CN108392498A - Mouse model is immunized in the bacterium attenuated live vaccine liquid aersol lung delivering of cloth Shandong - Google Patents
Mouse model is immunized in the bacterium attenuated live vaccine liquid aersol lung delivering of cloth Shandong Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/095—Neisseria
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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Abstract
It is delivered the invention discloses cloth Shandong bacterium attenuated live vaccine liquid aersol lung and mouse model is immunized.The present invention is based on cloth Shandong bacterium 104M vaccine strains; using BALB/c mouse as research object; construct a kind of new mouse immune model; safety of the cloth Shandong bacterium 104M vaccine strains in Mice Body and protection are studied and assessed, BALB/c mouse is used to demonstrate cloth Shandong bacterium 104M vaccine strains as the immunogenicity and effect of vaccine.The result shows that：Cloth Shandong bacterium solution aerosol lung route of delivery is a kind of effective immunization route, 105CFU cloth Shandong bacterium 104M can excite the immune response based on BALB/c mouse cellular immunity, and poison performance significant protective effect is attacked to cloth Shandong bacterium solution aerosol.For seek cloth Shandong bacterium 104M vaccines be further improved measure and to its immunologic mechanism research lay the foundation.
Description
Technical field
The invention belongs to biotechnologies, and in particular to bacterium attenuated live vaccine liquid aersol lung delivering in cloth Shandong is immune small
Mouse model.
Background technology
Cloth Shandong bacterium is a kind of gram negative bacilli, Arbo infectious disease cloth caused by infection people, domestic animal and wild animal
Shandong bacterium disease (abbreviation cloth disease).Treatment for cloth disease, still lacks effective measures at present, and vaccine inoculation is a kind of control and reduction
The popular most effective method of cloth disease.The use of animal vaccine largely reduces the propagation of cloth disease, the control to cloth disease
It plays an important role.The cloth Shandong bacteria vaccine of existing research has：Inactivated vaccine, DNA vaccination, subunit vaccine, carrier bacterin, recombinant protein
Vaccine, attenuated live vaccine etc., wherein that prevention animal brucellosis used at present is attenuated live vaccine S19, S2, M5, RB51,
People has 19-B and 104M with attenuated live vaccine.The introducing of 104M is instead of 19-B.The existing attenuated live vaccine used has and causes to move
Logistics production is caused a disease, and the side effects such as interference serodiagnosis, the research and development for carrying out new generation vaccines such as safely and effectively overcoming the deficiency are current
Research hotspot, there are many drawbacks, protecting effect drawn game of the different immunization routes to body using vaccine itself for existing
The research of the difference of portion's side effect, transformation and best immunization route to existing bacterial strain is to improve immune effect, mitigates side effect
Two important methods.
Cloth Shandong bacterium 104M is separated derived from Soviet Studies person from miscarriage ox tire, and carries out the attenuated strain of reduction preparation,
Once used in China group of people at high risk, the vaccine in actual use and China to cloth Shandong bacterium 104M to the related Research Literature of people
Report thinks that cloth Shandong bacterium 104M can provide people certain protection, but has the serious toxic side effect such as sensitization, pathogenic, in recent years
To deactivate.The vaccine for man for not obtaining international endorsement still at present reports relatively the animal immune experimental study of 104M
It is few.
Invention content
It is an object of the present invention to provide a kind of methods preparing animal model.
The method provided by the invention for preparing animal model includes the following steps：
A1) brucellosis vaccine in a manner of liquid aersol is delivered to the lung of animal, obtain animals following immunization mould
Type；
A2) animals following immunization is carried out in a manner of liquid aersol with cloth Shandong bacterium to attack poison, obtains animal model.
In the above-mentioned method for preparing animal model, the A1) in, the brucellosis vaccine is cloth Shandong bacterium attenuated live epidemic disease
Seedling；The cloth Shandong bacterium attenuated live vaccine concretely cloth Shandong bacterium attenuated live vaccine 104M；
The A2) in, cloth Shandong bacterium is specially cloth Shandong bacterium A19 bacterial strains.
