CN104771754B - A kind of porcine circovirus 2 type inactivated vaccine aqueous adjuvants and application - Google Patents
A kind of porcine circovirus 2 type inactivated vaccine aqueous adjuvants and application Download PDFInfo
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Abstract
The invention discloses a kind of aqueous adjuvants of porcine circovirus 2 type inactivated vaccine and application, adjunvant composition of the invention can significantly improve the immune effect of porcine circovirus 2 type inactivated vaccine, and can efficient protection of the excitating organism generation up to 150 days.Preparing process with time vaccines is stable, and after the vaccine immunity caused by side reaction is small etc. is respectively provided with unique advantage.Adjunvant composition preparation technology is simple, it is not necessary to which large scale equipment, cost is cheap, and is easy to the large-scale production of vaccine.
Description
Technical field
The invention mainly relates to pig vaccines arts.More particularly to a kind of water-based assistant of porcine circovirus 2 type inactivated vaccine
Agent and application.
Background technology
Porcine circovirus 2 type (Porcine circovirus type 2, PCV2) is postweaning multisystemic exhaustion synthesis
The main pathogen of (PMWS) is levied, the disease is mainly characterized by immunosupress and postweaning multisystemic exhaustion.This disease is main to be occurred
In the weanling pig of 6-15 week old, sick pig is mainly manifested in retarded growth, expiratory dyspnea, anaemia, diarrhoea, weak, lymph
The clinical symptoms such as enlargement, the slow, jaundice that increases weight are tied, and the immunologic function of infection piglet can also be made to suffer damage, cause machine
Body immunity degradation, so as to trigger secondary infection, aggravate the generation of the state of an illness.Major pathologic features are that lymph is thin in lymphoid tissue
Born of the same parents' missing, the infiltration of inflammatory granulocyte etc..In addition, PCV2 also with sow breeding difficulty, Hypertrophic necrotizing pneumonia, pigskin inflammation nephrosis
The illnesss such as syndrome and PRDC are related.In recent years, China swinery also largely broke out PMWS, with PMWS
It is a large amount of popular, also cause extremely serious consequence to China's pig industry.Control PCV2 infection in vaccine immunity room causes relevant disease
Important means.And in the development of vaccine, viral antigen content influences very big with adjuvant types on immune effect.
Current China's live vaccine is mainly based on oily adjuvant, i.e., with nonionic surfactant (Tween80, Span80)
Water-In-Oil dose vaccine is prepared under high shear as stable dosage forms agent and antigen.The viscosity of the vaccine is big, and injection is tired
Difficulty, side reaction is big, and is difficult to absorb, and easily carcass quality is had undesirable effect.In recent years, also successfully prepare both at home and abroad
W/O/W vaccine and oil-in-water vaccine.These adjuvants are mainly prepared by different surfactants and appropriate ratio
Form.But due to these adjuvant less stables and expensive so that intradermal vaccine producer of state is difficult to promote the use of.
Many factors are must take into consideration when selecting appropriate adjuvants.Adjuvant should make disengaging and inhaling for antigen in a manner of most effective
It is slow to receive speed, and the toxicity to host, allergenicity, excitant and other undesired side effects.In order to make one satisfied, assistant
Agent should be non-and kill viral, biodegradable, the high-caliber immunity of sustainable generation, cross protection can be stimulated to make
With, can it is compatible with a variety of antigens, in a variety of species effectively and to host without any toxicity and side effects (such as：Without injection site
Reaction).Other desired adjuvant characteristics for can micro-administration, it is dose-saving, have excellent shelf stabilities, can bear to do
It is dry, may be manufactured without oil-containing, solid or liquid can be used as to exist, it is easily simple prepare, technique is simple and its manufacture is not expensive.Most
Afterwards, it is therefore highly desirable to adjuvant can be configured to induction body fluid immune response or cellular immunity according to the needs of vaccination protocols
Response or the two have concurrently.However, it may conform to the adjuvant limited amount of above-mentioned condition.
From the saponin(e of South America Quillaia saponaria (Quillaja saponaria) bark extraction as adjuvant for some time.
(Lacaille-Dubois, M and Wagner H. (A review of the biological and
pharmacological activities of saponins.) current many live vaccine adjuvants include QuilA, QuilA
From the saponin(e cut of South America Chile Gleditsia officinalis (Quillaja saponaria molina) bark.QuilA is further fractionated
Then produce time cut, including QS-7, QS-17, QS-18 and QS-21 (United States Patent (USP) No：5057540).
