阅读说明：本技术 一种副猪嗜血杆菌佐剂疫苗及其制备方法 (Haemophilus parasuis adjuvant vaccine and preparation method thereof ) 是由 李晓华 刘栋 赖连杰 林晨宇 薛少华 林寰 郑新添 于 2021-09-15 设计创作，主要内容包括：本发明公开了一种副猪嗜血杆菌佐剂疫苗及其制备方法。属于植物材料的药物制品技术领域。本发明将银杏叶黄酮苷与副猪嗜血杆菌疫苗原液相结合,制备出副猪嗜血杆菌佐剂疫苗。与现有技术相比,本发明取得的有益效果为：该疫苗能够有效提高副猪嗜血杆菌的IgG抗体效价,有效提高细胞因子IL-2,IL-4、TNF-α、IFN-γ水平,仔猪免疫副猪嗜血杆菌佐剂疫苗二免后2周,仔猪T细胞亚群CD4～(+)、CD8～(+)数量显著增高,实现了对副猪嗜血杆菌血清4型、血清5型和血清13型攻击的60～100％的保护效果,为致病性副猪嗜血杆菌提供了新型疫苗和免疫方法。(The invention discloses a haemophilus parasuis adjuvant vaccine and a preparation method thereof. Belongs to the technical field of medicinal products of plant materials. The invention combines the ginkgo leaf flavonoid glycoside and the haemophilus parasuis vaccine stock solution to prepare the haemophilus parasuis adjuvant vaccine. Compared with the prior art, the invention has the following beneficial effects: the vaccine can effectively improve the IgG antibody titer of the haemophilus parasuis, effectively improve the levels of cytokines IL-2, IL-4, TNF-alpha and IFN-gamma, and can effectively improve the T cell subgroup CD4 of piglets 2 weeks after the immunization of the haemophilus parasuis adjuvant vaccine on the piglets + 、CD8 + The quantity is obviously increased, the protection effect of 60-100% of attack on the haemophilus parasuis serotype 4, serotype 5 and serotype 13 is realized, and a novel vaccine and an immunization method are provided for the pathogenic haemophilus parasuis.)

1. The haemophilus parasuis adjuvant vaccine is characterized by comprising a vaccine stock solution and an adjuvant;

the vaccine stock solution consists of (4+5+13) type bacterial solution antigens, and the volume ratio of the (4+5+13) type bacterial solution antigens to the (4+5+13) type bacterial solution antigens is 1: 1: 1;

the adjuvant is ginkgo leaf flavonoid glycoside, and the final concentration of the ginkgo leaf flavonoid glycoside in the adjuvant vaccine is 1-4 mg/mL.

2. The Haemophilus parasuis adjuvant vaccine of claim 1, wherein the final concentration of ginkgo biloba flavonoid glycoside in the adjuvant vaccine is 4 mg/mL.

3. The Haemophilus parasuis adjuvant vaccine of claim 1, wherein the bacterial concentration of 4, 5 type bacterial liquid antigen in the adjuvant vaccine is 2.0 x 10⁹CFU/mL, bacterial concentration of 13 type bacterial liquid antigen is 2.5 × 10⁹CFU/mL。

4. The method for preparing the haemophilus parasuis adjuvant vaccine according to any one of claims 1 to 3, comprising the steps of:

(1) preparing ginkgo leaf flavonoid glycoside;

(2) preparing a bacterial liquid antigen: culturing type 4, 5 and 13 haemophilus parasuis by liquid fermentation, adding formaldehyde solution with the volume of 0.25% of the bacterial solution, inactivating, and storing for later use;

(3) preparing a vaccine stock solution: preparing a vaccine stock solution according to a proportion;

(4) preparing an adjuvant vaccine: and (3) preparing the vaccine stock solution and an adjuvant into an adjuvant vaccine.

