CN105251002B - A kind of oil-in-water type nano-emulsion adjuvant and its MRSA nano emulsion adjuvant vaccine and preparation method - Google Patents
A kind of oil-in-water type nano-emulsion adjuvant and its MRSA nano emulsion adjuvant vaccine and preparation method Download PDFInfo
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Abstract
The invention discloses a kind of oil-in-water type nano-emulsion adjuvant and its MRSA nano emulsion adjuvant vaccine and preparation methods, by mass percentage include following component: surfactant 1-30%；Cosurfactant 0.1-15%；Oily phase 0.1-15%；Water 40%-98.8%；The adjuvant appearance clear liquid, for partial size in 1-100nm range, viscosity is low, belongs to height thermodynamic stable system, and high speed centrifugation is stablized not stratified；Also direct physical mixed can be wrapped up when with vaccine compatibility to use, it can be immune by administration routes such as intramuscular injection, collunariums.With at low cost, convenient drug administration, avoid cross-infection, small toxicity, good fluidity, it is small to body irritation the advantages that, to effectively improve the immune effect and stability of vaccine, have broad application prospects.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of oil-in-water type nano-emulsion adjuvant further relates to MRSA nanometers
The preparation method of newborn Adjuvanted vaccines and its nano emulsion adjuvant and vaccine.
Background technique
With the continuous popularization of vaccine product, application limitation caused by natural quality is also constantly exposed.Greatly
For most vaccine products under independent state, half-life period is all shorter, especially under the catalysis of temperature, half-life period be even more with
Temperature raising and straight line decline.Thus, most of vaccines such as MRSA vaccine requires a suitable preservation condition, with dimension
Its stability is held, wherein locating liquid environment more plays particularly critical effect to the maintenance of stability.And liquid environment
PH value, ionic strength and it is some other maintain stable ingredient (such as some carbohydrate molecules, amino acid composition, esters)
Addition may all influence vaccine product stable period, therefore, find a suitable vaccine adjuvant and its carrier to protect vaccine
Stability in production, storage, transportational process has particularly important meaning with popularization for the application of vaccine itself.Together
When, due to influence of the single effective component of vaccine during carrying out immune offer by various biological approaches, so that its
It cannot achieve expected immune effect, it is therefore desirable to some to enhance body to the immune response of vaccine antigen or change immune answer
The adjuvant of type is answered as auxiliary.And the function of adjuvant mainly has: recruiting immunocyte using position and immune molecule, enhancing are exempted from
Epidemic disease response；Enhance vaccine antigen transmitting；By changing the physical form of antigen, extend antigen retention time in body；Promote
Immune contact；Enhance the immunogenicity and immunological memory of poor antigen, such as highly purified antigen or recombinant antigen；Antigen is reduced to connect
Kind dosage and inoculation times；Promote immune effect of the vaccine in the weak group of immune response ability；Stimulate mononuclear phagocytic cells
Ability is offered to antigen；Lymphocyte differentiation is stimulated, increases and expands the mechanism such as immune response ability to enhance immune answer；Accelerate
The speed of immune response and extension duration；Change the configuration of antigen；Change type, IgG subclass and the antibody of humoral antibody
Compatibility and cellular immunity and mucosa-immune effect.Ideal adjuvant not only without immunogenicity and can enhance body
Immune response can more make body obtain optimal protective immunity and immunological memory.
FDA approval at present can the adjuvant of people only have that aluminium hydroxide is the aluminium adjuvant of representative, MF59, AS series of adjuvants are mesh
The preceding main adjuvant for being generally approved for human vaccination.Aluminium salt absorbed diphtheria toxoid is applied first extremely from nineteen twenty-six Glenny
The present is widely used to vaccine, there is two kinds of Alum adjuvants of aluminum phosphate and aluminium hydroxide.Document report shows that aluminum hydroxide adjuvant is made
There are many vaccine safety hidden danger in use for vaccine adjuvant: the stimulation of local allergic reaction, local organization is anti-
It answers, causes cutaneous lymphoid hyperplasia's disease, acute sarcoid shape disease, causes macrophage myofascitis and confirmed fatigue, nerve
System toxicity, Gulf War Syndrome correlation, self-closing disease correlation, sieve engler's disease correlation etc..With vaccine inoculation model
The continuous popularization enclosed, infant becomes the object of inoculation of many vaccines, and the development of infant's body system is not perfect, is bound to
Increase the sensibility of its side reaction to aluminum hydroxide adjuvant, especially there may be adverse effect to infant's cognitive development, thus
It is possible that breeding a tragedy.Aluminium adjuvant is unable to inducing cellular immune and mucosal immune response effect based on humoral immune response simultaneously
It answers, especially discovery in recent years, which is used for multiple times, immunosupress and progressive poisoning occurs and use limited and anti-to some vaccines
It is former invalid.Italy took the lead in ratifying the Infuenza subunit vaccine containing MF59 adjuvant in 1997 and is stood again up to listing；European Union in 2009
H1N1 vaccine listing of the approval containing AS03 (AS03 of vitamin E preparation is added in MontanideW/O oil-in-water adjuvant) adjuvant；
The Subclinical papillomavirus infection of adjuvant containing AS04 in 2009 (the AS04 adjuvant of aluminium hydroxide and the preparation of 3-O- deacylation Monophosphate Lipid A)
(HPV) vaccine lists in the U.S..Two kinds of O/W type Submicron Emulsion adjuvants of AF03 and W805EC are in clinical test at present.?
In the adjuvant for ratifying listing, MF59 and AS03 are oil-in-water type Submicron Emulsion, WO9014837, EP0399843A2,
The patents such as US6299884B1, US6451325B1, US20090191226A2 disclose prescription, preparation method and its use of MF59
On the way.The adjuvant contains 4.0%~5.0% squalene, 0.5% Tween 80,0.5% span 85, and average grain diameter is about 160nm.But
MF59 can only use filtration sterilization, if will lead to Submicron Emulsion layering, thus entire production process using terminal pressure sterilizing
Sterile working is required harsh.The patents such as US2007141078A1, US2010189741A1, US2010183667A1 disclose
Prescription, the preparation method and its usage of AS03.AS03 10.68mg containing squalene, the DL- alpha-tocopherol 11.86mg of single dosage,
Tween 80 4.85mg, average grain diameter are 150~155nm.During H1N1 is popular within 2009~2010 years, the multiple Nation Newspapers in Europe
It accuses vaccine recipient and nervous system adverse reaction occurs, be mainly shown as drowsiness and hypersomnia.According to Finnish National health with
A survey report that welfare research institute announces shows that 4~19 years old teenager is in the Hazard ratio for being vaccinated with vaccine future trouble hypnosia
Teenager with the non-vaccine inoculation of age group is higher by 9 times.Hypnosia is a kind of dyssomnias phenomenon, and patient can feel extremely tired
It is tired, it is drowsy, it often falls asleep in the case where having no early warning, even aprowl way is fallen asleep suddenly.Patent document
US2007014805A1, US2007191314A1, EP2080522A1 disclose prescription of AF03 and preparation method thereof.The adjuvant
Containing 2.5% squalene, about 0.48% ceteareth -12 (Ceteareth-12), about 0.37% sorbester p17, furthermore still contain
A kind of Toll-like protein receptor 4 (TLR-4) agonist, wherein 90% emulsion droplet size≤200nm.Preparation method is first to prepare
At w/o type Submicron Emulsion, 50~65 DEG C of experience phase transition are then heated to, obtain O/W type Submicron Emulsion after cooling.Preparation process is multiple
Miscellaneous, quality is more difficult to control.In addition, contained surfactant Ceteareth-12 has certain toxicity, it is typically only used for part
In external preparation.Patent document US2009304799A1, US2012003277A1 disclose the prescription of W805EC, preparation method and
Its purposes.The adjuvant contains 64% soybean oil, 8% ethyl alcohol, 5% Tween 80,1% hexadecylpyridinium chloride (CPC), average grain diameter
About 400nm.Since ethyl alcohol in prescription and Tween 80 content are higher, it is unsuitable for drug administration by injection, is limited only at present viscous as nose
The adjuvant of film immune vaccine.It is a kind of nano-emulsion assistant disclosed in patent document CN02144678.4 and CN201010103495.2
Agent, influenza vaccines need to undergo high-speed stirred and nanometer knock-on process to partial size between 10~100nm, but during the preparation process, hold
Easily leading to antigen valence reduces even inactivation.
