CN1706494A - Method of preparing dust mite allergen vaccine with nano particle carrier - Google Patents
Method of preparing dust mite allergen vaccine with nano particle carrier Download PDFInfo
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- CN1706494A CN1706494A CN 200510033959 CN200510033959A CN1706494A CN 1706494 A CN1706494 A CN 1706494A CN 200510033959 CN200510033959 CN 200510033959 CN 200510033959 A CN200510033959 A CN 200510033959A CN 1706494 A CN1706494 A CN 1706494A
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Abstract
The present invention relates to method of preparing dust mite allergen vaccine with nanometer particle as carrier. Dust mite is one of the main allergen causing allergic diseases. The present invention applies biodegradable nanometer PLGA particle as the allergen carrier to coat recombinant dust mite allergen Der p2/Der f2 in preparing nanometer level dust mite allergen vaccine. Specifically, by means of gene engineering technology, recombinant Der p2/Der f2 protein is prepared through cloning and expression in colibacillus and PLGA coated nanometer particle is prepared through re-emulsifying and solvent volatizing process. The form, average size, coating rate, medicine carrying amount and extracorporeal accumulation and release curve of the nanometer particle are obtained, and test shows that Der p2/Der f2-PLGA nanometer particle has certain curative effect on allergic asthma.
Description
Technical field
The present invention relates to the new generation vaccine invention field, specifically a kind of applying nano particle is the method for preparing carriers dust acarid allergen vaccine.
Background technology
Anaphylactic disease (as allergic asthma, allergic rhinitis etc.) is commonly encountered diseases and a frequently-occurring disease clinically, points out that according to WHO/IAACI in 2000 report pathogenesis of asthma rate and mortality rate are in rising trend in recent years.Part city, the whole nation is 3.3%-5.1% to teenager asthma epidemiology statistics asthma prevalence, wants high far beyond 1% before 10 years; China has at least 10,000,000 above children to suffer from asthma at present.
Anaphylactic disease is owing to various contacts or suck allergen and cause that the allergen major part is a protein, also can be the material of polypeptide or saccharide and synthetic such as medicine etc.Wherein dust demodicid mite and dermatophagoides pteronyssinus all are intensive allergens, can cause that the general allergy comprises diseases such as allergic asthma, allergic dermatitis, allergic urticaria.Corresponding about 70% of the various allergic effect diseases that account for of demodicid mite property allergic effect disease.Global asthma control strategy committee in 2003 proposes global asthma patient and estimates at 300,000,000 people.State-owned asthmatic children more than 1,000 ten thousand according to a preliminary estimate adds that adult's asthma whole nation has the asthma patient more than 2,500 ten thousand.
About the allergen problem of demodicid mite as asthma, German Dekker just proposed as far back as nineteen twenty-eight, but just was that the metabolite of confirmation dirt demodicid mite such as Dutch Voorhorst is the main allergen in the room dirt until 1964.Allergen immunotherapy is unique etiological treatment method.China since last century early seventies the allergic series of studies of dirt demodicid mite, carried out the investigation of a large amount of bases and application, developed and produced the thick immersion vaccine of dust demodicid mite for immunization therapy mite sensitivity's disease and obtain certain curative effect.But also there is the quality control difficulty in this thick immersion dirt demodicid mite vaccine, uses weak points such as loaded down with trivial details.The research of allergen immunotherapy has entered molecular level now, studies show that deleterious Th2 reaction to be turned to the Th1 direction by regulating the Th1/Th2 balance-loss state of allergic disease, helps the immunization therapy allergic asthma.
The invention of novel biomaterial and application cause people's very big interest, as have the Biodegradable material polylactic acid of thermoplasticity and solubility-poly-alcohol copolymer (PLGA) and have good biological tissue's compatibility, avirulence and biodegradability.Since Allen in 1984 etc. confirm that first polylactic acid (PLGA) microgranule can be used as antigen vectors, used the research that PLGA material preparation micron order medicine carrying particle carries out oral administration and be seen in many reports.Use the PLGA material and have advantages such as protective effect of drug and sustained release as the dust mite allergen carrier, can reduce the anaphylaxis of dirt demodicid mite vaccine, the chronicity and the loaded down with trivial details property of the traditional administering mode of elimination.In addition, can regulate the Th1/Th2 balance-loss state of allergic disease according to the PLGA administration nano-drug administration system of discovering of Moore and Gutierro etc., and deleterious Th2 reaction is turned to the Th1 direction, this will be than Al (OH)
3Adjuvant more helps the immunization therapy allergic asthma.Therefore, the advantage of, safety novel for the preparing carriers dust acarid allergen vaccine has and effectiveness with PLGA.
