CN109224069A - A kind of preparation method of eggs crack detection disease oil emulsion inactivated vaccine - Google Patents
A kind of preparation method of eggs crack detection disease oil emulsion inactivated vaccine Download PDFInfo
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Abstract
The invention belongs to the preparation fields of inactivated vaccine, and in particular to a kind of preparation method of eggs crack detection disease oil emulsion inactivated vaccine.The preparation method is the following steps are included: 1) prepared by secondary seed solution；2) bacterium solution culture: culture that secondary seed solution is ventilated in the culture tank of sterilizing controls condition of culture, obtains bacterium solution, bacterium solution, which is carried out, with centrifuge, hollow fiber membrane ultrafiltration device or cross-flow ultrafiltration concentration systems cleans concentration, suspension is made with PBS again, inactivation treatment obtains antigen liquid；3) preparation of oil emulsion inactivated vaccine: white oil, Si Ben -80 and aluminum stearate are mixed, and sterilized rear obtained oily phase；Tween-80 and antigen liquid are mixed to prepare water phase；It is mutually emulsified oily with water phase, and thimerosal solution is added, obtain oil emulsion inactivated vaccine.Preparation method of the invention is at low cost, and obtained antigenic component is uniform, and emulsifying effectiveness is good.
Description
Technical field
The invention belongs to the preparation fields of inactivated vaccine, and in particular to a kind of eggs crack detection disease is oil emulsion inactivated
The preparation method of vaccine.
Background technique
Avian cholera is by eggs crack detection (AvianPasteurellamultocida) caused by a kind of poultry
The multiple sexually transmitted disease of contact, also known as eggs crack detection disease.The disease is popular in China's fowl bacterial infectious disease
More serious one kind, chicken group infect the death rate with higher after being ill, and infection prevalence drastically influences China birds and supports
The sound development for growing industry causes biggish economic loss to China's aviculture, it is therefore desirable to actively take effective measures control
Make the disease.
What control that China is directed to this disease at present was still mainly taken is the method for antibiotic treatment, and common antibiotic is such as
Sulfa drugs has preferable therapeutic effect, however antibiotic long-term or repeatedly application can generate medicament residue and cause of disease
The problems such as bacterium drug resistance.Therefore popular to control the infection of the disease from source, it certainly will need to take effective precautionary measures.
Currently used for the disease prevention vaccine mainly include attenuated live vaccines and with standard adjuvant prepare inactivated vaccine, such as white-oil adjuvant
Inactivated vaccine, Freund's adjuvant inactivated vaccine etc..
During the preparation process, antigen method for concentration uses existing eggs crack detection disease oil emulsion inactivated vaccine
Aluminium glue concentration, aluminium glue is still in antigen after concentration, is affected when being emulsified to the emulsifying effectiveness of vaccine, and is used
The method of aluminium glue concentration, higher cost.
Summary of the invention
For above situation, the object of the present invention is to provide a kind of eggs crack detection disease oil emulsion inactivated vaccines
Preparation method.Preparation step through reasonable settings, antigen concentration therein is using centrifuge or hollow fiber membrane ultrafiltration device or cuts
It is concentrated to stream ultrafiltration concentration system, at low cost and antigenic component is uniform, and emulsifying effectiveness is good.
