Neutrophil apolipoprotein preservation solution and preparation process thereof
By modifying the complex modification of bovine serum albumin and dextran and trehalose, combined with antioxidants and antifreeze, the problem that existing preservation liquid cannot effectively ensure the activity of neutrophil apolipoprotein, significantly improving Protein stability and preservation effect.
Patent Information
- Application Number
- CN202410995194.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-24
- Publication Date
- 2025-02-14
- Estimated Expiration
- 2044-07-24
AI Technical Summary
The existing storage solution for preserving neutrophil apolipoprotein cannot effectively ensure protein activity. Simply adding bovine serum albumin can only prevent structural damage and cannot prevent activity loss.
By modifying the complex modification of bovine serum albumin and dextran and trehalose, a complex that can closely bind to protein molecules is formed, which enhances the stability and preservation ability of the preservation solution, and adds antioxidants and antifreeze to further improve the Improve the preservation effect.
It significantly improves the stability of neutrophil apolipoprotein during low temperature storage, reduces the probability of protein structure and activity damage, and enhances the preservation ability of the preservation solution.
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Figure CN118546236B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and in particular to a neutrophil apolipoprotein preservation solution and a preparation process thereof. Background Art
[0002] Neutrophil Lipocalin (HNL) is a protein found in the secondary granules of neutrophils. When the human body is infected with bacteria, activated neutrophils release HNL into the blood, causing the HNL concentration in the blood to increase significantly within 1-2 hours. The increase in HNL content in the blood can help doctors determine whether there is an acute bacterial infection and can also reflect the severity of the infection. Therefore, more in-depth research on neutrophil apolipoprotein is needed.
[0003] Bovine serum albumin is generally added to the preservation solution for preserving neutrophil apolipoprotein to enhance protein activity and prevent the protein from being destroyed during freezing. However, simply adding bovine serum albumin can only ensure that the protein structure is not destroyed, but the protein activity cannot be guaranteed. Therefore, the present invention provides a neutrophil apolipoprotein preservation solution and a preparation process thereof to solve the above-mentioned deficiencies in the prior art. Summary of the invention
[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a neutrophil apolipoprotein preservation solution and a preparation process thereof.
[0005] A preparation process of a neutrophil apolipoprotein preservation solution comprises the following steps:
[0006] S1: Bovine serum albumin modification
[0007] preparing a 1,6-hexanediamine solution and a BSA solution, adding the 1,6-hexanediamine solution to the BSA solution, and then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and EDAC to obtain an amination reaction solution; removing the precipitate in the amination reaction solution, dialyzing and freeze-drying to obtain the amination BSA;
[0008] S2: Dextran compound trehalose modified
[0009] The dextran and trehalose are mixed and dissolved in dimethyl sulfoxide to prepare a mixed sugar solution, succinic anhydride is dissolved in dimethyl sulfoxide to prepare a succinic anhydride solution, the succinic anhydride solution is added dropwise to the mixed sugar solution, and a catalyst is added to obtain a modified liquid, three times the volume of deionized water is added to the obtained modified liquid for dialysis and freeze-drying to obtain a modified polysaccharide;
[0010] S3: Preparation of bovine serum albumin-polysaccharide complex
[0011] The aminated BSA is dissolved in a PBS buffer solution to obtain an aminated BSA solution, the modified polysaccharide is dissolved in deionized water to prepare a modified polysaccharide solution, and then the aminated BSA solution is added dropwise to the modified polysaccharide solution, the mixture is fully stirred and mixed, and the mixture is filtered to obtain a composite reaction solution, the composite reaction solution is dialyzed, and then freeze-dried to obtain a bovine serum albumin-polysaccharide complex;
[0012] S4: Preparation of preservation solution
[0013] 10-20% by mass of bovine serum albumin-polysaccharide complex, 20-40% of antifreeze agent, 1-3% of antioxidant and phosphate buffered saline as the remainder are mixed, stirred evenly, and stored in a vacuum to obtain a neutrophil apolipoprotein storage solution.