Dirt demodicid mite II allergoid Der p2 and Der f2 are extracted from the demodicid mite body by Lind, and Mr is 14KD, and pI is 5.0 to 8.3.Der p2 and Der f2 aminoacid sequence and physicochemical property all similar all show as the stability to heat and acid.The most of homology of cDNA sequence analysis and epididymal proteins, with the combination rate of allergic asthma patients serum IgE be 80%～100%.They are one of topmost compositions of dust mite allergen.
The present invention adopts technique for gene engineering to clone and express preparation reorganization Derp2 and Der f2 albumen in escherichia coli, has overcome a large amount of difficulties of extracting from the demodicid mite body.Adopt PLGA nanometer adjuvant to prepare dirt demodicid mite recombinant allergen (Der p2/Der f2) vaccine simultaneously, be used for the treatment of allergic asthma.The present invention does not appear in the newspapers both at home and abroad as yet.
Summary of the invention
Purpose of the present invention be exactly utilize have good biological tissue's compatibility, avirulence, biodegradability, can raise the immunoreactive polylactic acid of Th1-poly-alcohol copolymer PLGA prepares novel, safety and effective dirt demodicid mite recombinant allergen vaccine as nanoparticulate carriers.
The present invention adopts technique for gene engineering to clone and express preparation reorganization Derp2 and Der f2 albumen in escherichia coli.Adopt emulsion-solvent volatile diffusion method to prepare polylactic acid-poly-alcohol copolymer (PLGA) nanoparticle simultaneously, make the nanoscale dust acarid allergen vaccine with nanoparticle parcel reorganization Der p2/Der f2 dust mite allergen, and be used for the treatment of allergic asthma.This vaccine has overcome the shortcoming that the thick immersion vaccine quality control of traditional dirt demodicid mite is difficult, the administration cycle is long, in addition, also can reduce the generation of allergy.This production technology is simple, has advantages such as economy and practicality.
The present invention includes and to implement in the following manner.
The specific embodiment
Content of the present invention specifies by following embodiment:
Embodiment 1: prepare recombinant dust mite allergen Der f2 with technique for gene engineering
Genetic engineering recombinant expression plasmid reorganization pET-24a (+)/Der f2 is made up by Liu Zhigang professor seminar of life sciences institute of Shenzhen University.With this plasmid Transformed E coli Bl21 (DE3), picking list colony inoculation contains among the LB of 50ug/ml kanamycin in 20ml, and 37 ℃, 200rpm shakes bacterium and spends the night.Inoculum concentration by 2% will be inoculated in the 1L LB/kam culture fluid, 260rpm shake bacterium to OD600 be 0.6 o'clock, it is 0.2mM that adding IPTG makes its final concentration, after inducing 5 hours, 4 ℃ of centrifugal 10min of 5000g, remove supernatant, precipitation is washed one time with 1 * PBS, antibacterial is resuspended among 40ml 1 * PBS/5mM EDTA, adds lysozyme to final concentration 0.5mg/ml, DTT is to final concentration 10mM, PMSF is to final concentration 1mmol/L, Triton X-100 is to final concentration 1%, and in room temperature frequently oscillation action place-80 ℃ of preservations after half an hour.
Prepare inclusion body by following method.(1) about 10 times of low power ultrasound under the ice bath, stops 30s at super 30s, and till thalline was not sticking, 4 ℃ of centrifugal 15min of 12000g removed supernatant.
(2) under ice bath, with the resuspended precipitation of 100ml inclusion body washing liquid I (50mmol/L PB PH8.0,100mmol/L NaCl, 5mmol/L EDTA), and magnetic agitation 30min, 12000g4 ℃ of centrifugal 15min removes supernatant; Use inclusion body washing liquid II (50mmol/L PB PH8.0 more successively, 100mmol/L NaCl, 5mmol/L EDTA, 2%Triton X-100) inclusion body washing liquid III (50mmol/L PB PH8.0,100mmol/L NaCl, 5mmol/L EDTA, 4mol/L carbamide) resuspended, ice bath magnetic agitation, the centrifugal supernatant that goes, at last precipitation is dissolved in 20ml solubilization of inclusion bodies liquid (50mmol/L PB PH8.0,300mmol/L NaCl, 20mmol/L imidazoles, 6mol/L carbamide) in, the centrifugal 30min of 12000g abandons precipitation.