5. The method according to claim 4, wherein the medium used in the step (2) comprises trypticase soy broth with a mass concentration of 3%, newborn bovine serum and NAD solution with a mass concentration of 0.04%, in a volume ratio of 5000:250: 1.

6. The method according to claim 4, wherein the culture conditions in the step (2) are: culturing at 37 ℃ and 180r/min for 22-24 h.

7. The method according to claim 4, wherein the step (2) further comprises diluting the bacterial suspension antigen to a bacterial suspension antigen type 4, 5 with a bacterial concentration of 2.0 x 10 or more⁹The bacterial concentration of CFU/mL and 13 type bacterial liquid antigen is more than or equal to 2.5 multiplied by 10⁹CFU/mL。

Technical Field

The invention relates to the technical field of medicinal products of plant materials, in particular to a haemophilus parasuis adjuvant vaccine and a preparation method thereof.

Background

Haemophilus parasuis is a gram-negative bacillus pumilus that often colonizes the upper respiratory tract of pigs. The bacterium is mainly harmful to piglets and adult pigs, is mainly characterized by fibrinous polyhedrosis, arthritis and meningitis, and can also cause sudden death of the pigs. A total of 15 haemophilus parasuis serotypes have been identified in recent years, of which serotypes 4 and 5 are the most prevalent.

Currently, the pathogenesis of haemophilus parasuis infection is known only in a limited way, and preventive measures cannot completely control the pathogenesis. In general, the key factor controlling the occurrence of haemophilus parasuis disease is the innate immunity of the piglets and their immunity from maternal antibodies, which is controlled primarily by vaccination methods.

However, the haemophilus parasuis vaccine has the following problems: (1) because the serotype is more and a large proportion of strains are not typed, the development of the high-efficiency cross-protection vaccine is extremely difficult; (2) the limitation of the adjuvant causes the type and the dosage of the adjuvant to influence the subsequent vaccine effect.

In conclusion, how to provide a haemophilus parasuis adjuvant vaccine with high-efficiency cross protection is a problem which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a haemophilus parasuis adjuvant vaccine and a preparation method thereof. The effective component ginkgo biloba flavonoid glycoside of the adjuvant is separated, extracted and prepared from Ginkgobibilobal of Ginkgoaceae, and then the adjuvant vaccine of the haemophilus parasuis is prepared by combining with the stock solution of the haemophilus parasuis vaccine. Has good immunoregulation function, can regulate the activity of lymphocytes and phagocytes, regulate the level of cell factors in serum of an organism and the like, and has the advantages of naturalness, low toxicity, no drug residue and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

a haemophilus parasuis adjuvant vaccine, comprising a vaccine stock solution and an adjuvant;

the vaccine stock solution consists of (4+5+13) type bacterial solution antigens, and the volume ratio of the (4+5+13) type bacterial solution antigens to the (4+5+13) type bacterial solution antigens is 1: 1: 1;

the adjuvant is ginkgo leaf flavonoid glycoside, and the final concentration of the ginkgo leaf flavonoid glycoside in the adjuvant vaccine is 1-4 mg/mL.

Further, the final concentration of ginkgo biloba flavonoid glycoside in the adjuvant vaccine is 4 mg/mL.

Furthermore, the bacterial concentration of 4 and 5 type bacterial liquid antigen in the adjuvant vaccine is 2.0 multiplied by 10⁹CFU/mL, bacterial concentration of 13 type bacterial liquid antigen is 2.5 × 10⁹CFU/mL。

A preparation method of a haemophilus parasuis adjuvant vaccine comprises the following steps:

(1) preparing ginkgo leaf flavonoid glycoside;

(2) preparing a bacterial liquid antigen: culturing type 4, 5 and 13 haemophilus parasuis by liquid fermentation, adding formaldehyde solution with the volume of 0.25% of the bacterial solution, inactivating, and storing for later use;

(3) preparing a vaccine stock solution: preparing a vaccine stock solution according to a proportion;

(4) preparing an adjuvant vaccine: and (3) preparing the vaccine stock solution and an adjuvant into an adjuvant vaccine.

