CN111358944A

CN111358944A - Composite vegetable oil vaccine adjuvant and application thereof

Info

Publication number: CN111358944A
Application number: CN202010132740.6A
Authority: CN
Inventors: 胡松华
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-02-29
Filing date: 2020-02-29
Publication date: 2020-07-03
Anticipated expiration: 2040-02-29
Also published as: CN111358944B

Abstract

The invention discloses a composite vegetable oil vaccine adjuvant which is prepared according to the following method: 1) the composite vegetable oil adjuvant comprises the following components in percentage by volume: 1.50-3.5% of oleic acid, 9.5-12.5% of span-85, 2.50-3.50% of polyglycerol ricinoleate and the balance of vegetable oil; 2) and adding a ginsenoside solution into the composite vegetable oil adjuvant according to the dosage ratio of 2-8 mg of ginsenoside to 100ml of composite vegetable oil adjuvant to obtain the composite vegetable oil vaccine adjuvant. After being injected into animals, the vaccine prepared by the adjuvant is easy to be absorbed by organisms, vaccine residues are not easy to form at the injection part, the duration time of the antibody can be prolonged, and the immune animals can be better protected.

Description

Composite vegetable oil vaccine adjuvant and application thereof

Technical Field

The invention relates to the technical field of vaccine adjuvants, in particular to a vegetable oil vaccine adjuvant containing ginsenoside, a preparation method and application thereof.

Background

Livestock and poultry breeding provides important protein sources for human beings, and is an indispensable important industry for human beings. In a large-scale farm, because animals are raised at high density, the animals are frequently contacted, so that epidemic diseases are more easily spread in the animals. The vaccine is injected into animals to enable the animals to generate specific immune response, the resistance of the animals to epidemic diseases is improved, the occurrence of the epidemic diseases can be reduced, the economic loss of farmers caused by the epidemic diseases is reduced, and the vaccine is an important measure for preventing the occurrence of the animal epidemic diseases. In order to improve the immune effect of the vaccine, an adjuvant needs to be added into the vaccine to enhance the strength of the immune response induced by the vaccine and prolong the duration of the immune response.

The animal vaccine adjuvant adopted in China at present is white oil which is mainly prepared from mineral oil. The white oil mainly contains a mixture of n-isoparaffin of C16-C31, and is difficult to be absorbed and metabolized by animals. Animal experiments prove that the polycyclic aromatic hydrocarbon components as impurities in the white oil have carcinogenic effect on organisms. Therefore, the use of mineral oil-based adjuvants can pose a potential threat to food safety. In addition, the vaccine emulsion prepared from mineral oil is not easy to be absorbed after being injected into animals, and can be retained at the injection part for a long time to cause side effects such as local tissue inflammation, suppuration, necrosis and the like, thereby influencing the quality of animal products. Thus, the use of oil-adjuvanted vaccines has been restricted in some developed countries, for example, the U.S. government has prohibited animals from injecting oil-adjuvanted vaccines within 42 days of marketing as early as more than 20 years ago.

The existing mineral oil vaccine adjuvant mainly has the following defects:

1. polycyclic aromatic hydrocarbon components remained in the mineral oil adjuvant have carcinogenic effect, and the adjuvant can pose potential threat to food safety after being prepared into vaccines to immunize animals;

2. the traditional mineral oil adjuvant is not easy to absorb and metabolize in animals, and the quality of animal products is influenced;

3. the traditional mineral oil adjuvant has the advantages of lower flash point, easy combustion and unsafe storage.

In view of the above, there is a need for further improvements in the prior art.

Reference documents:

(1) the annual pig slaughtering amount in 2012 is estimated to be 7.1 hundred million;

(2) the state of the broiler industry and chicken safety in China, livestock and veterinary medicine today, 2013, 05, 40-42;

(3) the third Chinese veterinary drug congress corpus, 2010: 34-39.

(4) Lilieright, white oil and white oil production technology, lubricating oil, 2003, 18 (4): 1-6.

(5) 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;

(6) chengshihao, Haohuoyang, Schgrand, etc. oil emulsion adjuvants and progress in safety studies thereof, Chinese poultry, 2013, 35 (16): 44-46.

(7) Zeng, design and selection of vaccine adjuvant: foreign medicine (biological product for prevention, diagnosis, treatment), 2003, 26 (2): 67-70.

(8) White Manying, Zhangjincheng, adulterated grain and oil food identification and inspection, Beijing: china standard press, 1996.