In order to overcome Quil A haemolysis and property of killing the virus, it is formed referred to as with cholesterol and phospholipid combination
The special construction of immunostimulating compound (ISCOM) or ISCOM matrix (ISCOMATRIX).(Ozel M., et al.；
J.Ultrastruc.and Molec.Struc.Re 102,240-248 (1989).When with antigen combination, ISCOM would generally
Induce Th1 cytotoxic T cell responses.However, although significantly decrease Quil A haemolysis property, but Quil A and cholesterol group
Such property will not be completely eliminated in merging.ISCOM another proteantigen that is limited to must have sufficiently large hydrophobic structure
Domain come with ISCOM reciprocations, so as to be merged in ISCOM.The protein of highly-hydrophilic can not be merged in ISCOM.Therefore,
ISCOM can produce autologous immune response in subject's body.
GERBU Adjuvant 100 (hereinafter referred to as " DDA ") and La Weiding.DDA is lipophilicity quaternary ammonium compound
(amine), it carries the two 18 carbon alkyl chains and two methyl groups with reference to positive charge quat molecules, molecular weight 631.It is made
Found for the purposes of adjuvant by Gall (Immunol.V.11, p.369,1966).It has been reported that DDA can stimulate it is strong through cell
The immune response of mediation, also show inducible humoral immune response.Many papers have been delivered, it was demonstrated that DDA resists as protein
Original, haptens (hapten), tumour, virus, protozoan and bacterium adjuvant effect.(Korsholm, K S., et al.,
Immunology, vol.121, pp.216-226,2007).
Montanide GEL 01 (being purchased from French SEPPIC companies) are a kind of highly stable polymer (polyacrylic acid
Ester) gel particle dispersion.Slow release characteristic caused by storing effect and heteropolymer absorption, improve intrinsic being immunized and answer
Answer system.
Preferable polymer is the acrylic or methacrylic acid polymer being crosslinked by the polyalkenyl ether of sugar or polyalcohol.
These polymer are known well with the entitled people of C974P (carbomer).These polymer are typically by alkene
What propyl group sucrose or Allyl pentaerythritol were crosslinked.Wherein it is possible to mention974p、934p、971p。
Levamisol is a kind of broad-spectrum de-worming medicine, is first synthetics for being found to have immunoregulation effect,
With extensive immunoregulatory activity, the extensive use in animal husbandry.Levamisol can make anti-thymus-dependent and anti-thymus gland non-
The antibody titer of dependence antigen is significantly raised.Its immunoregulatory activity mechanism has three aspects：First, have Cholinergic activity active
(relevant with imidazole group)；Second, body can be induced to produce various lymphokines, third, its metabolite, which has, removes free radical
Function.
Adjunvant composition is had no before adjunvant composition of the present invention and possesses ideal expectation feature.We have been working hard seek
Ask in porcine circovirus 2 type inactivated vaccine, the new adjunvant composition of standard adjuvant defect can be overcome.Especially, the height phase
Adjunvant composition of the present invention is hoped, can be in animal through cell-mediated cellullar immunologic response and humoral immune response, but without routine
The advantages that side effect and difficult preparation of adjuvant.Adjunvant composition prepared by the application is used for porcine circovirus 2 type inactivated vaccine.
The application of the vaccine can effectively reduce the porcine circovirus 2 type incidence of disease, or slow down the pig as caused by Infection of Porcine circovirus and break
Multisystem exhaustion syndrome after milk.
The content of the invention
It is an object of the invention to provide a kind of aqueous adjuvants composition, the adjunvant composition includes polymer, poly- carboxylic second
Alkene polymer, saponin(e, sterol, quaternary ammonium compound and levamisol.The present invention adjunvant composition optimization formula be：
Montanide GEL 01, Carbopol 974p, Quil A, cholesterol, GERBU Adjuvant 100 (DDA) and a left side
Revolve imidazoles.
It is another object of the present invention to provide a kind of aqueous adjuvants composition to prepare porcine circovirus 2 type inactivation epidemic disease
Application in seedling.Using the adjunvant composition can significantly improve porcine circovirus 2 type inactivated vaccine humoral immune response and
Cellullar immunologic response, not only it can just be produced at 14 days compared with High antibody level, and can height of the excitating organism generation up to 150 days
Imitate protection.The adjunvant composition does not need large-scale emulsifying device when preparing vaccine, after vaccine stable preparation process, vaccine immunity
The advantages that side reaction is small.
