CN108114279B - Preparation method of pseudomonas aeruginosa vaccine assisted by rod-shaped nano aluminum hydroxide - Google Patents
Preparation method of pseudomonas aeruginosa vaccine assisted by rod-shaped nano aluminum hydroxide Download PDFInfo
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- CN108114279B CN108114279B CN201810184115.9A CN201810184115A CN108114279B CN 108114279 B CN108114279 B CN 108114279B CN 201810184115 A CN201810184115 A CN 201810184115A CN 108114279 B CN108114279 B CN 108114279B
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- 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
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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
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
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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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
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Abstract
The invention discloses a method for preparing pseudomonas aeruginosa vaccine assisted by rod-shaped nano aluminum hydroxide, which comprises the following steps of firstly preparing the rod-shaped nano aluminum hydroxide by a bicontinuous reverse microemulsion method: mixing benzalkonium bromide, glycerol and cyclohexane in the same proportion, and adding appropriate amount of AlCl3Mixing with sodium hydroxide or ammonia water solution, mixing the two single-phase systems to obtain bicontinuous reverse microemulsion system, and maintaining the pH of the reaction system>7, reacting for 2.5 hours, adding absolute ethyl alcohol, demulsifying and centrifuging, centrifuging and washing precipitates for three times, and suspending the centrifuged precipitates in sterile water to obtain the rod-shaped nano aluminum hydroxide. Then, the pseudomonas aeruginosa and the rod-shaped nano aluminum hydroxide are mixed in the same volume, and the pseudomonas aeruginosa vaccine assisted by the rod-shaped nano aluminum hydroxide is obtained. The pseudomonas aeruginosa vaccine prepared by the invention has the advantages of good dispersibility, no impurities, simple equipment and process and the like, and is a preparation method with good development prospect.
Description
Technical Field
The invention relates to a preparation method of rod-shaped nano aluminum hydroxide and application of the rod-shaped nano aluminum hydroxide in pseudomonas aeruginosa bacterins, belonging to the technical field of biology.
Background
Pseudomonas aeruginosa, named as Pseudomonas aeruginosa, is an acapsular, spore-free and movable gram-negative obligate aerobic bacillus, has long (1.5-5.0) um multiplied by wide (0.5-1) um in cell size, is slender and different in length, is sometimes arranged in a ball rod shape or a linear shape, is a pair or a short chain shape, is widely present in soil, water, dirt, plants and animals, is often planted in upper respiratory tract, urinary tract, digestive tract, skin and other places of organisms, is an important opportunistic pathogen of immunosuppression individuals, and can cause acute infection of various animals such as mink, fox, chicken, sheep and the like. Pseudomonas aeruginosa remains a long standing problem due to its natural resistance and extraordinary adaptability to acquire resistance through a variety of mechanisms.
Vaccines have long been of interest as an alternative strategy to prevent susceptible populations from becoming infected with pseudomonas aeruginosa. A number of P.aeruginosa candidate vaccine antigens and delivery systems for the antigens have been tested in laboratory studies and are being evaluated in phase I-III clinical trials. Despite the wide and profound significance of developing P.aeruginosa vaccines, no effective vaccine against P.aeruginosa infection has been marketed to date.
Currently, the research of related pseudomonas aeruginosa antigens mainly depends on the pathogenesis of the pseudomonas aeruginosa antigens and pathogen-related virulence factors, which also helps people to recognize and discover potential immunogens for pseudomonas aeruginosa vaccines, such as flagella, pili, outer membrane proteins, lipopolysaccharides or thalli secretion products, such as mucoid exopolysaccharides, exotoxin a, protease and the like. Because the antigen composition of the pseudomonas aeruginosa is complex and the related serotypes are more, the serotypes of pathogenic bacteria in different regions can be different, and whether the serotype of the vaccine is consistent with the serotype of the epidemic disease or not directly influences the immune prevention effect of the vaccine. Therefore, the serotype of pathogenic bacteria in the disease area can be known and mastered timely and accurately, and the method has important reference value for selecting the vaccine. Only one vaccine can not obtain ideal immunoprophylaxis effect in multiple regions, and different multivalent vaccines aiming at epidemic serotypes in different regions must be developed to obtain good immunoprophylaxis effect.
