CN112451660A

CN112451660A - Oil adjuvant for preparing vaccine emulsion with low shearing force and preparation method and application thereof

Info

Publication number: CN112451660A
Application number: CN202011461695.5A
Authority: CN
Inventors: 胡松华
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-12-12
Filing date: 2020-12-12
Publication date: 2021-03-09

Abstract

The invention discloses a preparation method of an oil adjuvant for preparing a vaccine by low shearing force, which comprises the following steps: the oil adjuvant comprises a component A1 and a component A2, wherein the component A1: the component A2 is 60-70: 30-40 in volume ratio; white oil, span-80 and span-85 are mixed according to the weight ratio of 85-87: 8-10: mixing the components in a volume ratio of 4-6 to obtain a component A1; mixing Tween-80 and polyethylene glycol-200 in a volume ratio of 22-24: 75-78 to obtain a component A2. The invention also provides the application of the oil adjuvant: mixing an oil adjuvant and an antigen-containing water phase at a volume ratio of 1: 1; and emulsifying the obtained mixed solution at room temperature by a shaking table to obtain the vaccine emulsion.

Description

Oil adjuvant for preparing vaccine emulsion with low shearing force and preparation method and application thereof

Technical Field

The invention relates to a novel vaccine oil adjuvant and a preparation method and application thereof.

Background

China is a big country in the breeding industry, the number of livestock and poultry is huge, about 6.88 hundred million live pigs are grown in the market in 2017, and 113.7 hundred million broilers are grown (1, 2). Animal infectious diseases frequently occur in livestock farms, which are harmful to the health of animals and cause huge economic loss to animal husbandry production. Some zoonosis infections can be transmitted from animals to people, and harm human health. The injection of vaccine to livestock and poultry is an important measure for preventing the infectious diseases of animals at present. In order to improve the immune effect of vaccines, adjuvants are often added to animal vaccines. The oil adjuvant is a common veterinary vaccine adjuvant. The classical inactivated vaccine is prepared by mixing an adjuvant oil phase and an antigen water phase, emulsifying, and finally forming a vaccine emulsion.

When the traditional oil adjuvant is adopted to prepare the veterinary vaccine, the adjuvant oil phase and the antigen water phase can form a stable emulsion under the action of high-speed shearing force of the emulsion, and the emulsion has high technical requirements on emulsification equipment and high energy consumption.

Therefore, the novel oil adjuvant which can form a stable vaccine emulsion with an antigen water phase under the condition of low shearing force is developed, the preparation process of the veterinary vaccine can be greatly simplified, the energy consumption is reduced, and the safety of the preparation process of the vaccine is improved.

Reference to the literature

(1) Agricultural product date cargo net national statistical office: 6.88 hundred million heads of the live pigs are raised in the year 2017, and the number of the live pigs is increased by 0.5 percent. http:// finish. sina. com. cn/money/future/agri/2018-01-23/doc-ifyqtycx2309650. shtml.

(2) 2017, 42 hundred million white feather broilers are listed in China, and 37 hundred million Chinese chickens are listed in China. http:// www.xinm123.com/html/ex-info/475770. html.

(3) Qinyuyimin, Zhao hoe, Liyu, Wangman, evaluation of foreign vaccine adjuvant, Chinese veterinary magazine, 2005, 36: 34-36.

(4) Heng, zingiber officinale, mellowia, soneliapine, yuejiangxin, quality analysis of white oil adjuvants from different sources and research on safety and immune efficacy of avian influenza vaccines prepared from the same. 15-18.

(5) The river husbandry, the chicken industry, and http:// www.hemumuye.com are important issues that plague epidemic diseases.

(6)Stone HD.Efficacy of Experimental Animal and Vegetable Oil-Emulsion Vaccines for Newcastle Disease and Avian Influenza.Avian Diseases，1993,37(2)：399-405。

Disclosure of Invention

The invention aims to solve the problem of providing an oil adjuvant for preparing a vaccine by low shearing force and a preparation method and application thereof; the invention can overcome the defect that the traditional vaccine oil adjuvant can not be emulsified under low shearing force to form stable emulsion.

