CN109395076A - A kind of aftosa immunopotentiator, preparation and its application - Google Patents
A kind of aftosa immunopotentiator, preparation and its application Download PDFInfo
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Abstract
The present invention provides a kind of aftosa immunopotentiator, the aftosa immunopotentiator is obtained after astragalus polyose, triphenyl phosphate, hyaluronic acid and muramyl dipeptide are successively dissolved in solvent, the concentration of muramyl dipeptide is 80-220 μ g/mL in the immunopotentiator, the concentration of triphenyl phosphate is 20-60mg/mL, the concentration of hyaluronic acid is 1-6 μ g/mL, and the concentration of astragalus polyose is 1-6mg/mL.Aftosa vaccine the present invention also provides the preparation method of the aftosa immunopotentiator and containing the immunopotentiator.Aftosa immunopotentiator of the present invention, preparation method is convenient, the effect that can effectively improve vaccine is used after mixing with inactivated foot-and-mouth disease vaccine, the qualification rate of mean antibody levels, antibody not only can be improved, extend the duration to 6 months or more, and after saving 1 year, it still is able to significantly improve the antibody titer after aftosa vaccine is immunized.
Description
Technical field
The present invention relates to field of biological pharmacy, and in particular to a kind of aftosa immunopotentiator, preparation and its application.
Background technique
Aftosa (Foot-and-mouth disease, FMD) is by foot and mouth disease virus (Foot-and-mouth
Disease virus, FMDV) caused by the artiodactyls such as a kind of infected cattle, sheep and pig, with animal epidemic disease highly infectious
Disease.The virus has 7 serotypes: A, 1 type of O, C, SAT1, SAT2, SAT3 and Asia, and each serotype contains again
Several hypotypes.Vaccine immunity is one of the key measure for controlling the disease, and in China, aftosa vaccine belongs to compulsory immunization epidemic disease
Seedling, inactivated vaccine are vaccine main bodys used at present, and the most effective means of prevention and control at present, but there is also Antibody qualification rates
Technical problem lower, immune duration is shorter.
Immunopotentiator is the quick method for improving vaccine immunity effect, does not change existing production technology, and the R&D cycle is short,
Preparation method is simple, easy to use.Although the immunopotentiator for being currently used for aftosa can significantly improve inactivated foot-and-mouth disease vaccine
Immune efficacy, but immunopotentiator storage life is short, and vaccine formulation is used further to after preservation, and it is significant to will lead to Immune-enhancing effect effect
Decline.
Summary of the invention
The main object of the present invention is to provide a kind of aftosa immunopotentiator, can not only significantly improve aftosa vaccine
Antibody titer and immune duration after immune, and after saving 1 year, still be able to significantly improve aftosa vaccine it is immune after
Antibody titer.
Another object of the present invention is to provide the preparation method of the aftosa immunopotentiator, can be obtained using this method
The immunopotentiator obtained still is able to significantly improve the antibody titer after aftosa vaccine is immunized after saving 1 year.
It is a further object of the present invention to provide the applications of the aftosa immunopotentiator, and immune one is only needed for piglet
A secondary needle, which can be protected, to be delivered for sale, and the antibody duration extends to 6 months or more, significantly reduces pig raising cost.
The purpose of the present invention adopts the following technical scheme that realization:
A kind of aftosa immunopotentiator, the aftosa immunopotentiator are by astragalus polyose, triphenyl phosphate, transparent
Matter acid and muramyl dipeptide, which are successively dissolved in, to be obtained after solvent, and in the immunopotentiator concentration of muramyl dipeptide is 80-
220 μ g/mL, the concentration of triphenyl phosphate are 20-60mg/mL, and the concentration of hyaluronic acid is 1-6 μ g/mL, the concentration of astragalus polyose
For 1-6mg/mL.
In the present invention, the solvent be physiological saline, water for injection, PBS buffer solution, Tris-HCl buffer or
Hank ' s liquid.
In preferred technical solution, the solvent is the Tris-HCl buffer of pH8.0.
