Application of astragalus polysaccharide extracted by optimized alcohol precipitation process as H3N2 influenza vaccine adjuvant

Abstract

The invention relates to the technical field of biological medicine, and discloses an application of astragalus polysaccharide extracted by optimizing an alcohol precipitation process as an H3N2 influenza vaccine adjuvant, the application comprises the following steps: mixing the astragalus polysaccharide and an H3N2 influenza vaccine to prepare an immune preparation, extracting the astragalus polysaccharide by an ethanol precipitation method with the final concentration of 80%, the yield of the astragalus polysaccharide is 7.0-9.0% (based on the dry weight of raw materials), and the content of the astragalus polysaccharide in the H3N2 influenza vaccine adjuvant is 1-5%. In addition, the immune preparation can be used for remarkably improving the serum H3N2HI titer (Plt; 0.05) of the method. According to the application of the astragalus polysaccharide extracted by the optimized alcohol precipitation process as the H3N2 influenza vaccine adjuvant, the yield is 7.0-9.0% through the optimized alcohol precipitation process such as a precise extraction process and an ethanol precipitation method with the final concentration of 80%, and the prepared astragalus polysaccharide remarkably improves the serum H3N2HI titer (Plt; 0.05), the early-stage enhancement of the immune effect of the vaccine is assisted, the immune enhancement effect is synergistically exerted by scientifically matching the vaccine and combining 800 [mu] g of each animal with 15 [mu] g of H3N2 vaccine for each animal by determining the additive amount, and the effectiveness of matching of the adjuvant and the vaccine is guaranteed.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of astragalus polysaccharide extracted by an optimized alcohol precipitation process as an adjuvant for H3N2 influenza vaccine. Background Art

[0002] The H3N2 influenza vaccine is a vaccine used to prevent H3N2 subtype influenza virus infection. It is an inactivated vaccine. Influenza vaccines include trivalent inactivated vaccine (IIV3), trivalent live attenuated vaccine (LAIV3) and quadrivalent inactivated vaccine (IIV4). All contain H3N2 subtype influenza virus antigen components and can be used to prevent influenza A (H3N2) virus infection. The trivalent vaccine contains a lineage of influenza A (H1N1), H3N2 and type B viruses, while the quadrivalent vaccine adds a type B influenza virus to this. The trivalent inactivated influenza vaccine is suitable for people aged 6 months and above; the nasal spray trivalent live attenuated vaccine is suitable for people aged 3-17 years; and the quadrivalent inactivated vaccine is suitable for people aged 36 months and above.

[0003] In recent years, natural polysaccharide adjuvants have attracted widespread attention as vaccines due to their excellent immunomodulatory ability and low toxicity. Astragalus polysaccharide (APS) is an important active ingredient in Astragalus, a traditional Chinese medicine with immunomodulatory, blood sugar regulating, anti-aging, anti-tumor, antiviral, anti-fibrosis, antimicrobial, anti-radiation and lipid-lowering effects. As a dietary bioactive polysaccharide, APS is often supplemented in dietary administration to improve the growth performance and immune response of broilers and fish. So far, APS has shown potential as an effective adjuvant for a variety of vaccines, such as hepatitis B virus, foot-and-mouth disease virus, and Newcastle disease virus. Summary of the Invention

[0004] (1) Technical problems solved

[0005] In response to the shortcomings of the existing technology, the present invention provides an astragalus polysaccharide extracted by an optimized alcohol precipitation process for use as an adjuvant for H3N2 influenza vaccine. It has the advantages of efficient extraction, significantly improved immunogenicity, and high safety, and solves the problems of low yield and unclear adjuvant effect of the traditional alcohol precipitation process.

[0006] (2) Technical solution

[0007] In order to achieve the above-mentioned purposes of efficient extraction, significantly improved immunogenicity and high safety, the present invention provides the following technical solution: an application of astragalus polysaccharide extracted by an optimized alcohol precipitation process as an H3N2 influenza vaccine adjuvant, comprising mixing astragalus polysaccharide with H3N2 influenza vaccine to prepare an immune preparation, wherein the astragalus polysaccharide is extracted by 80% final concentration ethanol precipitation method, and its yield is 7.0-9.0% (based on the dry weight of the raw material), and the immune preparation significantly improves the serum H3N2HI titer during the initial immunization (P<0.05).

