Application of nostoc sphaeroides polysaccharide as immunologic adjuvant

Abstract

The invention provides application of nostoc sphaeroids kutz polysaccharide as an immunologic adjuvant. The nostoc sphaeroids kutz polysaccharide is prepared from galactose, glucose, xylose, mannose and glucuronic acid, can increase the expression quantity of macrophage surface molecules (CD80, CD86 and MHC-II) and the secretion level of cell factors (TNF-alpha and IL-6) in vitro, and can stimulate spleen cell proliferation and improve the activation proportion of B cells; the specific antibody level can be remarkably improved in vivo, the immune response of the body is enhanced, and continuous humoral immunity can be stimulated. The compound can be used for preparing an immunologic adjuvant of a vaccine and improving the immunogenicity of the vaccine.

Description

Technical Field

[0001] The invention relates to the field of immunological technology, and in particular to the application of scutellaria baicalensis polysaccharide as an immune adjuvant. Background Art

[0002] Vaccination is an effective measure to prevent and control infectious diseases. Vaccines are composed of antigens and adjuvants. Antigens are composed of proteins or carbohydrates from pathogens and are used to stimulate adaptive immune responses. Adjuvants are substances added to vaccines that can non-specifically enhance the intensity and persistence of adaptive immune responses and play a key role in vaccine development. According to the mechanism of action, adjuvants are roughly divided into three categories: delivery vehicles, immune agonists, and composite adjuvants. Delivery vehicle adjuvants can effectively present vaccine antigens, thereby enhancing antigen-specific immune responses, such as aluminum adjuvants, MF59, emulsions, liposomes, etc.; immune agonists enhance the body's immune response to antigens by acting intrinsically on the immune system, such as CpG oligodeoxynucleotides (CpG ODN).

[0003] Aluminum adjuvant is the most widely used vaccine adjuvant on the market. It mainly captures antigens at the injection site by forming macromolecular aggregates, promotes the uptake of antigen-presenting cells and promotes the slow release of antigens, thereby prolonging the action time of the antigen. Aluminum adjuvants also have some shortcomings and side effects. They can cause allergic reactions in the body and are ineffective against seasonal and pandemic influenza vaccines. Adjuvants such as MF59 and AS03 have been used in commercial vaccines. However, these adjuvants usually require booster doses to obtain a more sustained immune response, and their mechanism of action has not yet been clarified and has not been widely used. Based on the above problems, it is of great significance to develop new adjuvants that are efficient, safe, and can continuously stimulate humoral immunity. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the above-mentioned technologies to a certain extent at least, namely to provide a Nostoc sphaeroids kützpolysaccharide (NSKP) immune adjuvant which is low in toxicity, safe and can effectively enhance the body's immune response.

[0005] To this end, in the first aspect of the present invention, the present invention proposes the use of kudzu vine polysaccharide in the preparation of immune adjuvants. The kudzu vine polysaccharide is obtained by crushing the kudzu vine and extracting it by water extraction and alcohol precipitation.

[0006] According to the application of the present invention, the polysaccharide extracted from the kudzu vine is composed of galactose, glucose, xylose, mannose and glucuronic acid, which can increase the expression of macrophage surface molecules (CD80, CD86, MHC-II) and the secretion level of cytokines (TNF-α, IL-6) in vitro, and can stimulate spleen cell proliferation and increase the proportion of B cell activation; it can significantly increase the level of specific antibodies in vivo, enhance the body's immune response, and stimulate sustained humoral immunity. It can be used to prepare immune adjuvants for vaccines and improve the immunogenicity of vaccines.

[0007] Optionally, the kudzu vine root is crushed, mixed with water at a solid-liquid ratio of 1:10g / mL-1:80g / mL, water bathed at 75℃-95℃ for 4h, centrifuged to obtain the supernatant, 3-6 times the volume of 95% ethanol is added for alcohol precipitation, the precipitate is redissolved and freeze-dried to obtain the kudzu vine root polysaccharide.

