Subunit vaccine adjuvant and preparation method thereof

Abstract

The invention provides a subunit vaccine adjuvant and a preparation method thereof, 100 parts by weight of the vaccine adjuvant comprises 25-40 parts by weight of plant essential oil, 10-20 parts by weight of a plant polysaccharide solution, 30-50 parts by weight of a surfactant, and the balance of vitamin E, the plant essential oil comprises sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil, and the plant polysaccharide comprises ginseng polysaccharide, corncob polysaccharide and lycium barbarum polysaccharide. According to the invention, humoral immunity and cellular immunity of an organism can be rapidly activated after vaccine injection, the generation speed of an immunized antibody is high, and the immune irritation is small. The vaccine adjuvant and the inactivated antigen can be prepared into a stable O / W type vaccine, and the O / W type vaccine is small in viscosity, convenient to inject and good in immunogenicity. The plant essential oil and the surfactant in the agent are easily absorbed by animals and have no toxic or side effect on the animals.

Description

Technical Field

[0001] The invention relates to a subunit vaccine adjuvant and a preparation method thereof, and belongs to the technical field of biomedicine. Background Art

[0002] The main pathogenic circovirus prevalent in China is porcine circovirus type 2 (PCV2), which is considered to be one of the main pathogens causing porcine circovirus-associated disease (PCVAD). Although the harm caused by the disease can be reduced by strengthening feeding management, reducing feeding density, and rationally formulating feed, a large number of industry breeding status and experimental studies have shown that the main effective means of controlling PCV2 infection is still vaccination of piglets.

[0003] Subunit vaccines have advantages that whole virus inactivated vaccines and whole virus attenuated live vaccines do not have: there is no possibility of virus shedding, homologous recombination, and mutation, it is easy to prepare high-purity and high-titer antigens, and the prepared vaccines are safe, reliable, and efficient. In recent years, it has become a research trend for PCV2 vaccines in my country. In the preparation of subunit vaccines, the correctness of adjuvant selection has an important impact on the immune efficacy of the vaccine.

[0004] At present, aluminum salt adjuvants are still the main adjuvants for animal vaccines in my country. In addition, oil emulsions and propolis adjuvant vaccines have also been approved for production and put into use. Adjuvants are non-specific immunopotentiators that can assist specific antigens to enter the body and enhance the body's immune response to the target antigen. It is generally believed that adjuvants promote the effect of vaccines in two main aspects: first, they increase the stability of vaccines, thereby prolonging the duration of vaccine action; second, they activate the body's immune system and increase the intensity of vaccine action.

[0005] Patent CN114917334A discloses a water-in-oil-in-water composite vaccine adjuvant, a preparation method and an application thereof, which is composed of an inner water phase, an oil phase and an outer water phase wrapped in sequence from the inside to the outside, and the vaccine adjuvant is prepared from the following raw materials in mass percentage: white oil: 85-92wt%; first composite surfactant: 6-11wt%; second composite surfactant: 3-8wt%; brown algae polysaccharide sulfate aqueous solution: 0.1-1wt%. The water-in-oil-in-water composite vaccine adjuvant eventually forms a high-stability composite vaccine adjuvant with sufficient mechanical strength and no particle aggregation. However, the preparation process still requires the investment of high-value equipment, and the preparation process still requires the provision of high mechanical power. For animal vaccines, the production cost is still high.

[0006] Patent CN109675028A discloses a vaccine adjuvant, a preparation method and application thereof, and a porcine reproductive and respiratory syndrome vaccine. The vaccine adjuvant is a solution obtained by adding double-stranded nucleic acid and diethylaminoethyl glucose to an alkaline buffer solution; the concentration of double-stranded nucleic acid in the solution is 0.25-5 mg / mL, and the concentration of diethylaminoethyl glucose is 1-2.5 g / mL. The vaccine adjuvant combined with porcine reproductive and respiratory syndrome virus (PRRSV) can improve the body's cellular immunity, induce interferon, and reduce the immunosuppression produced by wild-type PRRSV, thereby allowing the body to quickly produce cytokines and quickly produce high levels of neutralizing antibodies, thereby enhancing the safety and immune efficacy of the vaccine. However, in terms of clinical application, cytokine adjuvants currently still have the problems of short half-life and high price. In addition, the grasp of dosage is also a problem that cytokine adjuvants need to be solved in the application process, because improper use of dosage may lead to potential autoimmune safety hazards.