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

(10) The river grazing and animal husbandry and the chicken raising industry take an important way for the plague of epidemic diseases;

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

201310754903.4 patent of "a vegetable oil adjuvant containing ginsenoside and its preparation method and application" teaches the preparation method of vegetable oil adjuvant: dissolving ginsenoside in dimethyl sulfoxide, and mixing to obtain ginsenoside solution; adding the ginsenoside solution into vegetable oil, and directly and uniformly mixing; or heating the vegetable oil to 20-40 ℃, adding the sorbitan monooleate and the ginsenoside solution into the vegetable oil, and uniformly mixing.

201910518452.1 the invention vegetable oil vaccine adjuvant, its preparation method and application, informs a preparation method of vegetable oil containing ginsenoside and vitamin E: adding span-80 and tween-80 into vegetable oil, and adding vitamin E and ginsenoside solution to obtain component A; mixing glycerol and 1, 2-propylene glycol to obtain a component B; and mixing the component A and the component B to obtain the vegetable oil vaccine adjuvant.

The vegetable oil adjuvant obtained in the two patents is used for preparing the vaccine, is an oil-in-water (O/W) emulsion, and has weaker adjuvant effect.

Disclosure of Invention

The invention aims to solve the technical problem of providing a ginsenoside-containing composite vegetable oil vaccine adjuvant, and a preparation method and application thereof.

In order to solve the technical problems, the invention provides a composite vegetable oil vaccine adjuvant which is prepared according to the following method:

1) the composite vegetable oil adjuvant comprises the following components in percentage by volume:

1.50-3.5% of oleic acid, 9.5-12.5% of span-85, 2.50-3.50% of polyglycerol ricinoleate (PGPR), and the balance of vegetable oil;

2) and adding a ginsenoside solution into the composite vegetable oil adjuvant according to the dosage ratio of 2-8 mg of ginsenoside to 100ml of composite vegetable oil adjuvant to obtain the composite vegetable oil vaccine adjuvant.

As an improvement of the composite vegetable oil vaccine adjuvant of the invention:

the vegetable oil is camellia oil, peanut oil, linseed oil, sunflower seed oil, rice oil, corn oil, soybean oil, blend oil, grape seed oil or rapeseed oil;

the preparation method of the composite vegetable oil adjuvant in the step 1) comprises the following steps: adding oleic acid, span-85 and polyglycerol ricinoleate (PGPR) into vegetable oil at 35-50 ℃, and uniformly mixing to obtain a composite vegetable oil adjuvant;

the preparation method of the ginsenoside solution in the step 2) comprises the following steps: and adding 5-15 mg of ginsenoside into 1 ml of dimethyl sulfoxide, and uniformly mixing to obtain a ginsenoside solution.

The invention also provides the application of the composite vegetable oil vaccine adjuvant: taking a composite vegetable oil vaccine adjuvant (a composite vegetable oil adjuvant containing ginsenoside) as an oil phase, and taking an aqueous solution containing antigen as a water phase;

heating the oil phase and the water phase to 35-40 ℃ respectively, and then mixing and emulsifying according to the volume ratio of 2:1 to obtain the water-in-oil (W/O) vaccine emulsion.

The vaccine prepared by the invention can be used according to the conventional use method.

Compared with the prior art, the invention has the following technical advantages:

(1) the invention adopts edible vegetable oil as raw material to prepare the vaccine adjuvant, thus greatly improving the safety of the vaccine and overcoming the potential harm of the traditional mineral oil adjuvant to food sanitation.

(2) The composite vegetable oil adjuvant is superior to the traditional adjuvant in the aspects of promoting the generation of the antibody and prolonging the effective period of the antibody.

(3) The vaccine prepared by the oil adjuvant is of a water-in-oil (W/O) type, and the natural vegetable oil is adopted, so that the vaccine has better compatibility with the conventional mineral oil and animal tissues, is easier to absorb and metabolize and is easy to absorb at an injection part, the defect that the conventional mineral oil adjuvant vaccine is not easy is overcome, and the quality of animal products is improved.

(4) The polycyclic aromatic hydrocarbon component in the adjuvant is greatly reduced, and the adjuvant has good food safety; the flash point is small, the material is not easy to burn, and the safety is good in the transportation, storage and processing processes;

in conclusion, the vaccine prepared by the adjuvant of the invention is easy to be absorbed by the body after being injected into animals, vaccine residue is not easy to form at the injection part, the duration time of the antibody can be prolonged, and the immune animals can be better protected.