[0008] Furthermore, the added dose of the astragalus polysaccharide is 800 μg per animal, and is used in combination with the H3N2 vaccine (15 μg HA / animal).

[0009] Furthermore, the astragalus polysaccharide is prepared by a water extraction and alcohol precipitation process, which specifically includes raw material processing, water extraction, concentration, alcohol precipitation optimization and purification. The specific operations are as follows:

[0010] Raw material processing: Astragalus slices were crushed and passed through a 40-mesh sieve, degreased with 80% ethanol at 60°C for 2 h, and then dried.

[0011] Water extraction: Add purified water to the defatted powder at a solid-liquid ratio of 1:16 (g / mL), extract at 100°C for 2 h, and centrifuge to obtain the supernatant (repeat once and combine the filtrates);

[0012] Concentration: The filtrate is concentrated under reduced pressure to 1 / 4 of the original volume;

[0013] Alcohol precipitation optimization: add ethanol to the concentrate to a final concentration of 70%, and let it stand at 4°C for 24 hours; add ethanol to the concentrate to a final concentration of 75%, and let it stand at 4°C for 24 hours; add ethanol to the concentrate to a final concentration of 80%, and let it stand at 4°C for 24 hours;

[0014] Purification: The precipitate was collected, washed twice with anhydrous ethanol, redissolved in distilled water, dialyzed (3.5 kDa MWCO) for 24 h, and freeze-dried to obtain APS.

[0015] Furthermore, the vaccination method of the immune preparation is intramuscular injection, and the immunization program includes primary immunization (day 0) and booster immunization (day 21).

[0016] Furthermore, the effect of the astragalus polysaccharide as an adjuvant is better than that of the traditional aluminum adjuvant (aluminum hydroxide), especially inducing higher antibody levels in the primary immunization stage.

[0017] Furthermore, the immune enhancement effect of the astragalus polysaccharide was detected by micro-hemagglutination inhibition (HI) method, and its geometric mean titer (GMT) was significantly higher than that of the control group.

[0018] Furthermore, the astragalus polysaccharide is suitable for mammalian models, including but not limited to mice, rats and poultry.

[0019] Furthermore, the molecular weight of the astragalus polysaccharide is above 3.5 kDa and is used for adjuvant preparation after purification by dialysis.

[0020] (3) Beneficial effects

[0021] Compared with the prior art, the present invention provides an application of astragalus polysaccharide extracted by an optimized alcohol precipitation process as an adjuvant for H3N2 influenza vaccine, which has the following beneficial effects:

[0022] 1. The application of astragalus polysaccharide extracted by the optimized alcohol precipitation process as an adjuvant for H3N2 influenza vaccine. Through precise extraction technology and optimized alcohol precipitation technology such as 80% final concentration ethanol precipitation method, the yield is 7.0-9.0%. Based on the dry weight of the raw material, the prepared astragalus polysaccharide significantly increases the serum H3N2HI titer during the first immunization (P<0.05), helping to enhance the initial immune effect of the vaccine.

[0023] 2. The application of astragalus polysaccharide extracted by the optimized alcohol precipitation process as an adjuvant for H3N2 influenza vaccine is achieved by clearly adding 800μg per animal, combined with 15μgHA / animal H3N2 vaccine, scientifically adapting the vaccine to synergistically exert an immune-enhancing effect and ensure the effectiveness of the combination of adjuvant and vaccine.

[0024] 3. The application of astragalus polysaccharide extracted by the optimized alcohol precipitation process as an adjuvant for H3N2 influenza vaccine is achieved through the improvement of the preparation process, the water extraction and alcohol precipitation process, including raw material processing, water extraction, concentration, alcohol precipitation optimization and purification, etc., and strict control from raw materials to finished products to ensure the stable quality of astragalus polysaccharide, laying a solid foundation for the application of adjuvants.

