CN108114279A - A kind of preparation method of the P. aeruginosa bacteria vaccine assisted by rod-like nano aluminium hydroxide - Google Patents
A kind of preparation method of the P. aeruginosa bacteria vaccine assisted by rod-like nano aluminium hydroxide Download PDFInfo
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- CN108114279A CN108114279A CN201810184115.9A CN201810184115A CN108114279A CN 108114279 A CN108114279 A CN 108114279A CN 201810184115 A CN201810184115 A CN 201810184115A CN 108114279 A CN108114279 A CN 108114279A
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- 229960005486 vaccine Drugs 0.000 title claims abstract description 60
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 title claims abstract description 44
- 229910021502 aluminium hydroxide Inorganic materials 0.000 title claims abstract description 40
- 241000894006 Bacteria Species 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 38
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 22
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 20
- 229960002233 benzalkonium bromide Drugs 0.000 claims abstract description 19
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 claims abstract description 19
- 235000011187 glycerol Nutrition 0.000 claims abstract description 19
- 239000004530 micro-emulsion Substances 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims abstract description 7
- 230000002441 reversible effect Effects 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
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- 238000005119 centrifugation Methods 0.000 claims abstract description 4
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- 239000002671 adjuvant Substances 0.000 claims description 23
- 238000005457 optimization Methods 0.000 claims description 22
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 11
- 229910052782 aluminium Inorganic materials 0.000 claims description 11
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- 238000013019 agitation Methods 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- NWFNSTOSIVLCJA-UHFFFAOYSA-L copper;diacetate;hydrate Chemical compound O.[Cu+2].CC([O-])=O.CC([O-])=O NWFNSTOSIVLCJA-UHFFFAOYSA-L 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 4
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium chloride Substances Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 4
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- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 description 1
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Classifications
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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
- A61K39/39—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
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
-
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of preparation method of the P. aeruginosa bacteria vaccine assisted by rod-like nano aluminium hydroxide, this method prepares rod-like nano aluminium hydroxide first with co-continuous reverse microemulsion process：Suitable AlCl is separately added into after the identical benzalkonium bromide of two parts of ratios, glycerine, hexamethylene are mixed in a certain ratio3With sodium hydroxide or ammonia spirit, then two single_phase systems are mixed into co-continuous Reverse Microemulsion System, keep reaction system pH>7 reaction 2.5h, then plus absolute ethyl alcohol demulsification centrifuges, and three times, the precipitation after centrifugation is suspended in sterile water for sediment centrifuge washing, up to rod-like nano aluminium hydroxide.Then pseudomonas aeruginosa is mixed into the P. aeruginosa bacteria vaccine assisted to get rod-like nano aluminium hydroxide in equal volume with rod-like nano aluminium hydroxide.P. aeruginosa bacteria vaccine produced by the present invention has many advantages, such as good dispersion, free from foreign meter and device therefor, simple for process, is a kind of preparation method with good development prospect.
Description
Technical field
It is false in verdigris the present invention relates to a kind of preparation method of rod-like nano aluminium hydroxide and the rod-like nano aluminium hydroxide
Application on monad vaccine, belongs to biological technical field.
Background technology
Pseudomonas aeruginosa once claims Pseudomonas aeruginosa, is a kind of no pod membrane, obligate without gemma, movable Gram-negative
Aerobic dialister bacterium, thalline size grows (1.5-5.0) um × wide (0.5-1) um, elongated and different in size, sometimes in club-shaped or line
Shape, paired or short catenation are widely present in soil, water, dirt, plant and animal body, are often colonized on organism and exhale
Road, the urinary tract are inhaled, alimentary canal and skin etc. are the important opportunists of immunosuppressed individuals, can cause mink, fox
The many animals acute infection such as leopard cat, chicken, sheep.Pseudomonas aeruginosa because with natural resistance and outstanding adaptability,
It can obtain drug resistance by number of mechanisms, therefore its effectively treatment and control the problem of being still a long-term existence.
