Vaccine adjuvant for poultry as well as preparation method and application of vaccine adjuvant

Abstract

The invention relates to a vaccine adjuvant for poultry as well as a preparation method and application thereof, and belongs to the technical field of poultry breeding. The invention provides a vaccine adjuvant for poultry. The vaccine adjuvant has dual functions of adjuvant and antigen presentation. The vaccine adjuvant for poultry is formed by compounding various natural components such as saponin, cholesterol, polysaccharide, amino acid, flavonoid, squalene, antibacterial peptide and protein hydrolysate, has a good immune synergistic effect, and can cause effective humoral immunity, cellular immunity and mucosal immunity responses in various animal bodies. According to the vaccine adjuvant for poultry, the transcription expression of immune related genes is regulated and controlled by promoting the proliferation of specific and non-specific cells, and the vaccine adjuvant for poultry has a good immune improvement effect on various poultry vaccines such as avian influenza vaccines and Newcastle disease vaccines. Moreover, through experimental verification, the vaccine adjuvant for the poultry can obviously advance the immune response time of the vaccine for the poultry, improve the expression level of immune antibodies and prolong the protection period of the vaccine antibodies.

Description

Technical Field

[0001] The invention belongs to the technical field of poultry breeding, and particularly relates to a poultry vaccine adjuvant and a preparation method and application thereof. Background Art

[0002] In modern poultry farming, poultry vaccines play a vital role. They are a key means to prevent and control poultry infectious diseases, ensure the health of poultry flocks, and improve farming efficiency. However, existing poultry vaccines have many disadvantages. On the one hand, after vaccination with some vaccines, the immune response takes a long time to start. During the peak period of the epidemic, it is impossible to provide effective immune protection for the poultry flocks in time, resulting in the poultry flocks being vulnerable to pathogens in the early stage. On the other hand, the expression level of immune antibodies is limited, and it is difficult to produce sufficient resistance to highly virulent pathogens. Moreover, the protection period of vaccine antibodies is short, and frequent vaccination is required, which not only increases the cost of breeding and manpower investment, but also may cause stress reactions to poultry flocks, affecting their growth and development and production performance. Therefore, for poultry vaccines, how to advance the immune response time, significantly increase the expression level of immune antibodies, and extend the protection period of vaccine antibodies has become a key technical problem that needs to be solved in the field of poultry vaccines. Summary of the invention

[0003] The purpose of the present invention is to provide a poultry vaccine adjuvant and its preparation method and application. The poultry vaccine adjuvant of the present invention can advance the immune response time of poultry vaccines, improve the expression level of immune antibodies, and prolong the protection period of vaccine antibodies.

[0004] The present invention provides a poultry vaccine adjuvant. The raw materials for preparing the poultry vaccine adjuvant include saponin, cholesterol, polysaccharide, amino acid, flavonoid, squalene, antimicrobial peptide, protein hydrolyzate and pharmaceutically acceptable auxiliary materials; the saponin includes ginsenoside and / or American ginsenoside; the polysaccharide includes one or more of yeast polysaccharide, astragalus polysaccharide and polygonatum polysaccharide.

[0005] Preferably, the amino acids include methionine, tryptophan, valine, lysine, leucine, isoleucine, phenylalanine, glutamine, threonine, arginine, cysteine ​​and histidine; the flavonoids include buckwheat flavonoids; and the protein hydrolysate includes wheat protein hydrolysate.

[0006] Preferably, the concentration of the saponin in the vaccine adjuvant is 0.02% to 0.10%;

[0007] The concentration of cholesterol in the vaccine adjuvant is 0.005% to 0.015%;

[0008] The concentration of the polysaccharide in the vaccine adjuvant is 0.01% to 0.15%;

[0009] The concentration of the amino acid in the vaccine adjuvant is 200 to 1150 μmol / L;

[0010] The concentration of the flavonoid in the vaccine adjuvant is 0.05% to 0.20%;

[0011] The concentration of the squalene in the vaccine adjuvant is 0.3% to 1.1%;

[0012] The concentration of the antimicrobial peptide in the vaccine adjuvant is 0.005% to 0.05%;

[0013] The concentration of the proteolysate in the vaccine is 0.05% to 0.3%.

[0014] Preferably, the excipient includes phosphate buffer and / or absolute ethanol.

[0015] Preferably, the phosphate buffer includes a phosphate buffer with a total phosphate content of 0.1 mol / L to 0.2 mol / L.

[0016] The present invention also provides a preparation method of the avian vaccine adjuvant according to the above technical solution, including the following steps:

[0017] Mix saponin, polysaccharide, antimicrobial peptide and proteolysate with phosphate buffer to obtain a first mixed solution;

[0018] Mix the amino acid with phosphate buffer to obtain a second mixed solution;

[0019] Mix cholesterol, flavonoid and squalene with absolute ethanol to obtain a third mixed solution;

[0020] Mix the first mixed solution, the second mixed solution and the third mixed solution to obtain the avian vaccine adjuvant.