Because recombinant protein N-end has the 6-His label, so available Ni
2+Metal sequestration chromatography carries out purification.Chelating sepharose Fast Flow is available from enamel Ma Xiya company.By the recombinate affinitive layer purification of Der f2 of following method
Concrete steps are as follows:
(1) gel pre-treatment:
Chelating?Sepharose?F.F
↓
ddH2O?5xCV
↓
0.5N?NaOH?1Xcv
↓
ddH2O?10xCV
↓
50mM?PB、300mM?NaCL?pH8.0?2xCV
↓
0.2M NiSO4 (turing green) up to gel
↓
ddH2O?5xCV
↓
50mM?PB、300mM?NaCL?pH4.0?3-5xCV
↓
50mM?PB、300mM?NaCL?pH8.0?6M?Urea?3-5xCV
(2) go up sample:
20ml Der P2 solubilization of inclusion bodies liquid
↓
50mM PB, 300mM NaCL pH8.0,20mM imidazoles, 6M Urea 3-5xCV
(falling back to baseline) up to the UV value
↓
50mM PB, 300mM NaCL pH8.0,200mM imidazoles, 6M Urea 2xCV
↓
50mM PB, 300mM NaCL pH8.0,400mM imidazoles, 6M Urea 2xCV
↓
Collect the recombiant protein peak by uv absorption.
(3), gel post processing:
50mM?PB、300mM?NaCL?pH8.0?5-10xCV
↓
0.2M?EDTA、300mM?NaCL?pH8.0?5-10xCV
↓
ddH2O?5-10xCV
↓
20％ethanol?5-10xCV
Embodiment 2: prepare recombinant dust mite allergen Der p2 with technique for gene engineering
Genetic engineering recombinant expression plasmid reorganization pET-24a (+)/Der p2 is made up by Liu Zhigang professor seminar of life sciences institute of Shenzhen University.Other preparation methoies are identical with embodiment 1.
Embodiment 3: the preparation of dust mite allergen nanoparticle
Getting 50mg PLGA is dissolved among the 1ml DCM, add 100 μ l recombinant allergen Derp2/Der f2 solution (100mg/ml), ultrasonic 1 minute (40w), as seen the milky colostrum forms, add 2%PVA solution 2ml, ultrasonic once more 1 minute, form emulsion, then this emulsion is transferred among the 50ml ddH2O (pH4.6), stirring at room makes organic solvent volatilization fully more than 4 hours, centrifuge washing three times (the back collecting precipitation of 5000rpm * 15min), again disperse with ddH2O, place the interior frozen drying of freezer dryer 24 hours, and collected exsiccant allergen-PLGA nanoparticle powder, 4 ℃ of preservations.
Embodiment 4: nanoparticle morphologic detection and nano particle diameter analysis
According to solution appearance scoring and scanning electron microscope observation method judge tentatively that particle shape and particle diameter solution appearance are that colloid is aqueous, homogenizing, translucent, milky, placement do not have precipitation in 24 hours, this is qualified nanoscale colloid solution.Mark according to the precipitation situation: do not have precipitation, 0 minute; Precipitation (+), 1 minute; Precipitation (++), 2 minutes; Precipitation (+++), 3 minutes; Precipitation (++ ++), 4 minutes.Scanning electron microscopic observation particle shape size: the lyophilizing particle that takes a morsel, disperse with distilled water, suspension is dropped on the microscope slide, treat its natural drying after the surperficial metal spraying of row handle, scanning electron microscope detects.Measure the maximum intersection length of 100 particulate each particles on stereoscan photograph, grain diameter is the arithmetic mean of instantaneous value of these intersection length.The scanning electron microscopic observation particle shape, method is with aforementioned.Adopt the full-automatic grain analyser of Multisizer to measure the mean diameter and the particle size distribution of nanoparticle.
Embodiment 5: the mensuration of nanoparticle envelop rate
Adopt the BCA method to measure the concentration of dust mite allergen in the supernatant.