In order to solve the technical problems, the invention provides a preparation method of an oil adjuvant E515-A for preparing a vaccine with low shearing force, wherein the oil adjuvant E515-A comprises a component A1 and a component A2, and the weight ratio of the component A1: the volume ratio of the component A2 is 60-70: 30-40;

s1, preparation of component A1:

adding and dissolving span-80 and span-85 into white oil at 25-45 ℃ under a stirring state to obtain a component A1;

the volume ratio of the white oil to the span-80 to the span-85 is 85-87: 8-10: 4-6;

s2, preparation of component A2:

mixing Tween-80 and polyethylene glycol-200 in a volume ratio of 22-24: 75-78 (preferably 23: 75-78) to obtain a component A2.

Description of the drawings: s2 is carried out at room temperature (10-30 ℃).

The invention also provides the application of the oil adjuvant for preparing the vaccine with low shearing force, which comprises the following steps: firstly, mixing the component A1 and the component A2 in a volume ratio of 60-70: 30-40 to form an oil adjuvant E515-A, and then mixing the oil adjuvant E515-A and an antigen-containing water phase in a volume ratio of 1: 1; and (3) emulsifying the obtained mixed solution in a shaking table at 50-200 rpm at room temperature for 1-5 minutes to obtain the vaccine emulsion.

Description of the drawings: the oil adjuvant needs to be ready for use, i.e. the component a1 and the component a2 are mixed just before the vaccine emulsion is prepared.

The white oil is colorless, tasteless and transparent oily liquid, and is mineral oil after deep refining, and the basic component of the mineral oil is saturated hydrocarbon.

The invention has the beneficial effects that: the adjuvant oil E515-A and the water phase containing the antigen can complete the emulsification process under low shearing force, simplify emulsification equipment and reduce energy consumption in the emulsification process. The low-shearing force emulsification can stabilize the temperature of the system, maintain the integrity of the antigen and improve the quality of the vaccine.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a photograph of an emulsion stained with the liposoluble dye Sudan III (A) and the water-soluble dye Sudan-Y (B);

FIG. 2 is a particle size distribution of an oil emulsion vaccine containing adjuvant E515-A;

FIG. 3 is a comparison of the stability of vaccines;

(A) conventional white oil adjuvants have low shear (100 rpm); (B) high shear force of conventional white oil adjuvant (18000 rpm); (C) E515-A adjuvant low shear (100 rpm);

FIG. 4 is NK cell killing activity;

FIG. 5 is a graph of mouse serum IFN- γ levels;

FIG. 6 is mouse serum IgG levels;

FIG. 7 is mouse serum IgG subclass levels;

FIG. 8 is spleen lymphocyte stimulation index.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

no. 10 white oil, available from Hangzhou oil refineries of the petrochemical group of China;

span-80, available from Guangzhou super Industrial Co., Ltd;

span-85, available from Aladdin reagents (Shanghai) Inc.;

tween-80, available from pharmaceutical group chemicals, ltd;

polyethylene glycol 200, available from alatin reagent (shanghai) ltd.

The component A1 is prepared at 25-45 ℃; the component A2 is prepared at room temperature (10-30 ℃).

Example 1-1: component A1 preparation scheme I

85mL of No. 10 white oil, 85mL of span-8010 mL of span-855 mL are taken and mixed evenly (stirred for 2 minutes at the rotating speed of 60 rpm) to obtain 100mL of a component A1.

Examples 1 to 2: component A1 preparation scheme two

Taking 86mL of No. 10 white oil, span-809 mL and span-855 mL, and mixing uniformly to obtain 100mL of component A1.

Examples 1 to 3: component A1 preparation scheme III

Taking 87mL of No. 10 white oil, span-807 mL and span-856 mL, and mixing uniformly to obtain 100mL of a component A1.

Example 2-1: component A2 preparation scheme I

Mixing Tween-8022 mL and polyethylene glycol 20078 mL (stirring at 120rpm for 1 min) to obtain 100mL of component A2.

Example 2-2: component A2 preparation scheme two

Mixing Tween-8023 mL and polyethylene glycol 20077 mL to obtain 100mL of component A2.

Examples 2 to 3: component A2 preparation scheme III

Mixing Tween-8024 mL and polyethylene glycol 20076 mL to obtain 100mL of component A2.

