Chitosan-based vaccine adjuvant and preparation method thereof
By introducing hydrophilic and hydrophobic groups into chitosan adjuvant and using quaternary ammonium salts to form a stable micelle structure, the problem of short duration of chitosan adjuvant is solved, and the effect of stable sustained release and enhanced immune effect is achieved.
Patent Information
- Application Number
- CN202510217895.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-26
- Publication Date
- 2025-05-30
AI Technical Summary
When chitosan or its derivatives are used directly as vaccine adjuvant, the duration is short and the vaccine immunity effect is limited.
By introducing hydrophilic and hydrophobic groups and combining the positively charged properties of the quaternary ammonium salt, chitosan quaternized derivatives are prepared to form a stable micelle or membrane structure, encapsulating and immobilizing antigen molecules, and achieving stable sustained release.
This chitosan-based vaccine adjuvant has excellent sustained release and stability, which can effectively ensure the stability of the antigen during the release process, enhance the immune effect and avoid the toxic side effects of crosslinking agents.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of vaccine adjuvants, and particularly relates to a chitosan-based vaccine adjuvant and a preparation method thereof. Background Art
[0002] An adjuvant is a substance that can accelerate, prolong or enhance the antigen-specific immune response and jointly form a vaccine preparation with a vaccine. The adjuvant itself has no antigenicity, can enhance the antigen-specific immune response, can reduce the amount of antigen used when added to the vaccine, enhance the immune effect, and can determine the quality of the vaccine to a certain extent.
[0003] Chitosan is a natural biodegradable polymer, and studies have shown that it can stimulate the acquired immune response. Chitosan has also been reported to enhance Th1 immunity, thus becoming a potential adjuvant for live poultry vaccines. However, chitosan has low solubility in aqueous solutions above pH 6.5, is insoluble in organic solvents, does not melt when heated, and is directly carbonized at high temperatures, which greatly limits its application.
[0004] In order to obtain better solubility, hydrophilicity and potential biological activity, chitosan is usually modified by methods such as quaternization, acylation, carboxylation, and sulfation. However, when chitosan or its derivatives are directly used as adjuvants, the duration is short and the vaccine immune effect is limited. Therefore, it is necessary to provide a chitosan-based vaccine adjuvant with a stable slow-release function and a preparation method thereof. Summary of the Invention
[0005] The purpose of the present invention is to provide a chitosan-based vaccine adjuvant and a preparation method thereof to solve the problem that when chitosan or its derivatives are directly used as adjuvants in the background art, the duration is short and the vaccine immune effect is limited.
[0006] The purpose of the present invention can be achieved by the following technical solutions:
[0007] A chitosan-based vaccine adjuvant, comprising the following raw materials in parts by weight:
[0008] 6.25 - 10 parts of a negatively charged chitosan derivative and 1 - 4 parts of a positively charged chitosan derivative;
[0009] Among them, the positively charged chitosan derivative is a chitosan quaternization derivative, and the synthesis steps are as follows:
[0010] Add chitosan and a quaternary ammonium salt containing an epoxy group to distilled water, and stir at 70 - 80 °C for 24 h; after the obtained reaction solution is dialyzed with distilled water in a dialysis bag for 72 h, it is freeze-dried at -80 °C in a freeze-dryer to obtain the chitosan quaternization derivative.
[0011] Furthermore, the molecular weight of the chitosan is 30 - 200 kDa; the mass ratio of chitosan to the quaternary ammonium salt containing epoxy groups is 1:2.
[0012] Furthermore, for the stirring, the stirring speed is 200 - 400 r / min.
[0013] Furthermore, the preparation steps of the quaternary ammonium salt containing epoxy groups are as follows:
[0014] Step (1): Add dodecyl bromide and N-methyl-2-hydroxyethylamine into absolute ethanol, stir at 100 °C for 12 h. After the reaction is completed, remove ethanol by rotary evaporation under reduced pressure to obtain a crude product. The obtained crude product is recrystallized with acetone, and then washed successively with sodium hydroxide aqueous solution and water to obtain a solid, which is dried in vacuum for standby.
[0015] Step (2): Add the solid prepared in step (1) into absolute ethanol, heat to 60 °C, stir, take epichlorohydrin in a constant pressure dropping funnel, and titrate completely within 30 min. After the reaction is completed, remove absolute ethanol by distillation under reduced pressure, perform recrystallization with ethyl acetate, and vacuum dry at 45 °C for 12 h after filtration, thus obtaining the quaternary ammonium salt containing epoxy groups.