Drawings

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

FIG. 1 comparison of IgG levels of antibody induced by patterned antigen (BSA) with complex vegetable oil adjuvants (prepared by methods of examples 1, 5, 9, and 13) (groups 2-5) and No. 10 white oil (group 1).

FIG. 2 comparison of IgG levels of antibody induced by patterned antigen (BSA) with the complex vegetable oil adjuvant (prepared by the methods of examples 2, 6, 10, and 14) (groups 2-5) and No. 10 white oil (group 1).

FIG. 3 comparison of IgG levels of antibody induced by patterned antigen (BSA) with the complex vegetable oil adjuvant (prepared by methods of examples 3, 7, 11, and 15) (groups 2-5) and No. 10 white oil (group 1).

FIG. 4 comparison of IgG levels of antibody induced by patterned antigen (BSA) with the complex vegetable oil adjuvant (prepared by methods of examples 4, 8, 12, and 16) (groups 2-5) and No. 10 white oil (group 1).

FIG. 5 comparison of IgG level of induced antibodies of foot-and-mouth disease vaccine by composite vegetable oil adjuvant (prepared by methods of examples 1, 5, 9 and 13) (groups 2-5) and No. 10 white oil (group 1).

FIG. 6 comparison of NDV vaccine induced HI titers by camellia oil, linseed oil, peanut oil, corn oil, grapeseed oil, sunflower oil, blend oil, rapeseed oil, soybean oil, cottonseed oil, rice oil type composite vegetable oil adjuvants (groups 3-13) and Mc52 (group 2).

Detailed Description

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

The raw materials used in the preparation process of the composite vegetable oil vaccine adjuvant can be obtained by a conventional and commercially available mode.

For example:

oleic acid (Aladdin Biotechnology, Inc., Shanghai, China),

span-85 (Aladdin Biotechnology, Inc., Shanghai, China),

polyglycerol polyricinoleate (cameisu biotechnology limited, shanghai, china),

camellia oil (Yihaijiali food industry Co., Ltd., Shanghai, China),

dimethyl sulfoxide (Aladdin Biotechnology Ltd., Shanghai, China),

ginsenoside (macrojiu group, Jilin, China).

Example 1, a method for preparing a composite vegetable oil (camellia oil) vaccine adjuvant, sequentially performing the following steps:

1) preparing a composite vegetable oil adjuvant:

according to the volume ratio, 1.5 percent of oleic acid, 12.5 percent of span-85, 2.5 percent of polyglycerol ricinoleate and the residual camellia oil are uniformly mixed at 35 ℃ to obtain the composite vegetable oil adjuvant.

2) Preparing the ginsenoside-containing composite vegetable oil adjuvant:

firstly, 10mg of ginsenoside is added into each ml of dimethyl sulfoxide and evenly mixed to obtain a ginsenoside solution. Then adding the ginsenoside solution into the composite vegetable oil adjuvant according to the dosage ratio of 2mg of ginsenoside to 100ml of composite vegetable oil adjuvant to obtain the composite vegetable oil (camellia oil) vaccine adjuvant.

Example 2 composite vegetable oil (Camellia oil) vaccine adjuvant

The composite vegetable oil adjuvant comprises the following components in percentage by volume: 3.5% of oleic acid, 9.5% of span-85 and 3.5% of polyglycerol ricinoleate; the balance being camellia oil.

The rest is equivalent to embodiment 1.

Example 3 composite vegetable oil (Camellia oil) vaccine adjuvant

The composite vegetable oil adjuvant is changed into a composite vegetable oil adjuvant which comprises the following components in percentage by volume: 2.5% of oleic acid, 11.5% of span-85 and 2.5% of polyglycerol ricinoleate; the balance being camellia oil.

The rest is equivalent to embodiment 1.

Example 4 composite vegetable oil (Camellia oil) vaccine adjuvant

In the step 2), the dosage ratio of the ginsenoside of 8mg to the composite vegetable oil adjuvant of 100ml is changed;

the rest is equivalent to embodiment 1.

Example 5-example 8, composite vegetable oil (peanut oil) vaccine adjuvant:

changing the camellia oil in the embodiments 1 to 4 into peanut oil; the rest are equal; thus obtaining the composite vegetable oil (peanut oil) vaccine adjuvant described in the example 5-8.

