Fungal galactan with immune adjuvant activity and its preparation method and application

Abstract

The invention discloses a galactan with immune adjuvant activity and a preparation method and application thereof. The invention uses chanterelle fruiting bodies as raw materials, extracts, purifies and refines to prepare water-soluble galactan with immune adjuvant function. The galactan involved in the invention can be developed into a medicine or health food with immune regulation effect.

Description

Technical Field

[0001] The invention belongs to the field of biochemical pharmacy, and specifically relates to fungal galactan with immune adjuvant activity and a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy is to fight tumors by regulating the body's immune defense mechanism. Due to its advantages of good targeting, few side effects, and safety, it has become the fourth type of anti-tumor therapy after surgery, chemotherapy, and radiotherapy, and has become a hot topic of global research. Vaccines are active immunotherapy in tumor immunotherapy, which can enable the body to produce a memory effect against tumor antigens and prevent tumor recurrence. Adjuvants are usually defined as entities added to vaccine formulations to increase the immunogenicity of antigens. They promote the formation of antigen pools, recruit immune cells at the injection site, activate the pattern recognition receptors of recruited antigen-presenting cells before or during antigen endocytosis, and then process and present antigens to T cells, resulting in cellular and / or humoral immune responses. Therefore, strengthening the research on adjuvants will surely promote the clinical application of vaccines and accelerate the research process of tumor immunotherapy.

[0003] Biomira combines the natural STn antigen with the carrier keyhole limpet hemocyanin (KLH) to construct the vaccine STn-KLH for the prevention of colorectal cancer and breast cancer metastasis However, the results of the Phase III clinical trial showed that failed to reduce the time to disease progression and increase overall survival. When combined with hormone therapy, the clinical efficacy is not obvious. One possible reason is that STn has insufficient immunogenicity and cannot produce a satisfactory immune response in the body. Therefore, it is crucial to find a vaccine adjuvant with strong immune adjuvant effect, safety and no side effects for the development and application of tumor vaccines.

[0004] The adjuvants that have been approved for use in humans include aluminum salt adjuvants, MF59, AS03, AS04, and Montanide ISA51. The most widely used is aluminum salt adjuvant, but it mainly promotes the body to produce Th2 type immune response, and long-term deposition in the body can induce local allergic reactions in the body; other emulsion adjuvants can induce the body to produce cellular immune responses, but they are toxic to the body to varying degrees, and cannot induce sufficient protective immune responses in antigens with weak immunogenicity. The above adjuvants are mainly used in vaccines such as hepatitis virus, influenza virus, and pneumococcus. Montanide ISA51, as an anti-tumor vaccine adjuvant, has been registered in Cuba in recent years for adjuvant vaccine treatment of non-small cell lung cancer. Although it can improve the immunogenicity of the vaccine, it still has varying degrees of local or systemic reactivity. Safety and immunomodulatory activity are the main obstacles in the development of adjuvants. Therefore, the development of safe and immunomodulatory adjuvants is the core task of future adjuvant research and development.

[0005] Polysaccharides have anti-tumor, immunomodulatory, antioxidant and anti-inflammatory activities. They have the advantages of low toxicity, fast degradation in the body, high biocompatibility, strong immunostimulatory activity and low reactogenicity. In particular, plant polysaccharides and edible fungal polysaccharides have the advantages of wide sources and low costs, making them one of the most promising candidate molecules for vaccine adjuvants. At present, the most studied are delta-inulin (AdvaxTM) from Asteraceae plants, glucan from edible fungi, arabinogalactan (ResistAidTM) from larch, and heteropolysaccharides or crude polysaccharides from Chinese herbal medicines, which are widely used as adjuvants in clinical trials or preclinical studies of anti-tumor, antiviral and antibacterial activities. Polysaccharides can play their adjuvant function by specifically binding to PRRs on the surface of antigen-presenting cells, promoting antigen presentation and upregulating costimulatory molecules on the cell surface, or activating complement, chemokines and activating inflammatory signals. Polysaccharides regulate immune activity with the characteristics of multiple pathways and multiple targets, which means that even in individuals who are genetically deficient in specific immune receptors or pathways, they are superior to compounds that rely only on single receptors. The research and development of polysaccharides as adjuvants has broad application prospects.