The present invention also provides the preparation method of the aftosa immunopotentiator, be by astragalus polyose, triphenyl phosphate, thoroughly
Bright matter acid and muramyl dipeptide obtain the aftosa immunopotentiator after being successively dissolved in solvent.
In the present invention, the solvent be physiological saline, water for injection, PBS buffer solution, Tris-HCl buffer or
Hank ' s liquid.
In preferred technical solution, the solvent is the Tris-HCl buffer of pH8.0.
The present invention also provides the aftosa vaccines for containing the immunopotentiator.
In the present invention, immunopotentiator is uniformly mixed with Tween-80 according to volume ratio for 96:4, obtains water phase；It is white
Oil is uniformly mixed with Si Ben -80 according to volume ratio for 96:4, obtains oily phase；Water phase is mutually mixed with oily according to volume ratio for 1:2,
Emulsification, obtains companion's vaccine；Companion's vaccine is mixed with commercialization aftosa vaccine according to volume ratio for 1:25~100, is stirred
Uniformly, the aftosa vaccine is obtained.
It in the present invention, is that 1:100 is mixed according to volume ratio with the O-shaped hoof-and-mouth disease venom of the pig of inactivation by immunopotentiator
Uniformly, aqueous phase solution is obtained；Aqueous phase solution is mixed with ISA206 according to volume ratio for 1:1, emulsifies, obtains the aftosa epidemic disease
Seedling.
The present invention compared with the existing technology, has the advantage that
1, aftosa immunopotentiator of the present invention, use can effectively improve vaccine after mixing with inactivated foot-and-mouth disease vaccine
Effect, not only can be improved the qualification rate of mean antibody levels, antibody, extend the duration to 6 months or more, and save one
Nian Hou still is able to significantly improve the antibody titer after aftosa vaccine is immunized.
2, the preparation method of aftosa immunopotentiator of the present invention is convenient, immunopotentiator matching while using or with postponing 4
Degree is saved 1 year and is ok, and addition is small in size, as long as 1%, it is remarkably improved immune efficacy, extends the antibody duration to 6
Month with.
3, immunopotentiator cooperation aftosa vaccine of the present invention only needs piglet immune primary, so that it may which protection arrives
Column, antibody duration extend to 6 months or more, significantly reduce pig raising cost, reducing swinery stress.
Detailed description of the invention
Fig. 1 shows the immune effect of each immunopotentiator.Abscissa is inactivation aftosa vaccine number, and ordinate is liquid
Mutually block the average value of ELISA antibody titer.
(addition saves the aftosa epidemic disease of immunopotentiator after different time to the storage life potency test of Fig. 2 immunopotentiator
The evaluation of seedling immune efficacy), wherein abscissa is each vaccine number, and ordinate is the average value of Liquid-phase blocking ELISA antibody titer.
Fig. 3 shows companion's vaccine+trivalent vaccine immune rear antibody duration, and wherein abscissa is the immune rear time, single
Position is day, in (A), (B), (C) ordinate be respectively A type, the average value of O-shaped and sub- I type Liquid-phase blocking ELISA antibody titer.
Fig. 4 shows the antibody duration after companion's vaccine+O-shaped vaccine immunity, and abscissa is the immune rear time, and ordinate is
The average value of O-shaped Liquid-phase blocking ELISA antibody titer.
Fig. 5 shows companion's vaccine+polypeptide vaccine immune rear antibody duration, and abscissa is immune vaccine grouping, left
Side ordinate is the OD value of O-shaped Liquid-phase blocking ELISA antibody test, and right side ordinate is O-shaped Liquid-phase blocking ELISA antibody titer
Average value.
Specific embodiment
The present invention will be further explained with reference to the accompanying drawing.
Embodiment 1: the method for preparing immunopotentiator and inactivating aftosa vaccine
1, experimental material
Muramyl dipeptide (being abbreviated as MDP) and hyaluronic acid (500~800kDa) are purchased from InvivoGen company.
Triphenyl phosphate (TPP) is purchased from Chinese medicines group.
Astragalus polyose is purchased from the honest Biotechnology Co., Ltd in Shaanxi.
ISA206 is purchased from match hectogram company.
