CN110079473A - A kind of preparation method and applications of pair chicken poultry bacillus outer membrane vesicles - Google Patents
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Abstract
The present invention provides a kind of preparation method and applications of secondary chicken poultry bacillus outer membrane vesicles, using the TSB fluid nutrient medium culture pair chicken poultry bacillus strain after optimization, cell-free culture supernatant is obtained after centrifugation, 0.22 μm of filtration treatment, then ultracentrifugation, gradient density centrifugal prepare the outer membrane vesicles of bacterium release.Outer membrane vesicles prepared by the present invention are used as subunit vaccine for being inoculated with chicken; the experimental results showed that outer membrane vesicles are a kind of efficient immunologic stimulants; can irritant test chicken serum generate high-caliber IgG; it is can effectively protect the infection that chicken resists A type pair chicken poultry bacillus, shows outer membrane vesicles as the preferable application prospect of subunit vaccine.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of preparation method and its epidemic disease of pair chicken poultry bacillus outer membrane vesicles
Seedling application.
Background technique
Secondary chicken poultry bacillus (Avibacterium paragallinarum, Apg) is tiny Gram-negative bacteria, bacterium
It is about 1-3 μm, 0.4-0.8 μm wide, atrichia does not form gemma, and virulent strain often has pod membrane.The bacterium amphimicrobian, solid
When cultivating on body culture medium, anaerobic environment or the CO of 3%-10% are needed2。
Secondary chicken poultry bacillus is the pathogenic bacteria of infectious coryza of chicken (infectious coryza, IC), and one kind of chicken is caused to be exhaled
Inhale road transmission disease.Cause chicken sneezing, flow out thin water sample nasal mucus, nose liquid is gradually sticky and has unpleasant stink, after dry
Yellow crusts are frozen into nares, influence to breathe, occurs eye conjunctiva and facial swelling later or even upper lower eyelid is bonded in
Together, thin, diarrhea makes chicken egg productivity decline of laying eggs, and economic loss is larger.
Vaccine immunity is prevention and the most effective approach that control infectious coryza of chicken occurs, and rear antibody level is immunized
Height reflects actual immune effect of vaccine, is directly related to vaccinated flock to the resistance of secondary chicken poultry bacillus.It is infected about chicken
The prevention of property coryza, was widely used that in the past and obtained the ablation methods such as the thallus of Apg formalin or thimerosal
Vaccine, but this method also has certain defect, and when giving these vaccines, it is secondary to there is the transient strong poison such as sit
Effect.Raiser is desirable to develop a kind of highly-safe vaccine strongly.In this case, it is desirable to develop toxicity at
Divide less subunit vaccine.
Compare inactivated vaccine, and subunit vaccine has better safety, and subunit vaccine only contains a kind of or a few
Kind antigen object can further increase the immunogenicity of subunit vaccine with combination adjuvant, the coupling modes such as polysaccharide or antigen controlled release.
It is handled by antigen controlled-release technology, obtains the atomic small particle for being integrated with antigen object, can preferably stimulate immunity of organism system
System, shows tempting application prospect.How the subunit vaccine of anti-infectious coryza of chicken be urgent need to resolve one is obtained
Technical problem.
Summary of the invention
In order to solve the above-mentioned technical problem, the object of the present invention is to provide the preparations of one logical kind of secondary chicken poultry bacillus outer membrane vesicles
Method.
A kind of preparation method of pair chicken poultry bacillus outer membrane vesicles comprising following steps:
S1, in vitro culture pair chicken poultry bacillus to growth period bacteria log phase are obtained in cell-free culture by being centrifuged, filtering
Clearly；
S2, process at ultracentrifugation is carried out to cell-free culture supernatant, obtains secondary chicken poultry bacillus outer membrane vesicles.
Preparation method as described above, it is preferable that in step sl, the in vitro culture pair chicken poultry bacillus, training used
Supporting base is the TSB fluid nutrient medium containing NAD and serum.