[0016] Among them, the molar ratio of dodecyl bromide to N-methyl-2-hydroxyethylamine in step (1) is 1 - 1.2:1.
[0017] Among them, the mass fraction of the sodium hydroxide aqueous solution in step (1) is 5%.
[0018] Among them, the molar ratio of the solid to 1,4-dibromobutane in step (2) is 2 - 2.1:1; the dosage ratio of the solid, absolute ethanol, epichlorohydrin and ethyl acetate is 69.3 g:60 - 80 ml:27.7 - 33.3 g:50 - 70 ml.
[0019] Among them, the synthesis steps of the quaternary ammonium salt containing epoxy groups in step (2) are as follows:
[0020]
[0021] Furthermore, the chitosan negatively charged derivative is 0-position substituted carboxymethyl chitosan, which is specifically synthesized by the following steps:
[0022] Mix water and isopropanol to form a mixed solution a. Take the chitosan and sodium hydroxide and add them into the mixed solution a. Under the condition of 50 °C, perform alkaline reaction with water bath stirring for 1 h to form a reaction solution;
[0023] Dissolve chloroacetic acid in isopropanol solution to form mixture b. Drop mixture b into the reaction solution within 30 min and react for 4 h. Add aqueous ethanol solution to terminate the reaction. Dialyze and freeze-dry to obtain 0-position substituted carboxymethyl chitosan.
[0024] Among them, the volume ratio of water to isopropanol in the mixed solution a is 1:4.
[0025] Among them, the molecular weight of chitosan is 200 kDa; the mass ratio of chitosan to sodium hydroxide is 10:13.5.
[0026] Among them, the dosage ratio of isopropanol to chloroacetic acid is 4 ml:3 g.
[0027] Among them, the volume ratio of the aqueous ethanol solution is 70%, and the dosage is 200 ml.
[0028] A preparation method of a chitosan-based vaccine adjuvant includes the following steps:
[0029] Mix the chitosan negatively charged derivative with water to obtain solution a, mix the chitosan positively charged derivative with water to obtain solution b. Mix solution a and solution b at room temperature and stir magnetically for 20 - 40 min to obtain the chitosan-based vaccine adjuvant.
[0030] Among them, the concentration of the solution a is 1 - 2 mg / ml; the concentration of the solution b is 1 - 2 mg / ml.
[0031] Among them, the chitosan-based vaccine adjuvant is stored at 4°C.
[0032] Advantages of the present invention:
[0033] By simultaneously introducing hydrophilic groups and hydrophobic groups and combining the positively charged property of the quaternary ammonium salt, the chitosan-based vaccine adjuvant of the present invention wraps around the antigen and forms a protective micelle and membrane structure, ensuring the stability during the antigen release process. It has the characteristics of safety and stable slow release.
[0034] The chitosan-based vaccine adjuvant prepared by the present invention has excellent slow release property. The reason is that the chitosan-based vaccine adjuvant prepared by the present invention contains both hydrophilic hydroxyl groups and hydrophobic alkyl long chains. This balance of hydrophilicity and hydrophobicity enables the chitosan-based vaccine adjuvant to form a stable micelle or membrane structure in aqueous solution, thereby effectively wrapping and fixing antigen molecules. Coupled with the stability and positive charge property of the quaternary ammonium salt itself, the chitosan-based vaccine adjuvant can ensure the stability of the antigen during the release process, thus achieving the effect of stable slow release.
[0035] A chitosan-based vaccine adjuvant prepared by the present invention has excellent stability. The reason is that the present invention uses a positively charged quaternary ammonium salt to prepare a chitosan quaternized derivative as the chitosan-based vaccine adjuvant. The positive charge enables the quaternary ammonium salt to generate electrostatic attraction with negatively charged antigen molecules, thereby enhancing the binding force between them. Moreover, the chemical structure of the quaternary ammonium salt is relatively stable and is not easily destroyed by enzymatic or hydrolytic actions in the organism. Therefore, the chitosan quaternized derivative can maintain the integrity of its structure and function for a long time, thereby ensuring the stable binding and release of antigen molecules.
