阅读说明：本技术 荔枝状纳米氧化锌团簇佐剂及其制备方法与作为疫苗佐剂的应用 (Litchi branch-shaped nano zinc oxide cluster adjuvant, preparation method thereof and application of litchi branch-shaped nano zinc oxide cluster adjuvant as vaccine adjuvant ) 是由 吴扬 刘英帅 郑良益 石宝兰 谢红玲 漆世华 刘希健 王治江 秦红刚 李婷婷 于 2021-08-12 设计创作，主要内容包括：本发明公开了一种荔枝状纳米氧化锌团簇佐剂及其制备方法与作为疫苗佐剂的应用,所述方法包括：将锌盐加入多元醇溶剂中混匀,获得锌盐多元醇溶液；将所述锌盐多元醇溶液进行第一加热,后加入去离子水,进行第二加热至溶液出现浑浊,去除热源待自然冷却,获得反应产物；其中,所述去离子水与所述锌盐多元醇溶液的体积比为1：15～100；将所述反应产物进行固液分离,获得氧化锌纳米团簇沉淀,后清洗,获得荔枝状纳米氧化锌团簇。该荔枝状纳米氧化锌团簇佐剂表面凹凸不平,有效提高抗原的吸附能力；该氧化锌纳米团簇与病毒、细菌的颗粒相似,可增加免疫细胞的识别和吞噬,提高抗原递呈效率。(The invention discloses a litchi-shaped nano zinc oxide cluster adjuvant, a preparation method thereof and application of the litchi-shaped nano zinc oxide cluster adjuvant as a vaccine adjuvant, wherein the method comprises the following steps: adding zinc salt into a polyalcohol solvent, and uniformly mixing to obtain a zinc salt polyalcohol solution; carrying out first heating on the zinc salt polyalcohol solution, then adding deionized water, carrying out second heating until the solution is turbid, removing a heat source, and naturally cooling to obtain a reaction product; wherein the volume ratio of the deionized water to the zinc salt polyalcohol solution is 1:15 to 100 parts; and carrying out solid-liquid separation on the reaction product to obtain a zinc oxide nano cluster precipitate, and then cleaning to obtain the litchi-shaped nano zinc oxide cluster. The litchi-shaped nano zinc oxide cluster adjuvant has uneven surface, so that the adsorption capacity of the antigen is effectively improved; the zinc oxide nano cluster is similar to particles of viruses and bacteria, so that the recognition and phagocytosis of immune cells can be increased, and the antigen presentation efficiency is improved.)

1. The preparation method of the litchi-shaped nano zinc oxide cluster adjuvant is characterized by comprising the following steps:

adding zinc salt into a polyalcohol solvent, and uniformly mixing to obtain a zinc salt polyalcohol solution;

carrying out first heating on the zinc salt polyalcohol solution, then adding deionized water, carrying out second heating until the solution is turbid, removing a heat source, and naturally cooling to obtain a reaction product; wherein the volume ratio of the deionized water to the zinc salt polyalcohol solution is 1:15 to 100 parts;

and carrying out solid-liquid separation on the reaction product to obtain a zinc oxide nano cluster precipitate, and then cleaning to obtain the litchi-shaped nano zinc oxide cluster.

2. The preparation method of the litchi-like nano zinc oxide cluster adjuvant according to claim 1, wherein the mass-to-volume ratio of the zinc salt to the polyol solvent is (1: 20-200) g/L.

3. The preparation method of the litchi-like nano zinc oxide cluster adjuvant according to claim 1, wherein the zinc salt comprises at least one of an inorganic zinc salt and an organic zinc salt; wherein the inorganic zinc salt comprises at least one of zinc chloride, zinc sulfate, zinc nitrate, zinc formate and zinc acetate.

4. The method for preparing the litchi-like nano zinc oxide cluster adjuvant according to claim 1, wherein the polyol solvent comprises polyhydric alcohol and at least one of oligomer and polymer thereof, and the polyhydric alcohol comprises at least one of ethylene glycol, propylene glycol, diethylene glycol, glycerol and polyethylene glycol.

5. The method for preparing the litchi-shaped nano zinc oxide cluster adjuvant according to claim 1, wherein the first heating comprises: preheating for 5-15 min at 120-180 ℃.

