CN103305474B - Porcine pseudorabies poison strain and its inactivated vaccine and application - Google Patents
Porcine pseudorabies poison strain and its inactivated vaccine and application Download PDFInfo
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Abstract
The invention discloses porcine pseudorabies poison strain and its inactivated vaccine and application, belong to separation and the application of porcine pseudorabies virus strain.Present invention firstly provides one plant of detached PRV (Pseudorabies virus) BJ strain from morbidity porcine tissue, its microbial preservation number is CGMCC No.7351.The invention discloses applying the method that described PRV (Pseudorabies virus) BJ strain prepares inactivated vaccine, including：Culture virus stain, obtains virus liquid；Add inactivator, virus liquid is inactivated and concentrates；Mix adjuvant and virus liquid, emulsifying, obtain final product.The present invention is optimized to the technological parameter of inactivated vaccine preparation method further, improves immune protection effectiveness and the safety of inactivated vaccine.Immune protection effectiveness and safety testing show, the swine pseudorabies vaccine of present invention preparation has good immune protection effectiveness and safety, can be clinically used for prevention or the treatment of porcine pseudorabies.
Description
Technical field
The present invention relates to one plant of detached viral prevalence strain, more particularly, to one plant detached pseudorabies prevalence virus
Strain and the swine pseudorabies vaccine being prepared with this strain, belong to the separation of porcine pseudorabies virus strain and answer
Use field.
Background technology
Pseudorabies viruses (Porcine pseudorabies virus, PRV) can cause multiple domestic animals and wild animal
To generate heat, very to itch（Except pig）And encephalomyelitiss are the disease of cardinal symptom.Immunity inoculation is the main plan of anti-pseudorabies processed
Slightly, what China's application at present was more is to prepare attenuated vaccine with Bartha-K61 strain.Although PRV gene delection attenuated vaccine energy
The appearance of clinical symptoms after effectively preventing infections, but Pigs Inoculated still may be by poison infection by force, and infection can be formed hides, subsequently dives
The virus of volt infection can be activated and cause and disseminate.
Prepare the research emphasis that inactivated vaccine is porcine pseudorabies vaccination areas, such inactivation with epidemic isolates at present
The use of vaccine is considered as prevention porcine pseudorabies, raising sow breeding potential, the effective ways of control piglet mortality rate.
Existing PRV (Pseudorabies virus) inactivated vaccine is primarily present the problem of following several respects：One is that strain is aging, viral
Titre is not high；Two is that inactivation technology falls behind, and is easily caused inactivator residual or inactivation is not thorough；Three is to use Traditional adjuvants, leads
Cause immune induction effect on driving birds is not good.Pseudorabies viruses difference strain has differences at the aspect such as virulence and biological property, and inactivates
The immunogenicity of Seedling is highly dependent with strain virulence.Additionally, research confirms, inactivator and immunological adjuvant are also impact PRV inactivation
The key factor of immune effect of vaccine, wherein, the species of inactivator and adjuvant use, ratio, dosage and inactivation time etc. are directly
The immune effect of impact PRV (Pseudorabies virus) inactivated vaccine.
Content of the invention
One of the object of the invention is to provide one plant of newly detached porcine pseudorabies virus (Porcine pseudorabies
Virus) strain；
The two of the object of the invention are that described porcine pseudorabies virus strain is prepared into inactivated vaccine, are applied to pig puppet mad
The prevention of dog disease disease or treatment；
The third object of the present invention is each technological parameter in the preparation method of inactivated vaccine to be optimized with screening to carry
The immune protection effectiveness of high inactivated vaccine or safety.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
Separate and by passing on the tissues such as the brain of the dead pig of present invention morbidity in Beijing morbidity pig farm 15 days, tonsil
Adapt to obtain one plant of PRV (Pseudorabies virus) BJ strain；The mechanism that this BJ strain submits patent accreditation to is carried out preservation by the present invention, its
Microbial preservation number is：CGMCC No.7351；Classification And Nomenclature：Porcine pseudorabies virus；The preservation time is：March 11 in 2013
Day；Depositary institution is：China Committee for Culture Collection of Microorganisms's common micro-organisms center；Preservation address is：Court of Beijing
Positive area North Star West Road 1 institute 3, Institute of Microorganism, Academia Sinica.
5～10 age in days PRV negative antibody piglets are inoculated in detached for present invention PRV (Pseudorabies virus) BJ strain, body temperature liter occurs
Up to more than 41.5 DEG C, obvious nervous symptoms and diarrhoea etc., 1～2d quick death, cut open inspection death pig visible typical case PRV
Infection change, it is possible to be separated to PRV virus from pathological tissues, illustrates that the separated BJ strain virulence of the present invention is strong.
