CN103305474A - Porcine pseudorabies virus strain as well as inactivated vaccine and applications thereof - Google Patents
Porcine pseudorabies virus strain as well as inactivated vaccine and applications thereof Download PDFInfo
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Abstract
The invention discloses a porcine pseudorabies virus strain as well as an inactivated vaccine and applications thereof, belonging to the field of separation and application of the porcine pseudorabies virus strain. The invention firstly provides a porcine pseudorabies virus BJ strain separated from diseased pig tissues, and the microbial preservation number of the porcine pseudorabies virus BJ strain is CGMCC (China General Microbiological Culture Collection Center) No.7351. The invention discloses a method for preparing the inactivated vaccine by applying the porcine pseudorabies virus BJ strain. The method comprises the steps of culturing a virus strain to obtain a virus solution; adding an inactivator, and inactivating and concentrating the virus solution; and evenly mixing an adjuvant and the virus solution, and emulsifying to obtain the inactivated vaccine. The technological parameters of the inactivated vaccine preparation method are further optimized, and the immune protection efficacy and safety of the inactivated vaccine can be improved. Shown by the immune protection efficacy and safety tests, the porcine pseudorabies inactivated vaccine prepared has good immune protection efficacy and safety, and can be clinically used for preventing or treating porcine pseudorabies.
Description
Technical field
The present invention relates to the viral prevalence strain that a strain separates, relate in particular to the pseudorabies prevalence virus stain of strain separation and the swine pseudorabies vaccine for preparing with this strain, belong to separation and the Application Areas of porcine pseudorabies virus strain.
Background technology
Pseudorabies virus (Porcine pseudorabies virus, PRV) can cause multiple domestic animal and wildlife take heating, very itch (except the pig) and encephalomyelitis as the disease of cardinal symptom.Immunization is the main policies of anti-pseudoabies processed, and what at present application of China was many is to prepare attenuated vaccine with the Bartha-K61 strain.The appearance of rear clinical symptom although PRV genetically deficient attenuated vaccine can prevent infections effectively, Pigs Inoculated still may be infected by strong poison, infect can form hide, the virus of latent infection can be activated and cause and disseminate subsequently.
At present prepare the research emphasis that inactivated vaccine is porcine pseudorabies immunization field with epidemic isolates, the use of this type of inactivated vaccine is considered to prevent the effective ways of porcine pseudorabies, raising sow breeding potential, control piglet mortality ratio.
Mainly there is the problem of following several respects in existing PRV (Pseudorabies virus) inactivated vaccine: the one, and strain is aging, virus titer is not high; The 2nd, inactivation technology falls behind, and causes easily that inactivator is residual or deactivation is not thorough; The 3rd, use traditional adjuvant, cause the immune induction poor effect.The different strains of Pseudorabies virus there are differences at aspects such as virulence and biological properties, and the immunogenicity of deactivation vaccine is very relevant with the strain virulence.In addition, studies confirm that, inactivator and immunological adjuvant also are the important factors that affects PRV inactivated vaccine immune effect, and wherein, kind, ratio, dosage and the inactivation time etc. that inactivator and adjuvant use directly affect the immune effect of PRV (Pseudorabies virus) inactivated vaccine.
Summary of the invention
One of the object of the invention provides new porcine pseudorabies virus (the Porcine pseudorabies virus) strain that separates of a strain;
Two of the object of the invention is that described porcine pseudorabies virus strain is prepared into inactivated vaccine, is applied to prevention or the treatment of porcine pseudorabies disease;
Three of purpose of the present invention is that each processing parameter among the preparation method of inactivated vaccine is optimized screening to improve immune protection effectiveness or the security of inactivated vaccine.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Separation and obtain a strain PRV (Pseudorabies virus) BJ strain by adaptation of virus the tissues such as the brain of the present invention Mortality pig in Beijing morbidity pig farm 15 days, tonsilla; The present invention submits to the mechanism of patent approval to carry out preservation this BJ strain, and its microbial preservation number is: CGMCC No.7351; Classification And Nomenclature: porcine pseudorabies virus; The preservation time is: on March 11st, 2013; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
5～10 age in days PRV negative antibody piglets are inoculated in the PRV (Pseudorabies virus) BJ strain that the present invention separates, occur more than the fervescence to 41.5 ℃, significantly nervous symptoms and diarrhoea etc., 1～2d is dead fast, cut open the visible typical PRV of the dead pig of inspection and infect variation, and can from pathological tissues, be separated to PRV virus, illustrate that the BJ strain virulence that the present invention separates is strong.