[0025] 4. The application of astragalus polysaccharide extracted by the optimized alcohol precipitation process as an adjuvant for H3N2 influenza vaccine induced higher antibody levels in the primary immunization stage. The immune enhancement effect was detected by the micro-hemagglutination inhibition method, and the geometric mean titer was significantly higher than that of the control group, breaking through the limitations of traditional adjuvants and improving the immune efficacy of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the experimental evaluation of the adjuvant effect of the present invention;

[0027] Figure 2 This is a schematic diagram of the rank sum test results before booster immunization (21 days) of the present invention;

[0028] Figure 3 This is a schematic diagram of the rank sum test results after booster immunization (35 days) of the present invention;

[0029] Figure 4 This is a comparison chart of polysaccharide yields obtained by 70%, 75%, and 80% alcohol precipitation of the present invention;

[0030] Figure 5 It is a bar graph of serum H3N2HI titer of the present invention. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] See also Figures 1-5 , an application of astragalus polysaccharide extracted by an optimized alcohol precipitation process as an adjuvant for H3N2 influenza vaccine, comprising mixing astragalus polysaccharide with H3N2 influenza vaccine to prepare an immune preparation, wherein the astragalus polysaccharide is extracted by 80% final concentration ethanol precipitation method, with a yield of 7.0-9.0% (based on the dry weight of the raw material), and the immune preparation significantly increases the serum H3N2HI titer during the first immunization (P<0.05).

[0033] In the case implementation, the added dose of APS was 800 μg per animal, combined with H3N2 vaccine (15 μg HA / animal);

[0034] Among them, the additive dosage was determined through screening in a large number of animal immunization tests. In the early stage, different astragalus polysaccharide dosage gradients (such as 200μg / animal, 400μg / animal, 600μg / animal, 800μg / animal, and 1000μg / animal) were set, and a comparative study of immune effects was carried out in combination with H3N2 vaccine. The serum antibody levels, immune organ index, cellular immune response and other indicators of animals after immunization were monitored. It was found that when the astragalus polysaccharide was 800μg / animal combined with 15μg HA / animal H3N2 vaccine, the animal body could produce more efficient and longer-lasting immune responses in the primary and booster immunization stages. The antibody production speed was fast, the peak was high and the maintenance time was long. The development of immune organs and cellular immune activation were also more ideal. Therefore, this was determined to be the optimal additive dosage to ensure the maximum effect of the vaccine adjuvant, and to provide an accurate dosage reference for the subsequent optimization of the H3N2 influenza vaccine immune effect with the help of this astragalus polysaccharide in mammalian models (mice, rats, poultry, etc.) and practical applications.

[0035] In this case implementation, astragalus polysaccharide was prepared through a water extraction and alcohol precipitation process, which specifically included raw material processing, water extraction, concentration, alcohol precipitation optimization and purification. The specific operations are as follows:

[0036] Raw material processing: Astragalus slices are crushed and passed through a 40-mesh sieve, degreased in 80% ethanol at 60°C for 2 hours, and then dried. This step can effectively remove fat-soluble impurities in the Astragalus raw material to prevent them from affecting the subsequent polysaccharide extraction. The particle size of the 40-mesh sieve ensures sufficient contact between the raw material and the ethanol. The mild degreasing temperature of 60°C removes impurities while reducing the loss of active ingredients. Drying creates dry and stable material conditions for subsequent water extraction.

[0037] Water extraction: Add purified water to the defatted powder at a solid-liquid ratio of 1:16 (g / mL), extract at 100°C for 2 hours, centrifuge and collect the supernatant (repeat once, and combine the filtrates). A solid-liquid ratio of 1:16 has been proven in multiple experiments to fully dissolve astragalus polysaccharides. High-temperature extraction at 100°C can accelerate the release of polysaccharides from cells. A 2-hour extraction time balances extraction efficiency and component integrity. Repeated extraction and combined filtrates can further improve the polysaccharide extraction rate and maximize the recovery of astragalus polysaccharides in the raw material.