The present invention provides a kind of preparation method of eggs crack detection disease oil emulsion inactivated vaccine, the preparation methods
The following steps are included:
1) prepared by secondary seed solution: by strain streak inoculation on improvement Martin's agar plate, selecting 8 ~ 12 colonies typicals, mixes
In a small amount of martin's bouillon, inoculation slant medium is several, sets 36 ~ 37 DEG C and cultivates 18 ~ 24 hours, as first order seed；It takes
The first order seed is inoculated in the martin's bouillon containing 0.1% cracking blood cell whole blood, is set 36 ~ 37 DEG C of ventilations and is cultivated 10 ~ 12 hours,
Obtain secondary seed solution；
2) bacterium solution culture: being packed into fermentation medium and defoaming agent in the fermenter, and after sterilizing, 0.1% cracking is added by culture base unit weight
Blood cell whole blood, tune pH value is 7.2, temperature is 37 DEG C, and ventilatory capacity control is 100L/h, and revolving speed control is 100r/min, and is being led to
It is 100% that oxygen dissolving value is corrected when tolerance is 600L/h, revolving speed is 200r/min, at this point, by the secondary seed solution with 1% ~ 2%
Ratio is inoculated in fermentation medium, is 7.2 ~ 7.4 by mending acid and mending alkali to control pH value, according to oxygen dissolving value tune in fermentation process
Ventilatory capacity and revolving speed are saved, the adjusting range of ventilatory capacity is 100 ~ 600L/h, and the adjusting range of revolving speed is 100 ~ 200r/min, fermentation
Start feed supplement after 6 hours, the material of benefit is the fermentation mediums of 4 times of concentrations, is increased suddenly after oxygen dissolving value drops to 20% or less stabilization
When start feed supplement, and feed supplement is no more than 100mL every time, cultivates 10 ~ 12 hours at 36 ~ 37 DEG C, obtains bacterium solution, with centrifuge, in
Fibre ultrafiltration device or cross-flow ultrafiltration concentration systems carry out bacterium solution cleaning concentration, then suspension is made with the PBS that pH value is 7.2,
Inactivation treatment obtains antigen liquid；
3) preparation of oil emulsion inactivated vaccine: white oil, Si Ben -80 and aluminum stearate are mixed, and sterilized rear obtained oily phase；It will
Tween-80 and the antigen liquid are mixed to prepare water phase；The oil is mutually subjected to cream according to 1: 1 ~ 3: 1 volume proportion with water phase
Change, and thimerosal solution is added, obtains the oil emulsion inactivated vaccine.
Preferably, 0.1% cracking blood cell whole blood and 4% healthy animal serum are contained on improvement Martin's agar plate.
According to the present invention, in step 1), first order seed warp is pure after the assay was approved, into subsequent step.First order seed needs
It is saved at 2 ~ 8 DEG C, validity period is no more than 14, and passage was no more than for 5 generations on culture medium.
Secondary seed warp is pure after the assay was approved, into subsequent step.Secondary seed need to set 2 ~ 8 DEG C of preservations, and validity period is not
More than 3 days.
According to the present invention, in step 2, the culture medium used is eggs crack detection special culture media, the culture medium
Using well known to a person skilled in the art culture mediums.
Fermented and cultured specifically includes: being packed into suitable fermenting culture medium and defoaming agent (usage amount by fermenter volume
For the conventional usage amount of this field), after sterilizing, 0.1% cracking blood cell whole blood is added by fermenting culture base unit weight, tune pH value is
7.2,37 DEG C of temperature, before starting fermentation, correction oxygen dissolving value be 100%(ventilatory capacity control be 600L/h, revolving speed control be 200r/
100%) min, oxygen dissolving value are corrected to when stablizing, ventilatory capacity control is 100L/h, revolving speed control is 100r/min；By the second level
Seed liquor is inoculated in fermentation medium with 1% ~ 2% ratio, in fermentation process, by mend acid mend alkali control pH value be 7.2 ~
7.4, after starting fermentation, according to DO value, it is gradually increased ventilatory capacity and revolving speed, until constant, ventilatory capacity maximum is adjusted to 600L/h,
Revolving speed maximum is adjusted to 200r/min, and feed supplement is carried out by the way of manual flow feeding, and it is 4 times of concentration hairs that feed supplement, which uses,
Ferment culture medium, feed supplement time carry out after the 6th hour, start to mend when dropping to after 20% or less stabilization and increase suddenly according to DO value
Material, each feed supplement are no more than 100mL, until culture terminates.
Bacterium solution after the completion of culture is through purely after the assay was approved, carrying out cleaning concentration.Cleaning concentration at this can use up can
Can the culture medium and endotoxin that go in bacteria-removing liquid, and then culture medium is avoided to generate foam, emulsification is impacted and endogenous toxic material
Influence of the element to animal body, moreover, realizing the unfavorable object that will not be introduced while concentration to post-processing steps such as emulsifications
Matter.
Specifically, the cleaning concentration includes: bacterium solution through centrifuge, hollow fiber membrane ultrafiltration device or cross-flow ultrafiltration concentration system
System concentration removal supernatant, the PBS that pH value is 7.2 is added in concentrate, suspension is made, repeat the above steps 2 ~ 3 times, wherein
Centrifuge, hollow fiber membrane ultrafiltration device and cross-flow ultrafiltration concentration systems be all made of existing equipment and correlation parameters.