[0021] The present invention also provides the application of the avian vaccine adjuvant according to the above technical solution or the vaccine adjuvant prepared by the preparation method according to the above technical solution in the preparation of avian vaccines.

[0022] Preferably, the vaccine includes avian influenza vaccine and / or Newcastle disease vaccine.

[0023] Preferably, in the avian vaccine, the dosage of the adjuvant is 600 to 1200 doses / mL.

[0024] The present invention also provides the application of the avian vaccine adjuvant according to the above technical solution or the vaccine adjuvant prepared by the preparation method according to the above technical solution in any one or two or more of the following functions:

[0025] ① Advance the immune response time;

[0026] ② Improve the expression level of immune antibodies;

[0027] ③ Prolong the protection period of vaccine antibodies.

[0028] The present invention provides an adjuvant for avian vaccines. The adjuvant for avian vaccines of the present invention is an immune stimulating composition with dual functions of adjuvant and antigen presentation. The adjuvant for avian vaccines of the present invention is composed of a variety of natural ingredients such as saponins, cholesterol, polysaccharides, amino acids, flavonoids, squalene, antimicrobial peptides and proteolysates, and has good immune synergy, and can cause effective humoral immunity, cellular immunity and mucosal immune responses in various animals. The adjuvant for avian vaccines of the present invention has a good immune enhancing effect on a variety of avian vaccines such as avian influenza vaccine and Newcastle disease vaccine by promoting the proliferation of specific and non-specific cells and regulating the transcriptional expression of immune-related genes. Moreover, the adjuvant for avian vaccines of the present invention has been verified by experiments to be able to significantly advance the immune response time, enabling the avian flock to generate immune responses faster after vaccination. At the same time, it greatly improves the expression level of immune antibodies to enhance the resistance to various pathogens, prolongs the protection period of vaccine antibodies, reduces the number of vaccinations, and lowers the breeding cost and stress of the avian flock. Brief Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 It is a graph of the experimental results of treatment group 1 of the small-scale test on the influence of the adjuvant for avian vaccines provided by the present invention on the change of the anti-titer of Newcastle disease vaccine;

[0031] Figure 2 It is a graph of the experimental results of treatment group 2 of the small-scale test on the influence of the adjuvant for avian vaccines provided by the present invention on the change of the anti-titer of Newcastle disease vaccine;

[0032] Figure 3 It is a graph of the experimental results of treatment group 3 of the small-scale test on the influence of the adjuvant for avian vaccines provided by the present invention on the change of the anti-titer of Newcastle disease vaccine;

[0033] Figure 4 It is a graph of the experimental results of treatment group 4 of the small-scale test on the influence of the adjuvant for avian vaccines provided by the present invention on the change of the anti-titer of Newcastle disease vaccine;

[0034] Figure 5 It is a graph of the experimental results of treatment group 5 of the small-scale test on the influence of the adjuvant for avian vaccines provided by the present invention on the change of the anti-titer of Newcastle disease vaccine;

[0035] Figure 6 Experimental result graph of treatment group 1 in the pilot experiment on the influence of the avian vaccine adjuvant provided by the present invention on the growth and decline of the antibody titer of the avian influenza vaccine;

[0036] Figure 7 Experimental result graph of treatment group 2 in the pilot experiment on the influence of the avian vaccine adjuvant provided by the present invention on the growth and decline of the antibody titer of the avian influenza vaccine;

[0037] Figure 8 Experimental result graph of treatment group 3 in the pilot experiment on the influence of the avian vaccine adjuvant provided by the present invention on the growth and decline of the antibody titer of the avian influenza vaccine;

[0038] Figure 9 Experimental result graph of treatment group 4 in the pilot experiment on the influence of the avian vaccine adjuvant provided by the present invention on the growth and decline of the antibody titer of the avian influenza vaccine;

[0039] Figure 10 Experimental result graph of treatment group 5 in the pilot experiment on the influence of the avian vaccine adjuvant provided by the present invention on the growth and decline of the antibody titer of the avian influenza vaccine;

[0040] Figure 11 Result comparison graph of the influence of the avian vaccine adjuvant provided by the present invention and the conventional commercially available vaccine adjuvant on the growth and decline of the antibody titer of the Newcastle disease vaccine;

[0041] Figure 12 Result comparison graph of the influence of the avian vaccine adjuvant provided by the present invention and the conventional commercially available vaccine adjuvant on the growth and decline of the antibody titer of the avian influenza vaccine;

[0042] Figure 13 State graph of the avian vaccine adjuvant provided by the present invention placed at room temperature (18°C - 25°C) for 1 month;

[0043] Figure 14 State graph of the avian vaccine adjuvant provided by the present invention placed at 2°C - 8°C for 1 month;

[0044] Figure 15 State graph of the avian vaccine adjuvant provided by the present invention placed at 37°C ± 2°C for 1 month. Detailed implementation mode

[0045] The present invention provides an avian vaccine adjuvant. The raw materials used for preparing the avian vaccine adjuvant include saponins, cholesterol, polysaccharides, amino acids, flavonoids, squalene, antibacterial peptides, protein hydrolysates, and pharmaceutically acceptable excipients; the saponins include ginsenosides and / or panax quinquefolius saponins; the polysaccharides include one or more of zymosan, astragalus polysaccharide, and polygonatum polysaccharide.