In order to achieve the above object, the present invention takes following technical measures：
A kind of aqueous adjuvants composition, its formula include：
Described Carbopol 974p concentration is 10mg/ml；Quil A concentration is 60mg/ml；The concentration of cholesterol
For 20mg/ml；The concentration of GERBU Adjuvant 100 (DDA) is 20mg/ml；The concentration of levamisol is 20mg/ml.
Above-described aqueous adjuvants composition, optimization formula include：
Above-described aqueous adjuvants composition, optimum formula include：
The method for preparing inactivated vaccine using above-mentioned aqueous adjuvants composition, including：
A) PH7.2 PBS is utilized, inactivation of viruses liquid viral level is adjusted, prepares virus stock solution used；
B) Carbopol 974p are added in step a composition and fully mixed；
C) Montanide GEL 01 are added in step b composition and fully mixed；
D) GERBU Adjuvant 100 (DDA) is added in step c composition and fully mixed；
E) Quil A are added in step d composition and fully mixed；
F) cholesterol is added in step e composition and fully mixed；
G) levamisol is added in step f composition and fully mixed；
H) by well mixed preparation, handle, produce finally by homogenizer.
Preferably, described virus is porcine circovirus 2 type-WH strains, CCTCC NO：V201333；
Preferably, the volume ratio of virus stock solution used and adjunvant composition is 13:7；
Preferably, effective viral level in final products is 107TCID50/ml。
The addition of above each component is added according to the proportioning of aqueous adjuvants composition of the present invention.
A kind of application of aqueous adjuvants composition in pig circular ring virus inactivated vaccine is prepared, is included in and prepares pig circular ring virus 2
Application in malicious 2 type inactivated vaccines.By obtained vaccine direct immunization animal, specific humoral immune response can be significantly improved
And cellullar immunologic response, excellent effect.
Compared with prior art, the present invention has advantages below：
(1) it is simple to prepare vaccine art, it is not necessary to large-scale emulsifying device, can greatly reduce on pollution probability and production
Cost.
(2) vaccine that the aqueous adjuvants are prepared has unique stability, is placed at 4 DEG C, 25 DEG C and 37 DEG C all than other
Vaccine will be stablized；
(3) vaccine that the aqueous adjuvants are prepared is without side reaction, and injection site is without granuloma or inflammatory reaction；
(4) aqueous adjuvants prepare vaccine immunity protection period is longer and also antibody level compared with other adjuvants epidemic disease
Height of seedling；
(5) vaccine that under special circumstances prepared by this adjuvant can be used for the emergence therapeutic use of the disease；
(6) caused specific antibody is very fast and high after the vaccine immunity that the adjunvant composition is prepared, and can be same
When induction body fluid is immune and cellular immunity；
Brief description of the drawings
Fig. 1 is that 1 vaccine immunity absorbing state schematic diagram after 28 days is organized in embodiment 5.
Absorbing state schematic diagram after Fig. 2 is immunized 28 days for group 3 in embodiment 5.
Fig. 3 is 201 adjuvant immunity absorbing state schematic diagram after 28 days in embodiment 5.
Fig. 4 is Marcol52 adjuvant immunities absorbing state schematic diagram after 28 days in embodiment 5.
Fig. 5 is the immune rear antibody average value schematic diagram of different adjuvant groups in embodiment 3.
Fig. 6 is the immune rear 28 days antibody average value (serum 1 of different adjuvant groups in embodiment 4:320 doubling dilutions) signal
Figure.
Embodiment
The present invention is described further and proved with reference to specific implementation, but present invention is not limited thereto.
Agents useful for same of the embodiment of the present invention, virus liquid and positive controls vaccine are as follows, are ability if not otherwise specified
The conventional scheme or reagent in domain.
Mulser：Germany, model：IKA companies RW20D models
By Quil A (Accurare Chemical＆Scientific Corp.) dissolvings in deionized water, it is prepared into
60mg/ml stock solution.Then, Quil A dissolving stock solutions are filtered using 0.22 micron membrane filter.
Cholesterol (Sigma companies) is dissolved in absolute ethyl alcohol, is prepared into 30mg/ml stock solution.Then, utilize
0.22 zut filter cholesterol stock solution.
