CN103520219B - A kind of innate immunity adjuvant and its vaccine composition and preparation method - Google Patents
A kind of innate immunity adjuvant and its vaccine composition and preparation method Download PDFInfo
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- CN103520219B CN103520219B CN201310481946.XA CN201310481946A CN103520219B CN 103520219 B CN103520219 B CN 103520219B CN 201310481946 A CN201310481946 A CN 201310481946A CN 103520219 B CN103520219 B CN 103520219B
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Abstract
The invention discloses a kind of innate immunity adjuvant and its vaccine compositions and preparation method.The innate immunity adjuvant is the compound adjuvant including at least two kinds of components of saponin and propolis, saponin is dissolved in water and prepares saponin solution, the ethyl alcohol that propolis is dissolved in 95% is prepared into propolis ethanol solution, then saponin solution is mixed in proportion with antigen, propolis solution after adding propolis ethanol solution or being diluted with water, obtains the vaccine composition.The present invention is prepared into natural compound adjuvant using Particular craft for the first time by saponin and propolis compatibility by a certain percentage, and it is horizontal to greatly improve immune response with the natural adjuvant of single component compared with, enhances immune effect.
Description
Technical field
The present invention relates to animal vaccine fields, in particular to animal vaccine adjuvant.
Background technique
" Adjuvant ", i.e. adjuvant derive from Greek " adjuvare " earliest, that is, the meaning helped.Immune assistant
Agent refers to applies simultaneously or in advance with antigen, can enhance the immune response ability that body is directed to antigen, or changes immune response class
The substance of type.There are many common immunologic adjuvant, there is emulsifier, metallic compound, natural or artificial synthesized bacterial product, core
Acid sequence, liposome and immunostimulating complex etc., but there are different defects (such as immune effect is undesirable or toxic side effect is big
Deng), it limits its application and develops.
Veterinary vaccines used in the country are still based on traditional oil-adjuvant vaccine at present, poor, production that there is immune effects
A series of serious problems such as complex process, side effect be strong.And natural extract adjuvant is wide may be selected, and it is low in cost, have water
The advantages that dissolubility, toxic effect is small, green natural is paid more and more attention, and this kind of new Adjuvanted vaccines certainly will will replace traditional oil to help
Vaccinating agent.Highly-safe, the small investment of production equipment of new Adjuvanted vaccines, simple production process, every part cost of estimation are not higher than
Existing oil adjuvant killed vaccine will generate huge social and economic benefits as can promoting.
For natural extract adjuvant, studying relatively broad substance at present includes propolis, saponin and various lipid polysaccharides
Etc..
Saponin (saponin(e/saponarin/saponin/saponin/sapogenin/saponaretin/ground Austria saponin), is from various natural plants
The ingredient with adjuvanticity is screened in object.A large number of studies show that: saponin can be such that exogenous antigen both stimulated to be currently the only
Body Th1 immune response, and can induce the adjuvant of CTL response.This unique property becomes subunit vaccine, intracellular
The ideal adjuvant of pathogen vaccines and cancer vaccine.Application range mainly includes that aftosa vaccine, rabies vaccine etc. are for animals at present
Vaccine.
Propolis has the biological activity of wide spectrum, is a kind of natural immunopotentiator.Draw using propolis or cooperation antigen
Enter body, specific immune response can be caused and start nonspecific defense mechanism, increase antibody production, enhances complement
The phagocytic activity of activity and phagocyte.The fowl cholera vaccine made of propolis has safe and reliable and has no toxic side effect etc. excellent
Point.Ingredient contained by propolis is extremely complex, and whether itself has antigenicity to be worth noting.Poland some scholars studies have shown that
For propolis itself without obvious antigenicity and anaphylaxis, toxic effect is smaller.
But these adjuvant components are immunoreacted there is induction as anti-exclusive use under existing technology of preparing and generate water
Put down low, a series of problems, such as immune protective effect is poor.
Summary of the invention
For the deficiency of current natural adjuvant, the purpose of the present invention is to provide a kind of compound adjuvant of natural extract,
And the vaccine composition using the natural compound adjuvant preparation, to improve the immune response level and immunoprotection effect of vaccine
Fruit.
In order to achieve the above object, the present invention adopts the following technical scheme:
A kind of innate immunity adjuvant includes saponin and propolis.
Innate immunity adjuvant of the invention is the compound adjuvant including at least two kinds of components of saponin and propolis, is first prepared respectively
Then saponin solution and propolis solution mix them with antigen when preparing vaccine by a certain percentage, saponin and bee in vaccine
The mass ratio of glue is 1: 0.8~1: 2.