[0046] In a specific embodiment, the concentration of the saponin in the vaccine adjuvant can be 0.02% to 0.10%; further, it can be 0.04% to 0.06% of ginsenoside or 0.03% to 0.08% of panax quinquefolius saponin; it can also be a combination of 0.02% to 0.10% of ginsenoside and panax quinquefolius saponin.

[0047] In a specific embodiment, the concentration of the cholesterol in the vaccine adjuvant can be 0.005% to 0.015%; further, it can be 0.008% to 0.010%.

[0048] In a specific embodiment, the concentration of the polysaccharide in the vaccine adjuvant can be 0.01% to 0.15%; further, it can be 0.03% to 0.06% of yeast polysaccharide, 0.04% to 0.06% of astragalus polysaccharide, or 0.05% to 0.08% of polygonatum polysaccharide; it can also be any mass ratio mixture of yeast polysaccharide, astragalus polysaccharide, and polygonatum polysaccharide that ensures the concentration of the polysaccharide in the vaccine adjuvant is 0.01% to 0.15%.

[0049] In a specific embodiment, the amino acids include methionine, tryptophan, valine, lysine, leucine, isoleucine, phenylalanine, glutamine, threonine, arginine, cysteine and histidine. In a specific embodiment, the concentration of the amino acids in the vaccine adjuvant can be 200 - 1125 μmol / L; further, it can be 10 μmol / L - 50 μmol / L methionine, 1.0 μmol / L - 8.0 μmol / L tryptophan, 10 μmol / L - 85 μmol / L valine, 20 μmol / L - 80 μmol / L lysine, 10 μmol / L - 95 μmol / L leucine, 15 μmol / L - 95 μmol / L isoleucine, 3 μmol / L - 50 μmol / L phenylalanine, 60 μmol / L - 400 μmol / L glutamine, 15 μmol / L - 80 μmol / L threonine, 40 μmol / L - 150 μmol / L arginine, 8 μmol / L - 20 μmol / L cysteine and 9 μmol / L - 20 μmol / L histidine, one or more than two of them; still further, it can be 20 μmol / L - 35 μmol / L methionine, 2.5 μmol / L - 5.0 μmol / L tryptophan, 40 μmol / L - 56 μmol / L valine, 40 μmol / L - 50 μmol / L lysine, 38 μmol / L - 45 μmol / L leucine, 30 μmol / L - 55 μmol / L isoleucine, 9 μmol / L - 25 μmol / L phenylalanine, 100 μmol / L - 200 μmol / L glutamine, 20 μmol / L - 45 μmol / L threonine, 60 μmol / L - 100 μmol / L arginine, 12 μmol / L - 18 μmol / L cysteine and 9.5 μmol / L - 15 μmol / L histidine.

[0050] In a specific embodiment, the flavonoids include tartary buckwheat flavonoids. In a specific embodiment, the concentration of the flavonoids in the vaccine adjuvant can be 0.05% - 0.20%; further, it can be 0.08% - 0.15% tartary buckwheat flavonoids.

[0051] In a specific embodiment, the concentration of squalene in the vaccine adjuvant can be 0.3% - 1.1%; further, it can be 0.6 - 0.9%.

[0052] In a specific embodiment, the concentration of the antimicrobial peptide in the vaccine adjuvant can be 0.005% - 0.05%; further, it can be 0.005 - 0.03%.

[0053] In a specific embodiment, the protein hydrolysate includes wheat protein hydrolysate. In a specific embodiment, the concentration of the protein hydrolysate in the vaccine can be 0.05% to 0.3%; further, it can be 0.08% to 0.15% of wheat protein hydrolysate.

[0054] In a specific embodiment, the excipients include phosphate buffer and / or absolute ethanol. In a specific embodiment, the excipients are solvents for each component.

[0055] The present invention also provides a preparation method of the avian vaccine adjuvant according to the above technical solution, comprising the following steps:

[0056] Mix saponin, polysaccharide, antibacterial peptide and protein hydrolysate with phosphate buffer to obtain a first mixed solution; mix amino acid with phosphate buffer to obtain a second mixed solution; mix cholesterol, flavonoid and squalene with absolute ethanol to obtain a third mixed solution; mix the first mixed solution, the second mixed solution and the third mixed solution to obtain the avian vaccine adjuvant.

[0057] The present invention mixes saponin, polysaccharide, antibacterial peptide and protein hydrolysate with phosphate buffer to obtain a first mixed solution. In a specific embodiment, the phosphate buffer includes a phosphate buffer with a total phosphate content of 0.1 mol / L to 0.2 mol / L. In a specific embodiment, the saponin includes ginsenoside and / or panax quinquefolius saponin; the polysaccharide includes one or more of yeast polysaccharide, astragalus polysaccharide and polygonatum polysaccharide; the protein hydrolysate includes wheat protein hydrolysate.

