CN106310249A - Preparation method and use method of novel adjuvant of oil emulsion inactivated vaccine - Google Patents
Preparation method and use method of novel adjuvant of oil emulsion inactivated vaccine Download PDFInfo
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- CN106310249A CN106310249A CN201510332905.3A CN201510332905A CN106310249A CN 106310249 A CN106310249 A CN 106310249A CN 201510332905 A CN201510332905 A CN 201510332905A CN 106310249 A CN106310249 A CN 106310249A
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Abstract
The invention discloses a preparation method of a novel adjuvant of an oil emulsion inactivated vaccine and a use method of the novel adjuvant in an oil emulsion inactivated vaccine and belongs to the technical field of preparation and use of the veterinary oil emulsion inactivated vaccine. The preparation method comprises taking a transfer factor diluent, carrying out filtration and degerming, adding tween-80 into the diluent according to a ratio to obtain a water phase, preparing an oil phase from white oil for injection and Span-80 according to a ratio, and carrying out emulsification on the water phase and the oil phase according to a ratio of 1: 2-1: 3 at a temperature of 15-35 DEG C at a rotation rate of 2500-3500rpm for 15-30min. The invention discloses a method for preparing an oil emulsion inactivated vaccine from the adjuvant. The method comprises carrying out emulsification mixing on the adjuvant and a routine vaccine according to a ratio of 1: 3 to 1: 4 to obtain a novel adjuvant vaccine. The novel adjuvant vaccine has immunization effects obviously better than those of the routine vaccine and has a short immunization blanking period and a uniform immunization antibody level higher than that of the routine vaccine.
Description
Technical field
The present invention relates to the preparation of a kind of oil-emulsion inactivated vaccine novel adjuvant, and this adjuvant is oil emulsion inactivated
Application process in Seedling, this invention can realize strengthening the technology requirement of inactivated vaccine immune effect.
Background technology
China breeding environment is complicated, and animal epidemic and pesticide, residual shine into meat product outlet of veterinary drug medicine are seriously damaged
Lose.Reduce Animal diseases, it is to avoid prolonged application antibiotic, develop high-quality, nuisance free meat products, Cai Nengbao
The sustainable and healthy development of card China aquaculture.
At present, prevent and control zoonosis in addition to safety measure, relying primarily on vaccine immunity, particularly
By the immunoprophylaxis of oil emulsion vaccine.For oil-emulsion inactivated vaccine, normal conditions can after immune 14 days
Produce antibody, how can evoke immunity in the shorter time, improve immune effect, need to add adjuvant with
Strengthen them and induce immunoreactive ability.
In inactivated vaccine, adjuvant is applied with antigen simultaneously, it is possible to strengthen collective's immunne response energy for antigen
Power, or change immunoreation type.Its function mainly has: use position to solicit immunocyte and immune molecule,
Strengthen immunne response；Strengthen vaccine antigen transmission；Promote immunity contact, strengthen poor antigen immunogenicity and
Immunological memory, changes the configuration of antigen；Change the kind of humoral antibody, IgG hypotype and the affinity of antibody,
And cellular immunization and mucosa-immune effect.Preferably adjuvant does not only have immunogenicity also energy enhancing body
Immunne response, it is possible to make body obtain optimal protective immunity and immunological memory.Relevant immunostimulant side
The research in face, the most, it is concentrated mainly on herbal polysaccharide, cytokine, interferon, gathers
Flesh born of the same parents etc., the most also one is commonly recognized immunostimulant---and transfer factor, current veterinary drug industry is
There are many families in batch production.Transfer factor (Thansfer Factor is called for short TF) is that human or animal's sensitization is the thinnest
A kind of lower-molecular substance that can shift sensitization information in the multiple factor of born of the same parents' release.Since nineteen ninety-five
Since Lawrence finds transfer factor (Transfer factor).The research of transfer factor is got more and more by people.