The O-shaped hoof-and-mouth disease venom of the pig of inactivation (strain is 98 plants of ZhuOXing foot and mouth disease virus Burma), goes out through binary ethylenimine
Living, 146s content is 5.87 μ g/mL.
Aftosa O, A, the Asia I trivalent vaccine of commercialization are purchased from Jin Yubaoling biologics Co., Ltd.
Aftosa Liquid-phase blocking ELISA antibody titer≤1:8 of the susceptible piglet of 6-7 week old health.
The present invention detects Liquid-phase blocking ELISA using aftosa Liquid-phase blocking ELISA kit (Lanzhou veterinary institute)
Antibody titer.
2. the configuration of immunopotentiator
Immunopotentiator contain 200 μ g/mL MDP, 50mg/mL triphenyl phosphate (TPP), 5 μ g/mL hyaluronic acids and
5mg/mL astragalus polyose, solvent are the Tris-HCl buffer of 0.1M, pH8.0.
Immunopotentiator the preparation method is as follows: solute is successively dissolved in solvent according to sequencing, finally use
Solvent constant volume, packing is into penicillin bottle after filtering (0.22 μm of filter) degerming, 4 DEG C of preservations.In order to investigate in immunopotentiator
Influence of the dissolution sequence of solute to its stability and immune effect devises 24 kinds of dissolution sequences, specific as shown in table 1.
Wherein the step of method 13 are as follows: hyaluronic acid is dissolved in the Tris-HCl buffer of 0.1M, pH8.0 by the first step
In；Astragalus polyose, stirring to dissolution is added in second step；Third step is added triphenyl phosphate (TPP), stirring to dissolution；4th
Muramyl dipeptide, stirring to dissolution is added in step；Using the Tris-HCl buffer constant volume of 0.1M, pH8.0.
Wherein the step of method 21 are as follows: astragalus polyose is dissolved in the Tris-HCl buffer of 0.1M, pH8.0 by the first step
In；Second step is added triphenyl phosphate (TPP), stirring to dissolution；Hyaluronic acid, stirring to dissolution is added in third step；4th
Muramyl dipeptide, stirring to dissolution is added in step；Using the Tris-HCl buffer constant volume of 0.1M, pH8.0.
Solute dissolution sequence, corresponding immunopotentiator and vaccine number in 1 24 kinds of preparation methods of table
24 kinds of immunopotentiators that method in table 1 is prepared are according to the number of preparation method, and number is immune increasing respectively
Strong agent 1-24.
3, method for preparing aftosa vaccine
Prepare the first method of aftosa vaccine: using immunopotentiator as companion's vaccine, it is specific the preparation method is as follows:
Immunopotentiator is mixed with Tween-80 according to volume ratio for 96:4, is stirred evenly as water phase；White oil and Si Ben -80 according to
Volume ratio is 96:4 mixing, is stirred evenly as oily phase；Water phase is mutually mixed with oily according to volume ratio for 1:2, emulsifies, obtains companion
Companion's vaccine.Companion's vaccine and commercialized vaccine (aftosa O, A, Asia I trivalent vaccine) is mixed for 1:25~100 according to volume ratio
It closes, is directly inoculated with after mixing evenly.
Prepare the second method of aftosa vaccine: by the O-shaped hoof-and-mouth disease venom of the pig of immunopotentiator and inactivation according to
Volume ratio is sufficiently mixed for 1:100, is prepared as aqueous phase solution.ISA206 and aqueous phase solution are respectively placed in about 30 points of room temperature
Then clock ISA206 is added in emulsion tank, aqueous phase solution is added in emulsion tank under 200 revs/min of speeds of agitator, 2000
It is stirred 10 minutes under the conditions of rev/min, obtains vaccine.Wherein water phase and the volume ratio of ISA206 are 1:1.
The vaccine prepared respectively according to above two method using immunopotentiator, the solute concentration phase in immunopotentiator
It is almost the same on immune effect under conditions of, only facilitate the user for producing upper different demands to use.
The present embodiment is by immunopotentiator 1-24 in title 2 using second method preparation inactivation aftosa vaccine, vaccine
Number is shown in Table 1.