Above-mentioned saponin solution is saponin to be dissolved in water and degerming is made；And propolis solution is the second that propolis is first dissolved in 95%
Alcohol prepares propolis ethanol solution, which may be directly applied to vaccine formulation, after can also being further diluted with water
It reapplies in vaccine formulation.
In general, the concentration of propolis is in 100-350mg/ml in above-mentioned propolis ethanol solution.
Based on above-mentioned innate immunity adjuvant, the present invention provides a kind of vaccine composition, including immunologic adjuvant and antigen, wherein
Immunologic adjuvant includes at least two kinds of natural materials of saponin and propolis.
In above-mentioned vaccine composition, the content of saponin is 4mg/ml~30mg/ml；The content of propolis be 5mg/ml~
30mg/ml。
Vaccine composition of the invention is prepared by the following method:
1) saponin is configured to certain density saponin solution and degerming with aqua sterilisa；
2) ethyl alcohol that propolis is dissolved in 95% is prepared into propolis ethanol solution, then directly or after being diluted with aqua sterilisa is used to walk
It is rapid 3)；
3) saponin solution made from step 1) and antigen are mixed in a certain ratio uniformly, are then added made from step 2
Bee
Sol solution obtains the vaccine composition.
Above-mentioned steps 2) prepare propolis ethanol solution concentration be 100-350mg/ml.
First saponin and antigen are mixed in a certain concentration ratio when preparing vaccine, are then added by a certain concentration range
The propolis ethanol solution (or propolis solution after dilution) handled well, after mixing, does not need to emulsify, time saving province with seedling process
Power save the cost.Since adjuvant of the present invention is to be formulated by two kinds of natural materials by certain concentration, and two kinds of ingredients can match
At the concentrate of high concentration, in the case, it is shared in vaccine that antigen can be greatly improved with seedling using antigen liquid as solute
Ratio plays the effect of concentrated vaccine, and the immune response for improving vaccine is horizontal.
The present invention for the first time by saponin (saponin(e/saponarin/saponin/saponin/sapogenin/saponaretin/ground Austria saponin) and
Propolis compatibility by a certain percentage, and novel natural compound adjuvant is prepared into using unique processing technology, with it is single at
Divide natural adjuvant compared to immune response level is greatly improved, enhances the immune effect of natural adjuvant, and preparation process
It is environmentally protective, the pure natural structure of adjuvant component is not destroyed, at home and abroad still belongs to pioneering.
Detailed description of the invention
Fig. 1 is that the HI antibody titer experimental result after chicken is immunized in the different adjuvant newcastle disease vaccines of embodiment 1, wherein a, b, c
Immune latter 1 week, 2 weeks, 3 weeks and 4 weeks experimental result is respectively corresponded with d.
Fig. 2 is the HI antibody titer experiment knot that 2 compound natural adjuvant ewcastle disease antigen concentrated vaccine of embodiment is immunized after chicken
Fruit, wherein a, b, c and d respectively correspond immune latter 1 week, 2 weeks, 3 weeks and 4 weeks experimental result.
Fig. 3 is the HI antibody titer experimental result after the immune chicken of adjuvant newcastle disease vaccine of 3 various concentration of embodiment, wherein
A, b, c and d respectively correspond immune latter 1 week, 2 weeks, 3 weeks and 4 weeks experimental result.
Specific embodiment
Following embodiment is merely illustrative, it is to be understood that the present invention is not limited to these embodiments restrictions.
Embodiment l
After bee glue purifying block (flavones content > 98%) 20g is worn into fine powder, it is added in 95% ethyl alcohol 60ml that (this example is only
Ratio) the propolis ethanol solution that concentration is 280mg/ml is made, make propolis granular size stable homogeneous, takes above-mentioned solution
10ml is slowly added in low temperature sterilization water (temperature is lower than 8 DEG C), and keeps stirring, and is settled to 200ml, final concentration of 14mg/
Ml is spare as the propolis ingredient of composite adjuvant.The saponin for separately weighing 280mg, is 20ml with aqua sterilisa constant volume, final concentration of
14mg/ml, the saponin component after aseptic filtration as adjuvant are spare.
In order to prove the better than ever natural adjuvant of the natural adjuvant immunity effect of novel compound of the invention, we are with newcastle disease
(ND) for inactivated vaccine, immunology comparison is carried out, the immune effect of adjuvant is compared.
