CN112618710B - Plant glycogen pig oral vaccination nanometer adjuvant and preparation method and application thereof - Google Patents
Plant glycogen pig oral vaccination nanometer adjuvant and preparation method and application thereof Download PDFInfo
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Abstract
The application discloses a plant glycogen pig oral vaccination nanometer adjuvant and a preparation method and application thereof. The method comprises the following steps: (1) Adding an antigen into the plant sugar aqueous solution, and carrying out vibration adsorption to obtain a mixed solution; (2) Under ice bath condition, adding fatty acid into the mixed solution, performing ultrasonic treatment to obtain emulsion, and performing dialysis to obtain the phytoglycogen pig oral vaccination nano adjuvant; in the phytoglycogen pig oral vaccinating nano adjuvant, the content of phytoglycogen is 0.5-10wt%, the content of antigen is 0.01-2wt% and the content of fatty acid is 0-20wt%. The prepared phytoglycogen-based nano adjuvant not only can delay the release of antigen by tolerating degradation, but also has stronger oxidation resistance with the oil-in-water emulsion formed by mixing with fatty acid, can improve the overall stability, can better protect the antigen, is safe and reliable in source, takes phytoglycogen as a vaccine adjuvant, has low cost, is safe and efficient, and has good preventive and protective capability on animals.
Description
Technical Field
The application belongs to the field of biological products for livestock, and particularly relates to a plant glycogen pig oral vaccination nanoadjuvant, a preparation method and application thereof.
Background
Oral immunization is the simplest and easiest way of immunization in the modern aquaculture industry. However, the oral immunization route is challenging, limiting its wide application. Because of the need to pass through the digestive tract, vaccine components are often degraded and therefore less immunogenic, and it is difficult to elicit an effective immune response, or even sometimes no immune response, and a dose of safe and effective adjuvant is required. Vaccine immunoadjuvants have become an integral part of the composition of new vaccines. Adjuvants have been used to improve the immunogenicity of partially inactivated vaccines, to enhance the immune response in low-response individuals, to improve the immunogenicity of vaccines vaccinated via mucosal routes, to modulate inappropriate immune responses, etc., and thus to improve protective immunity. Substances that have been demonstrated to be active by adjuvants include aluminum adjuvants, liposomal emulsions, proteoglycans, bacteria-like particles, and the like.
While many substances have proven to be adjuvanting, the selection or development of an advantageous adjuvant requires a combination of factors. What is needed is the safety of the adjuvant first. Due to their own pharmacological properties, adjuvants may affect the immunogenicity and safety of vaccine antigens. The most widely used aluminum adjuvants, although having better immune effect, have limitations and disadvantages. The structure of the frozen aluminum adjuvant is changed, and aggregation phenomenon occurs. Because of the freezing sensitivity of the aluminum adjuvant, the aluminum adjuvant cannot be frozen for storage, and a certain amount of cryoprotectant needs to be added, so that the safety risk is increased. It has also been reported that aluminum adjuvants can induce IgE production, possibly causing hypersensitivity, and in severe cases, the injected part is ulcerated and inflammatory reaction occurs locally. In addition to conventional aluminum adjuvants, oil-in-water emulsions are also increasingly used. The squalene oil is commonly used for preparing the oil-in-water emulsion, can form a drop-shaped emulsion with better uniformity and higher stability under the high-pressure condition. However, the side effects of oil-in-water emulsions are more severe than aluminum adjuvants, and more likely to cause inflammatory reactions at the injection site, and also cause liver damage over prolonged use. Other adjuvants, such as nucleic acid adjuvants, have been found to induce autoimmune diseases in animal models, presenting safety issues. Thus, the stimulation of the animal's immune system by the adjuvant must be within a suitable, predictable range. The development of novel adjuvants that are biosafety has become a research trend in vaccine development. Global vaccine safety actions were initiated by the world health organization in 2012 in order to enhance vaccine drug vigilance and to implement global vaccine safety. At present, cyclodextrin derivatives are reported to be used as vaccine adjuvants, however, the steps of separation and purification after cyclodextrin derivatization and modification are complex, the workload is large, and the mass production is difficult.