It can be seen that existing adjuvant adverse reaction is more, while the humidification of vaccine immunogenicity is unobvious, and
Granularity is greater than 100nm mostly, in preparation process using organic solvent, residual solvent toxicity is big, manufacturing process is complicated, condition requirement
It is high, processing operation time is too long, destroy protein stability, thermodynamic instability, to be unable to long-term preservation, encapsulation rate low, easy to leak,
The major defects such as energy consumption is big are exempted to cause vaccine that need to repeatedly be inoculated with, store low with easy in inactivation, absorption efficiency in transportational process
The serious problems such as epidemic disease protecting effect difference.Meanwhile the object used in view of vaccine product is human body, in matching for selection vaccine formulation
5, during matching, not only to consider the effect of institute's adding ingredient stabilizer and adjuvant, more to consider whether it has human body
Poison, nocuousness, if bad, the allergic reaction of human body can be caused.Therefore, seek it is a kind of it is safe, cheap, have stabilizer and adjuvant
The vaccine adjuvant of function has great Social benefit and economic benefit for the promotion and application of vaccine product, also becomes vaccine
The great realistic problem learned.
The emulsion by partial size less than 100nm is known as nano-emulsion at present, it helps surface living by oily phase-water phase-surfactant-
Property agent composition, the nano-emulsion that continuous phase is water phase, dispersed phase is oily phase is O/W type, and continuous phase is oily phase, dispersed phase is water phase
Nanoemulsion is w/o type.Nano-emulsion has partial size small and uniform, and easily prepared, stability, mobility are preferable, are easy to inject
The advantages that, it can be used as pharmaceutical carrier applied in the pharmaceutical preparation of a variety of absorption patterns.Appearance is transparent or translucent nanometer
Cream be the Thermodynamically stable spontaneously formed after being mixed in a certain ratio by oily phase, water phase, surfactant and cosurfactant,
Isotropism, appearance transparent or translucent dispersion.Nano-emulsion can be used as a kind of novel vaccine carrier, promote vaccine protein
The absorption of molecule in vivo the advantages that stability is good, is increasing to improve the immune effect of vaccine, while with high security
Unique advantage will be shown in terms of the immune effect of strong vaccine.
Therefore, it is badly in need of a kind of w/o type nanoemulsion of the partial size less than 100nm, there is good whole body and mucosa-immune to imitate
Fruit.
Summary of the invention
To overcome adverse reaction existing for existing vaccine adjuvant, especially aluminium adjuvant, the adjuvants such as MF59, AS series are to vaccine
The deficiencies of humidification of immunogenicity is unobvious, and stability is poor, the present invention provide a kind of oil-in-water type nano-emulsion adjuvant, also mention
It for vaccine and preparation method containing this nanometer of adjuvant, can greatly enhance immune effect of vaccine, reduce adverse reaction, while at
Divide simple, stable dosage forms, it is easy to use；The vaccine that the adjuvant and antigen are configured to, stability is good, and side reaction is small, immune effect
Good, the protection period is long.
The purpose of the present invention adopts the following technical scheme that realization.
A kind of oil-in-water type nano-emulsion adjuvant includes following component by mass percentage:
Preferably, the emulsion droplet size of the nano emulsion adjuvant is 1~100nm.It is furthermore preferred that the surfactant with help
The mass ratio of surfactant is 2:1~5:1.
In the present invention, the surfactant be Tween-80, Tween-85, polysorbate60, Crodaret and
Any one or more combinations in Emulsifier EL-60；The cosurfactant are as follows: sorbitan fatty acid ester, C2~C4
Monohydric alcohol and C2~C4 any one or more of combination of polyalcohol；The oil is mutually paraffin oil, ethyl acetate, nutmeg
Any one or more combinations in isopropyl ester, saualane, squalene, ethyl oleate, octanoic acid/certain herbaceous plants with big flowers acid triglyceride.
In the present invention, the sorbitan fatty acid ester is any one of Arlacel-85, sorbester p17 or sorbester p18 or more
The polyalcohol of kind of combination, the monohydric alcohol of the C2~C4 and C2~C4 are dehydrated alcohol, glycerol, 1,3-BDO or 1,3- the third two
Any one or more of combination of alcohol.
2, the nano emulsion adjuvant vaccine prepared using the oil-in-water type nano-emulsion adjuvant.
Preferably, the protein concentration of the nano emulsion adjuvant vaccine is 100~600 μ g/ml.
It is furthermore preferred that the albumen of the nano emulsion adjuvant vaccine and nano emulsion adjuvant package or direct physical mixed.
Most preferably, the nano emulsion adjuvant vaccine is MRSA nano emulsion adjuvant vaccine.
3, the preparation method of the nano emulsion adjuvant vaccine, includes the following steps:
B. emulsifier, assistant for emulsifying agent and oily phase are taken by formula, after mixing, temperature is 4~37 DEG C, revolving speed is 50~500
It is stirred evenly under the conditions of rev/min；
B. protein vaccine is added into the mixed liquor of step a to final concentration of 100~600 μ g/ml of protein vaccine, in temperature
Under conditions of being 50~150 revs/min for 4~37 DEG C, revolving speed, stir evenly；
C. it is added under stirring into the mixed liquor of step b and is equivalent to 70% distilled water of formula ratio, mixed, obtain clear molten
Liquid；
E. excess water is added to clear solution obtained by step c, mixes, obtains the nano emulsion adjuvant vaccine of clear.
Preferably, in step b, the protein vaccine is MRSA protein vaccine.