It is a further object to provide a kind of methods of screening brucellosis vaccine.
The method of screening brucellosis vaccine provided by the invention includes the following steps：
B1) brucellosis vaccine to be screened is delivered to the lung of animal in a manner of liquid aersol, after being immunized
Animal；
B2) animals following immunization is carried out in a manner of liquid aersol with cloth Shandong bacterium to attack poison, is imitated according to immunoprotection
Fruit, judges whether the brucellosis vaccine to be screened can provide effective protective effect.
In the method for above-mentioned screening brucellosis vaccine, the B1) in, the brucellosis vaccine to be screened is cloth Shandong bacterium
Attenuated live vaccine；The cloth Shandong bacterium attenuated live vaccine concretely cloth Shandong bacterium attenuated live vaccine 104M；
The B2) in, cloth Shandong bacterium is specially cloth Shandong bacterium A19 bacterial strains.
In the above-mentioned method for preparing animal model and screening brucellosis vaccine, the delivering is quantitative delivery；It is described
The delivering amount of cloth Shandong bacterium attenuated live vaccine 104M is 105CFU/ weight is the animal of 18-20g.
In the above-mentioned method for preparing animal model and screening brucellosis vaccine, the cloth Shandong bacterium attenuated live vaccine 104M's
It is 1 time to deliver number；The number for attacking poison is 1 time.
In the above-mentioned method for preparing animal model and screening brucellosis vaccine, by brucellosis vaccine or cloth Shandong to be screened
The lung that bacterium disease vaccine is delivered to animal in a manner of liquid aersol is by brucellosis vaccine or brucellosis epidemic disease to be screened
Seedling is delivered to the lung of animal by liquid aersol lung delivery apparatus；
The method for attacking poison is that cloth Shandong bacterium is delivered to animals following immunization mould by liquid aersol lung delivery apparatus
The lung of type or animals following immunization.
In the above-mentioned method for preparing animal model and screening brucellosis vaccine, the animal is mouse.
In the above-mentioned method for preparing animal model and screening brucellosis vaccine, it is described attack the malicious time be it is immune after the 22nd
Week；The toxic bacterial strain of attacking is cloth Shandong bacterium A19 bacterial strains；It is described attack toxic dose be every mouse (weight 18-20g) 2.5 ×
108CFU。
It is a still further object of the present invention to provide a kind of brucellosis vaccines.
The unit mouse immune dosage of brucellosis vaccine provided by the invention is 105CFU cloth Shandong bacterium attenuated live vaccine
104M；The unit mouse immune dosage is the dosage for the mouse primary immunization for being 18-20g for weight.
It being used to prepare the bacterium solution aerosol lung delivering of cloth Shandong it is a still further object of the present invention to provide one kind and mouse mould is immunized
Type or the kit for screening brucellosis vaccine.
Kit provided by the invention includes brucellosis vaccine and liquid aersol lung delivery apparatus.
In mentioned reagent box, cloth Shandong bacteria vaccine is cloth Shandong bacterium attenuated live vaccine 104M.
The kit further includes that cloth Shandong bacterium attacks toxic bacterial strain.Cloth Shandong bacterium attacks toxic bacterial strain concretely cloth Shandong bacterium A19 bacterium
Strain.
Final object of the present invention is to provide following C1)-C5) any application：
C1 the animal model or above-mentioned vaccine or liquid aersol lung delivery apparatus that) prepared by the above method are in screening cloth Shandong bacterium
Application in disease vaccine；
C2 the animal model or the above method or above-mentioned vaccine or mentioned reagent box or liquid aersol that) prepared by the above method
Application of the lung delivery apparatus in studying brucellosis vaccine prevention mechanism；
C3 the animal model or the above method or above-mentioned vaccine or mentioned reagent box or liquid aersol that) prepared by the above method
Application of the lung delivery apparatus in evaluating brucellosis vaccine immunity protecting effect；
C4) liquid aersol lung delivery apparatus answering in preparing the immune mouse model of cloth Shandong bacterium solution aerosol lung delivering
With；
C5) liquid aersol lung delivery apparatus and brucellosis vaccine are to prepare the bacterium solution aerosol lung delivering of cloth Shandong immune
Application in mouse model.