The present invention detached PRV (Pseudorabies virus) BJ strain is in ST cell, PK-15 cell, mdck cell, BHK-21 cell etc.
Propagation on cell is fast, and titre is high（Virus titer minimum up to 107.5TCID50/mL）, there is good cell-proliferation activity,
Inducible animal produces high titre neutralizing antibody, has good immunogenicity.
The present invention detached PRV (Pseudorabies virus) BJ strain can with from 5 provinces such as Beijing, Tianjin, Henan, Shandong or Guangxi
City's PRV positive antibody is effectively neutralized, and illustrates that BJ strain of the present invention has wider spectrotype.
Gene analysiss find, the separated PRV (Pseudorabies virus) BJ strain of the present invention can represent the Major Epidemic of Chinese PRV
Strain, can be used as the seed culture of viruses of preparation PRV (Pseudorabies virus) inactivated vaccine.
Another object of the present invention is to prepare porcine pseudorabies with the separated PRV (Pseudorabies virus) BJ strain of the present invention to go out
Live vaccine.
The second object of the present invention is achieved through the following technical solutions：
A kind of method preparing swine pseudorabies vaccine with the strain of PRV (Pseudorabies virus) BJ, including：
（1）Cultivate separated PRV (Pseudorabies virus) BJ strain, obtain virus liquid；
（2）Add inactivator in virus liquid, virus liquid is inactivated；
（3）Add adjuvant, mix homogeneously, emulsifying in the virus liquid after inactivation, obtain final product.
For improving the immune protective effect of inactivated vaccine or safety, the present invention is to used in preparing in inactivated vaccine
The parameters such as the species of inactivator, consumption and inactivation time are optimized and screen.The present invention is found through experiments, compared to it
Its inactivator, using binary ethylenimine（BEI）As the inactivator preparing inactivated vaccine, have toxic and side effects little, inactivation more
For thoroughly advantage, therefore present invention preferably employs binary ethylenimine（BEI）As inactivator.Process in inactivation of viruses liquid
In, the final concentration of the binary ethylenimine being added and inactivation time are for the immune protective effect of inactivated vaccine and safety
Etc. the impact having highly significant；In order to obtain more preferable inactivating efficacy to lift immune protection effectiveness and the safety of inactivated vaccine
Property, the present invention is optimized to the final concentration of binary ethylenimine and inactivation time, and the present invention is by substantial amounts of optimization experiment
Finally found that, as 0.01%～1% (w/v) of the final concentration of virus liquid total amount of BEI, inactivating efficacy is preferable；The present invention is very
It is found surprisingly that, as 0.05% (w/v) of the final concentration of virus liquid total amount of BEI, not only inactivate very thorough, and made
Standby inactivated vaccine product has optimal safety simultaneously.
Additionally, inactivation time also has the impact of highly significant for inactivating efficacy, it is a discovery of the invention that adding in virus liquid
After BEI, inactivation 6～60h has certain inactivating efficacy；The present invention passes through further experiment and finds that inactivation time is energy during 48h
Enough virus is thoroughly inactivated.
The mode of heretofore described inactivation of viruses liquid is preferably：Vibrate in gas bath after adding BEI in virus liquid
Carry out inactivation of virus, the temperature of described gas bath vibration is preferably 32 DEG C, and the rotating speed of gas bath vibration is preferably l20r/ under mode
min.
It has also been found that, the virus liquid after inactivation and the ratio of adjuvant also have certain shadow for immune protective effect
Ring；The present invention is found by experiment that, counts by volume, and virus liquid after inactivation and the ratio of adjuvant are 85:When 15, one is viscous
Degree just, be easier inject；Two be the obtained vaccine of this ratio, not breakdown of emulsion, holding time longer, immunoprotection can be improved
Effect, is capable of the quality of effective improving product, and heretofore described adjuvant is preferably ISA15AVG adjuvant（France's match Bick
The product of company, trade name：MontanideTM）, after this ISA15AVG adjuvant is degerming, can be directly used for vaccine emulsifying preparation.
The present invention further finds, by the virus liquid after inactivation through being centrifuged and filtering, adds after filtering cell debriss again
Enter adjuvant and carry out emulsifying, immune protection effectiveness and the safety of vaccine product can be significantly improved.
The present invention passes through further test and finds, it is possible to obtain the emulsification procedure of optimum emulsification effect is：（1）To inactivate
Same batch of virus liquid defrosting mix homogeneously, according to aqueous phase antigen and oil phase adjuvant 85:15 volume proportion；（2）800～1000r/
Min continuous stirring 20～50min, obtains the vaccine of oil-in-water dosage form；（3）Subpackage after static 10～30min.
