CN106109414B - A kind of aftosa vaccine dilution and the preparation method and application thereof - Google Patents
A kind of aftosa vaccine dilution and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of aftosa vaccine dilutions and the preparation method and application thereof.With the aftosa vaccine diluted aftosa vaccine, not only may be implemented accurately to be inoculated with；It can also overcome the shortcomings of accurately obtain practical effect data in existing checkout procedure, improve the precision and accuracy of efficacy test.Furthermore, there is aftosa vaccine dilution of the invention, vaccine can be by the concentration of bigger multiple, reduce storage, transportation cost, the large-scale inoculation for being conducive to farm, immune station etc., can also reduce the volume of emulsion tank in subsequent emulsifying process, and the stirring and temperature when emulsification are more uniform, so as to improve emulsifying effectiveness, while emulsification cost is saved.
Description
Technical field
The present invention relates to field of veterinary, and in particular to be a kind of aftosa vaccine dilution and preparation method thereof and answer
With.
Background technique
Aftosa (Aftosa), popular name " aphtha ", " warding off Huang " are one kind artiodactylous as caused by foot and mouth disease virus
Acute, hot, highly contagious disease.Aftosa mainly encroaches on artiodactyl beast, occasionally in people and other animals, faces and examines feature
Blister occurs for oral mucosa, hoof and skin of breast.The disease route of transmission is more, speed is fast, and Zeng Duoci is worldwide sudden and violent
Hair is popular, causes huge politics, economic loss.In consideration of it, World Organization for Animal Health (OIE) be classified as A class infectious disease it
It is first.
With the fast development of China's animal husbandry large-scale cultivation, the epidemic prevention of aftosa epidemic disease becomes the pass of animal husbandry development
Key.Currently, one of prevention and control major measure of aftosa epidemic disease is vaccine inoculation.Therefore, the supply of high quality vaccine and accurately
Inoculation liquid measure is to guarantee that aftosa epidemic disease can obtain the key factor of effective prevention and control and China's animal husbandry and can be able to health
The guarantee of development.
Currently, the production technology of aftosa vaccine are as follows: suspension cell culture virus → inactivation → purifying, concentration → emulsification →
Packing.Wherein, in the emulsifying process of domestic aftosa vaccine production, the emulsification method generallyd use is to be aseptically filled into adjuvant
Adjuvant special cylinder or in adjuvant special cylinder through high pressure sterilization, be prepared into oily phase；Then antigen liquid is prepared by technique requirement
Water phase containing antigen is finally mutually emulsified into emulsion (dosage form is generally W/O/W), i.e. vaccine with oil by water phase.
Antigen dosage is fixed in the vaccine of existing conventional method emulsification, using conventional dilution liquid (such as physiological saline, glucose
Deng) will cause vaccine layering and such W/O/W dose vaccine can not be diluted.The patent Shen of Publication No. CN1413732A
Please in disclose a kind of diluent being made of sodium chloride, potassium chloride, calcium chloride, glucose, phosphate etc., which is to use
Dilute antigen, and non-vaccine, it will also result in vaccine layering with this dilution dilution agent vaccine and be not available.Therefore, because not having
There is suitable diluent, can only be injected according to set injection liquid measure in vaccine inoculation, for high concentration vaccine, is inoculated with small
Since error is larger when liquid measure, its accuracy not can guarantee, can not be diluted using the method for conventional dilution, to vaccine yet
Precisely inoculation and the preparation of vaccine are unfavorable.
Summary of the invention
The purpose of the present invention is being directed to technological deficiency existing in the prior art, in a first aspect, providing a kind of aftosa epidemic disease
Seedling dilution, phosphoric acid salt buffer and oily phase；It preferably, is mutually to be emulsified by phosphate buffer with oil
W/O/W (W/O/W) shape emulsion.
The phosphate buffer includes following component:
The oil is mutually 206 adjuvants.
Its preparation includes: that the phosphate buffer (30 ± 1 DEG C) after degerming is slowly added to the oily phase (30 ± 1 after degerming
DEG C) in, the volume ratio of the phosphate buffer and oily phase is (30-50): (50-70) (preferably 46:54), in emulsification stirring frequency
Rate is to be emulsified under 35-40Hz, after 30min, emulsification stirring frequency is adjusted to 3-5Hz, is cooled to 15 DEG C rapidly hereinafter, i.e.
Obtain aftosa vaccine dilution.