[0007] Patent CN115671276A discloses a vaccine adjuvant containing ginseng total polysaccharide and aluminum salt and a vaccine composition thereof, including ginseng total polysaccharide and aluminum salt, wherein the mass ratio of ginseng total polysaccharide to aluminum salt is 0.0001-1000:1, preferably 0.1-5:1. The vaccine adjuvant is a mixture of GPS and aluminum salt, which has the advantages of good immune effect and convenient use, and provides a new adjuvant option for vaccines. Although the vaccine adjuvant enhances the immunogenicity of the antigen, it is still not good in shortening the time for the immune response to take effect and inducing a sustained immune response.

[0008] In view of this, the present application can develop vaccine adjuvants in terms of enhancing the immunogenicity of antigens, shortening the time it takes for immune responses to take effect, inducing sustained immune responses, reducing the number of vaccinations, saving antigen usage, and improving the effectiveness of vaccines for those with poor responses while taking safety into consideration. Summary of the invention

[0009] In view of the shortcomings of the prior art, the first object of the present invention is to provide a subunit vaccine adjuvant and a preparation method thereof.

[0010] The second object of the present invention is to provide a method for preparing the above vaccine adjuvant.

[0011] The second object of the present invention is to provide the application of the above vaccine adjuvant.

[0012] In order to achieve the first purpose, the present invention is implemented through the following technical scheme: a subunit vaccine adjuvant, based on 100 parts by weight, includes 25-40 parts by weight of plant essential oil, 10-20 parts by weight of plant polysaccharide solution, 30-50 parts by weight of surfactant, and the remainder of vitamin E.

[0013] Preferably, the plant essential oils include sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil, and ginger essential oil, and the mass ratio of the sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil, and ginger essential oil is 1:1-3:1-3:2-3.

[0014] Preferably, the concentration of the plant polysaccharide solution is 1-3 mg / mL.

[0015] Preferably, the plant polysaccharides include ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide, and the mass ratio of the ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide is 1:3-5:3-5.

[0016] Preferably, the average molecular weight of the plant polysaccharide is 5-25 kDa.

[0017] Preferably, the surfactant is PEG400 or Tween-80.

[0018] Preferably, the surfactant includes PEG400 and Tween-80, and the mass ratio of the two is 1:1-3.

[0019] By adopting the above technical scheme, the present invention contains plant essential oil and surfactant, and can form a uniform system only by hand shaking or slight stirring; by adding plant polysaccharides and vitamin E, the nonspecific immune ability of the body is improved, and the immunogenicity of the antigen is enhanced.

[0020] In order to achieve the second object, the present invention is implemented by the following technical solution: a method for preparing a subunit vaccine adjuvant, comprising the following steps:

[0021] S1: weigh sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil respectively according to the proportion, stir them evenly, then add a surfactant to the plant essential oils, and continue stirring to obtain a mixture A;

[0022] S2: Preparation of plant polysaccharide solution

[0023] Weigh ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide according to the ratio to prepare a plant polysaccharide solution with a concentration of 1-3 mg / mL;

[0024] S3: Add vitamin E and plant polysaccharide solution to mixture A in sequence, and continue stirring to obtain a vaccine adjuvant.

[0025] Preferably, the stirring conditions are a rotation speed of 500-1000 r / min and a stirring time of 20-45 min.

[0026] In order to achieve the third purpose, the present invention is implemented through the following technical solution: an application of a subunit vaccine adjuvant, wherein the vaccine adjuvant and the inactivated antigen are mixed in a mass ratio of 1:5.

[0027] By adopting the above technical scheme, the vaccine adjuvant and the inactivated antigen can be made into a stable O / W vaccine with low viscosity, convenient injection and good immunogenicity. The plant essential oil and surfactant in the vaccine are easily absorbed by animals and have no toxic side effects on animals.

[0028] Beneficial effects of the present invention:

[0029] (1) The present invention can quickly activate the body's humoral immunity and cellular immunity after vaccine injection, so that the antibody production speed after immunization is fast and the immunostimulation is small.

[0030] (2) The raw materials prepared by the present invention are easy to obtain, have little toxicity and side effects, and have good biocompatibility.

[0031] (3) The vaccine adjuvant of the present invention and the inactivated antigen can be made into a stable O / W vaccine with low viscosity, convenient injection and good immunogenicity. The plant essential oil and surfactant in the vaccine are easily absorbed by animals and have no toxic side effects on animals. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0033] Example 1

[0034] A subunit vaccine adjuvant comprises 25 g of plant essential oil, 20 g of plant polysaccharide solution, 50 g of surfactant and 5 g of vitamin E.

