CN108904796A - Rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof - Google Patents
Rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to rabbit hemorrhagic disease virus baculovirus vectors, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof, belong to immunological technique field.Recombination rabbit hemorrhagic disease virus VP60 baculoviral is inoculated with Sf9 insect cell, is cultivated in 27~28 DEG C, when cytopathy is up to 85% or more, harvest cell culture is simultaneously inactivated, to inactivate object as rabbit hemorrhagic disease virus antigen；Bacterium solution is inactivated to, rabbit source capsular serotype A group's pasteurella multocida C51-17 bacterial strain amplification cultivation using inactivated bacterial liquid as pasteurella multocida antigen；Two kinds of antigens and adjuvant are mixed and made into rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine in proportion.The present invention can provide a kind of highly-safe, good immune effect, the rabbit hemorrhagic disease virus baculovirus vector of simple process, pasteurella multocida disease bivalent inactivated vaccine, be used for prevention and control rabbit hemorrhagic disease（Rabbit pest）With rabbit pasteurella multocida disease.
Description
Technical field
The present invention relates to a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and
Preparation method belongs to immunological technique field.
Background technique
Rabbit hemorrhagic disease (Rabbit hemorrhagic diease, RHD) is by rabbit hemorrhagic disease virus (Rabbit
Hemorrhagic disease viurs, RHDV) caused by one kind is acute, strong, highly contagious disease, be commonly called as " rabbit
Pest ".The normal break out and spread of this disease, disease incidence and case fatality rate are high, and the death rate is up to 90% or so.Rabbit pasteurella multocida
It is by rabbit more killing property that sick (Pasteurellosis), which is also known as rabbit hemorrhagic septicemia (Hemorrhagic septicaemia, HS),
One kind caused by Pasteurella (Pasteurella multocida, Pm) is acute, Septic blood infectious disease.The disease can be divided into nose
A variety of sick types such as scorching type, pneumonia type, tympanitis type, various disease type lesions are inconsistent, but often there are two types of and two or more sick types
Joint occurs.Rabbit hemorrhagic disease virus and Pasteurella are classified as two class pathogenic microorganisms and three classes pathogenic microorganism respectively by China.
Rabbit haemorrhagic disease and pasteurella multocida disease are the main epidemic disease for seriously endangering rabbit healthy aquaculture, current prevention and control
Measure is mainly vaccine immunity.Although the country is existing " rabbit hemorrhagic disease, pasteurella multocida disease bivalent inactivated vaccine ",
Its antigenic component is rabbit haemorrhagic disease tissue poison and pasteurella multocida.But there are significant deficiencies for the vaccine, it is main to show
It differs greatly in the animal individual for being used to prepare rabbit hemorrhagic disease virus, is unfavorable for the large-scale production of vaccine antigen, quality control
It is difficult to carry out, it is most important that vaccine preparation process be easy to cause virulent diffusion, and biological safety is poor.In addition, more killing property Pasteur
Bacillus preparation process is not sufficiently stable, and antigen immunogenicity differs greatly between batch, and it is lower that malicious protective rate is attacked in efficacy test.More than
Defect has severely impacted vaccine quality, constrains enterprises production efficiency.
The large-scale production of vaccine may be implemented in current cell scale suspension culture and bacterium high density fermentation culture, has
Conducive to vaccine quality control, reduce cost, simplified production technology.The application is with national a kind of novel chiral synthon " rabbit hemorrhagic disease virus bar
Based on shape viral vectors inactivated vaccine (BAC-VP60 plants) ", by technological improvement, vaccine antigen production technology, enhancing are promoted
Original vaccine immunity effect develops rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine, dashes forward
The technical bottleneck of broken traditional vaccine, the production and quality for not only contributing to vaccine are stablized, and the update of vaccine will be also accelerated.
Summary of the invention
The technical problem to be solved by the present invention is to：A kind of rabbit hemorrhagic disease virus baculovirus vector, killing property bar are provided more
Family name's bacillosis bivalent inactivated vaccine and preparation method thereof, this method are more easier large-scale production, quality controllable, preparation process is steady
Calmly, biological safety is good.The vaccine is relative to existing vaccine in safety, Immunization protective rate, duration of immunity and storage life etc.
Aspect has substantive raising.
In order to solve the above technical problems, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, killing property Pasteur more
Bacillosis bivalent inactivated vaccine.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, antigenic component is recombination rabbit hemorrhagic disease virus VP60 baculoviral and killing property Pasteur more in the bivalent inactivated vaccine
Bacillus.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, antigenic component is recombination rabbit hemorrhagic disease virus VP60 baculovirus vector cell culture in the bivalent inactivated vaccine
Object and pasteurella multocida.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, antigenic component is that recombination rabbit hemorrhagic disease virus VP60 baculovirus vector suspends training entirely in the bivalent inactivated vaccine
Support cell culture and pasteurella multocida.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, antigenic component is recombination rabbit hemorrhagic disease virus VP60 baculovirus vector cell culture in the bivalent inactivated vaccine
Object and pasteurella multocida zymocyte liquid.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, antigenic component is that recombination rabbit hemorrhagic disease virus VP60 baculovirus vector suspends training entirely in the bivalent inactivated vaccine
Support cell culture and pasteurella multocida zymocyte liquid.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the bigeminy vaccine are by recombination rabbit hemorrhagic disease virus VP60 baculovirus cell culture and killing property Pasteur's bar more
It is prepared after the inactivation of bacterium zymocyte liquid.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the recombination rabbit hemorrhagic disease virus VP60 baculovirus cell culture are received when sick cell is of about 85% or more
It obtains.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the pasteurella multocida zymocyte liquid use C51-17 plants of freeze-dried vaccines of pasteurella multocida for strain.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the pasteurella multocida zymocyte liquid use C51-17 plants of pasteurella multocida freeze-dryings after solid culture
Bacterium is first order seed.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the first order seed are saved in 2~8 DEG C, and validity period is no more than 7.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the pasteurella multocida zymocyte liquid is using the pasteurella multocida after solid culture and Liquid Culture
C51-17 plants of freeze-dried vaccines are secondary seed.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the secondary seed are saved in 2~8 DEG C, and validity period is no more than 4.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine has carried out pure inspection to the secondary seed.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the pasteurella multocida zymocyte liquid are the zymocyte liquid of ventilation culture.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the pasteurella multocida zymocyte liquid have carried out pure inspection after fermented and cultured.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, antigen are not less than 1 containing the VP60 albumen hemagglutinative titer before inactivation by every milliliter of vaccine:256 carry out with seedling.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, antigen are not less than 7.5 × 10 containing pasteurella multocida bacterium number before inactivating by every milliliter of vaccine9CFU standard into
Row matches seedling.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, antigen are not less than 1 containing the VP60 albumen hemagglutinative titer before inactivation by every milliliter of vaccine:256, killing property Pasteur bar more
Bacterium bacterium number is not less than 7.5 × 109CFU standard is carried out with seedling.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid set 2~8 DEG C of preservations, are no more than 6
Month.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine, the pasteurella multocida inactivation liquid are set 2~8 DEG C of preservations, are no more than 6 months.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine has carried out inactivation to rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid before with seedling and has examined.