The present invention is based on cloth Shandong bacterium 104M vaccine strains, using BALB/c mouse as research object, construct a kind of new mouse
Immune model is studied and is assessed to safety of the cloth Shandong bacterium 104M vaccine strains in Mice Body and protection, uses BALB/c
Mouse demonstrates immunogenicity and effect of the cloth Shandong bacterium 104M vaccine strains as vaccine.The result shows that：Cloth Shandong bacterium solution aerosol
Lung route of delivery is a kind of effective immunization route, 105CFU cloth Shandong bacterium 104M can be excited based on BALB/c mouse cellular immunity
Immune response attacks poison performance significant protective effect to cloth Shandong bacterium solution aerosol.It is further to seek cloth Shandong bacterium 104M vaccines
Corrective measure and to its immunologic mechanism research lay the foundation.
Description of the drawings
Fig. 1 changes for mouse weight.(a) it is mouse weight dynamic change after M1, M2 are immunized 4,8,16,24 weeks；(b) it is
Mouse weight dynamic change after M3, M4 are 4,8,16,24 weeks immune；(c) it is that mouse weight is dynamic after M5, M6 are immunized 4,8,16,24 weeks
State changes；(d) it is mouse weight dynamic change after M1, M3, M5 are immunized 4,8,16,24 weeks.Wherein, M1 is that liquid aersol lung is passed
Send inoculation 105CFU cloth Shandong bacterium 104M；M2 is the life of liquid aersol lung delivering 0.05% (percent by volume) poloxamer of inoculation
Manage brine；M3 is that collunarium is inoculated with 105CFU cloth Shandong bacterium 104M；M4 is that collunarium inoculation contains 0.05% (percent by volume) poloxamer
Physiological saline；M5 is inoculated with subcutaneous injections 105CFU cloth Shandong bacterium 104M；M6 is that inoculated with subcutaneous injections contains 0.05% (volume basis
Than) physiological saline of poloxamer.
Fig. 2 changes for mouse spleen weight.(a) it is mice spleen weight dynamic change after M1, M2 are immunized 4,8,16,24 weeks；(b)
Mice spleen weight dynamic change after being immunized 4,8,16,24 weeks for M3, M4；(c) it is mice spleen weight after M5, M6 are immunized 4,8,16,24 weeks
Dynamic change；(d) it is mice spleen weight dynamic change after M1, M3, M5 are immunized 4,8,16,24 weeks.
Fig. 3 is that mouse spleen carries bacterium amount variation.(a) it is that mouse spleen carries bacterium amount dynamic after M1, M2 are immunized 4,8,16,24 weeks
Variation；(b) it is that mouse spleen carries bacterium amount dynamic change after M3, M4 are immunized 4,8,16,24 weeks；(c) it is M5, M6 immune 4,8,16,
Mouse spleen carries bacterium amount dynamic change after 24 weeks；(d) it is that mouse spleen carries bacterium amount dynamic after M1, M3, M5 are immunized 4,8,16,24 weeks
Variation.
Fig. 4 changes for mice serum antibody level.(a) it is mice serum Antibody dynamics after M1, M2 are immunized 4,8,16,24 weeks
Variation；(b) it is that mice serum Antibody dynamics change after M3, M4 are immunized 4,8,16,24 weeks；(c) it is M5, M6 immune 4,8,16,24
Mice serum Antibody dynamics change after week；(d) it is that mice serum Antibody dynamics change after M1, M3, M5 are immunized 4,8,16,24 weeks.
