Genetically engineered protein preserving fluid and preparation method thereof

Abstract

The invention discloses a genetic engineering protein preserving fluid, and relates to the technical field of genetic engineering, the genetic engineering protein preserving fluid contains sterile deionized water and the following components: NaCl with a concentration of 8 to 40 g / L, KCl with a concentration of 200 to 1000 mg / L, Na2HPO4 with a concentration of 1.44 to 7.2 g / L, KH2PO4 with a concentration of 240 to 1200 mg / L, bovine serum albumin with a concentration of 1 to 10%, fetal bovine serum with a concentration of 1 to 10%, a protein stabilizer with a concentration of 0.05 to 0.5%, a preservative with a concentration of 0.1 to 0.5%, a bacteriostatic agent with a concentration of 0.1 to 0.5%, and a reducing agent with a concentration of 0.05%. The quality control product preserving fluid provided by the invention is low in preparation cost, the storage stability of the quality control product at 2-8 DEG C and the freeze-thaw stability at-20 DEG C can be obviously prolonged by dissolving a quality control product solid in the preserving fluid or adding the quality control product into the preserving fluid which is concentrated twice in equal proportion (V / V), and the detection result of the actual concentration of the to-be-detected quality control product is not influenced.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and more specifically, to a preservation solution for genetically engineered proteins and a preparation method thereof. Background Art

[0002] Genetically engineered proteins are proteins expressed and produced in host cells through genetic engineering technology. These proteins are widely used in the fields of medicine, biotechnology, and industry, such as drug development, vaccine production, diagnostic reagents, enzyme preparations, etc. Due to their high purity and specificity, genetically engineered proteins play an important role in modern biopharmaceuticals and research.

[0003] Although genetically engineered proteins have broad application prospects, they face many challenges during production and storage, especially the stability problem. Protein molecules are easily affected by external environmental factors, such as temperature, pH value, oxidative stress, etc., resulting in changes in their structure and function, thereby losing activity or degrading. Therefore, how to effectively preserve genetically engineered proteins has become a key technical problem.

[0004] Currently, the commonly used methods for preserving genetically engineered proteins mainly include the following several types:

[0005] Cryopreservation: The protein solution is placed in a low-temperature environment (usually -20°C or -80°C) for preservation. Although this method can effectively extend the shelf life of proteins, frequent freeze-thaw cycles will cause protein denaturation and aggregation, affecting its stability and activity.

[0006] Adding preservatives: Chemical preservatives (such as sodium azide, sodium benzoate, etc.) are added to the protein solution to inhibit the growth of microorganisms. However, these preservatives may have toxic effects on the proteins themselves, reducing their biological activity, and their use is restricted in some applications (such as pharmaceutical preparations).

[0007] Buffer system: Specific buffers (such as phosphate buffer, Tris-HCl buffer, etc.) are used to maintain the pH value and ionic strength of the protein solution. Although these buffers can improve the stability of proteins to a certain extent, it is still difficult to maintain the activity and integrity of proteins under extreme conditions (such as high temperature, long-term storage).

[0008] Lyophilization technology: The protein solution is converted into a solid powder through freeze-drying to reduce the water content, thereby improving the stability of the protein. However, the freeze-drying process is complex and energy-consuming, and problems such as poor protein solubility and decreased activity may occur after reconstitution.

[0009] Although the above methods can, to a certain extent, extend the storage time of genetically engineered proteins, the storage effect is limited: in long-term storage or extreme environments, the stability of proteins will still significantly decline, unable to meet the actual application requirements.

[0010] Therefore, it is necessary to propose a preservation solution for genetically engineered proteins and its preparation method to solve the above problems. Summary of the Invention

[0011] The object of the present invention is to solve the problem of poor stability of existing protein preservation solutions.

[0012] The present invention specifically adopts the following technical solutions to achieve the above object:

[0013] A preservation solution for genetically engineered proteins, each liter of the preservation solution includes components with the following concentrations: 8 - 40 g / L NaCl, 200 - 1000 mg / L KCl, 1.44 - 7.2 g / L Na 2 HPO 4 、240 - 1200 mg / L KH 2 PO 4 、1% - 10% bovine serum albumin, 1% - 10% fetal bovine serum, 0.05% - 0.5% protein stabilizer, 0.1% - 0.5% preservative, 0.1% - 0.5% bacteriostatic agent, and 0.05% reducing agent, with the balance being sterile deionized water.

