Preparation and application of new stream bivalent inactivated vaccine containing composite immunopotentiator

Abstract

The invention discloses preparation and application of a new Newcastle disease bivalent inactivated vaccine containing a composite immunopotentiator, and the vaccine is characterized by comprising the following components: an inactivated Newcastle disease virus antigen solution of which the virus titer is greater than or equal to 10 < 9 > EID50 / mL, an inactivated H9 subtype avian influenza virus antigen solution of which the virus titer is greater than or equal to 10 < 9 > EID50 / mL, and an inactivated H9 subtype avian influenza virus antigen solution of which the virus titer is greater than or equal to 10 < 9 > EID50 / mL. The virus titer is larger than or equal to 10 < 9 > EID50 / mL, the water-phase solution is composed of an inactivated antigen solution and the compound immunopotentiator, and the oil-phase solution is composed of white oil, sorbitan trioleate and aluminum stearate; according to the invention, by adding the composite immunopotentiator, the rapid recognition and reaction of an immune system on antigens can be enhanced, the activation and function of antigen presenting cells can be promoted, and the antigen uptake and treatment capabilities of the antigen presenting cells can be improved, so that the formation of immune response can be accelerated, and the long time of the antigen in vivo can be maintained, thereby prolonging the persistence of the immune response; therefore, the defects of the existing inactivated vaccine in prevention and control of avian influenza and Newcastle disease are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of immune adjuvants, and in particular to the preparation and application of a new type of dual inactivated vaccine containing a composite immune enhancer. Background Art

[0002] Newcastle disease (ND) is a highly contagious, acute, and highly pathogenic infectious disease caused by Newcastle disease virus (NDV) of the genus Avulavirus in the family Paramyxoviridae. It is also known as Asian fowl plague or pseudofowl plague. The disease can cause severe respiratory symptoms, neurological symptoms, and digestive tract lesions in poultry, manifested as difficulty breathing, loose stools, neurological disorders, and mucosal and serosal bleeding. The mortality rate is extremely high. Newcastle disease can be transmitted through direct contact, air transmission, and contaminated feed and water sources, posing a major threat to the poultry industry and causing huge economic losses.

[0003] Avian influenza (AI) is a highly contagious, cross-species poultry disease caused by influenza A virus (AIV) of the Orthomyxoviridae family. The H9N2 subtype of avian influenza (AIV H9) is one of the most widespread avian influenza subtypes. It can cause fever, breathing difficulties, pulmonary hemorrhage, muscle pain, decreased feed intake and other symptoms in chickens, and can be rapidly transmitted through air, feces and other contact routes, especially in winter and spring. The incidence rate is high. This virus not only has a huge impact on the poultry farming industry, but also has been found to infect mammals due to its cross-host infection ability, and even poses a potential threat to human health. Therefore, the prevention and control of avian influenza is of vital importance.

[0004] At present, the main means of controlling Newcastle disease and H9 subtype avian influenza is vaccination. Traditional inactivated vaccines are widely used in poultry immunization and prevention due to their high safety and good stability. However, existing inactivated vaccines have the following shortcomings:

[0005] 1. Slow immune protection: Compared with live vaccines, inactivated vaccines induce weaker immune responses, and the body's immune system takes a long time to activate, so they cannot provide effective immune protection in a short period of time. In addition, after the inactivated vaccine is injected, the antigen is quickly cleared, resulting in a limited duration of immune response. Multiple vaccinations are required to maintain an effective immune level.

[0006] 2. Existing inactivated vaccines mostly use mineral oil or white oil as adjuvants, but simple oil adjuvants are difficult to effectively activate cellular immunity and humoral immunity, and the immune enhancement effect is limited, resulting in low levels of antibodies produced by the body. Inactivated vaccines usually need to be combined with immunopotentiators to improve the immune effect, but there are few types of existing immunopotentiators, some of which may have safety risks or side effects, and the antibody titers of some vaccines are still low;

[0007] To solve the above problems, we proposed the preparation and application of a new type of bivalent inactivated vaccine containing a composite immune enhancer. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides the preparation and application of a new type of bivalent inactivated vaccine containing a composite immunopotentiator. By introducing a new type of composite immunopotentiator, the immune protection ability of the vaccine is improved, the antigen retention time is prolonged, and the immune response is enhanced, thereby solving the deficiencies of the existing inactivated vaccines in the prevention and control of avian influenza and Newcastle disease, and solving the problems raised in the background technology.