For a long time, vaccine as a kind of alternative strategy with prevent Susceptible population infection pseudomonas aeruginosa receive pass always
Note.The transport system research of numerous pseudomonas aeruginosa candidate vaccine antigens and antigen has been subjected to laboratory research detection,
Carry out I-III clinical trial phase assessments.Although this has extensively and profound significance to developing P. aeruginosa bacteria vaccine, so far
The vaccine there are no effective confrontation charrin disease appears on the market in the market.
At present, the research of related pseudomonas aeruginosa antigens depends on its pathogenesis and the relevant virulence of pathogen
The factor, this has also helped people to recognize and excavate for the potential immunogene of P. aeruginosa bacteria vaccine, such as flagellum, pili, outer
Memebrane protein, lipopolysaccharides or thalline secretory product, such as muciform exocellular polysaccharide, exotoxin A and protease etc..Since verdigris is false single
The antigen composition of born of the same parents bacterium is complicated and associated serum type is more, and the pathogenic bacteria serotype of different regions may be different, vaccine serotype
Whether the immunoprophylaxis effect for directly influencing vaccine consistent with the popular serotype of the disease.Therefore, timely and accurately understand and
Infected regions pathogenic bacteria serotype is grasped, there is important reference value to the selection of vaccine.It only cannot be more by a kind of vaccine
A area obtains preferable immunoprophylaxis effect simultaneously, it is necessary to develop the different polyvaccines for different regions prevalence serotype
Good immunoprophylaxis effect could be obtained.
At this stage, the vaccine of pseudomonas aeruginosa research mainly includes：Inactivated vaccine, attenuated live vaccine, DNA vaccination, knot
Close vaccine etc..1) inactivated vaccine：Also known as killed vaccine is made by being inactivated after mass propgation pathogen.The applications such as Cripps
Formalin prepares the P. aeruginosa bacteria vaccine of inactivation, and by oral, the approach difference immune rat such as hypodermic injection tests number
According to showing pseudomonas aeruginosa clearance rate control group significantly lower than active immunity group, and the survival of rats rate of active set is also
It improves, and certain protective effect is equally played in acute Haemophilus influenzae infection.2) attenuated live vaccine (live-
Attenuated vaccine) by after attenuation treatment or avirulent live pathogen itself is made.The similary attenuation such as Zaidi
Live vaccine nasal cavity immunity mouse, final immunization carry out challenge test after 4 weeks, have not found illness symptom appearance, have tieed up for a long time
Hold effective protection.In test, after injection of attenuated live vaccine, respectively using the P. aeruginosa of different serotype and virulence
The eye conjunctiva that bacterium infection scratches, similary injection of attenuated live vaccine, protective rate are notable after infection.3) DNA vaccination (DNA vaccine)
It is that will encode the recombinant eukaryon expression vector of certain proteantigen, is injected by the approach such as injecting in body, in foreign gene
It in vivo expresses, inducing producing specificity is immunized.Price etc. recombinates OprF into eukaryon expression plasmid pVR1020, utilizes
Bacillus coli expression successfully builds pVR1020-Opr F gene vaccines.ICR mouse are immunized in 1d, 7d, 14d respectively,
Virus monitory is carried out to mouse in 28d, the results show has antibody to generate simultaneously induction body fluid immune response；It is protected to chronic infection
In power experiment, the results show protection is not high.4) combined vaccine (conjugate vaccine) is by polysaccharide antigen and egg
Bai Zaiti is combined and is made.Lu Miaoquan etc. successfully prepares MEP-OMPC combined vaccines, and inducible generate largely resists after mouse is immunized
The antibody of MEP；Challenge test shows that 85% mouse can survive.Although the research of related P. aeruginosa bacteria vaccine
Compare more, but throw away that there are some insurmountable problems.