Owing to its molecular weight is little, nontoxic, no antigen, does not causes allergic reaction, do not produce neutralizing antibody and can surpass
Get over the advantages such as germline boundary application, and at treatment immunodeficiency, malignant tumor, various infectious disease etc.
All obtaining certain curative effect in the test of aspect, therefore transfer factor has broad prospects in clinical practice.
Transfer factor has dialyzability, can be combined and no antigen itself with antigen.Transfer factor with
The specific immunity information of primed lymphocyte, it is possible to the special antigenicity that donor is had and acumen thereof
Transspecific give immunoreation negative receptor, receptor produces a delayed hypersensitivity energy simultaneously
Power.Transfer factor has premunition information, excites immunologic cellular activity, regulation immunologic function, enhancing machine
Body non-specific immunity etc. act on, and are described as the triggering agent of T cell activity, the reinforcing agent of cellular immunization,
Cell immunomodulator and interferon produce and start agent.
Transfer factor solution coordinates attenuated vaccine to use, it is verified that the immune effect of vaccine can be improved, but
Not yet have been reported that transfer factor as the research report of oil-emulsion inactivated vaccine adjuvant.
Summary of the invention
The present invention is that inactivated vaccine provides a kind of new immunological adjuvant-transfer factor, and utilizes this adjuvant system
The new method of standby inactivated vaccine.This vaccine is not only able to shorten the blank phase of oil-emulsion inactivated vaccine, also can improve
The antibody titer of inactivated vaccine, has positive effect to improving vaccine protection.
The present invention is to be achieved through the following technical solutions:
The raw material of adjuvant of the present invention consists of:
Aqueous phase:
Transfer factor 94%～96%
Tween 80 4%～6%
Oil phase:
Si Ben-80 5%～6%
Injection white oil 95%～94%
Aluminium stearate 0.5%～2%
Wherein said Material Percentage is volume ratio；
Wherein the content of peptides of transfer factor is 2.5～5.5mg/ml, and Ribose concentration is 50～100ug/ml, and transfer factor comes
Source is broiler spleen, laying hen spleen or Lien Sus domestica.
Wherein aqueous phase is 1: 2～1: 3 with the ratio of oil phase.
The adjuvant prepared and conventional vaccine are prepared from according to 1: 3～1: 4 ratio mixing and emulsifying.
Take above-mentioned raw materials, be prepared as vaccine adjuvant according to following technique
The preparation method of vaccine adjuvant of the present invention is:
Taking transfer factor diluent through 0.22um filtration sterilization, add tween 80 according to the above ratio, 37 DEG C shake up,
Hatching 1～2h, be prepared as aqueous phase, oil phase boils according to aforementioned proportion, 121 DEG C of sterilizings 30 minutes.?
Carrying out emulsifying at 15～35 DEG C, rotating speed is 2500rpm～3500rpm, and the time is 15～30min.After emulsifying completes,
Mixing according to the ratio of 1: 3～1: 4 with conventional vaccine, continue emulsifying 10～15min, emulsifying rotating speed is
2500rpm～3500rpm, treats that vaccine dosage and stability reach requirement.
The oil emulsion vaccine that the present invention makes, joins in vaccine using transfer factor as immunostimulant, Ke Yiyou
Effect improves the response of immunocyte, the submission of quickening antigen, and then shortens the Blank immunization phase of antigen, carries
High antibody horizontal, it is possible to activating immune system, eliminates immunosuppressant.
Detailed description of the invention
Embodiment 1:
1. prepared by adjuvant
1.1. transfer factor diluent is done aseptic, many through 0.22um polyether sulfone material filter element filtering, sampling
Peptide content, Ribose concentration detect, and steriling test total negative, content of peptides is 2.8mg/ml, and Ribose concentration is
58ug/ml。
1.2. 4 parts of sterilizing tween 80s are joined in 96 parts of transfer factor diluents, mixing, 37 DEG C of conditions
Under shake up 1h, take out and be put in room temperature.
1.3. joining in 94 parts of white oils by 1 part of aluminium stearate, heating adds Si Ben-806 after white oil is bright
Part, 121 DEG C of sterilizing 30min.
