CN110420324B - Immunogenic composition containing rabbit staphylococcus antigen and preparation method and application thereof - Google Patents

Immunogenic composition containing rabbit staphylococcus antigen and preparation method and application thereof Download PDF

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CN110420324B
CN110420324B CN201910706127.8A CN201910706127A CN110420324B CN 110420324 B CN110420324 B CN 110420324B CN 201910706127 A CN201910706127 A CN 201910706127A CN 110420324 B CN110420324 B CN 110420324B
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rabbit
printing
powder
antigen
bacteria
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CN110420324A (en
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孙晴
刘言娟
张桂芝
葛艳艳
王美欣
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Linyi University
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Linyi University
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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/2063—Proteins, e.g. gelatin
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses an immunogenic composition containing a rabbit staphylococcus antigen, and a preparation method and application thereof, wherein the immunogenic composition comprises the rabbit staphylococcus antigen, a dispersion carrier and an aqueous printing adjuvant mixture which are used as printing spray liquid, and freeze-dried protective powder which is used as powder, and the printing spray liquid is repeatedly sprayed on each layer of powder by adopting a 3DP printing technology to obtain the immunogenic composition containing the rabbit staphylococcus antigen with a three-dimensional structure. The immunogenic composition prepared by the method has good immunogenic activity and immune protection, can be effectively used for preventing and treating rabbit staphylococcus aureus, has the immune protection rate of up to 100 percent, and has good vaccine stability and difficult inactivation.

Description

Immunogenic composition containing rabbit staphylococcus antigen and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to an immunogenic composition containing a rabbit staphylococcus antigen, and a preparation method and application thereof.
Background
Humans are natural storage hosts for staphylococcus aureus (s. Healthy individuals can colonize the skin, nostrils and throat with staphylococcus aureus, continuously (10-35%), intermittently (20-75%), or in a non-carrying state (5-70%), without associated disease. The vast human storage host increases the opportunity for the evolution and dissemination of adapted pathogenic clonal types. Invasive staphylococcal infections from the gram-positive cocci staphylococcus aureus and staphylococcus epidermidis (s. epidermidis) are of particular concern because they are an increasing global public health problem. In particular, staphylococcus aureus is responsible for most hospital-acquired (nosocomial) infections, and its prevalence increases in community-onset infections. For example, the incidence of invasive methicillin-resistant staphylococcus aureus (MRSA) in the united states was estimated to be 31.8 per 100000, including 18, 650 deaths in 2005. Staphylococcal disease has increased dramatically over the past 20 years, with this increase being accompanied by concomitant intravascular devices and invasive procedures. This increase in disease incidence is more disconcerting due to the concomitant increase in antibiotic resistance, and therefore there is an urgent need for immunogenic compositions for use in vaccines or for eliciting polyclonal or monoclonal antibodies to confer passive immunity as a means of preventing or treating staphylococcal infections and related diseases.
Staphylococcosis is a common disease of rabbits, is an infectious disease common in the rabbit industry caused by staphylococcus aureus, can cause the incidence of rabbit groups of all ages, and is generally treated by antibiotics in farms for reducing economic loss, but the drug resistance of bacteria is enhanced and spread due to the application of a large amount of antibiotics for a long time, so that the prevention difficulty is increased continuously. In addition, the treatment with antibiotics is easy to cause drug residues in meat, which affects food safety. The development and development of effective vaccines has become a hotspot for the prevention of infection by this bacterium.
Most vaccines in the prior art are injections and freeze-dried powder, have large volume and occupy space for storage, and easily cause the invalidation of vaccine strains in the storage process, thereby reducing the protection rate.
Disclosure of Invention
The technical scheme of the invention is as follows: an immunogenic composition containing a rabbit staphylococcus antigen comprises a rabbit staphylococcus antigen, a dispersion carrier and an aqueous printing adjuvant mixture which are used as printing spray liquid, and freeze-dried protective powder which is used as powder, wherein the printing spray liquid is repeatedly sprayed on each layer of powder by adopting a 3DP printing technology to obtain the immunogenic composition containing the rabbit staphylococcus antigen with a three-dimensional structure;
the rabbit staphylococcus antigen has an effective immunogenic amount of 109-1010CFU/ml of rabbit staphylococcal antigen strain;
the dosage ratio of the printing spraying liquid to the freeze-drying protection powder is 1 mg: 0.2-0.5 ml.
