CN107837393A - Poultry live vaccine dilution adjuvant and preparation method and application - Google Patents
Poultry live vaccine dilution adjuvant and preparation method and application Download PDFInfo
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Abstract
The invention provides a kind of poultry live vaccine dilution adjuvant and preparation method and application.The present invention is by specific screening and combination, prepare poultry live vaccine dilution adjuvant, the adjuvant of the acquisition is applied in poultry live vaccine, it is horizontal original freeze dried vaccine immune antiboidy to be strengthened, improve the immune effect of poultry live vaccine, and the preparation method of the adjuvant is simple and easy, cost is cheap, and using effect is good.
Description
Technical field
The present invention relates to poultry pharmaceutical technology field, especially a kind of poultry live vaccine dilution adjuvant and preparation method thereof
With application.
Background technology
Adjuvant (Adjuvant) is also known as immunomodulator (Immunomodulator) or immunopotentiator
(Immunoenhancement), refer to mix prior to antigen or with antigen or inject in animal body simultaneously, can non-specifically change
Become or strengthen specific immune response of the body to the antigen, play a kind of material of booster action.The English name of adjuvant
Adjuvant derives from Latin " adjuvare ", looks like for " help ".Its effect is main as the main component of vaccine for adjuvant
Have：(1) antigen is made slowly to discharge；(2) antigen residence time in vivo is extended；(3) surface area of antigen is expanded；(4) promotion office
The inflammatory reaction in portion；(5) phagocytosis for strengthening phagocyte is offered with antigen.
Since nineteen twenty-five France immunologist Ranmon has found to add in vaccine some materials having no truck with can be special
Since the immune response for strengthening body different in naturely, the development of immunologic adjuvant so far nearly 90 years.Adjuvant species is a lot, by its property
It can be divided into：Mineral salts adjuvant, saponins adjuvant, mycobacterial derivatives class adjuvant, oil emu adjuvant, particulate antigen presentation system
Adjuvant, Cytokine adjuvant, polysaccharide adjuvant etc., wherein Alum adjuvant and oil emu adjuvant are most commonly used that at present, but they
Also there is the shortcomings that some are inevitable, viscosity is big, stability is bad and injection site significant reaction as oily adjuvant is present, in machine
Degraded in vivo absorbs slow, residence time length, the outward appearance and edible safety for influenceing meat, it is impossible to which effective stimulus T cell is immunized, and causes
Immunosupress；Alum adjuvant is then that be not enough to immune response of the induction compared with poor antigen and be unable to the immune of inducing cell mediation should
Answer, hypodermic injection has continuation granuloma often, it is impossible to absorb well.Live vaccine uses physiology substantially on domestic market
Salt solution or phosphate buffer are diluted, and the vaccine solution after dilution is free of immunomodulator material, and viral antigen is not
Induction body that can be good produces non-specific and specific immune response, therefore new live vaccine immunologic adjuvant is commercially
Great application prospect.
The content of the invention
The purpose of the present invention is：A kind of poultry live vaccine dilution adjuvant and preparation method and application is provided, should by it
It is horizontal that original freeze dried vaccine immune antiboidy can be strengthened in poultry live vaccine, its preparation method is simple and easy.
What the present invention was realized in：Poultry live vaccine dilution adjuvant, it is often left containing 1mg in the 1mL dilution adjuvants
Imidazoles, 0.3~1mg active polysaccharides, 5mg transfer factor, 3mg Sodium New Houttuyfonates are revolved, remaining is phosphate buffer.
Described active polysaccharide is chitosan and Inulin polysaccharide.
The preparation method of poultry live vaccine dilution adjuvant, comprises the following steps：
A) compounding high concentration mother liquor：
1) levamisol mother liquor：Levamisole hydrochloride dry powder is dissolved in sterilized water for injection, being configured to concentration is
100mg/mL levamisole hydrochloride liquid, by levamisole hydrochloride liquid after 116 DEG C of moist heat sterilization 30min, obtain levamisol
Mother liquor, saved backup at 4 DEG C；
2) active polysaccharide mother liquor：Chitosan and Inulin polysaccharide powder are dissolved in sterile phosphate buffer solution, with obtained
It is respectively 100mg/mL, 30mg/mL active polysaccharide mother liquor to chitosan-containing, Inulin polysaccharide concentration, 4 DEG C save backup；
3) transfer factor mother liquor：Transfer factor dry powder is dissolved in sterile phosphate buffer solution, being configured to concentration is
500mg/mL transfer factor mother liquor, cross degerming latter 4 DEG C of 0.22 μm of filter membrane and save backup；
4) neohouttuynin mother liquid of sodium：Sodium New Houttuyfonate crystal powder is dissolved in sterile phosphate buffer solution, matched somebody with somebody
The neohouttuynin mother liquid of sodium that concentration is 300mg/mL is made, crosses degerming latter 4 DEG C of 0.22 μm of filter membrane and saves backup；
B working concentration dilution adjuvant) is prepared：
Levamisol mother liquor, active polysaccharide mother liquor, transfer factor mother liquor, new fish raw meat are taken respectively in above-mentioned mass concentration ratio
The plain mother liquid of sodium of grass and the phosphate buffer of sterilizing, by above-mentioned each component it is well mixed after, it is degerming to cross 0.22 μm of filter membrane, obtains every
1mL 1mg containing levamisol, 0.3~1mg of active polysaccharide, transfer factor 5mg and Sodium New Houttuyfonate 3mg vaccine diluent assistant
Agent.