The natural Adjuvanted vaccines preparation steps of novel compound are as follows: first by above-mentioned saponin component and inactivation ewcastle disease antigen with volume
It is carried out after mixing than 1: 2, the above-mentioned propolis ingredient solution for adding 1 volume can be completed vaccine formulation (A group), in vaccine
Propolis and saponin component concentration be 7mg/ml, antigenic content accounts for the 50% of vaccine volume.Simultaneously with independent propolis ingredient assistant
Vaccinating agent (B group, propolis constituent concentration are 7mg/ml, and antigenic content accounts for the 50% of vaccine volume) and independent saponin component adjuvant
Vaccine (C group, saponin component concentration are 7mg/ml, and antigenic content accounts for the 50% of vaccine volume) is control.At the 0th day immune 30
Age in days SPF chicken (5/group), immunization ways are intramuscular injection, and immunizing dose is 0.4ml/.7th day, 14 days, 21 after immune
It, blood sampling in 28 days is clear surveys HI antibody titer, experimental result is shown in Fig. 1.The result shows that newcastle disease compound natural adjuvant inactivated vaccine (A
Group) it is immune after can produce the HI antibody of higher level, and the antibody duration is long, and immune effect is significantly higher than current natural
Adjuvant (B group and C group).
Embodiment 2
Compound adjuvant of the invention is to be formulated by two kinds of natural materials by certain concentration, and two kinds of ingredients can be made into
The concentrate of high concentration can greatly improve antigen ratio shared in vaccine with seedling using antigen liquid as solute in the case
Example, plays the effect of concentrated vaccine, and the immune response for improving vaccine is horizontal.It is carried out as follows in fact to verify this advantage of adjuvant
It tests:
After bee glue purifying block (flavones content > 98%) 20g is worn into fine powder, it is added in 95% ethyl alcohol 60ml that (this example is only
Ratio) the propolis ethanol solution that concentration is 280mg/ml is made, make propolis granular size stable homogeneous, as composite adjuvant
Propolis ingredient it is spare.The saponin for separately weighing 2800mg, is 10ml with aqua sterilisa constant volume, final concentration of 280mg/ml, degerming
Saponin component after filtering as adjuvant is spare.
Herein by taking newcastle disease (ND) inactivated vaccine as an example, immunology comparison is carried out, the antigen concentrated effect of adjuvant is compared.Epidemic disease
Seedling preparation steps are as follows: first above-mentioned saponin component and inactivation ewcastle disease antigen are carried out after mixing with volume ratio 1: 40, then plus
The above-mentioned propolis ingredient solution for entering 1 volume can be completed vaccine formulation (A ' group), and the propolis and saponin component concentration in vaccine are equal
For 7mg/ml, antigenic content accounts for the 95% of vaccine volume.Furthermore control group is set up, control group vaccine is compound assistant in embodiment 1
Agent group (A group), i.e. propolis in vaccine and saponin component concentration are 7mg/ml, and antigenic content accounts for the 50% of vaccine volume.?
0th day immune 30 age in days SPF chickens (5/group), immunization ways are intramuscular injection, and immunizing dose is 0.4ml/.7th after immune
It, 14 days, 21 days, blood sampling in 28 days is clear surveys HI antibody titer, experimental result is shown in Fig. 2.The result shows that newcastle disease compound natural adjuvant
Antigen concentrated effect is obvious, can produce higher HI antibody level after A' group is immune, and the duration is longer.
Embodiment 3
Compound adjuvant of the invention is to be formulated by two kinds of natural materials by certain concentration, in order to illustrate the immune of adjuvant
Relationship in reinforcing effect and adjuvant between propolis and saponin concentration, is tested as follows:
After bee glue purifying block (flavones content > 98%) 20g is worn into fine powder, it is added in 95% ethyl alcohol 60ml that (this example is only
Ratio) the propolis ethanol solution that concentration is 280mg/ml is made, make propolis granular size stable homogeneous, takes above-mentioned solution
10ml is slowly added in low temperature sterilization water (temperature is lower than 8 DEG C), and keeps stirring, and is settled to 100ml, final concentration of 28mg/
Ml is spare as the propolis ingredient of composite adjuvant.The saponin for separately weighing 280mg, is 10ml with aqua sterilisa constant volume, final concentration of
28mg/ml, the saponin component after aseptic filtration as adjuvant are spare.By above-mentioned saponin component and inactivation ewcastle disease antigen with volume
It is carried out after mixing than 1: 2, the above-mentioned propolis ingredient solution for adding 1 volume can be completed vaccine formulation (A group), in A group
Antigenic content accounts for the 50% of vaccine volume, and the propolis and saponin component concentration in vaccine are 14mg/ml,
It takes the propolis ethanol solution 10ml of 280mg/ml to be slowly added in low temperature sterilization water (temperature is lower than 8 DEG C), does not stop
Stirring, is settled to 200ml, final concentration of 14mg/ml is spare as the propolis ingredient of composite adjuvant.Separately weigh the soap of 280mg
Glucoside is 20ml with aqua sterilisa constant volume, final concentration of 14mg/ml, and the saponin component after aseptic filtration as adjuvant is spare.It will be upper
Saponin component and inactivation ewcastle disease antigen is stated to carry out after mixing, adding the above-mentioned propolis ingredient of 1 volume with volume ratio 1: 2
Vaccine formulation (B group) can be completed in solution, and antigenic content accounts for the 50% of vaccine volume in B group, the propolis and saponin in vaccine at
Point concentration is 7mg/ml.