Further, the preparation method of the rabbit staphylococcus antigen strain comprises the following steps:
1) recovering intestinal bacteria and respiratory bacteria separately cultured from liver and lung of dead rabbit infected with Staphylococcus aureus, respectively inoculating the two strains to common broth culture medium after pure bacteria is checked to be qualified, and culturing at 37 deg.C for 18-24h to obtain bacterial liquid with predetermined concentration;
2) determining the concentration of respiratory tract bacteria to be 1.0-5.0 hundred million/mL by adopting a turbidimetric method and an ultraviolet spectrophotometry, adding a formaldehyde solution according to 0.3% of the amount of the bacteria, and inactivating the bacteria at 37 ℃ for 18-24 h; determining the concentration of intestinal bacteria to be 8.0-11 hundred million/mL, bathing at 100 deg.C for 2.5h, adding 0.3% formaldehyde solution, and inactivating at 37 deg.C for 18-24 h;
3) inoculating the intestinal bacteria and the respiratory bacteria treated in the step 2) to a common agar inclined plane and a blood agar inclined plane, culturing at 37 ℃ for 24-48h to confirm that no viable bacteria grow, and storing the bacteria for immunization in a refrigerator at 4 ℃ for later use after microscopic examination and aseptic test verification of the bacteria for immunization are qualified to obtain the rabbit staphylococcus antigen strain. The staphylococcus rabbit antigen bacteria can be propagated and cultured after being separated, and batch production is realized.
Furthermore, the dispersion carrier is chitosan nanoparticles loaded with the disperse protein B, and the disperse protein B can promote the integral diffusion of the biological membrane and is beneficial to the dispersion and release of the staphylococcus rabbit antigen strains.
Further, the preparation method of the dispersion carrier comprises the following steps: adding a solution of the disperse protein B into a chitosan acetic acid solution with the concentration of 4.0mg/ml until the final concentration of the disperse protein B is 2.0mg/ml, fully and uniformly mixing the solution at 30-35 ℃ under the condition of magnetic stirring, standing the solution for 20-24h at 20-25 ℃, adding phosphate with the final concentration of 1.5mg/ml under the condition of 100-plus-material 300rpm low-speed magnetic stirring for activating the chitosan nanoparticles so as to be convenient for loading the disperse protein B, and keeping stirring for 30-40min, wherein the obtained chitosan nanoparticles loaded with the disperse protein B have the characteristic of firm loading of the disperse protein B.
Further, the aqueous printing adjuvant comprises the following components in percentage by weight: 0.5-1% of hyaluronic acid, 16-19% of glucose, 4-6% of penetration-promoting peptide and the balance of 60-80% of ethanol water solution by volume fraction. The hyaluronic acid belongs to a human body safe injection component, has certain viscosity, can be used as a binder in a printing process, and ensures the firmness of a finished product of the composition; the penetration-promoting peptide is used as a biological penetration-promoting agent, is beneficial to the rapid release of staphylococcus rabbit antigen strains, is safer compared with a chemical penetration-promoting agent, and the ethanol aqueous solution can be used as a solvent for water-soluble and water-insoluble components in freeze-dried protective powder.
Further, the freeze-drying protection powder comprises the following components in parts by weight: 22% of microcrystalline cellulose, 16% of a-D-mannopyranose, 10% of procyanidine, 15% of collagen, 9% of sodium hydrogen phosphate, 17% of xylitol and 11% of soybean lecithin. The microcrystalline cellulose can be used as a binder for printing and forming, and can also be used as a disintegrant for dissolving and releasing. a-D-mannopyranose is used to protect proteins under freezing conditions; the procyanidine is used for resisting oxidation and prolonging the shelf life of the vaccine; collagen, xylitol and soybean phospholipid are used as culture substrates, so that the stability of the vaccine in a preservation state can be effectively ensured.