GERBU Adjuvant 100 (Sigma companies) is dissolved in absolute ethyl alcohol, is prepared into 20mg/ml storage
Standby solution.Utilize 0.22 zut filter DDA stock solutions.
By Carbopol 974p (Lubrizol Corp. of the U.S.) dissolvings in deionized water, the deposit for being prepared into 10mg/ml is molten
Liquid.Then 121 DEG C of autoclaving 15min are standby.
Montanide GEL 01 (SEPPIC companies) 121 DEG C of autoclaving 15min of solution are standby.
By levamisol (Sigma companies) dissolving in deionized water, it is prepared into 20mg/ml stock solution.Then, it is sharp
With 0.22 zut filter levamisol stock solution.
Antigen used in this experimental vaccine is porcine circovirus 2 type-WH strains (CCTCC NO：V201333) inactivation of viruses liquid.
201 adjuvants are purchased from French SIPPEC companies.
201 Adjuvanted vaccines preparation methods：Two-phase is heated to 31 DEG C respectively with water-bath, the beaker of adjuvant will be filled, put
Under agitator, then by porcine circovirus 2 type-WH strains (CCTCC NO：V201333) inactivation of viruses liquid, which is slowly added into, fills
In the beaker of adjuvant, wherein 201 adjuvants are 1 according to mass ratio with antigen：1, speed of agitator 500rpm/min.Lasting stirring
10min is produced, and viral level is 10 in the final vaccine product7TCID50/ ml, for the embodiment of the present invention.
Marcol52 white oils are purchased from mobil oil chemical company of the U.S..
The Adjuvanted vaccines preparation methods of Marcol 52：Will be added with Tween 80 4%v/v porcine circovirus 2 type-WH strains (CCTCC
NO：V201333) inactivation of viruses liquid is slowly added into (volume ratio 1 in the adjuvants of Marcol 52:1.5) high speed shear 2, is then passed through
Secondary to obtain the vaccine, viral level is 10 in the final vaccine product7TCID50/ ml, for the embodiment of the present invention.
Embodiment 1：
Porcine circovirus 2 type inactivated vaccine (WH strains) is prepared using adjunvant composition of the present invention
Matched according to heterogeneity content in adjunvant composition, entered respectively with 65ml porcine circovirus 2 type virus liquids
Row emulsification, is prepared into 100ml porcine circovirus 2 type inactivated vaccines (being shown in Table 1), and obtained vaccine is named as group 1, group 2, group 3, group
4, for following examples.
Table 1：Each composition (volume ml) of vaccine prepared by different adjuvant prescriptions
Concrete operation method is as follows：
A) PH7.2PBS buffer solutions, adjustment porcine circovirus 2 type-WH strains (CCTCC NO are utilized：V201333) inactivation of viruses
The viral level of liquid, virus stock solution used is made；
B) Carbopol 974p are added in step a composition and fully mixed；
C) Montanide GEL 01 are added in step b composition and fully mixed；
D) GERBU Adjuvant 100 (DDA) is added in step c composition and fully mixed；
E) Quil A are added in step d composition and fully mixed；
F) cholesterol is added in step e composition and fully mixed；
G) levamisol is added in step f composition and fully mixed.
The waterborne compositions that g steps obtain are handled by homogenizer.Then 0.01% sulphur is added according to vaccine cumulative volume
Willow mercury is well mixed, and viral level is 10 in this final vaccine product7TCID50/ml.Finally packing, inspection, gland, labeling, bag
Dress, end product examine outbound, for following examples.
The adjunvant composition of the present invention is not only able to significantly improve the humoral immune response of porcine circovirus 2 type inactivated vaccine
And cellullar immunologic response, and the preparing process of vaccine is stable, immune duration is long and the side reaction caused by vaccine immunity
Aspect is respectively provided with the advantage of uniqueness.Adjunvant composition preparation technology does not need large scale equipment simply, and cost is cheap, and is easy to epidemic disease
The large-scale production of seedling.
