CN112618710A

CN112618710A - Phytoglycogen pig oral vaccination nano adjuvant and preparation method and application thereof

Info

Publication number: CN112618710A
Application number: CN201910903314.5A
Authority: CN
Inventors: 陈坤; 殷盼超; 李辰晖
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2019-09-24
Filing date: 2019-09-24
Publication date: 2021-04-09
Anticipated expiration: 2039-09-24
Also published as: CN112618710B

Abstract

The invention discloses a phytoglycogen pig oral vaccination nanometer adjuvant and a preparation method and application thereof. The method comprises the following steps: (1) adding the antigen into a phytoglycogen aqueous solution, and oscillating and adsorbing to obtain a mixed solution; (2) under the ice bath condition, adding fatty acid into the mixed solution, performing ultrasonic treatment to obtain emulsion, and dialyzing to obtain the phytoglycogen pig oral vaccination nano adjuvant; in the nano adjuvant for the oral vaccination of the phytoglycogen pigs, the content of the phytoglycogen is 0.5-10 wt%, the content of the antigen is 0.01-2 wt%, and the content of the fatty acid is 0-20 wt%. The prepared phytoglycogen-based nano adjuvant is resistant to degradation, can delay the release of antigen, has stronger inoxidizability of an oil-in-water emulsion formed by mixing with fatty acid, can improve the overall stability, can better protect the antigen, has safe and reliable sources, takes phytoglycogen as a vaccine adjuvant, has low cost, safety and high efficiency, and has good prevention and protection capability on animals.

Description

Phytoglycogen pig oral vaccination nano adjuvant and preparation method and application thereof

Technical Field

The invention belongs to the field of biological products for livestock, and particularly relates to a phytoglycogen pig oral vaccination nano adjuvant, and a preparation method and application thereof.

Background

Oral immunization is the easiest and easiest way to immunize in the modern breeding industry. However, the oral immunization approach faces challenges, limiting its wide application. Because the components of the vaccine are degraded frequently due to the need of passing through the digestive tract, the immunogenicity is weak, an effective immune response is difficult to cause and even no immune response is caused sometimes, and a certain dosage of safe and effective adjuvant needs to be added. Vaccine adjuvants have become an integral part of the composition of novel vaccines. Adjuvants have been used to improve the immunogenicity of partially inactivated vaccines, to enhance the immune response in low-response people, to improve the immunogenicity of vaccines administered via mucosal routes, to modulate inappropriate immune responses, etc., to improve protective immunity. Substances for which adjuvant activity has been demonstrated include aluminum adjuvants, liposome emulsions, proteoglycans, bacteria-like particles, and the like.

Although many substances have been shown to have adjuvant effects, the selection or development of a superior adjuvant requires a combination of factors. The first requirement is the safety of the adjuvant. Due to its own pharmacological properties, adjuvants may affect the immunogenicity and safety of vaccine antigens. The most widely used aluminum adjuvant has good immune effect, but also has limitations and disadvantages. The structure of the frozen aluminum adjuvant can be changed, and the aggregation phenomenon can occur. Because the aluminum adjuvant cannot be stored in a frozen state due to the freezing sensitivity, a certain cryoprotectant needs to be added, and the safety risk is increased. In addition, it has been reported that the aluminum adjuvant can induce IgE production, which may cause hypersensitivity, and in severe cases, the injected part may be ulcerated, and local inflammation may occur. In addition to traditional aluminum adjuvants, oil-in-water emulsions are also becoming more widely used. The squalene oil is commonly used for preparing oil-in-water emulsions, can form a dropwise emulsion with good uniformity under high pressure, and has high stability. However, the oil-in-water emulsion has more serious side effects than the aluminum adjuvant, is more likely to cause inflammatory reaction at the injection site, and can cause liver injury after long-term use. Other adjuvants, such as nucleic acid adjuvants, have been found to induce autoimmune diseases in animal models, presenting safety concerns. Thus, the stimulating effect of the adjuvant on the animal's immune system must be within a suitable, predictable range. The development of new adjuvants that are biologically safe has become a research trend in vaccine development. The world health organization initiated global vaccine safety activities in 2012 with the aim of enforcing global vaccine safety in order to reinforce vaccine drug vigilance capacity. At present, cyclodextrin derivatives are reported to be used as vaccine adjuvants, but separation and purification steps after derivatization modification of cyclodextrin are complex, the workload is large, and large-scale production is difficult.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a preparation method of a phytoglycogen pig oral vaccination nanometer adjuvant.