6. The method for preparing the litchi-shaped nano zinc oxide cluster adjuvant according to claim 1, wherein the second heating comprises: preheating for 5-15 min at 120-180 ℃.

7. The preparation method of the litchi-like nano zinc oxide cluster adjuvant according to claim 1, wherein the first heating, the second heating and the heat source removal are performed with magnetic stirring in the whole process during natural cooling, and the rotation speed of the magnetic stirring is 200-1000 rpm.

8. The preparation method of the litchi-like nano zinc oxide cluster adjuvant according to claim 1, wherein the step of performing solid-liquid separation on the reaction product to obtain a precipitate of zinc oxide nano clusters and then cleaning the precipitate to obtain litchi-like nano zinc oxide clusters comprises the following steps:

diluting the reaction product with an isovolumetric organic solvent, centrifuging at 6000-12000 rpm for 5-10 min, collecting the precipitate of the zinc oxide nano clusters, and cleaning with the organic solvent to obtain the litchi-shaped nano zinc oxide clusters.

9. The litchi-shaped nano zinc oxide cluster adjuvant prepared by the method of any one of claims 1 to 8 is characterized in that the litchi-shaped nano zinc oxide cluster adjuvant is a litchi-shaped zinc oxide cluster with the diameter of 150-300 nm formed by assembling a plurality of nano zinc oxide spheres.

10. The litchi-like nano zinc oxide cluster adjuvant according to claim 9, which is used as a vaccine adjuvant.

Technical Field

The invention relates to the technical field of immunology, in particular to a litchi-shaped nano zinc oxide cluster adjuvant, a preparation method thereof and application thereof as a vaccine adjuvant.

Background

Adjuvants are non-specific immunopotentiators that, when injected or pre-injected into the body with an antigen, can enhance the body's immune response to the antigen or alter the type of immune response. The adjuvant delays the degradation and elimination of the antigen by changing the physical shape of the antigen, and prolongs the retention time of the antigen in the body; stimulating the antigen presenting ability of mononuclear phagocytes; stimulating lymphocyte differentiation, increasing immune response capacity and other mechanisms to enhance the immune response of the organism. The use of adjuvants can reduce the amount of antigen or the vaccination dose/frequency, and improve the immune efficacy of the vaccine.

The conventional adjuvants mainly comprise an aluminum gel adjuvant, an oil emulsion adjuvant, a propolis adjuvant and a liposome adjuvant, but the adjuvants still have the defects of serious local reaction, non-freeze-drying preservation, poor stability, high production technology difficulty, high cost, single function, poor loading and compatibility of the immunologic adjuvant and the like. Therefore, the development of a novel high-efficiency and safe immune vaccine adjuvant to improve the immune efficacy is an urgent problem to be solved at present.

The nanometer material loads antigens, the uptake rate of Antigen Presenting Cells (APC) is improved through different ways, and the nanometer carrier plays a key role; the nanoparticles can effectively protect the loaded antigen/adjuvant from the influence of the surrounding biological environment, increase the half-life of the antigen/adjuvant to trigger adaptive immune response, and become a research hotspot of vaccine adjuvants gradually. Because the nano particles have unique carrier structural characteristics, the antigen substance and the nano particles can adsorb and stabilize vaccine antigens and can delay the release of the antigens; in addition, because of their size comparable to pathogens, nanoparticles can allow immunogens to be presented to the immune system in the same manner as pathogens are presented, inducing an immune response similar to that of natural infections.

Therefore, the development of a vaccine adjuvant for improving antigen presentation efficiency is urgently needed, and is a technical problem to be solved.

Disclosure of Invention

The invention aims to provide a litchi-shaped nano zinc oxide cluster adjuvant, a preparation method thereof and application of the litchi-shaped nano zinc oxide cluster adjuvant as a vaccine adjuvant, wherein the litchi-shaped nano zinc oxide cluster adjuvant has uneven surface, so that the adsorption capacity of an antigen is effectively improved; the zinc oxide nano cluster is very similar to particles of viruses and bacteria, so that the recognition and phagocytosis of immune cells can be increased, and the antigen presentation efficiency is improved.