Disclosure of Invention
In order to solve the defects and the shortcomings of the prior art, the primary purpose of the application is to provide a preparation method of a plant glycogen pig oral vaccination nanoadjuvant.
The application also aims to provide the plant glycogen pig oral vaccination nanoadjuvant prepared by the method. The phytoglycogen pig oral vaccination nanoadjuvant is prepared from highly branched soluble polysaccharide, namely phytoglycogen.
The application also aims to provide the application of the plant glycogen pig oral vaccination nanoadjuvant in the field of preparation of biomedical materials.
The application aims at realizing the following technical scheme:
the preparation method of the plant glycogen pig oral vaccination nanometer adjuvant comprises the following steps:
(1) Adding an antigen into the plant sugar aqueous solution, and carrying out vibration adsorption for 0.5-12 h at the temperature of 4-30 ℃ to obtain a mixed solution;
(2) Under ice bath conditions, adding fatty acid into the mixed solution in the step (1), performing ultrasonic treatment for 1-4 hours to obtain emulsion, and performing dialysis to obtain the phytoglycogen pig oral vaccination nanoadjuvant;
in the phytoglycogen pig oral vaccinated nano adjuvant, the content of phytoglycogen is 0.5-10wt%, the content of antigen is 0.01-2wt% and the content of fatty acid is 0.5-5wt%.
Preferably, the antigen in the step (1) is at least one of a live vaccine for porcine ileitis, an inactivated vaccine for porcine reproductive and respiratory syndrome, an inactivated vaccine for porcine mycoplasma pneumonia, an inactivated vaccine for porcine pseudorabies, an inactivated vaccine for porcine foot-and-mouth disease, an inactivated vaccine for porcine circovirus type 2 baculovirus vector, a bivalent inactivated vaccine for streptococcus suis and an inactivated vaccine for haemophilus parasuis disease.
Preferably, the mass ratio of the phytoglycogen in the aqueous solution of the plant glycogen in the step (1) to the antigen to the fatty acid in the step (2) is (0.5-10): (0.01-2): (0.5 to 5), more preferably (2 to 3): (0.5-1): 3.2.
preferably, 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.
Preferably, the concentration of the plant sugar aqueous solution in the step (1) is 1-15 mg/mL.
Preferably, the branching density of phytoglycogen in the aqueous solution of the phytoglycogen 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 speed of the oscillation in the step (1) is 50-300 rpm.
Preferably, the fatty acid in step (2) is oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, and at least one of oleic acid, linoleic acid, linolenic acid, arachidonic acid, and derivatives of eicosapentaenoic acid.
Preferably, the dialysis bag used in the dialysis in the step (2) has a molecular weight cut-off of 100000 and a dialysis time of 10-20 hours. The dialysis temperature is ice bath.
Preferably, after the dialysis in the step (2), concentration can be further performed to obtain the phytoglycogen pig oral vaccination nanoadjuvant.
The plant glycogen pig oral vaccination nanometer adjuvant prepared by the method.
The application of the plant glycogen pig oral vaccination nanometer adjuvant in the field of preparation of biological medicine materials.
Preferably, the application is: the application in preparing the pig oral vaccination medicine.
The technical principle of the application is as follows: the phytoglycogen used is a polysaccharide nanoparticle of highly branched molecular structure formed in the endosperm of plants. The density of phytoglycogen branches is 4-5%, one branch appears at every 20-25 glucose residues on average, and the phytoglycogen branches are soluble in water and have lower solution viscosity. Phytoglycogen branches are short and much more compact, and thus can form nanosphere particles with a radius of about 39 nm. The speed of releasing glucose from phytoglycogen is less than that of waxy corn starch and common starch, the phytoglycogen cannot be rapidly digested in the digestive tract, and the nano form of the phytoglycogen can be maintained for a certain time so as to achieve the effect of protecting antigen, and the phytoglycogen can be gradually decomposed by amylase and has the function of slowly releasing antigen. According to the application, the interaction of fatty acid and phytoglycogen is utilized to generate the amphiphilic phytoglycogen which is both oleophylic and hydrophilic, and the phytoglycogen can improve the oxidation stability of fatty acid, so that the oxidation stability of the phytoglycogen pig oral vaccination nanoadjuvant is effectively improved. The phytoglycogen raw material is derived from plants such as corn, rice and the like, and the animal has good phytoglycogen tolerance and natural advantages in the aspect of safety.
