CN114366808B - Polysaccharide and virus antigen co-delivery nano vaccine, and preparation method and application thereof - Google Patents
Polysaccharide and virus antigen co-delivery nano vaccine, and preparation method and application thereof Download PDFInfo
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- CN114366808B CN114366808B CN202111527296.9A CN202111527296A CN114366808B CN 114366808 B CN114366808 B CN 114366808B CN 202111527296 A CN202111527296 A CN 202111527296A CN 114366808 B CN114366808 B CN 114366808B
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- polysaccharide
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- vitamin
- polyethylene glycol
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55583—Polysaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Nanotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Virology (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Communicable Diseases (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Medical Informatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Physics & Mathematics (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- General Physics & Mathematics (AREA)
- Manufacturing & Machinery (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
Abstract
The application discloses a polysaccharide and virus antigen co-delivery nano vaccine, a preparation method and application thereof, wherein the nano vaccine comprises polysaccharide, avian adenovirus-4 type Penton recombinant protein and vitamin E polyethylene glycol succinate, the mass ratio of the polysaccharide to the avian adenovirus-4 type Penton recombinant protein to the vitamin E polyethylene glycol succinate is 0.5:1:05-2:1:2, and the particle size of the nano vaccine is 1-100 nm. The co-delivery nano vaccine in the application obviously reduces the incidence rate and death rate of the avian adenovirus by preparing the vaccine and carrying out application detection of the avian adenovirus-4 type virus, can also stimulate the organism to quickly generate stronger immune response, maintain the higher level immune response of the organism, and can promote chickens to quickly expel toxin and relieve organ injury.
Description
Technical Field
The application relates to the technical field of therapeutic or preventive nano vaccine preparations, in particular to a polysaccharide and virus antigen co-delivery nano vaccine, a preparation method and application thereof.
Background
Vaccine refers to a generic term for biological products that, after inoculation into the body, can cause the body to develop immunity to a particular disease. Vaccination is the most important and effective means of preventing infectious diseases, and more than 20 vaccines are now used for human disease prevention, of which more than half are viral vaccines. Because of the wide use of vaccines, the prevalence of acute infectious diseases such as smallpox, poliomyelitis, measles, diphtheria, etc., which have once seriously endangered human life and health, is effectively controlled. Vaccines are mostly prepared from killed or attenuated pathogens, and several other types of vaccines have been developed, namely second generation vaccines based on components of the pathogen that act as immunity, and third generation vaccines based on RNA or DNA, which have shown encouraging results in terms of immunogenicity. Although vaccination shows protective immunity against different diseases, there are still problems that limit their effectiveness in clinical applications. For example, attenuated live vaccines have the potential to recover virulence; the inactivated vaccine has poor immune effect and short maintenance time, and needs to be inoculated for multiple times; subunit vaccines have only a few main antigens, are weak in immunogenicity and have slightly poor preventive inoculation effects; the DNA and RNA vaccines with larger dosage have less risk of infection, but are easily degraded, so that the DNA and RNA vaccines are difficult to be transported to a target site in a targeted manner.