Further, the culture medium used in the step (2) comprises a trypticase soy broth culture medium with a mass concentration of 3%, newborn bovine serum and a NAD solution with a mass concentration of 0.04%, and the volume ratio of the trypticase soy broth culture medium, the newborn bovine serum and the NAD solution is 5000:250: 1.

Further, the culture conditions in the step (2) are as follows: culturing at 37 ℃ and 180r/min for 22-24 h.

Further, the step (2) further comprises diluting the bacterial liquid antigen into 4 and 5 types of bacterial liquid antigen with bacterial concentration more than or equal to 2.0 multiplied by 10⁹The bacterial concentration of CFU/mL and 13 type bacterial liquid antigen is more than or equal to 2.5 multiplied by 10⁹CFU/mL。

Through the technical scheme, compared with the prior art, the invention has the beneficial effectsThe fruit is as follows: the vaccine can effectively improve the IgG antibody titer of the haemophilus parasuis, effectively improve the levels of cytokines IL-2, IL-4, TNF-alpha and IFN-gamma, and can effectively improve the T cell subgroup CD4 of piglets 2 weeks after the immunization of the haemophilus parasuis adjuvant vaccine on the piglets⁺、CD8⁺The quantity is obviously increased, the protection effect of 60-100% of attack on the haemophilus parasuis serotype 4, serotype 5 and serotype 13 is realized, and a novel vaccine and an immunization method are provided for the pathogenic haemophilus parasuis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the Hps antibody titer detection results of the immunized piglets in experiment 2 of the present invention, wherein P is less than 0.05 compared with the normal saline group; p < 0.01 in comparison to saline group; p < 0.001 in comparison to saline group; # shows P < 0.05 compared to the vaccine stock solution group; # indicates P < 0.01 compared to the vaccine stock solution group; # indicates that P is less than 0.001 compared with the vaccine stock solution group;

FIG. 2 is a graph showing the serum IL-2 levels in 3 piglets of the experiment of the present invention, wherein P is < 0.05 compared to normal saline; p < 0.01 in comparison to saline group; p < 0.001 in comparison to saline group; # shows P < 0.05 compared to the vaccine stock solution group; # indicates P < 0.01 compared to the vaccine stock solution group; # indicates that P is less than 0.001 compared with the vaccine stock solution group;

FIG. 3 is a graph showing the serum IL-4 levels in 3 piglets of the experiment of the present invention, wherein P is < 0.05 compared to normal saline; p < 0.01 in comparison to saline group; p < 0.001 in comparison to saline group; # shows P < 0.05 compared to the vaccine stock solution group; # indicates P < 0.01 compared to the vaccine stock solution group; # indicates that P is less than 0.001 compared with the vaccine stock solution group;

FIG. 4 is a graph showing the serum TNF-a levels in the sera of 3 piglets of experiment 3 of the present invention, wherein P is < 0.05 compared to normal saline; p < 0.01 in comparison to saline group; p < 0.001 in comparison to saline group; # shows P < 0.05 compared to the vaccine stock solution group; # indicates P < 0.01 compared to the vaccine stock solution group; # indicates that P is less than 0.001 compared with the vaccine stock solution group;

FIG. 5 is a graph showing IFN-r levels in serum of 3 piglets in experiment of the present invention, wherein P is < 0.05 compared to normal saline; p < 0.01 in comparison to saline group; p < 0.001 in comparison to saline group; # shows P < 0.05 compared to the vaccine stock solution group; # indicates P < 0.01 compared to the vaccine stock solution group; # indicates that P is less than 0.001 compared with the vaccine stock solution group;