Immune protection effectiveness and safety testing show, separate the porcine pseudorabies inactivation epidemic disease of BJ strain preparation with the present invention
Seedling has good immune protection effectiveness and safety, clinically can be used for prevention or the treatment of porcine pseudorabies.
Inactivated vaccine immunizing dose of the present invention is：For sheep, breeding boar, sow or common pig, intramuscular injection 2mL/ head,
Once inoculate inactivated vaccine, now duration of immunity was up to more than 5～6 months.It is spaced after first time immunity inoculation 3 weeks and carry out 2 times and connect
Plant immunity, consolidate immune effect.
Brief description
Fig. 1 PRV virus BJ isolation of strains culture（A is normal BHK-21 cell；B is that inoculation pathological material of disease appearance is cytopathic
Cell）.
The pathological changes situation that BHK-21 cell occurs inoculated by Fig. 2 variable concentrations BJ strain venom（A inoculates for PRV virus stock solution used
Cell；B dilutes the cell of PRV poison disease vaccination for 10-1；C dilutes the cell of PRV poison disease vaccination for 10-2）.
Fig. 3 PRV-BJ strain negative staining electron microscope morphological observation.
Fig. 4 virus immunity fluoroscopy result（A is to connect malicious 24h result；B is negative control）.
Fig. 5 BJ strain and the phylogenetic analysis of Reference strains gE gene；Wherein, gE_BJ is the detached PRV-BJ of the present invention
Strain；PRV-H-gE is the H strain described in patent application publication number CN102344912A.
Specific embodiment
Embodiments of the present invention be will be described in more by the following example, it should be understood that described embodiment is only model
Example property, any restriction is not constituted to the scope of the present invention.It will be understood by those skilled in the art that without departing from the present invention
Spirit and scope under to modify to the details of technical solution of the present invention and form or can replace, but these modification or replace
Each fall within protection scope of the present invention.Separation, culture and the identification of embodiment 1 PRV (Pseudorabies virus) BJ strain
1. the separation of PRV (Pseudorabies virus) BJ strain, cultural characters
（1）Pathological material of disease gathers：Aseptic collection takes the brain of fetal death of sow, tonsil etc. to organize.With 1:10 addition MEM, grind, system
Standby tissue suspension, after repeatedly 3 freeze thawing, 2000r/min is centrifuged 15min, collects supernatant, then through 0.2 μm of filter membrane filter mistake
Filter, filter liquor is put -40 DEG C of refrigerator Cryopreservations.
（2）Separation and Culture：10% content that above-mentioned pathological material of disease is pressed virus-culturing fluid accesses the inoculation use not yet forming monolayer
BHK-21 cell, 1h is made in 37 DEG C of senses, changes plus the MEM culture fluid containing 2% calf serum, 37 DEG C of culture 5d.Second culture 96h, receives
Obtain toxic culture fluid, after 2 freeze thawing, receive poison.Continuous passage is cultivated, observation of cell pathological changes（CPE）, result shows：Cell is swollen
Greatly, circle contracting, then starts shedding off and gradually forms plaque focus, and have " drawing in the net " phenomenon（Fig. 1）.Through Plaque Clone purification,
Obtain one plant of porcine pseudorabies poison strain eventually, be named as BJ strain.This BJ strain is preserved in Chinese microorganism strain preservation pipe
Manage committee's common micro-organisms center, deposit number is：CGMCC No.7351.
（3）Cultural characters：BJ strain fast breeding on BHK-21 cell, 24～36h CPE reaches more than 85%（Fig. 2）,
Virus titer can reach 108.2More than TCID50/mL；BJ strain fast breeding, 24～36h on the ST cell grow up to monolayer
CPE reaches more than 85%, and virus titer can reach 107.8More than TCID50/mL；BJ strain fast breeding on PK15 cell, 24～
36hCPE reaches more than 85%, and virus titer can reach 107.5More than TCID50/mL；BJ strain fast breeding on mdck cell,
24～36h CPE reaches more than 85%, and virus titer can reach 108.0More than TCID50/mL.
2. the identification of PRV (Pseudorabies virus) BJ strain
（1）Negative staining electron microscope is observed
Through cultivating 3～5d on BHK-21 cell, more than CPE85% occurs, 12000rpm centrifugation sick cell supernatant is carried out
Negative staining electron microscope is observed.Result such as Fig. 3 shows：Negative staining electron microscope observes that virion is in oval or circular appearance, the no disease of cyst membrane
Malicious particle diameter about 100～125nm, with the mature virion diameter about 175～190nm of cyst membrane.