The propagation of PRV (Pseudorabies virus) BJ strain on the cells such as ST cell, PK-15 cell, mdck cell, BHK-21 cell that the present invention separates is fast, and titre is high, and (virus titer is minimum to reach 10
7.5TCID50/mL), have good cell-proliferation activity, but induced animal produces high titre neutralizing antibody, has good immunogenicity.
The PRV (Pseudorabies virus) BJ strain that the present invention separates can with carry out efficient neutralization from 5 PRV of provinces and cities positive antibodies such as Beijing, Tianjin, Henan, Shandong or Guangxi, illustrate that BJ strain of the present invention has wider spectrotype.
The genetic analysis discovery, the PRV (Pseudorabies virus) BJ strain that the present invention separates can represent the Major Epidemic strain of Chinese PRV, 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 swine pseudorabies vaccine with the PRV (Pseudorabies virus) BJ strain that the present invention separates.
Two of purpose of the present invention is achieved through the following technical solutions:
A kind of method for preparing swine pseudorabies vaccine with PRV (Pseudorabies virus) BJ strain comprises:
(1) cultivates the PRV (Pseudorabies virus) BJ strain that separates, obtain virus liquid;
(2) in virus liquid, add inactivator, with the virus liquid deactivation;
(3) add adjuvant in the virus liquid after the deactivation, mix, emulsification, and get final product.
For improving immune protective effect or the security of inactivated vaccine, the present invention is optimized and screens the parameters such as kind, consumption and inactivation time of the used inactivator in the preparation inactivated vaccine.The present invention found through experiments, than other inactivator, adopt divinyl imines (BEI) as the inactivator of preparation inactivated vaccine, have that toxic side effect is little, a deactivation advantage more thoroughly, so the present invention preferably adopts divinyl imines (BEI) as inactivator.In the process of inactivation of viruses liquid, the final concentration of the divinyl imines that adds and inactivation time have the impact of highly significant for the immune protective effect of inactivated vaccine and security etc.; In order to obtain better inactivating efficacy to promote immune protection effectiveness and the security of inactivated vaccine, the present invention is optimized final concentration and the inactivation time of divinyl imines, the present invention finally finds by a large amount of optimization experiment, when the final concentration of BEI was 0.01%～1% (w/v) of virus liquid total amount, inactivating efficacy was better; The present invention finds very unexpectedly, and when the final concentration of BEI was 0.05% (w/v) of virus liquid total amount, not only deactivation was very thorough, and prepared inactivated vaccine product has best security simultaneously.
In addition, inactivation time also has the impact of highly significant for inactivating efficacy, and the present invention finds, add BEI in the virus liquid after deactivation 6～60h have certain inactivating efficacy; Can be with the thorough deactivation of virus when the present invention finds that by further experiment inactivation time is 48h.
The mode of the inactivation of viruses liquid described in the present invention is preferably: carry out inactivation of virus after adding BEI in the virus liquid under the mode of gas bath vibration, the temperature of described gas bath vibration is preferably 32 ℃, and the rotating speed of gas bath vibration is preferably l20r/min.
The present invention also finds, the virus liquid after the deactivation and the ratio of adjuvant also have certain impact for immune protective effect; The present invention finds by test, meter by volume, and when the ratio of the virus liquid after the deactivation and adjuvant is 85:15, the one, viscosity is just in time, inject than being easier to; The 2nd, vaccine that this ratio obtains, breakdown of emulsion, shelf time are not longer; can improve immune protection effectiveness; the effective quality of improving product; adjuvant described in the present invention is preferably the ISA15AVG adjuvant (product of France match BIC Corp; trade(brand)name: MontanideTM), can be directly used in vaccine emulsification preparation after this ISA15AVG adjuvant degerming.