Further, the present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bigeminy
Inactivated vaccine has carried out inactivation to pasteurella multocida inactivation liquid before with seedling and has examined.
Preferably, the rabbit hemorrhagic disease virus baculovirus vector is recombination rabbit hemorrhagic disease virus VP60 baculoviral
BAC-VP60 plants, it is preserved in China typical culture collection center on June 26th, 2018, in Wuhan, China, Wuhan is big for address
It learns, postcode 430072, deposit number is CCTCC V 201833；Pasteurella multocida is C51-17 plants（Pasteurella
multocida C51-17）, be preserved in China typical culture collection center on June 26th, 2018, address in Wuhan, China,
Wuhan University, postcode 430072, deposit number are：CCTCC M 2018401.
Preferably, the bivalent inactivated vaccine contains adjuvant.
Preferably, the adjuvant is aluminium hydroxide gel, and the ratio of antigen and aluminium hydroxide gel is 9:1.
The present invention provides a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine
Preparation method includes the following steps：
(1) preparation of rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid：Rabbit hemorrhagic disease virus will be recombinated
VP60 baculoviral inoculating cell is cultivated, and carries out inactivation treatment to the cell culture of harvest；
(2) preparation of pasteurella multocida inactivation liquid：Fermentor ventilation culture is carried out to pasteurella multocida, to hair
Yeast-like fungi liquid carries out inactivation treatment；
(3) preparation of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine：Antigen contains
Amount is not less than 1 containing the VP60 albumen hemagglutinative titer before inactivation by every milliliter of vaccine:256, pasteurella multocida bacterium number is not low
In 7.5 × 109CFU standard adjuvant is added and mixes well with seedling.
Further, suspension culture is carried out to the recombination rabbit hemorrhagic disease virus VP60 baculoviral.
Further, inoculating cell is Sf9 cell in above-mentioned bigeminy vaccine preparation method step (1), and used medium is
Insect cell medium, recombination rabbit hemorrhagic disease virus VP60 baculoviral inoculum concentration is the 1% of culture medium total amount, and condition of culture is
27~28 DEG C of temperature, pH value are 6.0~6.2, dissolved oxygen (DO) 50%~60%, 50~70r/min of speed of agitator；Work as sick cell
Cell culture can be harvested up to 85% or more.
Further, the revolving speed when Sf9 cell culture is inoculated with recombination rabbit hemorrhagic disease virus VP60 baculoviral with it
Culture speed afterwards is identical or different.
Further, reach 2 × 10 in Sf9 cell6Inoculation recombination rabbit hemorrhagic disease virus VP60 rod-shaped disease when a/ml or so
Poison.
Further, cell culture is harvested when sick cell is up to 90% or more.
Further, cell culture is harvested when sick cell is up to 95% or more.
Further, it after the step (1) obtains rabbit hemorrhagic disease virus baculovirus vector cell culture, needs to carry out
Hemagglutinative titer measurement, cell culture should be not less than 1 to 1% people " O " type erythrocyte agglutination potency:512.
Further, in the step (1), inactivation treatment is carried out using cell culture of the formaldehyde to harvest；And to going out
Rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid after work carries out inactivation inspection：Nothing after inactivation liquid inoculating cell
Cytopathy, erythrocyte agglutination are measured without coagulation.
Further, in the step (1), using final concentration of 0.2% formalin to the cell culture of harvest
Carry out inactivation treatment.
Further, in the step (1), when carrying out inactivation treatment to the cell culture of harvest using formalin into
Row stirring.
Further, in the step (1), when carrying out inactivation treatment to the cell culture of harvest using formalin
Mixing speed and mixing speed when recombination rabbit hemorrhagic disease virus VP60 baculoviral culture are identical or different.
Further, in the step (1), when carrying out inactivation treatment to the cell culture of harvest using formalin
Cultivation temperature is different from cultivation temperature when recombination rabbit hemorrhagic disease virus VP60 baculoviral culture.
Further, in the step (1), when carrying out inactivation treatment to the cell culture of harvest using formalin
Cultivation temperature is higher than cultivation temperature when recombination rabbit hemorrhagic disease virus VP60 baculoviral culture.
Further, in the step (1), the recombination rabbit hemorrhagic disease virus VP60 baculoviral for inactivation is examined in inactivation
Carrier cell culture carries out.
Further, in the step (1), the cultivation temperature of cell culture and recombination rabbit haemorrhagic disease disease when inactivation is examined
Cultivation temperature when malicious VP60 baculoviral is cultivated is identical.