Examples 9-12, composite vegetable oil (corn oil) vaccine adjuvant:

changing the camellia oil of the embodiments 1 to 4 into corn oil; the rest are equal; thus obtaining the composite vegetable oil (corn oil) vaccine adjuvant described in the example 9-12.

Examples 13-16, composite vegetable oil (soybean oil) vaccine adjuvant:

the camellia oil in the embodiments 1 to 4 is changed into soybean oil; the rest are equal; thus, the adjuvant of the composite vegetable oil (soybean oil) vaccine described in example 13 to example 16 was obtained.

The following

experiments

1,2, 6, and 8 used the ginsenoside-containing composite vegetable oil adjuvants prepared by the methods of examples 1, 5, 9, and 13.

Experiment 1: the content of polycyclic aromatic hydrocarbon in the ginsenoside-containing composite vegetable oil adjuvant is lower than that of the conventional mineral oil adjuvant

1. Materials and methods

1.1. Vaccine adjuvants: the following adjuvants were used in this experiment,

(1) mc 52: product of ESSO Esso Petroleum Inc., France;

(2) no. 10 white oil: hangzhou oil refinery products of China petrochemical group, Hangzhou and China;

(2) camellia oil type composite vegetable oil adjuvant: prepared according to the method of example 1;

(3) peanut oil type composite vegetable oil adjuvant: prepared according to the method of example 5;

(4) corn oil type composite vegetable oil adjuvant: prepared according to the method of example 9;

(5) soybean oil type composite vegetable oil adjuvant: prepared according to the method of example 13;

1.2. the six adjuvants are sent to Shanghai professional company to detect the content of polycyclic aromatic hydrocarbon.

2. Results and analysis are given in Table 1 below

TABLE 1 polycyclic aromatic Hydrocarbon content in various oil adjuvants (μ g/kg)

Adjuvant	Content of fused ring aromatic hydrocarbon
		Mc52	2019.11
No. 10 white oil	219.24
		Camellia oil type composite vegetable oil adjuvant (example 1)	30.05
Corn oil type composite vegetable oil adjuvant (example 9)	29.98

Experiment 2: adjuvant effect of ginsenoside-containing composite vegetable oil adjuvant on model antigen bovine serum albumin BSA

1. Materials and methods

1.1. Experimental animals: female ICR mice (18-22 g) were purchased from Shanghai Spiker laboratory animals, Inc. Feeding in IVC independent air supply feeding cage. Feeding sterile feed and water at indoor temperature of 25 + -1 deg.C and humidity of 50% + -10%. The test was started 1 week after the animals were acclimatized.

1.2. Antigen solution: adding Bovine Serum Albumin (BSA) as model antigen (Sigma) into physiological saline, wherein the final concentration of BSA is 100 μ g/ml.

1.3. Vaccine adjuvants: except for Mc52, the procedure was as in experiment 1.

1.4. The preparation method of the experimental vaccine comprises the following steps: and (2) respectively heating the adjuvant and the antigen BSA solution in the step (1.3) to 35-40 ℃, mixing the adjuvant and the antigen BSA solution according to the volume ratio of 2:1, and emulsifying the mixture for 2 minutes (16000 revolutions per minute) by using an emulsifying machine (Beijing Zhuochuan electronic technology Co., Ltd., model number: FF-ESB-300) to prepare the corresponding milky vaccine emulsion. The normal saline and the antigen BSA solution are uniformly mixed according to the volume ratio of 2:1 for control.

1.5. Animal grouping and treatment: 36 mice were randomly divided into 6 groups. Animals in each group were injected intramuscularly (0.2 ml/time) with the experimental vaccine twice, two weeks apart as in table 2.

TABLE 2 animal grouping and treatment

1.6. Blood sampling

Blood is collected 1 and 2 weeks after the second immunization, and serum is prepared for antibody IgG detection.

1.7. The antibody detection method comprises the following steps: serum-specific IgG levels against BSA were detected by ELISA as follows:

(1) antigen coating: a96-well plate was used, and 100. mu.L/well of BSA solution (5. mu.g/mL in carbonate buffer, pH 9.6) was added thereto, followed by shaking, mixing, and sealing at 4 ℃ overnight.

(2) Washing: the liquid in the enzyme label plate is discarded, and PBST washing liquid is added according to 300 mu L/hole for washing 3 times. Patting to dry.

(3) And (3) sealing: fetal calf serum diluted in PBS at 5% concentration was added at 300. mu.L/well and incubated at 37 ℃ for 2 h.