The present invention further finds, the virus liquid after the deactivation through centrifugal and filtration, is added adjuvant again and carries out emulsification behind the filtering cell debris, can significantly improve immune protection effectiveness and the security of vaccine product.
The present invention finds by further test, and the emulsification program that can obtain the optimum emulsification effect is: (1) thaws same batch of virus liquid of deactivation and mixes, according to the volume proportion of water antigen and oil phase adjuvant 85:15; (2) 800～1000r/min continuously stirring 20～50min obtains the vaccine of oil-in-water formulation; (3) packing behind static 10～30min.
Immune protection effectiveness and safety testing show, the swine pseudorabies vaccine that separates the preparation of BJ strain with the present invention has good immune protection effectiveness and security, can be used for clinically prevention or the treatment of porcine pseudorabies.
Inactivated vaccine immunizing dose of the present invention is: for sheep, herd boar, sow or common pig, intramuscular injection 2mL/ head is once inoculated inactivated vaccine, and the terrain duration of immunity can reach more than 5～6 months.3 weeks of interval carry out immunoprophylaxis 2 times after the immunization for the first time, consolidate immune effect.
Description of drawings
(A is normal BHK-21 cell to the cultivation of Fig. 1 PRV virus BJ isolation of strains; B cytopathic cell occurs for the inoculation pathological material of disease).
(A is the cell of PRV virus stock solution used inoculation to the pathology situation that Fig. 2 different concns BJ strain venom inoculation BHK-21 cell occurs; B is the cell of 10-1 dilution PRV virus liquid inoculation; C is the cell of 10-2 dilution PRV virus liquid inoculation).
Fig. 3 PRV-BJ strain negative staining electron microscope morphological observation.
(A is for meeting malicious 24h result for Fig. 4 virus immunity fluoroscopy result; The negative contrast of B).
The phylogenetic analysis of Fig. 5 BJ strain and Reference strains gE gene; Wherein, gE_BJ is the PRV-BJ strain that the present invention separates; PRV-H-gE is the H strain described in the patent application publication number CN102344912A.
Embodiment
By the following example embodiments of the present invention will be described more specifically, it should be understood that described embodiment only is exemplary, does not consist of any restriction to scope of the present invention.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications or replace and all fall into protection scope of the present invention.Separation, the culture ﹠ 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 collection: aseptic collection is got the tissues such as the brain, tonsilla of fetal death of sow.Add MEM with 1:10, grind, the preparation tissue suspension, after 3 freeze thawing repeatedly, the centrifugal 15min of 2000r/min collects supernatant liquor, filters through 0.2 μ m filter membrane filter again, filtrate is put-40 ℃ of refrigerator Cryopreservations.
(2) separation and Culture: above-mentioned pathological material of disease is not yet formed the inoculation BHK-21 cell of individual layer by the 10% content access of virus-culturing fluid, 1h is made in 37 ℃ of senses, changes to add the MEM nutrient solution that contains 2% calf serum, cultivates 5d for 37 ℃.The s-generation is cultivated 96h, gathers in the crops toxic nutrient solution, after 2 freeze thawing, receives poison.Continuous passage is cultivated, observation of cell pathology (CPE), and the result shows: cell expands, the circle contracting, then beginning to come off forms the plaque focus gradually, and " drawing in the net " phenomenon (Fig. 1) is arranged.Through the Plaque Clone purifying, finally obtain a strain porcine pseudorabies poison strain, called after BJ strain.This BJ strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is: CGMCC No.7351.
(3) cultural characters: the BJ strain is fast breeding on the BHK-21 cell, and 24～36h CPE reaches (Fig. 2) more than 85%, and virus titer can reach 10
8.2More than the TCID50/mL; The BJ strain is growing up to fast breeding on the ST cell of individual layer, and 24～36h CPE reaches more than 85%, and virus titer can reach 10
7.8More than the TCID50/mL; The BJ strain is fast breeding on the PK15 cell, and 24～36hCPE reaches more than 85%, and virus titer can reach 10
7.5More than the TCID50/mL; BJ strain fast breeding on mdck cell, 24～36h CPE reaches more than 85%, and virus titer can reach 10
8.0More than the TCID50/mL.