Embodiment 2:Vaccine stability contrast test
Pig prepared by 4 groups of porcine circovirus 2 type inactivated vaccines and 201 adjuvants, the Marcol52 adjuvants of the preparation of embodiment 1 is justified
The type inactivated vaccine stability contrast test of circovirus virus 2
Pig circle prepared by 4 groups of porcine circovirus 2 type inactivated vaccines and 201 adjuvants, the Marcol52 adjuvants of the preparation of embodiment 1
The type inactivated vaccine of circovirus virus 2 is individually positioned in 37 DEG C, 25 DEG C, 4 DEG C of observations.Stability (the experiment of the regular sampling observation vaccine
It the results are shown in Table 2, table 3, table 4).
Shown by result of the test, porcine circovirus 2 type inactivated vaccine stability prepared by the adjuvant of this research and development is fine, special
It is not first group of adjuvant, is all optimal under the conditions of 37 DEG C of conditions, 25 DEG C, 4 DEG C.
Stability number of days is observed under the conditions of 237 DEG C of table
Stability number of days is observed under the conditions of 325 DEG C of table
Stability number of days is observed under the conditions of 44 DEG C of table
"-" represents evengranular emulsion.
" -+" represents to have milky/on vaccine liquid level upper strata or has oil precipitation/or layering.
"+" represents that vaccine emulsions have precipitation, and there are a large amount of milky things/or demulsification in liquid level upper strata.
Embodiment 3：
Vaccine antibody Fluctuation experiment prepared by different adjunvant compositions
Pig prepared by 4 groups of porcine circovirus 2 type inactivated vaccines and 201 adjuvants, the Marcol52 adjuvants of the preparation of embodiment 1 is justified
Fluctuation contrast test is immunized in the type inactivated vaccine of circovirus virus 2
1st, after main pathogen and associated antibodies detection are carried out to certain pig farm, porcine circovirus 2 type from 21-25 ages in days,
The cause of disease feminine gender such as CSFV, porcine reproductive and respiratory syndrome virus, pig parvoviral pig, porcine circovirus 2 type antibody are negative
Pig.
2nd, 35 negative antibody pigs will be bought back, be randomly divided into 7 groups, every group 5,1 group -6 groups are inoculated with 1 through musculi colli
Part vaccine (107TCID50/ heads part).7th group (in feeding process, wherein there is a hair sick.Finally by this pig in data statistics
Eliminate.) it is used as blank control (i.e. nonimmune group).
3rd, before being immunized and it is immune after the 7th day, the 14th day, the 28th day, the 60th day, the 90th day, the 120th day, 150 days to every group
Piglet is taken a blood sample, and separation serum detects antibody level with ELISA method.
4th, detection reagent
Porcine circovirus 2 type ELISA antibody assay kits are by Wuhan diagnostic reagent portion of Ke Qian Biological Co., Ltd.
There is provided.
5th, antibody detection method
Detected in strict accordance with porcine circovirus 2 type ELISA antibody assay kit operation instructions.
6th, criterion
Each hole OD is determined on ELIASA630nmValue.Experiment establishment condition is Positive control wells OD630nmValue answers >=1.0, the moon
Property control wells OD630nmValue answers≤0.25.Sample well OD630nmDuring value >=0.40, it is determined as the positive；Sample well OD630nmValue≤0.40
When, it is determined as feminine gender.
7th, antibody test result (being shown in Table 5, table 6, Fig. 5) after porcine circovirus type 2 vaccines prepared by different adjuvants are immune.
Antibody test result after 5 different adjuvant immunities of table
As shown in table 5,3 vaccines of group 1 of the invention and group, 14 days generation antibody is most fast after immune, average OD630Reach
0.9688 and 0.9394.56 days antibody titer OD of group 1 after immune6301.8418 can be reached, and organize 1 and organize 3 antibody levels and hold
It is all high compared with other adjuvant groups when continuous 150 days.It can be illustrated by this experiment, adjuvant group 1 of the invention and group 3 have preferably
Adjuvant effect, it is horizontal optimal particularly to organize 1 vaccine antibody.It is therefore preferable that after group 1 is used for porcine circovirus 2 type inactivated vaccine
Standby adjuvant.
Embodiment 4：
Serum doubling dilution antibody detection test
Piglet blood sampling in 28 days, separation serum after immune.Doubling dilution 1/40,1/80,1/160,1/ is carried out to serum
320、1/640.Serum doubling dilution ELISA resists after antibody level (detection method is with embodiment 3) is immune 28 days after observation is immune
Body testing result (is shown in Table 6, Fig. 6).