At the present stage, the vaccine for the pseudomonas aeruginosa study mainly comprises: inactivated vaccines, attenuated live vaccines, DNA vaccines, conjugate vaccines, and the like. 1) And (3) inactivated vaccine: also known as killed vaccines, are prepared by inactivation of a large amount of cultured pathogens. Cripps and the like use formalin to prepare inactivated pseudomonas aeruginosa vaccines, rats are respectively immunized by oral administration, subcutaneous injection and other ways, experimental data show that the clearance rate of pseudomonas aeruginosa in a control group is obviously lower than that of an active immunization group, the survival rate of the rats in the active group is also improved, and the inactivated pseudomonas aeruginosa vaccines also play a certain role in protection in haemophilus influenzae infection. 2) Live-attenuated vaccines (live-attenuated vaccines) are made from live pathogens that are attenuated or are avirulent themselves. Zaidi and the like use the same attenuated live vaccine to immunize mice through nasal cavities, and after 4 weeks of last immunization, the virus challenge test is carried out, and no diseased symptom is found, so that the effective protective power is maintained for a long time. In the test, after the attenuated live vaccine is injected, different serotypes and virulence of pseudomonas aeruginosa are respectively applied to infect scratched eye conjunctiva, and the attenuated live vaccine is also injected after infection, so that the protection rate is obvious. 3) A DNA vaccine (DNA vaccine) is prepared by injecting a recombinant eukaryotic expression vector encoding a certain protein antigen into a living body by injection or the like, expressing the recombinant eukaryotic expression vector in a living body by an exogenous gene, and inducing the recombinant eukaryotic expression vector to generate specific immunity. Price et al successfully constructed pVR1020-Opr F gene vaccine by recombining OprF into eukaryotic expression plasmid pVR1020 and expressing the same with Escherichia coli. The ICR mice are immunized at 1d, 7d and 14d respectively, and serum detection is carried out on the mice at 28d, so that the results show that antibodies are generated and humoral immune response is induced; in the test of protective power against chronic infection, the results show that the protective power is not high. 4) Conjugate vaccine (conjugate vaccine) is prepared by combining polysaccharide antigen and protein carrier. Lu 28156Chun and so on successfully prepare MEP-OMPC combined vaccine, and can induce a large amount of antibodies against MEP after immunizing mice; challenge experiments showed that 85% of mice survived. Although the research on the related pseudomonas aeruginosa vaccines is more, some problems which cannot be solved exist.
The aluminum adjuvant mainly comprises three kinds of aluminum hydroxide, aluminum phosphate and alum, and the aluminum adjuvant is commonly referred to as aluminum hydroxide adjuvant. Aluminum hydroxide is an amphoteric compound with an isoelectric point pI of 11.4, exists in a cationic form in a solution at pH 7.4, and is a good adsorbent for anionic antigens. The aluminium hydroxide adjuvant is present in the form of fibrous particles having an average size of 4.5X 2.2X 10nm, which particles, when aggregated, are present in a loose form.
Aluminum adjuvants also have some adverse effects. Such as: antigen-specific immune responses are only weakly or moderately enhanced and are not considered to induce cellular immune responses, are not suitable for vaccine antigens against viruses, intracellular bacteria and parasites, induce IgE-mediated allergic reactions and locally inflammatory reactions upon injection, with the appearance of erythema, subcutaneous nodules, granulomas, etc.
Nanoparticles refer to polymer-formed microparticles, typically between 1-1000nm in diameter, which have unique small size effects and interfacial effects. And the adjuvant is prepared into a nanometer level, so that the adjuvant has new characteristics. The nano aluminum adjuvant has better effect than the common adjuvant not only in the unique physicochemical characteristics of the nano particles, including size, shape, surface chemistry, roughness and the like, but also can avoid the occurrence of carrier effect, protect the vaccine, prolong the existing time of the vaccine in vivo, and be the first-choice phagocytic target of macrophage and dendritic cells.