[0009] The above technical objectives of the present invention are achieved through the following technical solutions:

[0010] A new type of two-inactivated vaccine containing a composite immune enhancer, the vaccine comprises the following components:

[0011] Inactivated Newcastle disease virus antigen solution, with a virus titer of ≥10 9 EID 50 / mL, inactivated H9 subtype avian influenza virus antigen solution, with a virus titer of ≥10 9 EID 50 / mL, composite immune enhancer, aqueous solution and oily solution.

[0012] Preferably, the composite immunopotentiator comprises the following ingredients in the following weight concentration ranges:

[0013] Antimicrobial peptides: 1-1000 mg / mL;

[0014] Poly I:C: 1-1000 mg / mL;

[0015] N,N,N-trimethylglycine: 1-1000 mg / mL;

[0016] Aloe polysaccharide: 1-1000mg / mL.

[0017] Preferably, the aqueous phase solution consists of an inactivated antigen solution and a composite immunopotentiator, and the oil phase solution consists of white oil, sorbitan trioleate and aluminum stearate.

[0018] Preferably, the volume ratio of the aqueous phase solution to the oil phase solution is 1:2.

[0019] Preferably, the volume ratio of the composite immunoenhancer to the inactivated antigen solution in the aqueous solution is 1:6.5.

[0020] Preferably, the mass ratio of white oil, sorbitan trioleate and aluminum stearate in the oil phase solution is 19:1:0.15.

[0021] The present invention also provides a method for preparing a novel dual inactivated vaccine containing a composite immunopotentiator, comprising the following steps:

[0022] S1: Preparation of aqueous solution: dissolving the composite immunopotentiator in proportion and mixing it with the inactivated antigen solution and polysorbate to prepare an aqueous solution;

[0023] S2: Preparation of oil phase solution: mixing white oil, sorbitan trioleate and aluminum stearate to form a uniform oil phase solution;

[0024] S3: Emulsification: The aqueous solution obtained in step S1 and the oily solution obtained in step S2 are subjected to shear emulsification to form a water-in-oil inactivated vaccine, wherein the shear emulsification is performed using a high-speed emulsifier at 8000-13000 rpm / min for 2-6 minutes to obtain a stable emulsion;

[0025] S4: Vaccine stabilization: The emulsified vaccine should be sealed and stored at 2-8°C for at least 30 days. There should be no significant change in the uniformity of the vaccine particle size, and the emulsification system should be stable without visible precipitation or stratification.

[0026] The present invention also provides an application of a Newcastle disease bivalent inactivated vaccine containing a composite immunopotentiator, which is used to prevent and control the spread of Newcastle disease and H9 subtype avian influenza, and is particularly suitable for large-scale immunization in poultry farming.

[0027] In summary, the present invention mainly has the following beneficial effects:

[0028] 1. The present invention can enhance the rapid recognition and response of the immune system to antigens by adding a composite immunopotentiator. Antimicrobial peptides, poly I-C and other ingredients can promote the activation and function of antigen-presenting cells, enhance their ability to take up and process antigens, and thus accelerate the formation of immune responses. The use of composite immunopotentiators can also delay the clearance of antigens, keep antigens in the body for a longer period of time, and thus prolong the persistence of the immune response.

[0029] 2. The composite immunopotentiator can significantly enhance the body's cellular immunity and humoral immunity by activating different immune pathways. For example, poly I-cell can induce the production of interferon and promote natural immune response, while antimicrobial peptides can enhance the activity of T cells and further enhance specific immune response. Through these mechanisms, the present invention can greatly improve the immune effect of vaccines, so that the body can produce stronger immune defense when encountering Newcastle disease or avian influenza virus.

[0030] 3. Compound immune enhancers enhance the immunogenicity of antigens, enabling the immune system to recognize and respond more effectively, thereby improving the immune effect of vaccines. Compound immune enhancers can help vaccines form stronger immune responses in the body and promote the efficient production of antibodies. By accelerating the activation of the immune system, it enhances the intensity and breadth of the immune response, thereby further improving the protective effectiveness of vaccines.