Example 3: vaccine formulation with component A1 and component A2 (combination 1)

60mL of component A1 (example 1-2) and 40mL of component A2 (example 2-2) were mixed thoroughly (stirring at 120rpm for 1 minute) to give 100mL of E515-A. And mixing E515-A and inactivated epidemic diarrhea virus antigen (PEDV) (10)^6.75TCID₅₀and/mL) (the benefit of preventive veterinary research institute of animal science of Zhejiang university) at 30 ℃, mixing according to the volume ratio of 1:1, placing in a shaking table (SHKE6000-8CE, product of Thermo company, USA), shaking at 30 ℃ and 200rpm for 1 minute to obtain the PEDV oil emulsion vaccine containing the E515-A adjuvant.

After the vaccine emulsion is centrifuged at 3000rpm for 15 minutes, no oil-water separation phenomenon is found, which indicates that the emulsion is stable.

Example 4: vaccine formulation with component A1 and component A2 (combination 2)

60mL of component A1 (example 1-1) and 40mL of component A2 (example 2-3) were mixed thoroughly (stirring at 120rpm for 1 minute) to give 100mL of E515-A. And mixing E515-A and inactivated epidemic diarrhea virus antigen (PEDV) (10)^6.75TCID₅₀/mL) at 30 ℃ according to the volume ratio of 1:1, placing the mixture in a shaking table, shaking the mixture at 30 ℃ and 200rpm for 1 minute to obtain the PEDV oil emulsion vaccine containing the E515-A adjuvant.

Example 5: vaccine formulation with component A1 and component A2 (combination 3)

60mL of component A1 (example 1-3) and 40mL of component A2 (example 2-1) were mixed thoroughly (stirring at 120rpm for 1 minute) to give 100mL of E515-A. And mixing E515-A and inactivated epidemic diarrhea virus antigen (PEDV) (10)^6.75TCID₅₀/mL) at 30 ℃ according to the volume ratio of 1:1, placing the mixture in a shaking table, shaking the mixture at 30 ℃ and 200rpm for 1 minute to obtain the PEDV oil emulsion vaccine containing the E515-A adjuvant.

In the above examples, the reaction is carried out at room temperature (10 to 30 ℃ C.) without any specific limitation.

Experiment 1: emulsion type, stability, viscosity and particle size of E515-A containing vaccines

1. Method of producing a composite material

1.1. And (3) emulsion type identification: the vaccine emulsion (example 7) was stained with a water-soluble dye, eosin-Y (product of Shanghai Biotech Co., Ltd.) and a fat-soluble dye, Sudan III (product of Shanghai Biotech Co., Ltd.), and the staining of the inner and outer phases was observed.

1.2. And (3) stability detection: 10mL of the vaccine emulsion (example 3) was pipetted into a centrifuge tube and centrifuged at 5810R (product of Eppendorf, Germany) at 3000rpm for 15min to observe precipitation and separation.

1.3. And (3) viscosity measurement: the viscosity of the vaccine emulsion (example 3) was measured by means of an NDJ-8S type rotational viscometer (product of Shanghai Changji geological instruments Co., Ltd.).

1.4. And (3) particle size measurement: the size and distribution of the particles of the vaccine emulsion (example 3) was measured using a Nano ZS model nanosized particle size potential analyser (product of Malvern instruments, Inc., UK).

2. Results and analysis

2.1. Emulsion type:

FIG. 1 shows that the water-soluble dye eosin stains the aqueous phase at the periphery of the lipid droplet (B); the fat-soluble dye sudan iii dyes the oil phase lipid droplets (a). The vaccine is an oil-in-water emulsion.

2.2. Stability: after the vaccine is centrifuged at 3000rpm for 15 minutes, no oil-water stratification is found, which indicates that the emulsion stability is good.

2.3. Viscosity: the viscosity of the vaccine was 18. + -.2 mPa.s, indicating good flowability of the vaccine (veterinary code <200 mPa.s).

2.4. Particle size distribution: FIG. 2 shows that the particle size distribution of the emulsion is 106-122 nm (92%) and 6-8 nm (8%), and the average particle size is 106.9 nm.

Experiment 2: comparison of stability of E515-A adjuvant vaccine emulsion and conventional oil adjuvant vaccine emulsion

1. Materials and methods

Preparation of E515-A adjuvant vaccine: the same as in example 3.