[0014] A preparation method for a preservation solution for genetically engineered proteins, including the above-mentioned preservation solution for genetically engineered proteins. The preparation of each liter of the protein preservation solution further includes the following steps:

[0015] S1. Measure 500 milliliters of sterile deionized water, weigh and add 0.05% - 0.5% protein stabilizer, and stir at room temperature for 3 hours;

[0016] S2. After waiting for 3 hours, continuously stir the solution obtained in step S1 and add 0.1% - 0.5% preservative, 0.05% reducing agent, 1% - 10% fetal bovine serum, and the weighed 8 - 40 g / L NaCl, 200 - 1000 mg / L KCl, 1.44 - 7.2 g / LNa 2 HPO 4 、240 - 1200 mg / LKH 2 PO 4 and 1% - 10% bovine serum albumin. After stirring at room temperature for 0.5 hours, make up the volume to 1 L;

[0017] S3. Filter the solution with a volume made up in step S2 through a hydrophilic filter membrane with a pore size of 0.45 μm to obtain the preservation solution for genetically engineered proteins, and store it at 4°C for standby

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The protein preservation solution provided by the present invention has a low preparation cost. Dissolving the quality control product solid in this preservation solution or adding the quality control product in an equal proportion (V / V) to twice the concentrated preservation solution can significantly extend the storage stability of the quality control product at 2-8°C and the freeze-thaw stability at -20°C, and does not affect the test results of the actual concentration of the quality control product to be measured. Specific embodiments

[0020] The following will combine the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] A genetic engineering protein preservation solution, the protein preservation solution contains sterile deionized water and components with the following concentrations: 8-40 g / L NaCl, 200-1000 mg / L KCl, 1.44-7.2 g / L Na 2 HPO 4 、240-1200 mg / L KH 2 PO 4 、1%-10% bovine serum albumin, 1%-10% fetal bovine serum, 0.05%-0.5% protein stabilizer, 0.1%-0.5% preservative, 0.1%-0.5% bacteriostatic agent and 0.05% reducing agent.

[0022] A preparation method of a genetic engineering protein preservation solution, the preparation of each liter of protein preservation solution includes the following steps:

[0023] S1. Measure 500 ml of sterile deionized water, weigh and add 0.05%-0.5% protein stabilizer, and stir at room temperature for 3 hours;

[0024] S2. After waiting for 3 hours, continuously stir the solution obtained in step S1 and add 0.1%-0.5% preservative, 0.05% reducing agent, 1%-10% fetal bovine serum and weighed 8-40 g / L NaCl, 200-1000 mg / L KCl, 1.44-7.2 g / L Na 2 HPO 4 、240-1200 mg / L KH 2 PO 4 and 1%-10% bovine serum albumin, stir at room temperature for 0.5 hours and then make up the volume to 1 L;

[0025] S3. Filter the solution with a fixed volume in S2 through a hydrophilic filter membrane with a pore size of 0.45 μm to obtain the gene engineering protein preservation solution, and store it at 4 °C for later use.

[0026] Detection reagent:

[0027]

[0028] The criteria for determining the stability of the protein during storage at 2 - 8 °C and freeze - thaw stability in the preservation solution: The deviation of the detection result does not exceed ±15%, and 15% is the accuracy deviation of the reagent.

[0029] Detection result = (The detection result of this month - The concentration of the protein to be measured) / The concentration of the protein to be measured * 100%

[0030] Prepare NaCl, KCl, Na 2 HPO 4 、KH 2 PO 4 、bovine serum albumin, fetal bovine serum, protein stabilizer, preservative, bacteriostatic agent and reducing agent for experiments, which are divided into the following examples:

[0031] Example 1

[0032] Reagent Name Content NaCl 16 g / L KCl 400 mg / L <![CDATA[Sodium 2 HPO 4 > 2.88 g / L <![CDATA[KH 2 PO 4 > 480 mg / L Bovine Serum Albumin 5％ Fetal Bovine Serum 5％ Protein Stabilizer 0.05％ Preservative 0.1％ Bacteriostatic Agent 0.1％ Reducing Agent 0.05％

[0033] Detect the prepared preservation solution according to the requirements of the kit instructions:

[0034] Reagent Name Test Result Serum Amyloid A Detection Reagent (Fluorescence Immunochromatography) <5 mg / L Procalcitonin Detection Reagent (Fluorescence Immunochromatography) <0.3 ng / mL Myoglobin Detection Reagent (Fluorescence Immunochromatography) <10 ng / mL C-Reactive Protein Detection Reagent (Fluorescence Immunochromatography) <0.5 mg / L

[0035] The detection results are all lower than the lower limit of the detection linear range of the kit, and the preservation solution has no influence on the detection results of the analytes in the kit.