[0014] Furthermore, step S1 of modifying bovine serum albumin comprises the following steps:
[0015] S1.1: Weigh 1.5-2 parts by mass of 1,6-hexanediamine and add it to 10-20 parts by volume of deionized water to dissolve it, and adjust the pH of the solution to 6-6.5 to obtain a 1,6-hexanediamine solution;
[0016] S1.2: Then, 0.2-0.3 parts by mass of bovine serum albumin is weighed and dissolved in 10-20 parts by volume of deionized water, and the pH of the solution is adjusted to 6-6.5 to obtain a BSA solution;
[0017] S1.3: After adding the 1,6-hexanediamine solution to the BSA solution, add 0.1-0.2 parts by mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to initiate the coupling reaction to activate the carboxyl group, stir at room temperature for 4-5 hours, then add 0.05-0.08 parts by mass of EDAC to the system, use dilute hydrochloric acid to maintain the pH value of the solution at 6-6.5, react for 5-6 hours, and obtain an amination reaction solution;
[0018] S1.4: Use a 0.45μm aqueous protein filter to remove the precipitate in the amination reaction solution, dialyze for 70-72 hours, and then freeze-dry to obtain the amination BSA.
[0019] Furthermore, step S2, modification of the glucan-complexed trehalose, comprises the following steps:
[0020] S2.1: Mix dextran and trehalose in a mass ratio of (1-2):1 to obtain a mixed sugar, weigh 1-2 parts by mass of the mixed sugar, use 15-20 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonication to obtain a mixed sugar solution;
[0021] S2.2: Weigh 1.85-2 parts by mass of succinic anhydride, use 15-20 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a succinic anhydride solution;
[0022] S2.3: adding the succinic anhydride solution dropwise to the mixed sugar solution, and then adding 0.05 parts by mass of a catalyst to the mixed solution, reacting at 65-70° C. for 12-16 hours to obtain a modified liquid;
[0023] S2.4: Add three times the volume of deionized water to the obtained modified liquid, dialyze for 70-72 hours and freeze-dry to obtain modified polysaccharide.
[0024] Furthermore, the catalyst in step S2.3 is 4-dimethylaminopyridine.
[0025] Furthermore, the freeze drying in step S2.4 is performed at 0-4°C for 1-3 hours.
[0026] Furthermore, step S3 of preparing a bovine serum albumin-polysaccharide complex comprises the following steps:
[0027] S3.1: dissolving the aminated BSA in a PBS buffer solution, adjusting the pH to 7.5-8, and adjusting the concentration to 0.01 mol / L, to obtain an aminated BSA solution;
[0028] S3.2: Dissolve the modified polysaccharide in deionized water to prepare a modified polysaccharide solution with a concentration of 1 mg / mL;
[0029] S3.3: Then, the amino BSA solution is added dropwise to the modified polysaccharide solution, and after being fully stirred and mixed for 10-12 hours, it is filtered using a 0.45 μm filter membrane for aqueous proteins to obtain a composite reaction solution;
[0030] S3.4: The composite reaction solution is dialyzed and then freeze-dried to obtain a bovine serum albumin-polysaccharide complex.
[0031] Furthermore, in step S3.3, the volume ratio of the amino BSA solution to the modified polysaccharide solution is 1:(2-5).
[0032] Furthermore, the antifreeze agent in step S4 is glycerol.
[0033] Furthermore, the antioxidant in step S4 is glutathione.
[0034] A neutrophil apolipoprotein preservation solution is prepared by the preparation process of the neutrophil apolipoprotein preservation solution.
[0035] Compared with the prior art, the present invention has at least the following beneficial effects:
[0036] 1. The present invention utilizes bovine serum albumin in combination with dextran and trehalose to form clumps that can be filled between proteins, prevent interaction between protein molecules, reduce aggregation, thereby preventing structural damage to proteins during low-temperature storage, reducing the degree of protein structural damage, and thus improving the stability of neutrophil apolipoprotein during low-temperature storage.
[0037] 2. The present invention prepares amino-BSA by modifying bovine serum albumin. The additional amino group can increase the binding sites between bovine serum albumin and the modified polysaccharide, enhance its function as a carrier, and enable the modified polysaccharide to form a complex with bovine serum albumin, so that the three are tightly bound and not easily disintegrated under low temperature and long-term storage conditions, thereby achieving the effect of supporting protein molecules and reducing the probability of protein structure damage.