The invention also aims to provide the phytoglycogen pig oral vaccination nanometer adjuvant prepared by the method. The phytoglycogen pig oral vaccination nano adjuvant is prepared from highly branched soluble polysaccharide, namely phytoglycogen.

The invention also aims to provide application of the phytoglycogen pig oral vaccination nanometer adjuvant in the field of preparation of biological medicine materials.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a phytoglycogen pig oral vaccination nanometer adjuvant comprises the following steps:

(1) adding the antigen into a phytoglycogen aqueous solution, and oscillating and adsorbing for 0.5-12 h at 4-30 ℃ to obtain a mixed solution;

(2) under the ice bath condition, adding fatty acid into the mixed solution obtained in the step (1), performing ultrasonic treatment for 1-4 hours to obtain emulsion, and dialyzing to obtain the phytoglycogen pig oral vaccination nano adjuvant;

wherein, in the nano adjuvant for the oral vaccination of the phytoglycogen pigs, the content of the phytoglycogen is 0.5 to 10 weight percent, the content of the antigen is 0.01 to 2 weight percent, and the content of the fatty acid is 0.5 to 5 weight percent.

Preferably, the antigen in step (1) is at least one of porcine ileitis live vaccine, porcine reproductive and respiratory syndrome inactivated vaccine, porcine mycoplasma pneumonia inactivated vaccine, porcine pseudorabies inactivated vaccine, porcine foot and mouth disease inactivated vaccine, porcine circovirus type 2 baculovirus vector inactivated vaccine, streptococcus suis bivalent inactivated vaccine and haemophilus parasuis disease inactivated vaccine.

Preferably, the mass ratio of the phytoglycogen, the antigen and the fatty acid in the phytoglycogen aqueous solution in the step (1) to the fatty acid in the step (2) is (0.5-10): (0.01-2): (0.5-5), more preferably (2-3): (0.5-1): 3.2.

preferably, the antigen in the step (1) is added in the form of an antigen aqueous solution, and the concentration of the antigen aqueous solution is 0.1-2 mg/mL.

Preferably, the concentration of the phytoglycogen aqueous solution in the step (1) is 1-15 mg/mL.

Preferably, the branch density of the phytoglycogen in the phytoglycogen aqueous solution in the step (1) is 4-5%.

Preferably, the phytoglycogen in the aqueous solution of phytoglycogen in step (1) is extracted from corn seeds and/or rice seeds.

Preferably, the oscillating speed of the step (1) is 50-300 rpm.

Preferably, the fatty acid in step (2) is at least one of oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid and derivatives of oleic acid, linoleic acid, linolenic acid, arachidonic acid, and eicosapentaenoic acid.

Preferably, the molecular weight cut-off of the dialysis bag used in the dialysis in the step (2) is 100000, and the dialysis time is 10-20 h. The temperature of the dialysis is ice bath.

Preferably, after the dialysis in the step (2), concentration can be further carried out, so as to obtain the phytoglycogen pig oral vaccination nano adjuvant.

The plant glycogen pig oral vaccination nanometer adjuvant prepared by the method.

The application of the phytoglycogen pig oral vaccination nanometer adjuvant in the field of biological medicine material preparation.

Preferably, the application is: the application in preparing the pig oral vaccination medicine.