[0008] Further, the kudzu vine root is crushed, mixed with water at a solid-liquid ratio of 1:40 g / mL, and placed in a water bath at 95° C. for 4 hours. The supernatant is centrifuged and precipitated with 4 times the volume of 95% ethanol. The precipitate is redissolved and freeze-dried to obtain the kudzu vine root polysaccharide.

[0009] Optionally, the monosaccharides of the Herba Puerariae polysaccharides consist of galactose, glucose, xylose, mannose and glucuronic acid.

[0010] Furthermore, the molar ratio of the galactose, the glucose, the xylose, the mannose and the glucuronic acid is 1.04:2.16:1.01:0.92:0.47.

[0011] In the second aspect of the present invention, an immune adjuvant is provided, which includes kudzu vine polysaccharide. The kudzu vine polysaccharide can promote the phagocytosis of macrophages (the expression ratios of CD80, CD86 and MHC-II are increased by 46% to 58%, 37% to 50% and 16% to 22%, respectively; the release ratios of cytokines TNF-α and IL-6 are increased by 405% to 441% and 73% to 84%), increase the activation ratio of B cells by 52% to 171%, and enhance the immunogenicity of antigens; increase the level of specific antibodies in the body (the levels of IgG, IgG1 and IgG2a are increased by 136% to 177%, 56% to 104% and 269% to 390%, respectively), and continuously stimulate humoral immunity.

[0012] In the third aspect of the present invention, there is provided use of the above-mentioned immune adjuvant in preparing a vaccine.

[0013] In the fourth aspect of the present invention, a vaccine is provided, comprising an immune adjuvant and an immunogen, wherein the immune adjuvant is the immune adjuvant mentioned above.

[0014] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The microstructure of NSKP at different magnifications;

[0016] Figure 2 Effects of NSKP on the expression levels of macrophage-related surface molecules and cytokine secretion levels; (A) Expression of CD80; (B) Expression of CD86; (C) Expression of MHC-II; (D) Secretion level of TNF-α; (E) Secretion level of IL-6;

[0017] Figure 3 Effects of NSKP on spleen cell proliferation and B cell activation; (A) spleen cell proliferation index; (B) B cell clustering; (C) B cell activation ratio;

[0018] Figure 4 The adjuvant activity of NSKP in vivo; (A) Experimental scheme of mice; (B) Specific antibody level in serum after the third immunization; (C) Specific antibody level in serum 7 days after the third immunization;

[0019] Figure 2-Figure 4 All experiments were repeated three times, and the data were statistically analyzed using GraphPad 9.5 software. Significant differences were analyzed using conventional one-way analysis of variance; p < 0.05 indicated a significant difference, indicated by "*"; p < 0.01 indicated an extremely significant difference, indicated by "**"; p < 0.001 indicated an extremely significant difference, indicated by "***"; p < 0.0001 indicated an extremely significant difference, indicated by "****". DETAILED DESCRIPTION

[0020] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0021] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0022] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0023] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0024] Example 1 Preparation and characterization of kudzu vine polysaccharide

[0025] After the sphaeroids kütz is crushed, it is mixed with water at a solid-liquid ratio of 1:40 g / mL, and the mixture is placed in a water bath at 95°C for 4 hours. The supernatant is taken and added with 4 times the volume of 95% ethanol for alcohol precipitation to precipitate sphaeroids kütz polysaccharide. The precipitate is taken out by centrifugation and redissolved. After freezing at -80°C for 2 hours, vacuum freeze-drying (temperature -70°C, vacuum degree 10-40 Pa, time 24-48 hours) is performed to obtain sphaeroids kütz polysaccharide (NSKP).