FIG. 6 is a drawing showing the peripheral blood CD4 of the piglet according to experiment 4 of the invention⁺And CD8⁺Expression level of T lymphocytes, wherein P < 0.05 compared with physiological saline; p < 0.01 in comparison to saline group; p < 0.001 in comparison to saline group; # shows P < 0.05 compared to the vaccine stock solution group; # indicates P < 0.01 compared to the vaccine stock solution group; # indicates that P is less than 0.001 compared with the vaccine stock solution group;

FIG. 7 is a drawing showing the peripheral blood CD4 of the piglet according to experiment 4 of the invention⁺/CD8⁺T lymphocyte fraction, wherein P < 0.05 compared to saline group; p < 0.01 in comparison to saline group; p < 0.001 in comparison to saline group; # shows P < 0.05 compared to the vaccine stock solution group; # indicates P < 0.01 compared to the vaccine stock solution group; # indicates that P is less than 0.001 compared with the vaccine stock solution group;

FIG. 8 is a graph showing the T lymphocyte stimulation index of peripheral blood of piglets in experiment 5 of the present invention, wherein P is < 0.05 compared with normal saline; p < 0.01 in comparison to saline group; p < 0.001 in comparison to saline group; # shows P < 0.05 compared to the vaccine stock solution group; # indicates P < 0.01 compared to the vaccine stock solution group; # indicates that P is less than 0.001 compared with the vaccine stock solution group;

FIG. 9 is a graph showing the peripheral blood B lymphocyte stimulation index of piglets in experiment 5 of the present invention, wherein P is < 0.05 compared with normal saline; p < 0.01 in comparison to saline group; p < 0.001 in comparison to saline group; # shows P < 0.05 compared to the vaccine stock solution group; # indicates P < 0.01 compared to the vaccine stock solution group; # indicates P < 0.001 compared to the vaccine stock solution group.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The medicament required by the embodiment of the invention is a conventional experimental medicament purchased from a commercially available channel; the experimental methods not mentioned in the examples are conventional experimental methods, and are not described in detail herein.

Example 1

(1) Preparation of ginkgo leaf flavonoid glycoside:

extracting folium Ginkgo by ultrasonic-assisted extraction and ethanol reflux extraction to obtain folium Ginkgo flavonoid glycoside; wherein the extraction temperature is 70 ℃, the extraction time is 2 hours, the material-liquid ratio is 1:25 and the volume fraction of ethanol is 70 percent;

(2) preparing a bacterial liquid antigen:

(21) preparation of a culture medium: dissolving 30g of Trypticase Soy Broth (TSB) in deionized water, diluting to 1000mL, shaking to dissolve, sterilizing with high pressure steam at 121 deg.C for 15min, adding 50mL of filter-sterilized newborn bovine serum and 200 μ L of filter-sterilized NAD with mass concentration of 0.04%.

(22) Respectively culturing 4, 5 and 13 type bacteria liquid of haemophilus parasuis by adopting liquid fermentation culture, culturing at 37 ℃ and 180r/min for 22-24 h to obtain various types of bacteria liquid, adding a formaldehyde solution according to 0.25% of the volume of the bacteria liquid, stirring and inactivating at 37 ℃ for 24h, sampling, inactivating, checking, and storing at 2-8 ℃.

(3) Preparing a vaccine stock solution:

diluting the inactivated haemophilus parasuis type 4, 5 and 13 bacterial liquid antigens prepared in the step (2) into type 4 and type 5 which are more than or equal to 2.0 multiplied by 10⁹CFU/mL, type 13 ≥ 2.5X 10⁹Mixing the materials according to the following proportion after CFU/mL:

(type 4+ type 5+ type 13) ═ V/3+ V/3; (Note: V means volume).

(4) Preparing an adjuvant vaccine:

the prepared ginkgo biloba flavonoid glycoside and the vaccine stock solution are prepared into haemophilus parasuis adjuvant vaccines with different concentrations (the concentration of the ginkgo biloba flavonoid glycoside in the adjuvant vaccine is 1mg/mL, 2mg/mL and 4mg/mL respectively).