Because of the complex antigen composition and the large number of related serotypes of pseudomonas aeruginosa, whole bacterins are selected as antigen components. However, in consideration of the small-size effect of the nano aluminum adjuvant and the fact that the bacterin particles are large, the rod-shaped nano aluminum hydroxide is prepared by using a bicontinuous reverse microemulsion method, so that the rod-shaped nano aluminum hydroxide has the diameter of nano grade but the length of micron grade, and simultaneously has the advantage of nano and can adsorb the macromolecular bacterins. The pseudomonas aeruginosa vaccine adsorbed by the aluminum hydroxide is used for carrying out immune prevention and immune safety experiments on mice, and whether the rod-shaped nano aluminum hydroxide can enhance the cellular immune response and the humoral immune response of the pseudomonas aeruginosa vaccine induced in the mice is observed. Meanwhile, possible action mechanism and toxic and side effect of the medicine are researched, so that reliable experimental basis is provided for future clinical application.
Disclosure of Invention
The invention aims to provide a preparation method of a pseudomonas aeruginosa vaccine assisted by rod-shaped nano aluminum hydroxide.
The technical scheme is as follows: the preparation method of the pseudomonas aeruginosa vaccine assisted by the rod-shaped nano aluminum hydroxide comprises the following steps:
1) preparing rod-like nano aluminum hydroxide:
① aluminum salt microemulsionThe method comprises the following steps: according to public knowledge, benzalkonium bromide, glycerol and cyclohexane are put into a beaker, added emulsifier namely benzalkonium bromide and glycerol are prepared according to the proportion of 1:3, 2:5, 1:2, 2:3, 1:1, 3:2, 2:1, 5:2 and 3:1, and are uniformly dispersed by magnetic stirring at 1000rpm/min and 100w of ultrasound. Using a syringe pump to mix AlCl3Dripping the mixture into a beaker at the speed of 6mL/h, wherein the reaction temperature is 30-60 ℃, the optimized temperature is 50 ℃, the stirring speed is 500-1500rpm/min, the optimized speed is 600rpm/min, and the reaction time is 30 min;
sodium hydroxide or ammonia water microemulsion system: according to public knowledge, benzalkonium bromide, glycerol and cyclohexane are put into a beaker, added emulsifier namely benzalkonium bromide and glycerol are prepared according to the proportion of 1:3, 2:5, 1:2, 2:3, 1:1, 3:2, 2:1, 5:2 and 3:1, and are uniformly dispersed by magnetic stirring at 1000rpm/min and 100w of ultrasound. Dropping sodium hydroxide or ammonia water into a beaker at the speed of 6mL/h by using a syringe pump, wherein the reaction temperature is 30-50 ℃, the optimized temperature is 50 ℃, the stirring speed is 500-1500rpm/min, the optimized speed is 600rpm/min, and the reaction time is 30 min;
thirdly, placing the mixed solution in the first step into a beaker, wherein the mass ratio of benzalkonium bromide to glycerol is 3:2, heating and stirring the mixed solution on a constant-temperature magnetic stirrer, and dripping the system in the second step (the mass ratio of benzalkonium bromide to glycerol is 3:2) into the first step at the speed of 5-30mL/h by using an injection pump, wherein the optimized speed is 20mL/h, the pH value of the system is kept in the range of 8-11, and the optimized pH value is 9; the reaction temperature is 30-60 ℃, and the optimized temperature is 50 ℃; the stirring speed is 500-1500rpm/min, and the optimized speed is 600 rpm/min; the reaction time is 2.0-5.0h, the optimization time is 2.5h, then 2 times of volume of absolute ethyl alcohol is added for demulsification, and the mixture is ultrasonically purified by 95 percent of absolute ethyl alcohol, centrifuged at 10000rpm/min for 10min, and is suspended in sterile water after repeated 3 times, thus obtaining the rod-shaped nano aluminum hydroxide.