[0006] The genus Chanterelle belongs to the kingdom Fungi, phylum Basidiomycetes, class Homobasidiomycetes, order Aphyllae, family Cantharellaceae, and genus Chanterelle. It is named Chanterelle because its cap is shaped like a trumpet and is apricot yellow or egg yellow, like the fat of a hen. The fruiting body of Chanterelle has a delicious almond flavor, bright color, and tender quality. It is a popular edible fungus. Chanterelle is particularly widely distributed in China, mainly in Hubei, Hunan, Sichuan, Guizhou, Yunnan and other places. Chanterelle has high edible and medicinal value, rich in protein, amino acids, vitamins, carotene, carbohydrates, crude fiber and mineral nutrients such as calcium, iron, and phosphorus, and has the functions of clearing eyes and lungs, benefiting intestines and stomach. Chanterelle polysaccharide is one of the main biologically active components of Chanterelle. In recent years, the pharmacological research of Chanterelle polysaccharide has made new progress in anti-tumor, hypoglycemic, anti-aging and immune regulation, but there is little research on Chanterelle polysaccharide as an immune adjuvant. At present, polysaccharide adjuvants are mainly dextran and arabinogalactan. There are few reports on the research of galactan as an adjuvant, especially the research of chanterelle galactan as an adjuvant. Summary of the invention

[0007] The purpose of the present invention is to provide a galactan having immune adjuvant function.

[0008] The galactan with immune adjuvant function provided by the present invention is prepared according to a method comprising the following steps:

[0009] (1) Extraction of total polysaccharides from Chanterelles:

[0010] Extracting the fruiting bodies of chanterelles with boiling water, collecting the extract; adding ethanol to the extract for alcohol precipitation, standing overnight, centrifuging, collecting the precipitate, washing, and vacuum drying to obtain water-extracted chanterelle total polysaccharide WCCP;

[0011] (2) Preparation of Neutral Sugar Fraction of Chanterelles:

[0012] a. Add the total polysaccharide WCCP of Chanterelles to distilled water, stir and centrifuge overnight, and collect the supernatant 1;

[0013] b. fractionating the supernatant 1 using a DEAE-cellulose column, first eluting with distilled water, collecting the eluate, concentrating, and drying to obtain a chanterelle neutral sugar fraction WCCP-N;

[0014] c. Dissolve WCCP-N in water, centrifuge, take the supernatant, and grade it using a Sepharose Cl-6B column. Elute with 0.1-0.3M NaCl solution, collect the eluate, dialyze with distilled water to remove salt, and lyophilize to obtain the WCCP-Nb fraction.

[0015] In step (1) of the above method, the extraction can be performed at least once, such as twice; the time of each extraction can be 3-6 hours, and during each extraction, the material ratio (w / v) of the chanterelle fruiting body to boiling water is 1:(10-40), specifically 1:25.

[0016] In step (1) of the above method, when adding ethanol for alcohol precipitation, an ethanol aqueous solution with a volume fraction of 95% can be used; so that the final concentration of ethanol in the system is 50-80%.

[0017] In step (1) of the above method, the centrifugation condition is 4000 rpm for 15 min.

[0018] In step (1) of the above method, the precipitate is washed with an ethanol aqueous solution having a volume fraction of 95% and anhydrous ethanol in sequence.

[0019] In step (2) a of the above method, in the solution prepared by adding the total polysaccharide WCCP of chanterelles to distilled water, the concentration of the total polysaccharide WCCP of chanterelles is 6-10 mg / ml.