Envelop rate=(amount of the dust mite allergen that does not wrap up in dirt demodicid mite recombinant allergen Der p2 or the Der f2 input amount-supernatant)/dust mite allergen input amount * 100%
BCA method protein detection program:
(1) standard substance of preparation bSA serial dilution degree.
(2) prepare detection reagent.
(3) get 96 orifice plates, add standard substance and sample, the 5ul/ hole adds detectable 100ul/ hole by the hole then, hatches 30 minutes for 37 ℃ behind the mixing.
(4) take out orifice plate, to be cooled (optical density, OD) 550nm carries out at the place microplate reader detection in optical density to room temperature.
Embodiment 6: the detection of nanoparticle drug loading
Behind alkaline lysis cracking particle, measure the content of dust mite allergen in the cracking supernatant, directly reflect the drug loading of dust mite allergen-PLGA nanoparticle.
Dust mite allergen amount/particle weight * 100% of drug loading=particle parcel
Take by weighing 10mg dust mite allergen Der p2/Der f2-PLGA nanoparticle, be scattered in the 1ml 0.1M NaOH/1% SDS lysate, spend the night in 37 ℃, the jolting of 50rpm/min constant temperature, lysate is after 50000rpm * 5min is centrifugal, the BCA method detects the content of dust mite allergen in the supernatant, and method is with aforementioned.
Embodiment 7: dirt demodicid mite recombinant allergen Der f2-PLGA nanoparticle release in vitro
Take by weighing 10mg dirt demodicid mite recombinant allergen Der f2-PLGA nanoparticle, be scattered in 1ml phosphate buffer (phosphate buffer saline, PBS) (pH7.4) in, 37 ℃, the jolting of 50rpm/min constant temperature, in the centrifugal collection supernatant of different time points sample, and replenishing isopyknic fresh PBS, parameter of noncentricity is the same.Sample time: 0,2,4,8,24hr, 2,3,5,7,9,11,14 days.Supernatant sample-20 ℃ preservation, the BCA method was carried out protein determination after all sample collection finished, and drew the cumulative in vitro release profiles.
Embodiment 8: dirt demodicid mite recombinant allergen Der p2-PLGA nanoparticle release in vitro
Take by weighing 10mg dirt demodicid mite recombinant allergen Der p2-PLGA nanoparticle, be scattered in 1ml phosphate buffer (phosphate buffer saline, PBS) (pH7.4) in, 37 ℃, the jolting of 50rpm/min constant temperature, in the centrifugal collection supernatant of different time points sample, and replenishing isopyknic fresh PBS, parameter of noncentricity is the same.Sample time: 0,2,4,8,24hr, 2,3,5,7,9,11,14 days.Supernatant sample-20 ℃ preservation, the BCA method was carried out protein determination after all sample collection finished, and drew the cumulative in vitro release profiles.
Embodiment 9:Der p2-PLGA nanoparticle immunization therapy allergic asthma
Animal is adopted BALB/c mouse, 6-8 age in week is divided into 5 groups by the random digit method of dividision into groups, 6 every group, is respectively (1) normal group (2) model group (3) blank nanometer treatment group (4) Der p2 (5) Der p2-PLGA treatment group.At 0,7,14 day, the 2-5 group thick immersion of dirt demodicid mite (100ug/ml)+10mg/mlAl (OH)
3Intraperitoneal sensitization.At 28,29,30 days, the 2-5 group gave the reagent corresponding treatment.At 44-46 days, the 2-5 group gave 50ug/50ul Der p2 collunarium continuously and excites, and excites back 24 hours, with all sacrifice of animal, carried out the effect of experimental evaluation treatment asthma such as bronchoalveolar lavage, the inspection of lung tissue pathology.Experiment shows that Der p2-PLGA nanoparticle immunization therapy allergic asthma is respond well.Embodiment 10:Der f2-PLGA nanoparticle immunization therapy allergic asthma
Animal is adopted BALB/c mouse, 6-8 age in week is divided into 5 groups by the random digit method of dividision into groups, 6 every group, is respectively (1) normal group (2) model group (3) blank nanometer treatment group (4) Der f2 (5) Der f2-PLGA treatment group.At 0,7,14 day, the 2-5 group thick immersion of dirt demodicid mite (100ug/ml)+10mg/mlAl (OH)
3Intraperitoneal sensitization.At 28,29,30 days, the 2-5 group gave the reagent corresponding treatment.At 44-46 days, the 2-5 group gave 50ug/50ul Der p2 collunarium continuously and excites, and excites back 24 hours, with all sacrifice of animal, carried out the effect of experimental evaluation treatment asthma such as bronchoalveolar lavage, the inspection of lung tissue pathology.Experiment shows that Der f2-PLGA nanoparticle immunization therapy allergic asthma is respond well.