1.4. according to 3: 1 ratios, oil phase and aqueous phase being carried out emulsifying, emulsifying temperature is 20 DEG C, and emulsifying rotating speed is
3000rpm, emulsifying 20min.
2. prepared by conventional vaccine
2.1. the newcastle that HA titer is 1: 1024 and the bird flu (H9 that HA titer is 1: 1024 are taken
Hypotype) virus liquid is separately added into formalin and inactivates, and two kinds of inactivation liquid are respectively 48 parts.
2.2. take 4 parts of tween 80s to be put in sterilizing cabinet, after 121 DEG C of sterilizing 30min, be cooled to room temperature, by tween
-80 are added in newcastle and avian influenza inactivation liquid, and every kind adds 2 parts.
1 part of aluminium stearate is joined in 94 parts of white oils by 2.3, and heating adds Si Ben-806 after white oil is bright
Part, 121 DEG C of sterilizing 30min.
Oil phase and aqueous phase are carried out emulsifying according to 3: 1 ratios by 2.4, and emulsifying temperature is 20 DEG C, and emulsifying rotating speed is
3000rpm, emulsifying 20min.
3. prepare containing Adjuvanted vaccines
Being mixed according to 2: 7 ratios with conventional vaccine by adjuvant, emulsifying 10min, emulsifying emulsifying rotating speed is
3000rpm。
Reference examples 1
With conventional vaccine for comparison case, preparation technology is with " ' 2. prepared by conventional vaccine ' in case study on implementation 1 ".
Reference examples 2
1. prepared by blank oil emulsion
1.2. joining in 96 parts of sterile salines by 4 parts of sterilizing tween 80s, mixing, under the conditions of 37 DEG C
Shake up 1h, take out and be put in room temperature.
1.3. joining in 94 parts of white oils by 1 part of aluminium stearate, heating adds Si Ben-80 6 after white oil is bright
Part, 121 DEG C of sterilizing 30min.
1.4. according to 3: 1 ratios, oil phase and aqueous phase being carried out emulsifying, emulsifying temperature is 20 DEG C, and emulsifying rotating speed is
3000rpm, emulsifying 20min.
2. prepared by conventional vaccine
2.1. the newcastle that HA titer is 1: 1024 and the bird flu (H9 that HA titer is 1: 1024 are taken
Hypotype) virus liquid is separately added into formalin and inactivates, and two kinds of inactivation liquid are respectively 48 parts.
2.2. take 4 parts of tween 80s to be put in sterilizing cabinet, after 121 DEG C of sterilizing 30min, be cooled to room temperature, by tween
-80 are added in newcastle and avian influenza inactivation liquid, and every kind adds 2 parts.
1 part of aluminium stearate is joined in 94 parts of white oils by 2.3, and heating adds Si Ben-806 after white oil is bright
Part, 121 DEG C of sterilizing 30min.
Oil phase and aqueous phase are carried out emulsifying according to 3: 1 ratios by 2.4, and emulsifying temperature is 20 DEG C, and emulsifying rotating speed is
3000rpm, emulsifying 20min.
3. prepared by blank sample vaccine
Blank oil emulsion is mixed according to 2: 7 ratios with conventional vaccine, emulsifying 10min, emulsifying emulsifying rotating speed
For 3000rpm.
Relatively case study on implementation 1, reference examples 1, reference examples 2 mass effect
1 shape
1.1 outward appearances: three is milky Emulsion.
1.2 dosage forms: three is all in water-in-oil type.In the plate filling purified water, drip two Emulsions, observe
Its form, if dispersion, and scattered time.Result shows that both forms are regular circle shapes, no
Diffusion.
1.3 viscositys: detect with Digital Viscometer.Result is it can be seen that the present invention and conventional vaccine glutinous
Degree is without significant difference.The results are shown in Table 1.