[0026] The chemical composition of NSKP was determined by anthrone sulfuric acid method, 3,5-dinitrosalicylic acid method and barium chloride-gelatin method. As shown in Table 1, the total sugar content of Pueraria polysaccharide was 84.95±0.59%, indicating that the purity of the extracted NSKP was high; reducing sugar and sulfate were 5.51±0.11% and 2.58±8.90%, respectively. The monosaccharide composition of NSKP was further analyzed. As shown in Table 2, NSKP was composed of galactose, glucose, xylose, mannose and glucuronic acid, with a molar ratio of 1.04∶2.16∶1.01∶0.92∶0.47. Figure 1 This is a scanning electron microscope image of NSKP. The results show that the microstructure of Pueraria lobata polysaccharide at different magnifications is a lamellar structure with a smooth and dense surface.

[0027] Table 1 Chemical composition of kudzu vine polysaccharide

[0028]

[0029] Table 2 Monosaccharide composition of kudzu vine polysaccharide

[0030]

[0031] Example 2 Effect of Puerariae Polysaccharide on Macrophage Activation

[0032] Macrophages are often used to evaluate immunomodulatory activity. C57BL / 6 male mice were intraperitoneally injected with 1 mL of 6% starch broth for three consecutive days. On the fourth day, the mouse peritoneal cavity was lavaged with PBS, and peritoneal macrophages were extracted from the mice for experiments. The cells were cultured at 3×10 5 100 μL / well were plated in a 24-well plate (300 μL / well). In addition, 200 μg / mL NSKP and 400 ng / mL LPS were added to the sample group and LPS group, respectively, and the same volume of PBS was added to the PBS group. Three parallel controls were set up. The cytokine release level was detected after 24 hours of culture, and the surface molecule expression was detected after 48 hours of culture. The flow cytometry analysis results are shown in Figure 2. Figure 2 As shown in the figure, compared with the PBS group, the LPS group and the NSKP group significantly increased the expression of macrophage surface molecules CD80, CD86, and MHC-II, and increased the levels of cytokines TNF-α and IL-6. The above results indicate that NSKP can stimulate the polarization of macrophage M1, increase the secretion level of cytokines, and has immunomodulatory activity.

[0033] Example 3 Effect of Puerariae Polysaccharide on Spleen Cell Proliferation

[0034] ConA can specifically stimulate T cell proliferation, while LPS can stimulate B cell proliferation. 2×10 spleen cells were collected from C57BL / 6 male mice. 5 200 μL / well was plated in a 96-well plate, 5 μg / mL of concanavalin A (ConA) and 10 μg / mL of lipopolysaccharide (LPS) were added to stimulate spleen cells as positive controls, 100-800 μg / mL of kudzu vine polysaccharide was added to the sample group, and the same volume of PBS was added to the PBS group, and three parallel controls were set up. After 48 hours of culture, CCK-8 reagent was added and incubated at 37°C for 1-4 hours, and then the OD was detected using an enzyme reader. 450 The spleen cell proliferation index was calculated. Figure 3 As shown in A, compared with the PBS group, the 100-800 μg / mL concentration of kudzu vine polysaccharide can significantly stimulate the proliferation of spleen cells, and the proliferation index increases with the increase of concentration, which is dose-dependent. The effect of kudzu vine polysaccharide on B lymphocytes was further analyzed by flow cytometry. The results are shown in Figure 3 As shown in B, kudzu vine polysaccharide significantly increased the proportion of B lymphocytes in spleen cells. B lymphocytes can also act as antigen presenting cells to take up antigens and present them to T cells. Further analysis of the activation of B lymphocytes showed that kudzu vine polysaccharide significantly increased the expression of CD86 on the surface of B lymphocytes, indicating that kudzu vine polysaccharide can promote B cell activation ( Figure 3 C).