Further, the preparation method of the composition comprises the following steps:
s1: dissolving 1-2 parts of a dispersion carrier in 20-35 parts of PBS (phosphate buffer solution) solution according to the mass parts, dropwise adding 1 wt% of SMCC (small molecule cellulose) for activation, magnetically stirring for 1-2 hours, then adding 1 part of rabbit staphylococcus antigen and 0.2 wt% of glutaraldehyde, continuously magnetically stirring for 6-12 hours, and blowing nitrogen to a constant volume of 1-5ml to obtain a concentrated solution loaded with the rabbit staphylococcus antigen; the rabbit staphylococcus antigen is adsorbed on the carrier containing the disperse protein B, so that the immobilization degree is improved, and the disperse protein B can be conveniently diluted and injected to have a good dispersing effect on the rabbit staphylococcus antigen.
S2: mixing the concentrated solution with an aqueous printing adjuvant according to the mass ratio of 1: 5-20 to obtain printing spraying liquid;
s3: designing a final shape model of the composition by using CAD software, transmitting the information of the drug material to a model system designed by a computer, loading the printing spray liquid into a spraying mechanism, loading the freeze-dried protective powder into a feeding mechanism as spread powder, spreading the powder layer by using a 3DP technology, spraying and printing layer by layer to obtain a tablet composition, and finally freeze-drying, radiation sterilization and coating packaging.
Further, the parameters printed in step S3 are: the powder spreading thickness of each layer is 100 mu m, the printing thickness is too thin, the printing is difficult to dry, the printing spraying liquid and the freeze-drying protection powder cannot be fully combined when the printing thickness is too thick, the forming is not facilitated, and the floating powder and the flying powder are serious; spraying the printing spray liquid on each layer of powder, wherein the spraying diameter is 5-10mm, the final thickness is 1-3mm, the powder spreading time interval is 30s, and the total amount ratio of the freeze-drying protection powder to the printing spray liquid in each tablet composition is 1 mg: 0.2-0.5ml, the total amount ratio of the freeze-drying protective powder to the printing spray coating liquid is less than 1 mg: when the volume of the ink is 0.2ml, the printing liquid is insufficient and has poor forming degree, and the dosage ratio is more than 1 mg: when the amount of the ink is 0.5ml, too much ink is not preferable for drying, and the moldability is also poor.
Further, the composition is used for preparing a vaccine for preventing rabbit staphylococcus, and the vaccine is used for obtaining hyperimmune serum, so that an effective serological differential diagnosis reagent is provided for bacteriological detection.
The invention has the beneficial effects that:
(1) the preparation method also utilizes the aqueous printing adjuvant mixture containing the rabbit staphylococcus antigen as the printing spray liquid, takes the freeze-dried protective powder as the paving powder, and adopts the 3DP printing technology to repeatedly spray the printing spray liquid on each layer of the paving powder to obtain the immunogenic composition containing the rabbit staphylococcus antigen, which has a three-dimensional structure, small volume, high porosity, high dilution and dissolution speed, and is easier to store compared with liquid and powder, so that the prepared vaccine can keep good immunogen activity;
(2) according to the invention, the rabbit staphylococcus antigen is fixed on the chitosan nanoparticle loaded with the disperse protein B, and the rabbit staphylococcus antigen strain is dispersed and released into a rabbit body in the injection process by utilizing the characteristic that the disperse protein B can promote the integral diffusion of a biological membrane, so that the drug effect is better exerted;
(3) the aqueous printing adjuvant disclosed by the invention contains hyaluronic acid with certain viscosity, can be used as a binder in a printing process to ensure the firmness of a finished product of the composition, and also comprises a penetration promoting peptide serving as a biological penetration promoter, so that the rapid release of a rabbit staphylococcus antigen strain is facilitated.
In a word, the immunogenic composition prepared by the method has good immunogenic activity and immunoprotection, can be effectively used for preventing and treating rabbit staphylococcus aureus, has the immunoprotection rate up to 100 percent, and has good vaccine stability and difficult inactivation.
Detailed Description
The following examples are given as further illustration, but the present invention is not limited to these examples. The reagents and apparatus of the present invention are commercially available products unless otherwise specified.