The beneficial effects of the present invention are: the invention discloses oil-in-water type nano-emulsion adjuvant, the simple, dosage form with ingredient
Stablize, it is easy to use, it is easy to inject, and small toxicity, property is mild, and solubilized effect of drugs is obvious, good fluidity, to body
The adjuvant is used to prepare nano emulsion adjuvant vaccine, can enhance the immune effect of vaccine by the advantages that irritation is minimum.With MRSA
For vaccine, using adjuvant of the invention compared with the MRSA vaccine of no adjuvant, with that dosage is small, bioavilability is high is excellent
Point；Compared with the MRSA containing other adjuvants, have greatly enhancing vaccine bioavilability, reduction irritation, reduction bad anti-
It answers, extend vaccine release time, and enhancing vaccine targeting, reduce the proportion of goods damageds.Vaccine side is prepared using adjuvant of the invention,
Simple process and low cost, obtained stable dosage forms easily store, good immune effect, and the protection period extends, and are easy to inject, side reaction
It is small, at the same can be used for can intramuscular injection, subcutaneous injection or collunarium, can also the inoculation of sublingual instillation immunization route.Therefore, have very
Good application prospect.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing:
Formulation optimization (a Smix=2:1 of Fig. 1 nano emulsion adjuvant and its MRSA nano emulsion adjuvant vaccine；B is Smix=
3:1；C is Smix=4:1；D is Smix=5:1).
(a is various concentration nano-emulsion assistant to the optimum preparation condition of Fig. 2 nano emulsion adjuvant and its MRSA nano emulsion adjuvant vaccine
The variation of the average particle size of agent and its MRSA nano emulsion adjuvant vaccine；B is that various concentration nano emulsion adjuvant and its MRSA nano-emulsion are helped
The current potential of vaccinating agent；C is the variation of different process MRSA nano emulsion adjuvant vaccine average particle size；D is different process MRSA nano-emulsion
Adjuvanted vaccines potential change).
(a is transmission electron microscope picture for the essential characteristic identification of Fig. 3 MRSA nano emulsion adjuvant vaccine；B is atomic force microscope figure；C is
Particle size distribution figure；D is potential image).
(a is nano emulsion adjuvant and its MRSA for the quality stability research of Fig. 4 nano emulsion adjuvant and its MRSA nano-emulsion vaccine
The detection of nano emulsion adjuvant vaccine protein structural intergrity；B is that nano emulsion adjuvant and its MRSA nano emulsion adjuvant vaccine construct are special
Property detection；C is that the protein integrity that nano emulsion adjuvant and its MRSA nano emulsion adjuvant vaccine are placed under different temperatures detects；D is
Nano emulsion adjuvant and its MRSA nano emulsion adjuvant vaccine place the protein integrity detection of different time).
(a is serum for serology antibody test after the intramuscular injection of Fig. 5 nano emulsion adjuvant and MRSA nano-emulsion vaccine is immune
Middle IgG antibody titre；B is IgG1 Antibody Results in serum；C is IgG2a Antibody Results in serum；D is IgG2b antibody in serum
As a result).
IgG1/IgG2a, IgA for being generated after Fig. 6 nano emulsion adjuvant and MRSA nano-emulsion vaccine intramuscular injection, IFN-γ and
(a is serum IgG 1/IgG2a result: b is IgA result in serum: c is IFN-γ result in serum to IL-17A result；D is serum
Middle IL-17A result).
(a is in serum for serology antibody test after the Nasal immunization of Fig. 7 nano emulsion adjuvant and MRSA nano-emulsion vaccine
IgG antibody titre；B is IgG1 Antibody Results in serum；C is IgG2a Antibody Results in serum；D is IgG2b antibody knot in serum
Fruit).
IgG1/IgG2a, IgA, IFN-γ and the IL- that Fig. 8 nano emulsion adjuvant and MRSA nano-emulsion vaccine Nasal immunization generate
(a is serum IgG 1/IgG2a result to 17A result；B is IgA result in serum: c is IFN-γ result in serum；D is in serum
IL-17A result).
The survival rate and bacterium of Fig. 9 nano emulsion adjuvant and MRSA nano-emulsion vaccine to general infection and pneumonia MRSA
Field planting rate removes result, and (a is nano emulsion adjuvant vaccine survival rate result；B is that nano emulsion adjuvant vaccine pneumonia is colonized clearance rate).
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.It is not specified in embodiment specific
The experimental method of condition, usually according to conventional conditions or according to the manufacturer's recommendations.
The screening of embodiment 1, nano emulsion adjuvant prescription
Surfactant (SF), such as: Tween-80, Tween-85, polysorbate60, polysorbas20 and Emulsifier EL-60
(EL40, EL35), Crodaret etc., cosurfactant (CoSF), such as sorbester p17, span 85 and ethyl alcohol, sweet
Oil, propylene glycol, dehydrated alcohol, 1,3- butanol, 1,3-PD, Comprehensive Designing experimental group, by 9:1,8:2,7:3,6:4,5:5,
4:6,3:7,2:8,1:9 (mass ratio of SF and CoSF) weigh SF and CoSF, selected oily phase are added, such as: paraffin oil, second
Acetoacetic ester, nutmeg isopropyl ester, saualane, squalene, ethyl oleate, octanoic acid/certain herbaceous plants with big flowers acid triglyceride etc., be vortexed concussion, is added dropwise
Water observes the variation of each group, with centrifugation 30min stability under the conditions of 13,000rpm and emulsion droplet size for main evaluation criterion,
So that it is determined that going out to form the SF and CoSF of nano-emulsion.With surfactants/cosurfactants (mixed surfactant), oil
Phase and deionized water draw pseudo-ternary phase diagram as 3 vertex of equilateral triangle, and the point in phasor each edge respectively indicates phase
The proportionate relationship between two components is answered, phasor any point indicates the mass percentage of each component in corresponding system.According to oil,
Water, mixed surfactant draw pseudo-ternary phase diagram in the mass fraction of critical point.Using nano emulsion adjuvant evaluation criterion into
Row judgement.Evaluation criterion are as follows: the liquid of preparation is in clear or translucent；There is blue-opalescent；High speed centrifugation (13000r/m,
After 30min) being centrifuged, stablize not stratified；There is Tyndall phenomenon after directional light is incident；Pass through transmission electron microscope and laser particle size
Instrument detects emulsion droplet between 1~100nm.If the mixing liquid of preparation is creamy white, there is scattering phenomenon after directional light is incident,
It is then emulsion；If clear, stiff, for gel, while liquid crystal can be also generated in phase transition process.There is refractive power using liquid crystal
In the presence of in petrographic microscope detection, it may appear that light and shade changes, so often using it to distinguish liquid crystal and gel, is Ru shinny
Liquid crystal, not shinny gel.According to its maximum pseudo-ternary phase diagram region, finally determines its surfactant and help surface-active
Agent interworking optimum combination are as follows: Tween 80/sorbester p17, Tween 80/sorbester p18, Tween 80/span 85, polysorbate60/sorbester p17, tween
85/ span 85, RH40/ ethyl alcohol, RH40/ propylene glycol, EL35/ propylene glycol, EL35/ glycerol.And by mass percentage, surface
Activating agent is 1-30%, cosurfactant 0.1-15%, and oil is mutually 0.1-15%, and water is equal within the scope of 40%-98.8%
Emulsion droplet can be made between 1~100nm, liquid is in clear or translucent；Have blue-opalescent, under the conditions of 13000r/m from
Heart 30min stablizes not stratified oil-in-water type nano-emulsion adjuvant.Wherein, surfactant is Tween-80, Tween-85, tween
60, polysorbas20, one of Crodaret (EL35, EL40), Emulsifier EL-60 or multiple combinations；Help table
Face activating agent is one of sorbitan fatty acid ester, the monohydric alcohol of C2~C4 or polyalcohol or a variety of, the dehydration mountain
Pears alcohol fatty acid ester is one of Arlacel-85, sorbester p17 and sorbester p18 and multiple combinations；The monohydric alcohol or polyalcohol of C2~C4
For one or more combinations in dehydrated alcohol, n-butanol, glycerol, 1,3- butanol, 1,3-PD, oil be paraffin oil, wool grease,
In ethyl acetate, isopropyl myristate (IPM), saualane, squalene, ethyl oleate, octanoic acid/certain herbaceous plants with big flowers acid triglyceride (GTCC)
One or more combinations.
