CN109609468A - A kind of porcine pseudorabies virus of six gene delection, pseudorabies disease vaccine and preparation method - Google Patents
A kind of porcine pseudorabies virus of six gene delection, pseudorabies disease vaccine and preparation method Download PDFInfo
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Abstract
The invention discloses porcine pseudorabies virus, pseudorabies disease vaccine and the preparation methods of a kind of six gene delections, are related to animal biological product field.The deposit number that the present invention provides the porcine pseudorabies virus of six gene delections is CGMCC No:16290.The porcine pseudorabies virus of six gene delection lacks six genes, pathogenic to be removed, immunogenicity is effectively retained, the vaccine prepared using the porcine pseudorabies virus, inducible body generates the interferon of more advanced dosage, has better immune effect in terms of urgent immunity inoculation.
Description
Technical field
The present invention relates to porcine pseudorabies vaccines arts, in particular to a kind of porcine pseudorabies of six gene delections
Poison, pseudorabies disease vaccine and preparation method.
Background technique
Porcine pseudorabies (Pseudorabies, PR) are also known as aujeszky's disease (Aujeszky's disease, AD), are
A variety of domestic animals such as pig, ox, sheep caused by Pseudorabies virus (Pseudorabies virus, PRV) and wild animal are to send out
Heat, surprise are itched (except pig) and encephalomyelitis is a kind of highly contagious disease of cardinal symptom.Pig be the disease natural host,
Major storage host and the infection sources, either newborn piglet, growing and fattening pigs, or adult boar, all by the threat of PR.It is showed
Symptom are as follows: the death rate is very high after Infection in Piglets in 2 week old, up to 100%, may occur in which that diarrhea, vomiting, incoordination, angle bow are anti-
, the nervous symptoms such as four limbs swimming, finally paralysis, failure are dead；Weanling pig can also cause death, but be mainly shown as breathing
Systemic symptom, also has part pig nervous symptoms, diarrhea, vomiting etc. occur, and the often depauperation of the piglet of resistance to mistake becomes cad pig；
It is then most of after growing and fattening pigs infection to be increased with body temperature, it has difficulty in breathing, occasionally has nervous symptoms, death does not occur generally；Adult Pig
It does not fall ill after infection or is showed only as the light symptoms such as body temperature raising, in stealthy infection, resistance to mainly performance growth and development is slow later
It is slow, the price of deed reduces etc., and can be long-term with poison or toxin expelling, become the most dangerous infection sources；It will lead to after pregnant sow infection
Miscarriage, production stillborn foetus, the mummification of fetus etc.；The disease can also cause boar infertility, Testis of Boar Pig swelling, sow to return feelings, Repeat breeding etc..
Only one serotype of Pseudorabies virus, but different strain virulence and in terms of have differences.
Vaccine inoculation is that effective anti-porcine pseudorabies processed are most economical, effective method.But the immune effect of existing pseudorabies disease vaccine
Fruit is unsatisfactory.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of porcine pseudorabies virus of six gene delections, which is through people
It is obtained after work transformation, lacks six genes, pathogenic to be removed, immunogenicity is effectively retained, mad using the pig puppet
The vaccine of dog disease poison preparation, inducible body generate the interferon of more advanced dosage, have in terms of urgent immunity inoculation more preferable
Immune effect.
Another object of the present invention is to provide the applications of above-mentioned porcine pseudorabies virus.
Another object of the present invention is to provide a kind of pseudorabies disease vaccine, the vaccine by above-mentioned six gene delection pig
Pseudorabies virus is made, with preferable immune effect.
Another object of the present invention is to provide a kind of methods for preparing above-mentioned pseudorabies disease vaccine.Pass through party's legal system
The pseudorabies disease vaccine obtained has preferable immune effect.
