A kind of atrophic rhinitis inactivated vaccine immunopotentiator and preparation method thereof
Technical field
The invention belongs to veterinary biologicses technical field, and in particular to a kind of atrophic rhinitis inactivated vaccine is used immune
Reinforcing agent and preparation method thereof.
Background technology
Atrophic rhinitis is mainly as caused by bordetella branchiseptica and Toxigenic Pasteurella multocida
It is a kind of chronic and with communicable breathing problem.The World Health Organization is defined as B class diseases, and ill pig goes out
Now sneeze, the deformation of nose is bleeding, face, nose is crooked and the adverse reaction such as growthing lag, causing the feed conversion rate of pig reduces,
Production performance is low, and huge economic loss is caused to intensive industrialized piggery.Simultaneously as after pathogen infection pig, infringement is exhaled
The normal configuration and function in road are inhaled, makes the reduction of pig body resistance, easily infects other cause of diseases, cause respiratory system syndrome, increase
Plus the death and culling rate of pig.This disease often betides the pig at 2~5 monthly ages, now the almost flourishing area of pig industry all over the world, and China is permitted
Also there is this disease in many areas.
Specifically, atrophic rhinitis is pig caused branch when being infected by environmental stimulus or other pathogens
Tracheae sepsis bordetella bacilli is adsorbed onto the epithelial cell of schneiderian membrane, destruction cilium and schneiderian membrane, bordetella bacilli release by flagellum
Dermotoxin pass through turbinate, destroy Gegenbaur's cell, interference calcium uptake and ostosis;Pasteurella multocida resides in
Dermotoxin is discharged on the schneiderian membrance destroyed by bordetella bacilli, osteoclast activity is strengthened, and causes bone to decompose.Typically
In the case of Pasteurella be difficult on the schneiderian membrance of health pig breed, only by Bordetella bordetella bacilli or other
Factor invasion and attack, mucous membrane are possible to cause to propagate in pig farm when suffering damage, therefore prevent bordetella bacilli from being adsorbed in schneiderian membrance
The problem of being primary solve.
To avoid propagation of the atrophic rhinitis in swinery, pig farm would generally inject atrophic rhinitis epidemic disease to pig
Seedling, however be used alone vaccine immune effect it is not good, generally require with the use of can strengthen vaccine immunity stress, excite and exempt from
The adjuvant of epidemic disease response strengthens the immune effect of vaccine, and that one kind can strengthen the preferential definite value of atrophic rhinitis vaccine antigen is glutinous in nose
The immunopotentiator of film is very in short supply.Adjuvant does not provide immune in itself, it only when constituting a kind of preparation with immunizing antigen,
The range and effect by the immune response of vaccine-induced generation can be improved.The atrophic rhinitis applied in the market is gone out
Live vaccine adjuvant includes alocol, lipoid adjuvant and propolis adjuvant;Aluminium hydroxide gel adjuvanted immunogenic is good, side effect
It is small, but it is unable to inducing cellular immune;Lipoid adjuvant and propolis adjuvant immunogenicity are good, can induce humoral immunity and cell is exempted from
Epidemic disease, the immune duration is long, but the prices of raw materials are expensive, and cost is too high.Therefore in recent years, researcher application in various countries' is different
Raw material and use distinct methods have developed a series of new adjuvant, but because effect difference or toxicity are big, be expired without one kind
Meaning effect.Therefore, the stimulation of atrophic rhinitis vaccine immunity can be strengthened by needing research one kind badly, excite immune response, realization has
Effect prevents and treats the immunopotentiator of atrophic rhinitis.
The content of the invention
Strong Th1, Th2 reaction is induced it is an object of the invention to provide a kind of, is drenched while inducing and producing cytotoxic T
Bar cell and bone-marrow-derived lymphocyte, the pig for making antigen preferentially be colonized in schneiderian membrance with enhancing cellular immunity and humoral immune function wither
Contracting rhinitis inactivated vaccine immunopotentiator.
The object of the present invention is achieved like this, and the atrophic rhinitis inactivated vaccine is included containing dry with immunopotentiator
The liquid and solvent of powder immunity particle, the liquid of the immunity particle containing dry powder it is main by the aluminium hydroxide aqueous solution, ginsenoside,
Cholesterol, vitamin C, DEAE-dextran, KI and polyethylene glycol are constituted by proper ratio compatibility.