The optimum formula of oil-in-water type nano-emulsion adjuvant is further screened according to optimum results, specific formula is as follows:
Formula 1: nano-emulsion vaccine adjuvant (100g): Tween-80 20g, propylene glycol 10g, medicinal IPM 10g, distilled water
40g。
Formula 2: nano-emulsion vaccine adjuvant (100g): Tween-80 30g, glycerol 10g, GTCC 10g, distilled water 50g.
Formula 3: nano-emulsion vaccine adjuvant (100g): Tween-80 5g, glycerol 0.1g, ethyl acetate 0.1g, distilled water
94.8g。
Formula 4: nano-emulsion vaccine adjuvant (100g): Tween-60 20g, propylene glycol 0.5g, Medol 10g, distilled water
69.5g。
Formula 5: nano-emulsion vaccine adjuvant (100g): Tween-85 30g, 1,3 butanediol 10g, GTCC 15g, distilled water
45g。
Formula 6: nano-emulsion vaccine adjuvant (100g): Tween-80 1g, propylene glycol 0.2g, glycerol 0.1g, GTCC 0.1g,
IPM0.1g, distilled water 97.5g.
Formula 7: nano-emulsion vaccine adjuvant (100g): Tween-85 1g, ethyl alcohol 1g, paraffin oil 0.1g, distilled water 97.9g.
Formula 8: nano-emulsion vaccine adjuvant (100g): polysorbate60 1g, Tween 80 2g, sorbester p18 0.1g, IPM 0.1g, distillation
Water 96.8g.
Formula 9: nano-emulsion vaccine adjuvant (100g): EL 353.5g, ethyl alcohol 0.5g, GTCC 0.5g, distilled water 94.5g.
Formula 10: nano-emulsion vaccine adjuvant (100g): EL 3530g, 1,3 butanediol 0.5g, ethyl acetate 10g, distilled water
59.5g。
Formula 11: nano-emulsion vaccine adjuvant (100g): EL4030g, ethyl alcohol 10g, ethyl oleate 10g, distilled water 50g.
Formula 12: nano-emulsion vaccine adjuvant (100g): 3 propylene glycol 0.1g, EL353g, EL401g, 1 paraffin oil 0.5g, steam
Distilled water 56.5g.
Formula 13: nano-emulsion vaccine adjuvant (100g): polysorbate60 1g, sorbester p17 0.1g, squalene 0.1g, distilled water
98.8g。
Formula 14: nano-emulsion vaccine adjuvant (100g): Tween 80 5g, span 85 0.1g, squalene 0.5g, distilled water
94.4g。
Formula 15: nano-emulsion vaccine adjuvant (100g): Tween 80 25g, sorbester p18 10g, saualane 10g, distilled water 55g.
Formula 16: nano-emulsion vaccine adjuvant (100g): polysorbate85 5g, sorbester p17 1g, saualane 0.1g, squalene 0.5g,
Physiological saline 93.4g.
Formula 17: nano-emulsion vaccine adjuvant (100g): polysorbate85 25g, sorbester p17 15g, squalene 10g, distilled water 50g.
Formula 18: nano-emulsion vaccine adjuvant (100g): EL351g, sorbester p17 0.1g, squalene 0.1g, distilled water 98.8g.
Formula 19: nano-emulsion vaccine adjuvant (100g): Tween 80 30g, span 85 15g, squalene 5g, distilled water 50g.
Formula 20: nano-emulsion vaccine adjuvant (100g): EL3510g, propylene glycol 1g, ethyl oleate 0.5g, physiological saline
88.5g。
Embodiment 2, oil-in-water type nano-emulsion adjuvant prepare MRSA nano emulsion adjuvant vaccine
Since Vaccines classes are various, the present embodiment mainly proves nanometer prepared by the present invention with MRSA nano emulsion adjuvant vaccine
Effect brought by newborn Adjuvanted vaccines.
MRSA proteantigen prepares bibliography (Zuo QF, Yang LY, Feng Q, Lu DS, Dong YD, Cai
CZ,et al.Evaluation of the protective immunity of a novel subunit fusion
vaccine in a murine model of systemic MRSA infection.PloS one 2013；8:e81212)
It is prepared by method.The original concentration of its proteantigen is 1.5mg/mL, and the purity of albumen is 99.4%, the antigen-prepared
70 DEG C of refrigerators save.
According to the oil-in-water type nano-emulsion adjuvant that embodiment 1 is screened, the vaccine of the nano emulsion adjuvant containing oil-in-water type, tool are prepared
Body formula and the preparation method is as follows:
It is formulated 1:150 μ g/ml MRSA nano emulsion adjuvant vaccine (100g): propylene glycol 10g, Tween-80 30g, medicinal Pork and beans
The MRSA vaccine 10ml and distilled water 40g of cool isopropyl propionate (IPM) 10g, 1.5mg/ml.
The preparation method is as follows:
C. after emulsifier (Tween-80), assistant for emulsifying agent (propylene glycol) and the oily phase (IPM) in formula 1 being mixed, at 25 DEG C
At a temperature of, by 150 revs/min of revolving speed, stir evenly；
B. MRSA vaccine is added into mixed liquor to the final concentration of 150 μ g/ml of MRSA vaccine, at a temperature of 25 DEG C, by 150
Rev/min revolving speed, stir evenly；
C. it is slowly added to 70% distilled water of formula ratio into mixed liquor under stirring, mixes, obtains clear solution；
D. surplus water phase is finally added, mixes, obtains the MRSA nano emulsion adjuvant vaccine of clear.
Light yellow nano emulsion adjuvant vaccine appearance, clear, partial size are obtained between 1~100nm, in 13000rpm condition
Not stratified after lower centrifugation 10min, after being placed at room temperature for 1 year, nano-emulsion is precipitated without apparent flocculation, layering and protein vaccine.