(4) Washing the above, adding serum to be detected diluted by fetal calf serum into 100 μ L/hole, and incubating at 37 deg.C for 2 h.

(5) The cells were washed as above, and 100. mu.L/well of HRP-labeled goat anti-mouse antibody was added and incubated at 37 ℃ for 2 h.

(6) The same procedure was followed as above, and 100. mu.L/well of freshly prepared TMB substrate working solution (protected from light) was added and incubated at room temperature for 10 min.

(7) Adding 2M H2SO4 stop solution into 50 μ L/hole, mixing by gentle shaking, and measuring OD value at 450nm wavelength with enzyme label plate within 15 min.

2. Results and analysis

The result is shown in figure 1, the No. 10 white oil (group 1) and 4 ginsenoside-containing composite vegetable oils (groups 2-5) have adjuvant effect, and the No. 10 white oil is a vaccine adjuvant with good quality produced in China. The IgG level of the adjuvant group of the invention is higher than that of the No. 10 white oil group, which shows that the adjuvant effect of the invention is better than that of the No. 10 white oil.

Experiment 3: adjuvant effect of ginsenoside-containing composite vegetable oil adjuvant on model antigen bovine serum albumin BSA

1. Materials and methods

The composite vegetable oil adjuvant was prepared according to the methods of example 2, example 6, example 10 and example 14, respectively, and the rest of experiment 2.

2. Results and analysis

The results are shown in fig. 2, the No. 10 white oil (group 1) and 4 ginsenoside-containing compound vegetable oils (groups 2-5) both have adjuvant effect, and the IgG level of the adjuvant group of the invention is higher than that of the No. 10 white oil group, which indicates that the adjuvant effect of the invention is better than that of the No. 10 white oil.

Experiment 4: adjuvant effect of ginsenoside-containing composite vegetable oil adjuvant on model antigen bovine serum albumin BSA

1. Materials and methods

The composite vegetable oil adjuvant was prepared according to the methods of example 3, example 7, example 11 and example 15, respectively, and the rest of experiment 2.

2. Results and analysis

The results are shown in fig. 3, the number 10 white oil (group 1) and 4 ginsenoside-containing complex vegetable oils (groups 2-5) both had adjuvant effect, and the IgG level of the adjuvant group of the present invention was higher than that of the number 10 white oil group.

Experiment 5: adjuvant effect of ginsenoside-containing composite vegetable oil adjuvant on model antigen bovine serum albumin BSA (4)

1. Materials and methods

The composite vegetable oil adjuvant was prepared according to the methods of example 4, example 8, example 12 and example 16, respectively, and the rest of experiment 2.

2. Results and analysis

The results are shown in fig. 4, the number 10 white oil (group 1) and 4 ginsenoside-containing complex vegetable oils (groups 2-5) both had adjuvant effect, and the IgG level of the adjuvant group of the present invention was higher than that of the number 10 white oil group.

As can be seen from fig. 1 to 4, the ginsenoside-containing composite vegetable oil prepared from camellia oil, peanut, corn oil and soybean oil as main raw materials has a vaccine adjuvant effect on the model antigen BSA, and the adjuvant effect is higher than that of the number 10 white oil adjuvant. Through a subsequent experiment 7, the adjuvant effect of the compound vegetable oil containing ginsenoside prepared by using 11 vegetable oils as main raw materials on the antigen of the inactivated pathogen newcastle disease virus is further observed, and the adjuvant effects of the compound vegetable oil and the antigen of the inactivated pathogen newcastle disease virus are compared.

Experiment 6: immunity enhancement effect of ginsenoside-containing composite vegetable oil adjuvant on foot-and-mouth disease vaccine

1. Materials and methods

1.1. The antigen in experiment 2 was changed from "BSA" to "FMDV", i.e., the inactivated antigen of swine O-type Foot and Mouth Disease Virus (FMDV) was added to physiological saline, and the final concentration of the 146s antigen (foot and mouth disease virus particles with sedimentation coefficient of 146) was 9 μ g/ml, to obtain an antigen-containing aqueous phase, and the rest of experiment 2 was the same. Animals were grouped and treated as per table 3 with two injections of vaccine (0.2 ml/time) at two week intervals.