[0035] In this embodiment, the plant essential oils include sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil in a mass ratio of 1:1:3:2.

[0036] In this embodiment, the concentration of the plant polysaccharide solution is 1 mg / mL.

[0037] In this embodiment, the plant polysaccharides include ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide in a mass ratio of 1:3:3.

[0038] In this embodiment, the average molecular weight of the plant polysaccharide is 5 kDa.

[0039] In this embodiment, the surfactant is PEG400.

[0040] A method for preparing a subunit vaccine adjuvant comprises the following steps:

[0041] S1: weigh sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil respectively according to the proportion, stir them evenly, then add a surfactant to the plant essential oils, and continue stirring to obtain a mixture A;

[0042] S2: Preparation of plant polysaccharide solution

[0043] Weigh ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide according to the ratio to prepare a plant polysaccharide solution;

[0044] S3: Add vitamin E and plant polysaccharide solution to mixture A in sequence, and continue stirring to obtain a vaccine adjuvant.

[0045] In this embodiment, the stirring conditions are a rotation speed of 500 r / min and a stirring time of 45 min.

[0046] An application of a subunit vaccine adjuvant, wherein the vaccine adjuvant is mixed with an inactivated antigen in a mass ratio of 1:5.

[0047] Example 2

[0048] A subunit vaccine adjuvant comprises 25 g of plant essential oil, 10 g of plant polysaccharide solution, 50 g of surfactant and 15 g of vitamin E.

[0049] In this embodiment, the plant essential oils include sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil in a mass ratio of 1:3:3:2.

[0050] In this embodiment, the concentration of the plant polysaccharide solution is 3 mg / mL.

[0051] In this embodiment, the plant polysaccharides include ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide in a mass ratio of 1:5:3.

[0052] In this example, the average molecular weight of the plant polysaccharide is 10 kDa.

[0053] In this embodiment, the surfactant is Tween-80.

[0054] A method for preparing a subunit vaccine adjuvant is the same as that in Example 1.

[0055] In this embodiment, the stirring conditions are a rotation speed of 1000 r / min and a stirring time of 20 min.

[0056] The application of a subunit vaccine adjuvant is the same as Example 1.

[0057] Example 3

[0058] A subunit vaccine adjuvant comprises 40g of plant essential oil, 15g of plant polysaccharide solution, 30g of surfactant and 15g of vitamin E.

[0059] In this embodiment, the plant essential oils include sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil in a mass ratio of 1:3:3:3.

[0060] In this embodiment, the concentration of the plant polysaccharide solution is 2 mg / mL.

[0061] In this embodiment, the plant polysaccharides include ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide in a mass ratio of 1:5:5.

[0062] In this embodiment, the average molecular weight of plant polysaccharides is 25 kDa.

[0063] In this embodiment, the surfactant is PEG400 and Tween-80 in a mass ratio of 1:1.

[0064] A method for preparing a subunit vaccine adjuvant is the same as that in Example 1.

[0065] The application of a subunit vaccine adjuvant is the same as Example 1.

[0066] Example 4

[0067] A subunit vaccine adjuvant comprises 30 g of plant essential oil, 20 g of plant polysaccharide solution, 40 g of surfactant and 10 g of vitamin E.

[0068] In this embodiment, the plant essential oils include sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil in a mass ratio of 1:2:1:3.

[0069] In this embodiment, the concentration of the plant polysaccharide solution is 1 mg / mL.

[0070] In this embodiment, the plant polysaccharides include ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide in a mass ratio of 1:4:5.

[0071] In this example, the average molecular weight of the plant polysaccharide is 20 kDa.

[0072] In this embodiment, the surfactant is PEG400 and Tween-80 in a mass ratio of 1:3.

[0073] A method for preparing a subunit vaccine adjuvant is the same as that in Example 1.

[0074] The application of a subunit vaccine adjuvant is the same as Example 1.

[0075] Example 5

[0076] A subunit vaccine adjuvant comprises 30 g of plant essential oil, 10 g of plant polysaccharide solution, 50 g of surfactant and 10 g of vitamin E.

[0077] In this embodiment, the plant essential oils include sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil in a mass ratio of 1:1:3:3.

[0078] In this embodiment, the concentration of the plant polysaccharide solution is 3 mg / mL.