[0038] 3. The present invention modifies dextran by mixing it with trehalose. After modification, dextran can improve its binding ability with amino-BSA, and trehalose can serve as a macromolecular support for bovine serum albumin-polysaccharide complex, thereby supporting between protein molecules, stabilizing the protein and preventing protein structure damage. After dextran and trehalose are combined, they have the advantages of both dextran and trehalose, which not only improves the adhesion of the modified polysaccharide to amino-BSA, but also enhances the preservation ability of the preservation solution to neutrophil apolipoprotein.
[0039] 4. The present invention prevents oxidation of proteins during storage by adding an antioxidant glutathione, and the antifreeze agent glycerol can fill the gaps between proteins and act as a buffer to prevent structural damage of proteins at low temperatures. The combination of glutathione and glycerol can further improve the preservation ability of the preservation solution for neutrophil apolipoprotein. BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable those skilled in the relevant art to make and use the present disclosure.
[0041] Figure 1 The present invention is a flow chart of a process for preparing a neutrophil apolipoprotein preservation solution used in an embodiment of the present invention. DETAILED DESCRIPTION
[0042] The following is a detailed description of a neutrophil apolipoprotein preservation solution and a preparation process thereof provided by the present invention in conjunction with the accompanying drawings and specific embodiments. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art may also adopt other alternatives to implement some known technologies; and the accompanying drawings are only for a more specific description of the embodiments, and are not intended to specifically limit the present invention. Embodiment 1:
[0043] A preparation process of a neutrophil apolipoprotein preservation solution, such as Figure 1 As shown, the following steps are included:
[0044] S1: Bovine serum albumin modification
[0045] S1.1: Weigh 1.5 parts by mass of 1,6-hexanediamine and add it to 10 parts by volume of deionized water to dissolve it, and adjust the pH of the solution to 6.5 to obtain a 1,6-hexanediamine solution;
[0046] S1.2: Then, 0.2 parts by mass of bovine serum albumin was weighed and dissolved in 10 parts by volume of deionized water, and the pH of the solution was adjusted to 6.5 to obtain a BSA solution;
[0047] S1.3: After adding the 1,6-hexanediamine solution to the BSA solution, 0.1 parts by mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to initiate the coupling reaction to activate the carboxyl group, and the mixture was stirred at room temperature for 5 hours. Then, 0.05 parts by mass of EDAC was added to the system, and the pH value of the solution was maintained at 6.5 using dilute hydrochloric acid. The mixture was reacted for 6 hours to obtain an amination reaction solution.
[0048] S1.4: Use a 0.45 μm filter membrane for aqueous proteins to remove the precipitate in the amination reaction solution, dialyze for 70 hours, and then freeze-dry to obtain the amination BSA.
[0049] S2: Dextran modified with trehalose
[0050] S2.1: Mix dextran and trehalose in a mass ratio of 2:1 to obtain a mixed sugar, weigh 1 mass part of the mixed sugar, use 15 volume parts of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a mixed sugar solution;
[0051] S2.2: Weigh 1.85 parts by mass of succinic anhydride, use 15 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a succinic anhydride solution;
[0052] S2.3: adding the succinic anhydride solution dropwise to the mixed sugar solution, and then adding 0.05 parts by mass of 4-dimethylaminopyridine to the mixed solution, reacting at 65° C. for 12 h to obtain a modified liquid;
[0053] S2.4: Add three times the volume of deionized water to the obtained modified liquid, dialyze for 72 hours and dry at 0°C for 3 hours to obtain modified polysaccharide.
[0054] S3: Preparation of bovine serum albumin-polysaccharide complex
[0055] S3.1: dissolving the aminated BSA in a PBS buffer solution, adjusting the pH to 8 and the concentration to 0.01 mol / L, to obtain an aminated BSA solution;
[0056] S3.2: Dissolve the modified polysaccharide in deionized water to prepare a modified polysaccharide solution with a concentration of 1 mg / mL;
[0057] S3.3: Then, the aminated BSA solution is added dropwise to the modified polysaccharide solution, and the volume ratio of the aminated BSA solution to the modified polysaccharide solution is 1:5. After being fully stirred and mixed for 12 hours, the mixture is filtered using a 0.45 μm filter membrane for aqueous proteins to obtain a composite reaction solution;
[0058] S3.4: The composite reaction solution is dialyzed and then freeze-dried to obtain a bovine serum albumin-polysaccharide complex.
