CN106497890A - A kind of 1 plant of porcine pseudorabies virus variant XF and preparation method and application - Google Patents
A kind of 1 plant of porcine pseudorabies virus variant XF and preparation method and application Download PDFInfo
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Abstract
The present invention relates to PRV (Pseudorabies virus) technical field, a kind of 1 plant of porcine pseudorabies virus variant XF and preparation method and application, the deposit number of described variant is:CCTCC NO V201654.The application adopts porcine pseudorabies virus variant（1 plant of XF）Being prepared into inactivated vaccine carries out safety and immune protective test, as a result shows, porcine pseudorabies virus variant（1 plant of XF）Inactivated vaccine safety is good, and immunoprotection efficiency is also apparently higher than other immune group, it was demonstrated that the Strain has preferable immunogenicity, can be used for the sick vaccine research and exploitation, with good vaccine development prospect.
Description
Technical field
The invention mainly relates to swine pseudorabies vaccine technical field, more particularly to a kind of porcine pseudorabies virus change
Different strain XF-1 strains and preparation method and application.
Background technology
Pseudorabies be by Pseudorabies viruses (Pseudorabies virus, PRV) cause including multiple domestic animals and open country
A kind of acute infectious disease that lively thing is suffered from altogether.Pig is the natural host of primary disease virus and storage person, and primary disease is itched to generate heat, very, brain
Myelitis and serious breeding difficulty are principal character, and national many provinces have used gE gene deleted live vaccines since 2011
The large-scale pig farm sow of immunity occurs producing weak pigle, stillborn fetuses, mummy, miscarriage；Returning often occur in replacement gilt and nonpregnant sows
The situation of feelings, Repeat breeding or non-estrus；Often there is atrophy of testis after boar infection, sexual disorder loses kind ability；Newly
There are the clinical symptoms of the doubtful pseudorabies such as nervous symptoms and death in piglet；Growing and fattening pigs show as respiratory symptom and weightening is stagnant
Slow, larger economic loss is caused to pig industry.Therefore, whether the current pseudorabies epidemic isolates of more suspection morph, from
And cause vaccine shielding failure.This conjecture is subsequently also obtained confirmation.Central China agricultural university, Chinese agricultural university, Nanjing agricultural university, China are dynamic
Thing Blight control center etc. shows that by multinomial experimentation kainogenesis pseudorabies are caused by new strain.Central China agriculture
Sparetime university learns animal epidemic diagnostic center and detached pseudorabies open country poison in national each province morbidity swine diseasess material is carried out gene sequencing, ties
Fruit shows, currently detached epidemic isolates there occurs the significant mutation of multiple bases and continuous in genes such as TK, gB, gE, gC
Property disappearance, these mutation and disappearance are likely to be a major reason for causing current pseudorabies popular again.Current commodity
The antibody for changing vaccine immunity generation is poor to the neutralising capacity of currently a popular strain.The appearance of variation strain causes Adult Pig also table
Reveal the serious symptoms similar to piglet, pig farm sow, piglet, growing and fattening pigs mortality propose new difficulty to the sick prevention and control
Topic, also results in serious economic loss to current pig industry.
It is vaccination to prevent the most effective method of the disease.At present, commercially available pseudorabies disease vaccine is most absolutely
Number is porcine pseudorabies routine inactivated vaccine and attenuated vaccine.But as epidemic isolates constantly morph, exist with commercial available vaccines
Have differences in terms of immunogenicity, the vaccine for causing traditional strain to prepare can not the current prevalence of prevention and control well pseudorabies
Disease.Accordingly, it would be desirable to research and develop the vaccine for the Pseudorabies viruses that make a variation at present, it is to solve currently a popular porcine pseudorabies most
Good approach.
Content of the invention
Object of the present invention is to provide a kind of porcine pseudorabies virus variant XF-1 strains, the strain is in 2016
October 19 delivered to China typical culture collection center preservation, and deposit number is：CCTCC NO:V201654, Classification And Nomenclature：
Porcine pseudorabies virus XF-1 strains.Address：Wuhan, China Wuhan University.
Further object is that there is provided a kind of preparation method of porcine pseudorabies virus variant XF-1 strains,
Method is simple, easy, it is adaptable to large-scale production.
Final object of the present invention there are provided a kind of application of porcine pseudorabies virus variant XF-1 strains.
In order to achieve the above object, the present invention takes following technical measures：
A kind of porcine pseudorabies virus variant XF-1 strains, applicant is from Henan Xinxiang City Fengqiu County pig farm morbidity Medulla sus domestica
Separate tissue, obtains one plant of wild poison parent plant, is named as:XF strains, are analyzed by gene sequencing, as a result show the strain in gE/
The genes such as gC/gB there occurs that the significant mutation of multiple bases and seriality disappearance, these mutation and disappearance cause the poison of strain
Power and antigen change.The gE Gene Partials of wild poison parent plant XF strains are lacked by applicant using the method for homologous recombination, so
The recombinant viruses of gE gene delection are obtained with reference to PCR amplifications by plaque screening purification afterwards.Will be direct for the recombinant viruses liquid of purification
Immune gB-ELISA and gE-ELISA antibody is negative piglet, and after immunity 28 days, detection gE-ELISA antibody remains as the moon
Property.So as to, it is further characterized by obtaining pure gE gene-deleted strains, applicant is by the porcine pseudorabies street strain XF strains that make a variation
Strain after gE gene delections is named as porcine pseudorabies virus XF-1 strains (present invention or referred to as PRV XF-1 strains).The strain
China typical culture collection center preservation is delivered on October 19th, 2016, deposit number is：CCTCC NO:V201654,
Classification And Nomenclature：Porcine pseudorabies virus XF-1 strains.Address：Wuhan, China Wuhan University.