2. the evaluation of PRV (Pseudorabies virus) BJ strain
(1) negative staining electron microscope is observed
Through cultivating 3～5d, occur more than the CPE85% on the BHK-21 cell, the centrifugal sick cell supernatant of 12000rpm carries out negative staining electron microscope to be observed.Result such as Fig. 3 show: negative staining electron microscope is observed virus particle and is ellipse or circular appearance, without the virus particle diameter 100～125nm of cyst membrane, with the mature virion diameter 175～190nm of cyst membrane.
(2) immuno-fluorescence assay
Get on the BJ strain inoculation ST passage cell of separation, at 5%CO
2Cultivate 28h under 37 ℃ of conditions, when CPE reaches 80%, pass the s-generation, select s-generation culture after 2 freeze thawing, do fluorescence antibody and detect.Get the ST cell cover glass culture that oneself cultivates 24h, virus inoculation is cultivated through 24h again.Take out cover glass, with the PBS flushing, fix through acetone, again with dyeing 30min in the wet box of PRV (Pseudorabies virus) fluorescence antibody, flushing, sealing, microscopy.The result shows: sample infects the ST passage cell, and cell has pathology, in nucleus tenuigenin, as seen is the specificity fluorescent of apple green, and negative control (Fig. 4) occur without specificity fluorescent.
(3) phylogenetic analysis of BJ strain
BJ strain gE gene nucleotide series homology is relatively used DNASTAR7.1 software BJ strain gE Gene Partial sequence and the derivation aminoacid sequence of measuring is carried out the homology comparative analysis with the same corresponding sequence of PRV strain isolated both at home and abroad, the result as shown in Figure 5, the PRV strain isolated BJ strain that the present invention separates and the genetic distance of HNJZ, LA strain and AF207700.1 strain are nearest.The PRV strain isolated BJ strain that the present invention separates and H strain (PRV-H-gE) genetic distance among the patent application publication number CN102344912A are far away, do not belong to same evolutionary branching.
(4) animal Orthogonal Rotational Regressive Tests
Inoculation 5～10 age in days PRV negative antibody piglets, obvious clinical symptom appears behind the injection 48h, main manifestations is more than the fervescence to 41.5 ℃, obvious nervous symptoms appears, instability of gait, eat breast and reduce, move inharmonious, four limbs are the shape of striking, the vomiting of part pig or diarrhoea, dead behind general 24～48h.Contrast pig non-evident sympton.Cut open the intensive needle-like of the visible kidney of inspection blutpunkte, meninx is obviously congested, and the myelencephalon liquid measure is too much, the substantial viscera Chang Kejian canescence necrotic lesions such as liver, spleen, and the pathological changes such as pulmonary congestion, oedema and downright bad point, and can from cerebral tissue, be separated to PRV virus.
The preparation of embodiment 2 PRV (Pseudorabies virus) BJ strain kind poison
1. basic bacteria is criticized foundation: the original seed culture of viruses of learning from else's experience and separating and identifying, access in the cell cultures such as ST cell, PK15 cell, mdck cell or BHK-21 cell that form individual layer by 1% of virus culture liquid measure, and put 5%CO
2In cultivate under 37 ℃ of conditions, observe CPE every day.Observations shows: cell expands, the circle contracting, and then beginning to come off forms the plaque focus gradually, and " drawing in the net " phenomenon is arranged, and when pathology reaches 80%, gathers in the crops toxic cell culture fluid, after 2 freeze thawing, receives poison.6 generations of continuous passage.
2. seeding is criticized foundation: get basic bacteria, access in the cell cultures such as ST cell, PK15 cell, mdck cell or BHK-21 cell that form individual layer by 1% of virus culture liquid measure, put 5%CO
2In cultivate under 37 ℃ of conditions, when pathology reaches 80%, gather in the crops toxic cell culture fluid, after 2 freeze thawing, receive poison.6 generations of continuous passage.