1.2. Conventional white oil adjuvant low shear preparation of vaccines: oil phase, 10mL of No. 10 white oil (Hangzhou oil refinery product of China petrochemical group) containing 6% span-80 (product of Guangzhou super energy industry Co., Ltd.). Preparing water phase, and preparing inactivated epidemic diarrhea virus antigen (PEDV) (10) containing 3% Tween-80 (product of national drug group chemical reagent Co., Ltd.)^6.75TCID₅₀mL) (the gift from the institute of veterinary prevention institute of animal science, university of zhejiang). And mixing the oil phase and the water phase according to the volume ratio of 1:1, and oscillating at 30 ℃ and 200rpm for 1 minute to obtain the PEDV vaccine prepared by the conventional white oil adjuvant under low shear force.

The above% is volume%.

1.3. Vaccine preparation with conventional oil adjuvant high shear: oil phase, 10mL of No. 10 white oil (Hangzhou oil refinery product of China petrochemical group) containing 6% span-80 (product of Guangzhou super energy industry Co., Ltd.). Preparing water phase, and preparing inactivated epidemic diarrhea virus antigen (PEDV) (10) containing 3% Tween-80 (product of national drug group chemical reagent Co., Ltd.)^6.75TCID₅₀mL) (the gift from the institute of veterinary prevention institute of animal science, university of zhejiang). Mixing oil phase and water phase at a volume ratio of 1:1, emulsifying at 30 deg.C in a high speed emulsifying machine

T10 basic，ULTRA-

)18000rpm for 1 minute to obtain PEDV vaccine prepared by conventional white oil adjuvant with high shearing force.

1.3. And (3) stability detection: after centrifugation of the three vaccines at 3000rpm for 15 minutes, oil-water stratification was observed.

2. Results

FIG. 3 shows that the vaccine (A) prepared by the low-shear method using the conventional white oil adjuvant after centrifugation shows no delamination, and the vaccine (B) prepared by the high-shear method and the vaccine (C) prepared by the E515-A adjuvant under the low-shear method show no delamination.

Experiment 3: immune response induced by PEDV vaccine containing E515-A adjuvant (combination 1)

1. Method of producing a composite material

1.1. Animal grouping and treatment: male ICR mice, 6-8 weeks old, 18-20g, were purchased from Shanghai Schlenk laboratory animals, Inc. The 60 mice were randomly divided into 5 groups of 12 mice each, and injected with 0.2mL of each abdominal subcutaneous injection:

(1) blank group: 0.2mL of a physiological saline solution,

(2) non-adjuvant control group: PEDV antigen (10)^6.75TCID₅₀mL) + physiological saline (PEDV antigen and physiological saline mixed in a volume ratio of 1: 1),

(3) white oil group: PEDV antigen (10)^6.75TCID₅₀mL) + white oil (PEDV antigen and white oil mixed in a volume ratio of 1: 1), emulsified at 18000rpm (high shear),

(4) group E515-A: prepared as in example 3;

(5) aluminum glue group: PEDV antigen (10)^6.75TCID₅₀(iv)/mL) + an alumina gel adjuvant (PEDV antigen and 20% aluminum hydroxide physiological saline mixed in a volume ratio of 1: 1);

immunizations were performed twice, two weeks apart. In each group, 6 mice were randomly selected, blood was collected 24h after primary immunization, serum was separated for detection of IFN- γ level, and spleen was aseptically separated to prepare lymphocytes for detection of NK cell killing activity. An additional 6 mice in each group were bled at 1 and 2 weeks post-hyperimmunization for serum isolation for detection of PEDV-specific IgG antibody and its subclass IgG1 and IgG2a levels, and spleens were aseptically isolated 2 weeks post-hyperimmunization for lymphocyte proliferation assays.