Further, in the step (2), fermentor ventilation culture is carried out to pasteurella multocida, the specific steps are：
Pasteurella multocida single colonie is inoculated in Martin's agar slant containing serum and cracking whole blood, culture obtains killing property bar more
Family name's bacillus first order seed；First order seed is inoculated in the martin's bouillon containing serum, culture obtains second level pasteurella multocida
Seed；It is packed into 60%~70% fermentation medium and 0.01%~0.02% defoaming agent by fermenter volume, culture medium is carried out
By 5%~10% inoculation secondary seed of culture base unit weight, while serum is added, 200~300r/ of speed of agitator in sterilization treatment
Min, pH value 7.2~7.6, ventilation culture, harvest culture bacterium solution；Every ml culture bacterium solution containing viable count should not less than 1.2 ×
1010CFU。
Further, in the step (2), fermentor ventilation training is carried out to C51-17 plants of freeze-dried vaccines of pasteurella multocida
It supports.
Further, in the step (2), the first order seed is saved in 2~8 DEG C, and validity period is no more than 7.
Further, in the step (2), the secondary seed is saved in 2~8 DEG C, and validity period is no more than 4.
Further, in the step (2), the secondary seed is purely examined.
Further, inactivation treatment is carried out using culture bacterium solution of the formaldehyde to harvest in step (2), and carries out inactivation inspection
It tests, Martin agar and pasteurella multocida fermentation medium of the sampling inoculation containing 4% serum and 0.1% cracking whole blood after inactivation
It is cultivated, answers asepsis growth.
Further, inactivation treatment is carried out using culture bacterium solution of 0.4% formaldehyde to harvest in the step (2).
Further, culture temperature when inactivation treatment is carried out in the step (2) using culture bacterium solution of the formaldehyde to harvest
It spends identical as cultivation temperature when pasteurella multocida fermented and cultured.
Further, stirring speed when inactivation treatment is carried out in the step (2) using culture bacterium solution of the formaldehyde to harvest
It spends different from mixing speed when pasteurella multocida fermented and cultured.
Further, stirring speed when inactivation treatment is carried out in the step (2) using culture bacterium solution of the formaldehyde to harvest
Mixing speed when degree is lower than pasteurella multocida fermented and cultured.
Further, use aluminium glue for adjuvant in the step (3).
Further, rabbit hemorrhagic disease virus baculovirus vector cell culture inactivates liquid and kills more in the step (3)
Property Pasteurella inactivation liquid mix well after adjuvant is added.
Further, rabbit hemorrhagic disease virus baculovirus vector cell culture inactivates liquid and kills more in the step (3)
Property 10~30r/min stirring when mixing well of Pasteurella inactivation liquid.
Further, rabbit hemorrhagic disease virus baculovirus vector cell culture inactivates liquid and kills more in the step (3)
Property Pasteurella inactivation liquid mix well after, be added adjuvant continue to mix.
Further, rabbit hemorrhagic disease virus baculovirus vector cell culture inactivates liquid and kills more in the step (3)
Property Pasteurella inactivation liquid mix well after be added adjuvant, 30~50r/min stirring.
The invention has the advantages that compared with prior art, the production of rabbit hemorrhagic disease virus antigen uses full suspension cell
Culture technique, production process is without using virulent, and the antigen hemagglutinative titer of preparation is high, immunogenicity is good, production technology is advanced, can be
The large-scale production of the workshop GMP；Pasteurella multocida antigen fermentating culturing process is more mature, and what is prepared between different batches is anti-
Former immunogenicity is more stable, and the similar vaccine for attacking toxic effect force inspecting protective rate than prior art preparation further increases.Rabbit
Haemorrhagic virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine preparation procedure are simpler, are easy to advise greatly
Mould production and control of product quality, biological safety is good, and product quality is more excellent, and production cost further decreases.According to this side
The vaccine safety of method preparation is more preferable, and Immunization protective rate is higher, and duration of immunity and storage life are also superior to prior art preparation
Similar vaccine.
Detailed description of the invention
Fig. 1 is rabbit hemorrhagic disease virus baculovirus vector, the pasteurella multocida disease two according to embodiment of the present invention
Join the flow chart of inactivated vaccine preparation method.
Specific embodiment
In order to make those skilled in the art more fully understand the present invention, following examples, but following realities are inventor provided
It applies example to be not intended to restrict the invention, feature described in the present invention, step can be combined arbitrarily, and can be with not
Those of same sequence executes, and be not limited to following description.
The source of seed culture of viruses and strain：It recombinates rabbit hemorrhagic disease virus VP60 baculoviral (BAC-VP60 plants), it is hereinafter referred to as heavy
Group baculoviral, is preserved in China typical culture collection center on June 26th, 2018, address is in Wuhan, China, Wuhan
University, postcode 430072, deposit number are CCTCC V 201833；Pasteurella multocida is C51-17 plants, in June, 2018
It is preserved within 26th China typical culture collection center, address is compiled in Wuhan, China, Wuhan University, postcode 430072, preservation
Number it is：CCTCC M 2018401.
Embodiment 1 recombinates rabbit hemorrhagic disease virus VP 60 albumen cell antigen culture tank suspension culture method
By the Sf9 cell of culture that suspends, 27~28 DEG C, with 90~120r/min culture 24~48 hours, continuous passage was expanded
Increase, passage ratio is 1:2~1:4, inoculating cell culture tank reaches the minimum culture capacity of the culture tank first, continues later
Insect cell medium is cultivated and adds, in bioreactor culture cell processes, control each parameter of bioreactor exists
Normal range (NR)：27~28 DEG C of temperature, pH value are 6.0~6.2, dissolved oxygen (DO) 50%~60%, 50~70 r/ of speed of agitator
min.Reach certain density (about 2 × 10 to cell in bioreactor6A/ml), recombinant baculovirus is inoculated in 1% ratio
It carries out virus and maintains culture, the parameters such as control temperature, pH value, DO, speed of agitator are in OK range.It connects after poison 4~5, works as disease
Born of the same parents attenuate of about 85% or more, cell culture can be harvested.By above-mentioned multiple culture experiment, 1% is carried out to cultured products
The test of people " O " type erythrocyte agglutination potency, the results are shown in Table 1 for cultured products hemagglutinative titer, cell culture product hemagglutinative titer
(HA) it is not less than 1:1024.