[0031] 4. The composite immunopotentiator of the present invention can prolong the time that the vaccine provides immune protection by enhancing the memory function of the immune system and maintaining antigen stimulation for a longer period of time. The slow release and persistent stimulation of antigens enable the immune system to maintain a highly efficient immune state for a longer period of time, thereby reducing the need for frequent vaccinations, reducing immunization costs, and improving the overall benefits of vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a graph of NDV antibody titers of the present invention;

[0033] Figure 2 It is a graph of the H9AIV antibody titer of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings of the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0035] The following examples are used to illustrate the present invention, but they cannot be used to limit the scope of protection of the present invention. The conditions in the examples can be further adjusted according to specific conditions. Simple improvements to the method of the present invention under the premise of the concept of the present invention belong to the scope of protection claimed in the present invention.

[0036] Example 1

[0037] This embodiment provides a new type of two-inactivated vaccine containing a composite immunopotentiator, the vaccine comprising the following components:

[0038] Inactivated Newcastle disease virus antigen solution, with a virus titer of ≥10 9 EID 50 / mL, inactivated H9 subtype avian influenza virus antigen solution, with a virus titer of ≥10 9 EID 50 / mL, composite immune enhancer, aqueous solution and oily solution.

[0039] Furthermore, the composite immunopotentiator comprises the following components in the following weight concentration ranges:

[0040] Antimicrobial peptides: 1-1000 mg / mL;

[0041] Poly I:C: 1-1000 mg / mL;

[0042] N,N,N-trimethylglycine: 1-1000 mg / mL;

[0043] Aloe polysaccharide: 1-1000mg / mL.

[0044] Furthermore, the aqueous phase solution is composed of an inactivated antigen solution and a composite immunopotentiator, and the oil phase solution is composed of white oil, sorbitan trioleate and aluminum stearate.

[0045] Furthermore, the volume ratio of the aqueous phase solution to the oil phase solution is 1:2.

[0046] Furthermore, the volume ratio of the composite immunoenhancer to the inactivated antigen solution in the aqueous solution is 1:6.5.

[0047] Furthermore, the mass ratio of the white oil, sorbitan trioleate and aluminum stearate in the oil phase solution is 19:1:0.15.

[0048] The present invention also provides a method for preparing a novel dual inactivated vaccine containing a composite immunopotentiator, comprising the following steps:

[0049] S1: Preparation of aqueous solution: dissolving the composite immunopotentiator in proportion and mixing it with the inactivated antigen solution and polysorbate to prepare an aqueous solution;

[0050] S2: Preparation of oil phase solution: mixing white oil, sorbitan trioleate and aluminum stearate to form a uniform oil phase solution;

[0051] S3: Emulsification: The aqueous solution obtained in step S1 and the oily solution obtained in step S2 are subjected to shear emulsification to form a water-in-oil inactivated vaccine, wherein the shear emulsification is performed using a high-speed emulsifier at 8000-13000 rpm / min for 2-6 minutes to obtain a stable emulsion;

[0052] S4: Vaccine stabilization: The emulsified vaccine should be sealed and stored at 2-8°C for at least 30 days. There should be no significant change in the uniformity of the vaccine particle size, and the emulsification system should be stable without visible precipitation or stratification.

[0053] The present invention also provides an application of a Newcastle disease bivalent inactivated vaccine containing a composite immunopotentiator, which is used to prevent and control the spread of Newcastle disease and H9 subtype avian influenza, and is particularly suitable for large-scale immunization in poultry farming.

[0054] Example 2

[0055] refer to Figure 1-Figure 2 , Evaluation test plan for poultry immune enhancers

[0056] 1. Experimental purpose: To evaluate the enhancing effect of compound immunopotentiators on Newcastle disease (NDV) and H9 subtype avian influenza (AIV H9) inactivated vaccines.

[0057] 2. Experimental Materials

[0058] 2.1 Immunoenhancer: The enhancer is prepared into a suitable concentration, added to the antigen in a suitable proportion and dosage, and the aqueous phase is prepared.

[0059] 2.2 Antigen: The virus titers of Newcastle disease inactivated antigen and H9 avian influenza inactivated antigen before inactivation were both greater than 109EID50 / mL.

[0060] 2.3 Experimental animals: physiological saline group (negative control group), conventional vaccine group (white oil + antigen group), combined immune enhancer + conventional vaccine group (white oil + antigen group), each group with 10 14-day-old white-feathered broilers, a total of 3 groups.

[0061] 2.4 Other test items: White oil, polysorbate, sorbitan trioleate, aluminum stearate, physiological saline, etc. were purchased from Shanghai Maihong Company.

[0062] 2.5 Detection reagents: AIV H9 hemagglutination inhibition test standard antigen and NDV standard antigen were purchased from Harbin Guosheng Biotechnology Co., Ltd.