Application of the poultry vaccine dilution adjuvant in poultry live vaccine, by poultry live vaccine and described vaccine diluent
Adjuvant is 1 by volume:15~1:After 20 mixed dissolutions, the poultry live vaccine containing adjuvant is obtained, to improve poultry live vaccine
Immune effect.
Described poultry live vaccine is family's poultry infectious bursal disease live vaccine, and poultry live Newcastle disease vaccine or poultry are infected
Property bronchitis live vaccine.
Levamisol is that (Levamisolum) is a kind of wide spectrum antihelmintic, while is also first and is found to have and exempts from
The artificial-synthetic compound of epidemic disease adjustment effect, there is extensive immunoregulatory activity, cell and humoral immunity can be promoted to promote leaching
Bar cell propagation.Chitosan (Chitosan) is the polysaccharide obtained by chitin by deacetylation.Research finds that shell gathers
Sugar can improve absorbability, the mediate drug transmission of local mucous membrane as vaccine adjuvant, can also improve the locality antibody of machine in addition
Reaction, antibody is promoted to produce.Inulin (Inulin) belongs to line style synanthrin type levulan, can improve animal intestinal tract environment, promotes
Immune Organs of Body is developed, and improves immunoglobulin content in blood, adjusts body immunity.Transfer factor (Transfer
Factor, TF), also known as transmission factor, produced by the lymphocyte with cellular immunity function.Transfer factor can activate huge
Phagocyte, internal interleukin I I, interferon et al. the generation of cytokine profiles in vivo are improved, have and trigger and adjust cell
Immunologic function, it is a kind of new immune stimulating agent.Sodium New Houttuyfonate (Sodium new houttuyfonate, SNH) is
Synthetic material and the addition compound of sodium hydrogensulfite, its existing certain bacteriostasis, and can improve immunity of organisms, increase
The phagocytic activity of strong leucocyte, improves body non-specific immune function.
As a result of above technical scheme, the present invention prepares poultry live vaccine by specific screening and combination
Dilution adjuvant, the adjuvant of the acquisition is applied in poultry live vaccine, original freeze dried vaccine immune antiboidy level can be strengthened, carried
The immune effect of high poultry live vaccine, and the preparation method of the adjuvant is simple and easy, cost is cheap, and using effect is good.
Brief description of the drawings
Fig. 1 is NDV HI antibody titer the dynamic monitor result figures；
Fig. 2 is interferon dynamic detection result figure；
Fig. 3 is interleukin-4 dynamic detection result figure；
Fig. 4 is interleukin 10 dynamic detection result figure；
Fig. 5 is t lymphocyte subset group (CD3, CD4, CD8) dynamic detection result figure；
Fig. 6 is influence result figure of the immunomodulator of the present invention to IBDV antibody；
Fig. 7 is influence result figure of the immunomodulator of the present invention to new branch bigeminal live vaccine NDV antibody；
Fig. 8 is influence result figure of the immunomodulator of the present invention to new branch bigeminal live vaccine IBV antibody.
Embodiment
Embodiments of the invention 1：Poultry live vaccine dilution adjuvant, often contain the left-handed miaows of 1mg in the 1mL dilution adjuvants
Azoles, 0.3~1mg active polysaccharides, 5mg transfer factor, 3mg Sodium New Houttuyfonates, remaining is phosphate buffer.
Described active polysaccharide is chitosan and Inulin polysaccharide.