0th day immune 30 age in days SPF chickens (5/group), immunization ways are intramuscular injection, and immunizing dose is 0.4ml/.Exempt from
It the 7th day after epidemic disease, takes a blood sample within 14 days, 21 days, 28 days and surveys HI antibody titer clearly, experimental result is shown in Fig. 3.The result shows that newcastle disease compound day
Right adjuvant antigen concentrated effect is obvious, can produce higher HI antibody level after A group is immune, and the duration is longer.It proves
Increasing the concentration of propolis and saponin component in adjuvant within the scope of a certain concentration can be improved the immunoenhancement result of adjuvant.
Claims (3)
1. a kind of vaccine composition, including innate immunity adjuvant and antigen, wherein innate immunity adjuvant includes saponin and propolis, soap
The mass ratio of glucoside and propolis is 1: 0.8~1: 2；
The content of saponin is 4mg/ml~30mg/ml in the vaccine composition, and the content of propolis is 5mg/ml~30mg/ml；
The innate immunity adjuvant of the vaccine composition is grouped as by two kinds of groups of saponin and propolis, first respectively prepare saponin solution and
Then antigen and saponin(e solution are mixed in a certain ratio uniformly, then propolis solution and above-mentioned mixed solution are mixed by propolis solution
It closes uniformly, obtains the vaccine composition.
2. vaccine composition as described in claim 1, which is characterized in that the vaccine composition is prepared by following steps
It arrives:
1) saponin is configured to certain density saponin solution and degerming with aqua sterilisa；
2) ethyl alcohol that propolis is dissolved in 95% is prepared into propolis ethanol solution, then directly or after being diluted with aqua sterilisa is used for step
3)；
3) saponin solution made from step 1) and antigen are mixed in a certain ratio uniformly, propolis made from step 2) is then added
Solution obtains the vaccine composition.
3. vaccine composition as claimed in claim 2, which is characterized in that step 2) prepare propolis ethanol solution concentration be
100-350mg/ml。
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| CN1471973A (en) * | 2003-03-08 | 2004-02-04 | 浙江大学 | Notoginseng saponine immunological component and vaccin preparation containing it |
| CN101019940A (en) * | 2007-03-06 | 2007-08-22 | 广东省家禽科学研究所 | Organic composite Chinese medicine immunopotentiator/adjuvant and its prepn process |
| CN101019885A (en) * | 2007-01-17 | 2007-08-22 | 南京农业大学 | Compound ginseng-astragalus immunopotentiator |
| CN101152571A (en) * | 2007-10-09 | 2008-04-02 | 浙江大学 | Vaccine wine oil adjuvant containing momordica cochinchinensis soap and method for preparing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1471973A (en) * | 2003-03-08 | 2004-02-04 | 浙江大学 | Notoginseng saponine immunological component and vaccin preparation containing it |
| CN101019885A (en) * | 2007-01-17 | 2007-08-22 | 南京农业大学 | Compound ginseng-astragalus immunopotentiator |
| CN101019940A (en) * | 2007-03-06 | 2007-08-22 | 广东省家禽科学研究所 | Organic composite Chinese medicine immunopotentiator/adjuvant and its prepn process |
| CN101152571A (en) * | 2007-10-09 | 2008-04-02 | 浙江大学 | Vaccine wine oil adjuvant containing momordica cochinchinensis soap and method for preparing the same |
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Effective date of registration: 20200119 Address after: 100193 Beijing Old Summer Palace West Road, Haidian District, No. 2 Patentee after: China Agricultural University Address before: 100193, building 402, building 1, Cheng Xiu garden, Tian Xiu garden, Beijing, Haidian District, 1 Patentee before: Hu Yanxin |