（2）Immuno-fluorescence assay
Take on detached BJ strain inoculation ST passage cell, in 5%CO228h is cultivated under the conditions of 37 DEG C, when CPE reaches 80%,
Pass the second filial generation, from second filial generation culture after 2 freeze thawing, do fluorescent antibody detection.Take the ST cell coverslip of oneself culture 24h
Culture, virus inoculation, then through 24h culture.Take out coverslip, rinsed with PBS, fix through acetone, then use PRV (Pseudorabies virus)
Dyeing 30min in fluorescent antibody wet box, rinses, sealing, microscopy.Result shows：Specimen infects ST passage cell, and cell is ill
Become, it is seen that being in the specificity fluorescent of Fructus Mali pumilae green in nucleus Cytoplasm, negative control no specificity fluorescent occurs（Fig. 4）.
（3）The phylogenetic analysis of BJ strain
BJ strain gE gene nucleotide series tetraploid rice application DNASTAR7.1 software is by the BJ measuring strain gE gene
Partial sequence and derivation aminoacid sequence with carry out tetraploid rice analysis with the corresponding sequence of domestic and international PRV separation strains, knot
Fruit as shown in figure 5, the genetic distance of the separated PRV separation strains BJ strain of the present invention and HNJZ, LA strain and AF207700.1 strain
Closely.H strain in the separated PRV separation strains BJ strain of the present invention and patent application publication number CN102344912A（PRV-H-gE）Lose
Pass distant, be not belonging to same evolutionary branching.
（4）Animal Orthogonal Rotational Regressive Tests
, obvious clinical symptoms after injection 48h, are mainly shown as body in inoculation 5～10 age in days PRV negative antibody piglets
Up to more than 41.5 DEG C of temperature rise, occur obvious nervous symptoms, instability of gait, eat breast minimizing, motion inharmonious, extremity be in strike
Shape, the vomiting of part pig or diarrhoea, dead after general 24～48h.Comparison pig non-evident sympton.The intensive pin of the visible kidney of cut open inspection
Shape petechia, meninges are substantially congested, marrowbrain hypervolia, the substantial viscera Chang Kejian canescence necrotic lesion such as liver, spleen, and lung fills
The pathological changes such as blood, edema and necrosis point are it is possible to be separated to PRV virus from cerebral tissue.
The preparation of embodiment 2 PRV (Pseudorabies virus) BJ strain kind poison
1. basic bacteria criticizes foundation：The original seed culture of viruses learnt from else's experience separately and identify, is formed by 1% access of Virus culture liquid measure
In the cell cultures such as the ST cell of monolayer, PK15 cell, mdck cell or BHK-21 cell, put 5%CO2In under the conditions of 37 DEG C
Culture, observes to CPE daily.Observed result shows：Cell expands, circle contracting, then starts shedding off and gradually forms plaque disease
Stove, and have " drawing in the net " phenomenon, when pathological changes reach 80%, harvest toxic cell culture fluid, after 2 freeze thawing, receive poison.Continuously
Passed on for 6 generations.
2. produce seed lot to set up：Take basic bacteria, by Virus culture liquid measure 1% access formed monolayer ST cell,
In the cell cultures such as PK15 cell, mdck cell or BHK-21 cell, put 5%CO2In cultivate under the conditions of 37 DEG C, when pathological changes reach
During to 80%, harvest toxic cell culture fluid, after 2 freeze thawing, receive poison.Continuous passage 6 generation.
3. seed culture of viruses standard
（1）Viral level
With DMEM culture fluid, seed culture of viruses is made continuous 10 times to be serially diluted, each dilution factor takes 100 μ L to add 96 hole cell trainings
In foster plate, each dilution factor makees 8 repetitions, is added to the cell culture such as ST or BHK growing up to monolayer, every hole 100 μ L（Cell contains
Measure with 3 × 105/mL）, and set normal cell culture comparison, put 37 DEG C, the CO containing 5%2Cultivate in incubator, observe CPE day by day,
Cell pyknosis, assembles, granule increases, and some cell fusions, comes off, more space and be then judged to CPE.Observe 7d, record is thin
The hole count of born of the same parents' pathological changes, calculates the TCID50 of virus according to Reed-Muench method.Comprehensive determination：Every milliliter of virus liquid is not less than
107.5More than TCID50 is qualified.
（2）Virulence
With 6～18 monthly ages, the no sheep of pseudorabies viruses neutralizing antibody or rabbit 3, each intramuscular injection of every sheep
Malicious 1mL is (containing 10 by force3.0LD50), rabbit is (containing 101.0LD50), observe 14d.Should at least 2 morbidity death.