In the past ten years, the nanotechnology is widely applied in vaccinology, and the nano vaccine developed by the technology has wide application prospect in preventing and treating infectious diseases such as hepatitis B, influenza, AIDS virus, tumor and the like and serious diseases. The advantages of nanovaccines compared to pure antigen vaccines mainly include: (1) The nano carrier can avoid the antigen from being rapidly degraded, and improves the stability of the vaccine preparation; (2) Providing good adjuvant properties, promoting activation of antigen presenting cells; (3) The nano-size enhances the enrichment of the antigen in the lymph nodes and further improves the immune response capability of the antigen. In recent years, many new inorganic and organic Nanoparticles (NPs) (e.g., gold nanoparticles, silicon-based nanoparticles, lipoprotein nanodiscs, nanogels, etc.) have achieved remarkable results in the development of nanovaccines. However, the preparation technology of the nano vaccine needs to use a chemical synthesis method, or needs to further combine the synthesis method with a cell-based semisynthesis step, so that the preparation technology is complicated, and the activity of the antigen cannot be effectively ensured. The protein synthesis can be realized by using engineering bacteria or mammalian cells in one step, so that the developed protein nano vaccine for biosynthesis such as virus-like particles, ferritin and the like has the advantages of short production time, low cost and the like, and has higher biocompatibility and safety. However, this strategy still faces technical difficulties such as low antigen loading rate, difficulty in coupling polysaccharide antigens with complex structures, and the like.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
In view of the technical defects, the application provides a polysaccharide and virus antigen co-delivery nano vaccine, a preparation method and application thereof, and aims to provide a preparation method of the co-delivery nano vaccine by taking polysaccharide as an adjuvant and virus recombinant protein as an antigen and vitamin E polyethylene glycol succinate as a carrier. The vaccine has remarkable effect for preventing and treating viral diseases. In livestock breeding, the compound feed can be used for preventing and treating virulent infectious diseases such as avian adenovirus and the like, and brings great economic benefit for the livestock breeding industry.
Therefore, as one aspect of the application, the application overcomes the defects in the prior art and provides a polysaccharide and virus antigen co-delivery nano vaccine, a preparation method and application thereof.
In order to solve the technical problems, the application provides the following technical scheme: a nano vaccine for co-delivering the polyose and virus antigen is prepared from polyose, virus antigen and vitamine E polyethanediol succinate (0.5-2) through proportional mixing (0.5-2).
Preferably, the polysaccharide comprises one or more of probiotic extracellular polysaccharide and plant polysaccharide.
Preferably, the viral antigen is an adenovirus antigen
Preferably, the viral antigen is adenovirus-4 type Penton recombinant protein
Preferably, the particle size of the nano vaccine is in the range of 1-110 nm
Preferably, the particle size of the nano vaccine is in the range of 40-60 nm.
As one of the aspects of the application, the application provides a preparation method of a polysaccharide and virus antigen co-delivery nano vaccine, which comprises the following steps of mixing polysaccharide, virus antigen and vitamin E polyethylene glycol succinate to obtain a mixed solution, and carrying out self-assembly reaction for 0.5-2 h at the temperature of 25-37 ℃ under the condition of 400-1000 rpm/min.
Preferably, the self-assembly reaction is carried out by dripping the mixed solution at a constant speed, wherein the dripping speed is 2-4 mL/min.
As one of the aspects of the application, the application provides an application of the polysaccharide and virus antigen co-delivery nano vaccine in preventing and treating adenovirus infectious diseases.
Preferably, the adenovirus infectious disease is a disease caused by infection of an avian adenovirus-4 type JS strain.
The application has the beneficial effects that:
in one aspect, the application provides a polysaccharide and viral antigen co-delivery nanovaccine, which consists of polysaccharide, viral antigen and vitamin E polyethylene glycol succinate. Compared with the prior art, the application has the following advantages:
1. the co-delivery nanovaccine in the application significantly reduces the incidence and mortality of avian adenoviruses. The test chickens of each group were subjected to detoxification three weeks after immunization, and the chickens of the control group died within 2 days after detoxification, and the chickens of each immunized group were normally fed and drunk, and the mental state was also normal. The dead chickens are only subjected to the section examination, and obvious pericardial effusion can be seen after the section examination of the control chickens, and the color of the pericardial effusion is pale yellow and thicker; the liver is enlarged and denatured, the touch is fragile, the appearance is earthy yellow, and the liver and the heart of the immunized group chicken are normal.
2. The co-delivery nano vaccine provided by the application not only can reduce the incidence rate and death rate of the avian adenovirus, but also can stimulate the organism to rapidly generate stronger immune response and maintain the higher level of immune response of the organism.