Fig. 5 changes for cytokine concentrations.A is mice serum cell factor after M1, M3, M5 4,8,16,24 weeks immune
IFN-γ (a) and IL-18 (b) concentration dynamic changes；B be M1, M3, M5 after immune 4,8,16,24 weeks mouse lung homogenate cell because
Sub- IFN-γ (a) and IL-18 (b) concentration dynamic changes；C is that 24 weeks (attacking after poison 2 weeks) mouse are immunized in M1, M2, M3, M4, M5, M6
Serum cytokines IFN-γ (a) and IL-18 (b) concentration dynamic changes；D is that M1, M2, M3, M4, M5, M6 (attack poison in immune 24 weeks
2 weeks afterwards) mice serum cell factor IFN-γ (a) and IL-18 (b) concentration dynamic changes.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
Experimental animal in following embodiments and bacterium source：The BALB/c mouse of 6-8 week old, female, weight 18-24g,
Purchased from from Beijing Vital River Experimental Animals Technology Co., Ltd., experiment mice gives sufficient water and food.
Cloth Shandong bacterium 104M bacterial strains in following embodiments are purchased from Lanzhou Institute of Biological Products；Cloth Shandong bacterium A19 bacterial strains are purchased from
Xinjiang Tian Mu companies.
Reagent in following embodiments and instrument：BBLTMBrucella Agar culture mediums, BD companies, the U.S.；BBLTM
Brucella Broth culture mediums, BD companies, the U.S.；TSA Tryptic Soy Agar culture mediums, BD companies, the U.S.；TSB
Tryptic Soy Broth culture mediums, BD companies, the U.S.；Brucella selective supplement, OXOID companies,
Britain；PLURONICS F87 PlunronicF68, sigma companies, the U.S.；ELISA kit, Shenzhen have up to section for biotechnology
Limit company, Shenzhen；Cytokine detection kits, e Bioscience companies, the U.S.；Small animal laryngoscope, the intelligent flourish and section in Beijing
Skill Co., Ltd, Beijing；Hand-held liquid aerosol lung delivery apparatus, the intelligent flourish and Science and Technology Ltd. in Beijing, Beijing.
The physiological saline containing 0.05% (volumn concentration) poloxamer in following embodiments is by PLURONICS F87
The solution being uniformly mixed so as to obtain with physiological saline, and the volumn concentration of poloxamer in the solution is 0.05%, physiological saline is
The Nacl aqueous solutions that mass percentage is 0.9%.
Embodiment 1, a kind of delivering of liquid aersol lung prepare the method that mouse model is immunized
One, experimental method
1, the preparation of cloth Shandong bacterium 104M vaccine strains and animal inoculation pvaccination
(1) preparation of cloth Shandong bacterium 104M vaccine strains
The 50 μ l of glycerol stock containing cloth Shandong bacterium 104M bacterial strains are taken to be inoculated in brucella broth culture medium (brucella borth)
In, shaken cultivation obtains 104M bacterium solutions to mid-log phase (OD600 ≈ 1.0) under the conditions of 200rpm, 37 DEG C.By 104M bacterium solutions
10min is centrifuged under the conditions of 6000rpm, 4 DEG C, collects thalline.With the physiology containing 0.05% (volumn concentration) poloxamer
Brine washing thalline twice, is resuspended to final concentration of 105The 104M bacterium solutions of CFU/ml are used for subsequent experimental.
(2) animal inoculation pvaccination
This experiment is divided into lung delivering, collunarium, the immune mouse of three kinds of approach is subcutaneously injected, each immunization route is divided into 104M epidemic diseases
Seedling experimental group and the physiological saline blank control group for containing 0.05% (volumn concentration) poloxamer, are divided into 6 groups, every group 30
Mouse (setting 5 time points, 6 mouse of each time point altogether)：
M1 groups (lung delivery experiment group)：Use hand-held liquid aerosol lung delivery apparatus by 50 μ l final concentrations by laryngoscope
It is 2 × 106The 104M bacterium solutions of CFU/ml are delivered to mouse lung；
M2 groups (lung delivering blank control)：50 μ l are contained using hand-held liquid aerosol lung delivery apparatus by laryngoscope
The physiological saline of 0.05% (volumn concentration) poloxamer is delivered to mouse lung；
M3 groups (collunarium experimental group)：By 20 μ l pipettors, 20 μ l final concentration of 5 × 106The 104M bacterium solutions of CFU/ml deliver
To mouse nostril；
M4 groups (collunarium blank control group)：20 μ l are contained into 0.05% (volumn concentration) Bo Luosha by 20 μ l pipettors
The physiological saline of nurse is delivered at mouse nostril；
(experimental group is subcutaneously injected) in M5 groups：By 1ml syringes by 100 μ l final concentration of 1 × 106The 104M bacterium of CFU/ml
It is subcutaneous that liquid is delivered to mouse；
(blank control group is subcutaneously injected) in M6 groups：100 μ l are moored containing 0.05% (volumn concentration) by 1ml syringes
It is subcutaneous that the physiological saline of Luo Shamu is delivered to mouse.