[0079] In this embodiment, the plant polysaccharides include ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide in a mass ratio of 1:3:4.

[0080] In this example, the average molecular weight of the plant polysaccharide is 10 kDa.

[0081] In this embodiment, the surfactant is PEG400 and Tween-80 in a mass ratio of 1:2.

[0082] A method for preparing a subunit vaccine adjuvant is the same as that in Example 1.

[0083] The application of a subunit vaccine adjuvant is the same as Example 1.

[0084] It is worth noting that the vaccine involved in the following test examples is porcine circovirus type 2 (O / W type) vaccine, specifically: the vaccine adjuvant prepared by the present invention is mixed with inactivated porcine circovirus type 2 (LG strain) virus liquid in a mass ratio of 1:5.

[0085] Test Example 1 Physical and Chemical Test

[0086] Test groups: Example 1-Example 5;

[0087] Test method: The vaccines obtained in Examples 1 to 5 were tested for appearance, dosage form, stability, viscosity, particle size and other indicators;

[0088] (1) Dosage form test: Take a 250 mL beaker, put 100 mL of cold water, use a clean pipette to draw a small amount of vaccine and drop it on the surface of the cold water, and observe the mixing of the droplet and the water;

[0089] (2) Viscosity: The viscosity of the product was measured using a Brookfield viscometer;

[0090] (3) Particle size: Use a pipette to accurately take 10 μL of the emulsion into a test cup, add 290 μL of PBS and mix thoroughly to ensure that the emulsified particles are well dispersed. Place the test cup in a Malvern nanoparticle size analyzer for measurement, and set the measurement temperature to 25 °C.

[0091] (4) Centrifugal stability: 10 mL of the emulsion was added to a centrifuge tube and centrifuged at 3000 rpm / min for 15 min to see if there was any water phase precipitated at the bottom of the tube;

[0092] (5) 37°C stability: Place in a 37°C incubator and observe regularly every day.

[0093] Test results: See Table 1 for details.

[0094] Table 1 Physical and chemical test results of vaccines of Example 1-Example 5

[0095]

[0096] Test Example 2 Safety Test of Porcine Circovirus Type 2 (O / W Type) Vaccine

[0097] 1. Safety experiment of porcine circovirus type 2 (O / W) vaccine on mice

[0098] Experimental groups: experimental group 1-experimental group 5 and control group 1;

[0099] Test method: Clean mice were divided into 6 groups, each with 10 mice. The porcine circovirus type 2 (O / W type) vaccine obtained in Examples 1 to 5 was used to immunize one group of mice, and 0.5 ml of the corresponding vaccine was injected subcutaneously in the neck of each mouse, and they were recorded as test groups 1 to 5 respectively; control group 1 was not vaccinated.

[0100] Experimental results: The health performance and survival of the mice were observed daily for 7 consecutive days. No adverse reactions or deaths caused by vaccine injection occurred, and there was no inflammation at the injection site.

[0101] 2. Safety experiment of porcine circovirus type 2 (O / W) vaccine on piglets

[0102] Experimental groups: experimental group 6-experimental group 10 and control group 2;

[0103] Experimental method: 14-day-old healthy piglets with negative porcine circovirus type 2 antibodies were randomly selected and divided into 6 groups, with 4 pigs in each group. The piglets were injected with porcine circovirus type 2 (O / W type) vaccines obtained in Example 1-Example 5, respectively, recorded as experimental group 6-experimental group 10, and the control group 2 was not injected; after the injection, the piglets in each group were placed in the same management environment for feeding and management, the body temperature of the piglets was measured and recorded regularly every day, and the experiment was completed after 14 days of continuous observation.

[0104] Test results: After 14 days of observation after vaccination, no abnormalities were found in the feeding and weight loss of piglets in each group; no abnormal reactions or allergic reactions were found at the vaccination site of the piglets; the body temperature of piglets in each group did not rise, that is, there were no obvious clinical symptoms, and the safety of vaccine adjuvants in each group was good.

[0105] Test Example 3 Evaluation of piglet immune efficacy

[0106] Experimental groups: experimental group 11-experimental group 15 and control group 3;

[0107] Test method: 30 piglets were selected and randomly divided into 6 groups, 5 in each group. The porcine circovirus type 2 (O / W type) vaccine obtained in Examples 1 to 5 was used to immunize a group of piglets, with an immunization dose of 2.0 ml / head, respectively, and recorded as test group 11 to test group 15, and the control group 3 was not immunized. Blood was collected 4 weeks after immunization, serum was separated, and the antibody titer was detected using an ELISA detection kit, and the positive rate of antibodies at different dilutions 4 weeks after immunization was counted.