3. The co-delivery nano vaccine provided by the application can reduce the incidence rate and death rate of the avian adenovirus, stimulate the organism to rapidly generate immune response, reduce the toxin expelling of chickens and relieve organ injury.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a self-made nano-mixer in the preparation of co-delivery nano-vaccines;
FIG. 2 is a transmission electron microscope observation of a probiotic extracellular polysaccharide and avian adenovirus-4 type Penton recombinant protein co-delivered nanovaccine in example 1;
FIG. 3 is a particle size distribution assay of the probiotic extracellular polysaccharide and avian adenovirus-4 type Penton recombinant protein co-delivered nanovaccine of example 1;
FIG. 4 is a transmission electron microscope observation of the nanoparticle vaccine co-delivered by the bighead atractylodes rhizome polysaccharide and the avian adenovirus-4 type Penton recombinant protein in example 2;
FIG. 5 is a particle size distribution assay of the recombinant protein co-delivered nanovaccine of Bighead atractylodes rhizome polysaccharide and avian adenovirus-4 type in example 2;
FIG. 6 is a particle size result of 18 kinds of nanomaterial prepared in comparative example 1;
FIG. 7 is an encapsulation efficiency result of 18 kinds of nanomaterial prepared in comparative example 1;
FIG. 8 is an electron microscopy observation of the co-delivered nanovaccine of the self-assembled probiotic extracellular polysaccharide and avian adenovirus-4 type Penton recombinant protein prepared in comparative example 3;
FIG. 9 is an ELISA test for antibody levels in the serum of each test group chicken in example 3;
FIG. 10 shows the measurement of cytokine expression in serum only the next day after immunization in each test group of chickens in example 3;
FIG. 11 shows the detection of toxin expelling condition of chickens in each test group in example 3;
FIG. 12 shows the effect of co-delivery of nanovaccine in example 3 on organ injury in chicken infected with adenovirus-4, dissected immediately after death of the vaccinated group, and no death of the immunized group within the experimental period (35 days), so dissected on day 35, wherein FIG. 12A shows the damage of pericardial effusion, etc. in dissected chicken, and FIG. 12B shows the histological signs characteristic of pericardial effusion syndrome in chicken heart, liver and spleen.
Detailed Description
In order that the above-recited objects, features and advantages of the present application will become more apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present application is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1: probiotic extracellular polysaccharide and avian adenovirus-4 type Penton recombinant protein co-delivery nano vaccine
The co-delivery nanometer vaccine of the probiotics extracellular polysaccharide and the avian adenovirus-4 type Penton recombinant protein is prepared by using the probiotics extracellular polysaccharide, the avian adenovirus-4 type Penton recombinant protein and vitamin E polyethylene glycol succinate. Wherein,,
probiotic extracellular polysaccharide: extracting exopolysaccharide from a probiotic bacterial strain (strain number: ACCC 03956) according to an organic solvent precipitation method (reference: separation and screening of exopolysaccharide-producing lactobacillus plantarum, molecular characterization and application research [ D ]. Wang Ji. Jilin university 2015), and detecting that the exopolysaccharide has a molecular weight of about 100K Da and consists of galactosamine hydrochloride, arabinose, galactose, glucose and glucuronic acid, wherein the ratio of the components is 0.152:0.364:0.479:0.258:0.137;
avian adenovirus-4 type Penton recombinant protein: expression and purification of avian adenovirus-4 type Penton recombinant protein: constructing pET32a (+) -Penton recombinant plasmid, carrying out induced expression on the pET32a (+) -Penton recombinant plasmid, and purifying the avian adenovirus-4 type Penton recombinant protein.