The present invention is implemented as follows:
There is new porcine pseudorabies epidemic situation nationwide before and after 2011, is mainly shown as sudden large area
Boar miscarriage, growing and fattening pigs lethal infection, swinery gE antibody positive rate increase suddenly, and selected swine farms gE antibody positive rate is up to
100%.The different regions of Epidemic outbreak of disease, immune state is different and shows different clinical manifestations.The preferable pig of immune state
, mostly without obvious clinical symptoms, but swinery gE positive rate increases suddenly；The poor pig farm of immune state, is mainly shown as kind
Large area miscarriage, grice diarrhoea, growing and fattening pigs respiratory symptom and the acute death of pig.The multiple experiments in the whole nation after new Epidemic outbreak of disease
Room is separated to new porcine pseudorabies virus prevalence strain, by pathogenicity experiment, immunoprotection experiment and molecular epidemiology etc.
Research, unanimously think newfashioned pseudorabies strain in major virulence gene gE, gI gene, and it is relevant to immunoprotection
There are the variations in multiple sites on gene gB, gC, gD.The new popular strain of China different regions separation, and is widely used at present
Pseudorabies disease live-vaccine Bartha K61 strain be located at different gene hypotypes, animal experiment is demonstrate,proved with neutralizing antibody detection
Reality, porcine pseudorabies virus protecting effect is weaker currently popular to China for Bartha K61 strain, and present invention use newly separates
Variation strain constructs the pseudorabies living vaccines of 6 gene delection such as TK, gE, gI, 11K, 28K and gG, and uses in the market
The certain vaccine of TK/gE/gI, gE/gI gene compare, which can induce body to generate the interferon of more advanced dosage,
There is better effect in terms of urgent immunity inoculation.The gene-deleted vaccine constructed using popular strain can be provided for piglet
100% immunoprotection.
Based on this, on the one hand, the present invention provides a kind of porcine pseudorabies virus of six gene delections, the porcine pseudorabies virus
Lack following gene: TK, gE, gI, 11K, 28K and gG.
Further, in some embodiments of the present invention, deposit number is CGMCC No:16290.
Porcine pseudorabies virus provided by the invention be lacked on the basis of PRV-FJ plants of Pseudorabies virus TK, gE, gI,
Six genes of 11K, 28K and gG obtain the porcine pseudorabies virus of six gene delections of the invention after gene editing.
The strain was preserved in positioned at the Chinese micro- of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on 08 29th, 2018
Biological deposits administration committee common micro-organisms center (CGMCC), taxology name: porcine pseudorabies virus
(Pseudorabies virus)；Deposit number: CGMCC NO.16290.
By above-mentioned 6 gene delection porcine pseudorabies virus according to 108TCID50/ only dosage intramuscular injection mouse and rabbit it is equal
Animal morbidity and death are not caused.Use 5 × 108TCID501 age in days piglet of virus liquid collunarium and intramuscular injection, does not cause 1 age in days
Piglet morbidity, the six gene delection porcine pseudorabies poison strain do not have pathogenic.In addition, mad using the six gene delection pig puppet
Vaccine made of dog disease poison strain, is verified through Study On Immunogenicity, after by vaccine, be can induce body and is generated more advanced dosage
Interferon, gE antibody test result is feminine gender, and gB antibodies positive, neutralizing antibody value is improved.
On the other hand, the present invention provides the porcine pseudorabies virus of above-mentioned six gene delection in preparing pseudorabies vaccines
Application.
In another aspect, the present invention provides a kind of pseudorabies disease vaccines comprising six gene delection as described above
Pseudorabies virus and stabilizer.
Further, in some embodiments of the present invention, the stabilizer include: oligosaccharide, amino acid, gelatin and
Protein zymolyte.
Further, in some embodiments of the present invention, the oligosaccharide is the combination of sucrose and trehalose.
Further, in some embodiments of the present invention, amino acid is sodium glutamate.
Further, in some embodiments of the present invention, protein zymolyte is lactoalbumin hydrolysate.
Further, in some embodiments of the present invention, based on mass volume ratio, the stabilizer contains: 5%-
10% sucrose (i.e. the sucrose containing 5-10g in 100ml stabilizer, hereinafter herewith), 1%-5% trehalose, 1%-5% glutamic acid
Sodium and 2%-10% lactoalbumin hydrolysate, the gelatin of 0.5%-1%, surplus are water.