[0036] A chitosan-based vaccine adjuvant prepared by the present invention has no toxic and side effects of cross-linking agents. The reason is that the chitosan-based vaccine adjuvant is synthesized by electrostatic adsorption without the use of any cross-linking agents, eliminating the toxic and side effects caused by the use of cross-linking agents. Specific embodiments
[0037] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. 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 belong to the scope of protection of the present invention.
[0038] The chitosan used in the present invention was purchased from Shanghai Macklin Biochemical Co., Ltd.
[0039] Example 1
[0040] This example provides a chitosan quaternized derivative, which is prepared by the following steps:
[0041] 0.01 mol of dodecyl bromide and 0.01 mol of N-methyl-2-hydroxyethylamine were added to 50 ml of anhydrous ethanol, and stirred at 100 °C for 12 h. After the reaction was completed, ethanol was removed by rotary evaporation under reduced pressure to obtain a crude product. The obtained crude product was recrystallized with acetone and then washed successively with a 5% sodium hydroxide solution and water to obtain a solid, which was dried in vacuo for later use.
[0042] 69.3 g of the above solid was added to 60 mL of anhydrous ethanol, heated to 60 °C, and stirred. 27.7 g of epichlorohydrin was weighed into a constant pressure dropping funnel and titrated completely within 30 min. After the reaction was completed, anhydrous ethanol was removed by distillation under reduced pressure, recrystallized with 50 ml of ethyl acetate, and vacuum dried at 45 °C for 12 h after filtration, to obtain a quaternary ammonium salt containing epoxy groups.
[0043] 5 g of chitosan with a molecular weight of 30 kDa and 10 g of quaternary ammonium salt containing epoxy groups were added to 70 mL of distilled water, and stirred in a water bath at 70 °C with a rotation speed of 200 r / min for 24 h. The resulting reaction solution was dialyzed against distilled water in a dialysis bag (spectrum labs 6 series dialysis bag with a molecular weight cut-off of 2000 and a flat width of 38 mm) for 72 h, and then freeze-dried at -80 °C in a freeze dryer to obtain the quaternized chitosan derivative.
[0044] Example 2
[0045] This example provides a quaternized chitosan derivative, which is prepared by the following steps:
[0046] 0.011 mol of dodecyl bromide and 0.010 mol of N-methyl-2-hydroxyethylamine were added to 55 mL of absolute ethanol, and stirred at 100 °C for 12 h. After the reaction was completed, ethanol was removed by rotary evaporation under reduced pressure to obtain a crude product. The obtained crude product was recrystallized with acetone and then washed successively with a 5% sodium hydroxide solution and water to obtain a solid, which was dried in vacuo for standby.
[0047] 69.3 g of the solid was added to 75 mL of absolute ethanol, heated to 60 °C and stirred. 30.53 g of epichlorohydrin was weighed into a constant pressure dropping funnel and titrated completely within 30 min. After the reaction was completed, absolute ethanol was removed by distillation under reduced pressure, recrystallized with 60 mL of ethyl acetate, and vacuum dried at 45 °C for 12 h after filtration to obtain the quaternary ammonium salt containing epoxy groups.
[0048] 6 g of chitosan with a molecular weight of 100 kDa and 12 g of quaternary ammonium salt containing epoxy groups were added to 80 mL of distilled water, and stirred in a water bath at 75 °C with a rotation speed of 200 r / min for 24 h. The resulting reaction solution was dialyzed against distilled water in a dialysis bag (spectrum labs 6 series dialysis bag with a molecular weight cut-off of 2000 and a flat width of 38 mm) for 72 h, and then freeze-dried at -80 °C in a freeze dryer to obtain the quaternized chitosan derivative.
[0049] Example 3
[0050] This example provides a quaternized chitosan derivative, which is prepared by the following steps:
[0051] 0.012 mol of dodecyl bromide and 0.010 mol of N-methyl-2-hydroxyethylamine were added to 60 mL of absolute ethanol, and stirred at 100 °C for 12 h. After the reaction was completed, ethanol was removed by rotary evaporation under reduced pressure to obtain a crude product. The obtained crude product was recrystallized with acetone and then washed successively with a 5% sodium hydroxide solution and water to obtain a solid, which was dried in vacuo for standby.