As shown in fig. 1, the nano-mixer in the preparation of co-delivery nano vaccine is formed by assembling a constant temperature heating magnetic stirrer, a triangular flask, a constant speed sample injector, a transfusion tube and a dropper (or a centrifuge tube with an opening at the lower end), the dripping speed is controlled by a small opening below the centrifuge tube (the larger the opening is, the faster the dripping speed is), the speed is controlled to be 3ml/min, and the self-assembly of the probiotic extracellular polysaccharide, the avian adenovirus-4 type Penton recombinant protein and the vitamin E polyethylene glycol succinate is carried out under the constant temperature magnetic stirring condition, and the specific steps are as follows:
1) Uniformly mixing 7mL of 0.6mg/mL of probiotic extracellular polysaccharide, 0.3mg/mL of avian adenovirus-4-type Penton recombinant protein and 0.6mg/mL of vitamin E polyethylene glycol succinate, and pushing the mixture into a nano mixer (figure 1) through a constant-speed sample injector, wherein the weight ratio of the three is 2:1:2, and the pushing speed of the constant-speed sample injector is 21mL/min; in fig. 1, the liquid in the constant-speed sample injector (injector) is the liquid mixed by the probiotics extracellular polysaccharide, the avian adenovirus-4 type Penton recombinant protein and the vitamin E polyethylene glycol succinate, and is uniformly pushed into a centrifuge tube with an opening at the lower end by the control of the constant-speed sample injector, and the centrifuge tube with the opening at the lower end is used for controlling the mixed liquid to slowly drop into a triangular flask.
4) The nanometer mixer solution is reacted for 30min according to the procedure of 400rpm/min and 25 ℃, the reacted mixed solution is collected to obtain a reaction product, the reaction product is a probiotic extracellular polysaccharide and avian adenovirus-4 type Penton recombinant protein co-delivered nanometer vaccine, and a TEM image of the reaction product under a scale of 100nm is shown in fig. 2, from the image, small particles which are uniformly dispersed are self-assembled probiotic extracellular polysaccharide and avian adenovirus-4 type Penton recombinant protein co-delivered nanometer vaccine, and larger agglomerate particles show that the nanometer material has aggregation tendency, but the size of the nanometer vaccine is not influenced, and from fig. 3, the particle size of the vaccine is 1-100 nm and is mainly distributed at about 50nm (85%). The small-sized nano particles have better penetration effect in vivo, and can more easily pass through biological barriers, so that the nano vaccine is more reliable in the process of carrying antigen to a target site. Research shows that the polymer nano particles can accurately regulate and control the size, show stronger targeting property, can realize controllable release and better control the immune reaction process. In addition, vitamin E polyethylene glycol succinate is a water-soluble derivative of vitamin E, and is formed by esterifying carboxyl of vitamin E succinate with polyethylene glycol 1000. Vitamin E polyethylene glycol succinate has both lipophilic tocopherol groups and hydrophilic polyethylene glycol long-chain polar groups, and the larger molecular surface area ensures that the vitamin E polyethylene glycol succinate has excellent surface activity and can be used as an excellent emulsifier. Compared with the traditional emulsifier polyvinyl alcohol, the emulsifying efficiency of the vitamin E polyethylene glycol succinate is 67 times higher, the medicine encapsulation efficiency can reach 100 percent, and the encapsulation efficiency of the probiotic extracellular polysaccharide and the avian adenovirus-4 type Penton recombinant protein co-delivered nano vaccine prepared by the method is as high as 95 percent.
Example 2: atractylodes macrocephala sugar and avian adenovirus-4 type Penton recombinant protein co-delivery nano vaccine
A nanometer vaccine co-delivering with Atractylodis rhizoma polysaccharide and fowl adenovirus-4 type Penton recombinant protein is prepared from Atractylodis rhizoma polysaccharide, fowl adenovirus-4 type Penton recombinant protein, and vitamin E polyethylene glycol succinate. Wherein,,
white atractylodes rhizome polysaccharide: dried largehead atractylodes rhizome slices are purchased from a traditional Chinese medicine decoction piece library of a middle hospital in Nanjing, and the technical process for extracting largehead atractylodes rhizome polysaccharide comprises the following steps: crushing bighead atractylodes rhizome, slicing, sieving with a 40-mesh sieve, carrying out ultrasonic wall breaking water bath extraction, centrifuging, carrying out alcohol precipitation, carrying out suction filtration, carrying out water dissolution, and drying to obtain polysaccharide. The detection shows that the molecular weight of the bighead atractylodes rhizome polysaccharide is about 3K Da, the bighead atractylodes rhizome polysaccharide consists of glucose and mannose, and the proportion of the components is 0.582:0.418;
avian adenovirus-4 type Penton recombinant protein: as in example 1.