In a first aspect of the invention, a preparation method of a litchi-shaped nano zinc oxide cluster adjuvant is provided, and the method comprises the following steps:

adding zinc salt into a polyalcohol solvent, and uniformly mixing to obtain a zinc salt polyalcohol solution;

carrying out first heating on the zinc salt polyalcohol solution, then adding deionized water, carrying out second heating until the solution is turbid, removing a heat source, and naturally cooling to obtain a reaction product; wherein the volume ratio of the deionized water to the zinc salt polyalcohol solution is 1:15 to 100 parts;

and carrying out solid-liquid separation on the reaction product to obtain a zinc oxide nano cluster precipitate, and then cleaning to obtain the litchi-shaped nano zinc oxide cluster.

Further, the mass-volume ratio of the zinc salt to the polyol solvent is (1: 20-200) g/L.

Further, the zinc salt includes at least one of an inorganic zinc salt and an organic zinc salt; wherein the inorganic zinc salt comprises at least one of zinc chloride, zinc sulfate, zinc nitrate, zinc formate and zinc acetate.

Further, the polyol solvent comprises polyhydric alcohol and at least one of oligomer and polymer thereof, and the polyhydric alcohol comprises at least one of ethylene glycol, propylene glycol, diethylene glycol, glycerol and polyethylene glycol.

Further, the first heating includes: preheating for 5-15 min at 120-180 ℃.

Further, the second heating includes: preheating for 5-15 min at 120-180 ℃.

Further, magnetic stirring is carried out in the whole process of the first heating, the second heating and the heat source removing to be naturally cooled, and the rotating speed of the magnetic stirring is 200-1000 rpm.

Further, the solid-liquid separation is carried out on the reaction product to obtain zinc oxide nano cluster precipitate, and then the precipitate is cleaned to obtain litchi-shaped nano zinc oxide clusters, which comprises the following steps:

diluting the reaction product with an isovolumetric organic solvent, centrifuging at 6000-12000 rpm for 5-10 min, collecting the precipitate of the zinc oxide nano clusters, and cleaning with the organic solvent to obtain the litchi-shaped nano zinc oxide clusters.

In a second aspect of the invention, the litchi-shaped nano zinc oxide cluster adjuvant prepared by the method is litchi-shaped nano zinc oxide cluster adjuvant which is formed by assembling a plurality of nano zinc oxide spheres and has the diameter of 150-300 nm.

In a third aspect of the invention, the litchi-shaped nano zinc oxide cluster adjuvant is provided for application as a vaccine adjuvant.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

1. according to the preparation method of the litchi-shaped nano zinc oxide cluster adjuvant, a zinc salt polyalcohol solution is subjected to first heating through a polyalcohol heating method to dissolve zinc salt, deionized water is rapidly added into the preheated zinc salt polyalcohol solution to initiate hydrolysis of zinc salt, second heating is carried out until the solution is turbid, a heat source is removed, and natural cooling is carried out to obtain a reaction product; wherein the volume ratio of the deionized water to the zinc salt polyalcohol solution is 1: 15-100 aims to obtain litchi-shaped nano zinc oxide clusters with moderate clusters (the diameter is 150-300 nm) by moderate hydrolysis speed of the zinc salt. The method is simple and effective, has low production cost and is suitable for large-scale production;

2. the litchi-shaped nano zinc oxide cluster adjuvant provided by the invention is a litchi-shaped zinc oxide cluster assembled by nano zinc oxide spheres with the diameter of about 5nm, the diameter of the litchi-shaped nano zinc oxide cluster is 150-300 nm, the surface of the litchi-shaped nano zinc oxide cluster is uneven, and zinc oxide nano spheres can be seen; the litchi-shaped nano zinc oxide cluster adjuvant has uneven surface, so that the adsorption capacity of the antigen is effectively improved; the zinc oxide nano cluster is similar to particles of viruses and bacteria, so that the recognition and phagocytosis of immune cells can be increased, and the antigen presentation efficiency is improved. The nano particles mainly enter cells in an endocytosis and infiltration mode, the interaction between the nano particles and the cells is directly influenced by the size of about 200nm, the cluster-shaped appearance, the increased surface structure and the like of the nano particles, and the antigen presentation efficiency is improved.