[0058] After obtaining the first mixed solution, the present invention mixes amino acids with a phosphate buffer solution to obtain a second mixed solution. In a specific embodiment, the phosphate buffer solution includes a phosphate buffer solution with a total phosphate content of 0.1 mol / L to 0.2 mol / L. In a specific embodiment, the amino acids include one or more of methionine, tryptophan, valine, lysine, leucine, isoleucine, phenylalanine, glutamine, threonine, arginine, cysteine, and histidine. In a specific embodiment, the amino acids can be 20 μmol / L to 35 μmol / L methionine, 2.5 μmol / L to 5.0 μmol / L tryptophan, 40 μmol / L to 56 μmol / L valine, 40 μmol / L to 50 μmol / L lysine, 38 μmol / L to 45 μmol / L leucine, 30 μmol / L to 55 μmol / L isoleucine, 9 μmol / L to 25 μmol / L phenylalanine, 100 μmol / L to 200 μmol / L glutamine, 20 μmol / L to 45 μmol / L threonine, 60 μmol / L to 100 μmol / L arginine, 12 μmol / L to 18 μmol / L cysteine, and 9.5 μmol / L to 15 μmol / L histidine.

[0059] After obtaining the second mixed solution, the present invention mixes cholesterol, flavonoids, and squalene with absolute ethanol to obtain a third mixed solution. In a specific embodiment, the flavonoids include tartary buckwheat flavonoids.

[0060] After obtaining the third mixed solution, the present invention mixes the first mixed solution, the second mixed solution, and the third mixed solution to obtain the avian vaccine adjuvant.

[0061] In a specific embodiment, the avian vaccine adjuvant can also be filtered through a 0.22 μm filter. In a specific embodiment, the total substance concentration of the avian vaccine adjuvant can also be adjusted using a phosphate buffer solution. In a specific embodiment, the phosphate buffer solution includes a phosphate buffer solution with a total phosphate content of 0.1 mol / L to 0.2 mol / L.

[0062] The present invention also provides the application of the avian vaccine adjuvant described in the above technical solution or the vaccine adjuvant prepared by the preparation method described in the above technical solution in the preparation of avian vaccines. In a specific embodiment, the avian vaccine adjuvant can be mixed with the vaccine and injected together. In a specific embodiment, the vaccines include avian influenza vaccine and / or Newcastle disease vaccine. In a specific embodiment, in the avian vaccine, the dosage of the adjuvant is 600 to 1200 doses / mL.

[0063] The present invention also provides the application of the avian vaccine adjuvant described in the above technical solution or the vaccine adjuvant prepared by the preparation method described in the above technical solution in any one or more of the functions as follows:

[0064] ① Advance the immune response time;

[0065] ② Improve the expression level of immune antibodies;

[0066] ③ Prolong the protection period of vaccine antibodies.

[0067] To further illustrate the present invention, the following will describe in detail an avian vaccine adjuvant provided by the present invention, its preparation method and application in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0068] The specifications and sources of some reagents used in the present invention are shown in Table 1.

[0069] Table 1 Specifications and sources of some reagents

[0070]

[0071]

[0072] Example 1

[0073] Preparation of avian vaccine adjuvant.

[0074] An avian vaccine adjuvant of the present invention is a water-soluble solution prepared by mixing ginsenoside, cholesterol, astragalus polysaccharide, amino acid, tartary buckwheat flavonoid, squalene, antibacterial peptide and wheat protein hydrolysate in a certain ratio. The specific operation steps are as follows:

[0075] Prepare 10 L of 0.1 mol / L phosphate buffer with ultrapure water.

[0076] Precisely weigh 0.5 g of antibacterial peptide, 4.0 g of ginsenoside, 6.0 g of astragalus polysaccharide and 10 g of wheat protein hydrolysate, and dissolve them in 8000 mL of phosphate buffer to obtain a first mixed solution.

[0077] Weigh the corresponding mass of amino acid according to its concentration in the vaccine adjuvant, and dissolve it in 100 mL of phosphate buffer solution to obtain a second mixed solution. Among them, the specific amino acids used and their concentrations in the second mixed solution are as follows:

[0078] L-Methionine: 30 μmol / L, L-Tryptophan: 3 μmol / L, L-Valine: 40 μmol / L, L-Lysine: 60 μmol / L, L-Leucine: 40 μmol / L, L-Isoleucine: 35 μmol / L, L-Phenylalanine: 12 μmol / L, L-Glutamine: 150 μmol / L, L-Threonine: 30 μmol / L, L-Arginine: 60 μmol / L, L-Cysteine: 15 μmol / L, L-Histidine: 10 μmol / L.