Experiment 1 piglet immunization experiment

Selecting the haemophilus parasuis adjuvant vaccines with different concentrations prepared in example 1, immunizing 2mL of the haemophilus parasuis adjuvant vaccine by a neck intramuscular injection way at the age of 15 days of piglets, carrying out secondary immunization 21 days later, immunizing the same way and dosage, and attacking 4, 5 and 13 types of haemophilus parasuis serum for the piglets 14 days after the secondary immunization in a way that 3 mL/head of intraperitoneal injection bacterial liquid is injected, wherein the content of viable bacteria of 4 and 5 types of haemophilus parasuis serum is 7.5 multiplied by 10⁹cfu/mL, 3.75X 10 for Haemophilus parasuis serotype 13¹⁰cfu/mL, and a vaccine stock solution and a blank control (a control group is injected with normal saline in the abdominal cavity) are set simultaneously (see Table 1 specifically). Collecting blood before immunization, 7 th and 14 th days after second immunization and 7 th and 14 th days after toxin attack, centrifuging at 4 deg.C 4000r/min for 25min, and freezing at-70 deg.C for storage; meanwhile, anticoagulant blood is collected 14 days after the second immunization for flow detection of T cell subsets.

TABLE 1 vaccine immunization profiles

The results are as follows:

vaccine stock solution + ginkgo biloba flavonoid glycoside (high) group: after the haemophilus parasuis serotype 4 bacterial solution is subjected to toxin attack for 16 hours, symptoms such as heaping, lying quietly, lassitude and the like appear, 1 piglet is dead after the toxin attack is carried out for 124 hours, 4 piglets still survive after 2 weeks, and the protection rate is 80%; after the vaccine stock solution and the ginkgo biloba flavonoid glycoside (high) group haemophilus parasuis serum 5 type bacterial solution are subjected to virus attack for 23 hours, the patient lies still, the spirit is restored after 3 days, the patient completely survives, and the protection rate is 100 percent; after the vaccine stock solution and the haemophilus parasuis serous 13 type bacterial solution of the ginkgo leaf flavonoid glycoside (high) group are subjected to virus attack for 26 hours, the symptoms of heaping, lying still, lassitude, lameness and the like appear at 1 head, 2 piglets die after 53 hours of virus attack, the spirit is gradually recovered after 96 hours of virus attack, 3 piglets still survive after 2 weeks, and the protection rate is 60%.

Vaccine stock solution + ginkgo biloba flavonoid glycoside (middle) group: after the haemophilus parasuis serotype 4 bacterial solution is subjected to toxin attack for 13 hours, symptoms such as heaping, lying quietly, lassitude and the like appear, 2 piglets die after the toxin attack for 19 hours, 3 piglets still survive after 2 weeks, and the protection rate is 60%; after the vaccine stock solution and the haemophilus parasuis serous type 5 bacterial solution in the ginkgo leaf flavonoid glycoside (middle) group are subjected to virus attack for 5 hours, symptoms such as heaping, lying still, lassitude and the like appear, 2 piglets die after the virus attack for 46 hours, 3 piglets still survive after 2 weeks, and the protection rate is 60%; the vaccine stock solution and the haemophilus parasuis serous 13 type bacterial solution of the ginkgo leaf flavonoid glycoside (middle) group are subjected to toxin attack for 12 hours to generate mental depression and 1 head of the pig has dyspnea symptoms, 2 piglets die after 50 hours of toxin attack, 3 piglets still survive after 2 weeks, and the protection rate is 60%.