2) Preparing a rod-shaped nano aluminum hydroxide-assisted pseudomonas aeruginosa vaccine:
preparing the prepared rod-like nano aluminum hydroxide into 1mg/mL, ultrasonically dispersing for 30min, and keeping the rod-like nano aluminum hydroxide in a refrigerator at 4 ℃ for later use;
② activating the vaccine of Pseudomonas aeruginosa twice, taking log phase, and treating with 0.4% paraformaldehyde for 48 hrPBS/saline wash 2 times, resuspension with PBS to adjust bacterial cell concentration to 109cells/mL for standby;
thirdly, mixing the prepared bacterins according to the volume ratio of 1:1, self-prepared rod-shaped nano aluminum hydroxide is mixed according to a proportion, and is subjected to vortex oscillation for 30min at the speed of 5000rpm/min by using a vortex mixer, and the mixture is fully and uniformly mixed, so that the pseudomonas aeruginosa vaccine assisted by the rod-shaped nano aluminum hydroxide is obtained.
Compared with the prior art, the invention has the following differences:
①Al(OH)3the preparation method of the adjuvant is different from the traditional preparation method of a single-phase microemulsion system, and AlCl is firstly prepared3And sodium hydroxide or ammonia water are respectively prepared into a single-phase microemulsion system, then the two single-phase microemulsion systems are mixed to obtain a two-phase microemulsion, and the rodlike nano aluminum hydroxide is obtained according to the substance nucleation rule in the slow mixing process of the two-phase microemulsion system.
② the rodlike nano aluminum hydroxide adjuvant is prepared by adopting a bicontinuous reverse microemulsion method, the prepared rodlike nano adjuvant has uniform particle diameter, the average diameter is 50-300nm, and the length is 0.8-3 um. (see attached FIG. 1)
③ the rod-shaped nano aluminum hydroxide adjuvant obtained by the reaction is not only applicable to the substances prepared by virus or viroid components (protein, DNA, RNA), but also particularly applicable to macromolecular substances such as bacterins and the like. The reverse adsorption of the nano adjuvant due to the larger vaccine particles is avoided, and the immune effect of the vaccine is influenced to the greatest extent.
The invention has the beneficial effects that: compared with the traditional process, the adjuvant obtained by the invention is rod-shaped and nano-sized, the preparation method is simple and effective, and the production cost is controllable; al (OH) in the present invention3The adjuvant is generated by a bicontinuous reverse microemulsion reaction, the rod-shaped specification is between the nanometer size and the micrometer size, and the macromolecular antigen can be effectively loaded, so that the immunogenicity of the antigen is improved, the detection result of the ELISA method of the vaccine is also obviously superior to that of the common aluminum adjuvant and Freund adjuvant group, the organism can be more effectively caused to generate immunoreaction, and more protective antibodies and cytokines can be generated. The method shows that the rod-shaped nano aluminum hydroxide adjuvanted pseudomonas aeruginosa vaccine obtained by the method has the advantages of small inoculation amount, less adverse reaction and high efficiencyHigh effect and high safety.
Drawings
FIG. 1 is a scanning electron microscope image of the rod-shaped nano aluminum hydroxide prepared in example 1.
FIG. 2 is an X-ray diffraction pattern of the rod-like nano aluminum hydroxide prepared in example 1.
FIG. 3 is an atomic force microscope photograph of the nano aluminum hydroxide in rod form prepared in example 1.
Detailed Description
The scheme of the present invention is further specifically illustrated by the following examples.