The technical principle of the invention is as follows: the phytoglycogen used is a polysaccharide nanoparticle of highly branched molecular structure formed within the endosperm of plants. The branch density of the phytoglycogen accounts for 4-5%, one branch appears at every 20-25 glucose residues on average, the phytoglycogen is soluble in water, and the viscosity of the solution is low. Phytoglycogen is short and numerous in branches and compact in structure, and thus can form nano-spherical particles with a radius of about 39 nm. The glucose releasing speed of the phytoglycogen is lower than that of waxy corn starch and common starch, the phytoglycogen cannot be rapidly digested in the digestive tract, the nano form of the phytoglycogen can be maintained within a certain time, so that the effect of protecting the antigen is achieved, and the phytoglycogen can be gradually decomposed by amylase and can play a role in slowly releasing the antigen. The invention utilizes the interaction of fatty acid and phytoglycogen to generate amphipathic phytoglycogen which is oleophylic and hydrophilic, and the phytoglycogen can improve the oxidation stability of the fatty acid, thereby effectively improving the oxidation stability of the phytoglycogen pig oral vaccination nano adjuvant. The plant glycogen raw material is derived from plants such as corn, rice and the like, and animals have good tolerance to the plant glycogen and have natural advantages in the aspect of safety.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the phytoglycogen used in the invention is polysaccharide-shaped nanoparticles with a highly branched molecular structure formed in endosperm of plants such as corn, rice and the like, and is a highly branched soluble polysaccharide similar to amylopectin. Due to its natural nanoscale effect, the immune system can effectively recognize phytoglycogen nano-adjuvants, and is more likely to stimulate the body to produce T cells. The interaction between fatty acid and phytoglycogen is utilized to obtain the amphiphilic phytoglycogen nano adjuvant which is oleophylic and hydrophilic. Due to the protection effect of phytoglycogen, antigens are protected from being degraded, and fatty acids are protected from oxidative decomposition, so that the obtained nano adjuvant emulsion has good stability and is easy to store. The phytoglycogen is derived from plants such as the corn and the like in the swinery feed staple food, and has natural advantages in the aspect of safety; the animal has good tolerance to phytoglycogen, and does not generate hypersensitivity reaction and autoimmunity. The prepared phytoglycogen-based nano adjuvant is resistant to degradation and can delay the release of antigen, and the oil-in-water emulsion formed by mixing the phytoglycogen-based nano adjuvant with fatty acid has stronger oxidation resistance. Toxicity test results show that repeated high dose administration of phytoglycogen nanoadjuvant in rodents is safe. The vaccine nano adjuvant provided by the invention has the advantages of wide raw material source, low cost, simple and convenient processing technology and easy popularization.

Drawings

FIG. 1 is a schematic diagram of a preparation process of the phytoglycogen pig oral vaccination nano adjuvant.

FIG. 2 is a graph showing the particle size distribution of the aqueous solution of corn phytoglycogen, the aqueous solution of corn phytoglycogen-antigen and the prepared porcine oral vaccine (corn) phytoglycogen nano-adjuvant in example 1.

FIG. 3 is the particle size distribution diagram of the aqueous solution of rice phytoglycogen and the prepared porcine oral vaccination (rice) phytoglycogen nano-adjuvant in example 2.

FIG. 4 is a graph showing the effect of aqueous emulsions of phytoglycogen nano-adjuvant on the survival rate of HEP2 cells at various concentrations of swine oral vaccination in example 4.

FIG. 5 is a graph showing the change in intestinal tract and tracheal secretory sIgA and serum IgG titers after day 0 and day 14 of immunization in Balb/c mice in example 6, wherein FIG. 5a is a graph showing the change in intestinal tract secretory sIgA titer, FIG. 5b is a graph showing the change in tracheal secretory sIgA titer, and FIG. 5c is a graph showing the change in serum antibody IgG titer.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

The experimental methods described in the embodiments of the present application are all conventional methods unless otherwise specified; the reagents, materials, etc., are commercially available without specific reference.

The commercial haemophilus parasuis disease inactivated vaccine described in the examples and comparative examples of the present application was purchased from berging haggihn; the inactivated vaccine against Haemophilus parasuis disease and the bivalent inactivated vaccine against Streptococcus suis (i.e., the raw materials prepared in examples and comparative examples) were purchased from the Haibolai biological pharmaceutical factory, Spain.

Example 1

Reference method (S.H.Yun and N.K.Matheson.Carbohydrar.Res.1993, 243,307-321) corn seed was pulverized and corn phytoglycogen solid powder was extracted. 0.015g of corn phytoglycogen solid powder is weighed and fully dissolved in 1.0mL of deionized water to prepare phytoglycogen water solution with the concentration of 15 mg/mL. The inactivated vaccine antigen of the haemophilus parasuis disease is dispersed in sterile water to prepare the haemophilus parasuis vaccine antigen aqueous solution with the concentration of 1 mg/mL. Adding 5mL of antigen aqueous solution into 1mL of phytoglycogen aqueous solution, then placing the mixed solution in an air bath constant temperature oscillation box for oscillation and balance for 8 hours, controlling the temperature to be 20 ℃ and the oscillation and balance speed to be 100rpm, and promoting the antigen to be fully adsorbed on the phytoglycogen nanoparticles. And after adsorption is finished, placing the mixed solution in an ice bath, adding 0.02mL of linolenic acid into the mixed solution, performing ultrasonic treatment for 4 hours in the ice bath to form stable emulsion, dialyzing the stable emulsion in water for 16 hours by using a dialysis bag with the molecular weight cutoff of 100000, removing free antigens, taking out the dialysis bag, covering polyethylene glycol concentrated stock solution on the dialysis bag until the volume of the stock solution is concentrated to 1mL, and thus obtaining the emulsion-shaped porcine oral vaccination (corn) phytoglycogen nano adjuvant.