It is preferred that, aluminium hydroxide contains in the liquid of the immunity particle containing dry powder described in the liquid of immunity particle containing dry powder
Measure as 75~85wt%, the content of ginsenoside is 0.1~2wt%, and the content of cholesterol is 0.1~2wt%, diethylin second
The content of base glucan is 0.1~1wt%.
It is preferred that, the content of vitamin C and KI meets following condition in the liquid of the immunity particle containing dry powder:Dimension
Raw element C and KI total mole number/aluminium hydroxide molal quantity >=1, the mass ratio of the aluminium hydroxide and polyethylene glycol is 1:0.1
~1.
It is preferred that, the solvent is physiological saline or PBS.
Present invention also offers a kind of method for preparing the atrophic rhinitis inactivated vaccine immunopotentiator, including
Following steps:
A, it is slowly added to ginsenoside, cholesterol into the aluminium hydroxide aqueous solution, vitamin C, diethylin ethyl Portugal gathers
Sugar and KI, continue to add polyethylene glycol after stirring 3~5min, are further continued for obtaining containing for golden yellow after 10~20min of stirring
The liquid of dry powder immunity particle, sterile guarantor is in brown receiving flask, being filled with liquid nitrogen by the liquid-packing of the immunity particle containing dry powder
Deposit;
B, immunopotentiator preparation:The liquid of the immunity particle containing dry powder of appropriate metrology is taken to be placed in the reaction equipped with solvent
In container, the concentration control by the liquid of the immunity particle containing dry powder in reaction vessel is 15wt%, opens agitating device stirring 20
~40min, is 2~8 DEG C by the temperature control in reaction vessel, stands 10~20h, immunopotentiator is obtained after filtration sterilization.
It is preferred that, the solvent is physiological saline or PBS.
Compared with prior art, the atrophic rhinitis inactivated vaccine immunopotentiator that the present invention is prepared, effectively
The foundation for suppressing bordetella branchiseptica and pasteurella multocida latent infection with reactivating, antibody can be shortened
The window phase of generation, improves the immune effect of atrophic rhinitis vaccine, and immune duration extension effectively avoids pig atrophy
Property rhinitis latent infection, be atrophic rhinitis disease prevention and eradicate provide guarantee, can be widely used in pig farm
The preventing and treating of atrophic rhinitis is carried out, the economic benefit on pig farm is improved.
Brief description of the drawings
Fig. 1 takes a blood sample after 42 days for first immunisation and detects obtained average PMT titres schematic diagram;
Fig. 2 takes a blood sample after 42 days for first immunisation and detects obtained ELISA mean antibody levels schematic diagrames;
Fig. 3 turns positive rate schematic diagram for immune rear piglet;
Fig. 4 is immune rear piglet antibody level schematic diagram;
Embodiment
With reference to embodiment, the present invention is further illustrated, but the present invention must not be added in any way
To limit, based on present invention teach that any changes and modifications made, belong to protection scope of the present invention.While institute of the present invention
It is commercially available prod unless otherwise instructed with test material.
The preparation of atrophic rhinitis inactivated vaccine immunopotentiator
Embodiment 1
It is prepared by a, the liquid of the immunity particle containing dry powder:The aqueous solution that 30ml aluminum hydroxide concentrations are 100ug/ml is taken, slowly
The lower ginsenoside for adding 4ug is stirred, 4ug cholesterol, 4ug DEAE-dextran adds after continuing stirring 5min
Enter vitamin C and KI total mole number/aluminium hydroxide molal quantity in polyethylene glycol, the liquid of the immunity particle containing dry powder=
4, the mass ratio of the aluminium hydroxide and polyethylene glycol is 1:1, it is further continued for after stirring 20min obtaining the immune containing dry powder of golden yellow
The liquid of particle, is observed using atomic force microscope, a diameter of 50nm of obtained dry powder immunity particle, will contain dry powder immunity particle
Liquid-packing do Preservation in sterile condition in brown receiving flask, being filled with liquid nitrogen, the shelf-life is 2 years;
B, immunopotentiator preparation:The liquid of the immunity particle containing dry powder of appropriate metrology is taken to be placed in equipped with PBS
Reaction vessel in, by reaction vessel the liquid of the immunity particle containing dry powder concentration control be 15wt%, open agitating device
20min is stirred, the rotating speed of agitating device is advisable most very much not to produce bubble, is 2 DEG C by the temperature control in reaction vessel, stands
20h is stayed overnight, and immunopotentiator is obtained after filtration sterilization, in the environment that obtained immunopotentiator is stored in 0 DEG C, controls the PH to be