（3）Immunogenicity
Seed culture of viruses is synchronously inoculated the cells such as ST to be bred, harvest virus liquid, after BEI inactivation, plus MontanideTM
ISA15AVG adjuvant mixing and emulsifying makes oil-in-water type inactivated vaccine, with 6～18 monthly ages, no pseudorabies viruses neutralizing antibody
Sheep 7,4 each intramuscular injection 1mL, another 3 are only used as compareing.After inoculation 14d, malicious 1mL (contains each intramuscular injection of every sheep by force
103.0LD50), observe 14d.3 morbidities of comparison sheep are dead, and immune sheep is all protected.
（4）Pure inspection
The PRV (Pseudorabies virus) BJ strain set up has been carried out with antibacterial, mycete, mycoplasma inspection, and to the 11st, 12 and 24 generation
Secondary seed culture of viruses has carried out the inspection of exogenous viruses, and result shows, it is sick that strain of the present invention criticizes all no antibacterial, mycete, mycoplasma and external sources
Poison pollution, all pure.
（5）Specificity
With DMEM liquid, seed culture of viruses is diluted to 200TCID50/ 0.1mL, malicious specific serum etc. with the resisting pstudorabies of inactivation
Amount mixes, and puts 37 DEG C of neutralization 1h, 3 bottles of the well-grown ST cell of synchronization inoculation（Cell content is with 3 × 105/mL）, set disease simultaneously
Poison comparison, normal cell controls and negative serum control, put 37 DEG C, the CO containing 5%2In incubator, cultivate 5～7d.Neutralization virus
Group and normal cell controls group all occur without CPE；All CPE in virus control group and negative serum control group.
The preparation of embodiment 3 porcine pseudorabies viral disease inactivated vaccine
1. the preparation of seedling virus liquid：PRV (Pseudorabies virus) BJ strain seed culture of viruses separated for embodiment 1 is pressed embodiment 2
Mode cultivates production seed poison, will be thin to the ST cell of 0.1% access formation monolayer of Virus culture liquid measure, PK15 cell, MDCK
In the cell culture such as born of the same parents or BHK-21 cell, put 37 DEG C of rotating and culturing, when pathological changes reach 80%, harvest toxic cell culture
Liquid, after 3 freeze thawing, receives poison.
2. inactivate：By virus liquid total amount 0.05%（W/v in virus liquid) add 2%（W/v binary ethylenimine solution),
Under conditions of 32 DEG C, the vibration of 120r/min gas bath, inactivate 48h, be subsequently adding 2% hypo solution, terminate inactivation,
And do steriling test.
3. concentrate:Take the virus liquid after inactivation, through 4000r/min horizontal centrifugal, then membrane filter is filtered with 0.2um.
Virus liquid after microfiltration is concentrated by ultrafiltration 5 times with 100KD filter membrane filter.
4. the preparation of inactivated vaccine：By same batch of virus liquid defrosting mix homogeneously of inactivation, then according to aqueous phase antigens inactive
Virus liquid and oil phase adjuvant ISA15AVG are with 85:15 volume proportion carries out emulsifying.Emulsification procedure is first aqueous phase to be put into emulsifying
It is stirred in device, is then slowly added into oil phase adjuvant, with 800～1000r/min continuous stirring 20～50min, make water bag
Oil-type vaccine, subpackage after static 10～30min.
5. inspection of semifinished product quality standard
（1）Steriling test
By existing《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
（2）Viral level measures
With DMEM culture fluid, seed culture of viruses is made continuous 10 times to be serially diluted, each dilution factor takes 100 μ l to add 96 hole cell trainings
In foster plate, each dilution factor makees 8 repetitions, is subsequently added monolayer ST cell suspension, and sets normal cell culture comparison, puts 37
DEG C, containing 5% CO2Cultivate in incubator, by sky observation of cell pathological changes（CPE）, cell pyknosis, assemble, granule increases, and have is thin
Born of the same parents merge, come off, more space then is judged to infect.Record cytopathic hole count, calculate disease according to Reed-Muench method
The TCID50 of poison, every milliliter of virus liquid of result is not less than 107.5TCID50.
（3）Inactivation inspection
Take the virus liquid after inactivation, by virus liquid and growth-promoting media 1:10 ratio is inoculated in ST cell, and 37 DEG C of cultures are observed
5d, to no pathological changes person blind passage 2 generation again, sets virus control simultaneously.Pathological changes that result is acellular.3 batches of semi-finished product of Laboratory Production are equal
Qualified.