In the present invention, count plate, and the PBS for being 7.2 with pH value are carried out to suspension by existing " Chinese veterinary pharmacopoeia " annex
Adjust viable count.
Preferably, the inactivation treatment includes: to make by 1.5 ‰ addition formalins of bacterium solution amount total amount with adding with stirring
It is sufficiently mixed, and 37 DEG C inactivate 24 hours.
Antigen liquid is inactivated enters subsequent step after the assay was approved.Specifically, the bacterium solution after taking inactivation, with improvement Martin's fine jade
Rouge plate does inactivation and examines, and answers asepsis growth.
Preferably, mutually preparation includes: that the injection white oil of 79 ~ 80 parts by weight and the Si Ben -80 of 3.9 ~ 4 parts by weight is mixed to oil
After conjunction, the aluminum stearate of 0.5 ~ 1 parts by weight is added, heating stirring mixes, through 115 ~ 116 DEG C high pressure sterilization 30 ~ 50 minutes, it is cooling
It is spare after to room temperature.
It is further preferred that oil phase preparation includes: the Si Ben -80 by the injection white oil of 79.68 parts by weight and 4 parts by weight
After mixing, be added 1 parts by weight aluminum stearate, heating stirring mix, through 116 DEG C high pressure sterilization 40 minutes, after being cooled to room temperature
It is spare.
Preferably, water phase preparation includes: that the Tween-80 of 4 ~ 5 parts by volume is taken to be packed into Agitation Tank, and 120 ~ 121 DEG C of sterilizings 30 ~
It is 50 minutes, spare after cooling；Tween-80 after the antigen liquid of 95 ~ 96 parts by volume and sterilizing is stirred evenly, keeps Tween-80 complete
Fully dissolved.
Preferably, emulsifying step includes: that oil is added in emulsion tank, while stirring at low speed, is slowly added into water phase, adds
It is emulsified again after complete 10 ~ 45 minutes, stirs preceding addition thimerosal solution terminating, make its final concentration of a ten thousandth.
In addition, the preparation method further includes packing, gland, labeling, packaging and product inspection step.
The equipment and parameter not being limited in the present invention are all made of ordinary skill in the art means.
Compared with prior art, the present invention has following beneficial to point:
The preparation step of present invention eggs crack detection disease oil emulsion inactivated vaccine through reasonable settings, control were cultivated
The conditions such as dissolved oxygen amount, feed supplement in journey improve the bacterium amount of eggs crack detection, in addition, super using centrifuge, doughnut
While bacterium solution is concentrated for filter or cross-flow ultrafiltration concentration systems, bacterium solution is cleaned, improves the quality of inactivated vaccine, at
This low and antigenic component is uniform, and emulsifying effectiveness is good.
Detailed description of the invention
Fig. 1 is the preparation flow figure of eggs crack detection disease oil emulsion inactivated vaccine of the invention.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with the embodiment of the present invention, it is clear that retouched
The embodiment stated is only a part of the embodiment of the present invention, instead of all the embodiments.
Preparation example
A kind of preparation method of eggs crack detection bacterium solution, the preparation method is the following steps are included: 1) secondary seed solution system
It is standby: by strain streak inoculation on improvement Martin's agar plate, to select 10 colonies typicals, be mixed in a small amount of martin's bouillon, connect
Kind slant medium is several, sets 37 DEG C and cultivates 20 hours, as first order seed；It takes the first order seed to be inoculated in split containing 0.1%
In the martin's bouillon for solving blood cell whole blood, sets 37 DEG C of ventilations and cultivate 10 hours, obtain secondary seed solution；2) bacterium solution culture: by fermentation
Tank volume is packed into suitable fermenting culture medium and defoaming agent, and after sterilizing, 0.1% cracking is added by fermenting culture base unit weight
Blood cell whole blood, adjust pH value be 7.2,37 DEG C of temperature, correction oxygen dissolving value be 100%(ventilatory capacity control be 600L/h, revolving speed control be
100%) 200r/min, oxygen dissolving value are corrected to when stablizing, ventilatory capacity control is 100L/h, revolving speed control is 100r/min；It will be described
Secondary seed solution is inoculated in fermentation medium with 2% ratio, in fermentation process, by mend acid mend alkali control pH value be 7.2 ~
7.4, after starting fermentation, according to DO value, it is gradually increased ventilatory capacity and revolving speed, until constant, ventilatory capacity maximum is adjusted to 600L/h,
Revolving speed maximum is adjusted to 200r/min, and feed supplement is carried out by the way of manual flow feeding, and it is 4 times of concentration hairs that feed supplement, which uses,
Ferment culture medium, feed supplement time carry out after the 6th hour, start to mend when dropping to after 20% or less stabilization and increase suddenly according to DO value
Material, each feed supplement are no more than 100mL, cultivate 12 hours at 37 DEG C, and sampling in each hour carries out count plate, the results are shown in Table 1.