The second aspect of the invention is to provide the method for preparing above-mentioned aftosa vaccine dilution, slow with producing phosphate
Fliud flushing is used as dilution water phase after degerming；By oily phase degerming, as dilution oil phase；By dilution water phase and dilution oil phase
Emulsification, obtains the aftosa vaccine dilution of W/O/W (W/O/W) shape.
The following steps are included:
1) after following component being dissolved in water, preparation obtains the phosphate buffer of pH=8.0,
2) phosphate buffer for obtaining step 1) is through 0.2 μm of filter degerming, as dilution water phase, then keeps temperature
Degree is at 30 ± 1 DEG C；
3) 30 ± 1 DEG C are then mutually maintained the temperature at as dilution oil phase through 0.2 μm of filter degerming by oily；
4) the dilution water phase of step 2) is slowly added into the dilution oil phase of step 3), in the emulsification of 35-40Hz
It is emulsified under stirring frequency, after 30min, emulsification stirring frequency is adjusted to 3-5Hz, is cooled to 15 DEG C rapidly hereinafter, to obtain the final product
To aftosa vaccine dilution；The volume ratio of dilution water phase and dilution oil phase is (30-50)/(50-70), preferably 46:54.
The third aspect of the invention is to provide a kind of diluted aftosa vaccine, comprising aftosa vaccine stoste and upper
State the aftosa vaccine dilution that aftosa vaccine dilution or the above method are prepared.
The preparation of the aftosa vaccine stoste are as follows: will inactivation foot-and-mouth disease antigen successively pass through physical purification, ultrafiltration (UF),
Chemical purification is made, and cycles of concentration obtains the antigen of high-purity, high concentration between 500-1000 times, as vaccine water phase；It will
206 adjuvants are as vaccine oil phase；Keeping the temperature of vaccine water phase and vaccine oil phase is 30 ± 1 DEG C, and vaccine water phase is slowly added to
It in vaccine oil phase, is emulsified under the emulsification stirring frequency of 35-40Hz, after emulsifying 30min, emulsification stirring frequency is adjusted to 3-
5Hz is cooled to rapidly 15 DEG C hereinafter, aftosa vaccine stoste to obtain the final product.
The fourth aspect of the invention is to provide the method for preparing above-mentioned diluted aftosa vaccine, by aftosa vaccine
The aftosa vaccine dilution that stoste and above-mentioned aftosa vaccine dilution or the above method are prepared is uniformly mixed.
The fifth aspect of the invention is to provide above-mentioned aftosa vaccine dilution in dilution aftosa vaccine stoste
Application.
Aftosa vaccine dilution provided by the invention not only may be implemented accurately to be inoculated with for diluting aftosa vaccine；
It can also overcome the shortcomings of accurately obtain practical effect data in existing checkout procedure.This is because 2010 editions " middle Chinese
People republic veterinary drug allusion quotation " method of regulation foot-and-mouth disease antigen detection is that standby quarantine seedling will be divided into 1 part, 1/3 part, 1/9 part
Three dosage groups, each dosage group are inoculated in experimental animal respectively and carry out efficacy test (PD50), the effect being actually capable of detecting when
The upper limit is 15.59 PD50, however the effect of some vaccines may be far longer than 15.59, but cannot be by due to the no to scale upper limit
It detected.Vaccine can be diluted to after different multiples with dilution of the invention and carry out efficacy test again, obtained effect
The practical effect data of vaccine can be accurately obtained multiplied by extension rate.
With the development of vaccine concentration technique, cooperates aftosa vaccine dilution of the invention, highly enriched vaccine can be made
Using more extensively, the immune head number amount of Packing Unit each in this way increases, and the amount of storage of vaccine is just reduced, can reduce storage at
This.Such as: the vaccine of existing every bottle of 100ml contains only the amount of antigen of 100 parts, can only be inoculated with 100 animals；And 100ml is dense
Amount of antigen of the contracting vaccine containing 500 parts uses when use with aftosa vaccine dilution of the invention, can be inoculated with 500
Animal, i.e., the vaccine of same Packing Unit, inoculation head number amount will increase, and be conducive to the large-scale inoculation at farm, immune station etc..This
Outside, there is aftosa vaccine dilution of the invention, vaccine can be concentrated, and can reduce emulsion tank in subsequent emulsifying process
Volume, the stirring and temperature when emulsifying in this way are more uniform, so as to improve emulsifying effectiveness, while having saved emulsification cost.