Control vaccine 1 (is free of reinforcing agent): preparing control vaccine 1 according to the second method for preparing aftosa vaccine, no
It is only that with place and immunopotentiator is substituted using the Tris-HCl buffer of 0.1M, pH8.0.
4, it is grouped, immune and antibody test
Experimental group and immune: the susceptible piglet of health is randomly divided into 25 groups, every group 10.Wherein adopt for 24 groups every group of piglet
With one of table 1 vaccine immunity, immunizing dose is 2mL/ head；Another set piglet as a control group, immunized controls vaccine 1,
Immunizing dose is 2mL/ head.
Observed after vaccine immunity, after being immunized for 24 hours in, observation piglet is without apparent fever and stress show, do not occur diarrhea,
The constitutional symptoms such as fash, oedema, purpura, excited irritability, loss of appetite, vomiting；Test pig injection site is observed in 14 days without red
The local reaction of the symptoms such as swollen, scleroma, itch, lymph enlargement, it was demonstrated that the immunopotentiator is to piglet safety.
Effect after immune is monitored, the specific method is as follows: 28 days after immune, taking a blood sample to each group piglet and separate blood
Clearly, the antibody after each vaccine immunity is detected using aftosa Liquid-phase blocking ELISA kit (Lanzhou veterinary institute) generates feelings
Condition is (when Liquid-phase blocking ELISA antibody titer is more than or equal to 26For antibody qualification), calculate the liquid phase resistance of piglet after each vaccine immunity
The average value of disconnected ELISA antibody titer.
5, the antibody situation of each group piglet after being immunized
As shown in Figure 1, antibody level significantly improves after most of vaccine immunity for control vaccine 1, generally exist
2-4 times.Effect after wherein vaccine 13 and 21 is immune is best, and Mean antibody titers can be improved 6 for control vaccine 1
Times or more, nearly 3 times are improved for the vaccine containing other immunopotentiators.
In conclusion using astragalus polyose, TPP, hyaluronic acid and muramyl dipeptide are successively dissolved method preparation and
At immunopotentiator 21 can significantly improve the immune efficacy of aftosa vaccine, there is good exempt to inactivated foot-and-mouth disease vaccine
Epidemic disease humidification.
The storage life of 2 immunopotentiator of embodiment is tested
1, the preparation of immunopotentiator
The preparation method is the same as that of Example 1 for immunopotentiator 21.
The preparation method is the same as that of Example 1 for immunopotentiator 13.
Immunopotentiator 25: immunopotentiator 25 contains 100 μ g/mL MDP, 30mg/mL triphenyl phosphate (TPP), 2 μ g/
ML hyaluronic acid and 2mg/mL astragalus polyose, solvent are the Tris-HC buffer of 0.1M, pH8.0.The preparation of immunopotentiator 25
Method is as follows: astragalus polyose is dissolved in the Tris-HCl buffer of 0.1M, pH8.0 by the first step；Tricresyl phosphate is added in second step
Phenyl ester (TPP), stirring to dissolution；Hyaluronic acid, stirring to dissolution is added in third step；4th step is added muramyl dipeptide, stirs
It mixes to dissolution；Using the Tris-HCl buffer constant volume of 0.1M, pH8.0.
Compound immunoenhancer 1: being the molten of the MDP and 0.4mg/mL beta glucan of MPL, 20mg/mL containing 2mg/mL
The Tris-HCl buffer of liquid, solvent 0.1M, pH8.0 is molten.Preparation method is as follows: same container is added after weighing each substance
In, solvent dissolution, constant volume is then added.
Compound immunoenhancer 2: be containing 500 μ g/mL MPL, 500 μ g/mL MDP, 500 μ g/mL beta glucans and
The Tris-HCl buffer of the solution of 5mg/mL astragalus polyose, solvent 0.1M, pH8.0 is molten.Preparation method is as follows: weighing each object
It is added after matter in same container, solvent dissolution, constant volume is then added.