Compared with the prior art, the application has the following advantages:
the phytoglycogen used in the application is polysaccharide-shaped nano particles with highly branched molecular structures formed in endosperm of plants such as corn, rice and the like, and is soluble polysaccharide which is similar to amylopectin and is highly branched. Because of the natural nanoscale effect, the immune system can effectively identify phytoglycogen nanoadjuvants, and the T cells are more easily stimulated by organisms. The interaction of fatty acid and phytoglycogen is utilized to obtain the amphiphilic phytoglycogen nanoadjuvant which is both oleophilic and hydrophilic. Due to the protective effect of phytoglycogen, antigens are protected from degradation, 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 corn and the like in pig feed staple food, and has natural advantages in safety; the animal has good tolerance to phytoglycogen, and can not generate hypersensitivity reaction, autoimmune generation and the like. The prepared phytoglycogen-based nano adjuvant not only can resist degradation and delay the release of antigen, but also has stronger oxidation resistance when mixed with fatty acid to form oil-in-water emulsion. Toxicity test results show that repeated high dose administration of phytoglycogen nanoadjuvants by rodents is safe. The vaccine nanometer adjuvant provided by the application has the advantages of wide raw material sources, low cost, simple processing technology and easy popularization.
Drawings
Fig. 1 is a schematic diagram of a preparation process of the phytoglycogen pig oral vaccination nanoadjuvant of the application.
FIG. 2 is a graph showing particle size distribution of aqueous corn phytoglycogen, aqueous corn phytoglycogen-antigen solutions and prepared nano-adjuvants for oral vaccination (corn) of swine in example 1.
FIG. 3 is a graph showing the particle size distribution of the aqueous solution of rice phytoglycogen nanoadjuvant of example 2 and the prepared oral vaccination (rice) of swine.
FIG. 4 is a graph showing the effect of oral vaccination of swine phytoglycogen nanoadjuvant aqueous emulsions at different concentrations on HEP2 cell viability in example 4.
FIG. 5 is a graph showing the secretory sIgA in the intestinal tract and the secretory sIgA in the trachea and the serum IgG titer of Balb/c mice in example 6 after day 0 and day 14, wherein FIG. 5 (a) is a graph showing the secretory sIgA in the intestinal tract, FIG. 5 (b) is a graph showing the secretory sIgA in the trachea, and FIG. 5 (c) is a graph showing the serum antibody IgG titer.
Detailed Description
The present application will be described in further detail with reference to examples and drawings, but embodiments of the present application are not limited thereto.
The experimental methods in the embodiment of the application are all conventional methods unless specified otherwise; the reagents, materials, and the like, unless otherwise specified, are all commercially available.
The commercially available haemophilus parasuis inactivated vaccine described in the examples and comparative examples of the present application was purchased from Boringer John; the haemophilus parasuis inactivated vaccine and streptococcus suis bivalent inactivated vaccine (i.e. the raw materials prepared in the examples and comparative examples) were purchased from the large pharmaceutical factory of spanish sea bleacher organisms.
Example 1
Reference method (s.h. Yun and n.k. matheson. Carbohydrate, res. 1993, 243, 307-321) corn seeds were crushed and corn phytoglycogen solid powders were extracted. 0.015g of corn phytoglycogen solid powder is weighed and fully dissolved in 1.0 mL deionized water to prepare a phytoglycogen aqueous solution with the concentration of 15 mg/mL. The haemophilus parasuis inactivated vaccine antigen is dispersed in sterile water to prepare a haemophilus parasuis vaccine antigen aqueous solution with the concentration of 1 mg/mL. 5 mL antigen aqueous solution is added into 1ml of plant glycogen aqueous solution, and then the mixed solution is placed in an air bath constant temperature shaking box for shaking and balancing for 8 hours, the temperature is controlled to be 20 ℃, the shaking and balancing speed is 100 rpm, so that the antigen is fully adsorbed on the plant glycogen nano-particles. After the adsorption is completed, the mixed solution is placed under ice bath, 0.02 mL linolenic acid is added into the mixed solution, and ultrasonic treatment is carried out under ice bath for 4 hours to form stable emulsion, a dialysis bag with the molecular weight cut-off of 100000 is used for dialysis in water for 16 hours, free antigen is removed, then the dialysis bag is taken out, polyethylene glycol is covered on the dialysis bag to concentrate stock solution until the stock solution volume is concentrated to 1mL, and the emulsion-like pig oral vaccination (corn) phytoglycogen nanoadjuvant can be obtained.