Vaccine stock solution + ginkgo biloba flavonoid glycoside (low) group: after 6 hours of toxin attack, the haemophilus parasuis serum 4 type bacterial solution has the symptoms of heaping, lying quietly, lassitude and the like, 2 piglets die after 9 hours of toxin attack, 3 piglets still survive after 2 weeks, and the protection rate is 60 percent; after the vaccine stock solution and the ginkgo biloba flavonoid glycoside (low) group haemophilus parasuis serum 5 type bacterial solution are subjected to toxin attack for 4 hours, symptoms such as heaping, lying still, lassitude and the like appear, 2 piglets die after the toxin attack for 33 hours, 1 piglet dies after the toxin attack for 57 hours, 2 piglets still survive after 2 weeks, and the protection rate is 40%; after the vaccine stock solution and the ginkgo biloba flavonoid glycoside (low) group haemophilus parasuis serum 13 type bacterial solution are subjected to virus attack for 9 hours, symptoms such as heaping, lying still, lassitude and the like appear, 2 piglets die after 61 hours of virus attack, and the protection rate is 60%.

Experimental results show that the haemophilus parasuis adjuvant vaccine prepared by adding 4mg/mL of ginkgo leaf flavonoid glycoside has a good immune effect, the protection rate on haemophilus parasuis serum type 4 can reach 80%, the protection rate on haemophilus parasuis serum type 5 can reach 100%, and the protection rate on haemophilus parasuis serum type 13 can reach 60% (see tables 2 and 3 for details).

TABLE 2 challenge results of Haemophilus parasuis in different concentration adjuvant vaccines

TABLE 3 piglet vaccine immunoprotection experiments

Note: the pathogenic standard of haemophilus parasuis disease: the sick pigs have clinical symptoms of fever (body temperature of more than 40.5 ℃ and lasting for 1-3 days), listlessness, cough, dyspnea, emaciation, lameness, rough fur and the like. The autopsy of the dying pig shows the pathological changes of multiple serositis (pleuritis, pericarditis and peritonitis), arthritis, meningitis and the like, and serous or cellulosic exudates appear on each serosal surface (joint capsule, pericardium, pleura and peritoneum).

Experiment 2 detection of Hps antibody titer of immunized piglets

The immunization and challenge methods were the same as in experiment 1.

The antibody titer detection result of Hps antibody in piglet immune protection experiments shows that 2 weeks after the second immunization, the antibody levels of the vaccine stock solution and the ginkgo biloba flavonoid glycoside (high) group are obviously higher than those of the vaccine stock solution group and the normal saline control group, and the antibody levels of the vaccine stock solution and the ginkgo biloba flavonoid glycoside (medium) group and the vaccine stock solution and the ginkgo biloba flavonoid glycoside (low) group are not obviously different compared with those of the vaccine stock solution group; 1 week after the challenge, the antibody levels of the vaccine stock solution and the ginkgo biloba flavonoid glycoside (high) group are obviously higher than those of the vaccine stock solution group and the normal saline group, and the antibody levels of the vaccine stock solution and the ginkgo biloba flavonoid glycoside (medium) group and the vaccine stock solution and the ginkgo biloba flavonoid glycoside (low) group are lower than those of the vaccine stock solution group; after 2 weeks of challenge, the antibody levels of the vaccine stock solution + ginkgo biloba flavonoid glycoside (high) group were significantly higher than those of the vaccine stock solution group, and the antibody levels of the vaccine stock solution + ginkgo biloba flavonoid glycoside (medium) group and the vaccine stock solution + ginkgo biloba flavonoid glycoside (low) group were higher than those of the vaccine stock solution group (see fig. 1).

Experiment 3 detection of immune piglet cytokine levels

The immunization and challenge methods were the same as in experiment 1.