Aluminium adjuvant mainly has three kinds of aluminium hydroxide, aluminum phosphate and alum, and usually said aluminium adjuvant refers to aluminium hydroxide
Adjuvant.Aluminium hydroxide is amphoteric compound, and isoelectric point pI=11.4 exists with cationic form in the solution of pH=7.4, is
The good adsorption agent of anion antigen.Aluminum hydroxide adjuvant exists in the form of fibrous particle, average-size size for 4.5 ×
2.2 × 10nm exists with loose form after this particle buildup.
There is also some adverse reactions for aluminium adjuvant.Such as：Can only weaker or moderate enhancement antigen specific immune response, and
It is considered being unable to inducing cellular immune response, is not suitable for for virus, intracellular bacteria and the vaccine antigen of parasite, induction
The allergy and injection site of IgE mediations have inflammatory reaction, erythema, subcutaneous nodule, granuloma etc. occur.
Nano-particle refers to the particle that polymer of the diameter generally between 1-1000nm is formed, it has unique small ruler
Very little effect and interfacial effect.And adjuvant is prepared into Nano grade, it will assign adjuvant new characteristic.Why nano-class aluminum adjuvant has
There is the physicochemical characteristics that effect more better than ordinary adjuvants is not only in that nano-particle itself uniqueness, include size, shape, table
Face chemistry and roughness etc., and nano-class aluminum adjuvant can protect vaccine to avoid the generation of carrier effect extend vaccine in vivo
Existence time or macrophage, the preferred phagocytosis target of Dendritic Cells.
Since the antigen composition of pseudomonas aeruginosa is complicated and associated serum type is more, therefore whole vaccine is selected as antigen
Ingredient.But the fact that in view of the small-size effect of nano-class aluminum adjuvant and larger vaccine particle, we are micro- with co-continuous reverse phase
Newborn method is prepared for rod-like nano aluminium hydroxide, makes a diameter of Nano grade but length is other in the micron-scale, be provided simultaneously with nanometer
Advantage and adsorbable macromolecular vaccine.Pseudomonas aeruginosa vaccine is adsorbed with it, and immunoprophylaxis and immunological safety are carried out to mouse
Can experiment, observation rod-like nano aluminium hydroxide enhance the vaccine-induced Mice Body inner cell immune response of pseudomonas aeruginosa and body
Liquid immune response.Its possible mechanism of action and toxic side effect are studied simultaneously, to be provided reliably for later clinical practice
Experimental basis.
The content of the invention
It is an object of the invention to provide a kind of systems of the P. aeruginosa bacteria vaccine assisted by rod-like nano aluminium hydroxide
Preparation Method.
Technical solution：The preparation method for the P. aeruginosa bacteria vaccine that the rod-like nano aluminium hydroxide of the present invention assists, bag
Include following step：
1) rod-like nano aluminium hydroxide is prepared：
1. aluminium salt microemulsion system：By public's knowledge, benzalkonium bromide, glycerine, hexamethylene are taken in beaker, and makes the emulsification of addition
Agent, that is, benzalkonium bromide and glycerine, configuration is than being 1:3、2:5、1:2、2:3、1:1、3:2、2:1、5:2、3:1, pass through magnetic agitation
1000rpm/min adds ultrasonic 100w that it is made to be uniformly dispersed.Using syringe pump by AlCl3It is instilled with the speed of 6mL/h in beaker, instead
Temperature is answered as 30-60 DEG C, optimization temperature is 50 DEG C, and speed of agitator 500-1500rpm/min, the rotating speed of optimization is 600rpm/
Min reacts 30min；
2. sodium hydroxide or ammonia microemulsion system：By public's knowledge, benzalkonium bromide, glycerine, hexamethylene are taken in beaker, and is made
Emulsifier, that is, the benzalkonium bromide and glycerine of addition, configuration is than being 1:3、2:5、1:2、2:3、1:1、3:2、2:1、5:2、3:1, pass through
Magnetic agitation 1000rpm/min adds ultrasonic 100w that it is made to be uniformly dispersed.Using syringe pump by sodium hydroxide or ammonium hydroxide with 6mL/h's