Preparation method as described above, it is preferable that the weight containing NAD in the TSB fluid nutrient medium containing NAD and serum
Measuring percent concentration is 0.01%, and serum is chicken serum, volumetric concentration 10%.
Preparation method as described above, it is preferable that in step sl, the filtering uses 0.22 μm of membrane filtration.
Preparation method as described above, it is preferable that in step s 2, the ultracentrifugation is that cell-free culture supernatant exists
4 DEG C of 150000 × g ultracentrifugation 3h, discard supernatant, and precipitating PBS buffer solution is resuspended；Ibid again 4 DEG C of 150000 × g exceed the speed limit from
Heart 3h, discards supernatant, and precipitating is resuspended in PBS buffer solution, as prepares secondary chicken poultry bacillus outer membrane vesicles.
It is a further object to provide the applications that preparation method as described above obtains secondary chicken poultry bacillus outer membrane vesicles.
Preparation method as described above obtains secondary chicken poultry bacillus outer membrane vesicles in preparing infectious coryza of chicken subunit vaccine
Application.
A kind of infectious coryza of chicken subunit epidemic disease obtaining secondary chicken poultry bacillus outer membrane vesicles preparation with the preparation method
Seedling.
Preparation method as described above obtains secondary chicken poultry bacillus outer membrane vesicles and is used as immunologic stimulant.
The beneficial effects of the present invention are:
The preparation method of pair chicken poultry bacillus outer membrane vesicles provided by the invention, this method can be prepared effectively outside secondary chicken poultry bacillus
Membrane vesicle, purification efficiency is high, application of the secondary chicken poultry bacillus outer membrane vesicles of acquisition as antigen in terms of vaccine, after optimization
TSB fluid nutrient medium culture pair chicken poultry bacillus strain, through centrifugation, obtain cell-free culture supernatant after 0.22 μm of filtration treatment,
Then ultracentrifugation, gradient density centrifugal prepare the outer membrane vesicles of bacterium release, by the visible major part of transmission electron microscope observing
Between outer membrane vesicles diameter 20nm-300nm, using outer membrane vesicles as subunit vaccine for being inoculated with chicken, it can effectively protect chicken and supports
The infection of imperial A type pair chicken poultry bacillus.Outer membrane vesicles immunity test chicken increases weight for a long time and clinical manifestation and PBS immune group are without significance difference
It is different.Experiment shows that outer membrane vesicles are a kind of efficient immunologic stimulants simultaneously, can not only irritant test chicken serum generation high level
IgG, and generate high-caliber IgA in nasal membrane, effectively have stimulated the mucosa-immune of chicken, show outer membrane vesicle
Bubble is used as the preferable application prospect of subunit vaccine, and the present invention is that the function of the secondary chicken poultry bacillus outer membrane vesicles of further investigation establishes base
Plinth.
Detailed description of the invention
Fig. 1 is the transmission electron microscopy figure of secondary chicken poultry bacillus and its outer membrane vesicles.
Fig. 2 is the transmission electron microscopy figure of the secondary chicken poultry bacillus outer membrane vesicles of purifying.
Fig. 3 is that outer membrane vesicles act on different time to HD-11 cell NO generation influence.
Fig. 4 is that outer membrane vesicles influence HD-11 cell-related inflammation factor expression as a result, wherein A group is IL-1 β, and B group is
IL-6, C group are IL-2, and D group is iNOS, and E group is IL-10.
Fig. 5 is testing result of the outer membrane vesicles to serum IgG antibody levels.
Specific embodiment
Following embodiment should not be construed as limiting the invention for further illustrating the present invention.Without departing substantially from this
Under the premise of spirit and essence, modification or replacement made for the present invention belong to scope of the invention.
The acquirement approach of various biomaterials described in embodiment is to provide a kind of approach of experiment acquisition only to reach
To specifically disclosed purpose, the limitation to biological material source of the present invention should not be become.In fact, used biomaterial
Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according to mentioning in embodiment
Show and is replaced.