On the other hand, the present invention provides a kind of preparation methods of pseudorabies disease vaccine as described above comprising such as
Lower step:
Step (a): the porcine pseudorabies virus of six above-mentioned gene delections is inoculated with suitable cell, harvests virus liquid；
Step (b): the virus liquid of harvest is mixed with stabilizer.
Further, in some embodiments of the present invention, above-mentioned preparation method further includes step (c): by virus liquid
The pseudorabies disease vaccine of finished product is made through vacuum freeze drying with the mixed solution of stabilizer.
Further, in some embodiments of the present invention, the stabilizer include: oligosaccharide, amino acid, gelatin and
Protein zymolyte.
Further, in some embodiments of the present invention, the oligosaccharide is the combination of sucrose and trehalose.
Further, in some embodiments of the present invention, amino acid is sodium glutamate.
Further, in some embodiments of the present invention, protein zymolyte is lactoalbumin hydrolysate.
Further, in some embodiments of the present invention, by mass percentage, the stabilizer contains: 5%-
10% sucrose, 1%-5% trehalose, 1%-5% sodium glutamate and 2%-10% lactoalbumin hydrolysate, 0.5%~1% it is bright
Glue, surplus are water.
Further, in some embodiments of the present invention, in step (a), the virus liquid and stabilizer according to
The ratio of 1:1-1:10 mixes.
Further, in some embodiments of the present invention, the suitable cell is BHK21 cell, ST cell, 293T
Cell, PK15 cell, Vero cell or MDBK cell, preferably BHK21 cell.
The Preparation Method provided using the invention of this hair, can be made pseudorabies disease vaccine, which contains six above-mentioned bases
Pathogenic to be removed because of the porcine pseudorabies virus of missing, immunogenicity is effectively retained, and is had using the vaccine preferable
Immune effect.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is immune rear porcine alpha-IFN testing result.
Fig. 2 is immune rear gB/gE on the 28th and neutralizing antibody testing result, and note: gB/gE antibody test, S/N value is less than 0.6
For the positive, it is greater than 0.7 for feminine gender, 0.6 to 0.7 is suspicious.
Fig. 3 is protest test temperature curve figure.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
Artificial reconstructed Pseudorabies virus
1, strain
PRV-FJ plants, by livestock and poultry infectious disease Key Laboratory of Sichuan Province from Fujian pseudorabies morbid pig pathological material of disease in 2015
Separation obtains；Morbid pig is mainly shown as that body temperature increases, loss of appetite, the symptoms such as sneezing.PRV-FJ plants with 107TCID50/ head
Dosage infect 28 age in days weanling pigs, impassivity symptom, attack after poison 7 days it is all dead.
ST cell and 293T cell, to contain the DMEM culture solution culture (Gibco company) of 10% fetal calf serum.
2, construction of recombinant vector
Artificial synthesized SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, then by SEQ ID NO.1 directed cloning
To at the Ase1 restriction enzyme site of pEGFP-C1 carrier, it is named as pEGFP-gI, SEQ ID NO.2 orientation gram is arrived into pEGFP-
At the Mlu1 restriction enzyme site of gI, it is named as pEGFP-gI28K.By SEQ ID NO.3 directed cloning to pUC57 carrier
Between EcoR1 and HindIII restriction enzyme site, it is named as pUC-TK.
3, gene editing vector construction
TK, gE, gG are constructed according to LentiCRISPR v2 carrier (Addgene company) specification method using 1 primer of table
The carrier of gene editing is respectively designated as carrier psgRNA-TK, psgRNA-gE, psgRNA-gGA and psgRNA-gGB.
GRNA only cuts viral nucleic acid open, and the corresponding gRNA of TK is recombinated after cutting TK open with pUC-TK,
To lack TK Gene Partial sequence, gE gRNA is recombinated after cutting viral genome open with pEGFP-gI28K, thus
Lack gI, gE, 11K, 28K gene.PsgRNA-gGA and psgRNA-gGB two join together to cut away a segment of gG from
And lack gG gene.
TK and gG is individually in some position in Pseudorabies virus, and tetra- genes of gI, gE, 11K and 28K are adjacent, therefore make
4 genes can be lacked simultaneously with a gRNA and corresponding homologous sequence.