[0038] Concentration: The filtrate is concentrated to 1 / 4 of its original volume under reduced pressure. The reduced pressure environment lowers the boiling point of the solution and achieves concentration at a lower temperature to avoid high temperature destroying the structure and activity of astragalus polysaccharides. Concentration to 1 / 4 of the original volume provides a polysaccharide solution of appropriate concentration for the subsequent alcohol precipitation step, which is conducive to precipitation formation;

[0039] Alcohol precipitation optimization: add ethanol to the concentrate to a final concentration of 70%, and let it stand at 4°C for 24 hours; add ethanol to the concentrate to a final concentration of 75%, and let it stand at 4°C for 24 hours; add ethanol to the concentrate to a final concentration of 80%, and let it stand at 4°C for 24 hours. The ethanol concentration is increased step by step for alcohol precipitation. This is based on the difference in solubility of astragalus polysaccharides with different molecular weights and structures at different ethanol concentrations. 70% ethanol first precipitates some macromolecular impurities and polysaccharides for preliminary purification; 75% ethanol is used for further precipitation to remove more impurities; 80% ethanol is used to deeply precipitate the target astragalus polysaccharide, and it is left to stand at 4°C for 24 hours to allow the precipitate to fully form and stabilize. Through gradient alcohol precipitation, accurate classification of polysaccharides and effective removal of impurities are achieved, ensuring the purity of the final product.

[0040] Purification: The precipitate was collected, washed twice with anhydrous ethanol, reconstituted with distilled water, and dialyzed (3.5kDa MWCO) for 24 hours. APS was then freeze-dried to obtain the product. Washing with anhydrous ethanol removed residual ethanol and small molecule impurities on the surface of the precipitate. Reconstitution with distilled water restored the water solubility of the polysaccharide. The 3.5kDa MWCO dialysis bag retained the target APS, allowing small molecule impurities such as monosaccharides and inorganic salts to pass through the dialysis membrane and be removed. 24 hours of dialysis ensured that the impurities were fully removed. Freeze-drying was performed in a low-temperature vacuum environment to prevent denaturation of the polysaccharide due to high temperature. The final product, APS, was a stable and high-purity APS that met the quality requirements for use as an adjuvant for the H3N2 influenza vaccine.

[0041] Among them, the precise control of various process parameters, from raw material pretreatment to final purification, each step has been verified and confirmed through multiple groups of parallel experiments and comparative verification under different conditions to ensure that the prepared astragalus polysaccharide is suitable for use as an adjuvant in the H3N2 influenza vaccine in terms of molecular weight, purity, and activity, laying a reliable material foundation for the subsequent evaluation of its immune-enhancing effect, so that the polysaccharide can stably play a role in enhancing immune response and increasing antibody levels when used in combination with the vaccine.

[0042] In the case implementation, the vaccination method of the immune preparation is intramuscular injection, and the immunization program includes primary immunization (day 0) and booster immunization (day 21);

[0043] Among them, intramuscular injection is selected in the quadriceps femoris of the animal's hind leg. After disinfection with alcohol, the drug is accurately administered with a sterile syringe of appropriate specifications to ensure that the vaccination dose of each animal is uniform and consistent. The initial immunization is set to stimulate the body to produce a basic immune response. The booster immunization on the 21st day is based on the principle of immune memory, stimulating the body to quickly produce higher levels and more lasting antibodies and immune cells. By regularly collecting animal serum and detecting the dynamic changes of antibodies, it is verified that this immunization program can make the body's immune response to the H3N2 influenza vaccine highly efficient and long-lasting, providing a reasonable timeline for the synergistic effect of vaccine and adjuvant.

[0044] In case implementation, the effect of astragalus polysaccharide as an adjuvant is better than traditional aluminum adjuvant (aluminum hydroxide), especially inducing higher antibody levels in the primary immunization stage;

[0045] Among them, by constructing an animal group experiment with the same immune environment, one group used astragalus polysaccharide adjuvant combined with H3N2 vaccine, and the other group used aluminum hydroxide adjuvant combined with H3N2 vaccine. The variables such as vaccine dosage and vaccination method were strictly controlled. Serum was collected on the 7th, 14th and 21st days after the initial immunization, and antibody concentration was detected by ELISA and other methods. The results showed that the astragalus polysaccharide group showed a rapid upward trend in antibodies on the 7th day after the initial immunization, and the antibody level on the 14th day was significantly higher than that in the aluminum adjuvant group (P<0.05). The mechanism may be that astragalus polysaccharide can more efficiently activate the maturation of dendritic cells and promote T cell proliferation and differentiation, providing a better immune initiation signal for the rapid production of antibodies from the level of immune cell activation.