6.5。
Embodiment 2
It is prepared by a, the liquid of the immunity particle containing dry powder:The aqueous solution that 30ml aluminum hydroxide concentrations are 100ug/ml is taken, slowly
The lower ginsenoside for adding 80ug of stirring, 80ug cholesterol, 40ug DEAE-dextran continues to stir after 3min
Add vitamin C and KI total mole number/aluminium hydroxide molal quantity in polyethylene glycol, the liquid of the immunity particle containing dry powder
=1, the mass ratio of the aluminium hydroxide and polyethylene glycol is 1:0.1, be further continued for stir 10min after obtain golden yellow contain dry powder
The liquid of immunity particle, is observed using atomic force microscope, a diameter of 120nm of obtained dry powder immunity particle, will be contained dry powder and be exempted from
The liquid-packing of epidemic disease particle does Preservation in sterile condition in brown receiving flask, being filled with liquid nitrogen, and the shelf-life is 2 years;
B, immunopotentiator preparation:The liquid of the immunity particle containing dry powder of appropriate metrology is taken to be placed in equipped with physiological saline
In reaction vessel, the concentration control by the liquid of the immunity particle containing dry powder in reaction vessel is 15wt%, opens agitating device and stirs
40min is mixed, the rotating speed of agitating device is advisable most very much not to produce bubble, is 8 DEG C by the temperature control in reaction vessel, stands
Immunopotentiator is obtained after 10h, filtration sterilization, in the environment that obtained immunopotentiator is stored in 4 DEG C, it is 7.5 to control PH.
Embodiment 3
It is prepared by a, the liquid of the immunity particle containing dry powder:The aqueous solution that 30ml aluminum hydroxide concentrations are 100ug/ml is taken, slowly
The lower ginsenoside for adding 40ug of stirring, 40ug cholesterol 20ug DEAE-dextran continues to stir after 4min
Add vitamin C and KI total mole number/aluminium hydroxide molal quantity in polyethylene glycol, the liquid of the immunity particle containing dry powder
=2, the mass ratio of the aluminium hydroxide and polyethylene glycol is 1:0.5, be further continued for stir 15min after obtain golden yellow contain dry powder
The liquid of immunity particle, is observed using atomic force microscope, a diameter of 100nm of obtained dry powder immunity particle, will be contained dry powder and be exempted from
The liquid-packing of epidemic disease particle does Preservation in sterile condition in brown receiving flask, being filled with liquid nitrogen, and the shelf-life is 2 years;
B, immunopotentiator preparation:The liquid of the immunity particle containing dry powder of appropriate metrology is taken to be placed in equipped with physiological saline
In reaction vessel, the concentration control by the liquid of the immunity particle containing dry powder in reaction vessel is 15wt%, opens agitating device and stirs
40min is mixed, the rotating speed of agitating device is advisable most very much not to produce bubble, is 8 DEG C by the temperature control in reaction vessel, stands
Immunopotentiator is obtained after 10h, filtration sterilization, in the environment that obtained immunopotentiator is stored in 8 DEG C, it is 7.5 to control PH.
Embodiment 4
It is prepared by a, the liquid of the immunity particle containing dry powder:The aqueous solution that 30ml aluminum hydroxide concentrations are 100ug/ml is taken, slowly
The lower ginsenoside for adding 60ug of stirring, 60ug cholesterol, 24ug DEAE-dextran continues to stir after 5min
Add vitamin C and KI total mole number/aluminium hydroxide molal quantity in polyethylene glycol, the liquid of the immunity particle containing dry powder
=1.4, the mass ratio of the aluminium hydroxide and polyethylene glycol is 1:0.8, it is further continued for obtaining doing containing for golden yellow after stirring 20min
The liquid of powder immunity particle, is observed using atomic force microscope, a diameter of 100nm of obtained dry powder immunity particle, will contain dry powder
The liquid-packing of immunity particle does Preservation in sterile condition in brown receiving flask, being filled with liquid nitrogen, and the shelf-life is 2 years;
B, immunopotentiator preparation:The liquid of the immunity particle containing dry powder of appropriate metrology is taken to be placed in equipped with physiological saline
In reaction vessel, the concentration control by the liquid of the immunity particle containing dry powder in reaction vessel is 15wt%, opens agitating device and stirs
40min is mixed, the rotating speed of agitating device is advisable most very much not to produce bubble, is 8 DEG C by the temperature control in reaction vessel, stands
Immunopotentiator is obtained after 10h, filtration sterilization, in the environment that obtained immunopotentiator is stored in 35 DEG C, it is 7 to control PH.