1.4 stability:
(1) take 10ml vaccine to be placed in centrifuge tube and be centrifuged 15min with 3000r/min, the aqueous phase separated out at the bottom of pipe
It is respectively less than 0.5ml.
(2) taking vaccine and place 21d at about 37 DEG C, result shows not stratified, does not has demulsifying phenomenon.
2 steriling tests
Test by the relevant regulations of " People's Republic of China's veterinary drug allusion quotation " 2010 editions annex, comply fully with,
All without bacterial growth.
3 safety examinations
With 21 age in days SPF chickens, every kind of vaccine immunity 10, every intramuscular injection vaccine 1ml, separately set blank
Compare 5.Every day observes, and inoculates latter 14 days and cuts open inspection, observes local and general reaction and observes local epidemic disease
Seedling absorbing state.Result shows, vaccine of the present invention, compared with conventional vaccine, untoward reaction does not the most occur.
The results are shown in Table 1
1 three kinds of vaccine physical forms of table and safety verification contrast
4 efficacy tests
4.1 newcastle parts
Serological method: with 30-60 age in days SPF chicken, 10 chickens of every kind of vaccine immunity, every subcutaneous or flesh
Meat injection 20ul, blank 5.Within after inoculation 7,14,21,28 days, take a blood sample, separate serum,
Carry out HI antibody titer mensuration.Result shows that vaccine of the present invention to exceed 1 than conventional vaccine HI antibody and drip
About degree, conventional vaccine was difficult to evoke humoral immunization at 7 days, produces immunoprotection, and the present invention was at 7 days
Having obvious immune antibody to produce, exceed 0.9 titre than reference examples, additionally antibody dispersion is also significantly lower than
Reference examples, shows that vaccine protection of the present invention is substantially better than conventional vaccine.The results are shown in Table 2.
2 three kinds of vaccine newcastle part efficacy test Comparative result of table
4.2 bird flu parts
Serological method: with 3～4 week old SPF chickens, 10 chickens of every kind of vaccine immunity, every subcutaneously or intramuscularly
Injection 0.3ml, blank 5.Within after inoculation 7,14,21,28 days, take a blood sample, separate serum, enter
Row HI antibody titer measures.Result shows that vaccine of the present invention to exceed 1 titre than conventional vaccine HI antibody
Left and right, and antibody dispersion is relatively low, shows that vaccine protection of the present invention is apparently higher than conventional vaccine.Evaluate
The results are shown in Table 3.
3 three kinds of vaccine bird flu part efficacy test Comparative result tables of table
The above results clearly shows, the immunization shown by novel adjuvant vaccine of the present invention and conventional vaccine phase
Ratio has 3 advantages: one is that antibody plays soon, and the Blank immunization phase is short；Two is that antibody titer is high, than routine
Vaccine exceeds about 1 titre；Three is that antibody dispersion is low, and antibody horizontal ratio is more uniform.
Overall merit, novel adjuvant of the present invention is obvious to the immunological enhancement of traditional vaccine, to improving vaccine matter
Amount, to protection animal health, significant to the sustainable and healthy development ensureing China's aquaculture.
Above-mentioned detailed description system illustrating for one of present invention possible embodiments, only this case study on implementation is not
It is to limit the scope of the claims of the present invention, all without departing from skill spirit of the present invention institute equivalence enforcement or change for it,
It is both contained in the scope of the claims of present case.
Claims (6)
1. the application process of an oil emulsion vaccine novel adjuvant for animals, it is characterized in that, by volume ratio 1: 3～1: 4, with conventional oil emulsion vaccine, novel adjuvant is carried out emulsifying to mix, be wherein mainly composed of transfer factor diluent, tween 80, vaccine antigen, Si Ben-80, injection white oil, aluminium stearate.
2. novel adjuvant as claimed in claim 1, it is characterised in that aqueous phase and oil are in a ratio of 1: 2～1: 3.
3. the application process of novel adjuvant as claimed in claim 1, it is characterised in that novel adjuvant and conventional oil emulsion vaccine volume ratio are 1: 3～1: 4.