[0020] In step (2) a of the above method, the centrifugation conditions in the overnight stirring centrifugation are: a rotation speed of 4500 rpm / min and a centrifugation time of 15 min.

[0021] In step (2) b of the above method, the filler of the DEAE-cellulose column may be DE-52, and the specification of the DEAE-cellulose column may be 1.5×15 cm or 7.5×30 cm.

[0022] The elution volume of the distilled water elution is 2-5 times (specifically 3 times) the column volume of the DEAE-cellulose column.

[0023] The elution volume of the NaCl solution during evaporation is 2-5 times (specifically 3 times) the column volume of the DEAE-cellulose column.

[0024] In step (2) c of the above method, WCCP-N is dissolved in a solution prepared in water, and the concentration of WCCP-N is 25-75 mg / ml.

[0025] In step (2) c of the above method, the centrifugation condition is: centrifugation at 10,000 rpm for 5 minutes.

[0026] In step (2) c of the above method, the elution volume may be 500-700 mL.

[0027] The present invention also provides the application of the galactan having the immune adjuvant function.

[0028] The application of the galactan with immune adjuvant function provided by the present invention is its application in preparing vaccine adjuvants, or its application in preparing vaccine compositions.

[0029] The present invention also protects a vaccine adjuvant.

[0030] The vaccine adjuvant of the present invention comprises, as its active ingredient, the above-mentioned galactan (WCCP-Nb fraction) having immune adjuvant function.

[0031] The vaccine adjuvant may further be an anti-tumor vaccine adjuvant.

[0032] The present invention also protects a vaccine composition.

[0033] The vaccine composition of the present invention comprises the above-mentioned galactan (WCCP-Nb fraction) having immune adjuvant function and an immunogen or antigen, or a polynucleotide encoding the immunogen or antigen.

[0034] The immunogen or antigen may be an anti-tumor vaccine or an anti-tumor carbohydrate vaccine, such as the vaccine STn-KLH.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The WCCP-Nb fraction can serve as a vaccine adjuvant to assist the vaccine in inhibiting the growth of tumor cells, while effectively enhancing the specific immune response to antigens and promoting the activation of antigen-presenting cells; specific mechanism studies have shown that the immune response may be regulated through the TLR2 pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The polysaccharide fraction of chanterelles can assist the vaccine in delaying the growth of tumor cells and increasing the titer of IgG antibodies. (A) Tumor tissue was peeled off and weighed 14 days after the fourth immunization. (B) Anti-STn antibodies in serum were detected by ELISA. One-way ANOVA was used for statistical analysis, **p<0.01, ***p<0.001.

[0038] Figure 2 The WCCP-Nb fraction of chanterelle polysaccharide can assist the vaccine in promoting the activation of DCs and B cells. Splenocytes were collected 14 days after the fourth immunization, and the activation of DCs and B cells in spleen lymphocytes was detected by flow cytometry. One-way ANOVA was used for statistical analysis, **p<0.01.

[0039] Figure 3The WCCP-Nb polysaccharide fraction of Chanterelles enhances immune response through the TLR2 pathway. Blood was collected 14 days after the second immunization. Anti-STn antibodies in serum were detected by ELISA. One-way ANOVA was used for statistical analysis, ***p<0.001. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified.

[0041] Example 1. Preparation of galactan with immune adjuvant function

[0042] (1) Weigh 1.5 kg of chanterelle fruiting bodies (purchased from a local market in Jilin Province), add 37.5 L of distilled water (material ratio is 1:25, w / v), and soak overnight. Extract at 100°C for 4 h, and filter to obtain extract I. Add 30 L of distilled water to the residue and extract at 100°C for 2 h, extract II. The water-extracted residue is placed in a 40°C oven for drying. Combine the two extracts, vacuum decompression concentrate to 5 L, centrifuge (4500 rpm, 10 min), add 95% ethanol aqueous solution to the supernatant to precipitate the ethanol to a final concentration of 65%, let stand overnight, centrifuge (4000 rpm, 15 min), collect the precipitate, wash with 95% ethanol aqueous solution and anhydrous ethanol in turn, and vacuum dry to obtain water-extracted chanterelle total polysaccharide WCCP (52.5 g), with a yield of 3.5%.