[0035] Example 4 Effect of Puerariae Polysaccharide on Humoral Immunity

[0036] like Figure 4 As shown in A, a mouse adjuvant model was constructed to explore the in vivo adjuvant activity of Pueraria polysaccharide. Nine BALB / c female mice were randomly divided into three groups, namely OVA group, Alum group, and NSKP group. The OVA group was immunized with 100 μL / mouse of 1 mg / mL OVA, the Alum group was immunized with 100 μL of Alum and 100 μL of 1 mg / mL OVA for each mouse, and the NSKP group was immunized with 100 μL of 5 mg / mL NSKP and 100 μL of 1 mg / mL OVA for each mouse. Each group was immunized three times on days 0, 7, and 14. The level of OVA-specific antibodies was detected by taking tail vein blood from mice one day after the third immunization, and the eyeball blood of mice was analyzed on the 21st day.

[0037] The specific antibody level in mouse serum was determined by ELISA. In a 96-well plate, each well was coated with 100 μL of OVA at a concentration of 100 μg / mL and placed at 4°C overnight. Use 5% skim milk and block at 37°C for 2 hours. Add the pre-diluted serum sample and incubate for 2.5 hours. Then add the secondary antibody (sheep anti-mouse IgG, IgG1 and IgG2a diluted at 1:5000) pre-diluted with 1% skim milk at a certain ratio and incubate at 37°C for 2 hours. Add TMB colorimetric solution and incubate at 37°C for 20 minutes. Continue to add 2 mol / L H2SO4 to each well, 50 μL / well, to terminate the reaction. Use an enzyme reader to detect and obtain the OD of each well. 450 . Figure 4 B is the specific antibody level in the tail vein blood after the third immunization. Compared with the OVA group, the sIgG, sIgG1, and sIgG2a specific antibody levels in the serum of mice in the NSKP group were significantly increased. The ratio of IgG2a to IgG1 in serum is usually used to reflect the ratio of Th2 to Th1 cells. The NSKP group was significantly higher than the other groups, indicating that the humoral immunity induced by the NSKP group was more biased towards Th1 immunity. The specific antibody level of the eyeball blood was further tested 7 days after the third immunization ( Figure 4 C) It was found that the mice in the kudzu vine polysaccharide group still maintained a high antibody level, proving that kudzu vine polysaccharide can stimulate sustained humoral immunity and has immune adjuvant activity.

[0038] In summary, according to the embodiments of the present invention, the present application extracts kudzu vine polysaccharide from kudzu vine dried product by water extraction and alcohol precipitation, and the kudzu vine polysaccharide can significantly stimulate macrophage activation and spleen cell proliferation in vitro, and can effectively enhance the level of specific antibodies in vivo, and has immune adjuvant activity. Therefore, the kudzu vine polysaccharide can be developed for immune adjuvants and vaccine preparations.

[0039] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0040] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. The application of Puerariae polysaccharide as an immune adjuvant, characterized in that: The kudzu vine polysaccharide is obtained by crushing the kudzu vine and extracting it by water extraction and alcohol precipitation.

2. The use according to claim 1, characterized in that The kudzu vine root is crushed, mixed with water at a solid-liquid ratio of 1:10g / mL-1:80g / mL, and water-bathed at 75°C-95°C for 4-6h. The supernatant is centrifuged and 95% ethanol is added for alcohol precipitation. The precipitate is redissolved and freeze-dried to obtain the kudzu vine root polysaccharide, which is composed of galactose, glucose, xylose, mannose, and glucuronic acid in molar ratios of 1.04:2.16:1.01:0.92:0.47, respectively.

3. An immune adjuvant, characterized in that It includes kudzu vine polysaccharide, which can promote the phagocytosis of macrophages and enhance the immunogenicity of antigens; Increase the activation ratio of B cells, improve the level of specific antibodies in the body, and continuously stimulate humoral immunity.

4. Use of the immune adjuvant according to claim 3 in preparing vaccines.

5. A vaccine comprising an immune adjuvant and an immunogen, characterized in that: The immune adjuvant is the immune adjuvant according to claim 3.

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