[0063] 3. Test methods

[0064] 3.1 Preparation of immunopotentiator: Add poly I-C, trimethylglycine and aloe polysaccharide to the antimicrobial peptide according to the required amount to prepare a composite immunopotentiator, and ensure that the final concentration of the antimicrobial peptide is 20 mg / mL.

[0065] 3.2 Vaccine preparation: Vaccines with or without added immunopotentiators were prepared with reference to conventional formulas. The formulas of each component are detailed in Table 1:

[0066]

[0067] Table 1 Vaccine composition

[0068] Take 2 portions of oil phase (30 mL, containing 1.5 mL sorbitan trioleate + 0.15 mg aluminum stearate + 28.5 mL white oil) for emulsification;

[0069] Shear emulsification was performed at 8000 rpm / min for 2 min, and 1 portion of water phase (15 mL) was slowly added at the same time. Shear emulsification was performed at 13000 rpm / min for 6 min. The mixture was sealed with a cap, labeled, and stored at 2-8°C.

[0070] 3.3 Inspection

[0071] 3.3.1 Properties: Appearance: white uniform emulsion, appearance: white uniform emulsion, dosage form: water-in-oil, qualitative: 10 mL of vaccine was added into a centrifuge tube and centrifuged at 3000 rpm / min for 15 min. No stratification was observed.

[0072] 3.3.2 Sterility test: The test is carried out in accordance with the appendix of the current "Chinese Veterinary Pharmacopoeia", and sterile growth is prohibited.

[0073] 3.4 Evaluation of the efficacy of white-feathered broiler chickens

[0074] 3.4.1 Newcastle disease-H9N2 avian influenza inactivated vaccine: 14-day-old white-feathered broilers were numbered and randomly divided into 16 groups, with 10 chickens in each group. Each chicken was injected with 0.3 mL of vaccine subcutaneously in the neck. Ten chickens were immunized with normal saline as the negative control group. The grouping is detailed in Table 2:

[0075]

[0076] Table 2 Grouping and immunization of vaccine AI part efficacy test

[0077] Blood was collected 7, 14, 21, 28 and 35 days after immunization, and serum was separated to determine the AI ​​and HI antibody titers of serum. The hemagglutination inhibition titer in serum was determined according to the national standard GB / T 17999.2-2008.

[0078] 4. Test results and analysis

[0079] See Table 3 for details:

[0080]

[0081]

[0082] Table 3 Average antibody titer data

[0083] Test conclusion:

[0084] Compared with the NDV vaccine control group, the average antibody titer of chicken serum in the antimicrobial peptide enhancer group at 21, 28 and 35 days after immunization was increased by 1.1log2, 1.9log2 and 2.3log2 respectively, which fully meets the industry requirement of enhancing the antibody titer by at least 0.5log2.

[0085] Compared with the H9 AIV vaccine control group, the average antibody titer of chicken serum in the antimicrobial peptide enhancer group at 28 and 35 days after immunization was increased by 1.3log2 and 0.8log2 respectively, which fully meets the industry requirement of enhancing the antibody titer by at least 0.5log2.

[0086] Example 3

[0087] 1. Experimental Materials

[0088] The inactivated Newcastle disease virus solution and the inactivated avian influenza H9N2 subtype virus solution were inactivated by 1% formaldehyde solution.

[0089] 14-day-old healthy white broiler chickens.

[0090] 2. Vaccine preparation

[0091] Poly I-C, trimethylglycine and aloe polysaccharide are added to the antimicrobial peptide according to the required amount to prepare a composite immune enhancer; the antimicrobial peptide content is ensured to be 20 mg / mL, and the mixture is filtered and sterilized using a syringe filter membrane.

[0092] An aqueous phase solution was prepared by mixing the immunopotentiator, inactivated Newcastle disease virus solution, inactivated avian influenza H9N2 subtype virus solution and polysorbate in a volume ratio of 2:6.2:6.2:0.6. After adding polysorbate, the solution was immediately placed in a water bath for about 5 minutes and oscillated to mix. Sorbitan trioleate and white oil were mixed in a volume ratio of 1:19, and 0.15 mg of aluminum stearate was added to prepare an oil phase. The solution was sheared and emulsified at 13000 rpm by a high-speed emulsifier for 6 minutes to obtain experimental group 1 (i.e., group 1).