[0108] Test results: See Table 2 for details.

[0109] Table 2 Results of antibody positive rates in different groups

[0110]

[0111] According to the requirements of the "Compilation of Quality Standards for Veterinary Biological Products (2010)", an antibody positive rate of ≥80% is qualified. An antibody titer of ≥1:64 can provide more than 99% immune protection to the pig body. It can be seen from Table 2 that four weeks after the piglets were immunized with the vaccines prepared in Examples 1 to 5, the proportion of antibody titers ≥1:64 was above 80%, thereby providing effective immune protection for the animals.

[0112] Experimental Example 4 Effect of the combination of plant essential oil, plant polysaccharide solution and vitamin E on immune efficacy

[0113] Test groups: test groups 16 to 22 and control group 4, wherein the vaccine adjuvant used in test group 16 is the same as in Example 1, the vaccine adjuvants of test groups 17 to 19 are not added with plant essential oils, plant polysaccharide solutions, and vitamin E, respectively, the vaccine adjuvant of test group 20 is not added with plant essential oils and plant polysaccharide solutions, the vaccine adjuvant of test group 21 is not added with plant essential oils and vitamin E, the vaccine adjuvant of test group 22 is not added with plant polysaccharide solutions and vitamin E, and the rest is the same as in Example 1;

[0114] Experimental method: 40 piglets were randomly divided into 8 groups, 5 in each group. The vaccines obtained from experimental groups 16 to 22 were used to immunize piglets (0.2 mL / head), and the control group 4 was injected with sterile PBS (0.2 mL / head). After 21 days, the second immunization was carried out using the same method and dosage. Blood was collected from each group of piglets before the first immunization, after the first immunization, and after the second immunization, and the serum was separated. The antibody level in the serum of each group of piglets was determined by the in vivo ELISA method.

[0115] Test results: See Table 3 for details.

[0116] Table 3 Effects of different adjuvant vaccines on piglet antibody levels

[0117]

[0118] Table 3 shows that 21 days after the first immunization, the serum antibody levels of piglets in test groups 16-22 were higher than those before immunization and control group 4, among which the antibody level of test group 16 was significantly increased; 14 days after the second immunization, compared with the antibody level after the first immunization, the antibody level of test groups 17-22 did not increase significantly, while the antibody level of test group 16, which contained plant essential oil, plant polysaccharide solution and vitamin E in the adjuvant, increased significantly. This shows that the combination of plant essential oil, plant polysaccharide solution and vitamin E can induce immunized piglets to produce higher antibody levels; at the same time, after the second immunization, its antibody level was significantly higher than that after the first immunization.

[0119] Experimental Example 5 Effect of the Compatibility of Sandalwood Seed Essential Oil and Sandalwood Essential Oil on Immune Efficacy

[0120] Test groups: test groups 23-26 and control group 5, wherein the vaccine adjuvant used in test group 23 is the same as in Example 1, the vaccine adjuvants of test groups 24-25 are not added with sandalwood seed essential oil and sandalwood essential oil, respectively, and the vaccine adjuvant of test group 26 is not added with sandalwood seed essential oil and sandalwood essential oil, and the rest is the same as in Example 1;

[0121] Experimental method: 25 piglets were randomly divided into 5 groups, 5 in each group. The vaccines obtained from experimental groups 23 to 26 were used to immunize piglets (0.2 mL / head), and the control group 5 was injected with sterile PBS (0.2 mL / head). After 21 days, the second immunization was performed using the same method and dosage. Blood was collected from each group of piglets before the first immunization, after the first immunization, and after the second immunization, and serum was separated. The antibody level in the serum of each group of piglets was determined by the in vivo ELISA method.

[0122] Test results: See Table 4 for details.

[0123] Table 4 Effects of different adjuvant vaccines on piglet antibody levels

[0124]

[0125] Table 4 shows that 21 days after the first vaccination, the serum antibody levels of piglets in test groups 23-26 were higher than those before vaccination and control group 5, among which the antibody level of test group 23 was significantly increased; 14 days after the second vaccination, compared with the antibody level after the first vaccination, test groups 24-26 were significantly increased, but the increase rate was significantly lower than that of test group 23. This shows that the combination of sandalwood seed essential oil and sandalwood essential oil can induce the immunized piglets to produce higher antibody levels; at the same time, after the second vaccination, the antibody level was significantly higher than that after the first vaccination.