[0059] S4: Preparation of preservation solution
[0060] 20% by mass of bovine serum albumin-polysaccharide complex, 40% by mass of glycerol, and 3% by mass of glutathione were mixed with phosphate buffered saline as the remainder, stirred evenly, and stored in a vacuum to obtain a neutrophil apolipoprotein storage solution.
[0061] Embodiment 2:
[0062] A preparation process of a neutrophil apolipoprotein preservation solution, such as Figure 1 As shown, the following steps are included:
[0063] S1: Bovine serum albumin modification
[0064] S1.1: Weigh 2 parts by mass of 1,6-hexanediamine and add it into 20 parts by volume of deionized water to dissolve it, and adjust the pH of the solution to 6.5 to obtain a 1,6-hexanediamine solution;
[0065] S1.2: Then, 0.3 parts by mass of bovine serum albumin was weighed and dissolved in 20 parts by volume of deionized water, and the pH of the solution was adjusted to 6.5 to obtain a BSA solution;
[0066] S1.3: After adding the 1,6-hexanediamine solution to the BSA solution, 0.2 parts by mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to initiate the coupling reaction to activate the carboxyl group, and the mixture was stirred at room temperature for 5 hours. Then, 0.05 parts by mass of EDAC was added to the system, and the pH value of the solution was maintained at 6.5 using dilute hydrochloric acid. The mixture was reacted for 6 hours to obtain an amination reaction solution.
[0067] S1.4: Use a 0.45 μm filter membrane for aqueous proteins to remove the precipitate in the amination reaction solution, dialyze for 70 hours, and then freeze-dry to obtain the amination BSA.
[0068] S2: Dextran modified with trehalose
[0069] S2.1: Mix dextran and trehalose in a mass ratio of 1:1 to obtain a mixed sugar, weigh 2 parts by mass of the mixed sugar, use 20 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a mixed sugar solution;
[0070] S2.2: Weigh 2 parts by mass of succinic anhydride, use 20 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a succinic anhydride solution;
[0071] S2.3: adding the succinic anhydride solution dropwise to the mixed sugar solution, and then adding 0.05 parts by mass of 4-dimethylaminopyridine to the mixed solution, reacting at 65° C. for 12 h to obtain a modified liquid;
[0072] S2.4: Add three times the volume of deionized water to the obtained modified liquid, dialyze for 72 hours and dry at 0°C for 3 hours to obtain modified polysaccharide.
[0073] S3: Preparation of bovine serum albumin-polysaccharide complex
[0074] S3.1: dissolving the aminated BSA in a PBS buffer solution, adjusting the pH to 8 and the concentration to 0.01 mol / L, to obtain an aminated BSA solution;
[0075] S3.2: Dissolve the modified polysaccharide in deionized water to prepare a modified polysaccharide solution with a concentration of 1 mg / mL;
[0076] S3.3: Then, the aminated BSA solution is added dropwise to the modified polysaccharide solution, and the volume ratio of the aminated BSA solution to the modified polysaccharide solution is 1:2. After being fully stirred and mixed for 12 hours, the mixture is filtered using a 0.45 μm filter membrane for aqueous proteins to obtain a composite reaction solution;
[0077] S3.4: The composite reaction solution is dialyzed and then freeze-dried to obtain a bovine serum albumin-polysaccharide complex.
[0078] S4: Preparation of preservation solution
[0079] 10% by mass of bovine serum albumin-polysaccharide complex, 20% by mass of glycerol, and 1% by mass of glutathione were mixed with phosphate buffered saline as the remainder, stirred evenly, and stored in a vacuum to obtain a neutrophil apolipoprotein storage solution.
[0080] Embodiment 3:
[0081] A preparation process of a neutrophil apolipoprotein preservation solution, such as Figure 1 As shown, the following steps are included:
[0082] S1: Bovine serum albumin modification
[0083] S1.1: Weigh 1.5 parts by mass of 1,6-hexanediamine and add it to 10 parts by volume of deionized water to dissolve it, and adjust the pH of the solution to 6 to obtain a 1,6-hexanediamine solution;
[0084] S1.2: Then, 0.2 parts by mass of bovine serum albumin was weighed and dissolved in 10 parts by volume of deionized water, and the pH of the solution was adjusted to 6 to obtain a BSA solution;
[0085] S1.3: After adding the 1,6-hexanediamine solution to the BSA solution, 0.1 parts by mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to initiate the coupling reaction to activate the carboxyl group, and the mixture was stirred at room temperature for 4 hours. Then, 0.05 parts by mass of EDAC was added to the system, and the pH value of the solution was maintained at 6 using dilute hydrochloric acid. The mixture was reacted for 5 hours to obtain an amination reaction solution.