[0052] 69.3 g of solid was added to 80 mL of absolute ethanol, heated to 60 °C, stirred, and 33.30 g of epichlorohydrin was weighed into a constant-pressure dropping funnel and titrated completely within 30 min. After the reaction, absolute ethanol was removed by vacuum distillation, recrystallized with 70 mL of ethyl acetate, filtered by suction, and vacuum dried at 45 °C for 12 h to obtain a quaternary ammonium salt containing epoxy groups.
[0053] 7 g of chitosan with a molecular weight of 200 kDa and 14 g of the quaternary ammonium salt containing epoxy groups were added to 90 mL of distilled water, and stirred under a water bath condition at 80 °C with a rotation speed of 200 r / min for 24 h. The obtained reaction solution was dialyzed in a dialysis bag (spectrum l abs 6 series dialysis bag with a molecular weight of 2000 and a flat width of 38 mm) using distilled water for 72 h, and then freeze-dried at -80 °C in a freeze dryer to obtain a chitosan quaternized derivative.
[0054] Example 4
[0055] This example provides a preparation method of a chitosan-based vaccine adjuvant, including the following steps:
[0056] Prepare a mixed solution a of water and isopropanol with a volume ratio of 1:4. Take 10 g of chitosan with a molecular weight of 200 kDa and 13.5 g of sodium hydroxide and add them to the mixed solution a. Under the condition of 50 °C, carry out an alkalization reaction with water bath stirring for 1 h to form a reaction solution. Dissolve 15 g of chloroacetic acid in 20 mL of isopropanol solution to form a mixed solution b. Drop the mixed solution b into the reaction solution within 30 min and react for 4 h; add 200 mL of an ethanol aqueous solution with a volume ratio of 70% to terminate the reaction. Dialyze (the dialysis bag is a spectrum l abs 6 series dialysis bag with a molecular weight of 2000 and a flat width of 38 mm), and freeze-dry to obtain 0-position substituted carboxymethyl chitosan.
[0057] Mix 0-position substituted carboxymethyl chitosan and water to obtain an aqueous solution a with a concentration of 1 mg / mL. Mix the chitosan quaternized derivative prepared in Example 1 and water to obtain a solution b with a concentration of 1 mg / mL. Mix 6.25 mL of the aqueous solution a and 1 mL of the aqueous solution b at room temperature, and stir magnetically for 20 min to obtain a chitosan-based vaccine adjuvant, which is stored at 4 °C for standby.
[0058] Example 5
[0059] This example provides a preparation method of a chitosan-based vaccine adjuvant, including the following steps:
[0060] Prepare a mixture a of water and isopropanol with a volume ratio of 1:4. Take 10 g of chitosan with a molecular weight of 200 kDa and 13.5 g of sodium hydroxide and add them to the mixture a. Under the condition of 50 °C, carry out an alkalization reaction with water bath stirring for 1 h to form a reaction solution. Dissolve 15 g of chloroacetic acid in 20 ml of isopropanol solution to form a mixture b. Drop the mixture b into the reaction solution within 30 min and react for 4 h; add 200 mL of an ethanol aqueous solution with a volume ratio of 70% to terminate the reaction. Dialyze (the dialysis bag is a spectrum l abs 6 series dialysis bag with a molecular weight of 2000 and a flat width of 38 mm), and freeze-dry to obtain 0-position substituted carboxymethyl chitosan.
[0061] Mix 0-position substituted carboxymethyl chitosan and water to obtain a solution a with a concentration of 1.5 mg / ml. Mix the chitosan quaternary ammonium derivative prepared in Example 2 and water to obtain a solution b with a concentration of 1.5 mg / ml. Mix 8 ml of ligand aqueous solution a and 2.5 ml of ligand aqueous solution b at room temperature, and stir magnetically for 30 min to obtain a chitosan-based vaccine adjuvant, which is stored at 4 °C for standby.
[0062] Example 6
[0063] This example provides a preparation method of a chitosan-based vaccine adjuvant, including the following steps:
[0064] Prepare a mixture a of water and isopropanol with a volume ratio of 1:4. Take 10 g of chitosan with a molecular weight of 200 kDa and 13.5 g of sodium hydroxide and add them to the mixture a. Under the condition of 50 °C, carry out an alkalization reaction with water bath stirring for 1 h to form a reaction solution. Dissolve 15 g of chloroacetic acid in 20 ml of isopropanol solution to form a mixture b. Drop the mixture b into the reaction solution within 30 min and react for 4 h; add 200 mL of an ethanol aqueous solution with a volume ratio of 70% to terminate the reaction. Dialyze (the dialysis bag is a spectrum l abs 6 series dialysis bag with a molecular weight of 2000 and a flat width of 38 mm), and freeze-dry to obtain 0-position substituted carboxymethyl chitosan.