Embodiment 1
In vitro culture pair chicken poultry bacillus method are as follows:
1, it activates: the freeze-drying lactobacillus streak inoculation of A type pair chicken poultry bacillus Hp8 bacterial strain is consolidated in the TSA containing chicken serum and NAD
Body culture medium, 5%CO2Under the conditions of, 37 DEG C of culture 12h~16h are inoculated with 5~8 single bacteriums on solid medium and drop down onto 6mL containing NAD
With the TSB fluid nutrient medium of serum, 37 DEG C of shaken cultivation 6h~8h；
Wherein, contain the TSA solid medium of NAD (nicotinamide adenine dinucleotide) are as follows: 15g is added in every 1000mL TSB
Agar powder, 121 DEG C high pressure sterilization 15 minutes, when culture medium temperature is to 50 DEG C, add NAD to 0.01% (v/w), add sterile
The chicken serum 10% of processing, is poured into plate solidifies in right amount；
TSB fluid nutrient medium containing NAD are as follows: weigh 30g TSB, be dissolved in 1000mL deionized water, 121 DEG C of high pressure sterilizations
15 minutes, restore to culture medium to room temperature, add NAD to 0.01% (v/w), adds the chicken serum of aseptic process to final concentration
10%.PBS buffer solution are as follows: 8mM Na2HPO4*12H2O, 1.5mM KH2P04,2.7mM KCl, 136.7mMNaCl, pH 7.4,
121 DEG C high pressure sterilization 15 minutes；
2, expand culture: bacterial cultures being then inoculated with TSB Liquid Culture of the 400mL containing NAD with 1% ratio of volume ratio
Base continues 37 DEG C of shaken cultivations, is 0.4~0.6 to Bacteria Culture to OD600nm；
3, it purifying: collecting in vitro culture pair chicken poultry alphacterium culture, 4 DEG C of 10000 × g are centrifuged 15min, discard precipitating,
0.22 μm of filtering supernatant obtains cell free supernatant object；4 DEG C of 150000 × g ultracentrifugation 3h, discard supernatant, and precipitate with appropriate PBS
Buffer is resuspended；Ibid 4 DEG C of 150000 × g ultracentrifugation 3h again, discard supernatant, and precipitating is resuspended in 3mL PBS buffer solution, i.e.,
For the secondary chicken poultry bacillus outer membrane vesicles of the purifying of preparation.
It is secondary chicken poultry bacillus and its outer membrane vesicles by the secondary chicken poultry alphacterium culture of collection, and purifies secondary chicken poultry bacillus outer membrane
After vesica carries out uranyl acetate negative staining respectively, with transmission electron microscope observing outer membrane vesicles form.And purifying is calculated using BCA method
The concentration of secondary chicken poultry bacillus outer membrane vesicles.
As a result as depicted in figs. 1 and 2, illustrate that secondary chicken poultry bacillus outer membrane vesicles majority form at spherical shape, there is membrane structure,
Its diameter is between 20-300nm.The protein concentration for measuring the outer membrane vesicles of extraction through BCA method is 2.66mg/mL.
Embodiment 2
The present embodiment studies the outer membrane vesicles (Out membrane vesicles, OMVs) of the preparation of embodiment 1 to chicken macrophage
The influence of cell passage cell (HD-11) nitric oxide (nitricox-ide, NO) production quantity
Test is divided into OMVs processing group (5 μ g/ml) and control group, two groups of HD-11 cell (Beijing City Agriculture and Forestry Institute herdings
Veterinary institute save and provide) respectively after treatment for 24 hours, 48h when take culture supernatant, according to NO detection kit (green cloud
It, S0021) operating procedure and calculation formula[12], measure and calculate each group D550Value takes out Griess Reagent I and II,
Make to reply room temperature.Solution (cell culture fluid DMEM+10%FBS) the dilution standard product used in sample to be tested are to various concentration
(0,1,2,5,10,20,40,60,100μM).By 50 holes μ l/, standard items and culture solution Supernatant samples are added in 96 orifice plates.It presses
Room temperature Griess Reagent I is added in 50 holes μ l/ in each hole.By 50 holes μ l/, room temperature Griess is added in each hole
Reagent II.It is placed at room temperature for after five minutes, 540nm measures absorbance.It is horizontal to compare each group NO generation using SPSS.