A kind of preparation method of porcine pseudorabies virus variant XF-1 strain virus liquid, including：
By XF-1 strains (virus titer >=108.0TCID50/ml) BHK-21 cell suspension is inoculated in.Virus inoculation amount is
1.0%～3% (v/v), controls 36 DEG C～37 DEG C of cultivation temperature, pH6.0～7.0, speed of agitator 80rpm～100rpm, dissolving
Oxygen concentration 40%～60%, just obtains porcine pseudorabies venom when reaching 85% to cytopathy.Cultured XF-1 strains are led to
Cross 0.65 μm of doughnut and remove cell debriss, then obtained final product finally by gel chromatography column purification by eluting, concentration again
The XF-1 strain virus stock solutions of the Pseudorabies viruses antigen of purification, i.e. purification.
Approach described above, it is preferred that the preparation method of BHK-21 cell suspension includes：
It is 4.0-6.0 × 10 by 1000ml concentration6The BHK-21 cell suspension of individual/ml, is inoculated in 10L bioreactors
In, BHK culture medium 8L containing 1%～3% (v/v) new-born calf serum is added, 36 DEG C～37 DEG C of cell culture temperature, pH is controlled
6.5～7.5, speed of agitator 80rpm～100rpm, dissolved oxygen concentration 40%～60% carry out suspension culture 2～4 days, to cell
Density reaches 3.0～6.0 × 106Individual/ml, to obtain BHK-21 cell suspension.By above-mentioned acquisition BHK-21 cell suspension, connect
Plant in 100L bioreactors, add BHK culture medium 80L containing 1%～3% (v/v) new-born calf serum, control cell culture
36 DEG C～37 DEG C of temperature, pH 6.5～7.5, speed of agitator 80rpm～100rpm, dissolved oxygen concentration 40%～60% are hanged
Floating culture 2～4 days, reaches 3.0～6.0 × 10 to cell density6Individual/ml, to obtain BHK-21 cell suspending liquids.
A kind of application of porcine pseudorabies virus variant XF-1 strains, including the usual manner using this area by XF-1 strains
It is prepared into porcine pseudorabies virus inactivated vaccine；Or XF-1 strains are combined with other strains, it is prepared into multiple vaccines.
In approach described above, it is preferred that vaccine adjuvant is the acceptable aqueous adjuvants of veterinary, including but not limited to aluminum
Salt series of adjuvants, Montanide IMS series of adjuvants or Montanide GEL series of adjuvants, propolis, immunostimulating complex,
Cytokine class adjuvant, nucleic acid and its derivatives class adjuvant, lecithin lipid adjuvant.The Montanide IMS series of adjuvants bags
Include 1313VG, 251C VG, 2215VG；The Montanide GEL series of adjuvants is GEL 01PR or Montanide PET
GEL A；The oil-in-water series of adjuvants includes MF59, Montanide ISA15A VG etc.；The cytokine class adjuvant bag
Include interleukin (IL-1, IL-2, IL-4, IL-12), interferon (IFN-γ, IFN-α, IFN-β) etc.；The nucleic acid and its derivative
Species adjuvant includes immunostimulatory sequence DNA (CpG DNA) or CpG oligodeoxynucleotide etc..
In the above scheme, it is preferred that the vaccine is oil-in-water inactivated vaccine, and adjuvant used by vaccine is IMS
1313VG.
The bivalent inactivated vaccine adjuvant optimal way of the present invention is related to IMS1313N VG, IMS2215VG, Gel01, card
The compositionss of one or more of ripple nurse, aluminium hydroxide gel.
Compared with prior art, the present invention has advantages below：
(1) oil-in-water type inactivated vaccine is prepared into using porcine pseudorabies virus variant (XF-1 strains) after purification, with
Strain Ea inactivated vaccine, Bartha-K61 live vaccine, HB-98 strains live vaccine carry out pig body Immunoprotection test, as a result show, pig
Pseudorabies viruses variant inactivated vaccine (XF-1 strains), not only safety is good, and protective efficacy is bright in immuning effect test
Aobvious is higher than other immune group.Prove that the Strain has preferable immunogenicity, can be used for the sick vaccine research and exploitation.
(2) strain for combining the present invention using French import MONTANIDE IMS 1313VG adjuvants is prepared into pseudorabies
Inactivated vaccine, to pig injecting immune during, the stress of pig body is little, therefore, the present invention vaccine combination safety
More preferably, the untoward reaction that multiple immunoprophylaxis can be avoided to occur.The vaccine that the aqueous adjuvants are prepared has unique stability,
Place at 4 DEG C, 25 DEG C and 37 DEG C stable all than other vaccines；The vaccine that the aqueous adjuvants are prepared is without side reaction, injection site
Without granuloma or inflammatory reaction；Vaccine immunity protection period that the aqueous adjuvants are prepared is longer and antibody horizontal is compared with other adjuvants
The vaccine of preparation is high；
(3) bivalent inactivated vaccine that the present invention is provided confirms that XF-1 strains can be prepared with antigen known to other this research fields
Combined vaccine into two or more disease.Its convenience main, multiple-effect, low cost become the direction of current vaccine research.
Compared with single vaccine, combined vaccine can reduce cost of labor, reduce the inoculation times of vaccine, reduce pig because vaccine immunity is produced
Raw stress.
(4) bigeminy vaccine that the present invention is provided changes people to porcine circovirus 2 type and PRV (Pseudorabies virus) while sense
Porcine circovirus 2 type and PRV (Pseudorabies virus) antigen are used in combination according to suitable ratio and are taken by understanding prejudice during dye first
Obtain good result.