2, clinical sign is observed
Mouse symptom is observed and recorded within 0th, 4,8,16,24 week after immune (alarms hair, expiratory dyspnea, mandatory abdomen
Portion breathes, by slow in reacting after touch or outside stimulus).
3, weight, spleen weight and spleen carry the measurement of bacterium amount
It weighs each group mouse weight within 0th, 4,8,16,24 week after immune, records data.After plucking eyeball blood sampling, receive
It is spare to collect serum.Cervical dislocation is put to death, and mouse is dissected, and sterile separating spleen is weighed, and is homogenized, phosphate-buffered (PBS, PH7.2)
It is serially diluted into different concentration gradients, is coated on Bu Shi selective mediums (brucella selective
Supplement is purchased from OXOID companies of Britain, and when use takes 1 to draw deionized water dilution, shakes up, and 37 DEG C of placement 15min add
Enter 50 DEG C, 500ml TSA culture mediums, mixes well, be down flat plate, cooling), each dilution does 3 repetitions, places 37 DEG C of incubators
Middle inversion is cultivated 3~4 days, and Colony Forming Unit (CFU) is counted, and takes each cell mean, is calculated spleen and is carried bacterium amount CFU/g, spleen
Bacterium amount is carried with log10CFU/g means standard deviations indicate.
4, mice serum antibody determination
4th, 8,16,24 week after immune, pluck eyeball and take blood, blood is stood overnight in 4 DEG C, 3000rpm centrifugations
5min collects serum, and -20 DEG C save backup.According to ELISA kit specification operating method, immunized mice serum is measured
Antibody titer (IgG, IgG1, IgG2a and IgA).It is as follows：Adjust cloth Shandong bacterium 104M a concentration of 109CFU/ml, heating
Inactivation, 100 holes μ l/ of coated elisa plate, 37 DEG C of incubation 2h, board-washing pat dry, and the serum that multiple proportions is serially diluted, 37 DEG C of incubations are added
30min, board-washing pat dry, then are separately added into the secondary antibody (sheep anti mouse) of HRP labels, and 37 DEG C of incubation 20min, board-washing pats dry.Bottom is added
Object is protected from light and is incubated 15min, and the static 5min of terminate liquid is added, and the absorbance value of 450nm and 630nm is read in microplate reader.
5, mice serum and lung homogenate cytokines measurement
4th, 8,16, the 24 week sterile separation lungs after immune are homogenized, by the lung tissue 3000rpm after homogenate from
Heart 5min collects lung homogenate supernatant, and -20 DEG C save backup.According to cytokine detection kits specification, immune rear blood is measured
The variation of cell factor (IFN-γ and IL-18) concentration in cleer and peaceful lung homogenate supernatant.
6, aerosol attacks the protection experiment of poison
20th week after immune, mouse spleen cloth Shandong bacterium is fully erased, the 22nd week, and every group of mouse (n=6) gives cloth Shandong bacterium
A19 bacterium solutions (preparation method of the preparation method of cloth Shandong bacterium A19 bacterium solutions with the 104M bacterium solutions in (1) of step 1) are molten with liquid gas
Glue lung delivering mode carries out attacking poison, and every mouse is with final concentration 5 × 109The cloth Shandong bacterium A19 bacterium solutions of CFU/ml (attack 50 μ l of poison；
2 weeks (24 weeks immune) acquires sample after attacking poison, and sample collection method is same as above.And carry out mouse according to the method described above and weigh, it takes a blood sample,
It takes lung spleen to weigh homogenate, counts spleen lungs and carry bacterium amount, prepare serum and lung homogenate supernatant, detect serum and lung homogenate supernatant
In antibody and cell factor, detection method be same as above.