[0086] S1.4: Use a 0.45 μm filter membrane for aqueous proteins to remove the precipitate in the amination reaction solution, dialyze for 72 hours, and then freeze-dry to obtain the amination BSA.
[0087] S2: Dextran modified with trehalose
[0088] S2.1: Mix dextran and trehalose in a mass ratio of 2:1 to obtain a mixed sugar, weigh 1 mass part of the mixed sugar, use 15 volume parts of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a mixed sugar solution;
[0089] S2.2: Weigh 1.85 parts by mass of succinic anhydride, use 15 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a succinic anhydride solution;
[0090] S2.3: adding the succinic anhydride solution dropwise to the mixed sugar solution, and then adding 0.05 parts by mass of 4-dimethylaminopyridine to the mixed solution, reacting at 70° C. for 16 hours to obtain a modified liquid;
[0091] S2.4: Add three times the volume of deionized water to the obtained modified liquid, dialyze for 72 hours and dry at 4°C for 1 hour to obtain modified polysaccharide.
[0092] S3: Preparation of bovine serum albumin-polysaccharide complex
[0093] S3.1: dissolving the aminated BSA in PBS buffer solution, adjusting the pH to 7.5 and the concentration to 0.01 mol / L, to obtain an aminated BSA solution;
[0094] S3.2: Dissolve the modified polysaccharide in deionized water to prepare a modified polysaccharide solution with a concentration of 1 mg / mL;
[0095] S3.3: Then, the aminated BSA solution is added dropwise to the modified polysaccharide solution, the volume ratio of the aminated BSA solution to the modified polysaccharide solution is 1:5, and after being fully stirred and mixed for 10 hours, the mixture is filtered using a 0.45 μm filter membrane for aqueous proteins to obtain a composite reaction solution;
[0096] S3.4: The composite reaction solution is dialyzed and then freeze-dried to obtain a bovine serum albumin-polysaccharide complex.
[0097] S4: Preparation of preservation solution
[0098] 20% by mass of bovine serum albumin-polysaccharide complex, 40% by mass of glycerol, and 3% by mass of glutathione were mixed with phosphate buffered saline as the remainder, stirred evenly, and stored in a vacuum to obtain a neutrophil apolipoprotein storage solution.
[0099] Comparative Example 1:
[0100] Compared with Example 1, the difference of Comparative Example 1 is that, in step S2, no dextran is added, but all trehalose is added, specifically: “S2.1: weigh 1 part by mass of trehalose, use 15 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a trehalose solution;
[0101] S2.2: Weigh 1.85 parts by mass of succinic anhydride, use 15 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a succinic anhydride solution;
[0102] S2.3: adding the succinic anhydride solution dropwise to the trehalose solution, and then adding 0.05 parts by mass of 4-dimethylaminopyridine to the mixed solution, reacting at 65° C. for 12 h to obtain a modified liquid;
[0103] S2.4: Add three times the volume of deionized water to the obtained modified liquid, dialyze for 72 hours and dry at 0°C for 3 hours to obtain a modified polysaccharide. The prepared neutrophil apolipoprotein preservation solution is recorded as Comparative Example 1.
[0104] Comparative Example 2
[0105] Compared with Example 1, the difference of Comparative Example 2 is that trehalose is not added in step S2, but all dextran is added, specifically: "S2.1: weigh 1 part by mass of dextran, use 15 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a dextran solution;
[0106] S2.2: Weigh 1.85 parts by mass of succinic anhydride, use 15 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a succinic anhydride solution;
[0107] S2.3: adding the succinic anhydride solution dropwise to the dextran solution, and then adding 0.05 parts by mass of 4-dimethylaminopyridine to the mixed solution, reacting at 65° C. for 12 h to obtain a modified liquid;
[0108] S2.4: Add three times the volume of deionized water to the obtained modified liquid, dialyze for 72 hours and dry at 0°C for 3 hours to obtain a modified polysaccharide. The prepared neutrophil apolipoprotein preservation solution is recorded as Comparative Example 2.