Be formulated 2:300 μ g/ml MRSA nano emulsion adjuvant vaccine (100g): glycerol 10g, EL3530g, medicinal GTCC 5g,
1.5mg/ml MRSA protein vaccine 20ml and distilled water 35g.
Be formulated 3:450 μ g/ml MRSA nano emulsion adjuvant vaccine (100g): sorbester p17 5g, Tween-85 20g, squalene 1g,
1.5mg/ml MRSA protein vaccine 30ml and distilled water 44g.
It is formulated 4:600 μ g/ml MRSA nano emulsion adjuvant vaccine (100g): span 85 2g, Tween-80 20g, squalene
0.1g, saualane 0.1g, 1.5mg/ml MRSA protein vaccine 40ml and distilled water 37.8g.
Formula 5:100 μ g/ml MRSAMRSA nano emulsion adjuvant vaccine (100g): weigh span 85 2g, Tween-80 20g,
Saualane 0.1g, 1.5mg/ml MRSA protein vaccine 6.666ml, distilled water 71.2g.
Following embodiment is combined into the group of EL35/ propylene glycol represents the research for carrying out MRSA nano emulsion adjuvant vaccine.
1, the best prescription of package MRSA nano emulsion adjuvant vaccine is determined
It is surfactant (SF), the third two using EL35 for the best prescription for determining package MRSA nano emulsion adjuvant vaccine
Alcohol is cosurfactant (CoSF), isopropyl myristate (IPM) is oily phase, is 2:1 by the mass ratio of SF-CoSF；3:1；
4:1；5:1, MRSA protein vaccine ultimate density are 150 μ g/ml, prepare MRSA nano emulsion adjuvant vaccine according to the method described above, so
The best prescription of best nano emulsion adjuvant package MRSA nano emulsion adjuvant vaccine is determined afterwards.
Above-mentioned 4 ratios are formed shown in the result figure 1 of nano-emulsion pseudo-ternary phase diagram.SF-CoSF ratio is as the result is shown
2:1；3:1；4:1；When 5:1, nano-emulsion can be formed；But SF-CoSF ratio be 4:1 when, nano-emulsion forming region figure is bright
It shows greater than its ratio be 2:1,3:1 and 5:1.Therefore, the surfactant and surfactant optimal proportion of its nano-emulsion are selected
For 4:1.
2, MRSA nano emulsion adjuvant vaccine optimum protein concentration determines
In order to determine the optium concentration of albumen in MRSA nano emulsion adjuvant vaccine, 0 μ g/ml, 150 μ g/ml, 225 μ g/ is selected
Ml, 300 μ six various concentrations of g/ml, 450 μ g/ml and 600 μ g/ml.Most according to above-mentioned acquisition MRSA nano emulsion adjuvant vaccine
Good prescription carries out preparing its nano emulsion adjuvant vaccine to above-mentioned six various concentration MRSA albumen.By nano emulsion adjuvant obtained
Its average particle size and current potential are analyzed with granularity potentiometric analyzer, its best egg that can be carried is determined according to its granularity and potential change
White concentration.
Six various concentration MRSA albumen carry out the granularity for preparing its nano emulsion adjuvant vaccine and Fig. 2 (a) is shown in potentiometric analysis,
Fig. 2 (b).It is average to form nano emulsion adjuvant vaccine the results show that when protein concentration is in 0 to 300 μ g/mL range by Fig. 2 (a)
Granularity increases as protein concentration increases, and when continuing growing 450 μ g/mL range, it is bright to form nano emulsion adjuvant vaccine granularity
It is aobvious to reduce, become the protein concentration of 600 μ g/mL its average particle size and obviously increases.Fig. 2 b is the results show that when protein concentration is 0
When to 300 μ g/mL range, form nano emulsion adjuvant vaccine current potential reduces as protein concentration increases, when continuing growing 450 μ
When g/mL range, forms nano emulsion adjuvant vaccine current potential and obviously rise, become the protein concentration of 600 μ g/mL its current potential and obviously drop
It is low.Complex chart 2a and Fig. 2 b determines that form the optimal transporter concentration of nano emulsion adjuvant vaccine is 450 μ g/mL, but egg
White concentration within the scope of 0~600 μ g/ml nano emulsion adjuvant vaccine partial size between 1~100nm.
The determination of the best preparation process of 3.MRSA nano emulsion adjuvant vaccine
It is well known that order of addition is different, forming quality stabilization to nano emulsion adjuvant vaccine has particularly significant influence factor.
Different adjunct ingredients is needed according to nano emulsion adjuvant vaccine forming process, design is four kinds of addition protein order work as follows
Skill: (1) HSCO technique: protein, surfactant, cosurfactant, oily phase are named as technique 1；(2) SHCO technique: table
Face activating agent, protein, cosurfactant and oily phase, are named as technique 2；(3) SCHO technique: surfactant helps surface
Activating agent, protein and oily phase, are named as technique 3；(4) SCOH technique: surfactant is added, followed by helps surface-active
Agent, oil and protein are named as technique 4.Above-mentioned four kinds of technique is obtained with the best prescription of acquisition and best transporter
MRSA nano emulsion adjuvant vaccine its average particle size of granularity potentiometric analyzer analysis detection and current potential.Become according to its granularity and current potential
Its optimal preparation process is determined in change.
The MRSA nano emulsion adjuvant vaccine of above-mentioned four kinds of technique its average particle size of granularity potentiometric analyzer analysis detection and
Current potential as a result, seeing Fig. 2 (c) and Fig. 2 (d).
The result shows that protein order of addition has a great impact to average grain diameter and current potential, Fig. 2 (c) and Fig. 2 (d) are seen.
SOCH order of addition can reduce the average grain diameter (31nm) of MRSA nano emulsion adjuvant vaccine.SHCO order of addition improves MRSA
The average grain diameter (about 100nm) of nano emulsion adjuvant vaccine, is shown in Fig. 2 (c).It is average caused by both orders of addition of HSCO and SCOH
Partial size is shown in Fig. 2 (c) there are about the difference between two orders of magnitude, such as 80-100nm.SCOH and other orders of addition (HSCO,
SHCO and SCHO) there is statistical difference (p < 0.01) in terms of granular size.In addition, there is no exist as its partial size one for current potential
The significant difference of sample, is shown in Fig. 2 (d).In the preparation process of MRSA nano emulsion adjuvant vaccine, there are only aobvious in SCOH group
Sex differernce is write, sees Fig. 2 (c) and Fig. 2 (d).The above results demonstrate short grained nano emulsion adjuvant vaccine (about 30nm) be can be with
It is formed.In addition, above-mentioned experiment also demonstrates different orders of addition, there is no additional benefits, and these additional benefits are logical
It is often the industry of nano emulsion adjuvant vaccine to be manufactured there is positive effect.Therefore, SCOH order of addition is best preparation
Technique can generate preferable particle, and this is more efficient for the transhipment of nano emulsion adjuvant vaccine.
