0040] Example 2
[0041] Hydrophobic chromatography purification
[0042] The method of hydrophobic chromatography (Phenyl Beads 6FF) purification is adopted, 30 ml of hydrophobic chromatography filler is loaded into the column, 300 ml of purified water is used to wash the column after loading, and then 300 ml of buffer A is used to balance. After the balance is completed, the dialyzed protein solution B is added to the column along the wall, and the protein detector is connected. When all the samples enter the filler, the buffer A is used for balance and washing, and the balance penetration liquid with peaks is collected to obtain 4 tubes of penetration liquid, which contains 65%-85% of α1-antitrypsin antigen, and then the buffer B is used for washing, and a total of 45 ml of washing liquid with peaks is collected, which contains 10-15% of α1-antitrypsin antigen and 70-80% of lipoprotein, so as to achieve the effect of removing lipoprotein, and then the filler is washed with 25% ethanol with a volume of 2 times of the filler for storage.
[0043] Example 3
[0044] Processing of α1-antitrypsin antigen
[0045] Combine the above-mentioned equilibrium penetration solutions to obtain 80 ml of crude protein solution C. Weigh 35 g of solid ammonium sulfate and slowly add it to the combined solution, stirring while adding until the ammonium sulfate is completely dissolved, and place it in a 2-8°C refrigerator for 1-2 hours. Centrifuge the sample after standing at 4°C and 8000-9000 rpm for 20-30 minutes to remove some small molecular weight impurities, collect the precipitate, dissolve the precipitate with buffer C to obtain protein solution D, and dialyze the protein solution D in buffer C for 48 hours, during which the dialysate is changed 3 times.
[0046] Example 4
[0047] Ion exchange chromatography (Q Beads HP)
[0048] The ion exchange chromatography (Q Beads HP) purification method is adopted, 30 ml of ion exchange chromatography filler is loaded into the column, 300 ml of purified water is used to wash the column after loading, and then 300 ml of buffer C is used for balancing, after balancing, the dialyzed protein solution D is added to the column along the wall, and the protein detector is connected, and when all the samples enter the filler, the buffer C is used for balancing and washing, and the peak of the balancing penetration liquid is collected to obtain 80 ml, and sucrose is used to concentrate to 10 ml to obtain the protein solution E. The penetration liquid contains 60-70% of α1-antitrypsin antigen, and the purity reaches 75-85%.
[0049] Example 5
[0050] Gel filtration chromatography (S-300)
[0051] The gel resin chromatography purification method is adopted, and 300 ml of S-300 filler is loaded on the column. After loading, the column is first washed with 500-1000 ml of purified water, and then balanced with 300-600 ml of buffer D. After the balance is completed, the protein solution D is added to the column, and the protein detector is connected. When the protein solution D completely enters the filler, it is balanced and washed with buffer D. The protein solution with peaks is collected in separate tubes, and the purity is identified by SDS-PAGE. The protein solution with purity ≥95% is combined into a tube to obtain 200 ml of α1-antitrypsin antigen.
[0052] Example 6
[0053] α1-antitrypsin antigen concentration and dialysis
[0054] The syntubular protein solution with a purity of ≥95% in Example 5 was concentrated and dialyzed. The syntubular solution was placed in a dialysis bag and concentrated to a volume of 20 ml using sucrose. The solution was dialyzed with buffer D for 5 h, centrifuged at 4°C and 8000-9000 rpm for 20-30 min, and the supernatant was taken and filtered through a 0.22 μm filter membrane to obtain a highly pure α1-antitrypsin antigen.
[0055] Example 7
[0056] Exploration of lyophilization protective agent for α1-antitrypsin antigen
[0057] The α1-antitrypsin antigen obtained in Example 6 was added with 40 ml of freeze-drying protective agent, shaken well, and dispensed into freeze-drying bottles, 1 ml per bottle, and placed in a vacuum freeze dryer for freeze drying. The freeze-dried finished product was subjected to stability testing. Three finished products were taken for reconstitution testing and recorded as the 0-day results, and another 3 were placed in a 37°C constant temperature box for 21 days. After 21 days, they were taken out for testing, and the deviation from the 0-day test result was -2.5%.
[0058] The above disclosure is only a preferred embodiment of the method for extracting and separating α1 antitrypsin antigen from natural human serum of the present invention. Of course, it cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.
Claims
1. A method for extracting and separating α1 antitrypsin antigen from natural human serum, characterized in that: The following steps are involved: Using human serum as a raw material, adding ammonium sulfate for the first time to obtain a first protein solution, adding ammonium sulfate to the first protein solution for a second time to perform salting out precipitation, and centrifuging to obtain a precipitate; The precipitate is subjected to hydrophobic chromatography, ion exchange resin and gel chromatography in sequence to obtain α1-antitrypsin antigen with a purity of ≥95%; A lyophilization protective agent is added to the α1-antitrypsin antigen for freeze-drying and storage to obtain a stable α1-antitrypsin antigen.
2. The method for extracting and separating α1 antitrypsin antigen from natural human serum according to claim 1, characterized in that: The mass concentration of ammonium sulfate added for the first time is 110-120 g / L, and the pH of the first protein solution added for the first time is 6.2-6.
5. The mass concentration of ammonium sulfate added for the second time is 420-440 g / L, and the pH of the first protein solution added for the second time is 5.4-5.
6.
3. The method for extracting and separating α1 antitrypsin antigen from natural human serum according to claim 1, characterized in that: The specific method of sequentially subjecting the precipitate to hydrophobic chromatography, ion exchange resin and gel chromatography to obtain α1-antitrypsin antigen with a purity of ≥95% is: Dialyzing the precipitate using a first buffer to obtain a second protein solution; Purifying the second protein solution through hydrophobic chromatography and washing with a second buffer to obtain a third protein solution; The third protein solution is subjected to ion exchange chromatography to obtain a fourth protein solution, which is then subjected to gel chromatography to obtain α1-antitrypsin antigen with a purity of ≥95%.
4. The method for extracting and separating α1 antitrypsin antigen from natural human serum according to claim 3, characterized in that: The components of the first buffer solution include Tris-hcl and Nacl, the concentration of the Tris-hcl is 10-15 mmol / L, the pH is 7.8-8.2, and the concentration of the Nacl is 260-300 mmol / L.
5. The method for extracting and separating α1 antitrypsin antigen from natural human serum according to claim 1, characterized in that: The components of the freeze-drying protective agent include sugars, salts, surfactants and polyhydroxy compounds, wherein the sugars include any one of lactose, sucrose and trehalose, and the addition amount thereof is 1-2%, the salts include any one of sodium chloride and potassium chloride, and the addition amount thereof is 200-260 mmol / L, the surfactants include any one of glycerol, Tween 20, Tween 80 and Triton X-100, and the addition amount thereof is 0.05-0.1%, and the polyhydroxy compounds include any one of sorbitol and polyethylene glycol, and the addition amount thereof is 0.05-0.07%.