The preparation method of poultry live vaccine dilution adjuvant, comprises the following steps：
A) compounding high concentration mother liquor：
1) levamisol mother liquor：Levamisole hydrochloride dry powder is dissolved in sterilized water for injection, being configured to concentration is
100mg/mL levamisole hydrochloride liquid, by levamisole hydrochloride liquid after 116 DEG C of moist heat sterilization 30min, obtain levamisol
Mother liquor, saved backup at 4 DEG C；
2) active polysaccharide mother liquor：Appropriate chitosan, Inulin polysaccharide powder are dissolved in sterile phosphate buffer solution, matched somebody with somebody
Chitosan-containing is made, Inulin polysaccharide concentration be respectively 100mg/mL, 30mg/mL active polysaccharide mother liquor, 4 DEG C save backup；
3) transfer factor mother liquor：Transfer factor dry powder is dissolved in sterile phosphate buffer solution, being configured to concentration is
500mg/mL transfer factor mother liquor, cross degerming latter 4 DEG C of 0.22 μm of filter membrane and save backup；
4) neohouttuynin mother liquid of sodium：Sodium New Houttuyfonate crystal powder is dissolved in sterile phosphate buffer solution, matched somebody with somebody
The neohouttuynin mother liquid of sodium that concentration is 300mg/mL is made, crosses degerming latter 4 DEG C of 0.22 μm of filter membrane and saves backup；
B working concentration dilution adjuvant) is prepared：
When being prepared by taking 100mL adjuvants as an example, 100mg/mL levamisol mother liquor 1mL, 0.3~1mg/mL are taken respectively
Active polysaccharide mother liquor 1mL, 500mg/mL transfer factor mother liquor 1mL, 300mg/mL neohouttuynin mother liquid of sodium 1mL, with
Remaining is that the well mixed rear 0.22 μm of filter membrane of the phosphate buffer dilution of sterilizing is degerming, as per 1mL 1mg containing levamisol,
0.3~1mg of active polysaccharide, transfer factor 5mg, Sodium New Houttuyfonate 3mg vaccine diluent immunomodulator.
Embodiments of the invention 2：Application of the poultry vaccine dilution adjuvant in chicken Newcastle disease live vaccine
First, experiment material
1. experimental animal：SPF hatching eggs are purchased from Beijing Cimmeria Wei Tong experimental animals Technology Co., Ltd., hatch rearmounted Guizhou
Foster bio tech ltd Animal House negative pressure isolated area is raised.
2. vaccine：Chicken Newcastle disease live vaccine (La Sota strains) is produced by Guizhou Foster bio tech ltd.
3. adjuvant component：Levamisol used in preparation is purchased from Guilin Pharmaceutical (Shanghai) Co., Ltd.；Chitosan is public purchased from Sigma
Department, Inulin polysaccharide are purchased from Belgian Beneo-Orafit companies；Transfer factor is purchased from Hangzhou Shan Mu bioengineering Co., Ltd；Newly
Sodium Houttuyfonate is purchased from the big magnificent great achievement medication chemistry Co., Ltd in Wuhan.
2nd, experimental method
1. assist agent solution is prepared
Poultry vaccine dilution adjuvant is prepared by the methods described of embodiment 1.
2. the situation of packet
The basically identical plumage of SPF chick 120 of body weight is chosen, is randomly divided into 2 groups, every group of 60 plumages divide in negative pressure isolated area
Do not raise, in the newcastle disease that 7 ages in days are diluted by collunarium vaccine inoculation, one group of inoculation 0.03mL with sterile phosphate buffer solution
Live vaccine, another group of chicken Newcastle disease live vaccines of the inoculation 0.03mL containing immunologic adjuvant.
3. antibody test
It is prepared by 3.1 serum
Every group takes 5 plumage test chickens at random every time, weekly under heart or wing venous blood sampling once, 3500rpm centrifugations, transfer
The transparent serum in upper strata (at least needs 200 μ L), and -20 DEG C of Cord bloods are to be measured.
3.2 Serum Antibody Detection
With reference to existing《Republic of China Veterinary Pharmacopoeia》Hemagglutination inhibition test (HI test) method is detected：Determine in serum
Antibody IgY against chicken Newcastle Disease potency.
4.T Lymphocyte subtypes tests
It is measured with reference to t lymphocyte subset group (CD3, CD4, CD8) ELISA detection kit specification.
5. cytokines measurement
The chicken serum collected weekly is carried out to the detection of relevant cell factor, with reference to chicken serum IFN-γ cell factor
ELISA detection kit specification, chicken serum IL-4 cell factor ELISA detection kit specifications and chicken serum IL-10 are thin
Intracellular cytokine ELISA detection kit specification is carried out.
3rd, experimental result
1.NDV HI antibody titer the dynamic monitor results
As shown in Figure 1, immunomodulator can promote NDV protection antibody to produce earlier initial stage in vaccine immunity, and maintain
Higher antibody titer.