3. The litchi-shaped nano zinc oxide cluster adjuvant is applied as a vaccine adjuvant, and the porcine pseudorabies inactivated vaccine (C1201 strain) adjuvanted by the litchi-shaped nano zinc oxide cluster adjuvant has the characteristics of small inoculation amount, no adverse reaction and high efficiency. The adjuvant can be used for inactivated vaccines and subunit vaccines, can also be used for recombinant protein vaccines, and is beneficial to immune protection of animal vaccines. And the side effect is obviously reduced compared with the mineral oil adjuvant in the aspect of animal safety.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a scanning electron microscope photograph of litchi-shaped nano-zinc oxide clusters prepared by the invention;

FIG. 2 is an X-ray crystal diffraction (XRD) pattern of the litchi-shaped nano-zinc oxide cluster prepared by the invention;

FIG. 3 is a distribution diagram of hydrated particle size of litchi-shaped nano-zinc oxide clusters prepared by the invention;

FIG. 4 shows Zeta potential of litchi-shaped nano-zinc oxide cluster prepared by the invention;

FIG. 5 shows the appearance of inactivated vaccine solution of porcine pseudorabies inactivated vaccine (C1201 strain) with litchi-like nano-zinc oxide cluster as adjuvant;

FIG. 6 shows the results of a comparison of neutralizing antibodies in immunized mice;

fig. 7 is a flowchart of a method for preparing a litchi-shaped nano zinc oxide cluster adjuvant according to an embodiment of the present invention.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.

The embodiment of the invention provides a litchi-shaped nano zinc oxide cluster adjuvant, which has the following general idea:

according to an exemplary embodiment of the present invention, there is provided a method for preparing a litchi-shaped nano zinc oxide cluster adjuvant, as shown in fig. 7, the method including:

step S1, adding zinc salt into the polyol solvent and uniformly mixing to obtain a zinc salt polyol solution;

in the step S1:

the zinc salt comprises at least one of an inorganic zinc salt and an organic zinc salt; wherein the inorganic zinc salt comprises at least one of zinc chloride, zinc sulfate, zinc nitrate, zinc formate and zinc acetate. Other zinc salts may be selected in other embodiments, with zinc acetate being preferred in embodiments of the invention;

the polyhydric alcohol solvent comprises polyhydric alcohol and at least one of oligomer and polymer thereof, and the polyhydric alcohol comprises at least one of ethylene glycol, propylene glycol, diethylene glycol, glycerol and polyethylene glycol. In other embodiments, other alcohols containing multiple hydroxyl groups may be selected, with diethylene glycol being preferred in embodiments of the invention;

the mass-volume ratio of the zinc salt to the polyol solvent is (1: 20-200) g/L. Preferably 1: 50; the mass-to-volume ratio is favorable for the nucleation and assembly of zinc oxide to form clusters; too much addition of the polyol solvent is not beneficial to the generation of zinc oxide nuclei, and too little addition of the polyol solvent is not beneficial to the formation of clusters with uniform sizes;

step S2, carrying out first heating on the zinc salt polyalcohol solution, then adding deionized water, carrying out second heating until the solution is turbid, removing a heat source, and naturally cooling to obtain a reaction product; wherein the volume ratio of the deionized water to the zinc salt polyalcohol solution is 1:15 to 100 parts;

in the above technical scheme, the volume ratio of the deionized water to the zinc salt polyalcohol solution is 1: 15-100 aims to ensure that the hydrolysis speed of the zinc salt is moderate to obtain litchi-shaped nano zinc oxide clusters with moderate clusters (the diameter is 150-300 nm); a large number of experiments and theoretical analysis show that the interaction between the nano particles and cells is directly influenced by the size of about 200nm, cluster-shaped appearance, increased surface structure and the like of the nano particles, and the antigen presentation efficiency is improved.

If the volume ratio of the deionized water to the zinc salt polyalcohol solution is less than 1:100, the zinc salt hydrolysis speed is slow, and the cluster size is increased; if the ratio of the zinc salt to the zinc salt is more than 1:15, the hydrolysis speed is high, the nucleation quantity is large, and the cluster size is small; the ratio of the deionized water to the zinc salt polyol solution is preferably 1: 50.

In a preferred embodiment, during the first heating, the second heating and the heat source removing and natural cooling, magnetic stirring is performed in the whole process, and the rotation speed of the magnetic stirring is 200-1000 rpm.