Detecting the level of cytokine IL-2

After 2 weeks of secondary immunization and 1 week of challenge, the flavonoid glycoside adjuvant vaccine group is significantly higher than the normal saline group, and compared with the vaccine stock solution group, the difference is not significant. (see FIG. 2)

② detection of cytokine IL-4 level

1 week after the second immunization, the vaccine stock solution + ginkgo biloba flavonoid glycoside (high) group and the vaccine stock solution + ginkgo biloba flavonoid glycoside (low) group are obviously higher than the vaccine stock solution group and the normal saline group; the IL-4 level of the vaccine stock solution and the ginkgo biloba flavonoid glycoside (high) group is slightly reduced 2 weeks after the second immunization; differences between groups were not significant 1 week after challenge and 2 weeks after challenge. (see FIG. 3)

Level detection of cell factor TNF-a

After 1 week of second immunization, the flavonoid glycoside adjuvant vaccine group is obviously higher than the vaccine stock solution group and the normal saline group; after 1 week of challenge, the TNF-a level of the vaccine stock solution + ginkgo biloba flavonoid glycoside (high) group is higher than that of the vaccine stock solution group, and the difference between the vaccine stock solution + ginkgo biloba flavonoid glycoside (middle) group and the vaccine stock solution + ginkgo biloba flavonoid glycoside (low) group is not obvious compared with that of the vaccine stock solution group; the differences among the groups were not significant 2 weeks after challenge. (see FIG. 4)

Detecting the level of cytokine IFN-r

The IFN-r level of the vaccine stock solution + ginkgo biloba flavonoid glycoside (high) group and the vaccine stock solution + ginkgo biloba flavonoid glycoside (low) group is obviously higher than that of the vaccine stock solution group and the normal saline group 1 week and 2 weeks after the second immunization, and the difference between the vaccine stock solution + ginkgo biloba flavonoid glycoside (middle) group and the vaccine stock solution group is not obvious; 1 week after challenge, IFN-r level of vaccine stock solution and ginkgo leaf flavonoid glycoside (high) group is still obviously higher than that of other groups; IFN-r levels were not significantly different in each group 2 weeks after challenge. (see FIG. 5)

Experiment 4 immunized piglet peripheral blood CD4⁺、CD8⁺Expression of T lymphocytes

The immunization protocol was as in experiment 1, and was measured two weeks after the second immunization.

The detection result of the T lymphocyte subpopulation in the peripheral blood of the piglet, 2 weeks after the second immunization, the peripheral blood CD4 of the piglet is treated by the ginkgo leaf flavonoid glycoside adjuvant vaccine⁺The expression level is obviously higher than that of a vaccine stock solution group, wherein the vaccine stock solution and the ginkgo leaf flavonoid glycoside (high) group CD4⁺Is obviously higher than the vaccine stock solution and the ginkgo biloba flavonoid glycoside (middle) group and the vaccine stock solution and the ginkgo biloba flavonoid glycoside (low) group; compared with the vaccine stock solution and the ginkgo biloba flavonoid glycoside (middle), the vaccine stock solution and the ginkgo biloba flavonoid glycoside (high) group and the vaccine stock solution and the ginkgo biloba flavonoid glycoside (low) group can reduce the CD8⁺The proportion of T lymphocytes, but the difference between the vaccine stock solution group and the ginkgo leaf flavonoid glycoside group and the vaccine stock solution group is not obvious; meanwhile, the vaccine stock solution + ginkgo biloba flavonoid glycoside (high) group and the vaccine stock solution + ginkgo biloba flavonoid glycoside (low) group are CD4⁺/CD8⁺The ratio of T lymphocytes was also significantly increased (see fig. 6, fig. 7). Shows that the vaccine stock solution and the ginkgo biloba flavonoid glycoside (high) group can stimulate the differentiation of the lymphocytes of the organism.

Experiment 5 proliferation index of peripheral blood lymphocytes of piglets

The immunization and challenge methods were the same as in experiment 1.