6. with regard to product inspection quality standard
（1）Safety verification standard
In order to ensure the safety of vaccine, 3 batches of vaccine priorities that the present invention is prepared to laboratory have been carried out " to white mice
Safety testing ", " to the inoculation of piglet single dose, single dose repeated inoculation, disposable overdose（2 multiple doses）The safety of inoculation
Property test ", " to the inoculation of replacement gilt single dose, single dose repeated inoculation, disposable overdose（2 multiple doses）The safety of inoculation
Property test ", " to the inoculation of in-pig single dose, single dose repeated inoculation, disposable overdose（2 multiple doses）The safety of inoculation
Property test ".Result of the test shows：Each immune animal single dose inoculation, single dose repeated inoculation, disposably after heavy dose of inoculation,
Pig state is all good, and drinking-water of searching for food is normal, is as good as paradoxical reaction；Also no obvious to the breeding function of in-pig after vaccination
Impact.Above test all proves that the inactivated vaccine prepared by the present invention is safe.
Found by experimental observation, occur without in 21d after inoculating inactivated vaccine of the present invention local redness, ulcer, erosion with
And neuratrophia, the phenomenons such as minimizing of searching for food are it was demonstrated that vaccine of the present invention has good safety.
The present invention has further carried out following safety testing：With the susceptible piglet of 10～20kg health（PRV neutralizing antibody
Potency is not higher than 1:2）2, each deep intramuscular injection inactivated vaccine 4mL of the present invention, separately take 2 pigs of condition identical not inoculate work
For comparison, Continuous Observation 21d, any locally or systemically untoward reaction being caused by vaccine injection did not occur within the observation period.
（2）The formulation of efficacy test standard
The cells such as seed culture of viruses inoculation ST are bred, is harvested virus liquid, after BEI inactivation, plus
MontanideTMISA15AVG adjuvant mixing and emulsifying makes oil-in-water type inactivated vaccine, with 6～18 monthly ages, no pseudorabies disease
The sheep of malicious neutralizing antibody 7,4 each intramuscular injection 1mL, another 3 are only used as compareing.After inoculation 14d, every sheep each muscle note
Penetrate BJ by force malicious 1mL (containing 103.0LD50), observe 14d.3 morbidities of comparison sheep are dead, and immune sheep is all protected.
The process optimization test of test example 1 PRV (Pseudorabies virus) inactivated vaccine
（1）Binary ethylenimine（BEI）With formalin-inactivated Comparision Test
Select final concentration of 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 1% (w/v's)
Formalin and final concentration of 0.005%, the BEI of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 1% (w/v)
PRV (Pseudorabies virus) is carried out inactivate comparative test, result of the test shows, can be by pig after 0.05% BEI 48h under the conditions of 32 DEG C
Pseudorabies viruses complete inactivation, and, result of the test shows that BEI is less to the damage of cell；PRV antigen adds 0.2% formaldehyde
Solution, also can reach the purpose of complete inactivation virus, but formaldehyde has strong and stimulating, if residual in vaccine through 37 DEG C of inactivation 48h
Stay free formaldehyde to enter animal body, untoward reaction can be produced.
（2）Binary ethylenimine（BEI）Using dosage and concentration determine
It is respectively adopted final concentration of 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.08%, 0.1%, 0.2%,
The BEI of 1% (w/v) inactivates to virus liquid, under conditions of 32 DEG C, the vibration of 120r/min gas bath, inactivates 48h, after inactivation
To product carry out mice infection test and white mice safety testing.Result of the test is shown in Tables 1 and 2.
The different BEI concentration of table 1 inactivates the infection Experiment on white mice result of PRV
The different BEI concentration of table 2 inactivates the white mice safety testing of PRV
Visible according to the result of the test of Tables 1 and 2, the BEI using final concentration of 0.05% inactivates to virus liquid, no
Only inactivate thoroughly, and prepared inactivated vaccine has optimal safety；Therefore, specifically preferred according to the invention with final concentration
The BEI of 0.05% (w/v) inactivates to virus liquid.
（3）Binary ethylenimine（BEI）Action time determines
With the BEI of final concentration of 0.05% (w/v) 32 DEG C, under conditions of 120r/min gas bath vibrates, be respectively 6h,
The inactivation of the different times such as 12h, 18h, 24h, 30h, 36h, 42h, 48h, 54h, 60h, does the training of inactivation of viruses liquid cell after inactivation
Foster thing infectious virus check and mice infection test.Result of the test is shown in Table 3.
Table 3PRV inactivation test
According to result of the test, final determine with the BEI solution of final concentration 0.05% 32 DEG C, 120r/min gas bath vibrates
Under the conditions of inactivation 48h prepared by inactivated vaccine there is optimal inactivating efficacy.