[0065] Mix 0-position substituted carboxymethyl chitosan and water to obtain a solution a with a concentration of 2 mg / ml. Mix the chitosan quaternary ammonium derivative prepared in Example 3 and water to obtain a solution b with a concentration of 2 mg / ml. Mix 10 ml of ligand aqueous solution a and 4 ml of ligand aqueous solution b at room temperature, and stir magnetically for 40 min to obtain a chitosan-based vaccine adjuvant, which is stored at 4 °C for standby.
[0066] Comparative Example 1
[0067] This example provides a preparation method of a chitosan-based vaccine adjuvant, including the following steps:
[0068] Compared with Example 5, chitosan with a molecular weight of 200 kDa was replaced with chitosan with a molecular weight of 20 kDa, and the remaining raw materials and preparation process were the same as those in Example 5.
[0069] Comparative Example 2
[0070] This example provides a method for preparing a chitosan-based vaccine adjuvant, which includes the following steps:
[0071] Compared with Example 5, chitosan with a molecular weight of 200 kDa was replaced with chitosan with a molecular weight of 250 kDa, and the remaining raw materials and preparation process were the same as those in Example 5.
[0072] Performance tests were carried out on the chitosan-based vaccine adjuvants prepared in Examples 4 - 6 and Comparative Examples 1 - 2.
[0073] The test method was to test the cumulative release rate of the immunized vaccine and the toxicity to dendritic cells after the chitosan-based vaccine adjuvant was combined with the antigen to form an immunized vaccine. Among them, the preparation method of the immunized vaccine was:
[0074] The chitosan vaccine adjuvants prepared in Examples 4 - 6 and Comparative Examples 1 - 2 were respectively mixed evenly with 0.63 g of the model antigen OVA at room temperature, and after magnetic stirring for 30 min, the immunized vaccine was obtained and stored at 4°C for later use.
[0075] Among them, the steps for testing the cumulative release rate of the immunized vaccine were as follows: (1) Establish a low-concentration BSA standard curve. (2) The specific measurement conditions were: maintaining the temperature at 37°C and the rotation speed at 60 r / min, sampling 30 μL at time points of 20 min, 40 min, 60 min, 80 min, 100 min, 120 min, and 150 min respectively, measuring the absorbance at 562 nm by the BCA method, calculating the change in the concentration of the polypeptide antigen, and calculating the cumulative release rate. The specific measurement method was: preparing an immunized vaccine adsorbed with the model antigen OVA, measuring 5 mL and placing it into a dialysis bag, fixing it on a glass rod, stirring evenly, and sampling regularly. The change in the concentration of the polypeptide antigen was measured by the BCA method, and the cumulative release rate was calculated.
[0076] Among them, the toxicity test of the immunized vaccine was to test and compare the toxicity to dendritic cells by the CCK-8 method; the concentration of the immunized vaccine was 100 μg / ml.
[0077] Among them, the chitosan-based vaccine adjuvant prepared in Example 5 was selected as the blank control group.
[0078] The test results are shown in Table 1:
[0079] Table 1
[0080]
[0081] As can be seen from Table 1, the in vitro release rate of the antigen of the immune vaccine prepared with chitosan molecular weight in the range of 30-200 kDa is higher than 89.9%, while the in vitro release rate of the antigen of the immune vaccine prepared outside the range of 30-200 kDa of chitosan molecular weight is relatively low, which is related to the hydrophilicity and swelling properties of chitosan with different molecular weights.
[0082] The immune vaccines prepared with chitosan molecular weight in the range of 30-200 kDa have no toxicity to cells at 100 μg / ml. From the blank control group, it can be seen that the chitosan-based vaccine adjuvant has no toxicity to cells at 100 μg / ml, and the dendritic cells maintain good activity. However, the immune vaccines prepared outside the range of 30-200 kDa of chitosan molecular weight show slight inhibition to cells at 100 μg / ml.
[0083] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.