[0079] Accurately weigh 0.8 g of cholesterol, 70 g of squalene, and 8 g of tartary buckwheat flavonoids, and dissolve them in 50 mL of absolute ethanol to obtain the third mixed solution.

[0080] Mix the first mixed solution, the second mixed solution, and the third mixed solution, make up the volume to 10 L with 0.1 mol / L phosphate buffer solution, and then filter through a 0.22 μm filter to obtain the avian vaccine adjuvant.

[0081] Example 2

[0082] Preparation of avian vaccine adjuvant.

[0083] An avian vaccine adjuvant of the present invention is a water-soluble solution prepared by mixing ginsenoside RO, panax quinquefolius saponin, cholesterol, astragalus polysaccharide, zymosan, polygonatum polysaccharide, amino acids, tartary buckwheat flavonoids, squalene, antimicrobial peptides, and wheat protein hydrolysate in a certain ratio. The specific operation steps are as follows:

[0084] Prepare 10 L of 0.1 mol / L phosphate buffer solution with ultrapure water.

[0085] Accurately weigh 0.5 g of antimicrobial peptides, 3.0 g of ginsenoside, 5.0 g of astragalus polysaccharide, and 10 g of wheat protein hydrolysate, and dissolve them in 8000 mL of phosphate buffer solution to obtain the first mixed solution.

[0086] Weigh the corresponding mass according to the concentration of amino acids in the vaccine adjuvant and dissolve it in 100 mL of phosphate buffer solution to obtain the second mixed solution. Among them, the specific amino acids used and their concentrations in the second mixed solution are as follows:

[0087] L-Methionine: 30 μmol / L, L-Tryptophan: 3 μmol / L, L-Valine: 40 μmol / L, L-Lysine: 60 μmol / L, L-Leucine: 40 μmol / L, L-Isoleucine: 35 μmol / L, L-Phenylalanine: 12 μmol / L, L-Glutamine: 150 μmol / L, L-Threonine: 30 μmol / L, L-Arginine: 60 μmol / L, L-Cysteine: 15 μmol / L, L-Histidine: 10 μmol / L.

[0088] Accurately weigh 0.8 g of cholesterol, 70 g of squalene, and 8 g of tartary buckwheat flavonoids, and dissolve them in 50 mL of absolute ethanol to obtain the third mixed solution.

[0089] Mix the first mixed solution, the second mixed solution, and the third mixed solution, and make up the volume to 10 L with 0.1 mol / L phosphate buffer solution. After filtering through a 0.22 μm filter, the avian vaccine adjuvant is obtained.

[0090] Application Example 1

[0091] Pilot experiment on the effect of the avian vaccine adjuvant described in Example 1 on the antibody titer change of Newcastle disease vaccine.

[0092] Set 5 treatment groups with different dosages of the avian vaccine adjuvant as follows:

[0093] Treatment group 1: Use the avian vaccine adjuvant at 600 doses / mL (0.6 μL / dose);

[0094] Treatment group 2: Use the avian vaccine adjuvant at 800 doses / mL (0.8 μL / dose);

[0095] Treatment group 3: Use the avian vaccine adjuvant at 1000 doses / mL (1.0 μL / dose);

[0096] Treatment group 4: Use the avian vaccine adjuvant at 1200 doses / mL (1.2 μL / dose);

[0097] Treatment group 5: Use the avian vaccine adjuvant at 1400 doses / mL (1.4 μL / dose).

[0098] Select 50 healthy 1-day-old white leghorns in each treatment group, and randomly divide them into 5 groups, with 10 chickens in each group, namely: 4 replicate groups using the avian vaccine adjuvant (pilot test group 1, pilot test group 2, pilot test group 3, and pilot test group 4); 1 control group using normal saline at the same dosage as the avian vaccine adjuvant (normal saline group).

[0099] The grouped chicks are immunized with Newcastle disease and avian influenza virus (H9 subtype) bivalent inactivated vaccine. At the same time, according to the dosage settings of the above treatment groups, the avian vaccine adjuvant or normal saline of the present invention is jointly administered to each treatment group and its corresponding group. After immunization, blood is collected at 1 d, 7 d, 10 d, 14 d, 21 d, 28 d, 35 d, 42 d, and 49 d. The hemagglutination inhibition test is used to measure and record the antibody titer change rule of each group of experimental white leghorns, and the obtained antibody data is the average value of the experimental animal data of each group (see Table 2).