2, vaccine preparation and immune efficacy evaluation
Immunopotentiator 21, immunopotentiator 13 and immunopotentiator 25, compound immunoenhancer 1 and compound immune are increased
Strong agent 2 is configured to vaccine progress pig after preservation half a year, 1 year under the conditions of 4 DEG C respectively and is immunized, immune with animal experiment evaluation
The immune efficacy of reinforcing agent.
(1) prepared by 21 related vaccines of immunopotentiator
After immunopotentiator 21 and 4 DEG C after 21,4 DEG C of immunopotentiator preservation half a year of Fresh is saved 1 year
Immunopotentiator 21 is respectively adopted following method and is configured to vaccine: by the O-shaped hoof-and-mouth disease venom of the pig of immunopotentiator and inactivation
It is sufficiently mixed according to volume ratio for 1:100, is prepared as aqueous phase solution.ISA206 and aqueous phase solution are respectively placed in room temperature about 30
Minute, then ISA206 is added in emulsion tank, aqueous phase solution is added in emulsion tank under 200 revs/min of speeds of agitator,
It is stirred 10 minutes under the conditions of 2000 revs/min, obtains vaccine.Wherein water phase and the volume ratio of ISA206 are 1:1.Fresh is exempted from
The epidemic disease that the immunopotentiator 21 after immunopotentiator 21 and 4 DEG C preservation 1 year after 21,4 DEG C of epidemic disease reinforcing agent preservation half a year is configured to
Seedling is successively denoted as 21-FMD, 21-FMD- half a year, 21-FMD- 1 year.
(2) 13 related vaccines of immunopotentiator
After immunopotentiator 13 and 4 DEG C after 13,4 DEG C of immunopotentiator preservation half a year of Fresh is saved 1 year
Immunopotentiator 13 is respectively adopted following method and is configured to vaccine: by the O-shaped hoof-and-mouth disease venom of the pig of immunopotentiator and inactivation
It is sufficiently mixed according to volume ratio for 1:100, is prepared as aqueous phase solution.ISA206 and aqueous phase solution are respectively placed in room temperature about 30
Minute, then ISA206 is added in emulsion tank, aqueous phase solution is added in emulsion tank under 200 revs/min of speeds of agitator,
It is stirred 10 minutes under the conditions of 2000 revs/min, obtains vaccine.Wherein water phase and the volume ratio of ISA206 are 1:1.Fresh is exempted from
The epidemic disease that the immunopotentiator 13 after immunopotentiator 13 and 4 DEG C preservation 1 year after 13,4 DEG C of epidemic disease reinforcing agent preservation half a year is configured to
Seedling is successively denoted as 13-FMD, 13-FMD- half a year, 13-FMD- 1 year.
(3) 25 related vaccines of immunopotentiator
After immunopotentiator 25 and 4 DEG C after 25,4 DEG C of immunopotentiator preservation half a year of Fresh is saved 1 year
Immunopotentiator 25 is respectively adopted following method and is configured to vaccine: by the O-shaped hoof-and-mouth disease venom of the pig of immunopotentiator and inactivation
It is sufficiently mixed according to volume ratio for 1:100, is prepared as aqueous phase solution.ISA206 and aqueous phase solution are respectively placed in room temperature about 30
Minute, then ISA206 is added in emulsion tank, aqueous phase solution is added in emulsion tank under 200 revs/min of speeds of agitator,
It is stirred 10 minutes under the conditions of 2000 revs/min, obtains vaccine.Wherein water phase and the volume ratio of ISA206 are 1:1.Fresh is exempted from
The epidemic disease that the immunopotentiator 25 after immunopotentiator 25 and 4 DEG C preservation 1 year after 25,4 DEG C of epidemic disease reinforcing agent preservation half a year is configured to
Seedling is successively denoted as 25-FMD, 25-FMD- half a year, 25-FMD- 1 year.