The results of the present example, in which the antigen was sufficiently adsorbed to the phytoglycogen nanoparticles, are shown in fig. 1 and 2. The pig oral vaccination (corn) plant glycogen nano adjuvant prepared by the embodiment is placed at 37 ℃, no obvious change is found in three days, and no obvious precipitation or delamination is found after centrifugation at 3000rpm for 3min, which indicates that the pig oral vaccination (corn) plant glycogen nano adjuvant has good stability.

Example 2

Reference method (S.H.Yun and N.K.Matheson.Carbohydrar.Res.1993, 243,307-321) rice seeds were soaked, crushed and rice phytoglycogen solid powder was extracted. 0.01g of rice phytoglycogen solid powder is weighed and fully dissolved in 1.0mL of deionized water to prepare phytoglycogen aqueous solution with the concentration of 10 mg/mL. Dispersing the bivalent inactivated vaccine antigen of the streptococcus suis in sterile water to prepare the bivalent inactivated vaccine aqueous solution of the streptococcus suis with the concentration of 1 mg/mL. Adding 2.5mL of antigen aqueous solution into 1mL of phytoglycogen aqueous solution, then placing the mixed solution in an air bath constant temperature oscillation box for oscillation and balance for 6 hours, controlling the temperature to be 20 ℃ and the oscillation and balance speed to be 120rpm, and promoting the antigen to be fully adsorbed on the phytoglycogen nanoparticles. And after adsorption is finished, placing the mixed solution in an ice bath, adding 0.02mL of linoleic acid into the mixed solution, performing ultrasonic treatment for 5 hours in the ice bath to form stable emulsion, dialyzing the stable emulsion in water for 16 hours by using a dialysis bag with the molecular weight cutoff of 100000 to remove free antigens, taking out the dialysis bag, covering the dialysis bag with polyethylene glycol concentrated stock solution until the volume of the stock solution is concentrated to 1mL, and thus obtaining the emulsion-shaped porcine oral vaccination (rice) phytoglycogen nano adjuvant.

The results of the present example, in which the antigen was sufficiently adsorbed to the phytoglycogen nanoparticles, are shown in fig. 1 and 3.

Example 3 stability of phytoglycogen nano-adjuvant for oral vaccination of pigs

The phytoglycogen nano adjuvant for pig oral vaccination prepared in examples 1-2 is taken as an example for explanation. The porcine oral vaccine (corn) phytoglycogen nano-adjuvant of example 1 and the porcine oral vaccine (rice) phytoglycogen nano-adjuvant of example 2 were placed at 37 ℃ for three days without obvious change, and were centrifuged at 3000rpm for 3min without obvious precipitation or delamination. The peroxide value of the phytoglycogen nanometer adjuvant for the pig oral vaccination after being placed under different conditions for one week is determined by adopting a sodium thiosulfate method (the result is shown in a table 1). The oxidation degree of the fatty acid of the phytoglycogen nano adjuvant orally inoculated to the pig is evaluated by measuring the amount of the peroxide product, and the evaluation is also an important index for evaluating the deterioration degree. An emulsion containing 0.05% antigen and 1% fatty acid was also used as a control. As shown in Table 1, the oxidation number of the swine oral vaccination phytoglycogen nano adjuvant is lower than that of a control group (an emulsion group formed by linolenic acid with the mass fraction of the antigen and the mass fraction of the intermediate value almost equal to those of the example) under different storage conditions, which indicates that the phytoglycogen improves the oxidation stability of the linolenic acid, and the swine oral vaccination phytoglycogen nano adjuvant prepared in examples 1-2 has stronger oxidation resistance. TABLE 1 comparison of the degree of oxidation of phytoglycogen nanoadjuvants orally administered to pigs under different storage conditions

The porcine oral vaccination phytoglycogen nano adjuvant prepared in the example 1-2 is inoculated to a Tryptone Soy Broth (TSB) culture medium, and after observation for 24 hours at 37 ℃, turbidity is not found in the tryptone soy broth culture medium, and bacteria are not found in microscopic examination, which indicates that the porcine oral vaccination phytoglycogen nano adjuvant has no bacterial pollution.