Example 1: the invention relates to a preparation method of a rod-shaped nano aluminum hydroxide adjuvant, which comprises the following steps:
① aluminum salt microemulsion system is prepared by adding benzalkonium bromide, glycerol, and cyclohexane into a beaker according to public knowledge, adding benzalkonium bromide and glycerol as emulsifier at a ratio of 1:3, 2:5, 1:2, 2:3, 1:1, 3:2, 2:1, 5:2, and 3:1, magnetically stirring at 1000rpm/min, adding 100w ultrasound, and dispersing AlCl with injection pump3Dripping the mixture into a beaker at the speed of 6mL/h, wherein the reaction temperature is 30-60 ℃, the optimized temperature is 50 ℃, the stirring speed is 500-1500rpm/min, the optimized speed is 600rpm/min, and the reaction time is 30 min;
sodium hydroxide or ammonia water microemulsion system: according to public knowledge, benzalkonium bromide, glycerol and cyclohexane are put into a beaker, added emulsifier namely benzalkonium bromide and glycerol are prepared according to the proportion of 1:3, 2:5, 1:2, 2:3, 1:1, 3:2, 2:1, 5:2 and 3:1, and are uniformly dispersed by magnetic stirring at 1000rpm/min and 100w of ultrasound. Dropping sodium hydroxide or ammonia water into a beaker at the speed of 6mL/h by using a syringe pump, wherein the reaction temperature is 30-50 ℃, the optimized temperature is 50 ℃, the stirring speed is 500-1500rpm/min, the optimized speed is 600rpm/min, and the reaction time is 30 min;
thirdly, placing the mixed solution in the first step into a beaker, wherein the mass ratio of benzalkonium bromide to glycerol is 3:2, heating and stirring the mixed solution on a constant-temperature magnetic stirrer, and dripping the mixed solution into the first step at a speed of 5-30mL/h, wherein the optimized speed is 20mL/h, keeping the pH value of the system within the range of 8-11, and optimizing the pH value to be 9 by using an injection pump; the reaction temperature is 30-60 ℃, and the optimized temperature is 50 ℃; the stirring speed is 500-1500rpm/min, and the optimized speed is 600 rpm/min; the reaction time is 2.0-5.0h, the optimization time is 2.5h, then 2 times of volume of absolute ethyl alcohol is added for demulsification, and the mixture is ultrasonically purified by 95 percent of absolute ethyl alcohol, centrifuged at 10000rpm/min for 10min, and is suspended in sterile water after repeated 3 times, thus obtaining the rod-shaped nano aluminum hydroxide.
Example 2: the invention relates to a preparation method of a pseudomonas aeruginosa vaccine assisted by rod-shaped nano aluminum hydroxide, which comprises the following steps:
preparing the prepared rod-like nano aluminum hydroxide into 1mg/mL, ultrasonically dispersing for 30min, and keeping the rod-like nano aluminum hydroxide in a refrigerator at 4 ℃ for later use;
② activating P.aeruginosa vaccine twice, collecting log phase, treating with 0.4% paraformaldehyde for 48 hr, washing with PBS/normal saline for 2 times, and resuspending with PBS to adjust bacterial cell concentration to 109cells/mL for standby;
thirdly, mixing the prepared bacterins according to the volume ratio of 1:1, self-prepared rod-shaped nano aluminum hydroxide is mixed according to a proportion, and is subjected to vortex oscillation for 30min at the speed of 5000rpm/min by using a vortex mixer, and the mixture is fully and uniformly mixed, so that the pseudomonas aeruginosa vaccine assisted by the rod-shaped nano aluminum hydroxide is obtained.
Example 3 Pseudomonas aeruginosa infection in mice toxicity test
Inoculating Pseudomonas aeruginosa to liquid LB culture, culturing at 37 deg.C for 12 hr, and adjusting the concentration of bacterial liquid to 5.0 × 1010After CFU/mL, sterile PBS was diluted in multiples to four concentration titers, 5.0 × 10, respectively10CFU/mL、5.0×109CFU/mL、5.0×108CFU/mL and 5.0 × 107CFU/mL. 10 BALB/c mice (injected volume 0.1mL) were inoculated per concentration gradient. Mice were observed for 30d and mortality was recorded for each group.
Example 4: animal immunization of pseudomonas aeruginosa thallus inactivated vaccine
Experimental animals: 50 female BALB/c mice, 6-8 weeks old, were randomly divided into 5 groups, including 4 experimental groups and 1 control group, each group of 10 mice. The total volume of the immune antigen is 300uL, and the immune antigen is injected in the muscle of the leg and is immunized three times at 0d, 14d and 28d respectively.