Specific embodiment
The antigen concentration in aftosa vaccine can be improved 1000 times or more at present, although antigen purification enriched level is not
It is disconnected to improve, but the dilution due to not being suitable for aftosa vaccine, the concentration of aftosa vaccine can only be can be directly used for
The concentration of inoculation.Residual or liquid getting device tool if the vaccine directly prepared using high concentration antigen, in operating process in container
On contamination can all make actual injection amount and theoretical value deviation larger, not can guarantee the accuracy of inoculum concentration.If developing one
Kind is suitable for the dilution of aftosa vaccine, is being diluted the vaccine of high concentration antigen using preceding dilution, then be inoculated with,
On the one hand it can guarantee the accurate of inoculum concentration, improve the precision of inspection；The vaccine storage capacity of another aspect high concentration antigen will be big
It is big to reduce, alleviate storage, the transport pressure of vaccine.Therefore, a kind of research and development of aftosa vaccine dilution be highly desirable and
It is very forward-looking.
The invention proposes a kind of preparation methods for diluting the dilution and dilution of aftosa vaccine；Further
Ground obtains diluted aftosa vaccine with diluted aftosa vaccine.
Firstly, aftosa vaccine dilution is the phosphate buffer (PBS) (dosage form W/O/W) of emulsification, preparation packet
Include following steps:
1) after according to each substance is dissolved in water as following formula, preparation obtains phosphate buffer, is formulated as follows:
Agents useful for same is that analysis is pure, is available commercially.
2) it will be added in water phase tank after phosphate buffer degerming with 0.2 μm of filter, as dilution water phase, and will be dilute
The temperature for releasing liquid water phase is maintained at 30 ± 1 DEG C；
3) by 206 adjuvants (206 adjuvants are purchased from Sai Bike (Shanghai) special chemical articles Co., Ltd), 0.2 μm of filter
It is added in emulsion tank after degerming, as dilution oil phase, the temperature of dilution oil phase is equally maintained at 30 ± 1 DEG C；
4) the dilution water phase in water phase tank is slowly added into the dilution oil phase in emulsion tank, dilution water phase:
Dilution oil phase (volume ratio) is (30-50)/(50-70), preferably 46:54, and emulsification stirring frequency is set as 35-40Hz and carries out cream
Change, after emulsifying 30min, stirring frequency is adjusted to 3-5Hz, spent glycol cools down rapidly (The faster the better for the speed of cooling) to 15
DEG C hereinafter, i.e. obtain aftosa vaccine dilution, dispensed.
Aftosa vaccine is purchased from Jinyu Baoling Biology Drugs Co., Ltd, and specific preparation method is the following steps are included: logical
Cross inactivation antigen is successively passed through physical purification, ultrafiltration (UF), chemical purification purifying concentration technology be made, cycles of concentration exists
Between 500-1000 times, antigen after being concentrated, as vaccine water phase, 206 adjuvants are as vaccine oil phase；By vaccine water phase and
The temperature of vaccine oil phase is adjusted to 30 ± 1 DEG C respectively, vaccine water phase is slowly added in vaccine oil phase, stirring frequency is set as
35-40Hz is emulsified, and after emulsifying 30min, stirring frequency is adjusted to 3-5Hz, slow cooling is to 15 DEG C hereinafter, last point
Fill the aftosa vaccine to get high-purity, high concentration.
When preparing diluted aftosa vaccine, calculated according to the effective protection liquid measure of vaccine and quasi- inoculation size of animal
Then the quantity of required high-purity, high concentration vaccine calculates the extension rate of vaccine, proportionally by mouth according to inoculation liquid amount
Fever aphthous vaccine diluent and aftosa vaccine are uniformly mixed to arrive diluted aftosa vaccine, can be directly inoculated with.
Below in conjunction with specific embodiment, the content of the present invention will be explained in more detail, and the present invention is further elaborated, but
These embodiments limit the invention absolutely not.
Means used in embodiment use the means of this field routine unless otherwise instructed.It is used in embodiment
Aftosa vaccine (O/MYA98/BY/2010 plants+asial/JSL/ZK/06 plants+Re-A/WH/10 plants), lot number: S163501, purchase
From Jinyu Baoling Biology Drugs Co., Ltd.Emulsion tank and water phase tank are purchased from Changchun Fuji spy fluid device Co., Ltd.