The atrophic rhinitis inactivated vaccine that aforementioned four embodiment is successfully prepared is carried out surely with immunopotentiator sample
Qualitative test, obtained data such as table 1:
The stability test data of the immunopotentiator of table 1
Above-mentioned result of the test shows, atrophic rhinitis inactivated vaccine prepared by 4 embodiments that the present invention is provided is with exempting from
Epidemic disease reinforcing agent sample has good stability.
Effect test of the immunopotentiator to atrophic rhinitis inactivated vaccine immunological enhancement
(1) prepare using the immunopotentiator prepared, after filtration sterilization, withered by 2mg/ parts and the pig containing 5ug/ parts
Contracting rhinitis antigen is mixed, and is prepared into vaccine.
(2) 20 double-negative replacement gilts of atrophic rhinitis antigen-antibody are randomly divided into 4 groups by packet, every group 5, in detail
Thin experiment packet situation is shown in Table 2.
(3) vaccine that will be prepared is immunized, replacement gilt was immunized respectively at antenatal 6-8 weeks and antenatal 3-4 weeks 1 time.
(4) antibody test blood sampling detection ELISA antibody and neutralizing antibody after first immunisation 42 days, and use single factor test side
Difference is analysed and double tail paired t-test statistics group differences.
Table 2 tests grouping sheet one
As Figure 1-Figure 2, A, B group represent addition immunopotentiator group to result of the test in figure, and C, D group represent no added
Group, result of the test shows:1st, addition immunopotentiator can significantly improve the neutralizing antibody level and ELISA of atrophic rhinitis
Antibody level;2nd, the antibody level that the atrophic rhinitis inactivated vaccine of the antigen containing 5ug is produced after addition immunopotentiator is higher than
The atrophic rhinitis inactivated vaccine of the antigen containing 20ug of immunopotentiator is not added, therefore can reduce by 4 times of antigen usage amounts, is saved
About cost.
Application test of the immunopotentiator in atrophic rhinitis inactivated vaccine
(1) prepare using the immunopotentiator for preparing, after filtration sterilization, by 2mg/ parts respectively with containing 20ug/ parts
Atrophic rhinitis killed vaccine antigen is mixed, and is prepared into vaccine.
(2) the double-negative 12 week old piglet of 20 atrophic rhinitis antigen-antibodies is randomly divided into 4 groups by packet, every group 5,
Detailed packet situation is shown in Table 3.
(3) vaccine that will be prepared is immunized piglet is immunized using intramuscular injection by 2ml/ parts.
(4) antibody test before immune and it is immune after 7,14,28,60, blood samplings in 90 and 120 days, ELISA in detection blood
Antibody level.
Table 3 tests grouping sheet two
As Figure 3-Figure 4, result of the test shows result of the test, and immunopotentiator can stimulate piglet quickly to produce pig atrophy
Property rhinitis antibody, being immunized 7 days can be with 80% turn of sun, hence it is evident that higher than other adjuvant groups, in addition, the antibody level produced is also above it
Its adjuvant group.
Safety testing of the immunopotentiator to pig
The immunopotentiator prepared in Example 1, musculi colli injection, note are carried out to swinery by 10ml/ parts
1 after penetrating, observe swinery reaction within 3,7,14 days, result of the test result as shown in table 4 shows that immunopotentiator has good safety
Property.
The safety testing result of the large bolus injection immunopotentiator of table 4
The immunopotentiator prepared using embodiment 1 coordinates atrophic rhinitis inactivated vaccine to atrophic rhinitis
Negative pig is immunized, 24h body temperature after detection is immune, the result of variations such as table 5 of body temperature after being immunized.
Table 5 carries out the body temperature reaction test result after being immunized after coordinating using different adjuvants and different vaccines to pig
Above-mentioned result of the test shows that the immunopotentiator prepared using the present invention coordinates atrophic rhinitis inactivation epidemic disease
When seedling carries out immune to pig, stress reaction is small, does not interfere with the identification of antigen, does not also interfere with immunogenicity and immune lasting
Phase, at the same can effectively strengthen vaccine Double immune stimulate, excite immune response, be atrophic rhinitis disease prevention and
Eradicate to provide and ensure, can be widely used in the preventing and treating that pig farm carries out atrophic rhinitis, improve the economic effect on pig farm
Benefit.
Above is the description to better embodiment of the present invention, and it is not understood to the limitation present invention, those skilled in the art
A variety of changes or combination can be made according to the present invention, without departing from the spirit of the present invention, the protection of the present invention all should be belonged to
Scope.