Experimental group 1: immunizing 100uL pseudomonas aeruginosa thallus antigen vaccine by the hind leg muscle of each mouse;
experimental group 2: immunizing 100uL of pseudomonas aeruginosa bacterial antigen and rod-shaped nano aluminum hydroxide adjuvant vaccine by the hind leg muscle of each mouse;
experimental group 3: immunizing 100uL of pseudomonas aeruginosa bacterial antigen and common aluminum adjuvant vaccine by the hind leg muscle of each mouse;
experimental group 4: immunizing 100uL pseudomonas aeruginosa bacterial antigen + Freund's adjuvant vaccine by the hind leg muscle of each mouse;
control group 1: each mouse was immunized with 100uL of physiological saline in the hind leg muscle.
The immune groupings and antigen doses are specified in the following table:
example 5: antibody titer determination and tissue and organ testing
1) Respectively collecting tail vein blood of animals in each experimental group at 7d, 14d, 21d and 28d after first immunization, centrifuging at 8000rpm for 10min, collecting mouse serum, aseptically subpackaging and storing in a refrigerator at-20 deg.C for use.
2) After 1 week of the last immunization, each group of mice was bled from the eyeball, sacrificed by cervical dislocation, and spleen tissue was removed. Standing the blood sample at 37 deg.C for 30min, 8000rpm/min for 10min, collecting serum, and subpackaging at-20 deg.C for use.
3) Antibody titers were determined in the blood samples collected above by ELISA.
4) Isolated spleen and spleen cells were cultured and the proliferation effect of mouse spleen cells was observed.
Example 6 histopathological examination
The internal organs of the lungs, the livers, the kidneys and the like of the immune virus attacking mice, the non-immune virus attacking mice and the normal healthy mice are dissected and taken, fixed by 10 percent neutral formaldehyde solution, embedded in paraffin, stained by H.E., observed under an optical microscope and photographed.
Claims (6)
1. A preparation method of pseudomonas aeruginosa vaccine assisted by rod-shaped nano aluminum hydroxide is characterized in that the diameter of the rod-shaped nano aluminum hydroxide is 50-300nm, and the length of the rod-shaped nano aluminum hydroxide is 0.8-3 mu m;
the preparation method of the pseudomonas aeruginosa vaccine assisted by the rod-shaped nano aluminum hydroxide comprises the following steps:
(S1) preparation of rod-like nanometer aluminum hydroxide
① aluminum salt microemulsion system is prepared by placing benzalkonium bromide, glycerol, and cyclohexane in a beaker at a mass ratio of benzalkonium bromide to glycerol of 3:2, magnetically stirring at 1000rpm, adding ultrasound at 100w to disperse the benzalkonium bromide and glycerol uniformly, and injecting AlCl solution with a syringe pump3Dripping into a beaker at the speed of 6mL/h, reacting at the temperature of 30-60 ℃, and the stirring speed of 500-1500rpm for 30 min;
sodium hydroxide or ammonia water microemulsion system: putting benzalkonium bromide, glycerol and cyclohexane in a beaker, adding an emulsifier, namely benzalkonium bromide and glycerol, in a mass ratio of 3:2, uniformly dispersing the benzalkonium bromide and the glycerol by adding 100w of ultrasound at 1000rpm under magnetic stirring, and dripping sodium hydroxide or ammonia water into the beaker at the speed of 6mL/h by using a syringe pump, wherein the reaction temperature is 30-50 ℃, the stirring speed is 500-1500rpm, and the reaction time is 30 min;