2. interferon dynamic detection result
As shown in Figure 2, in the peripheral blood interferon content of chicken this index, the IFN-γ of the group containing immunomodulator moves
State content is above the detection level of plain vaccine group respective point.
3. interleukin-4 dynamic detection result
From the figure 3, it may be seen that in the content of peripheral blood 4 of chicken this index, the IL-4 of immunomodulator group is moved
State content is higher than the detection level of corresponding plain vaccine group respective point.
4. interleukin 10 dynamic detection result
As shown in Figure 4, in the interleukin 10 content of peripheral blood of chicken this index, addition immunomodulator group
IL-10 dynamics content was above the detection level of plain vaccine group respective point in addition to the 2nd and the 3rd week.
5.T lymphocyte subgroups (CD3, CD4, CD8) dynamic detection result
As shown in Figure 5, in the t lymphocyte subset group content of peripheral blood of chicken this index, immunomodulator group is added
Dynamic content be higher than plain vaccine group respective point detection level.
Embodiment 3：The application that poultry vaccine dilution adjuvant is in poultry infectious bursal disease live vaccine
First, experiment material
1. experimental animal：With embodiment 2.
2. vaccine：Infectious bursal disease live-vaccine (B87 strains) is produced by Guizhou Foster bio tech ltd.
3. adjuvant component：With embodiment 2.
2nd, experimental method
1. assist agent solution is prepared
Immunomodulator is prepared by the methods described of embodiment 1.
2. the situation of packet
The basically identical plumage of SPF chick 120 of body weight is chosen, is randomly divided into 2 groups, every group of 60 plumages divide in negative pressure isolated area
Do not raise, in 14 ages in days by collunarium vaccine inoculation, the chicken that one group of inoculation 0.03mL sterile phosphate buffer solutions dilute is infected
Property bursal disease live vaccine, another group of infectious bursal disease live-vaccines of the inoculation 0.03mL containing immunologic adjuvant.
3. antibody test
It is prepared by 3.1 serum
Every group takes 5 plumage test chickens at random every time, weekly under heart or wing venous blood sampling once, 3500rpm centrifugations, transfer
The transparent serum in upper strata (at least needs 200 μ L), and -20 DEG C of Cord bloods are to be measured.
3.2 Serum Antibody Detection
Infectious bursa of Fabricius virus antibody in serum is entered with reference to love moral scholar IBD ELISA antibody assay kits specifications
Row detection.
3rd, experimental result
It will be appreciated from fig. 6 that immunomodulator can promote IBDV protection antibody to produce earlier initial stage in vaccine immunity, tie up simultaneously
Higher antibody titer is held, and the decay of IBDV antibody can be slowed down.
Embodiment 4：Poultry vaccine dilution adjuvant answering in poultry ewcastle disease, infectiousness bronchitis bigeminy live vaccine
With.
First, experiment material
1. experimental animal：With embodiment 3.
2. vaccine：Newcastle disease, infectiousness bronchitis bigeminy live vaccine (La Sota strain+H120 strains) are by Guizhou FOX
Special bio tech ltd's production.
3. adjuvant component：With embodiment 3.
2nd, experimental method
1. assist agent solution is prepared
Immunomodulator is prepared by the methods described of embodiment 1.
2. the situation of packet
The basically identical plumage of SPF chick 120 of body weight is chosen, is randomly divided into 2 groups, every group of 60 plumages divide in negative pressure isolated area
Do not raise, in the new branch two that 7 ages in days are diluted by collunarium vaccine inoculation, one group of inoculation 0.03mL with sterile phosphate buffer solution
Join live vaccine, new branch bigeminal live vaccines of another group of inoculation 0.03mL containing immunologic adjuvant.
3. antibody test
It is prepared by 3.1 serum
Every group takes 5 plumage test chickens at random every time, weekly under heart or wing venous blood sampling once, 3500rpm centrifugations, transfer
The transparent serum in upper strata (at least needs 200 μ L), and -20 DEG C of Cord bloods are to be measured.
3.2 Serum Antibody Detection
With reference to existing《Republic of China Veterinary Pharmacopoeia》Hemagglutination inhibition test (HI test) method is detected：Determine in serum
NDV, IBV antibody titer.
3rd, experimental result
As shown in Figure 7, immunomodulator can promote new branch bigeminal live vaccine induction NDV to protect earlier initial stage in vaccine immunity
Protect antibody to produce, while maintain higher antibody titer, and the decay of NDV antibody can be slowed down.
As shown in Figure 8, immunomodulator can promote new branch bigeminal live vaccine induction IBV to protect earlier initial stage in vaccine immunity
Protect antibody to produce, while maintain higher antibody titer, and the decay of IBV antibody can be slowed down.