Speed is instilled in beaker, and reaction temperature is 30-50 DEG C, and optimization temperature is 50 DEG C, and speed of agitator 500-1500rpm/min is excellent
The rotating speed of change is 600rpm/min, reacts 30min；
3. by above-mentioned 1. middle mixed solution in beaker, wherein benzalkonium bromide and qualities of glycerin ratio are 3:2, it is positioned over constant temperature magnetic force
Heating stirring on blender, and by above-mentioned 2. middle system, (benzalkonium bromide is 3 with qualities of glycerin ratio with syringe pump:2) with 5-30mL/
1. the speed of h instills in, wherein optimal speed is 20mL/h, and keeping system pH, optimization pH value is 9 in 8-11 scopes；Reaction
Temperature is 30-60 DEG C, and optimization temperature is 50 DEG C；Speed of agitator is 500-1500rpm/min, optimization rotating speed 600rpm/min；Instead
It is 2.0-5.0h between seasonable, the optimization time is 2.5h, and the absolute ethyl alcohol for adding in 2 times of volumes afterwards is demulsified, and with 95% anhydrous second
The purifying of alcohol ultrasound, 10000rpm/min centrifugation 10min, are suspended in sterile water after repeating 3 times to get rod-like nano hydroxide
Aluminium.
2) the P. aeruginosa bacteria vaccine that rod-like nano aluminium hydroxide assists is prepared：
1. the rod-like nano aluminium hydroxide prepared is configured to 1mg/mL, ultrasonic disperse 30min, it is spare in 4 DEG C of refrigerators；
2. will take the logarithm the phase after pseudomonas aeruginosa vaccine re-activation, and with 0.4% paraformaldehyde processing 48h after, PBS/
Physiological saline cleans 2 times, and adjustment bacterial cell concentration is resuspended as 10 using PBS9Cells/mL is for use；
3. by the vaccine prepared according to volume ratio 1：1 ratio mixing self-control rod-like nano aluminium hydroxide, using eddy mixer,
With the speed of 5000rpm/min by its vortex oscillation 30min, the verdigris that abundant mixing assists to get rod-like nano aluminium hydroxide
Pseudomonad vaccine.
Compared with prior art, the present invention the difference is that：
①Al(OH)3The preparation method of adjuvant is different from traditional Monophase microemulsion system and prepares, but first by AlCl3And hydrogen-oxygen
Change sodium or ammonium hydroxide is each configured to Monophase microemulsion system, two Monophase microemulsion systems are mixed again obtain two-phase micro emulsion afterwards
Liquid is tied up to species nucleate rule during being slowly mixed together by two-phase Emulsions and obtains rod-like nano aluminium hydroxide.
2. preparing rod-like nano aluminum hydroxide adjuvant using co-continuous reverse microemulsion method, the rod-like nano adjuvant grain size of preparation is equal
Even, average diameter is in 50-300nm, and length is in 0.8-3um.(see attached drawing 1)
3. the rod-like nano aluminum hydroxide adjuvant for reacting acquisition is removed suitable for virus or viroid ingredient (albumen, DNA, RNA) institute
The substance of preparation is also particularly suitable the macromolecular substances such as vaccine.The attached nanometer adjuvant of back suction when vaccine particle is larger is avoided,
Influence the immune effect of vaccine to the greatest extent.
Beneficial effects of the present invention are：Compared with traditional handicraft, the present invention obtains adjuvant as rod-like nano size and system
It is simple and effective to make method, production cost is controllable；Al (OH) in the present invention3Adjuvant is the reaction generation of co-continuous reverse micro emulsion,
Rodlike specification can effectively load macromolecular class antigen between nanometer and micron-scale, so as to improve the immunogene of antigen
Property, the ELISA method detection result of vaccine is also significantly better than common aluminium adjuvant and Freund's adjuvant group, more effectively body can be caused to produce
Raw immune response, generates more protection antibodies and cell factor.Show that the method obtains rod-like nano aluminium hydroxide and assists
P. aeruginosa bacteria vaccine have inoculum concentration it is small, adverse reaction is few, it is efficient the characteristics of.