[0109] Comparative Example 3:
[0110] Compared with Example 1, the difference of Comparative Example 3 is that step S2 and step S3 are not performed, and step S4 is directly performed after step S1 is completed. Step S4 is specifically: "Mix 20% by mass of amino-BSA, 40% by mass of glycerol, and 3% by mass of glutathione with phosphate buffered saline as the remainder, stir evenly, and store in a vacuum to obtain a neutrophil apolipoprotein preservation solution." The prepared neutrophil apolipoprotein preservation solution is recorded as Comparative Example 3.
[0111] Comparative Example 4
[0112] Compared with Example 1, the difference of Comparative Example 4 is that the antifreeze agent glycerol is not added in step S4, specifically: "mix 20% by mass of bovine serum albumin-polysaccharide complex, 3% glutathione and phosphate buffered saline as the remainder, stir evenly, and store in vacuum to obtain neutrophil apolipoprotein preservation solution" and the prepared neutrophil apolipoprotein preservation solution is recorded as Comparative Example 4.
[0113] Comparative Example 5
[0114] Compared with Example 1, the difference of Comparative Example 5 is that the antioxidant glutathione is not added in step S4, specifically: "Mix 20% by mass of bovine serum albumin-polysaccharide complex, 40% by mass of glycerol and phosphate buffered saline as the remainder, stir evenly, and store in a vacuum to obtain a neutrophil apolipoprotein preservation solution." The prepared neutrophil apolipoprotein preservation solution is recorded as Comparative Example 5.
[0115] Comparative Example 6
[0116] Compared with Example 1, the difference of Comparative Example 6 is that the antifreeze agent glycerol and the antioxidant glutathione are not added in step S4, specifically: "mixing 20% by mass of bovine serum albumin-polysaccharide complex with phosphate buffered saline as the remainder, stirring evenly, and storing in a vacuum to obtain a neutrophil apolipoprotein preservation solution" and the prepared neutrophil apolipoprotein preservation solution is recorded as Comparative Example 6.
[0117] Comparative Example 7
[0118] Compared with Example 1, the difference of Comparative Example 7 is that step S1 is not performed, and bovine serum albumin is directly added in step S3 to replace the amino BSA, specifically: "S3.1: dissolving bovine serum albumin in a PBS buffer solution, adjusting the pH to 8, and adjusting the concentration to 0.01 mol / L, to obtain a bovine serum albumin solution;
[0119] S3.2: Dissolve the modified polysaccharide in deionized water to prepare a modified polysaccharide solution with a concentration of 1 mg / mL;
[0120] S3.3: Then, the bovine serum albumin solution is added dropwise to the modified polysaccharide solution, the volume ratio of the bovine serum albumin solution to the modified polysaccharide solution is 1:5, and after being fully stirred and mixed for 12 hours, the mixture is filtered using a 0.45 μm filter membrane for aqueous proteins to obtain a composite reaction solution;
[0121] S3.4: The composite reaction solution is dialyzed and then freeze-dried to obtain a bovine serum albumin-polysaccharide complex. The prepared neutrophil apolipoprotein storage solution is recorded as Comparative Example 7.
[0122] Comparative Example 8
[0123] Compared with Example 1, the difference of Comparative Example 8 is that the step S2 in which the dextran and trehalose are directly mixed and modified is not performed, and the mixed solution of dextran and trehalose is directly added in step S3 to replace the modified polysaccharide, specifically: "S2: dextran composite trehalose modification
[0124] S2.1: Mix dextran and trehalose in a mass ratio of 2:1 to obtain a mixed sugar, weigh 1 mass part of the mixed sugar, use 15 volume parts of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a mixed sugar solution;
[0125] S3: Preparation of bovine serum albumin-polysaccharide complex
[0126] S3.1: dissolving the aminated BSA in a PBS buffer solution, adjusting the pH to 8 and the concentration to 0.01 mol / L, to obtain an aminated BSA solution;
[0127] S3.3: Then, the aminated BSA solution is added dropwise to the mixed sugar solution, the volume ratio of the aminated BSA solution to the mixed sugar solution is 1:5, and after being fully stirred and mixed for 12 hours, the mixture is filtered using a 0.45 μm filter membrane for aqueous proteins to obtain a composite reaction solution;
[0128] S3.4: The composite reaction solution is dialyzed and then freeze-dried to obtain a bovine serum albumin-polysaccharide complex. The prepared neutrophil apolipoprotein storage solution is recorded as Comparative Example 8.