[0084] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A chitosan-based vaccine adjuvant, characterized in that: It includes the following raw materials in parts by weight: 6.25-10 parts of negatively charged chitosan derivatives and 1-4 parts of positively charged chitosan derivatives; Among them, the chitosan positively charged derivative is a chitosan quaternary ammonium derivative, and the synthesis steps are as follows: Chitosan and epoxy-containing quaternary ammonium salt are added to distilled water and stirred at 70-80° C. for 24 hours. The obtained reaction solution is dialyzed with distilled water in a dialysis bag for 72 hours and then freeze-dried in a freeze dryer at -80° C. to obtain a quaternary ammonium derivative of chitosan.
2. A chitosan-based vaccine adjuvant according to claim 1, characterized in that: The preparation steps of the epoxy-containing quaternary ammonium salt are: Step (1): adding dodecane bromide and N-methyl-2-hydroxyethylamine to anhydrous ethanol, stirring at 100° C. for 12 h, and after the reaction is completed, removing the anhydrous ethanol by rotary evaporation under reduced pressure to obtain a crude product; the obtained crude product is recrystallized from acetone, and then washed with sodium hydroxide aqueous solution and water in sequence to obtain a solid, which is vacuum dried for later use; Step (2): adding the solid prepared in step (1) to anhydrous ethanol, heating to 60° C., stirring, taking epichlorohydrin into a constant pressure dropping funnel, and titrating completely within 30 minutes; after the reaction is completed, removing the anhydrous ethanol by vacuum distillation, recrystallizing with ethyl acetate, filtering, and vacuum drying at 45° C. for 12 hours to obtain a quaternary ammonium salt containing an epoxy group.
3. A chitosan-based vaccine adjuvant according to claim 1, characterized in that: The molecular weight of the chitosan is 30-200 kDa; the mass ratio of the chitosan to the epoxy-containing quaternary ammonium salt is 1:2; and the stirring speed is 200-400 r / min.
4. A chitosan-based vaccine adjuvant according to claim 2, characterized in that: The molar ratio of the dodecane bromide and N-methyl-2-hydroxyethylamine in step (1) is 1-1.2:1; and the mass fraction of the sodium hydroxide aqueous solution is 5%.
5. A chitosan-based vaccine adjuvant according to claim 2, characterized in that: The usage ratio of the solid, anhydrous ethanol, epichlorohydrin and ethyl acetate in step (2) is 69.3 g: 60-80 ml: 27.7-33.3 g: 50-70 ml.
6. A chitosan-based vaccine adjuvant according to claim 1, characterized in that: The chitosan negative charge derivative is 0-substituted carboxymethyl chitosan, which is specifically synthesized by the following steps: Mix water and isopropanol to form a mixed solution a, add chitosan and sodium hydroxide to the mixed solution a, and stir in a water bath at 50° C. for alkalization reaction for 1 hour to form a reaction solution; Dissolve chloroacetic acid in an isopropanol solution to form a mixed solution b, add the mixed solution b dropwise to the reaction solution within 30 minutes, and react for 4 hours; add an ethanol aqueous solution to terminate the reaction; dialyze, and freeze-dry to obtain 0-substituted carboxymethyl chitosan; Among them, the molecular weight of chitosan is 200kDa; the dosage ratio of mixed solution a, chitosan, sodium hydroxide, isopropanol, chloroacetic acid and ethanol aqueous solution is 100ml:10g:13.5g:20ml:15g:200ml, the volume ratio of water and isopropanol in mixed solution a is 1:4, and the volume ratio of ethanol aqueous solution is 70%.
7. A method for preparing a chitosan-based vaccine adjuvant as claimed in any one of claims 1 to 6, characterized in that: The following steps are involved: The negatively charged derivative of chitosan and water are mixed to obtain solution a, and the positively charged derivative of chitosan and water are mixed to obtain solution b. Solution a and solution b are mixed at room temperature and magnetically stirred for 20 to 40 minutes to obtain a chitosan-based vaccine adjuvant.
8. The method for preparing a chitosan-based vaccine adjuvant according to claim 7, characterized in that: The concentration of solution a is 1-2 mg / ml; the concentration of solution b is 1-2 mg / ml.
9. The method for preparing a chitosan-based vaccine adjuvant according to claim 7, characterized in that: The chitosan-based vaccine adjuvant was stored at 4°C.