Description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of the rod-like nano aluminium hydroxide prepared in example 1.
Fig. 2 is the X-ray diffractogram of the rod-like nano aluminium hydroxide prepared in example 1.
Fig. 3 is the atomic force microscopy diagram of the rod-like nano aluminium hydroxide prepared in example 1.
Specific embodiment
The present invention program is further elaborated with below by embodiment.
Embodiment 1：The preparation method of rod-like nano aluminum hydroxide adjuvant according to the present invention：
1. aluminium salt microemulsion system：By public's knowledge, benzalkonium bromide, glycerine, hexamethylene are taken in beaker, and makes the emulsification of addition
Agent, that is, benzalkonium bromide and glycerine, configuration is than being 1:3、2:5、1:2、2:3、1:1、3:2、2:1、5:2、3:1, pass through magnetic agitation
1000rpm/min adds ultrasonic 100w that it is made to be uniformly dispersed.Using syringe pump by AlCl3It is instilled with the speed of 6mL/h in beaker, instead
Temperature is answered as 30-60 DEG C, optimization temperature is 50 DEG C, and speed of agitator 500-1500rpm/min, the rotating speed of optimization is 600rpm/
Min reacts 30min；
2. sodium hydroxide or ammonia microemulsion system：By public's knowledge, benzalkonium bromide, glycerine, hexamethylene are taken in beaker, and is made
Emulsifier, that is, the benzalkonium bromide and glycerine of addition, configuration is than being 1:3、2:5、1:2、2:3、1:1、3:2、2:1、5:2、3:1, pass through
Magnetic agitation 1000rpm/min adds ultrasonic 100w that it is made to be uniformly dispersed.Using syringe pump by sodium hydroxide or ammonium hydroxide with 6mL/h's
Speed is instilled in beaker, and reaction temperature is 30-50 DEG C, and optimization temperature is 50 DEG C, and speed of agitator 500-1500rpm/min is excellent
The rotating speed of change is 600rpm/min, reacts 30min；
3. by above-mentioned 1. middle mixed solution in beaker, wherein benzalkonium bromide and qualities of glycerin ratio are 3:2, it is positioned over constant temperature magnetic force
Heating stirring on blender, and by above-mentioned 2. middle system, (benzalkonium bromide is 3 with qualities of glycerin ratio with syringe pump:2) with 5-30mL/
1. the speed that h, wherein optimal speed are 20mL/h instills in, keeping system pH, optimization pH value is 9 in 8-11 scopes；Reaction
Temperature is 30-60 DEG C, and optimization temperature is 50 DEG C；Speed of agitator is 500-1500rpm/min, optimization rotating speed 600rpm/min；Instead
It is 2.0-5.0h between seasonable, the optimization time is 2.5h, and the absolute ethyl alcohol for adding in 2 times of volumes afterwards is demulsified, and with 95% anhydrous second
The purifying of alcohol ultrasound, 10000rpm/min centrifugation 10min, are suspended in sterile water after repeating 3 times to get rod-like nano hydroxide
Aluminium.
Embodiment 2：The preparation method for the P. aeruginosa bacteria vaccine that rod-like nano aluminium hydroxide according to the present invention assists：
1. the rod-like nano aluminium hydroxide prepared is configured to 1mg/mL, ultrasonic disperse 30min, it is spare in 4 DEG C of refrigerators；
2. will take the logarithm the phase after pseudomonas aeruginosa vaccine re-activation, and with 0.4% paraformaldehyde processing 48h after, PBS/
Physiological saline cleans 2 times, and adjustment bacterial cell concentration is resuspended as 10 using PBS9Cells/mL is for use；
3. by the vaccine prepared according to volume ratio 1：1 ratio mixing self-control rod-like nano aluminium hydroxide, using eddy mixer,
With the speed of 5000rpm/min by its vortex oscillation 30min, the verdigris that abundant mixing assists to get rod-like nano aluminium hydroxide
Pseudomonad vaccine.