As an alternative embodiment, the first heating includes: preheating for 5-15 min at 120-180 ℃. (optimal time is 10min), the solution is heated uniformly by magnetic stirring in the heating process, and the magnetic stirring speed is 200-1000 rpm; the first heating has the preheating function, if the preheating temperature is lower than 120 ℃, the zinc salt is slowly hydrolyzed, and if the preheating temperature is higher than 180 ℃, the polyol can be oxidized and discolored; if the preheating time is less than 5min, the solution can not be uniformly heated, and if the preheating time is more than 15min, the oxidative discoloration of the polyhydric alcohol can be accelerated;

as an alternative embodiment, the second heating includes: preheating for 5-15 min at 120-180 ℃. Under the condition, the litchi-shaped nano zinc oxide cluster is favorably and successfully synthesized.

As a specific embodiment, under the condition of continuous magnetic stirring at 200-1000 rpm (optimal stirring speed is 500rpm), the following components are mixed according to water: rapidly adding deionized water into the preheated solution according to the volume ratio of 1:25, 1:50, 1:75 and 1:100 (the optimal ratio is 1:50) of the polyol solvent to initiate hydrolysis of the zinc salt, continuously heating for 5-15 min (the optimal time is 10min) until the solution is turbid, and proving that the litchi-shaped nano zinc oxide cluster is successfully synthesized at the moment;

and step S3, carrying out solid-liquid separation on the reaction product to obtain a zinc oxide nano cluster precipitate, and then cleaning to obtain the litchi-shaped nano zinc oxide cluster.

The step S3 specifically includes:

diluting the reaction product with an isovolumetric organic solvent, centrifuging at 6000-12000 rpm for 5-10 min, collecting the precipitate of the zinc oxide nano clusters, and cleaning with the organic solvent to obtain the litchi-shaped nano zinc oxide clusters.

The organic solvent comprises one of ethanol, methanol, propanol, isopropanol and dimethylformamide, and the organic solvent is preferably ethanol;

the cleaning step specifically comprises: adding a proper amount of organic solvent, dispersing and precipitating under the assistance of ultrasound, and then centrifuging at 6000-12000 rpm for 5-10 min to collect zinc oxide clusters; preferably, the purified litchi-shaped nano zinc oxide cluster adjuvant is obtained by repeating the steps for 1-3 times.

According to another typical embodiment of the invention, the litchi-shaped nano zinc oxide cluster adjuvant prepared by the method is provided. The litchi-shaped nano zinc oxide cluster adjuvant is a litchi-shaped zinc oxide cluster formed by assembling a plurality of nano zinc oxide spheres with the diameter of about 5nm (the range can be 4-6 nm), the diameter of the litchi-shaped nano zinc oxide cluster is 150-300 nm, the surface of the litchi-shaped nano zinc oxide cluster is uneven, and zinc oxide nanospheres can be seen;

the single zinc oxide cluster consists of a plurality of smaller nano particles, the surface is uneven, the specific surface area is large, and the adsorption capacity of the antigen is effectively improved; more importantly, the nanoclusters are very similar to particles of viruses and bacteria, so that the recognition and phagocytosis of immune cells can be increased, and the antigen presentation efficiency is improved; a large number of experiments and theoretical analysis show that the interaction between the nano particles and cells is directly influenced by the size of about 200nm, cluster-shaped appearance, increased surface structure and the like of the nano particles, and the antigen presentation efficiency is improved. Our materials substantially conform to these descriptions.

According to another typical embodiment of the invention, the litchi-shaped nano zinc oxide cluster adjuvant is provided to be applied as a vaccine adjuvant.

According to the embodiment of the invention, litchi-shaped nano zinc oxide cluster adjuvant solution is diluted into a semi-finished product with the concentration of 1mg/mL for later use; then the litchi-shaped nano zinc oxide cluster adjuvant solution and the inactivated porcine pseudorabies virus solution (C1201 strain) (the content of antigen before inactivation is 10)^8.5TCID₅₀Per ml) according to the inactivated antigen volume ratio of 1:1, preparing the vaccine for later use. The porcine pseudorabies inactivated vaccine (C1201 strain) adjuvanted by the litchi-like nano zinc oxide cluster adjuvant has the characteristics of small inoculation amount, no good reaction and high efficiency, and is expected to be widely applied to the preparation of various vaccines such as bacteria, viruses, subunit vaccines and the like.