7, data analysis
It is for statistical analysis to data result using SAS statistical softwares.Splenomegaly is with the average value ± standard of spleen weight
Difference calculates.Compared respectively with variance analysis between each group of data, P<0.05, indicate that difference is statistically significant.
Two, experimental result
1, clinical manifestation
Blank control group M2, M4, M6 and 104M immune group M1, M3, M5 group mouse state is good, tired without towering hair, breathing
The performances such as difficult, mandatory abdominal respiration, slow in reacting.
2, changes of weight
The results are shown in Figure 1 for changes of weight.M1, M2 group mouse weight continued smooth rise, and (24 weeks) M1 goes out after attacking poison
Existing downward trend, each time point after immune, M1<M2, P>0.05, it is not statistically significant.In when detecting, M3 and M4, M5
With M6 mouse weights without significant difference, P>0.05, it is not statistically significant；M1, M3, M5 group difference are not statistically significant.
3, spleen changes again
Spleen weight result of variations is as shown in Figure 2.The 4th week and the 8th week, compared with blank control group M2, M4, M6,104M exempted from
Epidemic disease group M1, M3, M5 group spleen weight slightly increases, about 0.2g, P>0.05, it is not statistically significant；In 24 weeks immune, blank control
Group M2, M4, M6 spleen dramatically increases again, M1<M2, M3<M4, M5<M6, P<0.05, it is statistically significant.Illustrate to attack mice spleen after poison
Dirty weight dramatically increases, and 104M immunized mice spleen weight increases are not notable.4th week, 8 weeks, 16 weeks after immune, 104M
Immune group M1, M3, M5 group difference is not statistically significant, and 24th week after immune, M1 is significantly higher than M3, M5, P<0.05, there is system
Meter learns meaning, illustrates that the more other approach of lung route of delivery cause obvious splenomegaly.
4, spleen carries bacterium amount variation
It is as shown in Figure 3 that spleen carries bacterium amount result of variations.At the 4th week, 8 weeks, 16 weeks, with blank control group M2, M4, M6 phase
Than 104M immune group M1, M3, M5 mouse spleens carry bacterium amount and dramatically increase, M1>M2, M3>M4, M5>M6, P<0.05, there is system
Meter learns meaning；Extend with immunization time, spleen carries bacterium amount and declines, and is inoculated with the 20th week, all mouse spleen bacterium are fully erased；Exempting from
The 24th week after epidemic disease, 104M immune groups M1, M3, M5 are remarkably decreased compared with blank control, and spleen carries bacterium amount decrement 2.6-3.2
(log10CFU/g), M1<M2, M3<M4, M5<M6, P<0.05, it is statistically significant；Compare between M1, M3, M5, P>0.05,
It is not statistically significant.Illustrate that cloth Shandong bacterium 104M can provide preferable protection using three kinds of immunization routes for BALB/c mouse and make
With.
5, antibody level detects
Antibody level testing result is as shown in Figure 4.Three kinds of immunization route serum IgGs, IgG2a/IgG1, IgA antibody titre
There is almost the same variation tendency：There is serum antibody within immune 4 weeks, peak within 8 weeks, slight decline occurs within 16 weeks, attack poison
2 weeks mice serum antibody was increased compared with 16 weeks afterwards, and after attacking poison, immune group M1, M3, M5 obviously higher than blank control group M2,
M4, M6, P<0.05, difference is statistically significant；Without significant difference between three kinds of approach.It attacks after poison 2 weeks, 104M immune groups M1,
M3, M5 serum antibody IgM substantially less than blank control group M2, M4, M6, P<0.05, difference is statistically significant, it may be possible to by
More obvious time dependence is shown in IgM.Each time point serum IgG 2a/IgG1 after immune>1, IgG antibody 2a is horizontal
It is above IgG1, illustrates that cellular immunity plays more obviously effect.The antibody level of serum difference of three kinds of immunization routes is without system
Meter learns meaning.The result shows that attack after poison cloth Shandong bacterium 104M induce BALB/c mouse serum antibody IgG, IgG1, IgG2a, IgA compared with
Significant the acquired immune response；Wherein serum antibody IgG2 is higher than IgG1, and it is more aobvious to show that cell immune response persistently plays
The effect of work.