As a result as shown in figure 3, from the figure 3, it may be seen that for 24 hours when, the NO amount that OMVs group generates rapidly increases, for 10.5 μm of ol/L；
And the NO amount that control group generates is 2.5 μm of ol/L, difference is extremely significant (p < 0.01).When 48h, the NO amount that OMVs group generates is 11 μ
Mol/L, control group NO amount are 4 μm of ol/L, and difference is extremely significant (p < 0.01).Show that OMVs can stimulate HD-11 cell to generate NO,
And expression quantity is increased over time.Since NO is a kind of biological messenger molecule, generates and aoxidized dependent on induction type one
Nitrogen synthase (inducible nitric oxide synthase, iNOS), in microorganisms such as bacterium, the fungies to resist an invasion and
It is played a very important role in inflammatory reaction, the results show that cell generates NO's after OMVs and HD-11 cell co-culture
Horizontal extremely significant raising, prompts OMVs that can enhance HD-11 Cell-mediated Immunity.So the present invention provides OMVs and can be conducive to improve
The effect of the immunocompetence of body.
Embodiment 3
The outer membrane vesicles (OMVs) prepared in quantitative fluorescent PCR (qPCR) detection embodiment 1 are to HD-11 cell-related inflammation
The influence of factor expression, concrete operations are as follows:
In 25cm2Routine culture chicken macrophage (HD-11), makes cell grow to 85% in culture bottle.With 6 bottles of cells point
For vesica group, lipopolysaccharides (LPS) group and control group, every group 2 bottles.5 μ g OMVs are added in vesica group, and 1 μ g/ml is added in lipopolysaccharide group
LPS, control group is without any processing, co-cultures later for 24 hours and 48h.Cell total rna is extracted with Trizol method respectively[9], it is dissolved in
20 μ L DEPC water.Reverse transcription carries out to specifications, and specific reaction system is as follows: 1 μ g of RNA, random primer 1 μ L, 5 × M-
4 μ L of MLV TR Buffer, 1 μ L, M-MLV reverse transcriptase of ribonuclease inhibitor 1 μ L, dNTP (2.5mmol/L) 8 μ L.
Carry out reverse transcription by following procedure after mixing: it is standby to be immediately placed on -20 DEG C of preservations after reaction by 42 DEG C of 50min, 70 DEG C of 15min
With.
According to chicken inflammatory factor on Genbank with respect to conservative region sequence, above and below the specificity with Primer5 design gene
It swims primer (being shown in Table 1), and is synthesized by Shanghai Bio-engineering Corporation.Reaction system is as follows: 1 μ L cDNA, each 1 μ of upstream and downstream primer
L, 12.5 μ L Brilliant SYBR Green QPCR master Mix, 0.375 μ L ROX, 9.125 μ L DEPC water.Reaction
Condition: 95 DEG C of initial denaturations 10min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 1min, 72 DEG C of extension 60s are recycled 40 times, and 72 DEG C re-extend
8min.Solubility curve reaction condition is as follows: 95 DEG C of 1min, 58 DEG C of 30s, 95 DEG C of 30s.All test group samples are independent in triplicate
It is horizontal to compare each group inflammatory factor using SPSS for test.