[0093] The inactivated Newcastle disease virus solution, the inactivated avian influenza H9N2 subtype virus solution and polysorbate were prepared in a volume ratio of 6.2:6.2:0.6 to prepare an aqueous phase solution, and physiological saline was added to make up to 15 mL. After adding polysorbate, it was immediately bathed in water for about 5 minutes and oscillated to mix. Sorbitan trioleate and white oil were mixed in a volume ratio of 1:19, and 0.15 mg of aluminum stearate was added to prepare an oil phase. The solution was sheared and emulsified at 13000 rpm by a high-speed emulsifier for 6 minutes to obtain experimental group 2 (i.e., group 2).

[0094] An equal volume of physiological saline was directly added to obtain experimental group 3 (i.e., group 3).

[0095] 3. Grouping, Immunization and Antibody Detection

[0096] Healthy white-feathered broiler chickens were randomly divided into 3 groups, with 10 chickens in each group. Group 1, Group 2, and Group 3 were immunized one group each, and the immunization dose was 0.3 mL.

[0097] Blood was randomly collected from 15 chickens before immunization. On the 7th, 14th, 21st, 28th and 35th days after immunization, blood was collected from healthy white-feathered broilers in each group to separate serum, and the hemagglutination inhibition titer experiment was used to determine whether the antibodies were qualified.

[0098] The antibody titers before immunization and on days 7, 14, 21, 28, and 35 after immunization are shown in Table 1 below:

[0099]

[0100]

[0101] Table 1 Average antibody titer data

[0102] 4.1% Red Blood Cell Preparation

[0103] Blood collected from SPF chickens was concentrated into a 50 mL centrifuge tube, and 1% PBS buffer was added to 50 mL.

[0104] Centrifuge at 1800rpm for 10min, repeat twice, transfer the red blood cell pellet to a 15mL centrifuge tube, add 1% PBS buffer to 15mL, centrifuge at 1500rpm for 5min, add the red blood cells obtained by centrifugation to 1% PBS buffer at a ratio of 1:100 and mix well to obtain 1% red blood cells.

[0105] 5. Antigen hemagglutination titer determination

[0106] Add 30 μL of 1xPBS to the 1st to 12th wells of a 96-well microhemagglutination plate (the minimum range of the sample gun is 30 μL), add 30 μL of antigen to the first well of each row, make gradient dilutions from the 1st to the 11th wells, pipette and mix ten times, aspirate 30 μL from the 11th well, use the twelfth well as a control well, add 30 μL of 1xPBS to each well (this step is to replace serum), add 30 μL of 1% red blood cell suspension to each well, shake slightly, let stand at room temperature for 20 minutes, then read the results, tilt the hemagglutination plate 70 degrees, and the maximum dilution of the antigen at which the red blood cells are completely agglutinated is the hemagglutination titer of the antigen, and one unit is 1 unit.

[0107] 6. Preparation and Validation of Four-unit Antigen

[0108] Dilute the purchased antigen dry powder with 2mL of 1x PBS per ampoule, store the excess in -80℃, and store the dry powder at -20℃. Take the preparation of 4 hemagglutination units of antigen as an example. If the hemagglutination titer of the antigen stock solution is 9log2, the dilution factor is 29 / 22=27=128 times, that is, take 11.8mL of normal saline and add 1mL of antigen diluted 1 to 10. The titer of the stock solution in this experiment is 10, so it is diluted 256 times, that is, 76.8mL of 1xPBS, add 3mL of antigen diluted 1 to 10 (0.3 stock solution + 2.71xPBS, add 1x PBS 30μL to the 1st to 12th wells of the 96-well microhemagglutination plate (the minimum range of the row gun is 30μL), add 30μL of four units of antigen to the first well of each row, make gradient dilutions from the first well to the twelfth well, pipette and mix ten times, aspirate 30μL from the twelfth well, and add 1x PBS to each well. 30μL (this step is to replace serum), add 30μL of 1% red blood cell suspension to each well, shake slightly, let it stand at room temperature for 20 minutes and then read the readings. Tilt the blood coagulation plate 70 degrees and observe. The first well is 21, the second well is 22, and the third well is 23. The ideal result is that the first and second wells are completely agglutinated, and the third well begins to flow down, which is exactly 4 units.