6, cytokines measurement
The results are shown in Figure 5.Mouse is delivered by liquid aersol lung, collunarium and subcutaneous routes are inoculated with 105CFU cloth
There is almost the same variation tendency in Shandong bacterium 104M, immune group M1, M3, M5 serum cytokines IFN-γ, IL-18 concentration：Exempt from
There is peak within 4 weeks after epidemic disease, continued to decline later by 16 weeks, after being immunized 24 weeks (attacking after poison 2 weeks), cell factor IL-18 significantly increases
Add, and IFN-γ increase is not notable.105CFU cloth Shandong bacterium 104M immune groups M1, M3, M5 detected lung 4 weeks, 8 weeks, 16 weeks
It is horizontal to be homogenized high concentration cell factor IFN-γ, IL-18, attacks after poison 2 weeks, all occurs significantly increasing, P<0.05, significant difference has
Statistical significance.In immunologic process, three kinds of approach no significant differences, P>0.05.2 weeks after attacking poison, serum I FN- γ,
IL-18 concentration：104M immune groups M1, M3, M5 are significantly higher than blank control group M2, M4, M6, P<0.05, significant difference has statistics
Learn meaning.Lung homogenate IFN-γ, IL-18 concentration：M1<M2、M3<M4、M5<M6, P<0.05, significant difference is statistically significant.
Three kinds of immunization routes are without significant difference, P>0.05.4,8,16 weeks after immune, blank control group M2, M4, M6 serum and lung homogenate
Cytokine concentrations are relatively low, are not detected, and do not shown in figure.The result shows that it is small to attack the immune induction BALB/c of cloth Shandong bacterium 104M after poison
Mouse lung homogenate high level cell factor IFN-γ, IL-18 illustrate that attacking poison excites mouse locally stronger cell immune response.
Serum cytokine concentrations are less than blank control group instead, it may be possible to which larger due to attacking toxic dose, immune group swashs in a short time
Send out cell factor higher, cell factor largely consumes, and is less than blank control group within 2 weeks after attacking poison.
The application of mouse model is immunized in the bacterium 104M vaccine liquid aersol lungs delivering of cloth Shandong：Vaccine prevention Mechanism Study, epidemic disease
Seedling effect assessment is compared with etc..
Claims (10)
1. a kind of method preparing animal model, includes the following steps：
A1) brucellosis vaccine in a manner of liquid aersol is delivered to the lung of animal, obtain animals following immunization model；
A2) the animals following immunization model is carried out attacking poison in a manner of liquid aersol with cloth Shandong bacterium, obtains animal model.
2. according to the method described in claim 1, it is characterized in that：
The A1) in, the brucellosis vaccine is cloth Shandong bacterium attenuated live vaccine；The cloth Shandong bacterium attenuated live vaccine is specially cloth
Shandong bacterium attenuated live vaccine 104M；
Or, the A2) in, the Shandongs Jun Weibu, the cloth Shandong bacterium A19 bacterial strains.
3. method according to claim 1 or 2, it is characterised in that：The delivering is quantitative delivery；
Or, the lung that brucellosis vaccine is delivered to animal in a manner of liquid aersol；
Or, described carry out attacking poison being to lead to cloth Shandong bacterium in a manner of liquid aersol with cloth Shandong bacterium to the animals following immunization model
Cross the lung that liquid aersol lung delivery apparatus is delivered to animals following immunization model；
Or, the delivering amount of the cloth Shandong bacterium attenuated live vaccine 104M is 105CFU/ weight is the animal of 18-20g；
Or, described attack the malicious time as immune the 22nd week afterwards；
Or, the delivering number of the cloth Shandong bacterium attenuated live vaccine 104M is 1 time；
Or, the number for attacking poison is 1 time；
Or, the animal is mouse.