(4) compound immunoenhancer 1,2 related vaccines
1,4 DEG C of compound immunoenhancer after 1,4 DEG C of compound immunoenhancer preservation half a year of Fresh is saved one
Compound immunoenhancer 1 after year is respectively adopted following method and is configured to vaccine: the pig of compound immunoenhancer and inactivation is O-shaped
Hoof-and-mouth disease venom is after 1:9 is mixed, to obtain aqueous phase solution according to volume ratio.ISA206 and aqueous phase solution are respectively placed in room
Then ISA206 is added in emulsion tank, emulsion tank is added in aqueous phase solution under 200 revs/min of speeds of agitator by temperature about 30 minutes
In, it is stirred 10 minutes under the conditions of 2000 revs/min, obtained vaccine.Wherein water phase and the volume ratio of ISA206 are 1:1.By new
Vaccine made of the compound immunoenhancer 1 of fresh preparation is denoted as compound 1-FMD；By the compound immune enhancing after 4 DEG C of preservation half a year
Vaccine made of agent 1 is denoted as compound 1-FMD- half a year；Vaccine made of compound immunoenhancer 1 after being saved 1 year 4 DEG C point
It is not denoted as compound 1-FMD- 1 year.
2,4 DEG C of compound immunoenhancer after 2,4 DEG C of compound immunoenhancer preservation half a year of Fresh are saved one
Compound immunoenhancer 2 after year is respectively adopted following method and is configured to vaccine: the pig of compound immunoenhancer and inactivation is O-shaped
Hoof-and-mouth disease venom is after 1:100 is mixed, to obtain aqueous phase solution according to volume ratio.ISA206 and aqueous phase solution are respectively placed in
Room temperature about 30 minutes, then ISA206 is added in emulsion tank, aqueous phase solution is added under 200 revs/min of speeds of agitator and is emulsified
In tank, stirred 10 minutes under the conditions of 2000 revs/min, obtained vaccine.Wherein water phase and the volume ratio of ISA206 are 1:1.By
Vaccine made of the compound immunoenhancer 2 of Fresh is denoted as compound 2-FMD；Increased by the compound immune after 4 DEG C of preservation half a year
Vaccine made of strong agent 2 is denoted as compound 2-FMD- half a year；Vaccine made of compound immunoenhancer 2 after being saved 1 year 4 DEG C
It is denoted as compound 2-FMD- 1 year.
(5) FMD control vaccine
FMD control vaccine is prepared according to the preparation method of 21-FMD, the difference is that only the Tris- with 0.1M, pH8.0
The immunopotentiator 21 of HCl buffer substitution Fresh.
3, it is grouped, immune and antibody test
Experimental group and immune: the susceptible piglet of health is grouped at random, every group 10, totally 16 groups.By the present embodiment title 2
The susceptible piglet of each immune one group of health of 16 kinds of vaccines of middle preparation, immunizing dose is 2mL/ head.
Observed after vaccine immunity, after being immunized for 24 hours in, observation piglet is without apparent fever and stress show, do not occur diarrhea,
The constitutional symptoms such as fash, oedema, purpura, excited irritability, loss of appetite, vomiting；Test pig injection site is observed in 14 days without red
The local reaction of the symptoms such as swollen, scleroma, itch, lymph enlargement.Therefore, above-mentioned vaccine is safe for piglet.
28th day after immune, the susceptible piglet blood sampling separation serum of each group health, using aftosa Liquid-phase blocking ELISA reagent
Box (Lanzhou veterinary institute) detect vaccine immunity after antibody production (when Liquid-phase blocking ELISA antibody titer be greater than etc.
In 26For antibody qualification), calculate mean antibody levels.
Mean antibody levels after immune are as shown in Figure 2.As shown in Figure 2, by the immunopotentiator of Fresh 21,13,
The effect of vaccine immunity made of the compound immunoenhancer 1,2 of mean antibody levels and Fresh after vaccine immunity made of 25
Fruit is suitable, there is apparent immunological enhancement；After saving half a year, compound immunoenhancer 1,2 and immunopotentiator 13 are made
Immune effect of vaccine be decreased obviously, mean antibody levels are basically unchanged after vaccine immunity made of immunopotentiator 21,25, are
4 times or more of mean antibody levels after compound immunoenhancer 1,2 and the corresponding vaccine immunity of immunopotentiator 13；It saves 1 year
Afterwards, immune effect of vaccine made of compound immunoenhancer 1,2 substantially with FMD control vaccine no significant difference, immunopotentiator
21, the mean antibody levels after vaccine immunity made of 25 are declined slightly, but mean antibody levels are still that compound immune increases
4 times or more of the strong corresponding vaccine mean antibody levels of agent 1,2.
