CN103981151B - A kind of pseudorabies inactivated vaccine and preparation method thereof - Google Patents
A kind of pseudorabies inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of pseudorabies inactivated vaccine and preparation method thereof, the Strain of described pseudorabies inactivated vaccine is Pseudorabies virus PRV HB strain, its preserving number is CGMCC NO.8758, preservation date is on 01 13rd, 2014, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.Pseudorabies inactivated vaccine prepared by the present invention has good safety, and can produce preferable immune protective effect after immunity dog, can be as the candidate vaccine strain of prevention and control Canis animals pseudorabies.
Description
Technical field
The present invention relates to a kind of pseudorabies inactivated vaccine and preparation method thereof, be specifically related to a strain Pseudorabies virus
Inactivated vaccine for Canis animals pseudorabies prevention and control prepared by PRV-HB, belongs to biological technical field.
Background technology
Pseudorabies (Pseudorabies, PR) is the warp caused by Pseudorabies virus (Pseudorabies Virus, PRV)
Ji animal and a kind of acute infectious disease of domestic animal.It is characterized in that generating heat, very itch, encephalomyelitis etc..Dog and the puppet of pig
Rabies are appeared in the newspapers the most repeatly.This disease in the cows being popular in the U.S. for 1813, Hungary scholar in 1902
Ao Yeziji (Aujeszky) has been described in more detail for the first time this disease, therefore named Ao Yeziji disease (also known as
AujeszkyShi is sick, Aujeszky ' s disease, AD), 1910, Schmiedhofer was demonstrate,proved by filtration test
This disease pathogen real is that first virus isolate virus.
Since nineteen forty-seven Liu Yongchun finds the pseudorabies of cat, existing 20 Duo Ge provinces and cities of China successively report this disease.
Nineteen ninety-five, Nyctereutes procyonoides is had to infect the report of PRV first.Since 2012, Shandong, Henan, Hebei etc. goes out in succession
Existing dog, Vulpes and Nyctereutes procyonoides infect PRV dead report on a large scale.
Along with China economic animal and the expansion year by year of Fur Animal Feeding scale, conscientiously carry out PR prevention and control and compel
The eyebrows and eyelashes.The mortality rate caused due to PR is almost up to 100%, huge to aquaculture harm.Vaccine is anti-disease-control
The effective means that toxoinfection is sick, thus research and develop and a kind of have good novel of China's independent intellectual property right, protectiveness
Efficient vaccine has great importance.At present, at large-scale pig farm, general employing PRV gene-deleted vaccine enters
The anti-PR processed of row.But not for the PR vaccine of dog, Vulpes and Nyctereutes procyonoides, Given this present invention carries out related vaccines and grinds
Study carefully.
Summary of the invention
An object of the present invention is to provide a kind of pseudorabies inactivated vaccine and preparation method thereof, and described pseudorabies is gone out
Live vaccine be Pseudorabies virus PRV-HB strain preserving number be CGMCC NO.8758, Classification And Nomenclature is pseudorabies
Poison, preservation date is on 01 13rd, 2014, and depositary institution is China Committee for Culture Collection of Microorganisms
Common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro-life of the Chinese Academy of Sciences
Thing institute.
The two of the object of the invention are to provide the preparation method of a kind of pseudorabies (PR) inactivated vaccine.
The separation of pseudorabies strain and qualification: 2012, pathological material of disease collection is from economic animal plant of Hebei province
The dead Vulpes of morbidity and Nyctereutes procyonoides brain, tonsil and liver organization, pathological material of disease is after grinding, and inoculation has grown up to the Ren sus domestica of monolayer
Epithelial cell (PK15 cell), carries out PCR to tissue culture pseudorabies (PRV) specific primer
Amplification identifies that gE and gB gene, amplification gE gene primer are respectively gE F and gE R, its nucleotide sequence
SEQ ID No.1 and SEQ ID No.2 respectively；Amplification gB gene primer is respectively gB F and gB R, its
Nucleotide sequence SEQ ID No.3 and SEQ ID No.4 respectively.And carry out rabbit body pathogenicity, it was demonstrated that separate
Virus be PRV, name PRV-HB-12 strain, referred to as PRV-HB strain.