（4）Binary ethylenimine（BEI）Toxicity test
Set 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, the isocyatic BEI of 0.1%, 0.2%, 1% (w/v)
Solution, injection body weight is 18～22g white mice, observes 10d, record death toll and survival number.Determine that 0.05%BEI0.4mL is peace
Full dosage.
To sum up, the present invention finally determines with the binary ethylenimine solution of final concentration in virus liquid 0.05% at 32 DEG C, 120r/
Min, inactivates 48h porcine pseudorabies venom under conditions of gas bath vibration, is subsequently adding 2% hypo solution, terminate inactivation.
The Vaccine effectiveness test of test example 2 PRV (Pseudorabies virus) inactivated vaccine
First, experimental vaccine
1st, for examination inactivated vaccine：Produce inactivated vaccine with embodiment 3 methods described, specific as follows：
（1）The preparation of seedling virus liquid：PRV (Pseudorabies virus) BJ strain seed culture of viruses is cultivated production kind in the way of embodiment 2
Son poison, 0.1% access of Virus culture liquid measure is formed in the ST cell culture of monolayer, puts 37 DEG C of rotating and culturing, work as pathological changes
When reaching 80%, harvest toxic cell culture fluid, after 3 freeze thawing, receive poison.
（2）Inactivation：2% is added in porcine pseudorabies virus liquid（W/v binary ethylenimine solution) is final concentration of to it
0.05%（W/v), 32 DEG C, 120r/min gas bath vibrate under conditions of, inactivate 48h, be subsequently adding 2% sodium thiosulfate molten
Liquid, terminates inactivation；
（3）Concentrate:Take the virus liquid after inactivation, through 4000r/min horizontal centrifugal, then membrane filter is filtered with 0.2um.
Virus liquid after microfiltration is concentrated by ultrafiltration 5 times with 100KD filter membrane filter；
（4）The preparation of inactivated vaccine：By the virus liquid defrosting mix homogeneously of inactivation, then according to aqueous phase antigens inactive is viral
Liquid and oil phase adjuvant ISA15AVG are with 85:15 volume proportion carries out emulsifying；Emulsification procedure is first to put into aqueous phase in emulsator
It is stirred, is then slowly added into oil phase adjuvant, with 9000r/min continuous stirring 40min, make oil-in-water dose vaccine, quiet
Only 20min.
2nd, compare inactivated vaccine：Used strain is the H strain disclosed in patent application publication number CN102344912A
（Its microbial preservation number is：CGMCC No.5013）；Except strain is different, the preparation method of comparison inactivated vaccine is gone out with for examination
The preparation method of live vaccine is identical.
2nd, test method
（1）Immune protective efficiency test to pig
40 pigs are randomly divided into two groups, that is, test 1 group and 2 groups of test, every group of 20 pigs；Test 1 group with for examination inactivation
Vaccination, 2 groups of test is inoculated with comparison inactivated vaccine；Immunizing dose is pig muscle injection 2mL/ head, once inoculation inactivation
Vaccine；6 months latter two test group of immunity inoculation are all attacked with the PRV (Pseudorabies virus) virulent strain of same dose, immune protective efficiency
Data is shown in Table 3.
The protection that table 3 inactivated vaccine is attacked to PRV (Pseudorabies virus) intensity
From result of the test, the inactivated vaccine prepared by the present invention has good immune protective effect for pig, and this
The immune protection effectiveness of invention inactivated vaccine will be significantly better than the comparison immune protection effectiveness to pig for the inactivated vaccine.
（2）Immune protective efficiency test to sheep
24 sheep at 6～18 monthly ages, no pseudorabies viruses neutralizing antibody are randomly divided into 3 groups, that is,：Test 1 group, examination
Test 2 groups and matched group, every group 8；Test 1 group of intramuscular injection 1mL for examination inactivated vaccine, 2 groups of intramuscular injection 1mL comparisons of test are gone out
Live vaccine, matched group does not give vaccine injection；After inoculation 14d, each intramuscular injection PRV (Pseudorabies virus) of every sheep malicious 1mL by force
(containing 103.0LD50), observe 14d.It was found that 8 sheep of 1 group of test all obtain protection, 5 sheep of 1 group of test are obtained
Must protect, 3 morbidities are dead；7 sheep morbidities of matched group are dead.
The safety testing of test example 3 PRV (Pseudorabies virus) inactivated vaccine
By the inactivated vaccine prepared by embodiment 3 to inoculate 10mL/ head, inoculation using dose inoculation 2mL/ head, overdose
Replacement gilt, pregnant pig have no that immunity has any impact to reproductive function, and body temperature, feed, breeding, gestation, farrowing are all normal.Examination
Testing result proves, inactivated vaccine safety of the present invention is good.