The specific implementation steps are as follows:
1) 7mL of each of 0.3mg/mL of bighead atractylodes rhizome polysaccharide, 0.3mg/mL of avian adenovirus-4 type Penton recombinant protein and 0.3mg/mL of vitamin E polyethylene glycol succinate is pushed into a nano mixer (as in example 1) through a constant-speed sampler, the weight ratio of the bighead atractylodes rhizome polysaccharide to the avian adenovirus-4 type Penton recombinant protein is 1:1, and the pushing speed is 21mL/min.
4) The nanometer mixer solution is reacted for 30min according to the procedure of 400rpm/min and 25 ℃, the reaction product is collected and is the white atractylodes rhizome polysaccharide and the fowl adenovirus-4 type Penton recombinant protein co-delivered nanometer vaccine, the TEM image of the reaction product under the scale of 100nm is shown in the figure, the evenly dispersed particles are self-assembled white atractylodes rhizome polysaccharide and fowl adenovirus-4 type Penton recombinant protein co-delivered nanometer vaccine, the particle size range of the vaccine is 1-110 nm, and the vaccine is mainly distributed at 100nm (65 percent) as shown in the figure 5. The encapsulation rate of the nanometer vaccine co-delivered by the bighead atractylodes rhizome polysaccharide and the avian adenovirus-4 type Penton recombinant protein is up to 90%. In a word, the bighead atractylodes rhizome sugar and avian adenovirus-4 type Penton recombinant protein co-delivery nano vaccine prepared by the method still keeps smaller particle size and higher encapsulation efficiency, but the particle size is larger than that of the probiotic extracellular polysaccharide and avian adenovirus-4 type Penton recombinant protein co-delivery nano vaccine.
Comparative example 1: preparation procedure preferred experimental procedure.
According to the ratio (2:1:2, 1:1:1, 0.5:1:0.5) of polysaccharide, virus antigen and vitamin E polyethylene glycol succinate, the assembly time (0.5 h,1h,2 h) and the assembly temperature (25-37 ℃) are optimized to obtain 18 nano materials as shown in Table 1. The particle size (fig. 6) and the encapsulation efficiency (fig. 7) of 18 kinds of nanomaterials were respectively detected, and the result shows that the No. 13 nanomaterial has a lower particle size (50 nm) and a higher encapsulation efficiency (95%), and the ratio of polysaccharide, virus antigen and vitamin E polyethylene glycol succinate is 2:1:2, the assembly time is 0.5h, and the assembly temperature is 25 ℃.
TABLE 1 preparation optimization of probiotic extracellular polysaccharide and avian adenovirus-4 type Penton recombinant protein co-delivery nanovaccine.
* And (3) injection: the carrier is vitamin E polyethylene glycol succinate.
Comparative example 2: the vaccine (eps+penton+tpgs) obtained by direct mixing of the probiotic extracellular polysaccharide of example 1, avian adenovirus-4 type Penton recombinant protein, vitamin E polyethylene glycol succinate was used.
The probiotic extracellular polysaccharide (0.6 mg/mL), the avian adenovirus-4 type Penton recombinant protein (0.3 mg/mL) and the vitamin E polyethylene glycol succinate (0.6 mg/mL) are evenly mixed with 7mL each.