Table 1 recombinates rabbit hemorrhagic disease virus VP60 baculovirus cell culture tank cultured products hemagglutinative titer (HA) result
| Culture volume (liter) | Receive malicious time (day) | HA(log2) |
| 5 | 5 | 10 |
| 5 | 5 | 11 |
| 6 | 4 | 10 |
| 6 | 5 | 11 |
| 40 | 4 | 10 |
| 40 | 5 | 11 |
| 50 | 5 | 11 |
The preparation of 2 rabbit hemorrhagic disease virus baculovirus vector cell culture of embodiment inactivation liquid
Final concentration of 0.2% is added into the recombination rabbit hemorrhagic disease virus VP60 baculovirus cell culture of above-mentioned preparation
Formalin, mix, change 37 DEG C of tank postposition inactivate 18~24 hours, 50~70r/min of speed of agitator；Or change 37 DEG C of bottle postposition
Inactivation 24 hours, shaking in during which every 2~3 hours is primary, 3 minutes every time, obtains rabbit hemorrhagic disease virus baculovirus vector cell
Culture inactivates liquid, and inactivation liquid sets 2~8 DEG C of preservations, should be no more than 6 months.When inactivation is examined, the rod-shaped disease of rabbit hemorrhagic disease virus is taken
Poisonous carrier cell culture inactivates liquid, and the 1% well-grown Sf9 cell of inoculation in an amount sets 27~28 DEG C of cultures, continuously
Observation 5 days, cell-free lesion harvest cell culture, set -20 DEG C or less multigelations 3 times, a blind passage generation, and inoculation growth is good
Good Sf9 cell is cultivated 5, and cell is without lesion, and cell liquid is without coagulation.
3 pasteurella multocida fermentation culture method of embodiment
The preparation of 3 pasteurella multocida fermentation liquids
The breeding of 3.1 first order seeds and identification make C51-17 plants of freeze-dried vaccines of pasteurella multocida of sterile saline
Appropriate dilution, then streak inoculation is in Martin's agar plate containing 4% serum and 0.1% cracking whole blood, set 37 DEG C of cultures 18~
22 hours, at least five colonies typical is chosen, is inoculated in Martin's fine jade containing 4% or so serum and 0.1% or so cracking whole bloods respectively
Rouge inclined-plane is several, sets 37 DEG C and cultivates 18~22 hours, as first order seed.It is saved in 2~8 DEG C, validity period is no more than 7.
The breeding of 3.2 secondary seeds takes first order seed to be inoculated in the martin's bouillon containing 0.5% serum with identification, sets 37 DEG C,
200r/min, shaken cultivation 14~18 hours, sampling was purely examined, and is used as secondary seed after qualified, is saved in 2~8 DEG C,
Validity period is no more than 4.
3.3 bacterium solution cultures carry out ventilation culture using fermentor, are packed into 30%~70% fermented and cultured by fermenter volume
Base and 0.01%~0.02% defoaming agent, after culture medium steam high-voltage sterilizing, when temperature is down to 37 DEG C or so, by culture base unit weight
5%~10% inoculation secondary seed solution, while by culture base unit weight 0.5% be added serum, ventilatory capacity stablize in 15LPM, stir
Mix 200~300r/min of revolving speed, pH value 7.2~7.6, dissolved oxygen (DO) minimum 0,37 DEG C of ventilations cultures 10 hours or so, harvest
Cultivate bacterium solution.Purely examined, while carrying out count plate, every ml culture bacterium solution containing viable count should not less than 1.2 ×
1010CFU.The results are shown in Table 2 for Different vaccine dosage and fermentation time count of bacteria, and viable count is most within about 10 hours, reachable
To 1.2 × 1010CFU/ml or more.
2 Different vaccine dosage zymogenous bacteria count results of table
The preparation of 4 pasteurella multocida of embodiment inactivation liquid
By pasteurella multocida liquid total amount 0.4% be added formalin, mix, change 37 DEG C of tank postposition inactivation 30~
36 hours, 50~70r/min of speed of agitator, pasteurella multocida liquid is made and inactivates liquid.Inactivation liquid sets 2~8 DEG C of preservations, should not
More than 6 months.
Inactivation is examined：Sampling inoculation cracks Martin's agar of whole blood and more containing 4% or so serum and 0.1% or so after inactivation
Killing property Pasteurella fermentation medium, 37 DEG C are cultivated 24 hours, answer asepsis growth.
5 bigeminy vaccine antigen of embodiment proportion is calculated and is prepared
According to cell culture (VP60 albumen) hemagglutinative titer and Pasteurella bacterium solution bacterial content of actual production, by every
In ml vaccine containing the VP60 albumen hemagglutinative titer before inactivation be 1:256, C51-17 plants of bacterium number of pasteurella multocida containing rabbit is 7.5
×109CFU standard carries out vaccine each group distribution ratio and calculates.Then qualified rabbit hemorrhagic disease virus baculovirus vector will be examined thin
Born of the same parents' culture inactivates liquid and pasteurella multocida inactivation liquid is mixed by said ratio, and low speed (10~30r/min) stirring 10~
20 minutes, then by antigen and aluminium glue 9:Aluminium hydroxide gel adjuvant is added in 1 ratio, and 30~50 r/min are stirred 10~20 minutes,
It mixes well, obtains rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine.Quantitative separating,
It jumps a queue sealing, labeling.Table 3, which is shown, examines qualified rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid and inspection
Test the vaccine formulation that qualified pasteurella multocida inactivation liquid carries out different ratio, 4 batch rabbit hemorrhagic disease virus bars of acquisition
Shape viral vectors, pasteurella multocida disease bivalent inactivated vaccine.
3 vaccine each group distribution ratio of table and vaccine formulation (every milliliter)
6 rabbit hemorrhagic disease virus baculovirus vector of embodiment, pasteurella multocida disease bivalent inactivated vaccine without bacterial examination
It tests
Random sampling is carried out to the inactivated vaccine of above-mentioned 4 batch and pays attention to representativeness, every batch of is taken out by 1 the percent of bottle number
Sample, but no less than 5 bottles, no more than 10 bottles, every bottle is tested respectively.It is not processed after vaccine to be checked is shaken up, directly
It takes 1.0ml to be inoculated with 50ml sulphur glycollate culture medium (TG), sets 35~37 DEG C of cultures, culture is drawn after 3 days, be inoculated with TG tubule
2, every 0.2ml, 1 are set 35~37 DEG C of cultures, and 1 is set 23~25 DEG C of cultures, separately takes 0.2ml, are inoculated with 1 pancreas junket soya peptone
Fluid nutrient medium (TSB) tubule sets 23~25 DEG C of cultures, cultivates 7.The equal asepsis growth of sample of every batch of sampling observation.