NK cell killing activity assay: aseptically separating spleen, grinding in mortar, sieving with 200 mesh sieve, centrifuging the filtrate at 1500 rpm for 8min, discarding supernatant, adding erythrocyte lysate, treating on ice for 3min, centrifuging at 1500 rpm for 8min, discarding supernatant, washing with Hank's solution for 3 times, adding prepared cell culture solution (containing 10% fetal calf serum and 1% penicillin/streptomycin double antibody), and adjusting lymphocyte concentration to 1 × 10⁸And 5X 10⁷one/mL, ready for use as effector cells. Adjusting the concentration of K562 cells to 2X 10⁶one/mL was used as target cell. Adding 100 μ L of two different concentrations of effector cells and 100 μ L of target cells (effective target ratio is 50:1 and 25:1, and effective target ratio is the ratio of effector lymphocyte cell number to target cell K562 cell number) into 96-well cell culture plate, respectively, adding 100 μ L of 1640 culture solution into control wells of effector cells and target cells, respectively, placing in 5% CO₂Culturing at 37 deg.C for 5h in cell culture box, adding 50 μ L MTT solution (2mg/mL), culturing for 4h, centrifuging at 1500 rpm for 10min, discarding supernatant, adding 150 μ L DMSO, shaking in dark for 15min, and measuring OD₅₇₀Values, and calculating NK cell killing activity according to the formula: [1- (Effector cell + target cell hole OD value-Effector cell hole OD value)/target cell hole OD value]×100％。

1.3. Splenic lymphocyte proliferation assay: spleen lymphocytes were prepared as above, and cell concentration was adjusted to 5X 10⁶one/mL. mu.L of lymphocytes were added to each 96-well culture plate, and then 100. mu.L of 1640 culture solution and Con A (5. mu.g. mL) were added thereto^-1) Or LPS (4. mu.g/mL), in 5% CO₂Culturing at 37 deg.C for 44h in cell culture box, and detecting OD by MTT method₅₇₀Values were calculated and lymphocyte Stimulation Index (SI) ═ stimulated-blank-OD value)/(unstimulated-blank-OD value).

1.4. Detection of serum IgG antibodies and subclasses thereof: detection by an indirect ELISA method, 100. mu.L of a 1: 1600 diluted serum to be detected is added into a 96-well plate pre-coated with a PEDV antigen, incubation is carried out for 60min at 37 ℃, and 100. mu.L of goat anti-mouse IgG labeled with HRP (1: 40000 dilution), IgG1(1:1500 dilution) or IgG2a (1: 1500 dilution) antibody, incubating at 37 deg.C for 30min, washing plate, adding 100 μ L TMB developing solution for 10min, adding 50 μ L stop solution, and detecting OD₄₅₀The value is obtained.

1.5. Serum cytokine assay: the fELISA kit produced by Wuhan doctor Ded bioengineering GmbH is adopted to detect the level of IFN-gamma in mouse serum, and the specific operation is carried out according to the instruction.

1.6. And (3) data statistics: the test results are expressed as Mean ± standard deviation (Mean ± SE) and were significantly different when tested using the Duncan method of One-way ANOVA post-hoc multiple comparisons in SPSS 24.0 statistical software, P < 0.05.

2. Results and analysis

E515-A increased the killing activity of NK cells, see FIG. 4.

The NK cell killing activity of the vaccine added with the adjuvant group is obviously higher than that of a control group without the adjuvant (P < 0.05). According to the size of the killing activity of the NK cells, the arrangement order of each group is as follows: group E515-A > white oil group > alumina gel group > non-adjuvanted control group (PEDV antigen + physiological saline); the latter two are similar: under the present experimental conditions, the PEDV antigen needs to be in the presence of an adjuvant to function.

E515-A increased serum IFN- γ levels, see FIG. 5.

The serum IFN-gamma of the adjuvant-added group in the vaccine is obviously higher than that of the non-adjuvant control group (P < 0.05). According to the IFN-gamma level, the sequence of each group is as follows: group E515-A > white oil group > alumina gel group > no adjuvant control group.

E515-A increased serum specific antibody levels, see FIG. 6.

The serum IgG level in the adjuvanted group was significantly higher in the vaccine than in the non-adjuvanted control group (P < 0.05). According to the IgG level, the arrangement order of each group is as follows: group E515-A > white oil group > alumina gel group > no adjuvant control group.

E515-A increased serum-specific antibody subclass levels, see FIG. 7.

The serum IgG subclass level of the adjuvanted group in the vaccine was significantly higher than that of the non-adjuvanted control group (P < 0.05). According to the level of the IgG subclass, the arrangement order of each group is as follows: group E515-A > white oil group > alumina gel group > no adjuvant control group.