Comparative example 3: co-delivery of probiotic extracellular polysaccharide and avian adenovirus-4-type Penton recombinant protein nanovaccine without self-assembly
Mixing 0.6mg/mL of probiotic extracellular polysaccharide, 0.3mg/mL of avian adenovirus-4 type Penton recombinant protein, and 0.6mg/mL of vitamin E polyethylene glycol succinate 7mL each, stirring at 400rpm/min at 25deg.C for 30min. As shown in fig. 8, no ordered nano morphology was observed.
Example 3: application of probiotic extracellular polysaccharide and avian adenovirus-4 type Penton recombinant protein co-delivery nano vaccine
The application also provides application of the probiotic extracellular polysaccharide for preventing and treating the avian adenovirus and the avian adenovirus-4 type Penton recombinant protein co-delivery nanometer vaccine, which adopts 60 healthy SPF chickens which have no avian adenovirus immunization history and infection history and are 1 day old, and the vaccine is provided by a Jiangsu Nanjing certain chicken farm laboratory, and the vaccine is grouped after being cultured for 7 days in a Nanjing agricultural university laboratory animal center, wherein each group comprises 10 chickens. The vaccine was injected into 7 day old SPF chicks at a dose of 0.2mL (0.1 mg/mL). After 3 weeks of immunization, the poultry adenovirus-4 type JS strain is injected into muscle, and the toxin counteracting dosage is 2 multiplied by 10 6.5 TCID 50 . The vaccine application experiment groups and flows are as follows:
group 1 is an injected PBS control group (PBS);
group 2 is recombinant protein Penton alone immunization group (Penton): selecting 10 SPF chickens, subcutaneously inoculating recombinant Penton subunit vaccine into the neck, wherein the injection dose of the vaccine is 0.2 mL/chicken, and the dosage of recombinant Penton protein in the vaccine is 20 mug/chicken;
group 3 is recombinant protein Penton combined probiotic extracellular polysaccharide adjuvant combined immunization group (penton+eps): selecting 10 SPF chickens, subcutaneously inoculating subunit vaccine of the combination of the Penton protein and the extracellular polysaccharide of the probiotics on the neck, wherein the injection dosage of the vaccine is 0.2 mL/vaccine, the dosage of the recombinant Penton protein in the vaccine is 10 mug/vaccine, and the dosage of the extracellular polysaccharide of the probiotics is 20 mug/vaccine;
group 4 is a probiotic extracellular polysaccharide and avian adenovirus-4-type Penton recombinant protein co-delivery nanovaccine group (EPS-Penton-NP, example 1): 10 SPF chickens are selected, the neck is inoculated with the probiotic extracellular polysaccharide and the avian adenovirus-4 type Penton recombinant protein subcutaneously to co-deliver the nanometer vaccine, the vaccine injection dose is 0.2 mL/vaccine, the recombinant Penton protein dosage in the vaccine is 10 mug/vaccine, and the probiotic extracellular polysaccharide dosage is 20 mug/vaccine.
The 5 groups are probiotic extracellular polysaccharide, avian adenovirus-4 type Penton recombinant protein and vitamin E polyethylene glycol succinate mixed vaccine groups (EPS+Pento+TPGS, comparative example 2): 10 SPF chickens are selected, the neck is inoculated with the extracellular polysaccharide of the probiotics, the avian adenovirus-4 type Penton recombinant protein and the vitamin E polyethylene glycol succinic acid mixed solution subcutaneously, the vaccine injection dosage is 0.2 mL/vaccine, the recombinant Penton protein dosage in the vaccine is 10 mug/vaccine, and the extracellular polysaccharide dosage of the probiotics is 20 mug/vaccine.