[0129] Apolipoprotein activity assay
[0130] The neutrophil apolipoprotein was transferred into the neutrophil apolipoprotein storage solution of Examples 1-3 and Comparative Examples 1-8 through a test tube, stored at -20°C for 7 days, and then taken out and thawed.
[0131] The activity of thawed neutrophil apolipoprotein was detected using the latex kit of neutrophil apolipoprotein. Take a 96-well plate and follow the operating instructions of the kit. First, add 2 μL of sample, then add 100 μL of R1 (buffer), incubate at room temperature for 2 hours, detect at 450 nm, read the absorbance A1, then add 50 μL of R2 (neutrophil apolipoprotein antibody is fixed on the surface of latex microspheres), react at 37 ° C for 5 minutes, read the absorbance A2, and record the change of absorbance before and after 5 minutes, calculate the difference △A=A1-A2, △A represents the turbidity, that is, turbidity. Water was used as a blank control during the experiment, and the neutrophil apolipoprotein stored in different neutrophil apolipoprotein storage solutions was marked. The statistical results are shown in Table 1.
[0132] Table 1
[0133]
[0134] It can be seen that the closer the turbidity is to that of water, the lower the activity of neutrophil apolipoprotein. The tertiary structure of neutrophil apolipoprotein provides multiple shapes that can bind to lipids, allowing neutrophil apolipoprotein to carry lipid particles. When the structure of neutrophil apolipoprotein is destroyed and its activity decreases, its ability to carry lipid particles is weakened, and the turbidity displayed is lower, which is closer to water with no carrying capacity.
[0135] The turbidity of Example 1 is 0.79, that of Example 2 is 0.74, and that of Example 3 is 0.72, all of which are higher than that of the comparative example. The neutrophil apolipoprotein of the examples has higher activity, that is, the protein of the examples has better storage stability.
[0136] The turbidity of comparative example 1 is 0.26, comparative example 2 is 0.47, and comparative example 3 is 0.46. This is because the complex of trehalose and dextran has a synergistic effect, which not only provides the modified polysaccharide with adhesion to amino BSA, but also enhances the preservation ability of the preservation solution to neutrophil apolipoprotein.
[0137] The turbidity of Comparative Example 4 is 0.43, that of Comparative Example 5 is 0.49, and that of Comparative Example 6 is 0.36. It can be seen that the activity of neutrophil apolipoprotein in Comparative Examples 4-6 is lower than that in the embodiment, but still has a certain activity because the antifreeze agent glycerol and the antioxidant glutathione provide a certain storage stability to the preservation solution. The lack of antifreeze agent glycerol or antioxidant glutathione will lead to a decrease in storage stability, and the storage stability of the combined use is the highest.
[0138] The turbidity of Comparative Example 7 is 0.42, and the turbidity of Comparative Example 8 is 0.39. This is because the unmodified bovine serum albumin is insufficiently bound to the modified polysaccharide, and the trehalose component in the unmodified polysaccharide is also difficult to bind to bovine serum albumin before modification, resulting in the disintegration of protein molecules in Comparative Example 7 and Comparative Example 8 during long-term storage. It can be seen that Comparative Example 7 and Comparative Example 8 cannot support protein molecules, resulting in damage to the protein structure.
[0139] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.