The litchi-shaped nano zinc oxide cluster adjuvant, the preparation method thereof and the application thereof as a vaccine adjuvant are described in detail below by combining examples and experimental data.

Example 1 litchi-like nano zinc oxide cluster adjuvant and preparation method thereof

1. Adding 1.1g of zinc acetate into a round-bottom flask containing 50mL of diethylene glycol, and magnetically stirring at 300rpm for 30min to fully dissolve the zinc acetate for later use;

2. putting the round-bottom flask filled with the solution into 180 ℃ silicone oil for preheating for 10min, and heating the solution uniformly by magnetic stirring in the heating process, wherein the magnetic stirring speed is 500 rpm;

3. under continuous magnetic stirring at 500rpm, as water: quickly adding deionized water into the preheated solution according to the volume ratio of diethylene glycol of 1:50 to initiate hydrolysis of zinc salt, continuously heating the solution for 10min to enable the solution to be turbid, and proving that the litchi-shaped nano zinc oxide cluster is successfully synthesized at the moment;

4. the heat was removed and magnetic stirring was continued until the solution cooled to room temperature. Diluting the product with equal volume of ethanol, centrifuging at 10000rpm for 5min to collect zinc oxide nano cluster precipitate, adding appropriate amount of ethanol, dispersing and precipitating under the assistance of ultrasound, centrifuging at 10000rpm for 5min to collect zinc oxide cluster, and repeating the steps for 3 times to obtain the purified litchi-shaped nano zinc oxide cluster adjuvant.

5. The SEM image of the litchi-shaped nano zinc oxide cluster adjuvant prepared in the embodiment of the present invention is as shown in fig. 1, and as can be seen from fig. 1, the litchi-shaped nano zinc oxide cluster adjuvant of the present invention is a litchi-shaped zinc oxide cluster assembled by a plurality of nano zinc oxide spheres having a diameter of about 5nm, and the litchi-shaped nano zinc oxide cluster adjuvant has a diameter of 150 to 300nm, has a rugged surface, and is visible as zinc oxide nanospheres.

The X-ray crystal diffraction (XRD) pattern of the litchi-like nano-zinc oxide cluster adjuvant prepared in the example of the present invention is shown in fig. 2, which indicates that the litchi-like nano-zinc oxide cluster adjuvant is hexagonal wurtzite zinc oxide crystal;

the distribution diagram of the hydrated particle size of the litchi-shaped nano zinc oxide cluster adjuvant prepared by the embodiment of the invention is shown in fig. 3, which shows that the particle size distribution of the litchi-shaped nano zinc oxide cluster is 100-400 nm, and mainly is 200-300 nm;

the Zeta potential of the litchi-shaped nano zinc oxide cluster adjuvant prepared by the embodiment of the invention is shown in fig. 4, which shows that the particle surface has positive charges.

Example 2 litchi-like nano zinc oxide cluster adjuvant and preparation method thereof

1. Adding 1.1g of zinc acetate into a round-bottom flask containing 22mL of diethylene glycol, and magnetically stirring at 200rpm for 30min to fully dissolve the zinc acetate for later use;

2. putting the round-bottom flask filled with the solution into silicone oil at 120 ℃ for preheating for 15min, and heating the solution uniformly by magnetic stirring in the heating process, wherein the magnetic stirring speed is 500 rpm;

3. under continuous magnetic stirring at 500rpm, as water: quickly adding deionized water into the preheated solution according to the volume ratio of diethylene glycol of 1:15 to initiate hydrolysis of zinc salt, continuously heating the solution for 10min to enable the solution to be turbid, and proving that the litchi-shaped nano zinc oxide clusters are successfully synthesized at the moment;

4. the heat was removed and magnetic stirring was continued until the solution cooled to room temperature. Diluting the product with equal volume of ethanol, centrifuging at 10000rpm for 5min to collect zinc oxide nano cluster precipitate, adding appropriate amount of ethanol, dispersing and precipitating under the assistance of ultrasound, centrifuging at 10000rpm for 5min to collect zinc oxide cluster, and repeating the steps for 3 times to obtain the purified litchi-shaped nano zinc oxide cluster adjuvant. The SEM and XRD patterns of the litchi-shaped nano zinc oxide cluster adjuvant prepared in the embodiment of the invention are basically the same as those of the embodiment 1.