1 gRNA of table constructs primer information
4, recombinant virus constructs
Conventionally extract PRV-FJ pnca gene group DNA；By PRV-FJ genomic DNA (3 μ g), pUC-TK plasmid (5
μ g) and psgRNA-TK plasmid (5 μ g) mixing after referring to Lipofectamine 3000 (Thermo fisher scientific)
Specification transfects the good 293T cell of growth conditions, and 37 DEG C of cultures are to there is cytopathy after transfection.Viral supernatants are taken to be inoculated with
BHK21 cell selects the thermophilic spot of single virus.The thermophilic spot inoculation BHK21 cell of the virus selected is taken, virus liquid is harvested, according to routine
Method extracts viral DNA, uses primer TKF:CATCCTCCGGATCTACCTCGACGGC and TKR:
CACACCCCCATCTCCGACGTGAAGG is referring to Lyophilized HS Taq PCR Master Mix (TAKARA) kit
Specification carries out PCR amplification, and the sample that amplified fragments are about 680bp is selected to carry out sequencing.Disease will be purified after sequencing
Malicious rPRV-FJ-delTK.
RPRV-FJ-delTK genome is conventionally extracted, genomic DNA (3 μ g), the pEGFP- of extraction are taken
GI28K plasmid (5 μ g) and psgRNA-gE are referring to Lipofectamine 3000 (Thermo fisher scientific) explanation
Book transfects the good 293T cell of growth conditions, and 37 DEG C of cultures are to there is cytopathy after transfection.Take viral supernatants inoculation ST thin
Born of the same parents, the thermophilic spot of picking green fluorescence carry out the thermophilic spot purifying of three-wheel, purified virus are named as rPRV-FJ-del5gene-EGFP.
PCDNA3.1-CRE carrier is said referring to Lipofectamine 3000 (Thermo fisher scientific)
Bright book transfects well-grown 293T cell, and cell, occurs in 24 hours inoculation rPRV-FJ-del5gene-EGFP virus after transfection
Cell conditioned medium is taken to be inoculated with ST cell after lesion, the thermophilic spot of picking unstressed configuration carries out the thermophilic spot purifying of three-wheel, purified virus is named as
RPRV-TIE18 plants.
Plasmid psgRNA-gGA (2 μ g) and psgRNA-gGB (2 μ g) is taken to transfect well-grown 293T cell, 37 after transfection
DEG C culture, rPRV-TIE18 plants of inoculation in 24 hours continue 37 DEG C and cultivate to there is cytopathy.It is thermophilic to collect the progress of virus liquid supernatant
Spot clone selects single thermophilic spot inoculation BHK21 cell, harvests viral supernatants.Viral DNA is conventionally extracted, is then used
GGup:GCACCTGATCGACCTCATCC and gG-Down:AAGATGGACACCCGGTGAGA is referring to Lyophilized HS Taq
PCR Master Mix (TAKARA) kit specification carry out PCR amplification, select amplified fragments lower than 1860bp sample into
Row sequencing.The viral sample that SEQ ID NO.4 has been lacked after sequencing is named as PRV-TIE18G.
5, the preservation of recombinant virus
PRV-TIE18G virus after identification is conventionally inoculated with BHK21 cell, culture is harvested to after there is CPE
Viral supernatants, -80 DEG C of preservations, are denoted as F0 generation after packing.The strain was preserved in positioned at Beijing's southern exposure on 08 29th, 2018
The China Microbiological preservation administration committee common micro-organisms center (CGMCC) of the institute 3 of area North Star West Road 1, taxology name:
Porcine pseudorabies virus (Pseudorabies virus)；Deposit number: CGMCC NO.16290.
Embodiment 2
PRV-TIE18G cell adaptation and pure property are examined
The PRV-TIE18G deposited that goes bail for is viral, and conventional method is inoculated with BHK21 cell, and culture harvests virus to after there is CPE
Supernatant.By PRV-TIE18G virus continuous passage to F5.Virus TCID50 measurement is carried out with BHK21 cell.According to " Chinese veterinary drug
Allusion quotation " method recorded of (2015 editions) annex carries out sterile, mycoplasma to different generation viruses, exogenous virus is examined.