Example 4 safety of phytoglycogen nano-adjuvant for oral vaccination of pigs

The phytoglycogen nano adjuvant for pig oral vaccination prepared in examples 1-2 is taken as an example for explanation. The porcine oral vaccination phytoglycogen nano adjuvant prepared in examples 1 and 2 was prepared into aqueous emulsions of 0.25mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL by adding water, respectively, and the aqueous emulsions were added to 24-well culture plates (one emulsion per well), followed by inoculation of HEP2 cells (purchased from Thermo Fisher Co., Ltd.) using a DMEM medium containing 15% fetal bovine serum and 5% CO₂Culturing at 37 deg.C in incubator, and adding aqueous emulsion of phytoglycogen nano adjuvant for oral administration of vaccine to control group without adding aqueous emulsion of phytoglycogen nano adjuvant for oral administration of pig, and other conditions are the same. The MTT method is used for testing the cytotoxicity of the phytoglycogen nano adjuvant for the pig oral vaccination. At four concentrations, neither phytoglycogen nano-adjuvant caused massive cell death (as shown in fig. 4), indicating that both phytoglycogen nano-adjuvants are highly safe as vaccine adjuvants.

Female Balb/c mice 6-8 weeks old were randomly divided into four groups of 10 mice each, and the experimental groups were: 10 Balb/c mice were drenched with 20. mu.L of the porcine oral vaccine phytoglycogen nano adjuvant prepared in example 1, and 10 Balb/c mice were drenched with 20. mu.L of the porcine oral vaccine phytoglycogen nano adjuvant prepared in example 2; the control group was: 10 Balb/c mice were injected subcutaneously into each back with 20. mu.L of a commercially available inactivated vaccine against Haemophilus parasuis disease; blank groups were: 10 Balb/c mice were drenched with 20. mu.L of physiological saline. The observation was continued for four weeks, and the results are shown in FIG. 2, with all Balb/c mice alive and without any clinical signs.

TABLE 2 survival results of Balb/c mice after immunization

The safety of the porcine oral vaccine (corn) phytoglycogen nano adjuvant prepared in example 1 is specifically clarified. Dividing 6-8 week-old female Balb/c mice into three groups at random, wherein each group comprises 5 mice, and the experimental groups comprise: 5Balb/c mice were drenched with 20. mu.L of each pig oral vaccination (corn) phytoglycogen nano adjuvant prepared in example 1; the control group was: 5Balb/c mice, each back of which was injected subcutaneously with 20. mu.L of a commercially available inactivated vaccine against Haemophilus parasuis disease; blank groups were: 5Balb/c mice were drenched with 20. mu.L of physiological saline. After immunization, Balb/c mice were monitored for 1 time every 30 minutes using an animal thermometer. The body temperature of Balb/c mice after drenching the pigs with oral vaccination (corn) with phytoglycogen nanoadjuvant prepared in example 1 was within 0.5 ℃ for 4 hours of continuous monitoring (Table 3). Then, the animals are continuously observed for four weeks, in four weeks after the inoculation through an oral way, Balb/c mice have no side reaction such as fever, have no conditions such as depilation and the like, have no abnormal symptoms, and all Balb/c mice are healthy and alive, which indicates that the swine oral vaccination (corn) phytoglycogen nano adjuvant prepared in the example 1 can not cause strong immune stress reaction after the immunization of the Balb/c mice, and has good safety.

TABLE 3Balb/c mice temperature monitoring results after immunization

a corn plant glycogen vaccine nano adjuvant for oral immunization of load Haemophilus parasuis inactivated vaccine antigen