[0046] In the case implementation, the immune enhancement effect of Astragalus polysaccharide was detected by micro-hemagglutination inhibition (HI) method, and its geometric mean titer (GMT) was significantly higher than that of the control group;

[0047] Among them, the standard operating procedures of HI detection were strictly followed, standardized hemagglutination antigens, positive serum and other reagents were prepared, the sera of immunized animals were graded diluted and reacted with hemagglutination antigens, the antibody titer was determined by observing the hemagglutination phenomenon, and the geometric mean titer was calculated statistically. The control group was set as the group only vaccinated with H3N2 vaccine. The results showed that the GMT value of the astragalus polysaccharide adjuvant group was 2-3 titer units higher than that of the control group, and the GMT dispersion between individual animals in the group was small, indicating that astragalus polysaccharide can stably enhance the immune effect of the vaccine, allowing the animal body to produce a more uniform and efficient antibody response, and verifying the immune enhancement value of the adjuvant from the perspective of serological testing.

[0048] In case implementation, Astragalus polysaccharide is suitable for mammalian models, including but not limited to mice, rats and poultry;

[0049] Among them, in the mouse model, BALB / c mice were selected and verified through intraperitoneal inoculation and other methods, and it was found that it had a positive effect on the development of mouse immune organs (such as increased spleen index and thymus index) and specific antibody production; SD rats were selected as the rat model to simulate the immune response characteristics of large animals and evaluate the effect of astragalus polysaccharide adjuvant on the immune effect of H3N2 vaccine. The results were consistent with the mouse model; SPF chickens were used as an example in the poultry model. After intramuscular inoculation, respiratory mucosal immunity and humoral immunity indicators were monitored, proving that astragalus polysaccharide can adapt to the immune physiological characteristics of poultry, broadening the application scope of adjuvants in the field of veterinary vaccines and providing a variety of solutions for the selection of vaccine adjuvants in the prevention and control of different animal diseases.

[0050] In the case implementation, the molecular weight of astragalus polysaccharide is above 3.5 kDa and is used for adjuvant preparation after purification by dialysis;

[0051] Among them, gel permeation chromatography (GPC) and other methods are used to accurately determine the molecular weight. The molecular weight range above 3.5kDa is determined based on the study of the correlation between the structure of astragalus polysaccharides and immune activity. The polysaccharide molecules in this molecular weight range not only retain enough active groups to bind to the surface receptors of immune cells, but also have a suitable spatial structure to mediate immune signal transmission. During dialysis purification, a dialysis bag with a molecular weight cutoff (MWCO) of 3.5kDa can effectively retain the target polysaccharide and allow small molecule impurities (such as free monosaccharides and inorganic salts) to pass through. After dialysis for more than 24 hours, the purity of the polysaccharide is increased by more than 60%, which lays a material foundation for subsequent use as an adjuvant to ensure the stability and specificity of the immune enhancement effect, so that the prepared adjuvant can accurately activate the body's immune pathway with its suitable molecular weight and high purity when synergized with the H3N2 vaccine.

[0052] In summary, the application of astragalus polysaccharide extracted by the optimized alcohol precipitation process as an adjuvant for H3N2 influenza vaccine, through precise extraction process and optimized alcohol precipitation process such as 80% final concentration ethanol precipitation method, the yield is 7.0-9.0%, based on the dry weight of raw materials, the prepared astragalus polysaccharide significantly improves the serum H3N2HI titer (P<0.05) during the first immunization, helps to strengthen the initial immune effect of the vaccine, by clarifying the added dose, 800μg per animal, combined with 15μgHA / animal H3N2 vaccine, scientifically adapting the vaccine, synergistically exerting the immune enhancement effect, and ensuring the effectiveness of the combination of adjuvant and vaccine.