Example 3 litchi-like nano zinc oxide cluster adjuvant and preparation method thereof

1. Adding 1.1g of zinc acetate into a round-bottom flask containing 220mL of diethylene glycol, and magnetically stirring at 1000rpm for 30min to fully dissolve the zinc acetate for later use;

2. putting the round-bottom flask filled with the solution into 160 ℃ silicone oil for preheating for 12min, and heating the solution uniformly by magnetic stirring in the heating process, wherein the magnetic stirring speed is 500 rpm;

3. under continuous magnetic stirring at 500rpm, as water: quickly adding deionized water into the preheated solution according to the volume ratio of 1:100 of diethylene glycol to initiate hydrolysis of zinc salt, continuously heating the solution for 10min to enable the solution to be turbid, and proving that the litchi-shaped nano zinc oxide clusters are successfully synthesized at the moment;

4. the heat was removed and magnetic stirring was continued until the solution cooled to room temperature. Diluting the product with equal volume of ethanol, centrifuging at 10000rpm for 5min to collect zinc oxide nano cluster precipitate, adding appropriate amount of ethanol, dispersing and precipitating under the assistance of ultrasound, centrifuging at 10000rpm for 5min to collect zinc oxide cluster, and repeating the steps for 3 times to obtain the purified litchi-shaped nano zinc oxide cluster adjuvant. The SEM and XRD patterns of the litchi-shaped nano zinc oxide cluster adjuvant prepared in the embodiment of the invention are basically the same as those of the embodiment 1.

Example 4 preparation of inactivated vaccine against porcine pseudorabies (strain C1201)

1. Diluting the litchi branch nano zinc oxide cluster adjuvant prepared in the example 1 with physiological saline to prepare 1mg/m L, and placing the litchi branch nano zinc oxide cluster adjuvant in a refrigerator at 4 ℃ for later use;

2. inactivated porcine pseudorabies virus fluid (C1201 strain) (the content of antigen before inactivation is 10)^8.5TCID₅₀Per ml) in a volume ratio of 1: the mixture is fully mixed with litchi-shaped nano zinc oxide cluster adjuvant according to the proportion of 1, and the experimental adjuvant vaccine is obtained. (three batches of experimental adjuvant vaccines are prepared by using the prepared three batches of nano zinc oxide cluster adjuvants respectively and are marked as an experimental adjuvant vaccine 1, an experimental adjuvant vaccine 2 and an experimental adjuvant vaccine 3.

Example 5 safety testing of the Experimental adjuvant vaccine against mice

Mice were intraperitoneally injected with 20 mice per batch of vaccine, 0.5 ml/mouse, and 5 non-vaccinated control mice were set. After inoculation, the mice were observed continuously for 14 days, and the mental, appetite, death, etc. of the mice were recorded by daily observation. The safety test results for mice show that: the tested mice have normal spirit and appetite and are healthy and alive; the vaccine is well absorbed, and has no red swelling and nodulation when being observed at the injection part; the vaccine was completely absorbed at the injection site of mice in the white oil adjuvant control vaccine group (table 1).

TABLE 1 clinical observations of vaccination of mice with vaccine

As can be seen from the data in table 1, the vaccine prepared with the adjuvant of the present invention is safe to kunming mice.

Example 6 safety testing of Experimental adjuvant vaccines against rabbits

5 rabbits (5.0 ml) are injected into legs of each vaccine, and are raised under the same condition by simultaneously setting 5 blank control rabbits, continuously observing for 14 days, and observing and recording whether the rabbit injection parts have swelling bags, mental states, appetite, death conditions and the like every day.

TABLE 2 clinical observations of the vaccine after rabbit vaccination

The test results show that: after continuously observing for 14 days, the tested rabbits have normal spirit and appetite; the vaccine is well absorbed, and has no red swelling and nodulation when being observed at the injection part; the white oil adjuvant control vaccine injection site vaccine was not completely absorbed (table 2). The blank control group is healthy and alive, and has no abnormal clinical response. The experimental adjuvant prepared vaccine is proved to have good safety to rabbits and no residue in immunity.