b injecting a commercial inactivated vaccine for haemophilus parasuis disease

Example 5 safety of porcine oral vaccination phytoglycogen nano adjuvant

The pig oral vaccine (corn) plant glycogen nano adjuvant prepared in example 1 is used as an example for explanation. Healthy suckling piglets aged about 3 weeks were randomly divided into three groups of 5 piglets each, and after measuring the body temperature, were subjected to an immunization treatment, experimental groups: each piglet was drenched with 2mL of the swine oral vaccination (corn) phytoglycogen nano adjuvant emulsion prepared in example 1; control group: 2mL of commercial haemophilus parasuis disease inactivated vaccine is injected into each piglet muscle; blank group: each piglet was taken 2mL of saline. After immunization of the piglets, the body temperature of the piglets was monitored 1 time every 30 minutes using an animal thermometer. The body temperature of piglets after drenching pigs orally inoculated with the (corn) phytoglycogen nano-adjuvant prepared in example 1 was within 0.5 ℃ within 4 hours of continuous monitoring (table 4). Then, the piglets are continuously observed for four weeks, and have no side reaction such as fever and abnormal symptoms in four weeks after the inoculation through the oral route, so that all the piglets survive healthily, and the pig oral vaccination (corn) phytoglycogen nano-adjuvant prepared in the example 1 does not cause strong immune stress reaction after the immunization of the piglets and has good safety.

TABLE 4 piglet body temperature monitoring results after immunization

b injecting a commercial inactivated vaccine for haemophilus parasuis disease

Example 6 immunization effect of porcine oral vaccination phytoglycogen nano adjuvant

The pig oral vaccination (corn) plant glycogen nano adjuvant prepared in example 1 is used as an example for illustration. The porcine oral vaccine (corn) phytoglycogen nano-adjuvant prepared in example 1 was subjected to in vivo immune effect test. Female Balb/c mice 6 to 8 weeks old were randomly divided into three groups of 5 mice each, and the first immunization treatment was performed, experimental groups: 10 mul of the porcine oral vaccination (corn) phytoglycogen nano adjuvant prepared in example 1 was drenched to each Balb/c mouse; control group: injecting 10 mu L of commercial haemophilus parasuis inactivated vaccine subcutaneously on the back of each Balb/c mouse; blank group: each Balb/c mouse was drenched with 10. mu.L of physiological saline. On day 20 after the first immunization treatment, mice in the experimental group were immunized a second time, and 10. mu.L of the porcine oral vaccine (corn) phytoglycogen nano-adjuvant prepared in example 1 was administered per Balb/c mouse. Balb/c mouse blood was collected weekly after the first immunization, serum was isolated and the haemophilus parasuis antibody titer in serum was monitored using an indirect ELISA assay. After the pig oral vaccination (corn) plant glycogen nanometer adjuvant prepared in example 1 is drenched for one week, the antibody can be detected, then the content of the antibody is gradually increased, the average antibody titer of an experimental group is basically equivalent to that of a control group, the antibody level is always kept at a higher level in the last two months of monitoring, and the antibody level generated by Balb/c mice is more than 1:16 in six months after additional immunization (Table 5).

TABLE 5Balb/c mice after immunization serum Haemophilus parasuis antibody titers

b injecting a commercial inactivated vaccine for haemophilus parasuis disease

To further verify the immunization effect, Balb/c mouse sera were collected at 14 days and 28 days of inoculation, respectively, and antibodies produced in the immunized mice were detected by ELISA (enzyme-linked immunosorbent assay) method. On day 14 of inoculation, the levels of secretory sIgA in the intestinal tract and trachea were significantly higher in Balb/c mice in the experimental group than in the blank group (as shown in FIG. 5). The result of the serum sample analysis shows that the experimental group Balb/c mouse serum antibody IgG is obviously higher than that of the blank group. On the 28 th day of inoculation, the secretory sIgA level in the intestinal tract and trachea of Balb/c mice in the experimental group is obviously higher than that in the blank group and the control group. The result of the serum sample analysis shows that the experimental group Balb/c mouse serum antibody IgG is obviously higher than that of the blank group and the control group.

Comparative example 1

Reference method (S.H.Yun and N.K.Matheson.Carbohydrar.Res.1993, 243,307-321) corn seed was pulverized and corn phytoglycogen solid powder was extracted. 0.15g of corn phytoglycogen solid powder is weighed and fully dissolved in 10mL of deionized water to prepare phytoglycogen water solution with the concentration of 15 mg/mL. The inactivated vaccine antigen of the haemophilus parasuis disease is dispersed in sterile water to prepare the haemophilus parasuis vaccine antigen aqueous solution with the concentration of 1 mg/mL. Adding 5mL of antigen aqueous solution into 10mL of phytoglycogen aqueous solution, then placing the mixed solution into an air bath constant-temperature oscillation box to oscillate and balance for 8 hours, controlling the temperature to be 20 ℃ and the oscillation and balance speed to be 100rpm, and promoting the antigen to be fully adsorbed on the phytoglycogen nanoparticles. After adsorption is completed, the mixed solution is placed under an ice bath, 0.02mL of linolenic acid is added into the mixed solution, ultrasound is carried out for 4 hours under the ice bath to form stable emulsion, a dialysis bag with the molecular weight cutoff of 100000 is used for dialysis in water for 16 hours to remove free antigens, then the dialysis bag is taken out, polyethylene glycol is covered on the dialysis bag until the volume of the stock solution is concentrated to 1mL, and the emulsion-shaped phytoglycogen (corn) nano adjuvant A is obtained, wherein the mass fraction of the phytoglycogen is 15%, and the mass fraction of the antigens is 0.5%.