[0100] Table 2 Effect of avian vaccine adjuvant on the antibody titer change of Newcastle disease vaccine

[0101]

[0102]

[0103] The results of the effect of the avian vaccine adjuvant on the growth and decline of the antibody titer of Newcastle disease vaccine are shown in Table 2 and Figures 1 to 5 As shown, through the analysis by SPSS software, it can be known that after the avian vaccine adjuvant described in the present invention is used in combination with Newcastle disease vaccine and avian influenza vaccine, compared with the normal saline control group, the antibodies of Newcastle disease vaccine and avian influenza vaccine both have extremely significant expression on the 10th day after immunization (n = 10, p < 0.01); on the 14th day of immunization, the antibody levels of the two vaccines are extremely significantly higher than those of the normal saline control group (n = 10, p < 0.05); on the 21st and 35th days of immunization, although the antibody levels of each group have increased to a certain extent, it can be seen from the experimental data that the antibody level of the experimental group with this vaccine adjuvant has extremely significant improvement (n = 10, p < 0.01); on the 42nd and 49th days of immunization, the antibody expressions of the experimental group with this vaccine adjuvant and the normal saline control group both decline, but the decline value of the experimental group with this vaccine adjuvant is extremely small, and the antibody expression level is still extremely significantly higher than that of the normal saline group (n = 10, p < 0.05). It can be concluded from this that when the avian vaccine adjuvant described in the present invention is used in combination with Newcastle disease vaccine and avian influenza vaccine, it can significantly advance the antibody expression of Newcastle disease vaccine and avian influenza vaccine, improve the antibody expression level, and at the same time extend the vaccine protection period.

[0104] Application Example 2

[0105] Pilot experiment on the effect of the avian vaccine adjuvant described in Example 2 on the growth and decline of the antibody titer of avian influenza vaccine.

[0106] Set 5 treatment groups with different dosages of avian vaccine adjuvant used, specifically as follows:

[0107] Treatment group 1: Use the avian vaccine adjuvant at 600 doses / mL (0.6 μL / dose);

[0108] Treatment group 2: Use the avian vaccine adjuvant at 800 doses / mL (0.8 μL / dose);

[0109] Treatment group 3: Use the avian vaccine adjuvant at 1000 doses / mL (1.0 μL / dose);

[0110] Treatment group 4: Use the avian vaccine adjuvant at 1200 doses / mL (1.2 μL / dose);

[0111] Treatment group 5: Use the avian vaccine adjuvant at 1400 doses / mL (1.4 μL / dose).

[0112] Select 300 healthy 1-day-old white - feather chickens within each treatment group, and randomly divide them into 5 groups, with 60 chickens in each group, namely: 4 replicate groups using avian vaccine adjuvant (Pilot Test Group 1, Pilot Test Group 2, Pilot Test Group 3, and Pilot Test Group 4); 1 control group using normal saline according to the same dosage as the same avian vaccine adjuvant (Normal Saline Group).

[0113] The grouped chicks were immunized with Newcastle disease and avian influenza virus (H9 subtype) bivalent inactivated vaccine. At the same time, according to the dosage settings of the above - mentioned treatment groups, the avian vaccine adjuvant or normal saline of the present invention was jointly administered to each treatment group and its corresponding group. After the immunization, blood was collected at 1d, 7d, 10d, 14d, 21d, 28d, 35d, 42d, and 49d. The hemagglutination inhibition test was used to measure and record the growth and decline law of antibody titers of experimental white - feather chickens in each group. The obtained antibody data was the average value of the experimental animal data in each group (see Table 3).

[0114] Table 3 Influence of avian vaccine adjuvant on the growth and decline of anti - efficacy titer of avian influenza vaccine

[0115]

[0116]

[0117] The results of the influence of avian vaccine adjuvant on the growth and decline of anti - efficacy titer of Newcastle disease vaccine were obtained as shown in Table 3 and Figures 6 to 10 As shown. Through SPSS software analysis, it can be known that after the avian vaccine adjuvant of the present invention is used in combination with Newcastle disease vaccine and avian influenza vaccine, compared with the normal saline control group, the antibodies of Newcastle disease vaccine and avian influenza vaccine both have extremely significant expression at the 10th day after immunization (n = 60, p < 0.01); at the 14th day of immunization, the antibody levels of the two vaccines are extremely significantly higher than those of the normal saline control group (n = 60, p < 0.05); at 21d and 35d of immunization, although the antibody levels of each group have increased to a certain extent, it can be seen from the experimental data that the antibody levels of the experimental group with this vaccine adjuvant have extremely significant increases (n = 60, p < 0.01); at 42d and 49d of immunization, the antibody expressions of the experimental group with this vaccine adjuvant and the normal saline control group both decline, but the decline value of the experimental group with this vaccine adjuvant is extremely small, and the antibody expression level is still extremely significantly higher than that of the normal saline group (n = 60, p < 0.05). It can be concluded that: when the avian vaccine adjuvant of the present invention is used in combination with Newcastle disease vaccine and avian influenza vaccine, it can significantly advance the antibody expression of Newcastle disease vaccine and avian influenza vaccine, improve the antibody expression level, and at the same time extend the vaccine protection period.

[0118] Application Example 3

[0119] A commercially available conventional vaccine adjuvant (produced by Hangzhou Yisikang Medical Technology Co., Ltd., M101 vaccine adjuvant) was selected as the adjuvant control group to compare and verify the usage effect of the avian vaccine adjuvant described in the present invention. The specific experimental method is as follows:

[0120] Pilot experiment: 30 healthy 1-day-old white leghorn chickens were selected and randomly divided into groups of 10 each, namely, the normal saline group, the experimental group, and the adjuvant control group.