TABLE 3 animal grouping and treatment

Group of	Mouse	Adjuvant	146s (mug)/one injection
				1	6	No. 10 white oil	0.6
2	6	Camellia oil type composite vegetable oil adjuvant (example 1)	0.6
				3	6	Peanut oil type composite vegetable oil adjuvant (example 5)	0.6
4	6	Corn oil type composite vegetable oil adjuvant (example 9)	0.6
				5	6	Soybean oil type composite vegetable oil adjuvant (example 13)	0.6
6	6	Physiological saline	0.6

1.2. Blood collection: as in experiment 2.

1.3. The antibody detection method comprises the following steps: detecting anti-FMDV-specific antibodies in serum, comprising the steps of:

(1) coating: adding carbonate buffer solution (pH 9.6) into rabbit anti-O type FMDV serum according to the ratio of 1:8, uniformly mixing, adding 50 mu L of coating into each hole of a 96-hole enzyme label plate, and placing at 4 ℃ for incubation overnight;

(2) washing the plate: washing the 96-well plate 3-5 times with PBST (PBS containing 0.05% Tween-20), 300. mu.L per well, 2 minutes each time (the same below);

(3) and (3) sealing: adding 300 mu L of PBS blocking solution containing 5% (w/v) skim milk into each well, and incubating for 2h at 37 ℃;

(4) washing the plate, adding 50 mu L of 1:8 diluted O-type FMDV antigen into each hole, and incubating for 2h at 4 ℃;

(5) washing the plate, adding 50 mu L of serum to be detected and negative serum diluted by 1:200 into each hole, and incubating for 1h at 37 ℃;

(6) washing the plate, adding HRP-labeled goat anti-mouse IgG antibody diluted at the ratio of 1:1000, incubating at the temperature of 37 ℃ for 1h at the concentration of 50 mu L/hole;

(7) washing the plate, adding TMB substrate solution for color development, incubating at 50 μ L/well for 10min at 37 ℃;

(8) washing the plate, adding 50 μ L/well 2M H₂SO₄Terminating the reaction;

(9) measuring OD 450nm value by a microplate reader.

2. Results and analysis

The results are shown in fig. 5, the number 10 white oil (group 1) and 4 ginsenoside-containing composite vegetable oils (groups 2-5) both have adjuvant effect on FMDV vaccines, and the adjuvant effect of the invention is superior to that of the number 10 white oil.

Experiment 7: the compound vegetable oil adjuvant containing ginsenoside can prolong the antibody production time of animals

1. Materials and methods

1.1. Test animals: 1 day old three yellow broilers (zhenning poultry ltd., ningbo, china).

1.2. Vaccine adjuvants: the following adjuvants were used in this experiment:

(1) mc 52: product of ESSO Esso Petroleum Inc., France;

(2) the compound vegetable oil adjuvant containing ginsenoside comprises the following components: referring to example 2, 11 kinds of ginsenoside-containing composite vegetable oil adjuvants are respectively prepared from camellia oil, linseed oil, peanut oil, corn oil, grape seed oil, sunflower seed oil, blend oil, rapeseed oil, soybean oil, cottonseed oil, and rice oil (Yihai Jiali food industry, Ltd., Shanghai, China);

1.3. the preparation method of the experimental vaccine comprises the following steps: mixing the adjuvant obtained in the step 1.2 with an inactivated Newcastle disease virus antigen (NDV) solution according to a volume ratio of 2:1, and emulsifying for 2 minutes (16000 r/min) by using an emulsifying machine (model: FF-ESB-300, manufactured by Beijing Otrochwan electronic technology Co., Ltd.) to obtain a corresponding milky vaccine emulsion. The normal saline and the antigen NDV solution are uniformly mixed according to the volume ratio of 2:1 for comparison.

1.4. Animal grouping and treatment: 130 broilers were randomly divided into 13 groups and fed to 21 days of age, each chicken was given an intramuscular injection of NDV vaccine according to table 4.

TABLE 4 animal grouping and treatment

1.5. Blood collection: blood was collected 1,2, 3, 4, 5, 6 weeks after immunization.

1.6. The antibody detection method comprises the following steps: serum HI titers were measured using the Hemagglutination Inhibition (HI) assay, as follows:

1) and (3) adding 25 mu l of physiological saline into the 1 st to 10 th holes of each row of the 96-well plate by using a micropipette, wherein the 11 th hole and the 12 th hole are respectively used as 4 units of antigen control and positive serum control.

2) And sucking 25 mul of serum to be detected by a micropipette, placing the serum in the 1 st hole, then diluting the serum to the 10 th hole in a multiple ratio, and discarding 25 mul.

3) The prepared 4 units of antigen were pipetted 25. mu.l per well using a micropipette.