[0043] (2) Weigh 8 g of the total polysaccharide sample (WCCP) of chanterelles prepared in step (1), dissolve it in distilled water to prepare a solution of 8 mg / ml, stir overnight to fully dissolve it, centrifuge (4000 rpm, 15 min), take the supernatant, and classify it using a DEAE-cellulose (DE-52) column, first eluting it with three volumes of distilled water, and then eluting it with three column volumes of 0.3M NaCl solution, collecting different eluates (the neutral sugar WCCP-N fraction eluted with distilled water; the acid sugar WCCP-A fraction eluted with NaCl), concentrating it to a small volume under vacuum at 60° C., dialyzing it using a 1000Da dialysis bag, removing small molecule inorganic salt impurities, and then lyophilizing it under reduced pressure. The final total polysaccharide of Chanterelles was fractionated by DEAE-cellulose (DE-52) column to obtain neutral sugar WCCP-N fraction and acidic sugar WCCP-A fraction, with the yields of 50.2% and 29% respectively relative to the total polysaccharide of Chanterelles.

[0044] The neutral sugar WCCP-N fraction or acid sugar WCCP-A fraction of chanterelles obtained by DEAE-cellulose ion exchange column chromatography was divided into 50 mg / ml solution, centrifuged at 10000 rpm for 5 min, and the supernatant was fractionated using Sepharose Cl-6B chromatography column, the eluent was 0.15M NaCl solution, and the elution flow rate was 0.4 ml / min. The polysaccharides at the elution peak position were collected respectively, dialyzed with distilled water for desalination, and freeze-dried to obtain WCCP-Nb fraction and WCCP-Ab fraction.

[0045] (3) The total sugar content of the water-extracted total polysaccharide WCCP was 75.3% by the phenol-sulfuric acid method, and the uronic acid content was 5.8% by the m-hydroxybiphenyl method.

[0046] (4) The monosaccharide composition of each polysaccharide fraction was determined by high performance liquid chromatography (HPLC), as shown in Table 1.

[0047] Table 1 Monosaccharide composition of each fraction of chanterelle polysaccharide

[0048]

[0049] a Yield relative to WCCP-N; b Yield relative to WCCP-A

[0050] Embodiment 2,

[0051] (1) Study on the effect of the adjuvant vaccine STn-KLH of chanterelle polysaccharide fraction on improving immune response

[0052] The experimental groups are as follows:

[0053] BALB / c mice aged 6-8 weeks were randomly divided into 5 groups, with 6 mice in each group.

[0054] PBS control group: each mouse was given 200 μL PBS buffer;

[0055] STn-KLH / WCCP-Ab group: each mouse was given 200 μL of the STn-KLH+WCCP-Ab vaccine solution; the STn-KLH+WCCP-Ab vaccine solution was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen), 2000 μg of WCCP-Ab and PBS buffer to 200 μL;

[0056] STn-KLH / WCCP-Nb group: Each mouse was given 200 μL of the STn-KLH+WCCP-Nb vaccine solution; the STn-KLH+WCCP-Nb vaccine solution was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen), 2000 μg of WCCP-Nb and PBS buffer to 200 μL;

[0057] STn-KLH group: Each mouse was given 200 μL of STn-KLH vaccine solution; the STn-KLH vaccine solution was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen) with PBS buffer to 200 μL;

[0058] STn-KLH / FA group: Each mouse was given 200 μL of vaccine STn-KLH + Freund's adjuvant solution; the vaccine STn-KLH + Freund's adjuvant solution was prepared by mixing 100 μL of STn-KLH solution with 100 μL of Freund's adjuvant, and 100 μL of STn-KLH was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen) with PBS buffer to 100 μL; Note: Freund's complete adjuvant (Sigma F5881) was used for the first immunization, and Freund's incomplete adjuvant (Sigma F5506) was used for the second to fourth immunizations.