(5) tradition culture process before porcine pseudorabies virus variant (XF-1 strains) changes.Suspension culture is all adopted
Technique, removes cell debriss by the hollow fibre filtering in 0.65 μm of aperture, then after suspension culture high concentration antigen again
Eluting, concentration again, finally by gel filtration chromatography purification, obtains high-load purifying antigen.
(6) porcine pseudorabies virus of the invention and porcine circovirus 2 type antigen composition and prepared using said composition
Vaccine, prevent and treat the infection of porcine pseudorabies and Porcine circovirus desease, in prevention and treatment porcine circovirus 2 type disease
And the application of porcine circovirus 2 type subclinical infection, porcine circovirus 2 type subclinical infection can be effectively prevented to pig circular ring virus 2
The transformation of malicious 2 type clinical infections, contains spreading for the state of an illness.
(7) vaccine that this invention is prepared using aqueous adjuvants compositionss, the specific antibody produced after immunity are very fast
And high, and can induction body fluid immunity simultaneously and cellular immunization.
(8) porcine pseudorabies are simple with porcine circovirus 2 type bivalent inactivated vaccine preparation method, and vaccine valence content is high,
Immunity is convenient and swift, with multiple immunity of the prior art, at least needs to play 2 pins or 3 pins could prevent and treat above two kinds of diseases
Vaccine and its immunization method are compared, and the present invention only immunity can just prevent porcine pseudorabies and Porcine circovirus desease infection for 1 time.Two
Connection inactivated vaccine reduces immune cost, reduce immune programme for children simultaneously minimizing pig because immunity cause stress.
Description of the drawings
Fig. 1 is dead Carnis Sus domestica oculopathy change observation result after 2 counteracting toxic substances of embodiment control piglet counteracting toxic substances；
Wherein, a is lungs；B is brain；C is liver；D is spleen；E is kidney.
Fig. 2 is gB-ELISA average antibody Changing Pattern schematic diagrams after 3 different pseudorabies vaccine immunities of embodiment.
Fig. 3 is 7 different porcine pseudorabies virus vaccine gB-ELISA average antibody Changing Patterns of embodiment.
Fig. 4 is 9 different pig circular ring virus vaccine average antibody Changing Patterns of embodiment.
Specific embodiment
" per part " of the present invention or "/head part " refer to every pig vaccine dose used every time.It is not specifically noted it
Place, described in embodiments of the present invention " per part " or "/head part " is 2ml.
In technical scheme of the present invention, if not otherwise specified, the ordinary skill in the art is；The reagent or material
Material, if not otherwise specified, is purchased from commercial channel.
Mulser：Germany, model：IKA companies RW20D models
MONTANIDE IMS 1313VG adjuvants are purchased from SIPPEC companies of France, standby with 0.22 zut filter.
Embodiment 1：
Porcine pseudorabies virus (XF-1 strains) liquid and the preparation of vaccine：
1st, bioreactor suspension culture BHK-21 cell is used：
The BHK-21 seed cell 5ml of Liquid nitrogen storage are taken, 37 DEG C of water-baths are melted rapidly, 1000rpm is centrifuged 5 minutes, discards
Supernatant, resuspended with BHK culture medium 100ml containing the low serum of 2% (v/v), it is transferred in 250ml shaking flasks, in 37 DEG C of constant temperature trainings
Foster shaking table culture, rotating speed are to carry out suspension culture 2 days under 80～100rpm/min, reach 2.0～4.0 × 10 to cell density6
During individual/ml, according to 1：4 ratio, in 37 DEG C of temperature, carries out transferred species culture 2～3 days, cell is expanded under 80～100rpm rotating speeds
1000ml volumes are increased to, makes cell density reach 4.0～6.0 × 106Individual/ml, to obtain BHK-21 cell suspension.
The 1000ml BHK-21 cell suspension of above-mentioned acquisition is inoculated in 10L bioreactors, add and contain 2% (v/
V) BHK culture medium 8L of new-born calf serum, controls 36.5 DEG C of cell culture temperature, pH 7.2, speed of agitator 80rpm, dissolved oxygen
Concentration 45%, carries out suspension culture 4 days, reaches 3.4 × 10 to cell density6Individual/ml, to obtain BHK-21 cell suspension.
Above-mentioned acquisition BHK-21 cell suspension is inoculated in 100L bioreactors, add the BHK containing 2% (v/v) new-born calf serum
Culture medium 80L, controls 36.5 DEG C of cell culture temperature, and pH7.15, speed of agitator 100rpm, dissolved oxygen concentration 45% are hanged
Floating culture 3 days, reaches 4.5 × 10 to cell density6Individual/ml, to obtain BHK-21 cell suspension.Wherein, the BHK for being adopted
Culture medium is believed purchased from Beijing day and bio tech ltd.
2nd, the culture of porcine pseudorabies virus (XF-1 strains)
By XF-1 strains (virus titer >=108.0TCID50/ml) it is inoculated in bioreactor, virus inoculation amount is 2.0%
(v/v), 37 DEG C of cultivation temperature is controlled, and pH 7.2, speed of agitator 90rpm, dissolved oxygen concentration 45% carry out Virus culture 2 days, this
When cytopathy reach 85%, just obtain porcine pseudorabies virus liquid.
3rd, the purification of porcine pseudorabies virus (XF-1 strains)
Cultured porcine pseudorabies virus (XF-1 strains) are removed cell debriss, Ran Houzai by 0.65 μm of doughnut
By eluting, concentration, final gel column chromatography obtains the antigen of purification.Antigen poison valency after purification is made to exist
107.5More than TCID50/ml, after adding final concentration of 0.4% 37 DEG C of inactivation 48h of formalin, by existing《Chinese veterinary drug
Allusion quotation》Annex test qualified after, 4 DEG C of storages are standby.