[0121] The grouped chicks were immunized with a Newcastle disease and avian influenza virus (H9 subtype) bivalent inactivated vaccine. At the same time, according to the above grouping, the chicks were respectively jointly administered with the avian vaccine adjuvant described in the present invention at 1000 vaccine doses / mL (1.0 μL / vaccine dose), normal saline at 1000 vaccine doses / mL (1.0 μL / vaccine dose), or a commercially available control vaccine adjuvant at 1000 vaccine doses / mL (1.0 μL / vaccine dose). After the immunization was completed, blood was collected at 1d, 7d, 10d, 14d, 21d, 28d, 35d, 42d, and 49d. The hemagglutination inhibition test was used to measure and record the growth and decline pattern of the antibody titers of the white leghorn chickens in each group. The obtained antibody data was the average value of the data of the experimental animals in each group (see Table 4).

[0122] Pilot-scale experiment: 150 healthy 1-day-old white leghorn chickens were selected and randomly divided into groups of 50 each, namely, the normal saline group, the experimental group, and the adjuvant control group.

[0123] The grouped chicks were immunized with a Newcastle disease and avian influenza virus (H9 subtype) bivalent inactivated vaccine. At the same time, according to the above grouping, the chicks were respectively jointly administered with the avian vaccine adjuvant described in the present invention at 1000 vaccine doses / mL (1.0 μL / vaccine dose), normal saline at 1000 vaccine doses / mL (1.0 μL / vaccine dose), or a commercially available control vaccine adjuvant (please supplement the dosage of the control group adjuvant by the teacher). After the immunization was completed, blood was collected at 1d, 7d, 10d, 14d, 21d, 28d, 35d, 42d, and 49d. The hemagglutination inhibition test was used to measure and record the growth and decline pattern of the antibody titers of the white leghorn chickens in each group. The obtained antibody data was the average value of the data of the experimental animals in each group (see Table 4).

[0124] Table 4 Effects of different treatment groups on the growth and decline of vaccine antibody titers

[0125]

[0126] The results of the effects of different treatment groups on the growth and decline of vaccine antibody titers are shown in Table 4 and Figure 11 、 Figure 12 as shown. It can be known from the analysis by SPSS software that whether it is the pilot experiment on the growth and decline of the Newcastle disease vaccine antibody titer or the pilot-scale experiment on the growth and decline of the avian influenza vaccine antibody titer.

[0127] After the avian vaccine adjuvant of the present invention is used in combination with Newcastle disease vaccine and avian influenza vaccine, compared with the normal saline control group and the vaccine adjuvant control group, the results of the small-scale experiment show that: the antibodies of Newcastle disease vaccine and avian influenza vaccine both have extremely significant expression on the 10th day after immunization (n = 10, p < 0.01); on the 14th day of immunization, the antibody levels of the two vaccines are extremely significantly higher than those of the normal saline control group and the vaccine adjuvant control group (n = 10, p < 0.05); on the 21st and 35th days of immunization, although the antibody levels of each group have increased to a certain extent, it can be seen from the experimental data that the antibody level of the experimental group with this vaccine adjuvant has extremely significant increase (n = 10, p < 0.01), while the vaccine adjuvant group has a significant increase compared with the normal saline group (n = 10, p < 0.05); on the 42nd and 49th days of immunization, the antibody expressions of the experimental group with this vaccine adjuvant, the normal saline control group, and the adjuvant control group all decline, but the decline value of the experimental group with this vaccine adjuvant is extremely small, and the antibody expression level is still extremely significantly higher than that of the normal saline group and the adjuvant control group (n = 10, p < 0.05).

[0128] The results of the pilot-scale experiment show that: both have extremely significant expression on the 10th day after immunization (n = 50, p < 0.01); on the 14th day of immunization, the antibody levels of the two vaccines are extremely significantly higher than those of the normal saline control group and the vaccine adjuvant control group (n = 50, p < 0.05); on the 21st and 35th days of immunization, although the antibody levels of each group have increased to a certain extent, it can be seen from the experimental data that the antibody level of the experimental group with this vaccine adjuvant has extremely significant increase (n = 50, p < 0.01), while the vaccine adjuvant group has a significant increase compared with the normal saline group (n = 50, p < 0.05); on the 42nd and 49th days of immunization, the antibody expressions of the experimental group with this vaccine adjuvant, the normal saline control group, and the adjuvant control group all decline, but the decline value of the experimental group with this vaccine adjuvant is extremely small, and the antibody expression level is still extremely significantly higher than that of the normal saline group and the adjuvant control group (n = 50, p < 0.05).

[0129] It can be concluded that: when the avian vaccine adjuvant of the present invention is used in combination with Newcastle disease vaccine and avian influenza vaccine, it can significantly advance the antibody expression of Newcastle disease vaccine and avian influenza vaccine, improve the antibody expression level, and at the same time extend the vaccine protection period.