Example 1
(1) Preparation of Staphylococcus rabbit antigen strains
1) Recovering intestinal bacteria and respiratory bacteria separately cultured from liver and lung of dead rabbit infected with Staphylococcus aureus, respectively inoculating two strains to common broth culture medium after pure bacteria is checked to be qualified, and culturing at 37 deg.C for 20h to obtain a predetermined concentration of bacteria solution;
2) determining the concentration of respiratory tract bacteria to be 3.0 hundred million/mL by adopting a turbidimetric method and an ultraviolet spectrophotometry, adding a formaldehyde solution according to 0.3 percent of the bacteria solution amount, and inactivating the bacteria at 37 ℃ for 20 hours; determining the concentration of intestinal bacteria to be 10 hundred million/mL, firstly bathing for 2.5h at 100 ℃, then adding 0.3% formaldehyde solution, and inactivating for 20h at 37 ℃;
3) inoculating the intestinal bacteria and the respiratory bacteria treated in the step 2) to a common agar inclined plane and a blood agar inclined plane, culturing at 37 ℃ for 36h to ensure that no viable bacteria grow, and storing the bacteria for immunization in a refrigerator at 4 ℃ for later use after the bacteria for immunization pass microscopic examination and sterility test verification to obtain the rabbit staphylococcus antigen strain. The staphylococcus rabbit antigen bacteria can be propagated and cultured after being separated, and batch production is realized.
(2) Preparation of aqueous printing adjuvant
Mixing 0.7% of hyaluronic acid, 18% of glucose, 5% of permeation-promoting peptide and the balance of ethanol water solution with the volume fraction of 70% according to the weight percentage to obtain the aqueous printing adjuvant. The hyaluronic acid belongs to a human body safe injection component, has certain viscosity, can be used as a binder in a printing process, and ensures the firmness of a finished product of the composition; the penetration-promoting peptide is used as a biological penetration-promoting agent, is beneficial to the rapid release of staphylococcus rabbit antigen strains, is safer compared with a chemical penetration-promoting agent, and the ethanol aqueous solution can be used as a solvent for water-soluble and water-insoluble components in freeze-dried protective powder.
(3) Preparation of freeze-dried protective powder
Mixing 22% of microcrystalline cellulose, 16% of a-D-mannopyranose, 10% of procyanidine, 15% of collagen, 9% of sodium hydrogen phosphate, 17% of xylitol and 11% of soybean phospholipid by weight, grinding and crushing to obtain the product with the particle size of 75 μm for later use.
(4) A method of preparing an immunogenic composition comprising a rabbit staphylococcal antigen comprising the steps of:
s1: according to the mass portion, 1 portion of dispersion carrier (chitosan nano particles with the particle size of 35 nm) is dissolved in 22 portions of PBS solution, and then 1 portion of effective dose 10 is added9Continuously stirring the CFU/ml rabbit staphylococcus antigen strain for 9 hours by magnetic force, and blowing nitrogen to fix the volume to 2ml to obtain concentrated solution loaded with the rabbit staphylococcus antigen;
s2: mixing the concentrated solution with an aqueous printing adjuvant according to the mass ratio of 1: 12, mixing to obtain printing spraying liquid;
s3: designing a final shape model of the composition by using CAD software, transmitting the information of the medicinal material to a model system designed by a computer, then loading a printing spray liquid into a spraying mechanism, loading freeze-dried protective powder into a feeding mechanism as spread powder, and spreading the powder layer by layer and spraying and printing layer by using a 3DP technology, wherein the printing parameters are as follows: the powder spreading thickness of each layer is 100 mu m, the printing spraying liquid is sprayed on each layer of powder spreading once, the spraying diameter is 7mm, the powder spreading time interval is 30s, and the total amount ratio of the freeze-drying protection powder to the printing spraying liquid in each tablet composition is 1 mg: 0.35ml to obtain tablet composition, and finally freeze drying, radiation sterilizing, and coating and packaging.
Before use as a vaccine, the tablet composition needs to be rapidly diluted in 1ml of injection solution, one tablet for each serving.
Example 2
This embodiment is substantially the same as embodiment 1 except that:
the dispersion carrier of the embodiment is chitosan nanoparticles loaded with the disperse protein B, and the disperse protein B can promote the integral diffusion of the biological membrane and is beneficial to the dispersion and release of the staphylococcus rabbit antigen strains.