The results of the antigen in this example being sufficiently adsorbed on phytoglycogen nanoparticles are shown in FIGS. 1 and 2. The pig oral vaccinated (corn) phytoglycogen nanoadjuvant prepared in the embodiment is placed at 37 ℃ without obvious change for three days, and is centrifuged at 3000 rpm for 3min without obvious precipitation or layering, so that the pig oral vaccinated (corn) phytoglycogen nanoadjuvant has good stability.
Example 2
Reference method (s.h. Yun and n.k. matheson. Carbohydrate, res. 1993, 243, 307-321) rice seeds were soaked, crushed and powdered phytoglycogen solids of rice were extracted. 0.01g of rice phytoglycogen solid powder is weighed and fully dissolved in 1.0 mL deionized water to prepare a phytoglycogen aqueous solution with the concentration of 10 mg/mL. Dispersing the streptococcus suis bivalent inactivated vaccine antigen in sterile water to prepare the streptococcus suis bivalent inactivated vaccine water solution with the concentration of 1 mg/mL. 2.5. 2.5 mL antigen water solution is added into 1ml of plant glycogen aqueous solution, and then the mixed solution is placed in an air bath constant temperature shaking box for shaking and balancing for 6 hours, the temperature is controlled to be 20 ℃, the shaking and balancing speed is 120 rpm, so that the antigen is fully adsorbed on the plant glycogen nano-particles. After the adsorption is completed, the mixed solution is placed under ice bath, 0.02 mL linoleic acid is added into the mixed solution, and ultrasonic treatment is carried out under ice bath for 5 hours to form stable emulsion, a dialysis bag with the molecular weight cut-off of 100000 is used for dialysis in water for 16 hours to remove free antigen, then the dialysis bag is taken out, polyethylene glycol is covered on the dialysis bag to concentrate stock solution until the stock solution volume is concentrated to 1mL, and the emulsion-like pig oral vaccination (rice) phytoglycogen nanoadjuvant can be obtained.
The results of the antigen in this example being sufficiently adsorbed on phytoglycogen nanoparticles are shown in FIGS. 1 and 3.
Example 3 stability of oral vaccinated phytoglycogen nanoadjuvants for pigs
An example of an oral vaccinated phytoglycogen nanoadjuvant for pigs prepared in examples 1-2 is described. The swine oral vaccinated (corn) phytoglycogen nanoadjuvant of example 1 and the swine oral vaccinated (rice) phytoglycogen nanoadjuvant of example 2 were placed at 37 ℃ for three days, respectively, without significant changes, and were centrifuged at 3000 rpm for 3min without significant precipitation or delamination. The sodium thiosulfate method was used to determine the peroxide value of the oral vaccinated phytoglycogen nanoadjuvant in pigs after leaving for one week under different conditions (results are shown in table 1). The degree of oxidation of the swine orally vaccinated phytoglycogen nanoadjuvant fatty acid was also an important indicator for evaluating the degree of deterioration by measuring the amount of peroxide product. Meanwhile, an emulsion containing 0.05% antigen and 1% fatty acid was used as a control group. As shown in table 1, the peroxide value of the oral vaccinated phytoglycogen nanoadjuvant for pigs is lower than that of a control group (an emulsion group formed by the antigen with almost the same mass fraction as that of the examples and the linolenic acid with the intermediate mass fraction) under different storage conditions, which indicates that the phytoglycogen improves the oxidation stability of the linolenic acid, and the oral vaccinated phytoglycogen nanoadjuvant for pigs prepared in examples 1-2 has stronger oxidation resistance.