[0059] The effect of regulating immune response in each group was detected.

[0060] Mice were immunized subcutaneously four times, once every two weeks, and colon cancer cells CT-26 (2*10 5 14 days after the fourth immunization, spleen cells and serum were collected. The tumors were removed and weighed, and the anti-STn antibody titer in the serum was detected by ELISA. Figure 1 . Figure 1 The results showed that immunization with WCCP-Nb or FA as adjuvant could delay tumor growth and effectively inhibit tumor size; STn-KLH / FA and STn-KLH / WCCP-Nb induced a stronger specific immune response against STn and induced higher anti-STn IgG antibody titers than STn-KLH, and the results showed statistically significant differences.

[0061] (2) Study on the mechanism of improving immune response of the Chanterelle polysaccharide fraction WCCP-Nb adjuvant vaccine STn-KLH

[0062] WCCP-Nb was co-immunized with the vaccine STn-KLH, and a control group of PBS, STn-KLH+FA and STn-KLH were set up to detect the effect of regulating antigen presenting cells.

[0063] The experimental groups are as follows:

[0064] BALB / c mice aged 6-8 weeks were randomly divided into 4 groups, with 6 mice in each group.

[0065] PBS control group: each mouse was given 200 μL PBS buffer;

[0066] STn-KLH / WCCP-N-b1: Each mouse was given 200 μL of the vaccine STn-KLH+WCCP-Nb solution; the vaccine STn-KLH+WCCP-Nb solution was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen), WCCP-N-b 2000 μg and PBS buffer to 200 μL;

[0067] STn-KLH group: Each mouse was given 200 μL of STn-KLH vaccine solution; the STn-KLH vaccine solution was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen) with PBS buffer to 200 μL;

[0068] STn-KLH / FA group: Each mouse was given 200 μL of vaccine STn-KLH+FA (Freund's adjuvant) solution; the vaccine STn-KLH+Freund's adjuvant solution was prepared by mixing 100 μL of STn-KLH solution with 100 μL of Freund's adjuvant, and 100 μL of STn-KLH was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen) with PBS buffer to 100 μL; Note: Freund's complete adjuvant (Sigma F5881) was used for the first immunization, and Freund's incomplete adjuvant (Sigma F5506) was used for the second to fourth immunizations.

[0069] Mice were immunized subcutaneously four times, once every two weeks, and colon cancer cells CT-26 (2*10 5 14 days after the fourth immunization, spleen cells were collected. Flow cytometry was used to detect the activation of antigen-presenting cells in spleen lymphocytes and the co-stimulatory molecules on the surface of DCs. Figure 2 The results showed that immunization with STn-KLH / WCCP-Nb significantly increased the expression of CD86 molecules on the surface of DCs and B cells ( Figure 2 ); STn-KLH / WCCP-Nb immunization could increase the expression of MHCI, MHCII, CD40 and CD86 on the surface of DCs (Table 2).

[0070] Table 2 Regulation of the expression of related molecules on the surface of DCs by the Chanterelle polysaccharide WCCP-Nb

[0071]

[0072]

[0073] (3) WCCP-Nb was co-immunized with the vaccine STn-KLH, and a control group PBS and a STn-KLH group were set up. At the same time, a TLR2 antibody blocking group (TLR2 blocking antibody was injected one day before the injection of the immune adjuvant and vaccine (100 μL, containing 250 μg of antibody, was injected into the tail vein), STn-KLH / WCCP-Nb / BA) was set up to detect whether WCCP-Nb regulates the immune response through the TLR2 pathway.

[0074] The experimental groups are as follows:

[0075] BALB / c mice aged 6-8 weeks were randomly divided into 4 groups, with 6 mice in each group.