Embodiment 3:Charrin disease mouse virulence test
By pseudomonas aeruginosa switching in liquid LB cultures, when 37 DEG C of cultures 12 are small, adjustment bacterial concentration for 5.0 ×
1010CFU/mL.Afterwards, sterile PBS doubling dilutions are respectively into four concentration titres：5.0×1010CFU/mL、5.0×
109CFU/mL、5.0×108CFU/mL and 5.0 × 107CFU/mL.Each concentration gradient is inoculated with 10 BALB/c mouse (injection volumes
For 0.1mL).Mouse observes 30d, records every group of death condition.
Embodiment 4：Pseudomonas aeruginosa thalline inactivated vaccine animal immune
Experimental animal：50 6-8 week old female BAl BIc/c mouse are randomly divided into 5 groups, including 4 experimental groups and 1 control group, often
10 mouse of group.Immunizing antigen total volume is 300uL, and leg muscle injection is immunized three times respectively in 0d, 14d, 28d.
Experimental group 1：100uL pseudomonas aeruginosa somatic antigen vaccines are immunized in every mouse hind leg muscle；
Experimental group 2：100uL pseudomonas aeruginosas somatic antigen+rod-like nano aluminium hydroxide assistant is immunized in every mouse hind leg muscle
Vaccinating agent；
Experimental group 3：100uL pseudomonas aeruginosas somatic antigen+conventional aluminium Adjuvanted vaccines are immunized in every mouse hind leg muscle；
Experimental group 4：100uL pseudomonas aeruginosas somatic antigen+Freund's adjuvant vaccine is immunized in every mouse hind leg muscle；
Control group 1：100uL physiological saline is immunized in every mouse hind leg muscle.
Immune grouping and antigen dose specifically see the table below：
Embodiment 5：Antibody titer measures and histoorgan is examined
1) head exempt from after 7d, 14d, 21d, 28d, respectively to each test group of animals carry out tail vein blood, 8000rpm from
Heart 10min collects mice serum, and aseptic subpackaged to be stored in -20 DEG C of refrigerators spare.
2) final immunization after 1 week, pluck eyeball and take blood, then cervical dislocation is given to put to death, and takes spleen tissue by each group mouse.Blood preparation in
37 DEG C stand 30min, 8000rpm/min 10min, collect serum, are sub-packed in -20 DEG C and save backup.
3) antibody titer in above-mentioned acquisition blood sample is measured with ELISA method.
4) increment effect of mouse boosting cell is observed in the culture of spleen and splenocyte that separation is taken out.
Embodiment 6:Histopathology detects
Solution takes the internal organ such as lung, liver, the kidney of Immunization group mouse, non-Immunization group mouse and normal health mouse, neutral with 10%
Formalin is fixed, specimens paraffin embedding slices, and H.E. dyeing, optical microphotograph Microscopic observation is simultaneously taken pictures.
Claims (6)
1. a kind of preparation method of the P. aeruginosa bacteria vaccine assisted by rod-like nano aluminium hydroxide, which is characterized in that described
Rod-like nano aluminium hydroxide a diameter of 50-300nm, length 0.8-3um.
2. rod-like nano aluminium hydroxide according to claim 1, which is characterized in that length is in the range of 0.8-1.5um
Adjuvant is especially suitable for P. aeruginosa bacteria vaccine.
3. preparation method according to claim 1, which is characterized in that being prepared by for the rod-like nano aluminium hydroxide is double
Continuous Reverse Microemulsion System structure.