Table 1 comparison of oxidation degree of oral vaccinated phytoglycogen nanoadjuvants for pigs under different storage conditions
Inoculating the prepared pig oral vaccinated phytoglycogen nanoadjuvant in example 1-2 into a Trypticase Soy Broth (TSB) culture medium, observing 24 h at 37 ℃, and observing no turbidity in the trypticase soy broth culture medium, wherein no bacteria are found in microscopic examination, thus indicating that the pig oral vaccinated phytoglycogen nanoadjuvant has no bacterial pollution.
Example 4 safety of oral vaccination of pigs with phytoglycogen nanoadjuvants
An example of an oral vaccinated phytoglycogen nanoadjuvant for pigs prepared in examples 1-2 is described. The swine oral vaccinated phytoglycogen nanoadjuvants prepared in examples 1 and 2 were formulated into aqueous emulsions at concentrations of 0.25 mg/mL, 0.5 mg/mL, 1mg/mL, 2 mg/mL, respectively, and the aqueous emulsions were added to 24-well plates (one emulsion per well), followed by seeding HEP2 cells (purchased from Thermo Fisher Co.) using DMEM medium containing 15% fetal bovine serum and at 5% CO 2 The incubation was carried out at 37℃in an incubator without the addition of an aqueous solution of phytoglycogen nanoadjuvant for oral vaccination of pigs in the control group, the other conditions being identical. The cytotoxicity of swine oral vaccinated phytoglycogen nanoadjuvants was tested using the MTT method. At the four concentrations, neither of the two phytoglycogen nanoadjuvants caused massive cell death (as shown in fig. 4), demonstrating the higher safety of the two phytoglycogen nanoadjuvants as vaccine adjuvants.
Female Balb/c mice with 6-8 weeks of age are randomly divided into four groups of 10 mice each, and the experimental groups are as follows: 10 Balb/c mice, each being infused with 20. Mu.L of the swine oral vaccinated phytoglycogen nanoadjuvant prepared in example 1, 10 Balb/c mice, each being infused with 20. Mu.L of the swine oral vaccinated phytoglycogen nanoadjuvant prepared in example 2. The control group was: 10 Balb/c mice, each with 20. Mu.L of a commercially available haemophilus parasuis inactivated vaccine injected subcutaneously in the back; the blank groups are: 10 Balb/c mice were each filled with 20. Mu.L of physiological saline. Four weeks of continuous observation, results are shown in figure 2, with all Balb/c mice surviving without any clinical manifestation.
TABLE 2 survival results of Balb/c mice after immunization
The safety of the swine oral vaccinated (corn) phytoglycogen nanoadjuvant prepared in example 1 was specifically demonstrated by way of example. Female Balb/c mice with 6-8 weeks of age are randomly divided into three groups of 5 mice each, and the experimental groups are as follows: 5 Balb/c mice, each filled with 20. Mu.L of the swine oral vaccinated (corn) phytoglycogen nanoadjuvant prepared in example 1; the control group was: 5 Balb/c mice, each back injected subcutaneously with 20. Mu.L of commercially available haemophilus parasuis inactivated vaccine; the blank groups are: 5 Balb/c mice were each filled with 20. Mu.L of physiological saline. After immunization, balb/c mice were monitored 1 time at 30 minute intervals for body temperature using an animal thermometer. Within 4 hours of continuous monitoring, balb/c mice had body temperature changes within 0.5 ℃ after oral administration of the vaccine (corn) phytoglycogen nanoadjuvant to pigs prepared in example 1 (Table 3). And then continuously observing for four weeks, wherein in four weeks after oral inoculation, balb/c mice have no side reactions such as fever and the like, have no unhairing and other conditions, have no abnormal symptoms, and all Balb/c mice survive healthily, so that the pig oral vaccinated (corn) phytoglycogen nanoadjuvant prepared in the embodiment 1 does not cause strong immune stress response after Balb/c mice are immunized, and the safety is good.