Example 7 safety testing of Experimental adjuvant vaccines on piglets

The 25 piglets were randomly divided into 5 groups of 5, each group having 5 piglets vaccinated with each adjuvant vaccine, 4 ml/head injected into neck muscle, and the other group being a blank control. After immunization, body temperature was measured daily, and the mental state and appetite of the pigs were observed, and adverse reactions of piglets were recorded. After 14 days, local inflammatory reaction at the injection site and pathological changes of organs were examined by a biopsy. According to the influence degree of vaccines with different adjuvants on safety indexes such as body temperature, clinical manifestations, local inflammatory reactions, pathological changes of organs and the like after immunization of piglets, the safety of the vaccine prepared by using the nano zinc oxide clusters as the adjuvants is evaluated. The results of the piglet body temperature are shown in Table 3, and the results of the clinical observation are shown in Table 4.

TABLE 3 temperature recordings after immunization of piglets with test adjuvant formulated vaccines

TABLE 4-clinical observations of piglets immunized with vaccine formulated with experimental adjuvants

Injecting: "No abnormality in clinical manifestation" refers to normal mental, appetite and body temperature of piglets.

The test results show that: after the piglets are continuously observed for 14 days after the vaccination, the body temperature, the spirit and the appetite of the piglets are all normal, and the test pigs have no adverse reaction or death; after the test is finished, the pig is dissected, and the heart, lung, liver, spleen, kidney, brain, tonsil, lymph node and other organs have no pathological changes. White oil adjuvant vaccines have incomplete vaccine absorption on eye. The result shows that the safety of the experimental adjuvant vaccine is good and is superior to that of the white oil adjuvant vaccine.

Example 8 efficacy of the Experimental adjuvant vaccine on mice

And respectively inoculating 20 Kunming mice with 17-22 g of experimental adjuvant vaccines and white oil adjuvant control vaccines, and immunizing 0.3ml of each abdominal cavity. At the same time5 virus attacking control groups and 5 blank control groups are arranged. After immunization, 21 days of pseudorabies virus PRV HP-17 virulent strain is used for virus challenge, and 0.1ml (containing 200 LD) of pseudorabies virus PRV HP-11 virus liquid is injected into each abdominal cavity₅₀). The observation was continued for 10 days and the number of healthy mice per group was recorded. 3 experimental adjuvant vaccine groups 16-19 mice were healthy; white oil adjuvant vaccine group 12/20 healthy and alive; control 5/5 dead; blank 5/5 is healthy. The result is shown in table 5, and the litchi-shaped nano zinc oxide cluster adjuvant-assisted porcine pseudorabies inactivated vaccine can obviously improve the virus attack protection rate of an immunized mouse.

TABLE 5 immune challenge test results on mice

Group of	Death number (only)	Number of healthy activities/total number
			Experimental adjuvant vaccine 1	2	18/20
Experimental adjuvant vaccine 2	1	19/20
			Experimental adjuvant vaccine 3	4	16/20
White oil adjuvant control vaccine group	8	12/20
			Control group for counteracting toxic pathogen	5	0/5
Blank control group	0	5/5

Example 9 detection of neutralizing antibodies generated by immunized mice

Each mouse was immunized with 20 mice grouped as in table 6 (litchi-like nano-zinc oxide cluster adjuvant, commercial white oil adjuvant control, commercial alumina gel adjuvant control, and blank control), each injected intraperitoneally with 0.3ml of vaccine, and 14 days later, each injected intraperitoneally with 0.3ml of vaccine again. Collecting venous blood of mouse tail at 4, 8, 16, 24, and 28 weeks after primary immunization, separating serum, diluting the serum to be detected, adding 100TCID₅₀The pseudorabies venom is put at 37 ℃ for 60 minutes, inoculated to a 96-well ST cell culture plate, and the titer of the neutralizing antibody of the detected serum is calculated according to the CPE condition statistical data of each well. (Table 6) the experimental data obtained were analyzed for one-way anova using SPSS11.5 statistical software, with P < 0.05 statistically significant (FIG. 6).

TABLE 6 immunization of mice to generate neutralizing antibodies

As can be seen from the analysis of the data in table 6, the neutralizing antibodies were produced in each experimental group from the fourth week and generally peaked twelve weeks later. The litchi-shaped nano zinc oxide cluster adjuvant experiment group achieves the best immune effect, the level of neutralizing antibodies is remarkably higher than that of other control experiment groups P < 0.05, and meanwhile, the level is maintained to be higher in the whole detection period.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.