The preparation method of the dispersion carrier comprises the following steps: adding a solution of the disperse protein B into a chitosan acetic acid solution with the concentration of 4.0mg/ml until the final concentration of the disperse protein B is 2.0mg/ml, fully and uniformly mixing the solution at 30-35 ℃ under the condition of magnetic stirring, standing the mixture at 25 ℃ for 22h, adding phosphate with the final concentration of 1.5mg/ml under the condition of 200rpm low-speed magnetic stirring, activating chitosan nanoparticles, facilitating the loading of the disperse protein B, and keeping stirring for 35min to obtain the chitosan nanoparticles loaded with the disperse protein B, wherein the chitosan nanoparticles have the characteristic of firm loading of the disperse protein B.
Wherein the method of preparing an immunogenic composition comprising a rabbit staphylococcal antigen comprises the steps of:
s1: according to the mass part, 1 part of a dispersion carrier (chitosan nanoparticles loaded with disperse protein B and having the particle size of 35 nm) is dissolved in 30 parts of PBS solution, 1 wt% of SMCC is dripped for activation, magnetic stirring is carried out for 1.5 hours, then 1 part of rabbit staphylococcus antigen and 0.2 wt% of glutaraldehyde are added, magnetic stirring is continued for 9 hours, and the volume is fixed to 2ml by nitrogen blowing, so that concentrated solution loaded with the rabbit staphylococcus antigen is obtained; the rabbit staphylococcus antigen is adsorbed on the carrier containing the disperse protein B, so that the immobilization degree is improved, and the disperse protein B can be conveniently diluted and injected to have a good dispersing effect on the rabbit staphylococcus antigen.
S2: mixing the concentrated solution with an aqueous printing adjuvant according to the mass ratio of 1: 12, mixing to obtain printing spraying liquid;
s3: designing a final shape model of the composition by using CAD software, transmitting the information of the medicinal material to a model system designed by a computer, then loading a printing spray liquid into a spraying mechanism, loading freeze-dried protective powder into a feeding mechanism as spread powder, and spreading the powder layer by layer and spraying and printing layer by using a 3DP technology, wherein the printing parameters are as follows: the powder spreading thickness of each layer is 100 mu m, the printing spraying liquid is sprayed on each layer of powder spreading once, the spraying diameter is 7mm, the powder spreading time interval is 30s, and the total amount ratio of the freeze-drying protection powder to the printing spraying liquid in each tablet composition is 1 mg: 0.35ml to obtain tablet composition, and finally freeze drying, radiation sterilizing, and coating and packaging.
Example 3
This embodiment is substantially the same as embodiment 2 except that:
the aqueous printing adjuvant of this example was a commercially available MontanideTMGel's aqueous vaccine adjuvant.
Example 4
This embodiment is substantially the same as embodiment 2 except that:
the freeze-drying protection powder of the embodiment adopts mannitol, glucose and vitamin C according to a mass ratio of 3: 2: 1 are combined.
Example 5
Preparing antiserum: the tablet composition of example 2 was dissolved in a sodium chloride solution to prepare an injection bacterial solution having a predetermined concentration. The injection amount of the 1 st, 2 nd, 3 th, 4 th and 5 th needles is respectively 0.5mL, 1.0mL, 2.0mL, 2.5mL and 3.0mL, and the interval between every two needles is 3 d. Testing blood 6d after the injection of the 5 th needle, and determining the titer of immune serum by adopting an ELISA method to ensure that the immune serum meets the preset requirement, namely the blood test is qualified; if the serum agglutination titer does not meet the predetermined requirement, the booster immunization is carried out for 1 time, and the injection dose of the 5 th needle is repeated: if the serum titer is not qualified, the rabbit is discarded. And (3) bleeding the rabbits qualified in blood test through the common carotid artery, standing the harvested whole blood at room temperature overnight, centrifuging at 2000r/min for 10min, and separating serum. And (3) absorbing the prepared antiserum by using related bacteria, adding a thimerosal solution to enable the final concentration to be 0.01% after complete absorption, and freezing and storing to obtain the antiserum.