2 PRV-TIE18G cell adaptation of table and pure property are examined
| Viral generation | F0 | F1 | F2 | F3 | F4 | F5 |
| Viral level (TCID50/ml) | 107.6 | 108.25 | 108.0 | 108.4 | 108.4 | 108.16 |
| Steriling test | It is negative | It is negative | It is negative | It is negative | It is negative | It is negative |
| Mycoplasma is examined | It is negative | It is negative | It is negative | It is negative | It is negative | It is negative |
| Exogenous virus is examined | It is pollution-free | It is pollution-free | It is pollution-free | It is pollution-free | It is pollution-free | It is pollution-free |
PRV-TIE18G virus F0 is 10 for viral level7.6TCID50/ml；Continuous passage is to F5 generation, F1 generation to F5 generation
Viral level is 108.0TCID50/ ml or more.Steriling test, mycoplasma inspection result are feminine gender, exogenous virus inspection result
Display is polluted without exogenous virus.
Embodiment 3
The pathogenicity of PRV-TIE18G
1~3 age in days PRV negative antibody piglet 35, is randomly divided into 6 groups and is respectively labeled as A, B, C, D, E, F, G group, selects
PRV-TIE18G virus F1 generation carries out challenge test according to following table (table 3).It is observed continuously 14 after attacking poison.
Pathogenicity of table 3 PRV-TIE18G and PRV-FJ to newborn piglet
| Group | Attack poison strain | Virus passages | Attack toxic dose | Attack malicious mode | Remarks |
| A | PRV-TIE18G | F1 | 108TICD50 | Collunarium inoculation | |
| B | PRV-TIE18G | F1 | 107TICD50 | Collunarium inoculation | |
| C | PRV-TIE18G | F1 | 108TICD50 | Intramuscular injection | |
| D | PRV-TIE18G | F1 | 107TICD50 | Intramuscular injection | |
| E | DMEM culture solution | \ | 2.0ml | Collunarium+intramuscular injection | Blank control |
| F | PRV-FJ | F6 | 107TICD50 | Collunarium | |
| G | PRV-FJ | F6 | 107TICD50 | Intramuscular injection |
It attacks and observes and measure piglet body temperature after poison daily, see whether pseudoabies clinical symptoms and death occur.As a result
Such as the following table 3.PRV-TIE18G collunarium and intramuscular injection, which are attacked poison and organized, as the result is shown does not occur body temperature raising and other clinical symptoms,
And collunarium and intramuscular injection attack malicious PRV-FJ have within piglet second day after attacking poison piglet occur body temperature increase (see Fig. 3), play spray
The symptoms such as sneeze, attack piglet death in the 3rd day after poison, after attacking poison the 4th day it is all dead.Blank control group does not occur any clinical condition
Shape is tested normal in whole process.
Table 4 PRV-TIE18G and PRV-FJ counts the pathogenicity test results of newborn piglet
| Group | Fever | Respiratory symptom | Nervous symptoms | The dead quantity |
| A | 0/5 | 0/5 | 0/5 | 0/5 |
| B | 0/5 | 0/5 | 0/5 | 0/5 |
| C | 0/5 | 0/5 | 0/5 | 0/5 |
| D | 0/5 | 0/5 | 0/5 | 0/5 |
| E | 0/5 | 0/5 | 0/5 | 0/5 |
| F | 5/5 | 5/5 | 0/5 | 5/5 |
| G | 5/5 | 4/5 | 0/5 | 5/5 |
Embodiment 4
Pseudorabies disease live-vaccine PRV-TIE18G's the preparation method is as follows:
Routine passage BHK21 cell carries out passage BHK21 cell according to " Chinese veterinary pharmacopoeia " 2015 version record method
Steriling test, mould are examined, mycoplasma is examined and exogenous virus is examined.
It is cultivated to 15L rolling bottle, after cell covers with single layer using examining qualified BHK21 cell conventionally to expand
Culture medium is sucked out, according to 0.5% ratio inoculation PRV-TIE18G kind poison, 37 DEG C are adsorbed 30 minutes, and then each rolling bottle adds
Add 1000ml DMEM serum-free medium.