3. seed culture of viruses standard
(1) viral level
With the DMEM nutrient solution seed culture of viruses is made continuous 10 times of serial dilutions, each extent of dilution is got 100 μ L and is added in the 96 porocyte culture plates, and each extent of dilution is done 8 repetitions, joins the cell cultures such as the ST that grows up to individual layer or BHK, and every hole 100 μ L(cell contents are with 3 * 10
5/ mL), and establish normal cell and cultivate contrast, put 37 ℃, contain 5% CO
2Cultivate in the incubator, observe day by day CPE, the cell pyknosis is assembled, and particle increases, and some cytogamy, comes off, occurs more space and then be judged to CPE.Observe 7d, record cytopathic hole count, calculate the TCID50 of virus according to the Reed-Muench method.Comprehensive definite: every milliliter of virus liquid is not less than 10
7.5TCID50 is above to be qualified.
(2) virulence
With 6～18 monthly ages, without 3 of the sheep of pseudorabies virus neutralizing antibody or rabbits, every strong malicious 1mL of each intramuscular injection of sheep (contains 10
3.0LD50), rabbit (contains 10
1.0LD50), observe 14d.Should at least 2 Mortalities.
(3) immunogenicity
Seed culture of viruses being inoculated synchronously the cells such as ST breeds, the results virus liquid, after the BEI deactivation, add MontanideTM ISA15AVG adjuvant mixing and emulsifying and make the oil-in-water-type inactivated vaccine, with 6～18 monthly ages, without 7 of the sheep of pseudorabies virus neutralizing antibody, 4 each intramuscular injection 1mL, in addition 3 in contrast.Behind the inoculation 14d, every strong malicious 1mL of each intramuscular injection of sheep (contains 10
3.0LD50), observe 14d.3 Mortalities of contrast sheep, immune sheep is all protected.
(4) pure check
Bacterium, mould, mycoplasma check have been carried out in the PRV (Pseudorabies virus) BJ strain of setting up, and the 11st, 12 and 24 generation seeds culture of viruses have been carried out the check of exogenous virus, the result shows, strain of the present invention is criticized all and polluted without bacterium, mould, mycoplasma and exogenous virus, and is all pure.
(5) specificity
With DMEM liquid seed culture of viruses is diluted to 200TCID
50/ 0.1mL with the resisting pstudorabies poison specific serum equivalent mixing of deactivation, puts in 37 ℃ and 1h, and (cell content is with 3 * 10 to inoculate synchronously 3 bottles in well-grown ST cell
5/ mL), establish simultaneously virus control, normal cell contrast and negative serum contrast, put 37 ℃, contain 5% CO
2In the incubator, cultivate 5～7d.CPE does not all appear in neutralization virus group and normal cell control group; CPE all appears in virus control group and negative serum control group.
The preparation of embodiment 3 porcine pseudorabies viral disease deactivation vaccines
1. seedling is with the preparation of virus liquid: the PRV (Pseudorabies virus) BJ strain seed culture of viruses that embodiment 1 is separated is cultivated seeding with poison by the mode of embodiment 2,0.1% access of virus culture liquid measure is formed in the cell cultures such as ST cell, PK15 cell, mdck cell or BHK-21 cell of individual layer, put 37 ℃ of rotating and culturing, when pathology reaches 80%, gather in the crops toxic cell culture fluid, after 3 freeze thawing, receive poison.
2. deactivation: by the 0.05%(w/v of virus liquid total amount) in virus liquid, add 2%(w/v) the divinyl imide liquor, under 32 ℃, the condition of 120r/min gas bath vibration, then deactivation 48h adds 2% hypo solution, stop deactivation, and do steriling test.
3. concentrated: as to get the virus liquid after the deactivation, through the 4000r/min horizontal centrifugal, then filter with 0.2um filter membrane filter.Virus liquid behind the micro-filtration is with 5 times of 100KD filter membrane filter ultrafiltration and concentration.