Test examples
1. Testing the relative potency values of the immunogenic compositions of the invention for antibody production
1) Preparation of test and reference vaccines
Taking 1 tablet of the tablet composition prepared in the embodiment 2 of the invention, and putting the 1ml of sodium chloride injection for dissolving to be used as a test vaccine; 1ml of a commercially available rabbit staphylococcal vaccine was taken as a control sample and used as a reference vaccine.
2) Test method
50 healthy rabbits were selected and divided into 5 groups, and the test vaccine was repeated in 3 batches, and each rabbit was injected subcutaneously into the back with 0.5ml of test vaccine and reference vaccine. All test animals were re-immunized 1 time 2 weeks after the initial immunization, and all test animals were bled 2 weeks after 2 immunizations, tested for antibody titers by ELISA standard methods and analyzed for relative efficacy using Rel Pot 4.0 software. The average detection results of the 3 batches of the vaccines to be tested and the reference vaccine are shown in table 1, wherein the relative efficacy value is ≧ 1.0, and the test is qualified.
Table 1 relative efficacy value test results
Relative potency value of antibody
1 batch of test vaccines 1.60
2 batches of test vaccines 1.52
3 batches of test vaccines 1.57
Reference vaccine 1.20
As can be seen from table 1, the relative potency values of the three test vaccines were all greater than 1.0, indicating that the relative potency test was acceptable and that the potency values were also all higher than the reference vaccine.
2. Viral potency testing of immunogenic compositions of the invention
1) Determination of test vaccines
The vaccines prepared in examples 1 to 4 were used as test vaccine compositions and a commercially available rabbit staphylococcal vaccine was used as a control sample.
2) Test method
Selecting 60 healthy rabbits, dividing the rabbits into 6 groups, wherein the first group is injected with 0.5ml of the vaccine composition prepared in example 1, the second group is injected with 0.5ml of the vaccine composition prepared in example 2, the third group is injected with 0.5ml of the vaccine composition prepared in example 3, the fourth group is injected with 0.5ml of the vaccine composition prepared in example 4, the fifth group is injected with 0.5ml of the vaccine composition prepared in a control sample, and the vaccine of the control sample is diluted by PBS buffer; the sixth group was injected with 0.5 ml/tube of PBS buffer.
On 14 days after immunization, groups 1-5 were protected against challenge with a strain of Staphylococcus rabbit (commercially available) at a vaccination rate of 100LD50Group 6 reseeding with PBS buffer of equal amount, LD50The determination is referred to the quality standard of Chinese biological products. The death of the rabbits was counted 21 days after challenge with virulent virus, and the immunization effect of the comparative vaccine is shown in table 2.
TABLE 2 test results of immune effects
Figure BDA0002152171520000091
As can be seen from table 2, when the challenge test is performed on each group, all rabbits in the PBS blank group die 21 days after challenge, all rabbits injected with the vaccine composition prepared in example 2 of the present invention survive, the protection rate is 100%, and the vaccine protection rate of the control sample group is 60%, and the vaccine composition prepared in example 1 does not use chitosan nanoparticles loaded with the disperse protein B as a disperse carrier, thereby affecting the dispersion release of the staphylococcus rabbit antigen strains to a certain extent, and reducing the immune effect thereof; example 3 Montanide was usedTMThe Gel aqueous vaccine adjuvant has the protection rate of 80 percent; practice ofExample 4 the following materials were used, mannitol, glucose, and vitamin C in a mass ratio of 3: 2: 1, the vaccine is partially inactivated probably due to insufficient protection force on the vaccine, so that the protection rate is only 60%.