1 relevant inflammatory factors primer sequence of table
OMVs influences HD-11 cell-related inflammation factor expression result as shown in figure 4, wherein A group is IL-1 β, and B group is
IL-6, C group be IL-2, D group be iNOS, E group be IL-10, for 24 hours when, compared with the control group, IL-1 β, IL-10, iNOS vesica group
And lipopolysaccharide group difference is extremely significant (p < 0.01), IL-6 vesica group significant difference (p < 0.05) compared with the control group, lipopolysaccharide group
Difference is extremely significant (p < 0.01) compared with the control group, IL-2 group vesica group significant difference (p < 0.05) compared with the control group.48h
When, compared with the control group, inequality is heteropolar significant (p < 0.01) for above 5 kinds of cell factor vesica groups and lipopolysaccharide group.Due to IL-l
β, IL-6 etc. are mainly discharged by macrophage, the expression of relevant inflammatory factors are detected by qPCR, OMVs can promote as the result is shown
HD-11 cell secretes IL-1 β, IL-2, IL-6, IL-10, iNOS, prompts macrophage by extremely significant activation, illustrates system of the present invention
Standby outer membrane vesicles can be obviously promoted inflammatory reaction.
The preparation of 4 outer membrane vesicle vaccine of embodiment and immune
By the OMVs and MONTANIDE of the preparation purifying of embodiment 1TM71 VG adjuvant of ISA (SEPPIC Products) presses 3:7
Mass ratio (antigen: adjuvant) mixing and emulsifying, prepared vaccine is named as V-OMVs-A, and wherein OMVs protein concentration is 100 μ
g/ml.42 age in days SPF test chickens are divided into OMVs immune group (30) and PBS control group (30).OMVs group test chicken chest
Intramuscular injection V-OMVs-A vaccine, 0.5mL/ only, two exempt from after two weeks by interval；Control group is inoculated with manufactured epidemic disease after PBS substitution OMVs
Seedling, 0.5mL/ only, are spaced primary with dose inoculation again after two weeks.
The measurement of serum IgG: the blood sampling of chicken venous sinus is carried out after first immunisation weekly and separates serum, using indirect ELISA
Measure the serum IgG antibody levels of anti-OMVs；Full bacterium antigen is diluted to 1 μ g/ μ L with coating buffer, is coated in 96 hole elisa Plates, 4
DEG C overnight, using 1: 100 diluted serum as primary antibody, 1: 10 000 diluted goat anti-chicken IgG-HRP (Sigma company) conduct
Secondary antibody passes through tmb substrate chromogenic assay D450Value.Two exempt from rear 4th week measures each group chicken serum IgG level respectively, as a result such as Fig. 5
Shown, the IgG antibody of OMVs immune group is horizontal extremely significant higher than control group (p < 0.01), illustrates that machine can be stimulated after chicken is immunized in OMVs
Body generates stronger humoral immunity.In Fig. 5, * * is that t check analysis test group and control group difference are extremely significant (P < 0.01).
Immunoprotection test: after two exempt from 4 weeks, test chicken uses sinus inoculation under the secondary chicken poultry bacillus socket of the eye of 3 serotypes to attack respectively
It hits.A type (Hp8 plants), Type B (BJ plants) and the c-type (668 plants) of secondary chicken poultry bacillus (are ground by Beijing City Agriculture and Forestry Institute animal and veterinary
Study carefully and provided), dosage is followed successively by A type 5 × 105CFU/0.2mL, Type B 2 × 105CFU/0.2mL, c-type 5 × 105CFU/0.2mL。
It is observed continuously 1 week, records the clinical onset situation of test chicken, including have a running nose, eyelid is swollen, excessive tear etc..
The result shows that all test chicken of control group is fallen ill successively, serious Rhinitis Symptoms are all shown, disease incidence is
100%.Only 2 test chickens show Rhinitis Symptoms, protective rate 8/10 after OMVs group A type attacks poison, and Type B and c-type attack malicious guarantor
Shield rate is respectively 1/10 and 3/10, prompts vesica immunogenicity to have type specificity, the results are shown in Table 2.