Serum doubling dilution ELISA antibody tests after table 6 is immune 28 days
As shown in fig. 6, in adjuvant group 1 of the present invention-groups 4, serum doubling dilution 1：When 320 all than other adjuvant groups all
It is high.Particularly organize 1 antibody level highest.And can be illustrated again by this experiment, the adjuvant of group 1 of the invention has preferable
Adjuvant immunity effect.
Embodiment 5：
Porcine circovirus 2 type inactivated vaccine immune efficacy contrast test prepared by different adjuvants
Pig prepared by 4 groups of porcine circovirus 2 type inactivated vaccines and 201 adjuvants, the Marcol52 adjuvants prepared with embodiment 1
Circovurus type 2 inactivated vaccine does immune efficacy contrast test.
Main pathogen and associated antibodies detection, porcine circovirus 2 type, swine fever from 21-25 ages in days are carried out to certain pig farm
The cause of disease feminine gender such as virus, porcine reproductive and respiratory syndrome virus, pig parvoviral pig, pig japanese b encephalitis, porcine circovirus 2 type resist
Body feminine gender pig 40.Qualified piglet is randomly divided into 8 groups, 1-6 groups (every group of 5 pigs) are immune group, the 7th group (5
Pig) it is to attack malicious control group；8th group (5 pigs) is blank control group.1 part vaccine is immunized by musculi colli respectively in immune group
(107TCID50/ head part).To 1-7 groups while carry out PCV2 virus WH strains (viral level is within 28 days after immune
1.0x107TCID50/ ml, CCTCC NO：V201333) attack, every pig musculi colli injects 3ml, collunarium 2ml, isolated rearing.
In attacking the malicious same day, all experiment pig weights of weighing.And in 3 days before attacking poison (i.e. immune after 25 days) and attack after poison 3,6, institute
Have and attack the equal injecting immune stimulus material of poison group piglet (porous hemocyanin emulsion prepared by Fei Shi Freund's incomplete adjuvants), each every head
Musculi colli injects 4ml.After observation 28 days, all experiment piglets are weighed again；Challenge test piglet blood sample is gathered, separates blood
Clear detection viremia virusemia；Cut open and kill challenge test piglet, take inguinal lymph nodes and lymphonodi mesenterici, carry out histology and exempt from
Epidemic disease histochemistry is detected.The incidence of piglet is analyzed by above indicator-specific statistics, blood sample is gathered, is carried out by ELISA method
PCV2 serum antibodies are analyzed.28 days cut open inspection whole porklings after poison are attacked, meet two in following three, you can judge morbidity.
A body temperature symptoms：Piglet body temperature raises (>=40 DEG C), should at least continue 3 days；
B weight standards：Body weight increase rate, which declines, should be not less than 5.0%, and the average daily gain for attacking malicious piglet should be less than non-attack
The average daily gain of malicious control group piglet.All experiment pig weights are weighed by head respectively in attacking the malicious same day, attack after poison 28 again
It is secondary to weigh all experiment pig weights；Wherein, the calculating of body weight increase rate is carried out as follows in " B weight standards "：
Body weight increase rate (%) is=non- to attack malicious control group piglet average daily gain-to attack poison group piglet average daily gain/attack poison
Control group piglet average daily gain X 100
C virus antigen detections：Lymph node tissue is detected with immunohistochemistry technique, detects PCV2 viruses.
4 groups of porcine circovirus 2 type inactivated vaccines and 201 adjuvants, the Marcol52 adjuvants prepared with embodiment 1, which prepares pig, to be justified
The type inactivated vaccine of circovirus virus 2 attacks poison protection comparative test result (being shown in Table 7)
Each immune group protective rate can be seen that more than 4/5 by protest test result, it is particularly immune 1 group
Overall immune effect is all better than other each groups.Rather than immune group attacks malicious sequela rate 5/5, thus, it could be seen that prepared by the adjuvant
Piglet can be produced and be effectively protected after vaccine immunity, and the evaluation criterion that antibody level can be examined as vaccine potency.By
This is visible, and adjuvant of the invention can play a significant role in porcine circovirus type 2 vaccines, animal can be made effectively to be protected
Shield.And it is highly resistant to the infection of pig circular ring virus.
Table 7：Vaccine protest test result prepared by different adjuvants
Note："+" represents positive；"-" represents negative.