[0076] PBS control group: each mouse was given 200 μL PBS buffer;

[0077] STn-KLH group: Each mouse was given 200 μL of STn-KLH vaccine solution; the STn-KLH vaccine solution was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen) with PBS buffer to 200 μL;

[0078] STn-KLH / WCCP-Nb group: Each mouse was given 200 μL of the STn-KLH+WCCP-Nb vaccine solution; the STn-KLH+WCCP-Nb vaccine solution was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen), 2000 μg of WCCP-Nb and PBS buffer to 200 μL;

[0079] TLR2 antibody blocking group: each mouse was given 200 μL of the vaccine STn-KLH+WCCP-Nb solution; the vaccine STn-KLH+WCCP-Nb solution was prepared by mixing STn-KLH (containing 2 μg of STn saccharide antigen), WCCP-N-b 2000 μg and PBS buffer to 200 μL; TLR2 blocking antibody was injected one day before the injection of immune adjuvant and vaccine.

[0080] Mice were subcutaneously immunized twice, once every two weeks, and spleen cells and serum were collected 14 days after the second immunization. The anti-STn antibody titer in the serum was detected by ELISA. The results showed that the anti-STn antibody titer in the serum of the TLR2 antibody blocking group was significantly lower than that of STn-KLH / WCCP-Nb ( Figure 3 ); Flow cytometry was used to detect the expression of co-stimulatory molecules on the surface of DCs in splenic lymphocytes. The results showed that after the addition of TLR2 blocking antibodies, the effect of WCCP-Nb in promoting DCs activation was inhibited (Table 3), indicating that WCCP-Nb may play an immune adjuvant role through the TLR2 pathway on the surface of DCs.

[0081] Table 3 Chanterelle polysaccharide WCCP-Nb regulates the expression of related molecules on the surface of DCs through TLR2 pathway

[0082]

Claims

1. Application of galactan in the preparation of anti-tumor vaccine adjuvant; The anti-tumor vaccine is STn-KLH; The preparation method of galactan comprises the following steps: (1) Extraction of total polysaccharides from Chanterelles: Extracting the fruiting bodies of chanterelles with boiling water, collecting the extract; adding ethanol to the extract for alcohol precipitation, standing overnight, centrifuging, collecting the precipitate, washing, and vacuum drying to obtain water-extracted chanterelle total polysaccharide WCCP; (2) Preparation of Neutral Sugar Fraction of Chanterelles: a. Add the total polysaccharide WCCP of Chanterelles to distilled water, stir and centrifuge overnight, and collect the supernatant 1; b. fractionating the supernatant 1 using a DEAE-cellulose column, eluting first with distilled water and then with a NaCl solution, collecting the distilled water eluate, concentrating, and drying to obtain a chanterelle neutral sugar fraction WCCP-N; In step (2) b, the filler of the DEAE-cellulose column is DE-52; The elution volume of the distilled water elution is 2-5 times the column volume of the DEAE-cellulose column; c. WCCP-N was dissolved in water and centrifuged. The supernatant was fractionated using a Sepharose Cl-6B column and eluted with a 0.15 M NaCl solution. The polysaccharide at the elution peak was collected and dialyzed with distilled water for desalting. The WCCP-Nb fraction was obtained after freeze-drying.

2. The use according to claim 1, characterized in that: In the step (1), the extraction is performed at least once; the time of each extraction is 3-6 hours, and during each extraction, the material ratio of the chanterelle fruiting body to boiling water is 1: 10-40 (w / v).

3. The use according to claim 1 or 2, characterized in that: In the step (1), when adding ethanol for alcohol precipitation, an ethanol aqueous solution with a volume fraction of 95% is used; so that the final concentration of ethanol in the system is 50-80%; In the step (1), the centrifugation condition is 4000 rpm, 15 min; In the step (1), the precipitate is washed with an ethanol aqueous solution having a volume fraction of 95% and anhydrous ethanol in sequence.