Embodiment 3, the evaluation of the quality essential characteristic of MRSA nano emulsion adjuvant vaccine
A small amount of MRSA nano emulsion adjuvant vaccine is appropriate, after being diluted with water 100 times, drips on being covered with the copper mesh for supporting film, quiet
It is only blotted after 10min with filter paper, then phosphotungstic acid (pH 7.4) solution that mass fraction is 2% is added dropwise in negative staining on copper mesh
3min is volatilized naturally, with transmission electron microscope observation and photographs.A small amount of MRSA nano emulsion adjuvant vaccine is appropriate, uses water
After 100 times of dilution, naturally dry is directly observed with Atomic Mechanics microscope IPC-208B (University Of Chongqing).All images are logical
Automatic smoothing processing is crossed to eliminate the low frequency noise of slow-scan direction.Measurement is completed under the following conditions: (power is normal for tungsten tipped probe
Number, 0.06Nm)；Scanning range, 10.5 × 10.5nm；Imaging pattern, development mode；And scan method, at room temperature point by point
Scanning.G3DR software is used to processing data.
Transmission and atom Electronic Speculum result are shown in Fig. 3 (a) and 3 (b).
The result shows that MRSA nano emulsion adjuvant vaccine is rounded under transmission electron microscope, inside is oily phase, sees Fig. 3
(a).Under Electronic Speculum, no less than 500 drop measurement partial sizes are randomly selected, obtain MRSA nano emulsion adjuvant vaccine particle size range 1
~100nm, average grain diameter 30nm.It is aobvious by the check image of atomic force microscope shown in atom transmission electron microscope such as Fig. 3 (b)
Show that most of drop puts on display subsphaeroidal form and average diameter about 30nm.Drop occurs as solid object, while surface exhibits
Out be mostly it is rugged and display recess and wave crest.There is no coalescence of water droplets, especially after antigen is in conjunction with nano emulsion adjuvant, together
When prove that nanoemulsion improves the stability of drop, and although albumen contacts with each other between adjacent drops, distributed between drop
Well.
A small amount of MRSA nano emulsion adjuvant vaccine is appropriate, after being diluted with water 50 times, to its average grain diameter, current potential, viscosity and folding
The basic indexs such as light instrument 90 current potential of Nano ZS and Analyzer testing result.
The partial size and current potential of MRSA nano emulsion adjuvant vaccine, are shown in Fig. 3 (c) and Fig. 3 (d).
Fig. 3 (c) shows that the average grain diameter of MRSA nano emulsion adjuvant vaccine is 31.34 ± 0.49nm.In addition, MRSA nano-emulsion
Adjuvanted vaccines show a relatively limited size distribution, and PdI value is 0.270 ± 0.270, less than 0.3.Because PdI value is surveyed
What is measured is the diffusion of size distribution, and one small PdI value indicates a narrow particle size range.Eletrokinetic potential be it is average-
7.33mV, as shown in Fig. 3 (d).Viscosity, refractive index and dispersing agent RI are 0.8872cP, 1.5nD20 and 1.330 respectively.In high speed
The appearance of centrifugation front and back all occurs significantly to change, and apparent flocculation, layering and drug precipitation phenomenon also occurs without generation.These
As a result illustrate that MRSA nano emulsion adjuvant vaccine is stablized, meet the essential characteristic of nano emulsion adjuvant vaccine.
The quality stability research of embodiment 4, nano emulsion adjuvant and its MRSA nano emulsion adjuvant vaccine
1, the integrality and specific detection of nano-emulsion vaccine
Blank nano-emulsion, MRSA nano emulsion adjuvant vaccine are taken into 0.2ml respectively, shaken up after the water of 2 times of volumes is added,
13000rpm.10min centrifugation is taken out supernatant and is taken out, and precipitating is dissolved with water；In addition by blank nano-emulsion, MRSA nano emulsion adjuvant
Vaccine 0.2ml is added the dehydrated alcohol demulsification of 2 times of volumes, 10min is centrifuged under the conditions of 13000rpm, takes out supernatant and take out, sink
It is dissolved with water in shallow lake.Demulsification and the supernatant not being demulsified precipitating are respectively taken into 80 μ l, 5 × albumen sample-loading buffer, 20 μ l is added, boils 5
Minute, take 10 μ l 12%SDS-PAG electrophoresis.Pure protein is diluted to same concentrations same treatment method loading.Remaining
With SDS-PAGE electrophoresis and Western blotting measurement albumen composition palliating degradation degree and antigen reactivity, experimental method
It is carried out by " fine works molecular biology experiment guide " and document.
The external integrality of MRSA nano emulsion adjuvant vaccine and specific detection as a result, seeing Fig. 4 (a) and Fig. 4 (b) institute respectively
Show.
The sequence of Fig. 4 (a) from left to right is successively are as follows: blank nanoemulsion is not demulsified supernatant, and blank nanoemulsion is not demulsified
Precipitating, blank nanoemulsion demulsification supernatant, blank nanoemulsion demulsification precipitating, protein molecular Marker, MRSA nano emulsion adjuvant
Vaccine is not demulsified supernatant, and MRSA nano emulsion adjuvant vaccine is not demulsified precipitating, and MRSA nano emulsion adjuvant vaccine is demulsified supernatant, and MRSA receives
Rice milk Adjuvanted vaccines demulsification precipitating, protein solution.The band from right to left of Fig. 4 (b) is sequentially consistent with Fig. 4's (a).From figure
Protein solution supernatant is equal after 4 (a) SDS-PAGE electrophoresis can be seen that the MRSA nano emulsion adjuvant vaccine of preparation and be demulsified
An only band, protein band are the position 48KD in protein molecular weight, after this sufficiently shows the protein nano cream of preparation,
Integrality is not obviously destroyed.There is very dark band in albumen precipitation after demulsification, after illustrating demulsification, the surface of nano-emulsion is living
Property agent, cosurfactant have partly precipitated effect to albumen.But albumen is mainly concentrated in aqueous solution and the albumen of demulsification
In supernatant.Fig. 4 a the result shows that, the external integrality of the protein nano of preparation cream is good.From Fig. 4 (b) specific detection knot
Protein solution supernatant has apparent in the pre-dyed protein molecular position Marker 48KD after fruit, albumin nanometer rice milk and demulsification
Band illustrates that the specificity of the protein nano cream of preparation is good.The nano-emulsion of Fig. 4 (a) and Fig. 4 (b) water blanks and blank
No matter it is demulsified in being precipitated with the supernatant not being demulsified without protein band, illustrates with SDS-PAGE and western blotting method in solution
It detects external integrality and specific detection is noiseless.