4. a kind of method of screening brucellosis vaccine, includes the following steps：
B1) brucellosis vaccine to be screened in a manner of liquid aersol is delivered to the lung of animal, obtain animals following immunization；
B2) animals following immunization is carried out in a manner of liquid aersol with cloth Shandong bacterium to attack poison, according to immune protective effect, is sentenced
Whether the brucellosis vaccine to be screened that breaks can provide effective protective effect.
5. according to the method described in claim 4, it is characterized in that：
The B1) in, the brucellosis vaccine to be screened is cloth Shandong bacterium attenuated live vaccine；The cloth Shandong bacterium attenuated live vaccine tool
Body is cloth Shandong bacterium attenuated live vaccine 104M；
Or, the B2) in, the Shandongs Jun Weibu, the cloth Shandong bacterium A19 bacterial strains.
6. method according to claim 4 or 5, it is characterised in that：The delivering is quantitative delivery；
Or, the lung that brucellosis vaccine to be screened is delivered to animal in a manner of liquid aersol is by cloth to be screened
Shandong bacterium disease vaccine is delivered to the lung of animal by liquid aersol lung delivery apparatus；
Or, described carry out attacking poison being that cloth Shandong bacterium is passed through liquid in a manner of liquid aersol with cloth Shandong bacterium to the animals following immunization
Aerosol lung delivery apparatus is delivered to the lung of animals following immunization；
Or, the delivering amount of the cloth Shandong bacterium attenuated live vaccine 104M is 105CFU/ weight is the animal of 18-20g；
Or, described attack the malicious time as immune the 22nd week afterwards；
Or, the delivering immune time of the cloth Shandong bacterium attenuated live vaccine 104M is 1 time；
Or, it is 1 time that malicious number is attacked in the delivering of the cloth Shandong bacterium A19；
Or, the animal is mouse.
7. a kind of brucellosis vaccine, it is characterised in that：Unit mouse immune dosage is 105CFU cloth Shandong bacterium attenuated live vaccine
104M；The unit mouse immune dosage is the dosage for the mouse primary immunization for being 18-20g for weight.
8. a kind of reagent for being used to prepare the bacterium solution aerosol lung delivering of cloth Shandong and mouse model or screening brucellosis vaccine being immunized
Box comprising brucellosis vaccine and liquid aersol lung delivery apparatus.
9. kit according to claim 9, it is characterised in that：
The brucellosis vaccine is cloth Shandong bacterium attenuated live vaccine 104M；
Or, the kit, which further includes cloth Shandong bacterium, attacks toxic bacterial strain.
10. following C1)-C5) any application：
C1 the vaccine or liquid aersol described in animal model or claim 7 that) prepared by any the methods of claim 1-3
Application of the lung delivery apparatus in screening brucellosis vaccine；
C2 animal model or claim the 4-6 any method or right that) prepared by any the methods of claim 1-3
It is required that the kit or liquid aersol lung delivery apparatus described in vaccine or claim 8 or 9 described in 7 are in research brucellosis
Application in vaccine prevention mechanism；
C3 animal model or claim the 4-6 any method or right that) prepared by any the methods of claim 1-3
It is required that the kit or liquid aersol lung delivery apparatus described in vaccine or claim 8 or 9 described in 7 are in evaluation brucellosis
Application in vaccine immunity protecting effect；
C4) application of the liquid aersol lung delivery apparatus in preparing the immune mouse model of cloth Shandong bacterium solution aerosol lung delivering；
C5) liquid aersol lung delivery apparatus and brucellosis vaccine are preparing the immune mouse of cloth Shandong bacterium solution aerosol lung delivering
Application in model.
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