TABLE 3 Balb/c monitoring results of body temperature of mice after immunization
a corn phytoglycogen vaccine nanoadjuvant for oral immunization loading haemophilus parasuis disease inactivated vaccine antigen
b injection immunization commercial haemophilus parasuis disease inactivated vaccine
Example 5 safety of oral vaccination of pigs with phytoglycogen nanoadjuvants
An oral administration of a vaccine of swine (corn) phytoglycogen nanoadjuvant prepared in example 1 is exemplified. Healthy suckling piglets of about 3 weeks of age are randomly divided into three groups of 5 piglets, and after body temperature measurement, immune treatment is carried out, and the experimental groups: each piglet was drenched with 2 mL the swine oral vaccination (corn) phytoglycogen nanoadjuvant emulsion prepared in example 1; control group: each piglet was intramuscular injected with 2 mL of a commercially available haemophilus parasuis inactivated vaccine; blank group: each piglet was drenched with 2 mL saline. After immunization of the piglets, the temperature of the piglets was monitored 1 time with an animal thermometer every 30 minutes. Within 4 hours of continuous monitoring, the temperature change of piglets after oral administration of the vaccine (corn) phytoglycogen nanoadjuvant for pigs prepared in example 1 was within 0.5 ℃ (table 4). And then, continuously observing for four weeks, wherein within four weeks after oral inoculation, piglets do not have side effects such as fever and the like, no abnormal symptoms are seen, and all piglets are healthy and alive, which indicates that the pig oral vaccinated (corn) phytoglycogen nanoadjuvant prepared in the example 1 does not cause strong immune stress response after immunization of the piglets, and has good safety.
Table 4 results of monitoring body temperature of piglets after immunization
a corn phytoglycogen vaccine nanoadjuvant for oral immunization loading haemophilus parasuis disease inactivated vaccine antigen
b injection immunization commercial haemophilus parasuis disease inactivated vaccine
Example 6 immunization effect of oral Vaccination of pigs with phytoglycogen nanoadjuvants
An oral vaccination of swine (corn) phytoglycogen nanoadjuvant prepared in example 1 was exemplified. The swine oral vaccinated (corn) phytoglycogen nanoadjuvant prepared in example 1 was subjected to in vivo immune effect test. Female Balb/c mice of 6 to 8 weeks of age were randomly divided into three groups of 5 animals each, and subjected to a first immunization treatment, experimental groups: 10. Mu.L of the pig oral vaccinated (maize) phytoglycogen nanoadjuvant prepared in example 1 was infused into each Balb/c mouse; control group: 10 mu L of commercially available haemophilus parasuis inactivated vaccine is injected subcutaneously into the back of each Balb/c mouse; blank group: each Balb/c mouse was filled with 10. Mu.L of physiological saline. On day 20 after the first immunization treatment, the mice of the experimental group were immunized a second time, and 10. Mu.L of the swine oral vaccinated (maize) phytoglycogen nanoadjuvant prepared in example 1 was infused into each Balb/c mouse. From the first immunization, balb/c mouse blood was collected weekly, serum was isolated, and the serum was monitored for haemophilus parasuis antibody titers using an indirect ELISA assay. Antibodies were detected after one week of oral administration of the vaccine (corn) phytoglycogen nanoadjuvant in swine prepared in example 1, followed by a gradual increase in antibody content, with the average antibody titer of the experimental group being substantially equivalent to that of the control group, and the antibody levels remained high throughout the last two months of monitoring, and at levels above 1:16 in Balb/c mice for six months after additional immunization (Table 5).
TABLE 5 antibody titers of haemophilus parasuis in serum of Balb/c mice after immunization
a corn phytoglycogen vaccine nanoadjuvant for oral immunization loading haemophilus parasuis disease inactivated vaccine antigen
b injection immunization commercial haemophilus parasuis disease inactivated vaccine
To further verify the immune effect, balb/c mouse serum was collected at 14 and 28 days, respectively, and antibodies produced in immunized mice were detected by ELISA (enzyme-linked immunosorbent assay). On day 14 of inoculation, the experimental group Balb/c mice had significantly higher levels of secretory sIgA in the gut and trachea than the blank group (as shown in FIG. 5). The results of analysis of serum samples showed that the experimental group Balb/c murine serum antibody IgG was significantly higher than the blank group. On day 28 of inoculation, the intestinal and tracheal secretory sIgA levels were significantly higher in Balb/c mice than in the blank and control groups. The results of analysis on serum samples show that the Balb/c murine serum antibody IgG of the experimental group is significantly higher than that of the blank group and the control group.