The pseudorabies prepared by Canis animals (dog, Vulpes, Nyctereutes procyonoides etc.) pseudorabies inactivated vaccine PRV-HB strain is gone out
Live vaccine.Strain >=10 Han Pseudorabies virus PRV-HB in described pseudorabies inactivated vaccine6TCID50/ head part
The preparation method of pseudorabies inactivated vaccine of the present invention:
Carry out titration of virus after Pseudorabies virus PRV-HB strain propagation, according to contained immunizing dose, add inactivator,
And after adjuvant, prepare vaccine.
Further, in above-mentioned preparation method, the propagation of described Pseudorabies virus PRV-HB strain includes following step
Rapid:
In the PK15 cell growing up to monolayer, in 2% ratio inoculation pseudorabies seed culture of viruses poison, it is placed in 37 DEG C,
In 5%CO2 incubator, gather in the crops after cultivating 96h, multigelation 3 times, put-20 DEG C of preservation, 6 should be less than
Individual month.
Further, in above-mentioned preparation method, described inactivator is beta-propiolactone, and described adjuvant is
Montanide PET GEL A (is called for short GEL A).
The preparation of pseudorabies inactivated vaccine of the present invention, specifically includes following steps:
By the qualified Pseudorabies virus PRV-HB strain of inspection, breed in a large number in PK15 cell, through 0.2% β-
Propiolactone inactivation 48h, adds 8%GEL A, utilizes dasher, under shear rate 200rpm, and emulsifying
10min, the conventional inactivated vaccine of preparation.
Safety and the Study On Immunogenicity result of PR inactivated vaccine of the present invention show, dog is had well by the present invention
Safety, immunity dog after preferable immune protective effect and can be produced, can be as prevention and control Canis animals pseudorabies
Candidate vaccine strain.
Accompanying drawing explanation
Fig. 1 is PRV-HB strain gE and gB gene amplification result figure, and wherein, M is DL2000DNA Marker,
1 is negative control, and 2 is gB gene fragment amplification product, and 3 is negative control, and 4 is gE gene fragment amplification
Product；
After Fig. 2 is vaccine immunity, each group dog antibody horizontal figure.
Detailed description of the invention
Further describe the present invention, advantages of the present invention and feature below in conjunction with specific embodiment to will be with describing
And it is apparent.But embodiment is only exemplary, the scope of the present invention is not constituted any restriction.This area
Skilled artisans appreciated that, can be to technical solution of the present invention under without departing from the spirit and scope of the present invention
Details and form are modified or replace, but these amendments and replacement each fall within protection scope of the present invention.
The isolation identification of embodiment 1 PRV-HB of the present invention strain
Materials and methods
1 pathological material of disease and cell pathological material of disease gather from the dead Vulpes of economic animal plant of Hebei province morbidity and Nyctereutes procyonoides brain, almond
Body and liver organization, PK15 cell is provided by Harbin veterinary institute.
The 2 dead Vulpes of virus multiplication morbidity, the brain of Nyctereutes procyonoides, tonsil and liver organization, after grinding with Potter-Elvehjem Tissue Grinders,
Make l:5 dilution with Hanks ' liquid and make suspension, add green grass or young crops/streptomycin to 1000U/mL, multigelation 3 times, warp
12000rpm is centrifuged 10min, takes supernatant, and inoculation has grown up to the PK15 cell of monolayer, in 5%CO2, 37 DEG C
Incubator cultivates 96h, and multigelation 3 times, centrifuging and taking supernatant ,-70 DEG C save backup.