[0053] In addition, by improving the preparation process, the water extraction and alcohol precipitation process, including the steps of raw material processing, water extraction, concentration, alcohol precipitation optimization and purification, strict control is exercised from raw materials to finished products to ensure the stable quality of Astragalus polysaccharide, laying a solid foundation for the application of adjuvants, inducing higher antibody levels in the initial immunization stage, and the immune enhancement effect was detected by the micro-hemagglutination inhibition method. The geometric mean titer was significantly higher than that of the control group, breaking through the limitations of traditional adjuvants and improving the immune efficacy of vaccines.

[0054] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0055] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for using astragalus polysaccharide extracted by an optimized alcohol precipitation process as an adjuvant for H3N2 influenza vaccine, comprising mixing astragalus polysaccharide with H3N2 influenza vaccine to prepare an immune preparation, characterized in that: The astragalus polysaccharide is extracted by 80% final concentration ethanol precipitation method, and the yield is 7.0-9.0% (based on the dry weight of the raw material). The immune preparation significantly improves the serum H3N2HI titer during the initial immunization (P<0.05).

2. The use of the astragalus polysaccharide extracted by the optimized alcohol precipitation process according to claim 1 as an adjuvant for H3N2 influenza vaccine, characterized in that: The added dose of the astragalus polysaccharide was 800 μg per animal, and was used in combination with the H3N2 vaccine (15 μg HA per animal).

3. The use of the astragalus polysaccharide extracted by the optimized alcohol precipitation process according to claim 1 as an adjuvant for H3N2 influenza vaccine, characterized in that: The astragalus polysaccharide is prepared by a water extraction and alcohol precipitation process, which specifically includes raw material processing, water extraction, concentration, alcohol precipitation optimization and purification. The specific operations are as follows: Raw material processing: Astragalus slices were crushed and passed through a 40-mesh sieve, degreased with 80% ethanol at 60°C for 2 h, and then dried. Water extraction: Add purified water to the defatted powder at a solid-liquid ratio of 1:16 (g / mL), extract at 100°C for 2 h, and centrifuge to obtain the supernatant (repeat once and combine the filtrates); Concentration: The filtrate is concentrated under reduced pressure to 1 / 4 of the original volume; Alcohol precipitation optimization: add ethanol to the concentrate to a final concentration of 70%, and let it stand at 4°C for 24 hours; add ethanol to the concentrate to a final concentration of 75%, and let it stand at 4°C for 24 hours; add ethanol to the concentrate to a final concentration of 80%, and let it stand at 4°C for 24 hours; Purification: The precipitate was collected, washed twice with anhydrous ethanol, redissolved in distilled water, dialyzed (3.5 kDa MWCO) for 24 h, and freeze-dried to obtain APS.

4. The use of the astragalus polysaccharide extracted by the optimized alcohol precipitation process according to claim 1 as an adjuvant for H3N2 influenza vaccine, characterized in that: The vaccination mode of the immune preparation is intramuscular injection, and the immunization program includes primary immunization (day 0) and booster immunization (day 21).

5. The use of the astragalus polysaccharide extracted by the optimized alcohol precipitation process according to claim 1 as an adjuvant for H3N2 influenza vaccine, characterized in that: The effect of the astragalus polysaccharide as an adjuvant is better than that of the traditional aluminum adjuvant (aluminum hydroxide), and it can induce a higher antibody level especially in the primary immunization stage.

6. The use of the astragalus polysaccharide extracted by the optimized alcohol precipitation process according to claim 1 as an adjuvant for H3N2 influenza vaccine, characterized in that: The immune enhancement effect of the astragalus polysaccharide was detected by micro-hemagglutination inhibition (HI) method, and the geometric mean titer (GMT) thereof was significantly higher than that of the control group.

7. The use of the astragalus polysaccharide extracted by the optimized alcohol precipitation process according to claim 1 as an adjuvant for H3N2 influenza vaccine, characterized in that: The astragalus polysaccharide is suitable for mammalian models, including but not limited to mice, rats and poultry.

8. The use of the astragalus polysaccharide extracted by the optimized alcohol precipitation process according to claim 1 as an adjuvant for H3N2 influenza vaccine, characterized in that: The molecular weight of the astragalus polysaccharide is above 3.5 kDa and is used for adjuvant preparation after purification by dialysis.

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