Comparative example 1
Reference method (s.h. Yun and n.k. matheson. Carbohydrate, res. 1993, 243, 307-321) corn seeds were crushed and corn phytoglycogen solid powders were extracted. 0.15g of corn phytoglycogen solid powder is weighed and fully dissolved in 10 mL deionized water to prepare a phytoglycogen aqueous solution with the concentration of 15 mg/mL. The haemophilus parasuis inactivated vaccine antigen is dispersed in sterile water to prepare a haemophilus parasuis vaccine antigen aqueous solution with the concentration of 1 mg/mL. Adding 5 mL antigen aqueous solution into 10 mL plant glycogen aqueous solution, placing the mixed solution in an air bath constant temperature shaking box, shaking and balancing for 8 hours, controlling the temperature to be 20 ℃ and the shaking and balancing speed to be 100 rpm, so as to promote the antigen to be fully adsorbed on the plant glycogen nano-particles. After the adsorption is completed, placing the mixed solution under ice bath, adding 0.02 mL linolenic acid into the mixed solution, performing ultrasonic treatment under ice bath for 4 hours to form stable emulsion, dialyzing in water for 16 hours by using a dialysis bag with the molecular weight cut-off of 100000 to remove free antigen, then taking out the dialysis bag, covering the dialysis bag with polyethylene glycol until the original liquid volume is concentrated to 1mL, and obtaining the emulsion-like phytoglycogen (corn) nanoadjuvant A, wherein the mass fraction of the phytoglycogen is 15%, and the mass fraction of the antigen is 0.5%.
Comparative example 2
Reference method (s.h. Yun and n.k. matheson. Carbohydrate, res. 1993, 243, 307-321) corn seeds were crushed and corn phytoglycogen solid powders were extracted. Corn phytoglycogen solid powder 0.015. 0.015g is weighed and fully dissolved in 1.0. 1.0 mL deionized water to prepare a phytoglycogen aqueous solution with a concentration of 15 mg/mL. The haemophilus parasuis inactivated vaccine antigen is dispersed in sterile water to prepare a haemophilus parasuis vaccine antigen aqueous solution with the concentration of 1 mg/mL. Adding 0.08mL of antigen aqueous solution into 1mL of plant glycogen aqueous solution, placing the mixed solution in an air bath constant temperature shaking box, shaking and balancing for 8 hours, controlling the temperature to be 20 ℃ and the shaking and balancing speed to be 100 rpm, so as to promote the antigen to be fully adsorbed on the plant glycogen nano-particles. After the adsorption is completed, the mixed solution is placed under ice bath, 0.02 mL linolenic acid is added into the mixed solution, and ultrasonic treatment is carried out under ice bath for 4 hours to form stable emulsion, a dialysis bag with the cut-off molecular weight of 100000 is used for dialysis in water for 16 hours to remove free antigen, then the dialysis bag is taken out, polyethylene glycol is covered on the dialysis bag until the original liquid volume is concentrated to 1mL, and then the phytoglycogen (corn) nanoadjuvant B in emulsion form is obtained, wherein the mass fraction of the antigen is 0.008%.
Comparative example 3
Reference method (s.h. Yun and n.k. matheson. Carbohydrate, res. 1993, 243, 307-321) corn seeds were crushed and corn phytoglycogen solid powders were extracted. 0.015g of corn phytoglycogen solid powder is weighed and fully dissolved in 1.0 mL deionized water to prepare a phytoglycogen aqueous solution with the concentration of 15 mg/mL. The haemophilus parasuis inactivated vaccine antigen is dispersed in sterile water to prepare a haemophilus parasuis vaccine antigen aqueous solution with the concentration of 1 mg/mL. Adding 5 mL antigen aqueous solution into 1mL plant glycogen aqueous solution, placing the mixed solution in an air bath constant temperature shaking box, shaking and balancing for 8 hours, controlling the temperature to be 20 ℃ and the shaking and balancing speed to be 100 rpm, so as to promote the antigen to be fully adsorbed on the plant glycogen nano-particles. After the adsorption is completed, the mixed solution is placed under ice bath, 0.005-mL-linolenic acid is added into the mixed solution, and ultrasonic treatment is carried out under ice bath for 4 hours to form stable emulsion, a dialysis bag with the cut-off molecular weight of 100000 is used for dialysis in water for 16 hours to remove free antigen, then the dialysis bag is taken out, polyethylene glycol is covered on the dialysis bag until the original liquid volume is concentrated to 1mL, and then the phytoglycogen (corn) nanoadjuvant B in emulsion form is obtained, wherein the mass fraction of the antigen is 0.4%.