One, the preparation of aftosa vaccine dilution
A series of aftosa vaccine dilutions of the invention, each ginseng in preparation process are prepared according to the method described above
Number is shown in Table 1.
Table 1 prepares the parameter of 1L embodiment 1-9 aftosa vaccine dilution
Two, high-purity, high concentration aftosa vaccine
By Jinyu Baoling Biology Drugs Co., Ltd, lot number S163501, S163502, S163503, antigen O/
MYA98/BY/2010 plants+asial/JSL/ZK/06 plants+Re-A/WH/10 plants.
Three, the dilution of high concentration aftosa vaccine
It takes dilution to be uniformly mixed with aftosa vaccine under aseptic condition, high-purity, high concentration aftosa vaccine is carried out
The dilution of corresponding multiple.
The screening of phosphate buffer in experimental example, aftosa vaccine dilution
Mouth after aftosa vaccine is diluted 5 times with the aftosa vaccine dilution that embodiment 1-9 is obtained, after detection dilution
The physical behavior (such as dosage form, appearance, viscosity, granularity) of fever aphthous vaccine, and using traditional demulsification technology demulsification, detect water phase sample
146S.Comparative example 1-3 is the dilution obtained by the method for present invention preparation aftosa dilution, is only dilution water phase or dilute
The ingredient for releasing liquid oil phase is different from embodiment listed by the present invention；1 dilution water phase of comparative example is phosphate buffer, formula
Are as follows: 10g/L disodium hydrogen phosphate (Na2HPO4·12H2O), 0.3g/L potassium dihydrogen phosphate (KH2PO4), 8.1g/L sodium chloride (NaCl),
The volume ratio of dilution water phase and dilution oil phase is 46:54；2 dilution water phase of comparative example is also phosphate buffer, is matched
Side is identical as disclosed in CN1413732A, are as follows: 8.0g/L sodium chloride (NaCl), 0.125g/L disodium hydrogen phosphate (Na2HPO4·
12H2O), 0.4g/L potassium chloride (KCl), 0.06g/L potassium dihydrogen phosphate (KH2PO4), 0.2g/L magnesium sulfate (MgSO4·7H2O)、
1.0g/L glucose, 0.14g/L calcium chloride (CaCl2), the volume ratio of dilution water phase and dilution oil phase is 46:54；Compare
The formula of 3 dilution oil phase of example are as follows: what 0.9L/L white oil, 0.04L/L tween (TWEEN) and 0.06L/L Span (SPAN) formed
Mixture (i.e. press volumn concentration, containing 90% white oil, 4% tween and 6% Span), the formula of dilution water phase
Same as Example 5, the volume ratio of dilution water phase and dilution oil phase is 46:54.By taking embodiment 5 as an example, testing result is shown in
Table 2.
Table 2 dilutes the physical behavior and 146S content of the aftosa vaccine after 5 times
It can be seen that the vaccine obtained with the diluted of comparative example 1 and comparative example 2 by the experimental result of table 2
The 146S rate of recovery is low, and 146S is the Effective Antigens ingredient in aftosa vaccine, and the low expression dilution of the 146S rate of recovery is in vaccine
The protectiveness of effective component is bad, and degradation or inactivation are more, it is seen that the dilution of comparative example 1 and 2 is not suitable for the dilution of vaccine.
The vaccine one side viscosity obtained with the diluted of comparative example 3 is higher to be unfavorable for animal inoculation pvaccination, another aspect vaccination particles
Degree is not in normal distribution, and dosage form stability is poor, therefore comparative example 3 is not suitable for vaccine dilution yet.And with dilution of the invention
Diluting obtained vaccine, not only viscosity is suitable, granularity is good in normal distribution, dosage form stability, but also to effective anti-in vaccine
The ultimate constituent 146S rate of recovery is high, illustrates that dilution of the invention is good to the effective component protectiveness in vaccine, degrades or inactivates
Antigen is few, the dilution suitable for vaccine.
By comparing the quality of the vaccine after three kinds of vaccine diluent dilutions, in conjunction with cost comprehensive analysis, the present invention
Aftosa vaccine dilution be used to dilute aftosa vaccine and be more suitable for.
Other embodiments also have similar experiment as a result, no longer repeating one by one.