Contain 0.1% cracking blood cell whole blood and 4% healthy animal serum on improvement Martin's agar plate.
In step 1), first order seed warp is pure after the assay was approved, into subsequent step.It is qualified that secondary seed is purely examined
Afterwards, into subsequent step.
In step 2, the culture medium used is eggs crack detection special culture media.
Table 1 cultivates different time count plate result (unit: CFU/mL)
Compare preparation example
A kind of preparation method of eggs crack detection bacterium solution, the preparation method is the following steps are included: 1) secondary seed solution system
It is standby: by strain streak inoculation on improvement Martin's agar plate, to select 10 colonies typicals, be mixed in a small amount of martin's bouillon, connect
Kind slant medium is several, sets 37 DEG C and cultivates 20 hours, as first order seed；It takes the first order seed to be inoculated in split containing 0.1%
In the martin's bouillon for solving blood cell whole blood, sets 37 DEG C of ventilations and cultivate 10 hours, obtain secondary seed solution；2) bacterium solution culture: by fermentation
Tank volume is packed into suitable fermenting culture medium and defoaming agent, and after sterilizing, 0.1% cracking is added by fermenting culture base unit weight
Blood cell whole blood, adjust pH value be 7.2,37 DEG C of temperature, correction oxygen dissolving value be 100%(ventilatory capacity control be 600L/h, revolving speed control be
100%) 200r/min, oxygen dissolving value are corrected to when stablizing, ventilatory capacity control is 100L/h, revolving speed control is 200r/min；It will be described
Secondary seed solution is inoculated in fermentation medium with 2% ratio, in fermentation process, by mend acid mend alkali control pH value be 7.2 ~
7.4, after starting fermentation, according to DO value, it is gradually increased ventilatory capacity, until constant, ventilatory capacity maximum is adjusted to 600L/h, at 37 DEG C
10h is cultivated, sampling in each hour carries out count plate, the results are shown in Table 2.
Contain 0.1% cracking blood cell whole blood and 4% healthy animal serum on improvement Martin's agar plate.
In step 1), first order seed warp is pure after the assay was approved, into subsequent step.It is qualified that secondary seed is purely examined
Afterwards, into subsequent step.
In step 2, the culture medium used is eggs crack detection special culture media.
Table 2 cultivates different time count plate result (unit: CFU/mL)
Embodiment
A kind of preparation method of eggs crack detection disease oil emulsion inactivated vaccine, the preparation method include following step
Rapid: 1) prepared by antigen: the bacterium solution prepared by preparation example the method carries out bacterium solution with centrifuge and cleans concentration, then is with pH value
Suspension is made in 7.2 PBS, and inactivation treatment obtains antigen liquid；3) preparation of oil emulsion inactivated vaccine: by white oil, Si Ben -80 and
Aluminum stearate mixing, and sterilized rear obtained oily phase；Tween-80 and the antigen liquid are mixed to prepare water phase；By the oily phase
It is emulsified with water phase according to 2: 1 volume proportion, and thimerosal solution is added, obtain the oil emulsion inactivated vaccine.
Contain 0.1% cracking blood cell whole blood and 4% healthy animal serum on improvement Martin's agar plate.
In step 1), the bacterium solution after the completion of the culture is through purely after the assay was approved, carrying out cleaning concentration.Cleaning at this
Concentration can remove culture medium and endotoxin in bacteria-removing liquid as far as possible, and then culture medium is avoided to generate foam, cause to emulsification
The influence of influence and endotoxin to animal body, moreover, will not be introduced while realizing concentration to post-processings such as emulsifications
The unfavorable substance of step.
The cleaning concentration includes: bacterium solution through centrifuge concentration removal supernatant, and it is 7.2 that pH value is added in concentrate
Suspension is made in PBS, repeats the above steps 3 times.