The embodiment of the present invention being not limited to described in embodiment, those skilled in the art are according to the present invention
Technical scheme draw other embodiments, also belong to the present invention technological innovation scope.Obvious those skilled in the art
Member can carry out various changes and modification without departing from the spirit and scope of the present invention to the present invention.So, if the present invention
These modifications and variations belong in the range of the claims in the present invention and its equivalent technologies, then the present invention is also intended to comprising these changes
Including modification.
Claims (5)
- A kind of 1. poultry live vaccine dilution adjuvant, it is characterised in that：In per the 1mL dilution adjuvants containing 1mg levamisols, 0.3~1mg active polysaccharides, 5mg transfer factor, 3mg Sodium New Houttuyfonates, remaining is phosphate buffer.
- 2. poultry live vaccine dilution adjuvant according to claim 1, it is characterised in that：Described active polysaccharide gathers for shell Sugar and Inulin polysaccharide.
- A kind of 3. preparation method of poultry live vaccine dilution adjuvant as claimed in claim 1 or 2, it is characterised in that including Following steps：A) compounding high concentration mother liquor：1) levamisol mother liquor：Levamisole hydrochloride dry powder is dissolved in sterilized water for injection, it is 100mg/ to be configured to concentration ML levamisole hydrochloride liquid, by levamisole hydrochloride liquid after 116 DEG C of moist heat sterilization 30min, levamisol mother liquor is obtained, 4 DEG C save backup；2) active polysaccharide mother liquor：Active polysaccharide is dissolved in sterile phosphate buffer solution, it is more that preparation obtains chitosan-containing, inulin Sugared concentration is respectively 100mg/mL, 30mg/mL active polysaccharide mother liquor, and 4 DEG C save backup；3) transfer factor mother liquor：Transfer factor dry powder is dissolved in sterile phosphate buffer solution, it is 500mg/ to be configured to concentration ML transfer factor mother liquor, cross degerming latter 4 DEG C of 0.22 μm of filter membrane and save backup；4) neohouttuynin mother liquid of sodium：Sodium New Houttuyfonate crystal powder is dissolved in sterile phosphate buffer solution, is configured to Concentration is 300mg/mL neohouttuynin mother liquid of sodium, crosses degerming latter 4 DEG C of 0.22 μm of filter membrane and saves backup；B working concentration dilution adjuvant) is prepared：Levamisol mother liquor, active polysaccharide mother liquor, transfer factor mother liquor, neohouttuynin are taken respectively in above-mentioned mass concentration ratio Mother liquid of sodium and the phosphate buffer of sterilizing, after above-mentioned each component is well mixed, 0.22 μm of filter membrane excessively is degerming, obtains per 1mL 1mg containing levamisol, 0.3~1mg of active polysaccharide, transfer factor 5mg and Sodium New Houttuyfonate 3mg vaccine diluent adjuvant.
- 4. application of the poultry vaccine dilution adjuvant according to claim 1 in poultry live vaccine, it is characterised in that：Will Poultry live vaccine is 1 by volume with described vaccine diluent adjuvant:15~1:After 20 mixed dissolutions, the family containing adjuvant is obtained Fowl live vaccine, to improve the immune effect of poultry live vaccine.
- 5. application of the poultry vaccine dilution adjuvant according to claim 4 in poultry live vaccine, it is characterised in that：Institute The poultry live vaccine stated is family's poultry infectious bursal disease live vaccine, poultry live Newcastle disease vaccine or family's avian infectious bronchitis Live vaccine.
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| CN108837151A (en) * | 2018-09-30 | 2018-11-20 | 派生特（福州）生物科技有限公司 | The preparation method of one boar vaccine diluent |
| CN108904813A (en) * | 2018-09-30 | 2018-11-30 | 派生特（福州）生物科技有限公司 | A kind of preparation method of fowl vaccine diluent |
| CN111467488A (en) * | 2020-04-17 | 2020-07-31 | 内蒙古必威安泰生物科技有限公司 | Water-soluble composite immunologic adjuvant and application thereof |
| CN114306590A (en) * | 2021-12-31 | 2022-04-12 | 皖西学院 | Veterinary vaccine diluent with immunity enhancement effect and preparation method and application thereof |
| CN115444933A (en) * | 2022-09-14 | 2022-12-09 | 贵州福斯特生物科技有限公司 | Preparation method and application of novel water-soluble compound adjuvant for rabbits |
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| CN111467488B (en) * | 2020-04-17 | 2023-09-01 | 内蒙古必威安泰生物科技有限公司 | Water-soluble composite immunoadjuvant and application thereof |
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