37 DEG C are continued to cultivate, primary every observation in 12 hours.When observing that cytopathy reaches 90% or more, rolling bottle is harvested
Inner virus liquid.2~8 DEG C of preservations after cell fragment are filtered away using 10um filter core.According to " Chinese veterinary pharmacopoeia " 2015 editions
The TCID50 of the method measurement virus liquid of record.
It is prepared according to the ratio of sucrose 5%, trehalose 2%, lactoalbumin hydrolysate 6%, sodium glutamate 3%, gelatin 0.85%
Freeze drying protectant.115 DEG C of room temperatures after high pressure sterilization 30 minutes save backup.By freeze drying protectant and virus liquid according to the ratio of 1:1
Quantitative separating to 10ml cillin bottle, capping is placed in freeze dryer after example mixing, pre-cooled, drying process freeze dried vaccine.Freeze-drying
Gland is placed in 2~8 DEG C of preservations.
Vaccine after taking freeze-drying is according to " Chinese veterinary pharmacopoeia " 2015 editions progress steriling tests, mycoplasma inspection, exogenous virus
It examines and viral level measures.
Embodiment 5
The immunogenicity of pseudorabies disease live-vaccine PRV-TIE18G
3-4 week old porcine pseudorabies virus antigen-antibody feminine gender piglet 20 are selected, is randomly divided into tetra- groups of A, B, C, D, every group 5
Head.According to the form below (table 5) carries out intramuscular injection immunity inoculation, and 1-7 days after being immunized, daily blood sampling carries out porcine alpha-IFN detection；It is immune
The separation of blood sampling on the 28th serum carries out PRV gB (IDEXX), gE (IDEXX), neutralizing antibody (PRV-FJ neutralization) detection afterwards；28 after exempting from
After day blood sampling, every test pig collunarium is inoculated with PRV-FJ (107TICD50/ head) carry out protest test.
The grouping of 5 PRV-TIE18G Study On Immunogenicity of table
| Group | Strain | Generation | Immunizing dose | Attack poison strain | Attack toxic dose |
| A | PRV-TIE18G | F1 | 105.0TICD50 | PRV-FJ | 107TICD50 |
| B | PRV-TIE18G | F5 | 105.0TICD50 | PRV-FJ | 107TICD50 |
| C | Bartha-K61 | \ | 105.5TICD50 | PRV-FJ | 107TICD50 |
| D | DMEM | \ | 2.0ml | PRV-FJ | 107TICD50 |
2 PRV-TIE18G immune groups detect porcine alpha-IFN in the 2nd day to the 6th day after exempting from blood；Bartha-
K61 and control group inoculation are preceding to after being inoculated with the 7th day, do not detect in blood porcine alpha-IFN (Fig. 1).28 days, 2 after exempting from
The gB antibody of PRV-TIE18G immune group and Bartha-K61 immune group equal 5/5 is positive；GE antibody equal 5/5 is negative；Control group gB,
GE equal 5/5 is negative.The neutralizing antibody level ratio Bartha-K61 immune group to PRV-FJ of 2 PRV-TIE18G immune groups is more
Height, control group is without neutralizing antibody (Fig. 2).
Progresss protest test on the 28th after immune, 2 PRV-TIE18G immune groups do not occur heating paresthesia, spiritual,
Appetite is normal, and all survivals on the 14th are observed continuously；Bartha-K61 immune group 1/5 occurs generating heat and appetite stimulator, is observed continuously
It survives 4, dead 1 within 14th；All there is heating paresthesia in control group, lassitude, appetite stimulator occurs, observes 14 extremely
Die 3.
6 PRV-TIE18G Study On Immunogenicity protest test result of table statistics
| Group | Fever | Spiritual loss of appetite | Nervous symptoms | The dead quantity | As a result |
| A | 0/5 | 0/5 | 0/5 | 0/5 | 5/5 protection |
| B | 0/5 | 0/5 | 0/5 | 0/5 | 5/5 protection |
| C | 1/5 | 1/5 | 0/5 | 1/5 | 4/5 protection, 1/5 is dead |
| D | 5/5 | 5/5 | 0/5 | 5/5 | 5/5 morbidity, 5/5 is dead |
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Sichuan Huashen Animal Biolog Products Co., Ltd.