Embodiment 6：
Different adjuvants prepare porcine circovirus 2 type inactivated vaccine safety testing
Pig prepared by 4 groups of porcine circovirus 2 type inactivated vaccines and 201 adjuvants, the marcol52 adjuvants of the preparation of embodiment 1 is justified
The type inactivated vaccine security contrast test of circovirus virus 2.
Carry out main pathogen and associated antibodies detection to Wuhan pig farm, porcine circovirus 2 type from 21-25 ages in days,
The cause of disease feminine gender such as CSFV, porcine reproductive and respiratory syndrome virus, pig parvoviral pig, porcine circovirus 2 type antibody are negative
Pig 35.Qualified piglet is randomly divided into 7 groups, 1-6 groups (every group of 5 pigs) are immune group, and the 7th group is blank control
Group.Immune group is inoculated with 4ml by musculi colli overdose respectively, observes 14 days, and determines 7 days Temperature changings.
Pig prepared by 4 groups of porcine circovirus 2 type inactivated vaccines and 201 adjuvants, the marcol52 adjuvants of the preparation of embodiment 1 is justified
7 days body temperature result of variations (being shown in Table 8) after the type vaccine immunity of circovirus virus 2
Pig prepared by 4 groups of porcine circovirus 2 type inactivated vaccines and 201 adjuvants, the marcol52 adjuvants of the preparation of embodiment 1 is justified
The type inactivated vaccine security contrast test testing result (being shown in Table 9) of circovirus virus 2
Pig prepared by 4 groups of porcine circovirus 2 type inactivated vaccines and 201 adjuvants, the marcol52 adjuvants of the preparation of embodiment 1 is justified
Absorbing state contrasts after the type inactivated vaccine of circovirus virus 2 is immune 28 days.
Table 8：The Temperature changing (DEG C) of different time before and after inoculation
The result as shown in table 8-9 can be seen that the present invention the vaccine of group 1- groups 4 all without appearance it is any by vaccine immunity and
Caused adverse reaction.It can be said that bright, aqueous adjuvants composition of the invention is suitable for porcine circovirus 2 type inactivation epidemic disease
The standby adjuvant of seedling.
Vaccine safety testing inspection result prepared by 9 different adjuvants of table
It is visible by above-mentioned experiment, the porcine circovirus 2 type inactivated vaccine ratio prepared by aqueous adjuvants composition of the present invention
Other oil adjuvants all effects on permeability, side reaction and immune effect are distinguished.The vaccine is simultaneously highly resistant to pig circular ring virus 2
Poison infection, reduce swinery incidence of disease, improve swinery holistic health, particularly organize 1 and group 3 vaccines on immune effect more
With advantage.And the vaccine stability of adjuvant group 1 is best, it is adapted to the large-scale production of vaccine producer and storage.Therefore, 1 epidemic disease is organized
Seedling has good market application foreground at present.
Claims (3)
1. a kind of aqueous adjuvants composition suitable for porcine circovirus 2 type, its formula includes：
The concentration of the Carbopol 974p is 10mg/ml；Quil A concentration is 60mg/ml；The concentration of cholesterol is
30mg/ml；The concentration of GERBU Adjuvant 100 is 20mg/ml；The concentration of levamisol is 20mg/ml；
Described porcine circovirus 2 type is porcine circovirus 2 type-WH strains, CCTCC NO：V201333.
2. the preparation method of vaccine is prepared using the aqueous adjuvants composition described in claim 1, including：
A) PH7.2 PBS is utilized, inactivation of viruses liquid viral level is adjusted, prepares virus stock solution used；
B) Carbopol 974p are added in step a composition and fully mixed；
C) Montanide GEL 01 are added in step b composition and fully mixed；
D) GERBU Adjuvant 100 (DDA) is added in step c composition and fully mixed；
E) Quil A are added in step d composition and fully mixed；
F) cholesterol is added in step e composition and fully mixed；
G) levamisol is added in step f composition and fully mixed；
H) by well mixed preparation, handle, produce finally by homogenizer.
3. application of the aqueous adjuvants composition in porcine circovirus 2 type inactivated vaccine is prepared described in claim 1.
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| CN103667196A (en) * | 2012-09-25 | 2014-03-26 | 普莱柯生物工程股份有限公司 | Vaccine composition containing porcine circovirus-2 antigen and swine flu antigen |
| CN104248760A (en) * | 2013-12-16 | 2014-12-31 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and application thereof |
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