4. novel adjuvant preparation method as claimed in claim 1, it is characterised in that comprise the steps:
Taking transfer factor diluent through 0.22um filtration sterilization, add tween 80 according to the above ratio, 37 DEG C shake up, and hatch 1～2h, are prepared as aqueous phase, and oil phase proportionally boils, 121 DEG C of sterilizings 30 minutes.Carrying out emulsifying at 15～35 DEG C, rotating speed is 2500rpm～3500rpm, and the time is 15～30min.
5. transfer factor diluent as claimed in claim 1, it is characterized in that, described transfer factor diluent needs through 0.22um aseptic filtration, content of peptides is 2.5～4.5mg/ml, Ribose concentration is 30-100ug/ml, and transfer factor raw material sources is one or more in broiler spleen, laying hen spleen, Lien Sus domestica.
6. novel adjuvant as claimed in claim 1, the raw material that it is characterized by composition is as follows
Aqueous phase:
Transfer factor 94%～96%
Tween 80 4%～6%
Oil phase:
Si Ben-80 5%～6%
Injection white oil 95%～94%
Aluminium stearate 0.5%～2%.
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Cited By (6)
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| CN108144057A (en) * | 2018-01-10 | 2018-06-12 | 杭州洪桥中科基因技术有限公司 | A kind of vaccine diluent and preparation method thereof |
| CN110665379A (en) * | 2019-10-08 | 2020-01-10 | 浙江农林大学 | Water-in-oil type emulsification method aiming at vaccine antigen |
| CN111729081A (en) * | 2020-07-21 | 2020-10-02 | 天康生物股份有限公司 | Vaccine adjuvant, preparation method and application thereof, and prepared vaccine |
| CN112675299A (en) * | 2021-01-06 | 2021-04-20 | 黑龙江省百洲生物工程有限公司 | Preparation method of compound adjuvant inactivated vaccine for preventing and controlling goose astrovirus |
| CN114288403A (en) * | 2021-12-20 | 2022-04-08 | 浙江洪晟生物科技股份有限公司 | Newcastle disease and H9 subtype avian influenza bivalent inactivated vaccine containing immunopotentiator and preparation method and application thereof |
| CN114569715A (en) * | 2022-05-07 | 2022-06-03 | 中义(北京)健康研究院 | Vaccine composition, vaccine adjuvant, and preparation method and preparation system thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108144057A (en) * | 2018-01-10 | 2018-06-12 | 杭州洪桥中科基因技术有限公司 | A kind of vaccine diluent and preparation method thereof |
| CN110665379A (en) * | 2019-10-08 | 2020-01-10 | 浙江农林大学 | Water-in-oil type emulsification method aiming at vaccine antigen |
| CN111729081A (en) * | 2020-07-21 | 2020-10-02 | 天康生物股份有限公司 | Vaccine adjuvant, preparation method and application thereof, and prepared vaccine |
| CN111729081B (en) * | 2020-07-21 | 2024-05-24 | 天康生物制药有限公司 | Vaccine adjuvant, preparation method and application thereof and prepared vaccine |
| CN112675299A (en) * | 2021-01-06 | 2021-04-20 | 黑龙江省百洲生物工程有限公司 | Preparation method of compound adjuvant inactivated vaccine for preventing and controlling goose astrovirus |
| CN112675299B (en) * | 2021-01-06 | 2023-10-20 | 黑龙江省百洲生物工程有限公司 | Preparation method of goose astrovirus prevention and control compound adjuvant inactivated vaccine |
| CN114288403A (en) * | 2021-12-20 | 2022-04-08 | 浙江洪晟生物科技股份有限公司 | Newcastle disease and H9 subtype avian influenza bivalent inactivated vaccine containing immunopotentiator and preparation method and application thereof |
| CN114569715A (en) * | 2022-05-07 | 2022-06-03 | 中义(北京)健康研究院 | Vaccine composition, vaccine adjuvant, and preparation method and preparation system thereof |
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