The safety of embodiment 7 rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine
It examines
Above-mentioned vaccine is sufficiently shaken up, 35~42 age in days healthy rabbits 5 is taken to be only used as test group, selects rabbit skin of neck
Loose position carries out disinfection to injection site with 75% cotton ball soaked in alcohol, respectively vaccinates 2.0ml.Simultaneously with the same terms man
Rabbit 5 is only used as compareing, not vaccine inoculation, is observed continuously 14, the state of mind, diet, excrement, part and the whole body of two groups of record
Reaction etc..It lives the results show that all test rabbits are strong, the state of mind, diet, excrement, injection site absorb good, no lump,
Illustrate that vaccine safety is good.
The effect inspection of 8 rabbit hemorrhagic disease virus baculovirus vector of embodiment, pasteurella multocida disease bivalent inactivated vaccine
It tests
8.1 by the rabbit hemorrhagic disease virus baculovirus vector of above-mentioned preparation, pasteurella multocida disease bivalent inactivated vaccine
It sufficiently shakes up, taking 2~3 monthly ages susceptible rabbit of health, (antibodies against rabbit hemorrhagic disease virus HI potency is not higher than 1:2, weight 1.75~
2.0kg) 12,1.0ml is respectively vaccinated, while setting up control group rabbit 12.
After poison is attacked immune 14 days in 8.2 rabbit hemorrhagic disease virus parts, immune rabbit 6 is taken, together with control rabbit 6, each neck
Malicious (the viral level 10 of rabbit hemorrhagic disease virus Anhui mound strain liver is subcutaneously injected4 LD50/ ml) 1.0ml, it is observed continuously 7.
The single colonies typical of strain after the recovery of 8.3 pasteurella multocida toxicity test pickings is inoculated in meat containing Martin
Soup, is cultivated 12~16 hours, takes bacterial cultures 1.0ml to be serially diluted again with sterile saline 10 and carry out bacterium meter by 37 DEG C
Number.After 24 hours, according to count results, bacterium solution is diluted with sterile saline, is made in every milliliter of dilution respectively about
It is 4,6,8,10 containing bacterium number, each dosage neck subcutaneous injection test rabbit 6,1.0ml/ is only.While inoculation test rabbit
Poison of attacking against each other with bacterium solution carries out count of bacteria, and count results is actually attack toadstool number.It is observed continuously 7, and it is dead to record test rabbit
Quantity.
8.4, which attack toadstool, prepares according to toxicity test as a result, the single colonies typical of the strain after picking recovery is inoculated in horse
Fourth meat soup, is cultivated 12~16 hours by 37 DEG C, is taken bacterial cultures 1.0ml to be serially diluted again with sterile saline 10 and is carried out carefully
Bacterium counts.After 24 hours, according to count results, bacterium solution is diluted with sterile saline, adjusts pasteurella multocida
C51-17 plants are a minimum lethal dose (MLD).Poison of attacking against each other while inoculation test rabbit with bacterium solution carries out count of bacteria, counting
It as a result is actually to attack toadstool number.
After poison is attacked immune 21 days in 8.5 pasteurella multocida part, immune rabbit 6 is taken, together with control rabbit 6, each neck
The virulent bacterium solution 1.0ml of C51-17 plants of pasteurella multocida of one minimal lethal dose of portion's subcutaneous injection (bacterial content 5~
10CFU/ml), it is observed continuously 7.
8.6 result rabbit hemorrhagic disease virus parts：It is all dead to compare rabbit, immune rabbit is all strong to live；Pasteurella multocida
Part：It is all dead to compare rabbit, immune rabbit at least 5 is strong to live.
9 rabbit hemorrhagic disease virus baculovirus vector of embodiment, pasteurella multocida disease bivalent inactivated vaccine minimum exempt from
Epidemic disease dosage
9.1 by the rabbit hemorrhagic disease virus baculovirus vector of above-mentioned preparation, pasteurella multocida disease bivalent inactivated vaccine
It sufficiently shakes up, 2~3 monthly ages susceptible rabbit (rabbit of health is subcutaneously injected with 1.0ml, 0.5ml, 0.25ml, 0.125ml neck respectively
Haemorrhagic virus antibody HI potency is not higher than 1: 2,1.75~2.0kg of weight)；Control group rabbit is set up simultaneously.
9.2 rabbit hemorrhagic disease virus are attacked after poison inspection is immunized 14, take every batch of vaccine various dose that rabbit each 6 is immunized respectively
Only, rabbit 6 are compareed together with the same terms, respectively malicious (the viral level of neck subcutaneous injection rabbit hemorrhagic disease virus Anhui mound strain liver
104LD50/ ml) 1.0ml, it is observed continuously 7.The results show that 3 batches of vaccine immunity dosage are that 0.25ml/ only reaches the above immune rabbit,
It carries out attacking poison with rabbit hemorrhagic disease virus, immune rabbit, which is all good for, to live, the immune rabbit of 0.125ml/ only, and at least 4/6 strong work compares rabbit
All dead, there is the typical pathological change of rabbit hemorrhagic disease in dead rabbit, red thin to dead rabbit liver suspension " O " type of conducting oneself
Born of the same parents' agglutination test is positive (table 4).