[0126] Experimental Example 6 Effect of the combination of ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide on immune efficacy

[0127] Test groups: test groups 27-33 and control group 6, wherein the vaccine adjuvant used in test group 27 is the same as in Example 1, the vaccine adjuvants of test groups 28-30 are not added with ginseng polysaccharide, corn cob polysaccharide, and wolfberry polysaccharide, respectively, the vaccine adjuvant of test group 31 is not added with ginseng polysaccharide and corn cob polysaccharide, the vaccine adjuvant of test group 32 is not added with corn cob polysaccharide and wolfberry polysaccharide, and the vaccine adjuvant of test group 33 is not added with ginseng polysaccharide and wolfberry polysaccharide, and the rest is the same as in Example 1;

[0128] Experimental method: 40 piglets were randomly divided into 8 groups, 5 in each group. The vaccines obtained from experimental groups 27 to 33 were used to immunize piglets (0.2 mL / head), and the control group 6 was injected with sterile PBS (0.2 mL / head). After 21 days, the second immunization was carried out using the same method and dosage. Blood was collected from each group of piglets before the first immunization, after the first immunization, and after the second immunization, and the serum was separated. The antibody level in the serum of each group of piglets was determined by the in vivo ELISA method.

[0129] Test results: See Table 5 for details.

[0130] Table 5 Effects of different adjuvant vaccines on piglet antibody levels

[0131]

[0132] Table 5 shows that 21 days after the first immunization, the serum antibody levels of piglets in test groups 27-33 were higher than those before immunization and control group 6, among which the antibody level of test group 23 was significantly increased; 14 days after the second immunization, compared with the antibody level after the first immunization, test groups 27-33 were all increased, but the increase rate was significantly lower than that of test group 27. This shows that the combination of ginseng polysaccharides, corn cob polysaccharides and wolfberry polysaccharides, especially the addition of corn cob polysaccharides, can induce immunized piglets to produce higher antibody levels; at the same time, after the second immunization, the antibody level was significantly higher than that after the first immunization.

[0133] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0134] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A subunit vaccine adjuvant, characterized in that: Based on 100 parts by weight, the invention comprises 25-40 parts by weight of plant essential oil, 10-20 parts by weight of plant polysaccharide solution, 30-50 parts by weight of surfactant and the balance of vitamin E.

2. The subunit vaccine adjuvant according to claim 1, characterized in that The plant essential oils include sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil. The mass ratio of the sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil is 1:1-3:1-3:2-3.

3. The subunit vaccine adjuvant according to claim 2, characterized in that The concentration of the plant polysaccharide solution is 1-3 mg / mL.

4. The subunit vaccine adjuvant according to claim 3, characterized in that The plant polysaccharides include ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide, and the mass ratio of the ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide is 1:3-5:3-5.

5. The subunit vaccine adjuvant according to claim 4, characterized in that The average molecular weight of the plant polysaccharide is 5-25 kDa.

6. The subunit vaccine adjuvant according to claim 5, characterized in that The surfactant is PEG400 or Tween-80.

7. The subunit vaccine adjuvant according to claim 6, characterized in that The surfactant includes PEG400 and Tween-80, and the mass ratio of the two is 1:1-3.

8. A method for preparing a subunit vaccine adjuvant as claimed in any one of claims 1 to 7, characterized in that: The steps include: S1: weigh sandalwood seed essential oil, sandalwood essential oil, tea tree essential oil and ginger essential oil respectively according to the proportion, stir them evenly, then add a surfactant to the plant essential oils, and continue stirring to obtain a mixture A; S2: Preparation of plant polysaccharide solution Weigh ginseng polysaccharide, corncob polysaccharide and wolfberry polysaccharide according to the ratio to prepare a plant polysaccharide solution with a concentration of 1-3 mg / mL; S3: Add vitamin E and plant polysaccharide solution to mixture A in sequence, and continue stirring to obtain a vaccine adjuvant.

9. The method for preparing a subunit vaccine adjuvant according to claim 8, characterized in that: The stirring conditions are a rotation speed of 500-1000 r / min and a stirring time of 20-45 min.

10. Use of the subunit vaccine adjuvant as described in any one of claims 1-7, wherein the vaccine adjuvant and the inactivated antigen are mixed in a mass ratio of 1:5.

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