[0130] Application Example 4

[0131] Performance evaluation of vaccine adjuvant.

[0132] Perform the following performance evaluation on the vaccine adjuvant provided in Example 1 of the present invention:

[0133] 1. Safety test:

[0134] Healthy 1-day-old white - feather broilers were randomly selected and evenly divided into 6 groups, with 15 broilers in each group. Five of the groups were each injected with the avian vaccine adjuvant described in Example 1 of the present invention at a dose of 1.4 μL per broiler, and the other group was used as a normal control. After injection, each group was placed in the same management environment for feeding and management. The body temperature of the white - feather broilers was measured regularly every day and recorded. The experiment was completed after continuous observation for 14 days.

[0135] Experimental results: During the test period, the body temperatures of the white - feather broilers in each group did not show an increase in body temperature (body temperature exceeding 40 °C), and there were no obvious clinical symptoms, indicating that the safety of the vaccine adjuvants in each group was good.

[0136] 2. Stability test:

[0137] Nine samples of the vaccine adjuvant prepared in Example 1 of the present invention were randomly selected and divided into 3 groups. These 3 groups of samples were respectively placed at 2 °C - 8 °C, room temperature (18 °C - 25 °C), and 37 °C ± 2 °C for 1 month. Whether there was a layering phenomenon in each group of samples was observed. If there was no layering, it indicated good stability. The test results are as Figures 13 to 15 shown. The vaccine adjuvant provided in Example 1 of the present invention did not show a layering phenomenon after 1 month under the conditions of 2 - 8 °C ( Figure 14 ), room temperature ( Figure 13 ), and 37 °C ( Figure 15 ), that is, the stability was good.

[0138] Although the above - mentioned embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative labor, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A poultry vaccine adjuvant, characterized in that: The raw materials for preparing the poultry vaccine adjuvant include saponin, cholesterol, polysaccharide, amino acid, flavonoid, squalene, antimicrobial peptide, protein hydrolyzate and pharmaceutically acceptable auxiliary materials; The ginsenosides include ginsenoside Ro and / or American ginsenosides; the polysaccharides include one or more of yeast polysaccharides, astragalus polysaccharides and polygonatum polysaccharides.

2. The poultry vaccine adjuvant according to claim 1, characterized in that The amino acids include methionine, tryptophan, valine, lysine, leucine, isoleucine, phenylalanine, glutamine, threonine, arginine, cysteine ​​and histidine; the flavonoids include tartary buckwheat flavonoids; and the protein hydrolysate includes wheat protein hydrolysate.

3. The poultry vaccine adjuvant according to claim 1, characterized in that The concentration of the saponin in the vaccine adjuvant is 0.02% to 0.10%; The concentration of cholesterol in the vaccine adjuvant is 0.005% to 0.015%; The concentration of the polysaccharide in the vaccine adjuvant is 0.01% to 0.15%; The concentration of the amino acid in the vaccine adjuvant is 200-1150 μmol / L; The concentration of the flavonoids in the vaccine adjuvant is 0.05% to 0.20%; The concentration of squalene in the vaccine adjuvant is 0.3% to 1.1%; The concentration of the antimicrobial peptide in the vaccine adjuvant is 0.005% to 0.05%; The concentration of the protein hydrolysate in the vaccine is 0.05% to 0.3%.

4. The poultry vaccine adjuvant according to claim 1, characterized in that The auxiliary materials include phosphate buffer and / or anhydrous ethanol.

5. The poultry vaccine adjuvant according to claim 4, characterized in that The phosphate buffer comprises a phosphate buffer having a total phosphate content of 0.1 mol / L to 0.2 mol / L.

6. The method for preparing the poultry vaccine adjuvant according to any one of claims 1 to 4, comprising the following steps: Mixing saponin, polysaccharide, antimicrobial peptide and protein hydrolysate with phosphate buffer to obtain a first mixed solution; Mixing the amino acid with the phosphate buffer to obtain a second mixed solution; Mixing cholesterol, flavonoids and squalene with anhydrous ethanol to obtain a third mixed solution; The first mixed solution, the second mixed solution and the third mixed solution are mixed to obtain the poultry vaccine adjuvant.

7. Use of the poultry vaccine adjuvant according to any one of claims 1 to 4 or the poultry vaccine adjuvant prepared by the preparation method according to claim 5 or 6 in the preparation of poultry vaccines.

8. The use according to claim 7, characterized in that: The vaccine comprises an avian influenza vaccine and / or a Newcastle disease vaccine.

9. The use according to claim 7, characterized in that: In the poultry vaccine, the amount of adjuvant used is 600 to 1200 portions / mL.

10. Use of the poultry vaccine adjuvant according to any one of claims 1 to 4 or the vaccine adjuvant prepared by the preparation method according to claim 5 or 6 in any one or more of the functions ① to ③: ① Advance immune response time; ② Improve the expression level of immune antibodies; ③Extend the protection period of vaccine antibodies.

Images