E515-A promotes lymphocyte proliferation response, see FIG. 8.

The lymphocyte stimulation index of the adjuvant-added group in the vaccine is significantly higher than that of the non-adjuvant control group (P < 0.05). According to the stimulation index, the arrangement order of each group is as follows: group E515-A > white oil group > alumina gel group > no adjuvant control group.

Description of the drawings: after the antigen + white oil (high shearing force) emulsification, the temperature of the vaccine is increased, the immune response of the vaccine group is lower than that of the antigen + E515-A (low shearing force), and the immune response is probably related to the structure of the antigen which is changed by the temperature increase in the emulsification process.

Experiment 4: immune response induced by PEDV vaccine with E515-A adjuvant (combinations 2 and 3)

36 mice were randomly divided into 6 groups of 6 mice each, immunized twice, two weeks apart. Serum was isolated 24h after primary immunization for detection of IFN- γ levels and 1 and 2 weeks after secondary immunization for detection of PEDV-specific IgG antibodies. Other experimental methods follow experiment 3, but the vaccine of group E515-A was replaced with the vaccine obtained in examples 4 and 5, and the test items only detected serum IgG and serum IFN-. gamma.levels. The results are shown in Table 1.

Table 1 serum IgG and IFN- γ levels 1 week after the diimmune (n ═ 6)

Group of	Serum IgG level (OD450)	Serum IFN-gamma levels (pg/mL)
			Blank group	0.1237±0.0591	160.3±28.9
Non-adjuvanted control group	0.1245±0.0639	305.2±22.2
			White oil (high shearing force)	0.8887±0.0978	440.3±29.4
Group E515-A (example 4)	1.2078±0.1028	490.5±26.7
			Group E515-A (example 5)	1.1921±0.1031	501.2±27.2
Aluminum glue group	0.4081±0.0917	390.4±24.6

As can be seen from Table 1, the E515-A + antigen group showed significantly higher serum IgG and IFN-. gamma.levels than the other groups.

Comparative example 1-1, 60mL of "component A1 (example 1-2), 40mL of" component A2 (example 2-2) "was changed to" component A180 mL, component A220 mL "and the rest was identical to example 3.

Comparative example 1-2, 60mL of "component A1 (example 1-2), 40mL of" component A2 (example 2-2) "was changed to" component A150 mL, component A250 mL "and the rest was identical to example 3.

Comparative example 2-1, component a1 was modified to: 80mL of No. 10 white oil, span-8010 mL of the white oil, and span-8510 mL of the white oil, and the balance of the white oil is equivalent to example 3.

Comparative examples 2-2, component a1 was modified to: no. 90 white oil 80mL, span-805 mL, span-855 mL, the rest is equivalent to example 3.

The vaccine emulsions prepared in the 4 comparative examples were found to separate into oil and water layers after centrifugation at 3000rpm for 15 minutes, indicating that the emulsions were unstable and could not be used for vaccine applications.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims

1. The preparation method of the oil adjuvant for preparing the vaccine with low shearing force is characterized by comprising the following steps: the oil adjuvant comprises a component A1 and a component A2, wherein the component A1: the component A2 is 60-70: 30-40 in volume ratio;

s1, preparation of component A1:

adding span-80 and span-85 into white oil at 25-45 ℃ under stirring and dissolving to obtain a component A1;

s2, preparation of component A2:

mixing Tween-80 and polyethylene glycol-200 in a volume ratio of 22-24: 75-78 to obtain a component A2.

2. The method of claim 1, wherein the oil adjuvant is prepared by the following steps: the volume ratio of the Tween-80 to the polyethylene glycol-200 is 23: 75-78.

3. An oil adjuvant for a low shear formulated vaccine prepared according to the method of claim 1 or 2.

4. Use of an oil adjuvant according to claim 3 wherein: firstly, mixing the component A1 and the component A2 in a volume ratio of 60-70: 30-40 to form an oil adjuvant; mixing the oil adjuvant and the water phase containing the antigen in a volume ratio of 1: 1; and (3) emulsifying the obtained mixed solution in a shaking table at 50-200 rpm at room temperature for 1-5 minutes to obtain the vaccine emulsion.