4th, the preparation of swine pseudorabies vaccine (XF-1 strains)
The porcine pseudorabies virus (XF-1 strains) that step 3 is obtained are with IMS 1313VG adjuvants according to mass ratio 5:1 is carried out
Emulsifying mixes 30min, subpackage, inspection after the completion of emulsifying.Inspection according to《Chinese veterinary pharmacopoeia》Annex is carried out, through after the assay was approved
4 DEG C save backup, and obtain final product swine pseudorabies vaccine (XF-1 strains), and in the final product per 2ml, porcine pseudorabies virus has
Effect viral level is 107.5TCID50, every pig immunity 2ml every time.For following examples.
Embodiment 2：
The Study On Immunogenicity of swine pseudorabies vaccine (XF-1 strains)
1st, materials and methods
The piglet of Wuhan pig farm 21-25 ages in days is bought in 1.1 experimental animal markets, and main pathogen and associated antibodies are carried out
Detection, it is negative and pseudorabies to select to porcine circovirus 2 type, swine fever virus, porcine reproductive and respiratory syndrome virus cause of disease
Neutralizing antibody is negative, and (PRV NATs are not higher than 1:2) piglet, 30 altogether.
1.2 counteracting toxic substances strain PRV (Pseudorabies virus) street strains (XF strains) derive from Wuhan Ke Qian Biological Co., Ltd..
Test pig is randomly divided into 6 groups by 1.3 methods, and 5 per group, each group situation is as follows：
1st group is swine pseudorabies vaccine (XF-1 strains), i.e., from the vaccine of the preparation of embodiment 1；
2nd group be import live vaccine (Bartha-K61 strains), lot number：77BN, market purchase Spain Hai Bolai are biological big
Pharmaceutical factory；
3rd group be swine pseudorabies vaccine (Strain Ea), lot number：150601；
4th group be pseudorabies disease live-vaccine (HB98 strains), lot number：150612；
5th group is counteracting toxic substances matched group；
6th group is blank control group.
Wherein, the 3rd group and the 4th group of vaccine are the offer of Wuhan Ke Qian Biological Co., Ltd..All piglets are according to 1
Head part/head carries out immunity.After immunity 21 days, carry out two and exempt from.Take a blood sample after exempting from 14 days two, separate serum, be neutralized index
Determine.And all piglets are carried out with collunarium counteracting toxic substances, counteracting toxic substances dosage 1ml/ heads, counteracting toxic substances strain is PRV (Pseudorabies virus) street strain (XF
Strain), viral titer is 107.0TCID50/ml.Isolated rearing after counteracting toxic substances, free choice feeding.Daily observation experiment pig disease symptom is (such as
Lethargy, inappetence, heating, nervous symptoms, respiratory symptom and death etc.), to gathering the heart after dead pig Systematic anatomy
Dirty, liver, spleen, lungs, kidney, lymph node and cerebral tissue.Observation 14 days simultaneously records body temperature, respiratory system, digestive system, god
Clinical response through system.Counteracting toxic substances result is drawn according to piglet morbidity criterion.
Piglet morbidity criterion after 1.4 counteracting toxic substances
(1) breathing or digestive system clinical symptoms sneeze, nose have secretions, dyspnea；Have loose bowels, vomit.
(2) body temperature reaction body temperature >=40.5 DEG C, at least continue 2 days.
Nervous system clinical symptoms spasm, sialorrhea, fall down to the ground strike, the typical clinical symptom such as extreme agitation
(4) dead
Meet above (3rd), (4th) item or while meeting (1st) (2nd) item, you can judge morbidity.
2nd, result of the test
After 2.1 immunity, antibody horizontal is determined as shown in Table 1, and after exempting from two, PRV (Pseudorabies virus) variant (XF-1 strains) gB resists
Body and neutralizing antibody are apparently higher than other immune group, and all immune group of gE antibody are all negative.Nonimmune group of gB, gE, neutralization
Antibody is all negative.
After 2.2 counteracting toxic substances, clinical symptoms as shown in Table 1, are all fallen ill for the 5th group after counteracting toxic substances, wherein dead 4, and immune group
In in addition to the 1st group other each groups have morbidity performance.Wherein, the 2nd group 2, the 3rd group 2, the 4th group of 1 experiment pig appearance heating
(body temperature >=40.5 DEG C), spirit are depressed, cough, dyspnea, the clinical manifestation such as nervous symptoms and death, and the 1st group of experiment pig
The mental status is good, does not show any abnormalities phenomenon, and counteracting toxic substances protective rate is up to 100%.
After 2.3 counteracting toxic substances, dead pig dissects symptom (see Fig. 1)
Protest test result after 1 different strain immunity of table
Note：* represent that each vaccine strain body temperature after counteracting toxic substances meets or exceeds the percentage ratio of 40.5 DEG C of natural law
PRV (Pseudorabies virus) variant XF-1 strains neutralizing antibody highest as can be seen from the test results, next to that import
Bartha-K61 strain attenuated vaccines.From in terms of counteracting toxic substances protective rate, PRV (Pseudorabies virus) variant XF-1 strains are also highest.Therefore, may be used
To illustrate that PRV (Pseudorabies virus) variant XF-1 strains are more highly resistant to for current pig farm outburst variation porcine pseudorabies.