Immunization of SPF chicken at 28 days of age (21 days after inoculation) by intramuscular injection of FAdV-4JS strain for virus content of more than or equal to 10 6 TCID 50 Per mL, 0.2mL of each injection, and death and pathological changes are observed and recorded after toxin attack. The individual groups of chickens were collected and serum isolated 2, 7, 14, 28 and 35 days after immunization and antibody levels were monitored by enzyme-linked immunosorbent assay (ELISA). And extracting RNA from serum at the 2 nd day after immunization, and detecting the expression change of the cell factor by using a fluorescent quantitative PCR method after reverse transcription. Cloaca swabs of each test group chicken were collected 3 and 7 days after challenge,and (3) extracting DNA by using an ultrapure DNA extraction kit after treatment, and monitoring the toxin expelling condition of each test group chicken after toxin attack by using a real-time fluorescence quantitative PCR method.
Test results:
1. co-delivery of vaccine nanovaccine immune challenge protection results
The immune toxicity eliminating protection result is shown in table 2. Compared with a control group, after the recombinant Penton antigen is inoculated, the immune protection rate is improved, and the protection rate of 20 mug/Penton on SPF chicken is 40%, which shows that the recombinant Penton subunit vaccine has a certain immune protection effect. After the addition of the probiotic extracellular polysaccharide adjuvant, the immunoprotection effect of the vaccine is improved to 70%, and the co-delivery nano vaccine group can be used for immunizing at a low dose of Penton (10 mug/dose), the vaccine protection rate is 100%, which shows that the immunoprotection effect of subunit vaccines is obviously improved by the nano adjuvant and protein. In contrast, the protection effect of directly mixing the probiotic extracellular polysaccharide, the avian adenovirus-4 type Penton recombinant protein and the vitamin E polyethylene glycol succinic acid is only 80 percent.
Table 2. Results of immunoprotection test of avian adenovirus-4 type JS strain by each test group.
2. Effect of Co-delivered nanovaccine on chicken antibody levels
The results of the ELISA detection of antibodies in serum showed (FIG. 9) that the antibody levels in chickens were continuously increased with the extension of the immunization time, peaked at day 28 after immunization (7 d after challenge) and then gradually decreased. Wherein, the co-delivery nano vaccine group generates antibodies faster and has high level, the chicken of the co-delivery nano vaccine immune group has the highest level of antibodies after 28 days of immunization, and then the chicken of the co-delivery nano vaccine immune group is combined with the immune group and is singly immunized. In particular, co-delivered nanovaccine groups showed a significant increase in cytokine (IL-2, ifn- β, TNF- α, G-CSF) expression in serum the next day after immunization (P < 0.001), indicating that the body developed strong cellular immunity to enhance the antiviral ability of the body (fig. 10).
3. Monitoring of chicken detoxification by co-delivery nano vaccine
FIG. 11 shows the virus removal through the digestive tract. From the aspect of the toxin expelling amount of chickens in each test group, the co-delivery nano vaccine immune group chickens only have the lowest virus expelling amount due to strong antiviral immunity, and then the mixed liquid immune group is combined with the immune group, and the Penton single immune group chickens only have higher toxin expelling amount.
4. Effect of Co-delivered nanovaccine on avian adenovirus-4 infected chicken viscera injury
The animals of the immunized group were examined for no apparent damage such as pericardial effusion (fig. 12A), so we used histopathological examination to further analyze if the chicken tissue was damaged at a microscopic level. The chicken of the challenge control group observed histological signs characteristic of pericardial effusion syndrome in heart, liver and spleen (fig. 12B). Such as partial myocardial fiber rupture, myocardial interstitial congestion; the lobular structure of liver disappears, and the liver cells appear nuclear inclusion bodies, necrosis and hyperemia and nuclear fragmentation; spleen lymphocytes severely diminish and necrose, especially the white marrow. Chickens immunized with 20 μg of the mixture of Penton protein, or of the probiotic extracellular polysaccharide, penton recombinant protein, and vitamin E polyethylene glycol succinate showed slight pathological changes. Such as liver lobule structure, liver cell hemorrhagic necrosis; myocardial fibrous gap broadening; spleen lymphocytes have little necrosis. In contrast, no significant histological lesions were observed in the tissue sections of both the co-delivered nanovaccine group and the negative control chicken (fig. 12B).