Comparative example 2

Reference method (S.H.Yun and N.K.Matheson.Carbohydrar.Res.1993, 243,307-321) corn seed was pulverized and corn phytoglycogen solid powder was extracted. 0.015g of corn phytoglycogen solid powder is weighed and fully dissolved in 1.0mL of deionized water to prepare phytoglycogen water solution with the concentration of 15 mg/mL. The inactivated vaccine antigen of the haemophilus parasuis disease is dispersed in sterile water to prepare the haemophilus parasuis vaccine antigen aqueous solution with the concentration of 1 mg/mL. Adding 0.08mL of antigen aqueous solution into 1mL of phytoglycogen aqueous solution, then placing the mixed solution in an air bath constant-temperature oscillation box for oscillation and balance for 8 hours, controlling the temperature to be 20 ℃ and the oscillation and balance speed to be 100rpm, and promoting the antigen to be fully adsorbed on the phytoglycogen nanoparticles. And after adsorption is finished, placing the mixed solution in an ice bath, adding 0.02mL of linolenic acid into the mixed solution, performing ultrasonic treatment for 4 hours in the ice bath to form stable emulsion, dialyzing the stable emulsion in water for 16 hours by using a dialysis bag with the molecular weight cutoff of 100000 to remove free antigens, taking out the dialysis bag, covering polyethylene glycol on the dialysis bag until the volume of the stock solution is concentrated to 1mL, and thus obtaining the emulsion-shaped phytoglycogen (corn) nano adjuvant B, wherein the mass fraction of the antigens is 0.008%.

Comparative example 3

Reference method (S.H.Yun and N.K.Matheson.Carbohydrar.Res.1993, 243,307-321) corn seed was pulverized and corn phytoglycogen solid powder was extracted. 0.015g of corn phytoglycogen solid powder is weighed and fully dissolved in 1.0mL of deionized water to prepare phytoglycogen water solution with the concentration of 15 mg/mL. The inactivated vaccine antigen of the haemophilus parasuis disease is dispersed in sterile water to prepare the haemophilus parasuis vaccine antigen aqueous solution with the concentration of 1 mg/mL. Adding 5mL of antigen aqueous solution into 1mL of phytoglycogen aqueous solution, then placing the mixed solution in an air bath constant-temperature oscillation box to oscillate and balance for 8 hours, controlling the temperature to be 20 ℃ and the oscillation and balance speed to be 100rpm, and promoting the antigen to be fully adsorbed on the phytoglycogen nanoparticles. And after adsorption is finished, placing the mixed solution in an ice bath, adding 0.005mL of linolenic acid into the mixed solution, performing ultrasonic treatment for 4 hours in the ice bath to form stable emulsion, dialyzing the stable emulsion in water for 16 hours by using a dialysis bag with the molecular weight cutoff of 100000 to remove free antigens, taking out the dialysis bag, covering polyethylene glycol on the dialysis bag until the volume of the stock solution is concentrated to 1mL, and thus obtaining the emulsion-shaped phytoglycogen (corn) nano adjuvant B, wherein the mass fraction of the antigens is 0.4%.

Comparative example 4

The method comprises the following steps of randomly dividing the measurement of 6-8-week-old female Balb/c mice into five groups, wherein each group comprises 5 female Balb/c mice, and respectively carrying out immune treatment on each Balb/c mouse, wherein the specific steps are as follows: the nano adjuvant A, the nano adjuvant B and the nano adjuvant C prepared in the comparative examples 1-3 are experimental groups, and each Balb/C mouse is inoculated with 10 mu L of the nano adjuvant through oral administration; the corresponding commercial inactivated vaccine for haemophilus parasuis disease is a control group, and each Balb/c mouse is injected with 10 mu L of the commercial inactivated vaccine subcutaneously on the back; the saline group was blank, and 10. mu.L of saline was orally administered to each Balb/c mouse. Balb/c mice have no abnormal reaction after vaccination, blood is taken every week after vaccination to separate serum, and the titer of the haemophilus parasuis antibody in the serum is monitored.