4) Placing on a micro-oscillator, oscillating for 1min, and mixing.

5) Standing at room temperature for 20 min.

6) Pipette 1% erythrocyte suspension into each well, 25. mu.l each.

7) Placing on a micro-oscillator, oscillating for 1min, and mixing.

8) The reaction mixture was allowed to stand at room temperature for 40min, and the results were observed.

9) And (4) judging the result: the HI titer of the serum was expressed as the log of 2 at the maximum dilution of the serum that completely inhibited erythrocyte agglutination.

2. Results and analysis

When the avian serum HI titer is greater than 4Log2, the animal is considered to have an anti-NDV infection effect. Mc52 is a common adjuvant for poultry vaccines. As shown in FIG. 6, both Mc52 and the ginsenoside-containing composite vegetable oil adjuvant can promote the production of specific antibody. However, the HI titer of Mc52 group decreased faster, and at 6 weeks after immunization, the HI titer was lower than 4, which was less than the protective antibody level, while the HI titer of the adjuvant group of the present invention was still greater than 4, which had a protective effect on the animals, indicating that the duration of the antibody could be prolonged.

A good vaccine should be able to maintain high levels of antibodies for a long period of time after immunization, and to confer infection resistance to the animal. The vaccine can be made to achieve this function by selecting the appropriate adjuvant. As can be seen from FIG. 6, the adjuvants prepared from different vegetable oils have different adjuvant effects. The adjuvants that maintain the antibody at a higher level still 6 weeks after immunization, in terms of high to low antibody titers, are in turn: corn oil, linseed oil, peanut oil, blend oil, grape seed oil, rapeseed oil, camellia oil, soybean oil, rice oil, sunflower seed oil and Mc52 cottonseed oil.

Experiment 8: the ginsenoside-containing composite vegetable oil adjuvant is easier to be absorbed at injection site than conventional mineral adjuvants

1. Materials and methods

1.1. Experimental animals: 1 day old three yellow broilers (zhenning poultry ltd., ningbo, china).

1.2. Vaccine adjuvants: the adjuvant used in this experiment was the same as in experiment 2;

1.3. the preparation method of the vaccine comprises the following steps: the adjuvant and the antigen NDV solution are mixed according to the volume ratio of 2:1, and emulsified for 2 minutes (16000 r/min) by an emulsifying machine (model: FF-ESB-300, Tokyo Zhuochuan electronic technology Co., Ltd.) to prepare the corresponding milky vaccine emulsion.

1.4. Animal grouping and treatment: 72 chickens were raised to two weeks (14 days old) and divided into 6 groups of 12 chickens. Then 0.3ml of NDV vaccine was administered intramuscularly in the chest as per Table 5. After immunization, 2 chickens were dissected from each group per week to examine the injection site for vaccine emulsion residue.

2. Results and analysis

The results of the observation are shown in Table 5. Vaccine residues were still seen at week 5, i.e. 49 days of age after 10 white oil and Mc52 adjuvant vaccine injection. This is not suitable for fast-growing broilers that are on the market at about 50 days of age. And no vaccine residue is found in 5 weeks after 4 vaccine injections of the composite vegetable oil adjuvant, so that the vaccine is suitable for broiler vaccines.

TABLE 5 time to detection of residual vaccine at injection site after immunization

-: no vaccine residue was found in 2 chickens; +: only 1 chicken was observed to have vaccine residues; ++: vaccine residues remained in 2 chickens.

Experiment 9: the flash point of the ginsenoside-containing composite vegetable oil adjuvant is lower than that of the conventional mineral adjuvant

1. Materials and methods

1.1. The ginsenoside-containing composite vegetable oil adjuvant comprises the following components: referring to examples 1, 5, 9 and 13, camellia oil type, peanut oil type, corn oil type and soybean oil type composite vegetable oil adjuvants were prepared, respectively.

1.2. Flash point determination: measured using a flash point measuring instrument (model: SYD-261-I, Shanghai Seiki Seiko Seiki Ltd., Shanghai, China). According to the specification, an adjuvant oil sample is poured into the detection cup, so that the oil surface is consistent with the scale marks in the cup. Turning on the stirrer, switching on an air source and igniting; and when the reading of the thermometer is increased to 90 ℃, adjusting the temperature increase speed to 2-3 ℃ per minute, igniting twice per minute, turning off the stirrer during ignition, and recording the reading of the thermometer when the flash fire occurs, namely the flash point of the detection sample.