4. preparation method according to claim 3, which is characterized in that the preparation specifically includes following steps：
1. aluminium salt microemulsion system：By public's knowledge, benzalkonium bromide, glycerine, hexamethylene are taken in beaker, and makes the emulsification of addition
Agent, that is, benzalkonium bromide and glycerine, configuration is than being 1:3、2:5、1:2、2:3、1:1、3:2、2:1、5:2、3:1, pass through magnetic agitation
1000rpm/min adds ultrasonic 100w that it is made to be uniformly dispersed, using syringe pump by AlCl3It is instilled with the speed of 6mL/h in beaker, instead
Temperature is answered as 30-60 DEG C, optimization temperature is 50 DEG C, speed of agitator 500-1500rpm/min, and optimization rotating speed is 600rpm/
Min reacts 30min；
2. sodium hydroxide or ammonia microemulsion system：Benzalkonium bromide, glycerine, hexamethylene are taken in beaker, and makes the emulsification of addition
Agent, that is, benzalkonium bromide and glycerine, configuration is than being 1:3、2:5、1:2、2:3、1:1、3:2、2:1、5:2、3:1, pass through magnetic agitation
1000rpm/min adds ultrasonic 100w that it is made to be uniformly dispersed, and is instilled sodium hydroxide or ammonium hydroxide with the speed of 6mL/h using syringe pump
In beaker, reaction temperature is 30-50 DEG C, and optimization temperature is 50 DEG C, speed of agitator 500-1500rpm/min, and optimization rotating speed is
600rpm/min reacts 30min；
3. by above-mentioned 1. middle mixed solution in beaker, wherein benzalkonium bromide and qualities of glycerin ratio are 3:2, it is positioned over constant temperature magnetic force
Heating stirring on blender, and by above-mentioned 2. middle system, (benzalkonium bromide is 3 with qualities of glycerin ratio with syringe pump:2) with 5-30mL/
1. the speed of h instills in, wherein optimal speed is 20mL/h, and keeping system pH, optimization pH value is 9 in 8-11 scopes；Reaction
Temperature is 30-60 DEG C, and optimization temperature is 50 DEG C；Speed of agitator is 500-1500rpm/min, optimization rotating speed 600rpm/min；Instead
It is 2.0-5.0h between seasonable, the optimization time is 2.5h, and the absolute ethyl alcohol for adding in 2 times of volumes afterwards is demulsified, and with 95% anhydrous second
The purifying of alcohol ultrasound, 10000rpm/min centrifugation 10min, are suspended in sterile water after repeating 3 times to get rod-like nano hydroxide
Aluminium.
5. preparation method according to claim 1, which is characterized in that the verdigris that the rod-like nano aluminium hydroxide assists is false
The preparation of unit cell bacteria vaccine includes：
1. the rod-like nano aluminium hydroxide prepared is configured to 1mg/mL, ultrasonic disperse 30min, it is spare in 4 DEG C of refrigerators；
2. will take the logarithm the phase after pseudomonas aeruginosa vaccine re-activation, and with 0.4% paraformaldehyde processing 48h after, PBS/
Physiological saline cleans 2 times, and adjustment bacterial cell concentration is resuspended as 10 using PBS9Cells/mL is for use；
3. by the vaccine prepared according to volume ratio 1：1 ratio mixing self-control rod-like nano aluminium hydroxide, using eddy mixer,
With the speed of 5000rpm/min by its vortex oscillation 30min, the verdigris that abundant mixing assists to get rod-like nano aluminium hydroxide
Pseudomonad vaccine.
6. a kind of P. aeruginosa bacteria vaccine assisted by rod-like nano aluminium hydroxide, it is characterised in that, which is according to power
Profit requires 4 or 5 the methods to be prepared.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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2018
- 2018-03-06 CN CN201810184115.9A patent/CN108114279B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| XU LI等: "Aluminum (oxy)hydroxide nanosticks synthesized in bicontinuous reverse microemulsion have potent vaccine adjuvant activity", 《ACS APPL MATER INTERFACES》 * |
| YINGLI CHEN等: "Aluminum (oxy) Hydroxide Nanorods Activate an Early Immune Response in Pseudomonas aeruginosa Vaccine", 《ACS APPL. MATER. INTERFACES》 * |
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