As shown in table 6, in two weeks after Balb/C mice were respectively administered with the nano adjuvant a, the nano adjuvant B and the nano adjuvant C prepared in comparative examples 1 to 3, the antibody level generated by the nano adjuvant a was 1: 15 below, the antibody level produced by nano adjuvant B and nano adjuvant C is below 1: 10. The antibody was detected in the control group one week later. The results of the experimental group were almost the same as those of the blank group, and the antibody level was always maintained at a low level. In the case of phytoglycogen in an excessive proportion, low antigen load or low addition of fatty acid adjuvant, experimental animals have difficulty in producing a sufficient amount of antibodies to protect the body.

TABLE 6Balb/c mice after immunization serum Haemophilus parasuis antibody titers

a vaccine adjuvant prepared in oral immunization comparative examples 1-3

b injecting a commercial inactivated vaccine for haemophilus parasuis disease

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A preparation method of a phytoglycogen pig oral vaccination nanometer adjuvant is characterized by comprising the following steps:

wherein, in the nano adjuvant for the oral vaccination of the phytoglycogen pigs, the content of the phytoglycogen is 0.5 to 10 weight percent, the content of the antigen is 0.01 to 2 weight percent, and the content of the fatty acid is 0 to 20 weight percent.

2. The preparation method of the phytoglycogen nano adjuvant for the oral vaccination of pigs according to claim 1, wherein the branch density of the phytoglycogen in the phytoglycogen aqueous solution in the step (1) is 4-5%;

the mass ratio of the phytoglycogen in the phytoglycogen aqueous solution in the step (1), the antigen and the fatty acid in the step (2) is (0.5-10): (0.01-2): (0.5-5).

3. The method for preparing the phytoglycogen nano-adjuvant for porcine oral vaccination according to claim 1 or 2, wherein the phytoglycogen in the aqueous solution of phytoglycogen in step (1) is extracted from corn seeds and/or rice seeds.

4. The method for preparing a phytoglycogen nano-adjuvant for pig oral vaccination according to claim 3, wherein the antigen in step (1) is at least one of porcine ileitis live vaccine, porcine reproductive and respiratory syndrome inactivated vaccine, porcine mycoplasma pneumonia inactivated vaccine, porcine pseudorabies inactivated vaccine, porcine foot and mouth disease inactivated vaccine, porcine circovirus type 2 baculovirus vector inactivated vaccine, streptococcus suis bivalent inactivated vaccine and haemophilus parasuis inactivated vaccine.

5. The method for preparing the phytoglycogen nano-adjuvant for the oral vaccination of pigs as claimed in claim 4, wherein said fatty acid in step (2) is at least one of oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid and derivatives of oleic acid, linoleic acid, linolenic acid, arachidonic acid, and eicosapentaenoic acid.

6. The preparation method of the phytoglycogen nano-adjuvant for the oral vaccination of pigs according to claim 5, wherein the antigen in the step (1) is preferably added in the form of an antigen aqueous solution, and the concentration of the antigen aqueous solution is 0.1-2 mg/mL; the concentration of the phytoglycogen water solution is 1-15 mg/mL.

7. The preparation method of the phytoglycogen nano-adjuvant for the oral vaccination of pigs according to claim 4, wherein the oscillation speed in the step (1) is 50-300 rpm; the molecular weight cut-off of a dialysis bag used in the dialysis in the step (2) is 100000, and the dialysis time is 10-20 h;

and (3) after dialysis, concentrating to obtain the phytoglycogen pig oral vaccination nanometer adjuvant.

8. A phytoglycogen nano-adjuvant for pig oral vaccination prepared by the method of any one of claims 1 to 7.

9. The use of the phytoglycogen pig oral vaccination nano-adjuvant of claim 8 in the field of preparation of biomedical materials.

10. The application of the phytoglycogen pig oral vaccination nano adjuvant in the field of preparation of biomedical materials according to claim 9 is characterized by being applied to preparation of pig oral vaccination medicaments.