T lymphocyte stimulation index

2 weeks after the second immunization, the T lymphocyte stimulation index of the vaccine stock solution and the ginkgo biloba flavonoid glycoside (high) group is obviously higher than that of the vaccine stock solution group, and the difference between the vaccine stock solution and the ginkgo biloba flavonoid glycoside (medium) group and the vaccine stock solution and the ginkgo biloba flavonoid glycoside (low) group is not obvious; the T lymphocyte index of the vaccine stock solution and the ginkgo leaf flavonoid glycoside (high) group is reduced after 1 week of challenge, and is in an increasing trend after 2 weeks of challenge. (see FIG. 8)

(B lymphocyte stimulation index)

2 weeks after the second immunization, the B lymphocyte stimulation index of the vaccine stock solution and the ginkgo biloba flavonoid glycoside (high) group is obviously higher than that of the vaccine immunization group, and the B lymphocyte stimulation index of the vaccine stock solution and the ginkgo biloba flavonoid glycoside (medium) group and the B lymphocyte stimulation index of the vaccine stock solution and the ginkgo biloba flavonoid glycoside (low) group are slightly lower than that of the vaccine stock solution group; 1 week after the challenge, the B lymphocyte stimulation index of the vaccine stock solution group is obviously increased, and the B lymphocyte index of the vaccine stock solution and the ginkgo leaf flavonoid glycoside (high) group is reduced; after 2 weeks of challenge, the B lymphocyte stimulation index of the vaccine stock solution group is obviously reduced, and the B lymphocyte index of the vaccine stock solution and the ginkgo leaf flavonoid glycoside (high) group is obviously higher than that of the vaccine stock solution group (see figure 9).

Experiment 6 safety test of vaccine

Safety test of one-time single-dose inoculation of haemophilus parasuis adjuvant vaccine to target animals and non-use day-old animals

5 heads of 10-15 day-old piglets (purchased from a certain fine breed pig farm in Fujian Longyan city) are subjected to neck intramuscular injection by using the adjuvant vaccine (the final concentration of the flavonoid glycoside adjuvant is 4mg/mL) prepared in the embodiment 1 of the invention, each injection is 2 mL/head, and a normal saline control group is additionally arranged. The experimental pigs were observed for allergy, abnormality in spirit and diet, red swelling at the injection site, weight gain of piglets, and normal sow born status within 2 weeks after vaccination. The piglets have good spirit, no red swelling phenomenon appears at the injection part after inoculation, and the spirit and the food intake are normal; the saline control group test pigs were normal (see table 4).

TABLE 4 one-time vaccination safety test results for non-use day-old target animals

② safety test of one-time overdose inoculation of adjuvant vaccine of haemophilus parasuis on target animal

The adjuvant vaccine (the final concentration of the flavonoid glycoside adjuvant is 4mg/mL) prepared in the embodiment 1 of the invention is injected into 5 healthy and susceptible piglets of 10-15 days old through neck muscle respectively, 4 mL/head of the piglets is provided with a normal saline control group, and the safety of the pigs after the vaccine inoculation is observed and tested. The result shows that no red and swollen reaction appears at the injection part after the piglets of 10-15 weeks are inoculated, the spirit and the ingestion are normal, and the piglet does not have obvious difference with the piglets of a normal saline control group. It is demonstrated that the adjuvant vaccine of the present invention is safe for one overdose (see table 5).

TABLE 5 safety test for overdose of Haemophilus parasuis adjuvant vaccine

③ single-dose repeat inoculation safety test

The adjuvant vaccine (the final concentration of the flavonoid glycoside adjuvant is 4mg/mL) prepared in the embodiment 1 of the invention is used for vaccinating healthy susceptible piglets of 10-15 days old, the vaccination is repeated once after 14 days, the safety of the vaccination of the inoculated piglets through single-dose repeated vaccination is observed, and a normal saline control group is additionally arranged. The result shows that the adjuvant vaccine has no adverse effect on the breeding pigs, has no allergy phenomenon, has no adverse reaction such as red swelling at the inoculation part, and the like, has normal spirit and feed intake of the breeding pigs, and has no obvious difference between the weight gain of piglets and the weight gain of piglets of a normal saline solution control group. The adjuvant vaccine prepared according to the present invention proved safe for repeated single dose vaccination (see table 6).

Table 6 single dose repeat safety test results for experimental piglets

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.