[0036] 1.1 Verification of the preservation solution of the quality control product to ensure the stability of the protein to be measured during storage at 2 - 8 °C

[0037] Table 1 Detection results of the stability of the protein to be measured during storage at 2 - 8 °C in the preservation solution

[0038]

[0039] 1.2 Verification of the preservation solution of the quality control product to ensure the freeze - thaw stability of the protein to be measured

[0040] Table 2 Detection results of the freeze - thaw stability of the protein to be measured in the preservation solution

[0041]

[0042]

[0043] Example 2

[0044] Reagent Name Content NaCl 8 g / L KCl 200 mg / L <![CDATA[Sodium 2 HPO 4 > 1.44 g / L <![CDATA[KH 2 PO 4 > 240 mg / L Bovine Serum Albumin 5％ Fetal Bovine Serum 5％ Protein Stabilizer 0.05％ Preservative 0.1％ Bacteriostatic Agent 0.1％ Reducing Agent 0.05％

[0045] Detect the prepared preservation solution according to the requirements of the kit instruction manual:

[0046]

[0047]

[0048] The detection results are all lower than the lower limit of the detection linear range of the kit, and the preservation solution has no influence on the detection results of the analytes to be detected by the kit.

[0049] 2.1 Verification of the preservation solution of the quality control product to ensure the stability of the analyte protein stored at 2 - 8°C

[0050] Table 3 Detection results of the stability of the analyte protein stored at 2 - 8°C in the preservation solution

[0051]

[0052] 2.2 Verification of the preservation solution of the quality control product to ensure the freeze - thaw stability of the analyte protein

[0053] Table 4 Detection results of the freeze - thaw stability of the analyte protein in the preservation solution

[0054]

[0055]

[0056] Example 3

[0057]

[0058]

[0059] Detect the prepared preservation solution according to the requirements of the kit instruction manual:

[0060] Reagent Name Test Result Serum Amyloid A Detection Reagent (Fluorescence Immunochromatography) <5 mg / L Procalcitonin Detection Reagent (Fluorescence Immunochromatography) <0.3 ng / mL Myoglobin Detection Reagent (Fluorescence Immunochromatography) <10 ng / mL C-Reactive Protein Detection Reagent (Fluorescence Immunochromatography) <0.5 mg / L

[0061] The detection results are all lower than the lower limit of the detection linear range of the kit, and the preservation solution has no influence on the detection results of the analytes to be detected by the kit.

[0062] 3.1 Verification of the preservation solution of the quality control product to ensure the stability of the analyte protein stored at 2 - 8°C

[0063] Table 5 Detection results of the stability of the analyte protein stored at 2 - 8°C in the preservation solution

[0064]

[0065]

[0066] 3.2 Verification of the Preservation Solution for Quality Control Products to Ensure the Freeze-Thaw Stability of Proteins to Be Measured

[0067] Table 6 Detection Results of the Freeze-Thaw Stability of Proteins to Be Measured in the Preservation Solution

[0068]

[0069] Conclusion: The detection results of the three formula preservation solutions within the scope of the above three embodiments for preserving different recombinant proteins all meet the requirements, and all can stably preserve the proteins in the preservation solution. Among them, in the detection results of Example 2, the detection results of serum amyloid A, procalcitonin, and C-reactive protein in the samples prepared in the preservation solution are higher than the theoretical values, and in Example 3, the detection results of each protein concentration are lower than the theoretical values. The reason may be the influence of the matrix with different ion concentrations in the preservation solution.

[0070] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. The patent protection scope of the present invention is subject to the claims. Any equivalent structural changes made by using the content of the specification of the present invention should, by the same token, be included in the protection scope of the present invention.

Claims

1. A genetic engineering protein preservation solution, characterized in that: Each liter of preservation solution includes the following components in concentrations: 8-40g / L NaCl, 200-1000mg / L KCl, 1.44-7.2g / L Na2HPO4, 240-1200mg / L KH2PO4, 1%-10% bovine serum albumin, 1%-10% fetal bovine serum, 0.05%-0.5% protein stabilizer, 0.1%-0.5% preservative, 0.1%-0.5% antibacterial agent and 0.05% reducing agent, and the balance is sterile deionized water.

2. A method for preparing the genetic engineering protein preservation solution according to claim 1, characterized in that: The following steps are involved: The preparation of each liter of protein preservation solution also includes the following steps: S1. Measure 500 ml of sterile deionized water, weigh it, add 0.05% 0.5% protein stabilizer, and stir at room temperature for 3 hours; S2, after waiting for 3 hours, continuously stirring the solution obtained in step S1 and adding 0.1%-0.5% preservative, 0.05% reducing agent, 1%-10% fetal bovine serum and weighed 8-40g / L NaCl, 200-1000mg / L KCl, 1.44-7.2g / LNa2HPO4, 240-1200mg / LKH2PO4 and 1%-10% bovine serum albumin, stirring at room temperature for 0.5 hour, and then fixing the volume to 1L; S3. Filter the fixed volume solution in S2 through a hydrophilic filter membrane with a pore size of 0.45 μm to obtain a genetic engineering protein preservation solution, which is stored at 4°C for later use.

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