2, the stability study of nano-emulsion vaccine
By MRSA nano emulsion adjuvant vaccine deposit in respectively 4 DEG C, 25 DEG C and 40 DEG C, sample January, 2 months, March, sampling pair
The detection of its protein integrity.Meanwhile sample is placed in room temperature, respectively at 0,1,2,3,4,6,8,10, sampling in December to its egg
White integrity detection.Result is measured to its integrality and sees Fig. 4 c and Fig. 4 d.It can be seen that from Fig. 4 d, albumen only has one always
Band, protein band are the position 48KD in protein molecular weight, and all there is no substantially changeing for the position of albumen and concentration.On
Stating result sufficiently confirms, the MRSA nano emulsion adjuvant vaccine quality of preparation is highly stable.
The immunological evaluation generated after embodiment 5, the intramuscular injection of MRSA nano emulsion adjuvant vaccine and bronchia mucosal are immune
1, the grouping of animal and immune
BALB/c mouse (28): SPF grades, 6-8w age, weight 18-20g, female；C57 mouse (28): SPF grades, 6-
8w age, weight 18-20g, female.Experiment is divided into four groups: PBS, BNE, antigen and MRSA nano emulsion adjuvant vaccine group.Balb/c is small
Mouse uses intramuscular injection (bilateral；100 μ l volumes contain 15 μ g antigens) immunization ways, C57 mouse is using nasal-cavity administration (30 μ l volumes
Containing 30 μ g antigens) mode.0 day first immunisation, 7d, 14d, each booster immunization 1 time.MRSA nano emulsion adjuvant vaccine is matched
System: it is mixed with PBS dissolution antigen (30 μ g/ are only) with isometric nano emulsion adjuvant.Negative control group is for simple PBS and merely
Nano emulsion adjuvant.21st day after immune, take a blood sample with blood taking needle to every mouse, with IgG after ELISA detection mouse immune,
IgG1, IgG2 humoral response level and IFN-γ and IL-17A cellullar immunologic response are horizontal.Serum diluting multiple: IgG detects blood
Clear extension rate is between 1:500-512,000；It is 1:500 that IgG1, IgG2a, IgG2b and IgA, which detect serum diluting multiple,.
IgG in serum, IgG1, IgG2a, IgG2b and IgA's detects to obtain by ELISA.IFN-γ and IL-17A by cell because
Sub- detection kit is detected.
2, the immunological evaluation generated after the intramuscular injection of MRSA nano emulsion adjuvant vaccine is immune
The immunology serology antibody evaluation result generated after the intramuscular injection of MRSA nano emulsion adjuvant vaccine is immune, is shown in figure
5.From fig. 5, it can be seen that PBS, BNE, the IgG geometric mean titer of antigen group, MRSA nano emulsion adjuvant vaccine group are respectively
500,552.044,64000 and 156033.7.As shown in Fig. 5 (a), the IgG titre (Log of MRSA nano emulsion adjuvant vaccine2) be
It is highest in four groups, and there is statistical difference (P=0.0067, P < 0.01 with antigen group, BNE group；P=0.0001, P <
0.01).MRSA nano emulsion adjuvant vaccine serum IgG 1 OD450 absorbance value in four groups highest, see Fig. 5 (b).In addition,
Compared with antigen group, BNE group, there is statistical difference (P=0.0210, P < 0.05；P=0.0001, P < 0.01).Fig. 5 (c) is aobvious
Show, the serum IgG 2a absorbance value of MRSA nano emulsion adjuvant vaccine group is highest in four groups.In addition, with antigen group, BNE group phase
Than having statistical difference (P=0.0365, P < 0.05；P=0.0211, P < 0.05).The IgG2b absorbance value of antigen group is slightly
Higher than MRSA nanoemulsion Adjuvanted vaccines group, but not statistical difference (P > 0.05), but MRSA nanoemulsion adjuvant epidemic disease
The absorbance value of seedling group is higher than BNE group (P=0.0005, P < 0.01).
Immunology serology IgG1/IgG2a, IgA for being generated after the intramuscular injection of MRSA nano emulsion adjuvant vaccine group is immune,
IFN-γ and IL-17A are as a result, be shown in Fig. 6.
As can be seen from Figure 6, MRSA nano emulsion adjuvant vaccine group IgG1/IgG2a ratio result is significantly lower than proteantigen group,
See Fig. 6 (a), is higher than blank nano-emulsion group.The IgG1/IgG2a ratio of proteantigen is apparently higher than control group.Fig. 6 (b) display,
The serum IgA absorbance value of MRSA nano emulsion adjuvant vaccine group be four groups in highest, and be significantly better than antigen group (P=0.0128,
P < 0.05) and BNE group (P=0.0001, P < 0.01).Fig. 6 (c) display, the stimulation of MRSA nano emulsion adjuvant vaccine group are high-caliber
IFN-γ response, and it is significantly higher than antigen group (P=0.0137, P < 0.05) and BNE group (P=0.0001, P < 0.01).It is same with this
When, the IL-17A response level of MRSA nano emulsion adjuvant vaccine group significantly improves, and figure Fig. 6 (d) is significantly better than antigen group (P=
0.0040, P < 0.01) and BNE group (P=0.0007, P < 0.01).
3, the immunological evaluation generated after the Nasal immunization of nano emulsion adjuvant
The immunology serology antibody evaluation result generated after the Nasal immunization of MRSA nano emulsion adjuvant vaccine, is shown in Fig. 7.
Fig. 7 (a) shows that the IgG antibody titre of C57 mouse is significantly higher than antigen group after MRSA nano emulsion adjuvant vaccine immunity
(P=0.0050, P < 0.01) and BNE group (P=0.0001, P < 0.05).PBS, BNE group, antigen group and MRSA nano-emulsion assistant
The IgG geometric mean liter of vaccinating agent is respectively 125,609.506,1259.921 and 3482.202.Fig. 7 (b) the results show that
The IgG1 response level of MRSA nano emulsion adjuvant vaccine is much higher, while being significantly higher than antigen group (P=0.0001, P < 0.01)
With BNE group (P=0.0001, P < 0.01).Fig. 7 (c) is the results show that the IgG2a response level of MRSA nano emulsion adjuvant vaccine is wanted
It is much higher, while being significantly higher than antigen group (P=0.0003, P < 0.01) and BNE group (P=0.0001, P < 0.01).Fig. 7 (d) knot
Fruit shows that the IgG2b response level of MRSA nano emulsion adjuvant vaccine is much higher, while being significantly higher than antigen group (P=
0.0071, P < 0.01) and BNE group (P=0.0001, P < 0.01).
Immunology serology IgG1/IgG2a, IgA for being generated after the Nasal immunization of MRSA nano emulsion adjuvant vaccine, IFN-γ
With IL-17A as a result, seeing Fig. 8.