4. the preparation of inactivated vaccine: same batch of virus liquid of deactivation thawed to be mixed, and then carries out emulsification according to water antigen inactivation of viruses liquid and oil phase adjuvant ISA15AVG with the volume proportion of 85:15.The emulsification program stirs for first water being put into emulsor, then slowly adds the oil phase adjuvant, with 800～1000r/min continuously stirring, 20～50min, makes oil-in-water formulation vaccine, packing behind static 10～30min.
5. inspection of semifinished product quality standard
(1) steriling test
Test by existing " Chinese veterinary pharmacopoeia " appendix, answer asepsis growth.
(2) viral level is measured
With the DMEM nutrient solution seed culture of viruses is made continuous 10 times of serial dilutions, each extent of dilution is got 100 μ l and is added in the 96 porocyte culture plates, and each extent of dilution is done 8 repetitions, adds subsequently individual layer ST cell suspension, and establishes normal cell and cultivate contrast, puts 37 ℃, contains 5% CO
2Cultivate in the incubator, by a day observation of cell pathology (CPE), cell pyknosis, assemble, particle increases, and some cytogamy, comes off, occurs more space and then be judged to infection.Record cytopathic hole count, calculate the TCID50 of virus according to the Reed-Muench method, every milliliter of virus liquid of result is not less than 10
7.5TCID50.
(3) deactivation check
Get the virus liquid after the deactivation, be inoculated in the ST cell in the ratio of virus liquid and growth media 1:10, cultivate for 37 ℃ and observe 5d, to without pathology person 2 generations of blind passage again, establish simultaneously virus control.The acellular pathology of result.3 batches of work in-process of Laboratory Production are all qualified.
6. about the inspection after construction quality standard
(1) safety verification standard
In order to guarantee the security of vaccine, the present invention has successively carried out " to the safety testing of small white mouse ", " to the safety testing of the inoculation of piglet single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) inoculation ", " to the safety testing of the inoculation of replacement gilt single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) inoculation ", " to the safety testing of the inoculation of pregnant sow single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) inoculation " to 3 batches of vaccines of laboratory preparation.Test-results shows: after each immune animal single dose inoculation, single dose repeated inoculation, the disposable heavy dose of inoculation, the pig state is all good, and the drinking-water of searching for food is normal, without abnormal response; Breeding function to pregnant sow after the vaccine inoculation also has no significant effect.Above test proves that all the prepared inactivated vaccine of the present invention is safe.
Find by experimental observation, inoculate behind the inactivated vaccine of the present invention and the phenomenons such as part redness, ulcer, erosion and neuratorphy, the minimizing of searching for food do not occur in the 21d, prove that vaccine of the present invention has good security.
The present invention has further carried out following safety testing: with healthy susceptible piglet (the PRV NAT is not higher than 1:2) 2 of 10～20kg, each deep intramuscular injection inactivated vaccine 4mL of the present invention, other gets 2 identical pigs of condition and does not inoculate in contrast, the any part or the systemic adverse reactions that are caused by vaccination do not appear in Continuous Observation 21d within the observation period.
(2) formulation of efficacy test standard
Seed culture of viruses being inoculated the cells such as ST breeds, the results virus liquid, after the BEI deactivation, add MontanideTMISA15AVG adjuvant mixing and emulsifying and make the oil-in-water-type inactivated vaccine, with 6～18 monthly ages, without 7 of the sheep of pseudorabies virus neutralizing antibody, 4 each intramuscular injection 1mL, in addition 3 in contrast.Behind the inoculation 14d, every strong malicious 1mL of each intramuscular injection BJ of sheep (contains 10
3.0LD50), observe 14d.3 Mortalities of contrast sheep, immune sheep is all protected.
The process optimization test of test example 1 PRV (Pseudorabies virus) inactivated vaccine
(1) divinyl imines (BEI) and formalin-inactivated Comparision Test
Selecting final concentration is 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, the formaldehyde solution of 1% (w/v) and final concentration are 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, the BEI of 0.2%, 1% (w/v) carries out the deactivation comparison test to PRV (Pseudorabies virus), and test-results shows, 0.05% BEI can be with the PRV (Pseudorabies virus) complete inactivation behind 48h under 32 ℃ of conditions, and test-results 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 through 37 ℃ of deactivation 48h, but formaldehyde has strong and stimulating, if residual free formaldehyde enters animal body in the vaccine, can produce untoward reaction.