Count plate is carried out to suspension by existing " Chinese veterinary pharmacopoeia " annex, and the PBS for being 7.2 with pH value adjusts viable bacteria
Number.
The inactivation treatment includes: that formalin is added by the 1.5 ‰ of bacterium solution amount total amount, with adding with stirring, makes it sufficiently
Mixing, 37 DEG C inactivate 24 hours.
Antigen liquid is inactivated enters subsequent step after the assay was approved.Specifically, the bacterium solution after taking inactivation, with improvement Martin's fine jade
Rouge plate does inactivation and examines, and answers asepsis growth.
Oily mutually preparation includes: after mixing the injection white oil of 79.68 parts by weight and the Si Ben -80 of 4 parts by weight, to be added 1
The aluminum stearate of parts by weight, heating stirring mix, through 116 DEG C high pressure sterilization 40 minutes, be cooled to room temperature rear spare.
Water phase preparation includes: that the Tween-80 of 4 parts by volume is taken to be packed into Agitation Tank, and 121 DEG C sterilize 40 minutes, cooling standby
With；Tween-80 after the antigen liquid of 96 parts by volume and sterilizing is stirred evenly, Tween-80 is completely dissolved.
Emulsifying step includes: that oil is added in emulsion tank, while stirring at low speed, is slowly added into water phase, after adding again
Emulsification 30 minutes stirs preceding addition thimerosal solution terminating, makes its final concentration of a ten thousandth.It is not steeped in emulsion process
Foam phenomenon.
In addition, the preparation method further includes packing, gland, labeling, packaging and product inspection step.
Vaccine made from embodiment is subjected to effect evaluation: using 3 monthly age of vaccine immunity SPF chicken 10, each neck is subcutaneously infused
Penetrate vaccine 1mL, while it is not immune as control to set 6 chickens, 14 days and 21 days after exempting from after exempting from, take respectively 5 immune chickens and 3 it is right
It carries out attacking poison according to chicken, the virulent bacterium solution of C48-1 (1mL contains 3 ~ 10 viable bacterias) of each 1 lethal dose of intramuscular injection is observed 14, system
Meter attacks the death condition of malicious chicken.It the results are shown in Table 3, poison attacked within 14th after exempting from, control group is all dead, and immune group 80% is protected；21 after exempting from
Attack poison day, control group is all dead, and immune group 100% is protected.According to the vaccine of the preparation of preparation method described in the present embodiment after exempting from
It can produce good immune protective effect within 14th, can produce strong protecting effect within 21 days after exempting from.
Table 3 exempts from rear different time and attacks malicious result
By the data of table 1-3 it is found that present invention eggs crack detection disease oil emulsion inactivated vaccine through reasonable settings
Preparation step controls the conditions such as dissolved oxygen amount, the feed supplement in incubation, the bacterium amount of eggs crack detection can be improved, in addition,
While bacterium solution is concentrated using centrifuge, hollow fiber membrane ultrafiltration device or cross-flow ultrafiltration concentration systems, bacterium solution is cleaned,
At low cost and antigenic component is uniform, and emulsifying effectiveness is good, improves the quality of inactivated vaccine.
The embodiment of the present invention is described above, above description is exemplary, and non-exclusive, and also not
It is limited to disclosed embodiment.Without departing from the scope and spirit of illustrated embodiment, for the art
Many modifications and changes are obvious for those of ordinary skill.