Claims (10)
1. one plant of PRV (Pseudorabies virus) (Porcine pseudorabies virus) BJ strain is it is characterised in that its microorganism
Preserving number is：CGMCC No.7351.
2. use in the vaccine of preparation prevention or treatment porcine pseudorabies for the PRV (Pseudorabies virus) BJ strain described in claim 1
On the way.
3. the inactivated vaccine of a kind of prevention or treatment porcine pseudorabies is it is characterised in that include：Described in claim 1, pig puppet is mad
The inactivation of virus liquid of dog disease poison BJ strain preparation and adjuvant.
4. a kind of preparation method of swine pseudorabies vaccine is it is characterised in that comprise the following steps：
(1) the PRV (Pseudorabies virus) BJ strain described in culture claim 1, obtains virus liquid；
(2) inactivator, inactivation of viruses liquid are added in virus liquid；
(3) add adjuvant, mix homogeneously, emulsifying in the virus liquid after inactivation, obtain final product.
5. in accordance with the method for claim 4 it is characterised in that：The inactivator adding in virus liquid in step (2) is two
Aziridine；Based on w/v, add binary ethylenimine in virus liquid to the 0.01%～1% of its final concentration of virus liquid total amount.
6. in accordance with the method for claim 5 it is characterised in that：Binary ethylenimine is added extremely in virus liquid in step (2)
The 0.05% of its final concentration of virus liquid total amount.
7. in accordance with the method for claim 4 it is characterised in that：Inactivation time described in step (2) is 6～60 hours.
8. in accordance with the method for claim 7 it is characterised in that：Inactivation time described in step (2) is 48 hours.
9. in accordance with the method for claim 4 it is characterised in that：Step (2) adds after inactivator in gas bath in virus liquid
Carry out inactivation of virus under the mode of vibration；The temperature of described gas bath vibration is 32 DEG C, and the rotating speed of gas bath vibration is l20r/min.
10. in accordance with the method for claim 4 it is characterised in that：Count by volume, the virus liquid after inactivation in step (3)
Ratio with adjuvant is 85:15.
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| CN104328090B (en) * | 2013-09-22 | 2018-08-31 | 普莱柯生物工程股份有限公司 | A kind of porcine pseudorabies virus strain, vaccine composition and its preparation method and application |
| CN103756977B (en) * | 2013-12-11 | 2016-01-06 | 姜平 | Porcine pseudorabies variant gE and gI genetically deficient virus strain and application thereof |
| CN103627678B (en) * | 2013-12-19 | 2016-03-02 | 姜平 | A kind of porcine pseudorabies virus variant PRV-ZJ01 and application |
| CN103981151B (en) * | 2014-04-09 | 2016-10-26 | 中国农业科学院哈尔滨兽医研究所 | A kind of pseudorabies inactivated vaccine and preparation method thereof |
| CN104630153A (en) * | 2014-12-02 | 2015-05-20 | 湖北省农业科学院畜牧兽医研究所 | Porcine pseudorabies virus strain and application thereof |
| CN105732807B (en) * | 2016-02-24 | 2019-02-26 | 福州大北农生物技术有限公司 | A kind of preparation method of porcine pseudorabies virus positive serum |
| CN107137705B (en) * | 2017-05-08 | 2018-01-23 | 广东渔跃生物技术有限公司 | The production method of pseudorabies gE gene delection viral inactivation vaccines |
| CN108048413B (en) * | 2017-12-20 | 2021-06-25 | 哈药集团生物疫苗有限公司 | Pseudorabies virus canine animal isolate, inactivated vaccine prepared from pseudorabies virus canine animal isolate and application of pseudorabies virus canine animal isolate |
| CN109735510B (en) * | 2018-12-26 | 2023-02-17 | 河南农业大学 | Porcine pseudorabies virus variant HN-QYY, HN-QYY-gE-and construction method and application thereof |
| CN110283793B (en) * | 2019-08-05 | 2020-08-28 | 中国动物卫生与流行病学中心 | Method for separating and culturing porcine pseudorabies virus |
| CN111705040A (en) * | 2020-06-17 | 2020-09-25 | 国药集团动物保健股份有限公司 | Method for removing mycoplasma in pseudorabies virus liquid |
| CN113633765A (en) * | 2021-08-03 | 2021-11-12 | 金宇保灵生物药品有限公司 | Porcine pseudorabies gE gene deletion inactivated vaccine and production method thereof |
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