Such as Fig. 8 (a), in four groups, the IgG1/IgG2a ratio of nano emulsion adjuvant and MRSA nano emulsion adjuvant vaccine group is
It is highest.As shown in Fig. 8 (b), the absorbance value of nano emulsion adjuvant and MRSA nano emulsion adjuvant vaccine group IgA be also in four groups most
Height, furthermore it has statistical difference (P=0.0024, P < 0.01 with antigen group and BNE group；P=0.0007, P < 0.01).Fig. 6
(c) it shows, the high-caliber IFN-γ response of MRSA nano emulsion adjuvant boosting vaccine, and is significantly higher than antigen group (P=0.0137, P
< 0.05) and BNE group (P=0.0001, P < 0.01).At the same time, the IL-17A response level of MRSA nano emulsion adjuvant vaccine is aobvious
It writes and improves, such as Fig. 6 (d), be significantly better than antigen group (P=0.0040, P < 0.01) and BNE group (P=0.0007, P < 0.01).
The immunological evaluation generated after embodiment 6, the intramuscular injection of MRSA nano emulsion adjuvant vaccine and bronchia mucosal are immune
1, the foundation of lethal infection and pulmonary inflammation model
Blab/c mouse tail vein injection 109CFU (LD90) MRSA252, C57 mouse collunarium 2.5 × 108CFU (infectious agent
Amount) MRSA252.The survival state for observing and recording mouse daily, observes and records three times daily, until 10 days.C57 mouse lung exists
It collects within 1 day and 3 days after infection, detection bacterium field planting amount.
2, protective effect of the MRSA nano emulsion adjuvant vaccine to pyemia mouse
Protective effect of the MRSA nano emulsion adjuvant vaccine to pyemia mouse, is shown in Fig. 9 (a).Fig. 9 (a) is as can be seen that MRSA
The survival rate (100%) of nano emulsion adjuvant vaccine is apparently higher than antigen group and (28.6%, P=0.0082, P < 0.01) blank is received
Rice milk group (14.28%, P=0.0023, P < 0.01).The immune protective rate of MRSA nano emulsion adjuvant vaccine group reaches 100%, card
Real MRSA nano emulsion adjuvant vaccine has good protecting effect.
3, MRSA nano emulsion adjuvant vaccine acts on the clearance rate of MRSA pulmonary inflammation model
In order to which whether the MRSA nano emulsion adjuvant vaccine for determining new is protected from the internal of bacterial growth, MRSA is injected and has received
1 day and 3 days infection of staphylococcus aureus MRSA252 after the immune lung harvest with control-animal of rice milk Adjuvanted vaccines, then
The quantity of bacterium is counted.The results show that reduced levels are contained in mouse active immunity and MRSA nano emulsion adjuvant vaccine lung
Staphylococcus aureus, than immune inmature antigenic (P=0.0306 and P=0.0246, P < 0.05) and MRSA nanometers of blank
The control mice (P=0.0017 and P=0.0001, P < 0.01) of newborn Adjuvanted vaccines is low, such as Fig. 9 (b).In addition, active immunity with
Ratio contained by antigen Naive mice lung is immune golden yellow with PBS (P=0.0011 and P=0.0017, P < 0.01) from control mice
The essentially very low level of color staphylococcus.In addition, the lung active immunity of mouse and MRSA nanoemulsion Adjuvanted vaccines and antigen
The staphylococcus aureus that albumen all contains significantly extremely reduced levels 1 day and it is infectious (P=0.0001 and P=0.0005, P <
0.01) the last 3 days.The result shows that the novel nanoemulsion vaccines of immune response may be to Staphylococcus aureus clonal part
Ground protection.
The present invention by with preparation prescription, best transporter amount, best preparation process, successfully filter out with EL/
Propylene glycol/IPM is the nano emulsion adjuvant vaccine of representative, has successfully been made with transmission electron microscope, high speed centrifugation, current potential and Particle Size Analyzer
For standby partial size in 1-100nm, average grain diameter is the nano emulsion adjuvant vaccine of 31nm.Simultaneously it is placed at room temperature for 1 year and its it is outer
Seeing research confirms, nano emulsion adjuvant quality is extremely stable.After intramuscular injection and bronchia mucosal Nasal immunization, by serum
The IFN-γ and IL-17A cytokine content of the antibody levels such as IgG, IgG1, IgG2a, IgG2b and IgA and serum detection hair
Existing, nano emulsion adjuvant can induce extremely strong mucosa-immune and general immunity response.Pass through the lethal model of MRSA mouse sepsis and lung
Scorching model, nano emulsion adjuvant have good immunization
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (4)
1. a kind of nano emulsion adjuvant vaccine prepared using oil-in-water type nano-emulsion adjuvant, which is characterized in that the oil-in-water type
Nano emulsion adjuvant is grouped as by following group by mass percentage:
Surfactant 1-30%；
Cosurfactant 0.1-15%；
Oily phase 0.1-15%；
Water 40%-98.8%；
The surfactant is Crodaret；The cosurfactant is propylene glycol；The oil is mutually meat
The mass ratio of cardamom isopropyl ester, the surfactant and cosurfactant is 4:1, with resistance in the nano emulsion adjuvant vaccine
Methicillin s. aureus protein antigen is as antigen, and end of the proteantigen in the nano emulsion adjuvant vaccine
Concentration is 150 μ g/ml；
Preparation method is as follows:
A. surfactant, cosurfactant and oily phase are taken by formula, after mixing, temperature be 4 ~ 37 DEG C, revolving speed be 50 ~
It is stirred evenly under the conditions of 500 revs/min；
B. proteantigen is added into the mixed liquor of step a to the final concentration of 150 μ g/ml of proteantigen, temperature be 4 ~ 37 DEG C,
Under conditions of revolving speed is 50 ~ 150 revs/min, stir evenly；
C. it is added under stirring into the mixed liquor of step b and is equivalent to 70% distilled water of formula ratio, mixed, obtain clear solution；
E. excess water is added to clear solution obtained by step c, mixes, obtains the nano emulsion adjuvant vaccine of clear.
2. nano emulsion adjuvant vaccine according to claim 1, it is characterised in that: the emulsion droplet size of the nano emulsion adjuvant is 1
~100 nm.
3. nano emulsion adjuvant vaccine according to claim 1, it is characterised in that: the albumen of the nano emulsion adjuvant vaccine is anti-
The former and direct physical mixed of the nano emulsion adjuvant.
4. the preparation method of any one of the claim 1 ~ 3 nano emulsion adjuvant vaccine, which comprises the steps of:
A. surfactant, cosurfactant and oily phase are taken by formula, after mixing, temperature be 4 ~ 37 DEG C, revolving speed be 50 ~
It is stirred evenly under the conditions of 500 revs/min；
B. proteantigen is added into the mixed liquor of step a to the final concentration of 150 μ g/ml of proteantigen, temperature be 4 ~ 37 DEG C,
Under conditions of revolving speed is 50 ~ 150 revs/min, stir evenly；
C. it is added under stirring into the mixed liquor of step b and is equivalent to 70% distilled water of formula ratio, mixed, obtain clear solution；
E. excess water is added to clear solution obtained by step c, mixes, obtains the nano emulsion adjuvant vaccine of clear.
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