The protecting effect that chicken attacks poison to Apg bacterial strain is immunized in table 2
Protest test can be improved the tolerance that chicken attacks secondary chicken poultry bacillus after OMVs is immune as the result is shown, show
The OMVs contains Partial key protective antigens ingredient, can be used as infectious coryza of chicken subunit vaccine.Further, malicious examination is attacked
Test result demonstrate that A type pair chicken poultry bacillus OMVs inoculation after chicken can produce preferable immanoprotection action, be resistant to
The attack of homotype bacterial strain.
Sequence table
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Claims (8)
1. a kind of preparation method of pair chicken poultry bacillus outer membrane vesicles comprising following steps:
S1, in vitro culture pair chicken poultry bacillus to growth period bacteria log phase obtain cell-free culture supernatant by being centrifuged, filtering；
S2, process at ultracentrifugation is carried out to cell-free culture supernatant, obtains secondary chicken poultry bacillus outer membrane vesicles.
2. preparation method according to claim 1, which is characterized in that in step sl, the in vitro culture pair chicken poultry bar
Bacterium, culture medium used are the TSB fluid nutrient medium containing NAD and serum.
3. preparation method according to claim 2, which is characterized in that the TSB fluid nutrient medium containing NAD and serum
In the weight percent concentration containing NAD be 0.01%, serum is chicken serum, volumetric concentration 10%.
4. preparation method according to claim 1, which is characterized in that in step sl, 4 DEG C of 10000 × g of the centrifugation from
Heart 15min, discards precipitating, and the filtering uses 0.22 μm of membrane filtration.
5. preparation method according to claim 1, which is characterized in that in step s 2, the ultracentrifugation is will be without thin
Born of the same parents' culture supernatant is discarded supernatant in 4 DEG C of 150000 × g ultracentrifugation 3h, and precipitating PBS buffer solution is resuspended；Ibid again 4 DEG C
150000 × g ultracentrifugation 3h, discards supernatant, and precipitating is resuspended in PBS buffer solution, as prepares secondary chicken poultry bacillus outer membrane vesicles.
6. any one of -5 preparation methods obtain secondary chicken poultry bacillus outer membrane vesicles and are preparing avian infectious nose according to claim 1
Application in scorching subunit vaccine.
7. the chicken that a kind of preparation method described in any one of claim 1-5 obtains secondary chicken poultry bacillus outer membrane vesicles preparation is infected
Property rhinitis subunit vaccine.
8. a kind of immunologic stimulant, which is characterized in that immunologic stimulant is according to any one of the claim 1-5 preparation method
The secondary chicken poultry bacillus outer membrane vesicles of acquisition.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111440748A (en) * | 2020-05-15 | 2020-07-24 | 黑龙江八一农垦大学 | Method for separating, purifying and identifying necrobacillus outer membrane vesicles |
| CN111909900A (en) * | 2020-07-19 | 2020-11-10 | 东南大学 | Method for enhancing immune response based on in-situ self-assembly intelligent nanoparticles |
| CN112342156A (en) * | 2020-10-22 | 2021-02-09 | 浙江省农业科学院 | Preparation method and application of outer membrane vesicle of bordetella |
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| CN106220716A (en) * | 2016-07-28 | 2016-12-14 | 北京市农林科学院 | Infectious coryza of chicken subunit vaccine and preparation method thereof |
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| CN111440748A (en) * | 2020-05-15 | 2020-07-24 | 黑龙江八一农垦大学 | Method for separating, purifying and identifying necrobacillus outer membrane vesicles |
| CN111909900A (en) * | 2020-07-19 | 2020-11-10 | 东南大学 | Method for enhancing immune response based on in-situ self-assembly intelligent nanoparticles |
| CN112342156A (en) * | 2020-10-22 | 2021-02-09 | 浙江省农业科学院 | Preparation method and application of outer membrane vesicle of bordetella |
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