The embodiments of the present invention have been described in detail, but the present invention is only the preferred embodiments of the present invention, and is not limited thereto. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. An immunogenic composition containing a rabbit staphylococcus antigen is characterized by comprising a rabbit staphylococcus antigen, a dispersion carrier and an aqueous printing adjuvant mixture which are used as printing spray liquid, freeze-dried protective powder which is used as powder paving, and the printing spray liquid is repeatedly sprayed on each layer of the powder paving by adopting a 3DP printing technology to obtain the immunogenic composition containing the rabbit staphylococcus antigen with a three-dimensional structure;
the rabbit staphylococcus antigen has an effective immunogenic amount of 10 9 -10 10 CFU/ml of rabbit staphylococcal antigen strain;
the dosage ratio of the printing spray liquid to the freeze-drying protection powder is 1 mg: 0.2-0.5 ml;
the dispersion carrier is chitosan nano particles loaded with disperse protein B;
the aqueous printing adjuvant comprises the following components in percentage by weight: 0.5-1% of hyaluronic acid, 16-19% of glucose, 4-6% of penetration-promoting peptide and the balance of 60-80% of ethanol water solution by volume fraction;
the freeze-drying protection powder comprises the following components in parts by weight: 22% of microcrystalline cellulose, 16% of a-D-mannopyranose, 10% of procyanidine, 15% of collagen, 9% of sodium hydrogen phosphate, 17% of xylitol and 11% of soybean lecithin.
2. The immunogenic composition comprising rabbit staphylococcal antigen of claim 1 wherein the rabbit staphylococcal antigen species is prepared by a process comprising the steps of:
1) recovering intestinal bacteria and respiratory bacteria separately cultured from liver and lung of dead rabbit infected with Staphylococcus aureus, respectively inoculating the two strains to common broth culture medium after pure bacteria is checked to be qualified, and culturing at 37 deg.C for 18-24h to obtain bacterial liquid with predetermined concentration;
2) determining the concentration of respiratory tract bacteria to be 1.0-5.0 hundred million/mL by adopting a turbidimetric method and an ultraviolet spectrophotometry, adding a formaldehyde solution according to 0.3% of the amount of the bacteria, and inactivating the bacteria at 37 ℃ for 18-24 h; determining the concentration of intestinal bacteria to be 8.0-11 hundred million/mL, firstly performing water bath at 100 ℃ for 2.5h, then adding 0.3% formaldehyde solution, and inactivating at 37 ℃ for 18-24 h;
3) inoculating the intestinal bacteria and the respiratory bacteria treated in the step 2) to a common agar inclined plane and a blood agar inclined plane, culturing at 37 ℃ for 24-48h to confirm that no viable bacteria grow, and storing the bacteria for immunization in a refrigerator at 4 ℃ for later use after microscopic examination and aseptic test verification of the bacteria for immunization are qualified to obtain the rabbit staphylococcus antigen strain.
3. The immunogenic composition comprising rabbit staphylococcal antigen of claim 1 wherein the composition is prepared by a process comprising the steps of:
s1: according to the mass parts, 1-2 parts of a dispersion carrier is dissolved in 20-35 parts of PBS solution, 1 wt% of SMCC is dripped for activation, magnetic stirring is carried out for 1-2 hours, then 1 part of the rabbit staphylococcus antigen and 0.2 wt% of glutaraldehyde are added, magnetic stirring is continued for 6-12 hours, and nitrogen is blown to a constant volume of 1-5ml, so that concentrated solution loaded with the rabbit staphylococcus antigen is obtained;
s2: mixing the concentrated solution and the aqueous printing adjuvant according to a mass ratio of 1: 5-20 to obtain printing spraying liquid;
s3: designing a final shape model of the composition by using CAD software, transmitting medicine material information to a model system designed by a computer, loading the printing spraying liquid into a spraying mechanism, loading the freeze-dried protective powder into a feeding mechanism as spread powder, spreading the freeze-dried protective powder layer by using a 3DP technology, spraying and printing layer by layer to obtain a tablet composition, and finally freeze-drying, radiation sterilization and coating packaging.
4. The immunogenic composition comprising rabbit staphylococcal antigen of claim 3 wherein the parameters printed in step S3 are: the powder spreading thickness of each layer is 100 mu m, the printing spraying liquid is sprayed on each layer of powder spreading once, the spraying diameter is 5-10mm, the powder spreading time interval is 30s, and the total weight ratio of the freeze-drying protective powder to the printing spraying liquid in each tablet composition is 1 mg: 0.2-0.5 ml.
5. The immunogenic composition comprising rabbit staphylococcus antigens according to claim 1, wherein the composition is used for preparing a vaccine for preventing rabbit staphylococcus, and the vaccine is used for obtaining hyperimmune serum, so that effective serological differential diagnosis reagents are provided for bacteriological detection.
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