Embodiment 3：
Several different porcine pseudorabies vaccine antibody Fluctuation contrast tests
1st, test material and method
The piglet of Wuhan pig farm 21-25 ages in days is bought in 1.1 experimental animal markets, and main pathogen and associated antibodies are carried out
Detection, it is negative and pseudorabies to select to porcine circovirus 2 type, swine fever virus, porcine reproductive and respiratory syndrome virus cause of disease
Neutralizing antibody is negative, and (PRV NATs are not higher than 1:2) piglet, 25 altogether.
1.2 porcine pseudorabies specific positive serum neutralization titers are 1:256, by Wuhan Ke Qian Biological Co., Ltd.
There is provided.
Test pig is randomly divided into 5 groups by 1.3 test methods, 5 per group, and packet situation is as follows：
First group is vaccine of the swine pseudorabies vaccine (XF-1 strains) from the preparation of embodiment 1；
Second group be import live vaccine (Bartha-K61 strains), lot number：77BN markets purchase Spain Hai Bolai is biological big
Pharmaceutical factory；
3rd group be swine pseudorabies vaccine (Strain Ea), lot number：150601；
4th group be pseudorabies live vaccine (HB98 strains), lot number：150612；
5th group is blank control group.
All piglets carry out immunity according to 1 part/head.After immunity 21 days, carry out two and exempt from.After immunity, 14 days respectively,
Take a blood sample within 28 days, 42 days, 56 days, 90 days, separate serum.Detection pseudorabies gB antibody and detection porcine pseudorabies neutralizing antibody effect
Valency, calculates and turns positive rate, while each immune group of detection is in the average neutralizing antibody of different immunization times.
2nd, result of the test
2.1st, porcine pseudorabies vaccine antibody Fluctuation comparative test result (table 2 and Fig. 2)
2 porcine pseudorabies vaccine antibody Fluctuation comparative test result of table
From on table 2 it can be seen that each group vaccine is after immunity 28 days, blank control group antibody remains as feminine gender, other immunity
Group can produce higher antibody horizontal.
Result of the test can be illustrated：From the point of view of antibody horizontal, the vaccine that either prepared by embodiment 1, or marketing
Seedling can produce higher antibody horizontal.
Different time neutralizing antibody detection test (being shown in Table 3) after 2.2 each immune group immunity
Different time neutralizing antibody detection result of the test after 3 vaccine immunity of table
As can be seen from Table 3, the vaccine and other commercially available vaccines that prepared by embodiment 1 turn positive rate be all qualified, and
Without notable difference.Each group vaccine turns a positive rate after immune 14 days and has reached 50%.After immunity 28 days, during each immune group is average
With the basic zero difference of antibody, turn positive rate 100%.
Embodiment 4：
Pseudorabies disease vaccine (XF-1 strains) safety testing
1st, test material and method
The piglet of Wuhan pig farm 21-25 ages in days is bought in 1.1 experimental animal markets, to carrying out main pathogen and associated antibodies
Detection, it is negative and pseudorabies to select to porcine circovirus 2 type, swine fever virus, porcine reproductive and respiratory syndrome virus cause of disease
Neutralizing antibody is negative, and (PRV NATs are not higher than 1:2) piglet, 20 altogether.
Test pig is randomly divided into 4 groups by 1.2 methods, per group of 5 piglets.Wherein pig puppet of the group 1 prepared by embodiment 1 is mad
Dog disease inactivated vaccine；2 are organized for swine pseudorabies vaccine (Strain Ea), lot number：150401；Group 3 is pseudorabies disease live-vaccine
(98 plants), lot number：150540；Group 4 is blank.Wherein, group 2, group 3 are provided for Wuhan Ke Qian Biological Co., Ltd..
Group 1 and 2 every pig musculi colli of group inject 2 parts, and every pig musculi colli injects 10 parts to group 3 to specifications.Observation 14
It and log.
The all immune group of 2 results are good for without local response and all work.The results are shown in Table 4.
5 vaccine safety result of the test of table
As can be seen from Table 5, each group whole 5/5 has no adverse reaction, and each vaccine is respectively provided with good safety.
Embodiment 5：
The application of porcine pseudorabies virus variant XF-1 strains：
The preparation of porcine circovirus 2 type-WH strain vaccines：
1st, the PK-15 cells (purchased from ATCC) of monolayer will be covered with, remove cell culture fluid, by PCV2 seeds culture of viruses by 0.1～
0.2% inoculum concentration is inoculated on PK-15 cells, and it is 10 to plant poison poison valency8.0TCID50/ml, 37 DEG C adsorb 30 minutes, add thin
Born of the same parents' maintaining liquid, puts 37 DEG C of rotating and culturing.Daily observation 1～2 time, cell growth is good, and 36～37 DEG C of cultures were harvested after 4～7 days
After cell culture, freeze thawing 2-3 time, it is 10 to determine malicious valency result7.5Antigen is placed in less than -20 DEG C and is preserved by TCID50/ml.Will
Cultured virus liquid is filtered by 0.2 μm of filter post of doughnut, is removed cell debriss again through eluting, concentration, is finally passed through again
Ion exchange is obtained the antigen of purification.Antigen poison valency result is 10 after purification7.5More than TCID50/ml, adds final concentration
37 DEG C of inactivation 24h of formalin for 0.4%, by existing《Chinese veterinary pharmacopoeia》Annex test qualified after, 4 DEG C of storages are standby
With.
2nd, by porcine circovirus 2 type antigen after purification and IMS 1313VG adjuvants according to mass ratio 5:1 carries out emulsifying mixes
Even 30min, in the complete rear subpackage of emulsifying, inspection.Inspection according to《Chinese veterinary pharmacopoeia》Annex is carried out, and is preserved through 4 DEG C after the assay was approved
Standby, porcine circovirus 2 type inactivated vaccine is obtained final product, in the final product per 2ml, effectively viral level is 107.0TCID50, every pig
Each immunity 2ml.