(2) divinyl imines (BEI) using dosage and concentration are determined
Adopting respectively final concentration is 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.08%, 0.1%, the BEI of 0.2%, 1% (w/v) carries out deactivation to virus liquid, under 32 ℃, the condition of 120r/min gas bath vibration, deactivation 48h, the product that obtains after the deactivation carry out mice infection test and small white mouse safety testing.Test-results sees Table 1 and table 2.
The infection Experiment on white mice result of table 1 different B EI concentration deactivation PRV
The small white mouse safety testing of table 2 different B EI concentration deactivation PRV
According to the test-results of table 1 and table 2 as seen, adopting final concentration is that 0.05% BEI carries out deactivation to virus liquid, and not only deactivation is thorough, and prepared inactivated vaccine has best security; Therefore, the present invention particularly preferably carries out deactivation with the BEI of final concentration 0.05% (w/v) to virus liquid.
(3) divinyl imines (BEI) is determined action time
Be that the BEI of 0.05% (w/v) is under 32 ℃, the condition of 120r/min gas bath vibration with final concentration, do respectively the deactivation of the different times such as 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 54h, 60h, do the test of the infectious virus inspection of inactivation of viruses liquid cell culture and mice infection after the deactivation.Test-results sees Table 3.
Table 3PRV inactivation test
According to test-results, final determine to have best inactivating efficacy with the BEI solution of the final concentration 0.05% prepared inactivated vaccine of deactivation 48h under 32 ℃, the condition of 120r/min gas bath vibration.
(4) divinyl imines (BEI) toxicity test
Set 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, the isocyatic BEI solution of 1% (w/v), the injection body weight is 18～22g small white mouse, observes 10d, record death toll and survival number.Determine that 0.05%BEI0.4mL is safe dose.
To sum up, the present invention finally determines divinyl imide liquor with final concentration in the virus liquid 0.05% at 32 ℃, 120r/min, and then deactivation 48h porcine pseudorabies venom under the condition of gas bath vibration adds 2% hypo solution, stops deactivation.
The protection potency test of test example 2 PRV (Pseudorabies virus) inactivated vaccines
One, experimental vaccine
1, produce inactivated vaccine for examination inactivated vaccine: embodiment 3 described methods, specific as follows:
(1) seedling is with the preparation of virus liquid: PRV (Pseudorabies virus) BJ strain seed culture of viruses is cultivated seeding with poison by the mode of embodiment 2,0.1% access of virus culture liquid measure is formed in the ST cell culture of individual layer, put 37 ℃ of rotating and culturing, when pathology reaches 80%, gather in the crops toxic cell culture fluid, after 3 freeze thawing, receive poison.
(2) divinyl imide liquor deactivation: add 2%(w/v in porcine pseudorabies virus liquid) to its final concentration is 0.05%(w/v), under 32 ℃, the condition of 120r/min gas bath vibration, then deactivation 48h adds 2% hypo solution, stops deactivation;
(3) concentrated: as to get the virus liquid after the deactivation, through the 4000r/min horizontal centrifugal, then filter with 0.2um filter membrane filter.Virus liquid behind the micro-filtration is with 5 times of 100KD filter membrane filter ultrafiltration and concentration;
(4) preparation of inactivated vaccine: the virus liquid of deactivation thawed to be mixed, and then carries out emulsification according to water antigen inactivation of viruses liquid and oil phase adjuvant ISA15AVG with the volume proportion of 85:15; The emulsification program stirs for first water being put into emulsor, then slowly adds the oil phase adjuvant, with 9000r/min continuously stirring 40min, makes oil-in-water formulation vaccine, static 20min.
2, contrast inactivated vaccine: used strain is that (its microbial preservation number is: CGMCC No.5013) in disclosed H strain among the patent application publication number CN102344912A; Different except strain, the preparation method of contrast inactivated vaccine is with identical for the preparation method of examination inactivated vaccine.