<120>a kind of porcine pseudorabies virus, pseudorabies disease vaccine and the preparation method of six gene delections
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1151
<212> DNA
<213>artificial sequence
<400> 1
ggcgtgaaca tcctcaccga cttcatggtg gcgctccccg aggggcaaga gtgcccgttc 60
gcccgcgtgg accagcaccg cacgtacaag ttcggcgcgt gctggagcga cgacagcttc 120
aagcggggcg tggacgtgat gcgattcctg acgccgttct accagcagcc cccgcaccgg 180
gaggtggtga actactggta ccgcaagaac ggccggacgc tcccgcgggc ctacgccgcc 240
gccacgccgt acgccatcga ccccgcgcgg ccctcggcgg gctcgccgag gcccaggccc 300
cggccccggc ccaggccccg gccgaagccc gagcccgccc cggcgacgcc cgcgcccccc 360
ggccgcctgc ccgagccggc gacgcgggac cacgccgccg gggggcgccc cacgccgcga 420
cccccgaggc ccgagacgcc gcaccgcccc ttcgccccgc cggccgtcgt gcccagcggg 480
tggccgcagc ccgcggagcc gttcccgccc cggaccaccg ccgcgccggg cgtctcgcgc 540
caccgctcgg tgatcgtcgg cacgggcacc gcgatgggcg cgctcctggt gggcgtgtgc 600
gtctacatct tcttccgcct gaggggggcg aaggggtatc gcctcctggg cggtcccgcg 660
gacgccgacg agctaaaagc gcagcccggt ccgtagcctc cgcagtaccg gcgtcgatga 720
tgatggtggc gcgcgacgtg acccggctcc ccgcggggct cctcctcgcc gccctgaccc 780
tggccgccct gaccccgcgc gtcgggggcg tcctcttcag gggcgccggc gtcagcgtgc 840
acgtcgccgg cagcgccgtc ctcgtgcccg gcgacgcgcc caacctgacg atagacggga 900
cgctgctgtt tctggagggg ccctcgccga gcaactacag cgggcgcgtg gagctgctgc 960
gcctcgaccc caagcgcgcc tgctacacgc gcgagtacgc cgccgagtac gacctctgcc 1020
cccgcgtgca ccacgaagcc ttccgcggct gcctgcgcaa gcgcgagccg ctcgcccggc 1080
gcgcgtccgc cgcggtggag gcgcgccggc tattaatata acttcgtata gcatacatta 1140
tacgaagtta t 1151
<210> 2
<211> 999
<212> DNA
<213>artificial sequence
<400> 2
atctcccccg gctcgctggc cctgctgccg cgcgccgtgc gccccgtcgt gcggacgcgg 60
tccgacccca cggcgccgtt ctacatcacc accgagacgc acgagctgac gcggcgcccc 120
ccggcggacg gctcgaagcc cggggagccc ctcaggatca gcccaccccc gcggctggac 180
acggagtggt cgtccgtcct gaacgggatc cagtacctga actcgggggc ccggggcacg 240
gcccccgtcc acctgtggat cctgggcgcc gccgacctct gcgaccaggt gctcctggcc 300
gcctcccgca gcaccgccgc cggagcctcc cacgcccaga cgggcgcgcg cctgacccgg 360
cgccggcccg ggctgacgga cgccgacgcc ctggacgtga tcgtcgccgg gatccaggcg 420
acccgcgcca tgttcgcgcg ggtccacaac cgctcctggc gccacgccgg cgagtggacg 480
gaggccctgc actcccagat cgtgacccgg ggcgacgtgc gccggcgccg aggcgggcgc 540
ggcaacggac gcgagcgcgc cccgcgatgt accatctcct agacggcagg atctctccgc 600
gtcccccacc cccccaaaaa acaaacaata aacgctctcg ctctggcacc cgatgacacg 660
cctccgtcct ctctctccct cccactgacg ccacccctcc cctcgccgac aacgccatcg 720
tcgcccggcg tcggccggac cggcggttct ccccccaccc cgtccccccc caccccgtcc 780