Claims
1. A process for preparing a neutrophil apolipoprotein preservation solution, characterized in that: The steps include: S1: Bovine serum albumin modification preparing a 1,6-hexanediamine solution and a BSA solution, adding the 1,6-hexanediamine solution to the BSA solution, and then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and EDAC to obtain an amination reaction solution; removing the precipitate in the amination reaction solution, dialyzing and freeze-drying to obtain the amination BSA; S2: Dextran compound trehalose modified S2.1: Mix dextran and trehalose in a mass ratio of (1-2):1 to obtain a mixed sugar, weigh 1-2 parts by mass of the mixed sugar, use 15-20 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonication to obtain a mixed sugar solution; S2.2: Weigh 1.85-2 parts by mass of succinic anhydride, use 15-20 parts by volume of dimethyl sulfoxide as a solvent, and dissolve it by ultrasonic method to obtain a succinic anhydride solution; S2.3: adding the succinic anhydride solution dropwise to the mixed sugar solution, and then adding 0.05 parts by mass of catalyst 4-dimethylaminopyridine to the mixed solution, reacting at 65-70° C. for 12-16 hours to obtain a modified liquid; S2.4: Add three times the volume of deionized water to the obtained modified liquid, dialyze for 70-72 hours and freeze-dry to obtain a modified polysaccharide; S3: Preparation of bovine serum albumin-polysaccharide complex The aminated BSA is dissolved in a PBS buffer solution to obtain an aminated BSA solution, the modified polysaccharide is dissolved in deionized water to prepare a modified polysaccharide solution, and then the aminated BSA solution is added dropwise to the modified polysaccharide solution, the mixture is fully stirred and mixed, and the mixture is filtered to obtain a composite reaction solution, the composite reaction solution is dialyzed, and then freeze-dried to obtain a bovine serum albumin-polysaccharide complex; S4: Preparation of preservation solution 10-20% by mass of bovine serum albumin-polysaccharide complex, 20-40% of antifreeze agent, 1-3% of antioxidant and phosphate buffered saline as the remainder are mixed, stirred evenly, and stored in a vacuum to obtain a neutrophil apolipoprotein storage solution.
2. The process for preparing a neutrophil apolipoprotein preservation solution according to claim 1, characterized in that: Step S1: bovine serum albumin modification, comprising the following steps: S1.1: Weigh 1.5-2 parts by mass of 1,6-hexanediamine and add it to 10-20 parts by volume of deionized water to dissolve it, and adjust the pH of the solution to 6-6.5 to obtain a 1,6-hexanediamine solution; S1.2: Then, 0.2-0.3 parts by mass of bovine serum albumin is weighed and dissolved in 10-20 parts by volume of deionized water, and the pH of the solution is adjusted to 6-6.5 to obtain a BSA solution; S1.3: After adding the 1,6-hexanediamine solution to the BSA solution, add 0.1-0.2 parts by mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to initiate the coupling reaction to activate the carboxyl group, stir at room temperature for 4-5 hours, then add 0.05-0.08 parts by mass of EDAC to the system, use dilute hydrochloric acid to maintain the pH value of the solution at 6-6.5, react for 5-6 hours, and obtain an amination reaction solution; S1.4: Use a 0.45μm aqueous protein filter to remove the precipitate in the amination reaction solution, dialyze for 70-72 hours, and then freeze-dry to obtain the amination BSA.
3. The process for preparing a neutrophil apolipoprotein preservation solution according to claim 2, characterized in that: The freeze drying in step S2.4 is performed at 0-4°C for 1-3 hours.
4. The process for preparing a neutrophil apolipoprotein preservation solution according to claim 3, characterized in that: Step S3 prepares a bovine serum albumin-polysaccharide complex, comprising the following steps: S3.1: dissolving the aminated BSA in a PBS buffer solution, adjusting the pH to 7.5-8, and adjusting the concentration to 0.01 mol / L, to obtain an aminated BSA solution; S3.2: Dissolve the modified polysaccharide in deionized water to prepare a modified polysaccharide solution with a concentration of 1 mg / mL; S3.3: Then, the amino BSA solution is added dropwise to the modified polysaccharide solution, and after being fully stirred and mixed for 10-12 hours, it is filtered using a 0.45 μm filter membrane for aqueous proteins to obtain a composite reaction solution; S3.4: The composite reaction solution is dialyzed and then freeze-dried to obtain a bovine serum albumin-polysaccharide complex.
5. The process for preparing a neutrophil apolipoprotein preservation solution according to claim 4, characterized in that: The volume ratio of the amino-BSA solution to the modified polysaccharide solution in step S3.3 is 1:(2-5).
6. The process for preparing a neutrophil apolipoprotein preservation solution according to claim 5, characterized in that: The antifreeze agent in step S4 is glycerol.
7. The process for preparing a neutrophil apolipoprotein preservation solution according to claim 6, characterized in that: The antioxidant in step S4 is glutathione.
8. A neutrophil apolipoprotein storage solution, characterized in that: The neutrophil apolipoprotein storage solution is prepared by the preparation process of any one of claims 1 to 7.