Porcine pseudorabies virus and the preparation of porcine circovirus 2 type bivalent inactivated vaccine：
By inactivated porcine pseudorabies virus liquid (XF-1 strains) and porcine circovirus 2 type-WH strains (CCTCC NO：V201333)
Virus liquid, requires mix homogeneously according to malicious valency, then again with import adjuvant IMS1313N VG according to mass ratio 5：1 carries out emulsifying
30 minutes.Porcine pseudorabies and porcine circovirus 2 type bivalent inactivated vaccine (hereinafter referred to as bigeminy vaccine) are obtained, finally
So that PRV (Pseudorabies virus) antigenic content is 10 in per 2ml finished product Seedlings7.5TCID50, porcine circovirus 2 type antigenic content is
107.0TCID50, every pig immunity 2ml every time.
Embodiment 6：
Porcine pseudorabies are verified with porcine circovirus 2 type bivalent inactivated vaccine immune efficacy：
1 test material
The piglet of Wuhan pig farm 21-25 ages in days is bought in 1.1 experimental animal markets, and main pathogen and associated antibodies are carried out
Detection, it is negative and pseudorabies to select to porcine circovirus 2 type, swine fever virus, porcine reproductive and respiratory syndrome virus cause of disease
Neutralizing antibody is negative, and (PRV NATs are not higher than 1:2) piglet, 42 altogether.
1.2 test vaccines：
Group A is the bigeminy vaccine prepared by embodiment 5；
Group B is the porcine pseudorabies virus inactivated vaccine (XF-1 strains) prepared by embodiment 1；
Group C is the porcine circovirus 2 type inactivated vaccine prepared by embodiment 5.
1.3 counteracting toxic substances are with strain PRV (Pseudorabies virus) street strain (XF strains)；Pig circular ring virus counteracting toxic substances strain is pig circular ring virus 2
Malicious 2 type-WH strains, CCTCC NO：V201333.Derive from Wuhan Ke Qian Biological Co., Ltd..
2 test methods
Test pig is randomly divided into 6 groups by 2.1 test packets, and packet situation is as follows：
Group A is the bivalent inactivated vaccine vaccine prepared by embodiment 5, totally 12 piglets；
Group B is the swine pseudorabies vaccine (XF-1 strains) prepared by embodiment 1, totally 6 piglets；
Group C is the porcine circovirus 2 type inactivated vaccine prepared by embodiment 5, totally 6 piglets；
Group D is PRV (Pseudorabies virus) counteracting toxic substances matched group, totally 6 pigs；
Group E is porcine circovirus 2 type counteracting toxic substances matched group, totally 6 piglets.
Group F is nonimmune blank control group, totally 6 piglets.As negative control, isolated rearing is needed.
2.1.1 PRV (Pseudorabies virus) counteracting toxic substances part
(1) by bivalent inactivated vaccine and swine pseudorabies vaccine (XF-1 strains), respectively every pig musculi colli immunity 1
Head part.Counteracting toxic substances after immune 28 days.Wherein, from group A immune group 12,6 first tap PRV (Pseudorabies virus) of random choose, group B6 heads are complete
Attack PRV (Pseudorabies virus) in portion.Every piglet collunarium counteracting toxic substances PRV (Pseudorabies virus) street strain's (XF strains) virus liquid 1.0ml, virus liquid
Malicious valency is 107.0TCID50/ml, observes 14 days.Observation recording respiration system, digestive system, the clinical response of nervous system,
And survey body temperature 14 days.Counteracting toxic substances result is drawn according to piglet morbidity criterion.
(2) piglet morbidity criterion after counteracting toxic substances
(5) breathing or digestive system clinical symptoms sneeze, nose have secretions, dyspnea；Have loose bowels, vomit.
(6) body temperature reaction body temperature >=40.5 DEG C, at least continue 2 days.
Nervous system clinical symptoms spasm, sialorrhea, fall down to the ground strike, the typical clinical symptom such as extreme agitation
(8) dead
Meet above (3rd), (4th) item or while meeting (1st) (2nd) item, you can judge morbidity.
2.1.2 pig circular ring virus counteracting toxic substances part
(1) by bivalent inactivated vaccine and porcine circovirus 2 type inactivated vaccine, every pig musculi colli injecting immune 1 respectively
Head part.Counteracting toxic substances after immune 28 days.To pig circular ring virus immune group and counteracting toxic substances matched group, each group carries out PCV2 virus WH strains simultaneously
(viral level is 107.0TCID50/ ml, CCTCC NO：V201333) counteracting toxic substances, every pig musculi colli inject 3ml, collunarium 2ml,
Isolated rearing.In the counteracting toxic substances same day, all test pig weights of weighing.And 3 days before the counteracting toxic substances 3 after (i.e. 25 days after immunity) and counteracting toxic substances
Day, 6 days, the equal injecting immune stimulus material of all counteracting toxic substances group piglets (porous hemocyanin breast prepared by Fei Shi Freund's incomplete adjuvants
Agent), every pig musculi colli injects 2ml every time.After observation 28 days, again all test piglets are weighed；Collection challenge test
Piglet blood sample, separates Virus monitory viremia；Cut open and kill challenge test piglet, take inguinal lymph nodes and mesenteric lymph node,
Carry out histology and Immunohistochemical detection.The incidence that piglet is analyzed by above indicator-specific statisticss.28 days after counteracting toxic substances
The whole porklings of cut open inspection.