3 molecular biology reagents DNA extraction kit are purchased from Omega company；LA used by PCR amplification
Taq archaeal dna polymerase is purchased from precious biological engineering (Dalian) company；Glue reclaims (in a small amount) test kit purchased from Shanghai
Hua Shun biological engineering company limited.
4 carriers and strain pMD18-T cloning vehicle and DH5 α competent escherichia coli cell are purchased from precious biological
Engineering (Dalian) company.
5 design of primers utilize the specificity of gE and gB gene in Oligo6.0 software design synthesis amplification PRV
Primer, amplification gE gene primer is respectively gE F and gE R, its nucleotide sequence SEQ ID No.1 respectively
With SEQ ID No.2；Amplification gB gene primer is respectively gB F and gB R, its nucleotide sequence SEQ respectively
ID No.3 and SEQ ID No.4.
The PCR amplification PCR amplification program of 6gE and gB gene:
94 DEG C of denaturations 4min,
94 DEG C of degeneration 30 seconds,
Anneal 30 seconds for 55 DEG C,
72 DEG C extend 1min,
Cyclic amplification 30 times；
72 DEG C extend 10min.
PCR primer reclaims (in a small amount) test kit with Shanghai Hua Shun company glue and reclaims.
After the PCR primer that glue reclaims is connected with pMD18-T carrier by 7 connections with conversion, convert DH5 α big
Enterobacteria competent cell, then takes out 100 μ L bacterium solution and coats the LB containing ampicillin (50 μ g/mL)
On flat board, cultivate 16h for 37 DEG C.Picking white isolates bacterium colony, is inoculated in 5mL and contains ampicillin (50 μ g/mL)
LB liquid medium in, 37 DEG C cultivate 12h.
8 genes of interest order-checkings, by the bacterium solution through being accredited as the positive, send Invitrogen company to carry out sequencing.
9 rabbit body pathogenicities take 63 monthly age rabbit (by Harbin veterinary institute Experimental Animal Center
There is provided) divide 2 groups, counteracting toxic substances group 4, every rabbit neck part subcutaneous injection 1mL cells and supernatant Han PRV is right
According to group 2, every cervical region subcutaneous injection 1ml Hanks ' liquid, 48h observer's rabbit invasion situation.
Result of the test:
The size of 1PCR result gE and gB genetic fragment respectively may be about 0.4kb and 0.3kb, with intended
Size is consistent, and result is as shown in Figure 1.
After 2 rabbit body pathogenicity test results counteracting toxic substances 48h, counteracting toxic substances group rabbit lick sting hind leg until fall hair, breakage and
Hemorrhage, body temperature rising, quadriplegia, tic, opisthotonus or even paralysis occur then, last exhaustion is dead；
Matched group has no visible clinical symptoms.
The preparation of embodiment 2 Canis animals PR of the present invention inactivated vaccine and immune efficacy evaluation thereof
1 materials and methods
1.1 virus multiplications and titration are in the PK15 cell growing up to monolayer, with 2% ratio inoculation PRV-HB
Plant poison, be placed in 37 DEG C, 5%CO2In incubator, gather in the crops after cultivating 96h, multigelation 3 times, 12000rpm
Centrifugal 10min, takes supernatant, titrates rearmounted-20 DEG C of preservations, should be less than 6 months.
Cell is cultivated the beta-propiolactone that the supernatant prepared adds 0.2% by 1.2 inactivation of virus and emulsifying, inactivates 48h,
After testing after inactivation completely, add the GEL A of 8%, utilize dasher, under shear rate 200rpm,
Emulsifying 10min.
1.3 dog body immune efficacies are evaluated
1.3.1 8 10 week old PRV antibody test feminine gender beasle dogs (are ground purchased from Guangzhou medicine by immunity with counteracting toxic substances
Company limited of Jiu Zong institute Animal Experimental Study development centre) it is randomly divided into 2 groups.1st group (5) are that vaccine is exempted from
Epidemic disease group, subcutaneous injection single dose Adjuvanted vaccines, immunity 2 times, every minor tick 14d；2nd group (3) are right
According to group subcutaneous injection 1ml PBS.Two exempt from rear 14d, by intramuscular injection counteracting toxic substances 1ml (106TCID50/ml)
PRV-HB.Before counteracting toxic substances, all dogs are all anaesthetized by the dog dormancy treasured of intramuscular injection 0.15mL/kg.