Embodiment 11:Der p2/Der f2-PLGA nanoparticle combined immunization treatment allergic asthma
Animal is adopted BALB/c mouse, 6-8 age in week is divided into 5 groups by the random digit method of dividision into groups, 6 every group, is respectively (1) normal group (2) model group (3) blank nanometer treatment group (4) Der p2+Der f2 (5) Der p2-PLGA+Der f2-PLGA treatment group.At 0,7,14 day, the 2-5 group thick immersion of dirt demodicid mite (100ug/ml)+10mg/mlAl (OH)
3Intraperitoneal sensitization.At 28,29,30 days, the 2-5 group gave the reagent corresponding treatment.At 44-46 days, the 2-5 group gave 50ug/50ul Der p2 collunarium continuously and excites, and excites back 24 hours, with all sacrifice of animal, carried out the effect of experimental evaluation treatment asthma such as bronchoalveolar lavage, the inspection of lung tissue pathology.Experiment shows that Der p2/Der f2-PLGA nanoparticle combined immunization treatment allergic asthma is respond well.
Claims (6)
1, a kind of applying nano particle carrier prepares the method for dust acarid allergen vaccine.Specifically adopt technique for gene engineering in escherichia coli, to clone and express preparation reorganization Der p2/Derf2 albumen.Adopt emulsion-solvent volatile diffusion method to prepare the nanoparticle of PLGA parcel.Judge particle shape according to solution appearance scoring and scanning electron microscope observation method, measure the mean diameter of nanoparticle with grain analyser.The BCA method is measured envelop rate, the drug loading of nanoparticle and is drawn nanoparticle cumulative in vitro release profiles.By zoopery proof Der p2/Der f2-PLGA nanoparticle the allergic asthma mice there is therapeutical effect.
2, the Der p2 dirt demodicid mite recombinant allergen nanoparticle for preparing the PLGA parcel by emulsion-solvent volatile diffusion method according to claim 1.
3, the Der f2 dirt demodicid mite recombinant allergen nanoparticle for preparing the PLGA parcel by emulsion-solvent volatile diffusion method according to claim 1.
4, be used for the treatment of allergic asthma according to the described Der p2-PLGA of claim 1 nano vaccine.
5, be used for the treatment of allergic asthma according to the described Der f2-PLGA of claim 1 nano vaccine.
6, unite treatment according to claim 1 described Der p2-PLGA nano vaccine and Der f2-PLGA nano vaccine to allergic asthma.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008092902A1 (en) * | 2007-01-30 | 2008-08-07 | Alk-Abelló A/S | Use of a first house dust mite group 2 allergen for treating allergy to a second house dust mite group 2 allergen |
| CN102258780A (en) * | 2011-07-15 | 2011-11-30 | 北京新华联协和药业有限责任公司 | Lyophilized mite allergen vaccine and preparation method thereof |
| CN104524565A (en) * | 2014-12-24 | 2015-04-22 | 南华大学 | New Der p1 nano vaccine for treating lung cancer as well as preparation method and application thereof |
| WO2019170584A1 (en) * | 2018-03-05 | 2019-09-12 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and composition for the treatment of allergic asthma |
-
2005
- 2005-04-07 CN CN 200510033959 patent/CN1706494A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008092902A1 (en) * | 2007-01-30 | 2008-08-07 | Alk-Abelló A/S | Use of a first house dust mite group 2 allergen for treating allergy to a second house dust mite group 2 allergen |
| CN102258780A (en) * | 2011-07-15 | 2011-11-30 | 北京新华联协和药业有限责任公司 | Lyophilized mite allergen vaccine and preparation method thereof |
| CN104524565A (en) * | 2014-12-24 | 2015-04-22 | 南华大学 | New Der p1 nano vaccine for treating lung cancer as well as preparation method and application thereof |
| WO2019170584A1 (en) * | 2018-03-05 | 2019-09-12 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and composition for the treatment of allergic asthma |
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