[0109] 7. Hemagglutination inhibition test

[0110] One sample per row, 160 chickens means 160 rows, one hemagglutination plate has 8 rows, that is 20 plates, add 30μL of 1x PBS to all wells, draw serum to the first well of each row, dilute gradiently to the eleventh well, discard 30μL, the twelfth well is used as a blank control (dilute to the twelfth well, and the negative control is done separately at the end), add 30μL of four units of antigen to each well, shake slightly, let stand at room temperature for half an hour, add 30μL of 1% red blood cell suspension to each well, pay attention to the time of adding the first plate and the last plate, 20 plates are about 6min, follow the order, and read each plate in exactly 20min (this is the best time), the well with the highest dilution multiple that just flows down is the inhibition titer, the full agglutination of the negative control is normal, the serum hemagglutination inhibition titer of avian influenza and Newcastle disease is ≥24 and is positive.

[0111] refer to Figure 1-Figure 2 The present invention achieves the purpose of improving the immune effect of the combined inactivated vaccine of Newcastle disease and H9 subtype avian influenza by adding antimicrobial peptides, poly I-C, trimethylglycine and aloe polysaccharide to the combined inactivated vaccine of Newcastle disease and H9 subtype avian influenza and detecting the effects on the humoral immune response of chickens.

[0112] Finally, it should be noted that the present invention is not limited to the above embodiments, and there may be many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered to be within the scope of protection of the present invention.

[0113] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that, unless otherwise defined, the technical terms or scientific terms used in the present invention should have the usual meanings understood by persons with ordinary skills in the field to which the present invention belongs, and the words "include" or "comprise" and the like used in the present invention mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents.

[0114] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A new type of inactivated bivalent vaccine containing a composite immunopotentiator, characterized in that: The vaccine contains the following components: Inactivated Newcastle disease virus antigen solution, with a virus titer of ≥10 9 EID 50 / mL, inactivated H9 subtype avian influenza virus antigen solution, with a virus titer of ≥10 9 EID 50 / mL, composite immune enhancer, aqueous solution and oily solution.

2. A new type of inactivated dual vaccine containing a composite immunopotentiator according to claim 1, characterized in that: The composite immune enhancer comprises the following ingredients in the following weight concentration ranges: Antimicrobial peptides: 1-1000 mg / mL; Poly I:C: 1-1000 mg / mL; N,N,N-trimethylglycine: 1-1000 mg / mL; Aloe polysaccharide: 1-1000mg / mL.

3. A new type of inactivated dual vaccine containing a composite immunopotentiator according to claim 1, characterized in that: The aqueous phase solution consists of an inactivated antigen solution and a composite immunopotentiator, and the oil phase solution consists of white oil, sorbitan trioleate and aluminum stearate.

4. A new type of inactivated dual vaccine containing a composite immunopotentiator according to claim 1, characterized in that: The volume ratio of the aqueous phase solution to the oil phase solution is 1:

2.

5. A new type of inactivated dual vaccine containing a composite immunopotentiator according to claim 1, characterized in that: The volume ratio of the composite immunoenhancer to the inactivated antigen solution in the aqueous solution is 1:6.

5.

6. The preparation and use of a new type of dual inactivated vaccine containing a composite immune enhancer according to claim 1, characterized in that: The mass ratio of white oil, sorbitan trioleate and aluminum stearate in the oil phase solution is 19:1:0.

15.

7. A method for preparing the inactivated vaccine according to claim 1, characterized in that: The following steps are involved: S1: Preparation of aqueous solution: dissolving the composite immunopotentiator in proportion and mixing it with the inactivated antigen solution and polysorbate to prepare an aqueous solution; S2: Preparation of oil phase solution: mixing white oil, sorbitan trioleate and aluminum stearate to form a uniform oil phase solution; S3: Emulsification: The aqueous solution obtained in step S1 and the oily solution obtained in step S2 are subjected to shear emulsification to form a water-in-oil inactivated vaccine, wherein the shear emulsification is performed using a high-speed emulsifier at 8000-13000 rpm / min for 2-6 minutes to obtain a stable emulsion; S4: Vaccine stabilization: The emulsified vaccine should be sealed and stored at 2-8°C for at least 30 days. There should be no significant change in the uniformity of the vaccine particle size, and the emulsification system should be stable without visible precipitation or stratification.

8. The use of a new type of dual inactivated vaccine containing a composite immunopotentiator according to claims 1-7, characterized in that: The vaccine is used to prevent and control the spread of Newcastle disease and H9 subtype avian influenza, and is particularly suitable for large-scale immunization in poultry farming.

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