1.3.2 1d-10d after clinical symptoms counteracting toxic substances, every day, the clinical symptoms of each group of dog was observed in timing.
1.3.3 1d-10d after virus toxin expelling detection counteracting toxic substances, gathers the Nasopharyngeal swabs of each group of dog, passes through PCR every day
The toxin expelling situation of method detection dog.
1.3.4 antibody test respectively organize dog before immunity after 0d, and immunity 1w (1 week), 2w, 3w, 4w adopt
Blood, separates serum, measures antibody horizontal by IDEXX antibody blocking ELISA kit.
2 result of the tests
The antibody horizontal of dog after 2.1 vaccine immunities
After vaccine immunity group is exempted from one, there is low-level antibody in 2w；Two exempt from rear 1w occurs that relatively principle of readjustment, restructuring, consolidation and improvement is special to PRV
Opposite sex antibody levels (blocking-up rate ＜ 0.6 is positive), matched group dog does not produces antibody, as shown in Figure 2.
The clinical symptoms of dog after 2.2 counteracting toxic substances
In the whole counteracting toxic substances stage, vaccine immunity dog does not goes out to show visual clinical symptoms.Comparison dog 2d after counteracting toxic substances starts
Performance spirit depressed, appetite declines, sialorrhea, bite the symptoms such as injection site, and dog only death occurs in 3d, attacks
The 4th day matched group dog of poison is the most dead.
Toxin expelling situation after 2.3 counteracting toxic substances
The all dogs of vaccine immunity are not detected by virus discharge；Negative control group 2d after counteracting toxic substances starts toxin expelling occur, holds
After continuing counteracting toxic substances, 4d is dead to all dogs.
Claims (7)
1. a pseudorabies inactivated vaccine strain, it is characterised in that: described pseudorabies inactivated vaccine strain
For Pseudorabies virus PRV-HB strain, its preserving number is CGMCC NO.8758, and preservation date is 2014 01
The moon 13, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. the pseudorabies inactivated vaccine prepared by pseudorabies inactivated vaccine strain as claimed in claim 1.
Pseudorabies inactivated vaccine the most according to claim 2, it is characterised in that: described pseudorabies inactivation epidemic disease
Strain >=10 Han Pseudorabies virus PRV-HB in Seedling6TCID50/ head part.
4. the preparation method of pseudorabies inactivated vaccine as claimed in claim 2, it is characterised in that include following
Step:
Carry out titration of virus after Pseudorabies virus PRV-HB strain propagation, according to contained immunizing dose, add inactivator,
And after adjuvant, prepare vaccine.
Preparation method the most according to claim 4, it is characterised in that described Pseudorabies virus PRV-HB
The propagation of strain comprises the following steps:
In the PK15 cell growing up to monolayer, in 2% ratio inoculation pseudorabies seed culture of viruses poison, it is placed in 37 DEG C,
In 5%CO2 incubator, gather in the crops after cultivating 96h, multigelation 3 times, put-20 DEG C of preservation, 6 should be less than
Month.
Preparation method the most according to claim 4, it is characterised in that: described inactivator is beta-propiolactone,
Described adjuvant is Montanide PET GEL A.
Preparation method the most according to claim 4, it is characterised in that: specifically include following steps:
By the qualified Pseudorabies virus PRV-HB strain of inspection, breed in a large number in PK15 cell, through 0.2% β-
Propiolactone inactivation 48h, adds 8%Montanide PET GEL A, utilizes dasher, in shear rate
Under 200rpm, emulsifying 10min, the conventional inactivated vaccine of preparation.
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