The above examples show that the co-delivery nanovaccine prepared by the present patent can reduce the amount of applied viral antigen and has a very high protective effect. The application of the co-delivery nano vaccine can greatly reduce the use cost of the vaccine, simplify the immunization program, provide high-efficiency protection for virulent infectious diseases, and further improve the economic benefit for farms.
The application relates to the technical field of therapeutic or preventive nano vaccine preparations, in particular to a preparation and application of a co-delivery nano vaccine taking polysaccharide as an adjuvant and virus recombinant protein as an antigen. The polysaccharide and virus antigen co-delivery nano vaccine provided by the application is prepared by a self-assembly method of polysaccharide, virus antigen and vitamin E poly (hexanediol) succinate in a self-made nano mixer; the preparation greatly improves humoral immunity and cellular immunity of organism, maintains higher immunity, remarkably reduces morbidity and mortality of virus, and greatly improves survival rate of organism. In addition, the preparation can reduce the use amount of virus antigen, simplify the immunization program, reduce the culture cost and has great popularization significance.
It should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered in the scope of the claims of the present application.
Claims (6)
1. A polysaccharide and viral antigen co-delivery nanovaccine characterized in that: comprises polysaccharide, virus antigen and vitamin E polyethylene glycol succinate, wherein the mass ratio of the polysaccharide to the virus antigen to the vitamin E polyethylene glycol succinate is (0.5-2) 1 (0.5-2);
the polysaccharide comprises one or more of probiotic extracellular polysaccharide and plant polysaccharide;
the extracellular polysaccharide has a molecular weight of 100K Da and consists of aminogalactose hydrochloride, arabinose, galactose, glucose and glucuronic acid, wherein the proportion of the components is 0.152:0.364:0.479:0.258:0.137; the plant polysaccharide comprises bighead atractylodes rhizome polysaccharide, the molecular weight of the bighead atractylodes rhizome polysaccharide is 3K Da, the bighead atractylodes rhizome polysaccharide consists of glucose and mannose, and the proportion of the components is 0.582:0.418;
the virus antigen is adenovirus-4 type Penton recombinant protein;
the preparation method of the polysaccharide and virus antigen co-delivery nano vaccine comprises the following steps: mixing polysaccharide, virus antigen and vitamin E polyethylene glycol succinate to obtain a mixed solution, and performing self-assembly reaction on the mixed solution at the temperature of 25-37 ℃ for 0.5-2 hours at the speed of 400-1000 r/min; and the self-assembly reaction is carried out by dripping the mixed solution at a constant speed, wherein the dripping speed is 2-4 mL/min.
2. The polysaccharide and viral antigen co-delivery nanovaccine of claim 1, wherein: the particle size of the nano vaccine is 1-110 nm.
3. The polysaccharide and viral antigen co-delivery nanovaccine according to claim 2, wherein: the particle size of the nano vaccine is 40-60 nm.
4. The method for preparing the polysaccharide and virus antigen co-delivery nano vaccine according to claim 1, which is characterized in that: comprises the steps of,
mixing polysaccharide, virus antigen and vitamin E polyethylene glycol succinate to obtain a mixed solution, and performing self-assembly reaction on the mixed solution at the temperature of 25-37 ℃ for 0.5-2 hours at the speed of 400-1000 r/min;
and the self-assembly reaction is carried out by dripping the mixed solution at a constant speed, wherein the dripping speed is 2-4 mL/min.
5. Use of the polysaccharide and viral antigen co-delivery nanovaccine of claim 1 in the preparation of a vaccine for the prevention and treatment of infectious diseases of adenovirus.
6. The use of the polysaccharide and viral antigen co-delivery nano vaccine according to claim 1 in the preparation of a medicament for preventing and treating infectious diseases in poultry, characterized in that: the infectious disease is a disease caused by infection of an avian adenovirus-4 type JS strain.
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