It can be seen that higher immune efficacy can still be maintained after 4 DEG C save 1 year in immunopotentiator 21 and 25.
Embodiment 3: the immunopotentiator 21 after saving 1 year is immunized aftosa commercialized vaccine as companion's vaccine
Effect evaluation
1, immune vaccine preparation:
Immunopotentiator 21 after saving 1 year under the conditions of taking 4 DEG C carries out the preparation of companion's vaccine as research object.
It according to volume ratio is 96:4 by the immunopotentiator 21 after being saved 1 year under the conditions of 4 DEG C and Tween-80, mixing is equal
It is even, as water phase；White oil is uniformly mixed with Si Ben -80 according to volume ratio 96:4, as oily phase；By water phase and oil mutually according to body
For product than being that 1:2 is mixed, emulsification obtains companion's vaccine.
According to volume ratio it is 96:4 by the Tris-HCl buffer of 0.1M, pH 8.0 and Tween-80, is uniformly mixed, as
Water phase；White oil is uniformly mixed with Si Ben -80 according to volume ratio 96:4, as oily phase；Mutually it is according to volume ratio by water phase and oil
1:2 mixing, emulsification obtain control companion's vaccine.
Companion's vaccine, control companion's vaccine are uniformly mixed with each commercialized vaccine according to volume ratio 1:50 respectively, obtained
Immune vaccine, to investigate the immunopotentiator 21 after saving 1 year to the immunological enhancement of commercialized vaccine.Wherein commodity
Changing vaccine includes: (1) aftosa O, A, Asia I tervalence inactivated vaccine (being abbreviated as trivalent vaccine), date of manufacture 20170409, purchase
From Inner Mongol Jin Yubaoling biologics Co., Ltd；(2) O-shaped inactivated foot-and-mouth disease vaccine (being abbreviated as O-shaped vaccine), date of manufacture
20170316, it buys from middle peasant Witter biotech inc；(3) Schweineseuche O-shaped synthetic peptide vaccine (is abbreviated as more
Peptide vaccine), polypeptide vaccine lot number: (2017) 090297522, date of manufacture 20170421, purchase joins from Shanghai Shen.
2, it is grouped, immune and antibody test
Experimental group and immune: the susceptible piglet of health is grouped at random, every group 10, totally 6 groups.
Companion's vaccine, control companion's vaccine and each commercialized vaccine are prepared immune one group immune respectively with vaccine
The susceptible piglet of health, the trivalent seedling of addition companion's vaccine is named as companion's vaccine+trivalent vaccine, and adds control companion's vaccine
Trivalent vaccine is named as control trivalent vaccine；The name of other immune vaccines is according to said method analogized.Immunizing dose is 2mL/ head,
Specifically it is shown in Table 2.
2 vaccine immunity of table and grouping
| Grouping | Immune vaccine type | Immune head number |
| 1 | Companion's vaccine+trivalent vaccine | 10 |
| 2 | Compare trivalent vaccine | 10 |
| 3 | Companion's vaccine+O-shaped vaccine | 10 |
| 4 | Compare O-shaped vaccine | 10 |
| 5 | Companion's vaccine+polypeptide vaccine | 10 |
| 6 | Compare polypeptide vaccine | 10 |
Antibody production after each group piglet immunological is monitored: 28th, 60,90,120,150,180 day after immune
Take a blood sample separation serum to each group piglet respectively, using O-shaped aftosa Liquid-phase blocking ELISA kit (Lanzhou veterinary institute) and
Polypeptide vaccine VP1 antibody assay kit (purchased from Shanghai Shen join) detection vaccine immunity after antibody level and to the duration into
Row monitoring.O-shaped Liquid-phase blocking ELISA antibody titer is more than or equal to 26For antibody qualification, A and Asia I type Liquid-phase blocking ELISA antibody
Titre is more than or equal to 27For antibody qualification.