4. The use according to claim 1 or 2, characterized in that: In step (2) a, in the solution prepared by adding distilled water to the total polysaccharide WCCP of Chanterelles, the concentration of the total polysaccharide WCCP of Chanterelles is 6-10 mg / ml; In step (2) a, the centrifugation conditions in the overnight stirring centrifugation are: a rotation speed of 4500 rpm / min and a centrifugation time of 15 min.

5. The use according to claim 1 or 2, characterized in that: In step (2) b, the elution volume of the NaCl solution is 2-5 times the column volume of the DEAE-cellulose column.

6. The use according to claim 1 or 2, characterized in that: In step (2) c, WCCP-N is dissolved in a solution prepared in water, and the concentration of WCCP-N is 25-75 mg / ml; In step (2) c, the centrifugation condition is: 10,000 rpm for 5 min.

7. A vaccine composition comprising galactan and an anti-tumor vaccine; The anti-tumor vaccine is STn-KLH; The preparation method of galactan comprises the following steps: (1) Extraction of total polysaccharides from Chanterelles: Extracting the fruiting bodies of chanterelles with boiling water, collecting the extract; adding ethanol to the extract for alcohol precipitation, standing overnight, centrifuging, collecting the precipitate, washing, and vacuum drying to obtain water-extracted chanterelle total polysaccharide WCCP; (2) Preparation of Neutral Sugar Fraction of Chanterelles: a. Add the total polysaccharide WCCP of Chanterelles to distilled water, stir and centrifuge overnight, and collect the supernatant 1; b. fractionating the supernatant 1 using a DEAE-cellulose column, eluting first with distilled water and then with a NaCl solution, collecting the distilled water eluate, concentrating, and drying to obtain a chanterelle neutral sugar fraction WCCP-N; In step (2) b, the filler of the DEAE-cellulose column is DE-52; The elution volume of the distilled water elution is 2-5 times the column volume of the DEAE-cellulose column; c. WCCP-N was dissolved in water and centrifuged. The supernatant was fractionated using a Sepharose Cl-6B column and eluted with a 0.15 M NaCl solution. The polysaccharide at the elution peak was collected and dialyzed with distilled water for desalting. The WCCP-Nb fraction was obtained after freeze-drying.

8. The vaccine composition according to claim 7, characterized in that: In the step (1), the extraction is performed at least once; the time of each extraction is 3-6 hours, and during each extraction, the material ratio of the chanterelle fruiting body to boiling water is 1: 10-40 (w / v).

9. The vaccine composition according to claim 7 or 8, characterized in that: In the step (1), when adding ethanol for alcohol precipitation, an ethanol aqueous solution with a volume fraction of 95% is used; so that the final concentration of ethanol in the system is 50-80%; In the step (1), the centrifugation condition is 4000 rpm, 15 min; In the step (1), the precipitate is washed with an ethanol aqueous solution having a volume fraction of 95% and anhydrous ethanol in sequence.

10. The vaccine composition according to claim 7 or 8, characterized in that: In step (2) a, in the solution prepared by adding distilled water to the total polysaccharide WCCP of Chanterelles, the concentration of the total polysaccharide WCCP of Chanterelles is 6-10 mg / ml; In step (2) a, the centrifugation conditions in the overnight stirring centrifugation are: a rotation speed of 4500 rpm / min and a centrifugation time of 15 min.

11. The vaccine composition according to claim 7 or 8, characterized in that: In step (2) b, the elution volume of the NaCl solution is 2-5 times the column volume of the DEAE-cellulose column.

12. The vaccine composition according to claim 7 or 8, characterized in that: In step (2) c, WCCP-N is dissolved in a solution prepared in water, and the concentration of WCCP-N is 25-75 mg / ml; In step (2) c, the centrifugation condition is: 10,000 rpm for 5 min.

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