4 various dose bivalent inactivated vaccine rabbit hemorrhagic disease virus Anhui mound strain liver poison protest test result of table
9.3 rabbit pasteurella multocida are attacked after poison inspection is immunized 21, take every batch of vaccine various dose that rabbit is immunized respectively
Each 6, rabbit 6 are compareed together with the same terms, the killing property Pasteur bars of a minimum lethal dose (MLD) are subcutaneously injected in neck more respectively
C51-17 plants of bacterium virulent bacterium solution 1.0ml, are observed continuously 7.The results show that 3 batches of vaccine immunity dosage only reach for 1.0ml/
The immune rabbit of 0.5ml/ only carries out attacking poison with rabbit pasteurella multocida, and the strong work of rabbit at least 5/6, immune 0.25ml/only reach is immunized
The immune rabbit at least 2/6 of 0.125ml/ only is strong to live, and control rabbit is all dead, and it is typical that rabbit pasteurella multocida disease occurs in dead rabbit
Pathological change, separation identification is carried out to dead rabbit liver bacterium, is rabbit pasteurella multocida (table 5).
5 various dose bivalent inactivated vaccine C51-17 plants of protest test results of rabbit pasteurella multocida of table
9.4 conclusion
Rabbit hemorrhagic disease virus baculovirus vector, the measurement of pasteurella multocida disease bivalent inactivated vaccine minimum immune dosage
The result shows that immune rabbit can be protected to resist rabbit hemorrhagic disease virus virulent when vaccine immunity is that 0.25ml/ only reaches the above dosage
Attack；When vaccine immunity is that 0.5ml/ only reaches the above dosage, 5 immune rabbits is at least protected to resist rabbit pasteurella multocida
C51-17 plants of attacks illustrate that only (hemagglutinative titer containing VP60 albumen before inactivating is 1: 128 to 0.5ml/, pasteurella multocida bacterium
Number is 3.75 × 109It CFU) is the inactivated vaccine minimum immune dosage.
Since vaccine may have antigen losses (potency reduction) in storage life and guarantee that vaccine can in longer duration of immunity
Effectively to prevent rabbit hemorrhagic disease and rabbit pasteurella multocida disease, in practical applications by the rod-shaped disease of rabbit hemorrhagic disease virus
Poisonous carrier, pasteurella multocida disease bivalent inactivated vaccine (BAC-VP60 plants+C51-17 plants) immunizing dose be set to 1.0ml/
Only (hemagglutinative titer containing VP60 albumen before inactivating is 1:256, pasteurella multocida bacterium number is 7.5 × 109CFU)。
The duration of immunity of 10 rabbit hemorrhagic disease virus baculovirus vector of embodiment, pasteurella multocida disease bivalent inactivated vaccine
10.1 vaccine immunities inactivate 3 batch rabbit hemorrhagic disease virus baculovirus vectors, pasteurella multocida disease bigeminy
Vaccine (lot number：201501,201502,201503) and 1 batch commercially available rabbit hemorrhagic disease, pasteurella multocida disease bigeminy
Inactivated vaccine (lot number：2015004) 35~42 age in days healthy rabbits, are immunized respectively, neck subcutaneous injection, 1.0ml/ is only；Simultaneously
Set up control group rabbit.
10.2 attack poison and observation
10.2.1 rabbit hemorrhagic disease virus is attacked after poison inspection is immunized the 3rd, 4,5,6,7 month, takes every batch of vaccine immunity rabbit 6 respectively
Only, rabbit 6 are compareed together with the same terms, respectively malicious (the viral level of neck subcutaneous injection rabbit hemorrhagic disease virus Anhui mound strain liver
104LD50/ml) 1.0ml is observed continuously 7, and the dead size of animal with protection of record carries out dissect observation simultaneously to dead rabbit
Liver is taken to carry out people " O " type hemagglutination test (HA test).The results show that after 3 batches of vaccine immunities be immunized rabbit with rabbit hemorrhagic disease virus into
Row attacks poison, and rabbit is immunized and is all good for work, rabbit 7 months dead 1 after immune, the whole dead (tables of control rabbit are immunized in similar product
6), there is the typical pathological change of rabbit hemorrhagic disease in dead rabbit, conducts oneself " O " type erythrocyte agglutination to dead rabbit liver suspension
Test, is the positive.
Table 6 vaccine immunity duration test result (rabbit hemorrhagic disease virus attacks poison)
10.2.2 rabbit pasteurella multocida is attacked after poison inspection is immunized the 3rd, 4,5,6,7 month, and every batch of vaccine is taken to exempt from respectively
Epidemic disease rabbit 6, rabbit 6 are compareed together with the same terms, the killing property Pasteur of a minimum lethal dose (MLD) are subcutaneously injected in neck more respectively
C51-17 plants of bacillus virulent 1.0 ml of bacterium solution, are observed continuously 7, and the dead size of animal with protection of record carries out dead rabbit
Dissect is observed and carries out bacterium separation identification.The results show that when attacking poison with C51-17 plants of rabbit pasteurella multocida, the after being immunized
3, the strong work of immune rabbit at least 4/6 of poison is attacked in the strong work of immune rabbit at least 5/6 for attacking poison for 4,5,6 months for 7th month after immune, similar
Product is immunized rabbit and death occurs when duration of immunity attacks poison, attacks to poison with poison within the 7th month and dies 4, cannot generate enough protections,
Equal 6/6 death of rabbit is compareed, the typical pathological change of rabbit pasteurella multocida disease occurs in dead rabbit, to dead rabbit liver bacterium
Separation identification is carried out, is rabbit pasteurella multocida (table 7).
7 vaccine immunity duration of table test result (rabbit pasteurella multocida attacks poison)
10.2.3 before serum antibody titer measurement rabbit immunization and 1st, 2,3,4,5,6,7 month after immune, together with control
Group rabbit, acquires blood respectively, separates serum, measures rabbit hemorrhagic disease virus HI antibody titer and rabbit pasteurella multocida antibody
OD450Value.The antibody test result that the 1st~7 month all serum of malicious rabbit will be attacked for 7th month is for statistical analysis.As a result it shows
Show, 1 month after being immunized, the rabbit hemorrhagic disease virus HI antibody titer that is averaged reaches 1 in rabbit anteserum:23.17, 3 months after being immunized, family
Rabbit hemorrhagic disease virus HI antibody titer peaks in rabbit anteserum, is hereafter gradually reduced, and puts down in 7 months its serum after vaccinating
Equal antibody level is still maintained at 1:23More than, and control group Serum Antibodies of Rabbits is averaged HI potency 1:20~1:20.33Between；Exempt from
1 month after epidemic disease, rabbit pasteurella multocida antibody ELISA detects OD in rabbit anteserum450Value reaches 0.423,3 months after being immunized,
Rabbit pasteurella multocida antibody ELISA detects OD in rabbit anteserum450Value peaks, and is hereafter gradually reduced, after vaccinating
Average antibody ELISA detects OD in 6 months its serum450Value is still 0.4 or more, and control group Serum Antibodies of Rabbits is average
ELISA detects OD450Value is between 0.140~0.159 (table 8, table 9).