(2) piglet morbidity criterion after counteracting toxic substances, meets two in following three, you can judge morbidity.
A body temperature symptoms：Piglet body temperature raises (>=40 DEG C), should at least continue 3 days；
B weight standards：Body weight increase rate decline should be not less than 5.0%, and the average daily gain of counteracting toxic substances piglet should be less than non-attacking
The average daily gain of malicious matched group piglet.All test pig weights are weighed respectively by head in the counteracting toxic substances same day, after counteracting toxic substances, 28 again
Secondary weigh all test pig weights；Wherein, in " B weight standards ", the calculating of body weight increase rate is carried out as follows：
Body weight increase rate (%)=non- counteracting toxic substances matched group piglet average daily gain-counteracting toxic substances group piglet average daily gain/counteracting toxic substances
Matched group piglet average daily gain × 100
C virus antigen detections：Lymph node tissue is detected with immunohistochemistry technique, PCV2 viruses are detected.
3 result of the tests
3.1 bivalent inactivated vaccines and swine pseudorabies vaccine (XF-1 strains) counteracting toxic substances protection comparative test result (see
Table is 6)
Counteracting toxic substances protection comparative test result (being shown in Table 6) of 3.2 bivalent inactivated vaccines and porcine circovirus 2 type inactivated vaccine
6 each vaccine group efficacy test result of table
PRV (Pseudorabies virus) challenge test part：Observe after immune A groups and immunity B group piglet counteracting toxic substances and do not find clinical condition
Shape.And counteracting toxic substances matched group D there are 4, there are the symptoms such as body temperature rising, lethargy, loss of appetite, dyspnea.In morbidity
4 piglets in, have 3 death.
Pig circular ring virus challenge test part：Observe after immune A groups and immunity C group piglet counteracting toxic substances and do not find clinical condition
Shape.And counteracting toxic substances matched group E6 head piglets have morbidity in various degree.
Result of the test shows that (being shown in Table 6), each vaccine have immune protective effect, can resist the attack of PRV with PCV2's
Attack.Meanwhile, bivalent inactivated vaccine compare us with the immunity test of single Seedling it is seen that, the immune effect of bigeminy vaccine with each
Immune effect from single Seedling is substantially suitable.So as to be further characterized by, during two-strain antigen simultaneous immunity animal, the two is special
Being produced without of heterogenetic antibody interferes effect.Further challenge test confirms that bivalent inactivated vaccine has and respective list
Seedling identical resists the effect of strong virus attack.
Embodiment 7：
Several different porcine pseudorabies vaccine antibodies detect contrast test
2nd, test material
The piglet of Wuhan pig farm 21-25 ages in days is bought in 1.1 experimental animal markets, and main pathogen and associated antibodies are carried out
Detection, it is negative and pseudorabies to select to porcine circovirus 2 type, swine fever virus, porcine reproductive and respiratory syndrome virus cause of disease
Neutralizing antibody is negative, and (PRV NATs are not higher than 1:2) piglet, 25 altogether.
1.2 porcine pseudorabies specific positive serum neutralization titers are 1:256, by Wuhan Ke Qian Biological Co., Ltd.
Prepare
The bigeminy vaccine that 1.3 tests are prepared with vaccine embodiment 5；Swine pseudorabies vaccine prepared by embodiment 1
(XF-1 strains)；Pseudorabies disease live-vaccine (98 plants), lot number：150540, provided by Wuhan Ke Qian limited companies；Import
Bartha-K61 strain live vaccine, lot number：The biological big pharmaceutical factories of 77BN markets purchase Spain Hai Bolai
2nd, test method
Test pig is randomly divided into 5 groups, per group of 5 piglets.Packet situation is as follows：
Group 1 is bigeminy vaccine prepared by embodiment 5；
Group 2 is porcine pseudorabies list Seedling (XF-1 strains) prepared by embodiment 1；
3 are organized for pseudorabies live vaccine (98 plants) live vaccine, lot number：150540；
4 are organized for import Bartha-K61 strain live vaccine, lot number：77BN；
Group 5 is blank.
Every pig musculi colli 1 part of immunity of each immune group.After immunity, adopt within 14 days respectively, 28 days, 42 days, 56 days, 90 days
Blood system is from serum.Detection pseudorabies antibody and detection PRV (Pseudorabies virus) NAT, calculate and turn positive rate, while detection
Each immune group is in the average neutralizing antibody of different immunization times.
3rd, result of the test
3.1st, porcine pseudorabies vaccine antibody Fluctuation comparative test result (table 7 and Fig. 3)
7 porcine pseudorabies vaccine antibody Fluctuation comparative test result of table
As can be seen from Table 7, the bivalent inactivated vaccine and swine pseudorabies vaccine (XF-1 strains) that prepared by the present invention,
Without notable difference in immune antibody level.From on table 7 it can be seen that each group vaccine is after immunity 28 days, blank control group is still
For feminine gender, other immune group can produce higher antibody horizontal.
Result of the test can be illustrated：From the point of view of antibody horizontal, either bigeminy vaccine or porcine pseudorabies list Seedling (XF-1
Strain), or the commercial seedling of market purchase can produce higher antibody horizontal.
3.2 each immune group are in different immunization time neutralizing antibodies detection test (being shown in Table 8)
8 vaccine immunity different time neutralizing antibody of table detects result of the test
As can be seen from Table 8, it is all qualified, not notable difference that all vaccines turn positive rate.Each group vaccine exists
Immunity turns positive rate and has reached 50% after 14 days.After immunity 28 days, the basic zero difference of the average neutralizing antibody of each immune group turns positive rate
100%.On the whole, the Combined vaccine that prepared by the present invention is effective.