placing the mixed solution in the step one in a beaker, wherein the mass ratio of benzalkonium bromide to glycerol is 3:2, heating and stirring the mixed solution on a constant-temperature magnetic stirrer, dripping the system in the step one at the speed of 5-30mL/h by using an injection pump, keeping the pH value of the system in the range of 8-11, reacting the system at the temperature of 30-60 ℃, stirring the system at the rotation speed of 500 plus materials at 1500rpm for 2.0-5.0h, adding 2 times of volume of absolute ethyl alcohol for demulsification, suspending the mixture in 95% absolute ethyl alcohol for ultrasonic purification, centrifuging the mixture at 10000rpm for 10min, repeating the steps for 3 times, and then obtaining rod-shaped nano aluminum hydroxide;
(S2) method for preparing pseudomonas aeruginosa vaccine by using rod-shaped nano aluminum hydroxide as adjuvant
Preparing the prepared rod-like nano aluminum hydroxide into 1mg/mL, ultrasonically dispersing for 30min, and keeping the rod-like nano aluminum hydroxide in a refrigerator at 4 ℃ for later use;
② activating P.aeruginosa vaccine twice, taking log phase, and treating with 0.4% paraformaldehydeAfter 48h, the cells were washed 2 times with PBS/physiological saline and resuspended in PBS to adjust the bacterial cell concentration to 109cells/mL for standby;
thirdly, mixing the prepared bacterins with self-made rod-shaped nano aluminum hydroxide according to the volume ratio of 1:1, performing vortex oscillation on the bacterins for 30min at the speed of 5000rpm by using a vortex mixer, and fully and uniformly mixing to obtain the rod-shaped nano aluminum hydroxide assisted pseudomonas aeruginosa vaccine.
2. The method according to claim 1, wherein the rod-like nano aluminum hydroxide has a length of 0.8 to 1.5 μm.
3. The preparation method according to claim 1, wherein the reaction temperature is 50 ℃ and the rotation speed is 600rpm when the aluminum salt microemulsion system is prepared.
4. The preparation method according to claim 1, wherein the reaction temperature is 50 ℃ and the rotation speed is 600rpm when the sodium hydroxide or ammonia water microemulsion system is prepared.
5. The preparation method according to claim 1, wherein when the sodium hydroxide or ammonia water microemulsion system is dripped into the aluminum salt microemulsion system, the dripping speed is 20mL/h, the pH value of the system is 9, the reaction temperature is 50 ℃, and the stirring speed is 600 rpm; the reaction time was 2.5 h.
6. Pseudomonas aeruginosa vaccine obtainable by the process according to any one of claims 1 to 5.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2169193C2 (en) * | 1999-08-12 | 2001-06-20 | Научно-исследовательский институт пушного звероводства и кролиководства им. В.А. Афанасьева | Strain of bacterium pseudomonas aeruginosa 5142 used for preparing vaccine against fur wild animals pseudomonosis |
| CN102101687A (en) * | 2009-12-16 | 2011-06-22 | 国家纳米科学中心 | Equipment for preparing aluminium hydroxide nanorod and preparation method thereof |
| CN104189898A (en) * | 2014-06-27 | 2014-12-10 | 四川大学 | Pseudomonas aeruginosa vaccine and preparation method thereof |
| CN106390111A (en) * | 2016-11-25 | 2017-02-15 | 于彦强 | Preparation method of mink hemorrhagic pneumonia inactivated vaccine and application thereof |
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Patent Citations (4)
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|---|---|---|---|---|
| RU2169193C2 (en) * | 1999-08-12 | 2001-06-20 | Научно-исследовательский институт пушного звероводства и кролиководства им. В.А. Афанасьева | Strain of bacterium pseudomonas aeruginosa 5142 used for preparing vaccine against fur wild animals pseudomonosis |
| CN102101687A (en) * | 2009-12-16 | 2011-06-22 | 国家纳米科学中心 | Equipment for preparing aluminium hydroxide nanorod and preparation method thereof |
| CN104189898A (en) * | 2014-06-27 | 2014-12-10 | 四川大学 | Pseudomonas aeruginosa vaccine and preparation method thereof |
| CN106390111A (en) * | 2016-11-25 | 2017-02-15 | 于彦强 | Preparation method of mink hemorrhagic pneumonia inactivated vaccine and application thereof |
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