In summary, rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine are to rabbit
The duration of immunity of hemorrhagic disease was up to 7 months, to the duration of immunity of rabbit pasteurella multocida disease up to 6 months.
8 vaccine immunity phase of table rabbit hemorrhagic disease virus HI antibody titer result
9 vaccine immunity phase of table rabbit pasteurella multocida antibody ELISA testing result
The storage life of 11 bigeminy vaccine of embodiment
The vaccine saved to 2~8 DEG C carries out safety verification and effect inspection after 3,6,9,12,15,18,21 months respectively
It tests.The results show that the 3 batch vaccines for saving different time carry out overdose safety test to 35~42 age in days healthy rabbits, it is right
It saves 12 months pervious 1 batch similar products and carries out overdose safety test, be observed continuously 14, all rabbit are strong to live.
The result shows that 3 batch rabbit hemorrhagic disease virus baculovirus vectors, pasteurella multocida disease bivalent inactivated vaccine save 21
Good security after month, good security after similar product saves 12 months.With the 3 batch vaccines for saving different time and 1 batch
Secondary similar product distinguishes immune health susceptible rabbit 12,14 days after being immunized, immune rabbit 6 is taken respectively, together with the same terms pair
According to rabbit 6, carries out rabbit hemorrhagic disease virus and attack toxic effect force inspecting；21 days after immune, immune rabbit 6 is taken respectively, together with the same terms
Control rabbit 6 carries out pasteurella multocida and attacks toxic effect force inspecting.The results show that rabbit is immunized after 3 batches of vaccine immunities with rabbit bleeding
Syndrome virus carries out attacking poison, and rabbit is immunized and is all good for (table 10) living, and control rabbit is all dead；Immune rabbit with pasteurella multocida into
Row attacks poison, and the strong work of rabbit at least 5/6, control rabbit 6/6 dead (table 11) is immunized.As a result illustrate that rabbit hemorrhagic disease virus baculoviral carries
Body, pasteurella multocida disease bivalent inactivated vaccine are saved in 2~8 DEG C, and storage life is at least 18 months, and commercially available rabbit viral goes out
Mass formed by blood stasis, pasteurella multocida disease bivalent inactivated vaccine in 2~8 DEG C of storage lives be 12 months.
10 vaccine storage life efficacy test result of table (rabbit hemorrhagic disease virus attacks poison)
-：It is not tested.
11 vaccine storage life efficacy test result of table (rabbit pasteurella multocida attacks poison)
-：It is not tested.
These results suggest that rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine
Storage life has been more than the level of domestic corresponding similar product.
The basic principles, main features and advantages of the present invention have been shown and described above.Those skilled in the art
Member is it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this hairs
Bright principle, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these variations
It all fall within the protetion scope of the claimed invention with improvement.The claimed scope of the invention is by appended claims and its waits
Effect object defines.
Claims (11)
1. a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine, which is characterized in that institute
State antigenic component in bivalent inactivated vaccine is recombination rabbit hemorrhagic disease virus VP60 baculovirus cell culture and killing property Pasteur more
Bacillus fermentation bacterium solution.
2. rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine according to claim 1,
It is characterized in that, the bigeminy vaccine is by recombination rabbit hemorrhagic disease virus VP60 baculovirus cell culture and killing property Pasteur more
It is prepared after the inactivation of bacillus fermentation bacterium solution, antigen is not less than as in every milliliter of vaccine containing the VP60 albumen hemagglutinative titer before inactivation
1:256, rabbit pasteurella multocida bacterium number is not less than 7.5 × 109CFU standard is carried out with seedling.
3. rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine according to claim 1,
It is characterized in that, the rabbit hemorrhagic disease virus baculovirus vector is recombination rabbit hemorrhagic disease virus VP60 baculoviral BAC-
VP60 plants, deposit number is CCTCC V 201833；Pasteurella multocida is C51-17 plants, and deposit number is：CCTCC M
2018401。
4. rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine according to claim 1,
It is characterized in that, the bivalent inactivated vaccine contains adjuvant.
5. rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine according to claim 4,
It is characterized in that, the adjuvant is aluminium hydroxide gel, the ratio of antigen and aluminium hydroxide gel is 9:1.
6. the preparation method of a kind of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine, packet
Include following steps：
（1）The preparation of rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid：Rabbit hemorrhagic disease virus VP60 will be recombinated
Baculoviral inoculating cell is cultivated, and carries out inactivation treatment to the cell culture of harvest；
（2）The preparation of pasteurella multocida inactivation liquid：Fermentor ventilation culture is carried out to pasteurella multocida, to zymophyte
Liquid carries out inactivation treatment；
（3）The preparation of rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine：Antigenic content is pressed
It is not less than 1 containing the VP60 albumen hemagglutinative titer before inactivation in every milliliter of vaccine:256, pasteurella multocida bacterium number is not less than 7.5
×109 CFU standard adjuvant is added and mixes well with seedling.
7. rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine according to claim 1
Preparation method, which is characterized in that the step（1）Middle inoculating cell is Sf9 cell, and used medium is Insect cellculture
Base, recombination rabbit hemorrhagic disease virus VP60 baculoviral inoculum concentration be culture medium total amount 1%, condition of culture be 27~28 DEG C of temperature,
PH value is 6.0~6.2, dissolved oxygen (DO) 50%~60%, 50~70 r/min of speed of agitator；When sick cell is up to 85% or more
Harvest cell culture.
8. rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine according to claim 1
Preparation method, which is characterized in that the step（1）After obtaining rabbit hemorrhagic disease virus baculovirus vector cell culture,
Hemagglutinative titer measurement need to be carried out, cell culture should be not less than 1 to 1% people " O " type erythrocyte agglutination potency:512.
9. rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine according to claim 1
Preparation method, which is characterized in that the step（1）In, inactivation treatment is carried out using cell culture of the formaldehyde to harvest；And
Inactivation inspection is carried out to the rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid after inactivation：Inactivate liquid inoculating cell
Cell-free lesion afterwards, erythrocyte agglutination titration is without coagulation.
10. rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine according to claim 1
Preparation method, which is characterized in that the step（2）In, fermentor ventilation culture is carried out to pasteurella multocida, it is specific to walk
Suddenly it is：Pasteurella multocida single colonie is inoculated in Martin's agar slant containing serum and cracking whole blood, culture obtains more
Killing property Pasteurella first order seed；First order seed is inoculated in the martin's bouillon containing serum, culture obtains second level more killing property bar
Family name's bacillus seed；Be packed into 60%~70% fermentation medium and 0.01%~0.02% defoaming agent by fermenter volume, to culture medium into
By 5%~10% inoculation secondary seed solution of culture base unit weight, while serum is added, 200 ~ 300 r/ of speed of agitator in row sterilization treatment
Min, pH value 7.2~7.6, ventilation culture, harvest culture bacterium solution；Every ml culture bacterium solution containing viable count should not less than 1.2 ×
1010CFU。
11. rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine according to claim 1
Preparation method, which is characterized in that step（2）It is middle that inactivation treatment is carried out using culture bacterium solution of the formaldehyde to harvest, and go out
Biopsy is tested, Martin agar and pasteurella multocida fermented and cultured of the sampling inoculation containing 4% serum and 0.1% cracking whole blood after inactivation
Base is cultivated, and asepsis growth is answered.
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| RU2741643C1 (en) * | 2020-06-15 | 2021-01-28 | Федеральное государственное бюджетное учреждение "Федеральный центр охраны здоровья животных" (ФГБУ "ВНИИЗЖ") | Associated vaccine against myxomatosis, pasteurellosis and viral haemorrhagic disease 1 and type 2 of rabbits |
| CN112843228A (en) * | 2021-04-10 | 2021-05-28 | 福建省农业科学院畜牧兽医研究所 | Bivalent inactivated vaccine for rabbit pasteurellosis and preparation method thereof |
| CN113046271A (en) * | 2021-04-10 | 2021-06-29 | 福建省农业科学院畜牧兽医研究所 | Rabbit F-type pasteurella multocida and application thereof in preparation of inactivated vaccine |
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| CN113336858A (en) * | 2021-05-20 | 2021-09-03 | 江苏省农业科学院 | Rabbit hemorrhagic disease virus VP60 recombinant antigen with single-site chimeric Pasteurella pasteurella PlpE epitope and preparation and application thereof |
| CN113402615A (en) * | 2021-05-20 | 2021-09-17 | 江苏省农业科学院 | Rabbit hemorrhagic disease virus VP60 recombinant antigen with double-site chimeric Pasteurella pasteurella PlpE epitope and preparation and application thereof |
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| CN101732704A (en) * | 2008-11-21 | 2010-06-16 | 中牧实业股份有限公司 | Method for preparing inactivated bivalent vaccine against rabbit hemorrhagic disease and pasteurella multocida disease |
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| CN101215576A (en) * | 2008-01-18 | 2008-07-09 | 江苏省农业科学院 | Rabbit viral haemorrhagic virus capsid protein gene recombination baculovirus and bacterin |
| CN101732704A (en) * | 2008-11-21 | 2010-06-16 | 中牧实业股份有限公司 | Method for preparing inactivated bivalent vaccine against rabbit hemorrhagic disease and pasteurella multocida disease |
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| RU2741643C1 (en) * | 2020-06-15 | 2021-01-28 | Федеральное государственное бюджетное учреждение "Федеральный центр охраны здоровья животных" (ФГБУ "ВНИИЗЖ") | Associated vaccine against myxomatosis, pasteurellosis and viral haemorrhagic disease 1 and type 2 of rabbits |
| CN112843228A (en) * | 2021-04-10 | 2021-05-28 | 福建省农业科学院畜牧兽医研究所 | Bivalent inactivated vaccine for rabbit pasteurellosis and preparation method thereof |
| CN113046271A (en) * | 2021-04-10 | 2021-06-29 | 福建省农业科学院畜牧兽医研究所 | Rabbit F-type pasteurella multocida and application thereof in preparation of inactivated vaccine |
| CN113069539A (en) * | 2021-04-10 | 2021-07-06 | 福建省农业科学院畜牧兽医研究所 | Triple inactivated vaccine for A-type and D-type pasteurella multocida and staphylococcus aureus of rabbit and preparation method thereof |
| CN113336858A (en) * | 2021-05-20 | 2021-09-03 | 江苏省农业科学院 | Rabbit hemorrhagic disease virus VP60 recombinant antigen with single-site chimeric Pasteurella pasteurella PlpE epitope and preparation and application thereof |
| CN113402615A (en) * | 2021-05-20 | 2021-09-17 | 江苏省农业科学院 | Rabbit hemorrhagic disease virus VP60 recombinant antigen with double-site chimeric Pasteurella pasteurella PlpE epitope and preparation and application thereof |
| CN113402615B (en) * | 2021-05-20 | 2024-02-23 | 江苏省农业科学院 | Double-site chimeric Pasteurella multocida PlpE epitope rabbit hemorrhagic disease virus VP60 recombinant antigen, preparation and application thereof |
| CN113336858B (en) * | 2021-05-20 | 2024-02-23 | 江苏省农业科学院 | Rabbit hemorrhagic disease virus VP60 recombinant antigen with single site chimeric Pasteurella PlpE epitope, preparation and application thereof |
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