Antibody duration such as Fig. 3 after trivalent seedling is immune.As seen from the figure, adding in trivalent seedling can be with after immunopotentiator 21
O-shaped, A type and Asia I type Liquid-phase blocking ELISA antibody titer are significantly improved, and the antibody duration is no less than 6 months.
Antibody duration such as Fig. 4 after O-shaped inactivated foot-and-mouth disease vaccine is immune.As seen from the figure, in O-shaped inactivated foot-and-mouth disease vaccine
Immune rear O-shaped Liquid-phase blocking ELISA antibody titer can be significantly improved after addition immunopotentiator 21, and the antibody duration is many
In 6 months.
28 days antibody level such as Fig. 5 after polypeptide vaccine is immune.As seen from Figure 5, it can be shown after adding immunopotentiator 21
It writes and improves the antibody level after polypeptide vaccine is immunized, and the qualification rate peace of O-shaped Liquid-phase blocking ELISA antibody titer can be improved
Equal antibody titer.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of aftosa immunopotentiator, it is characterised in that the aftosa immunopotentiator is by astragalus polyose, tricresyl phosphate
Phenyl ester, hyaluronic acid and muramyl dipeptide, which are successively dissolved in, to be obtained after solvent, muramyl dipeptide in the immunopotentiator
Concentration is 80-220 μ g/mL, and the concentration of triphenyl phosphate is 20-60mg/mL, and the concentration of hyaluronic acid is 1-6 μ g/mL, Radix Astragali
The concentration of polysaccharide is 1-6mg/mL.
2. aftosa immunopotentiator according to claim 1, it is characterised in that the solvent is physiological saline, injection
Water, PBS buffer solution, Tris-HCl buffer or Hank ' s liquid.
3. aftosa immunopotentiator according to claim 2, it is characterised in that the Tris-HCl that the solvent is pH8.0 is slow
Fliud flushing.
4. the preparation method of aftosa immunopotentiator described in one of claim 1-3, it is characterised in that by astragalus polyose, phosphoric acid
Triphenylmethyl methacrylate, hyaluronic acid and muramyl dipeptide obtain the aftosa immunopotentiator after being successively dissolved in solvent.
5. the preparation method of aftosa immunopotentiator according to claim 4, it is characterised in that the solvent is physiology salt
Water, water for injection, PBS buffer solution, Tris-HCl buffer or Hank ' s liquid.
6. aftosa immunopotentiator according to claim 5, it is characterised in that the Tris-HCl that the solvent is pH8.0 is slow
Fliud flushing.
7. the aftosa vaccine containing immunopotentiator described in one of claim 1-3.
8. aftosa vaccine according to claim 7, it is characterised in that be according to volume ratio by immunopotentiator and Tween-80
96:4 is uniformly mixed, and obtains water phase；White oil is uniformly mixed with Si Ben -80 according to volume ratio for 96:4, obtains oily phase；By water phase with
Oily is mutually 1:2 mixing according to volume ratio, and emulsification obtains companion's vaccine；By companion's vaccine and commercialization aftosa vaccine according to body
Product stirs evenly than being that 1:25 ~ 100 is mixed, obtains the aftosa vaccine.
9. aftosa vaccine according to claim 7, it is characterised in that by the O-shaped hoof-and-mouth disease of the pig of immunopotentiator and inactivation
Venom is uniformly mixed according to volume ratio for 1:100, obtains aqueous phase solution；It according to volume ratio is 1:1 by aqueous phase solution and ISA206
Mixing, emulsification, obtains the aftosa vaccine.
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| CN107158374A (en) * | 2017-06-13 | 2017-09-15 | 江苏省农业科学院 | A kind of immunopotentiator, inactivated foot-and-mouth disease vaccine and preparation method thereof |
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| CN107158374A (en) * | 2017-06-13 | 2017-09-15 | 江苏省农业科学院 | A kind of immunopotentiator, inactivated foot-and-mouth disease vaccine and preparation method thereof |
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| 肖建雄等: "复合佐剂的研究进展", 《广东畜牧兽医科技》 * |
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