2. Results and analysis

The flash point is the lowest temperature at which a flame causes the sample to catch fire with steam and spread the flame to the surface of the liquid. The flash point value is used for indicating whether combustible substances exist in the combustible substances, and is one of important parameters of petroleum product transportation, storage, operation and safety management. Table 6 shows that the flash point of the adjuvant of the present invention is greatly Mc52 and that of No. 10 white oil. The product of the invention is safer than the traditional adjuvant in transportation, storage and operation.

TABLE 6 flash point assay results for several adjuvants

Adjuvant	Flash Point (. degree.C.)
		Mc52	158
White oil 10#	155
		Camellia oil type composite vegetable oil adjuvant	228
Peanut oil type composite vegetable oil adjuvant	225
		Corn oil type composite vegetable oil adjuvant	230
Soybean oil type composite vegetable oil adjuvant	227

Experiment 10: the vaccine emulsion prepared by adopting the ginsenoside-containing composite vegetable oil adjuvant is water-in-oil type emulsion (W/O)

1. Materials and methods

1.2. Preparing a vaccine: referring to experiment 7, NDV vaccines with camellia oil type, peanut oil type, corn oil type and soybean oil type composite vegetable oil adjuvants were prepared, respectively.

1.3. And (3) identifying the type of the vaccine emulsion: (1) the dilution method comprises the steps of respectively dropping 2-3 drops of the emulsion into clear water and vegetable oil, and observing the dispersion condition of the vegetable oil emulsion drops in water and oil. As a result, the emulsion cannot be dispersed in water, and a water-in-oil emulsion (W/O) is excellent in dispersion in oil; the emulsion is well dispersible in water, but is not dispersible in oil and is an oil-in-water emulsion (O/W). (2) The dyeing method comprises the step of respectively dripping water-soluble eosin Y dye solution and oil-soluble Sudan III dye solution into a vaccine emulsion. As a result, the vaccine did not stain when the eosin Y staining solution was dropped, while the vaccine stained an oil-in-water emulsion (O/W) when the Sudan III staining solution was dropped; the vaccine was stained when eosin Y staining solution was dropped, and the vaccine was not stained when Sudan III staining solution was dropped as a water-in-oil emulsion (W/O).

2. Results and analysis

The results of the identification are shown in Table 7. The NDV vaccine emulsion prepared with the adjuvant of the present invention can be dispersed in oil but not in water, can be stained by sudan III but not by eosin Y, and is a water-in-oil emulsion (W/O).

TABLE 7 identification of the type of emulsion of NDV vaccine containing ginsenoside-containing composite vegetable oil adjuvant

Comparative example 1, the use of "oleic acid" was eliminated, camellia oil was changed to corn oil, and the amount of corn oil was increased accordingly, the remainder being identical to example 2.

This case causes oil-water separation and fails to form a stable emulsion.

Comparative example 2, the use of "polyglycerol ricinoleate" was eliminated, the camellia oil was changed to corn oil, and the amount of corn oil was increased accordingly, and the rest was identical to example 2.

This case causes oil-water separation and fails to form a stable emulsion.

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 composite vegetable oil vaccine adjuvant is characterized by being prepared by the following method:

1.50-3.5% of oleic acid, 9.5-12.5% of span-85, 2.50-3.50% of polyglycerol ricinoleate and the balance of vegetable oil;

2. The composite vegetable oil vaccine adjuvant according to claim 1, which is characterized in that:

the vegetable oil is camellia oil, peanut oil, linseed oil, sunflower seed oil, rice oil, corn oil, soybean oil, blend oil, grape seed oil or rapeseed oil.

3. The composite vegetable oil vaccine adjuvant according to claim 1 or 2, which is characterized in that:

the preparation method of the composite vegetable oil adjuvant in the step 1) comprises the following steps: adding oleic acid, span-85 and polyglycerol ricinoleate into vegetable oil at 35-50 ℃, and uniformly mixing to obtain the composite vegetable oil adjuvant.

4. The composite vegetable oil vaccine adjuvant according to claim 3, which is characterized in that:

5. Use of the composite vegetable oil vaccine adjuvant according to any one of claims 1 to 4, wherein:

taking a composite vegetable oil vaccine adjuvant as an oil phase and an aqueous solution containing antigen as a water phase;

heating the oil phase and the water phase to 35-40 ℃ respectively, and then mixing and emulsifying according to the volume ratio of 2:1 to obtain the water-in-oil type vaccine emulsion.