Claims (10)
1. a kind of preparation method of eggs crack detection disease oil emulsion inactivated vaccine, which is characterized in that the preparation method packet
Include following steps:
1) prepared by secondary seed solution: by strain streak inoculation on improvement Martin's agar plate, selecting 8 ~ 12 colonies typicals, mixes
In a small amount of martin's bouillon, inoculation slant medium is several, sets 36 ~ 37 DEG C and cultivates 18 ~ 24 hours, as first order seed；It takes
The first order seed is inoculated in the martin's bouillon containing 0.1% cracking blood cell whole blood, is set 36 ~ 37 DEG C of ventilations and is cultivated 10 ~ 12 hours,
Obtain secondary seed solution；
2) bacterium solution culture: being packed into fermentation medium and defoaming agent in the fermenter, and after sterilizing, 0.1% cracking is added by culture base unit weight
Blood cell whole blood, adjusting pH value is 7.2, and temperature is 37 DEG C, and ventilatory capacity control is 100L/h, and revolving speed control is 100r/min, and logical
It is 100% that oxygen dissolving value is corrected when tolerance is 600L/h, revolving speed is 200r/min, at this point, by the secondary seed solution with 1% ~ 2%
Ratio is inoculated in fermentation medium, is 7.2 ~ 7.4 by mending acid and mending alkali to control pH value, according to oxygen dissolving value tune in fermentation process
Ventilatory capacity and revolving speed are saved, the adjusting range of ventilatory capacity is 100 ~ 600L/h, and the adjusting range of revolving speed is 100 ~ 200r/min, fermentation
Start feed supplement after 6 hours, the material of benefit is the fermentation mediums of 4 times of concentrations, is increased suddenly after oxygen dissolving value drops to 20% or less stabilization
When start feed supplement, and feed supplement is no more than 100mL every time, cultivates 10 ~ 12 hours at 36 ~ 37 DEG C, obtains bacterium solution, with centrifuge, in
Fibre ultrafiltration device or cross-flow ultrafiltration concentration systems carry out bacterium solution cleaning concentration, then suspension is made with the PBS that pH value is 7.2,
Inactivation treatment obtains antigen liquid；
3) preparation of oil emulsion inactivated vaccine: white oil, Si Ben -80 and aluminum stearate are mixed, and sterilized rear obtained oily phase；It will
Tween-80 and the antigen liquid are mixed to prepare water phase；The oil is mutually subjected to cream according to 1: 1 ~ 3: 1 volume proportion with water phase
Change, and thimerosal solution is added, obtains the oil emulsion inactivated vaccine.
2. preparation method according to claim 1, it is characterised in that: split on improvement Martin's agar plate containing 0.1%
Solve blood cell whole blood and 4% healthy animal serum.
3. preparation method according to claim 1, it is characterised in that: in step 1), first order seed and secondary seed difference
Through purely after the assay was approved, into subsequent step.
4. preparation method according to claim 1, it is characterised in that: in step 2, bacterium solution warp is pure after the assay was approved, into
Row cleaning concentration；Suspension carries out count plate, and the PBS for being 7.2 with pH value adjusts viable count；It is qualified that antigen liquid is examined through inactivation
Enter subsequent step afterwards.
5. preparation method according to claim 1 or 4, it is characterised in that: the cleaning concentration includes: bacterium solution through being centrifuged
Machine, hollow fiber membrane ultrafiltration device or cross-flow ultrafiltration concentration systems concentration removal supernatant, it is 7.2 that pH value is added in concentrate
Suspension is made in PBS, repeats the above steps 2 ~ 3 times.
6. preparation method according to claim 1, it is characterised in that: the inactivation treatment includes: by bacterium solution amount total amount
1.5 ‰ addition formalins are mixed them thoroughly with adding with stirring, and 37 DEG C inactivate 24 hours.
7. preparation method according to claim 1, it is characterised in that: mutually preparation includes: by the injection of 79 ~ 80 parts by weight to oil
After white oil and the Si Ben -80 of 3.9 ~ 4 parts by weight mixing, the aluminum stearate of 0.5 ~ 1 parts by weight is added, heating stirring mixes, warp
115 ~ 116 DEG C high pressure sterilization 30 ~ 50 minutes, be cooled to room temperature rear spare.
8. preparation method according to claim 1 or claim 7, it is characterised in that: water phase preparation includes: to take spitting for 4 ~ 5 parts by volume
Temperature -80 is packed into Agitation Tank, and 120 ~ 121 DEG C sterilize 30 ~ 50 minutes, spare after cooling；By the antigen liquid of 95 ~ 96 parts by volume with go out
Tween-80 after bacterium stirs evenly, and is completely dissolved Tween-80.
9. preparation method according to claim 8, it is characterised in that: emulsifying step includes: that oil is added in emulsion tank,
While stirring at low speed, it is slowly added into water phase, is emulsified again after adding 10 ~ 45 minutes, stirs preceding addition thimerosal solution terminating,
Make its final concentration of a ten thousandth.
10. preparation method according to claim 1, it is characterised in that: the preparation method further include packing, gland, labeling,
Packaging and product inspection step.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115216478A (en) * | 2022-06-01 | 2022-10-21 | 华南农业大学 | Construction method and application of pasteurella multocida endotoxin attenuated inactivated vaccine strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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