Two, test method
(1) immune protective efficiency of pig is tested
40 pigs are divided into two groups at random, namely test 1 group and the test 2 groups, every group of 20 pigs; Test 1 group with for the inoculation of examination inactivated vaccine, test 2 groups and use the inoculation of contrast inactivated vaccine; Immunizing dose is pig muscle injection 2mL/ head, once inoculates inactivated vaccine; 6 months latter two test group of immunization are all attacked with the PRV (Pseudorabies virus) virulent strain of same dose, and the immunoprotection force data sees Table 3.
The protection that table 3 inactivated vaccine is attacked PRV (Pseudorabies virus) intensity
From test-results as seen, the prepared inactivated vaccine of the present invention has good immune protective effect for pig, and the immune protection effectiveness of inactivated vaccine of the present invention will significantly be better than contrasting inactivated vaccine to the immune protection effectiveness of pig.
(2) immune protective efficiency of sheep is tested
With 6～18 monthly ages, be divided at random 3 groups without 24 sheep of pseudorabies virus neutralizing antibody, that is: test 1 group, test 2 groups and control group, 8 every group; Test 1 group of intramuscular injection 1mL for the examination inactivated vaccine, test 2 groups of intramuscular injection 1mL contrast inactivated vaccines, control group does not give vaccination; Behind the inoculation 14d, every strong malicious 1mL of each intramuscular injection PRV (Pseudorabies virus) of sheep (contains 10
3.0LD50), observe 14d.Found that, 8 sheep testing 1 group all obtain protection, and 5 sheep testing 1 group obtain protection, 3 Mortalities; 7 sheep Mortalities of control group.
The safety testing of test example 3 PRV (Pseudorabies virus) inactivated vaccines
Embodiment 3 prepared inactivated vaccines are inoculated the 10mL/ head with use dose inoculation 2mL/ head, overdose, and inoculation replacement gilt, pregnant pig have no immune has any impact to reproductive function, and body temperature, feed, breeding, gestation, farrowing are all normal.Experiment results proved, inactivated vaccine security of the present invention is good.
Claims (10)
1. a strain PRV (Pseudorabies virus) (Porcine pseudorabies virus) BJ strain is characterized in that, its microbial preservation number is: CGMCC No.7351.
2. the purposes of PRV (Pseudorabies virus) BJ strain claimed in claim 1 in the vaccine of preparation prevention or treatment porcine pseudorabies.
3. an inactivated vaccine that prevents or treat porcine pseudorabies is characterized in that, comprising: inactivation of virus liquid and the adjuvant of the described PRV (Pseudorabies virus) BJ strain preparation of claim 1.
4. the preparation method of a swine pseudorabies vaccine is characterized in that, may further comprise the steps:
(1) cultivates PRV (Pseudorabies virus) BJ strain claimed in claim 1, obtain virus liquid;
(2) in virus liquid, add inactivator, inactivation of viruses liquid;
(3) add adjuvant in the virus liquid after the deactivation, mix, emulsification, and get final product.
5. it is characterized in that in accordance with the method for claim 4: the inactivator that adds in the virus liquid in the step (2) is the divinyl imines; By w/v, adding divinyl imines to its final concentration in the virus liquid is 0.01%～1% of virus liquid total amount.
6. it is characterized in that in accordance with the method for claim 5: adding divinyl imines to its final concentration in the virus liquid in the step (2) is 0.05% of virus liquid total amount.
7. in accordance with the method for claim 4, it is characterized in that: the inactivation time described in the step (2) is 6～60 hours, is preferably 48 hours.
8. it is characterized in that in accordance with the method for claim 4: step (2) is carried out inactivation of virus after adding inactivator in the virus liquid under the mode of gas bath vibration; The temperature of described gas bath vibration is 32 ℃, and the rotating speed of gas bath vibration is l20r/min.
9. in accordance with the method for claim 4, it is characterized in that: count by volume, the virus liquid in the step (3) after the deactivation and the ratio of adjuvant are 85:15.
10. the inactivated vaccine of the prevention that is prepared by each method of claim 4～9 or treatment porcine pseudorabies.
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