Embodiment 8：
Checking of the Combined vaccine to porcine circovirus 2 type effectiveness
Porcine circovirus type 2 vaccines antibody dynamic regularity contrast test
1st, materials and methods
The piglet of Wuhan pig farm 21-25 ages in days is bought in 1.1 experimental animal markets, and main pathogen and associated antibodies are entered
Row detection, from mad for negative and pig puppet to porcine circovirus 2 type, swine fever virus, porcine reproductive and respiratory syndrome virus cause of disease
Dog neutralizing antibody is negative, and (PRV NATs are not higher than 1:2) piglet, 30 altogether.
It is negative pig that 1.2 methods will buy 30 antibody back, is randomly divided into 6 groups, 5 per group, and packet situation is as follows：
Group 1 is bivalent inactivated vaccine prepared by embodiment 5；
Group 2 is porcine circovirus 2 type inactivated vaccine prepared by embodiment 5；
Group 3 is the porcine circovirus 2 type inactivated vaccine of offer commercialization before the section of Wuhan；
Group 4 is that import Seedling is bought in market；
Group 5 is that domestic commercial seedling is bought in market；
Group 6 is blank control group (i.e. nonimmune group).
Every pig 1 part of immunity, organizes 1 group 5 through musculi colli 1 part vaccine of immunity to specifications.After immunity, observation
Clinical manifestation, and taking periodic blood detection antibody as requested.
1.3 detection kit porcine circovirus 2 type ELISA antibody assay kits are limited by biological share before the section of Wuhan
Corporate diagnosis reagent section is provided.
2nd, test method
Before 2.1 all test pigs immunity and the 7th day after immunity, the 14th day, the 28th day, the 60th day, the 90th day, 120 days right
Every group of piglet blood sampling, separates serum ELISA method detection antibody level.
2.2 antibody detection method
Detected in strict accordance with porcine circovirus 2 type ELISA antibody assay kit operation instructions.
2.3 criterion
Each hole OD is determined in microplate reader630nmValue.Test establishment condition is Positive control wells OD630nmValue answers >=1.0, the moon
Property control wells OD630nmValue answers≤0.25.Sample well OD630nmDuring value >=0.40, it is judged to the positive；Sample well OD630nmValue≤0.40
When, it is judged to feminine gender.
3rd, result of the test antibody test result (referring to table 9 and Fig. 4)
Antibody test result after 9 different pig circular ring virus vaccine immunities of table
As can be seen from Table 9, the porcine circovirus type 2 vaccines vaccine that prepared by bigeminy vaccine and embodiment 5, in immunity
The antibody horizontal of the commercial seedling detection of the antibody for detecting afterwards and market purchase is not significantly different from.And after immunity 14 days, antibody
All turn sun.When immune 42 days to 56 days, antibody horizontal reaches highest.Each vaccine group goes out outside indivedual pigs after immune 4 months
Antibody is still the positive.On the whole, bigeminy vaccine has good immune effect.
Embodiment 9：
PRV (Pseudorabies virus) and porcine circovirus 2 type bivalent inactivated vaccine safety testing
1 materials and methods
The piglet of Wuhan pig farm 21-25 ages in days is bought in 1.1 experimental animal markets, anti-to carrying out main pathogen and correlation
Health check-up is surveyed, from mad for negative and pig puppet to porcine circovirus 2 type, swine fever virus, porcine reproductive and respiratory syndrome virus cause of disease
Dog neutralizing antibody is negative, and (PRV NATs are not higher than 1:2) piglet, 20 altogether.
1.2 method
Divide 4 groups, 5/group at random by test pig.Bivalent inactivated vaccine, PCV2 Seedlings and embodiment 1 prepared by Example 5
The PRV Seedlings of preparation and each musculi colli injection of saline control group, 2 part/heads are observed 14 days.
The all test pigs of 2 results are anti-without local and are all good for work.Assay is shown in Table 10.
10 vaccine safety result of the test of table
As can be seen from Table 10, bivalent inactivated vaccine and embodiment 1 prepare XF-1 strain lists vaccine after inoculation piglet,
Whole 5/5 have no adverse reaction, and each vaccine is respectively provided with good safety.
Claims (6)
1. a kind of porcine pseudorabies virus variant, it is characterised in that：Described porcine pseudorabies virus variant is that pig puppet is mad
Dog disease virus XF-1 strains, deposit number is:CCTCC NO V201654.
2. application of the porcine pseudorabies virus variant XF-1 strains according to claim 1 in vaccine is prepared.
3. application according to claim 2, it is characterised in that the vaccine is inactivated vaccine.
4. application according to claim 2, it is characterised in that the immunizing dose of every pig is 107.5TCID50.
5. application according to claim 2, it is characterised in that the adjuvant in the vaccine is MONTANIDE IMS
1313VG.
6. the preparation method of the porcine pseudorabies virus variant described in claim 1, comprises the steps：
By the variant described in claim 1, virus titer >=108.0TCID50/ ml, are inoculated in BHK-21 cell suspension；Disease
Malicious inoculum concentration be 1.0%～3%, v/v, control 36 DEG C～37 DEG C of cultivation temperature, pH6.0～7.0, speed of agitator 80rpm～
100rpm, dissolved oxygen concentration 40%～60% just obtain porcine pseudorabies venom when reaching 85% to cytopathy；Will be cultured
XF-1 strains remove cell debriss by 0.65 μm of doughnut, then again by eluting, concentration, pure finally by gel chromatography column
Change the Pseudorabies viruses antigen for obtaining purification, i.e. the XF-1 strain virus stock solutions of purification.
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