The above is only a preferred embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (9)
1. a kind of aftosa vaccine dilution, which is characterized in that its raw material is by phosphate buffer and oily phase composition；
The phosphate buffer consists of the following compositions:
PH is 8；
The oil is mutually 206 adjuvants；
Its preparation includes: the phosphate buffer in the oily phase after the phosphate buffer after degerming to be slowly added to degerming
Volume ratio with oily phase is (30-50): (50-70), is emulsified under being 35-40Hz in emulsification stirring frequency, will after 30min
Emulsification stirring frequency is adjusted to 3-5Hz, is cooled to 15 DEG C rapidly hereinafter, obtaining aftosa vaccine dilution.
2. aftosa vaccine dilution according to claim 1, which is characterized in that it is mutually newborn with oil by phosphate buffer
The emulsion of W/O/W shape made of change.
3. aftosa vaccine dilution according to claim 1 or claim 2, which is characterized in that the phosphate buffer and oily phase
Volume ratio be 46:54.
4. the method for preparing any aftosa vaccine dilution of claim 1-3, which is characterized in that prepare phosphate-buffered
Liquid is used as dilution water phase after degerming；By oily phase degerming, as dilution oil phase；Dilution water phase is slowly added to dilution
In oily phase, the volume ratio of the dilution water phase and dilution oil phase is (30-50): (50-70), is in emulsification stirring frequency
It is emulsified under 35-40Hz, after 30min, emulsification stirring frequency is adjusted to 3-5Hz, is cooled to 15 DEG C rapidly hereinafter, obtaining water
The aftosa vaccine dilution of packet Water-In-Oil (W/O/W) shape.
5. method according to claim 4, which comprises the following steps:
1) after following component being dissolved in water, preparation obtains the phosphate buffer of pH=8.0,
2) then the phosphate buffer for obtaining step 1) is maintained the temperature at through 0.2 μm of filter degerming as dilution water phase
30±1℃；
3) 30 ± 1 DEG C are then mutually maintained the temperature at as dilution oil phase through 0.2 μm of filter degerming by oily；
4) the dilution water phase of step 2) is slowly added into the dilution oil phase of step 3), is stirred in the emulsification of 35-40Hz
It is emulsified under frequency, after 30min, emulsification stirring frequency is adjusted to 3-5Hz, is cooled to 15 DEG C rapidly hereinafter, obtaining mouth
Fever aphthous vaccine diluent；The volume ratio of dilution water phase and dilution oil phase is (30-50)/(50-70).
6. a kind of diluted aftosa vaccine, which is characterized in that any described comprising aftosa vaccine stoste and claim 1-3
The aftosa vaccine dilution that aftosa vaccine dilution or any the method for claim 4-5 are prepared.
7. diluted aftosa vaccine according to claim 6, which is characterized in that the preparation of the aftosa vaccine stoste
Are as follows: will inactivation foot-and-mouth disease antigen successively pass through physical purification, ultrafiltration (UF), chemical purification be made, cycles of concentration is in 500-1000
Between times, the antigen of high-purity, high concentration is obtained, as vaccine water phase；Using 206 adjuvants as vaccine oil phase；Keep vaccine water
Mutually and the temperature of vaccine oil phase is 30 ± 1 DEG C, vaccine water phase is slowly added in vaccine oil phase, frequency is stirred in the emulsification of 35-40Hz
It is emulsified under rate, after emulsifying 30min, emulsification stirring frequency is adjusted to 3-5Hz, is cooled to 15 DEG C rapidly hereinafter, mouth to obtain the final product
Fever aphthous vaccinogen liquid.
8. the method for preparing the diluted aftosa vaccine of claim 6 or 7, which is characterized in that by aftosa vaccine stoste
The mouth hoof being prepared with any aftosa vaccine dilution of claim 1-3 or any the method for claim 4-5
Epidemic disease vaccine diluent is uniformly mixed.
9. application of any aftosa vaccine dilution of claim 1-3 in dilution aftosa vaccine stoste.
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| CN107441522B (en) * | 2017-09-26 | 2021-02-19 | 金宇保灵生物药品有限公司 | Degerming method of adjuvant for foot-and-mouth disease vaccine |
| CN108079288B (en) * | 2018-02-09 | 2022-04-26 | 天康生物制药有限公司 | Vaccine, emulsification process of vaccine and application |
| CN109464662B (en) * | 2018-11-16 | 2021-09-14 | 中牧实业股份有限公司 | Vaccine antigen diluent |
| CN114904006B (en) * | 2022-05-27 | 2023-10-17 | 中农威特生物科技股份有限公司 | Foot-and-mouth disease antigen thermal stability protective agent and preparation method and application thereof |
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