Comparative example 4
The female Balb/c mice with 6-8 weeks of age are randomly divided into five groups, 5 mice in each group are subjected to immune treatment respectively, and the specific steps are as follows: the nanometer adjuvant A, the nanometer adjuvant B and the nanometer 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 nanometer adjuvant through oral administration; the corresponding commercial haemophilus parasuis inactivated vaccine is used as a control group, and 10 mu L of the commercial inactivated vaccine is injected into each Balb/c mouse through the back in a subcutaneous mode; the saline group was a blank group, and 10. Mu.L of saline was orally administered to each Balb/c mouse. Balb/c mice were vaccinated without abnormal reaction, serum was collected weekly after vaccination, and serum was monitored for haemophilus parasuis antibody titers.
As shown in table 6, the antibody level produced by the nanoadjuvant a was 1 in two weeks after the nanoadjuvant a, nanoadjuvant B and nanoadjuvant C prepared in comparative examples 1 to 3 were taken respectively in Balb/C mice: below 15, the antibody level produced by the nanoadjuvant B and the nanoadjuvant C is below 1:10. And the antibody can be detected after one week in the control group. The results of the experimental group were almost the same as the blank group, and the antibody level was maintained at a lower level all the time. In the case of a low amount of phytoglycogen, antigen load or fatty acid adjuvant addition in an excessive proportion, it is difficult for the experimental animal to produce a sufficient amount of antibody to protect the body.
TABLE 6 antibody titers of Haemophilus parasuis in serum of Balb/c mice after immunization
a vaccine adjuvant prepared by oral immunization comparative examples 1-3
b injection immunization commercial haemophilus parasuis disease inactivated vaccine
The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present application should be made in the equivalent manner, and the embodiments are included in the protection scope of the present application.
Claims (3)
1. The preparation method of the oral vaccination of the plant glycogen pigs is characterized by comprising the following steps:
(1) Adding an antigen into the plant sugar aqueous solution, and carrying out vibration adsorption for 0.5-12 h at the temperature of 4-30 ℃ to obtain a mixed solution;
(2) Under ice bath conditions, adding fatty acid into the mixed solution obtained in the step (1), performing ultrasonic treatment for 1-4 hours to obtain emulsion, and performing dialysis to obtain phytoglycogen pig oral vaccination;
wherein, the content of phytoglycogen is 0.5-10wt%, the content of antigen is 0.01-2wt% and the content of fatty acid is 0.5-5wt%;
the branching density of phytoglycogen in the plant sugar aqueous solution in the step (1) is 4-5%;
the mass ratio of the phytoglycogen in the aqueous solution of the plant sugar in the step (1) to the antigen to the fatty acid in the step (2) is (0.5-10): (0.01-2): (0.5-5);
extracting phytoglycogen in the aqueous solution of the phytoglycogen in the step (1) from corn seeds and/or rice seeds;
the antigen in the step (1) is haemophilus parasuis vaccine antigen;
the antigen is 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 plant sugar aqueous solution is 1-15 mg/mL;
the fatty acid in the step (2) is at least one of linoleic acid and linolenic acid.
2. The method for preparing an oral vaccination of a plant glycogen pig according to claim 1, wherein the shaking speed in step (1) is 50 to 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 hours;
and (3) after the dialysis in the step (2), concentrating to obtain the phytoglycogen pig oral vaccination.
3. A plant glycogen pig oral vaccination made by the method of any one of claims 1-2.
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