ccccccaccc ctgcccccgc ttcgtccgac tctcgccccc cgcgggaggg ttccgcggct 840
cgctccccgt ctcatccccc cgtctcatcc ccccgtctca ctcccatctc cctccctcca 900
ccccgtctca tccccccatc tcccttcccc acgagggccg ggaggggaaa aaacgcccga 960
gagacgagag agttgaggtt cgagcggcgg gccgccgtg 999
<210> 3
<211> 1000
<212> DNA
<213>artificial sequence
<400> 3
ccactgcccg ggtgatggcg ctcggcgggg cgctgtacgt gcccgagccg atggcgtact 60
ggcgcactct gttcgacacg gacacggtgg ccggtattta cgatgcgcag acccggaagc 120
agaacggcag cctgagcgag gaggacgcgg ccctcgtcac ggcgcagcac caggccgcct 180
tcgcgacgcc gtacctgctg ctgcacacgc gcctggtccc gctcttcggg cccgcggtcg 240
agggcccgcc cgagatgacg gtcgtctttg accgccaccc ggtggccgcg acggtgtgct 300
tcccgctggc gcgcttcatc gtcggggaca tcagcgcggc ggccttcgtg ggcctggcgg 360
ccacgctgcc cggggagccc cccggcggca acctggtggt ggcctcgctg gacccggacg 420
agcacctgcg gcgcctgcgc gcccgcgcgc gcgccgggga gcacgtggac gcgcgcctgc 480
tcacggccct gcgcaacgtc gacctggggc cctcgccgcg cgtctgcgcc gcggccgtgg 540
cggcgcagac gcgcggcatg gaggtgacgg agtccgcgta cggcgaccac atccggcagt 600
gcgtgtgcgc cttcacgtcg gagatggggg tgtgaccctc gcccctccca cccgcgccgc 660
ggccagatgg agaccgcgac ggaggcaacg acgacggcgt gggagggggc tcggggcgcg 720
tataaagcta tgtgtatgtc atcccaataa agtttgccgt gcccgtcacc atgcccgcgt 780
cgtccgtgcg cctcccgctg cgcctcctga ccctcgcggg cctcctggcc ctcgcggggg 840
ccgccgccct cgcccgcggc gcgccgcagg gtgggccgcc ctcgccgcag gggggtcccg 900
cgcccaccgc ggcgcccgcg cgcgggccca ccctgttcgt cctggtcggc gacggctccg 960
cgtggttcgt cttccagctc ggcgggctgg gggcgctcaa 1000
<210> 4
<211> 618
<212> DNA
<213>artificial sequence
<400> 4
tcgtggtccg caccgtcgtg gccagagagg cccctcggga gctctgctac ggccaccccg 60
tccacgacga ccggcggccc gtcgggcccg cgaccgacgc ccagcccgtg aacccgctcg 120
cccccgccaa cgccaccggg acggactact ctcgcggctg cgagatgcgc ctcctggatc 180
cgcctctcga cgtatcgtcc cgctcctcgg accccgtcaa cgtgaccgtc gcctggttct 240
ttgacggcgg ccactgcaag gtgcccctcg tccaccgcga gtactacggc tgccccgggg 300
acgccatgcc ctccgtcgag acgtgcaccg gcgggtactc gtacacccgc acgcgcatcg 360
acaccctgat ggagtacgcc ctcgtgaacg ccagcctcgt gctgcagccc gggctgtacg 420
acgccggcct gtacatcgtc gtgctcgtct ttggcgacga cgcctacctc ggcaccgtct 480
ccctgtcggt ggaggccaac ctggactacc cctgcggcat gaagcacggg ctcacgatca 540
cccgccccgg ggccaccctc ccacccatcg cccccacggc cggcgaccac cagcgctggc 600
gcgggtgctt cccctcga 618
Claims (10)
1. a kind of porcine pseudorabies virus of six gene delections, which is characterized in that the following gene of porcine pseudorabies virus missing: TK,
GE, gI, 11K, 28K and gG.
2. the porcine pseudorabies virus of six gene delection according to claim 1, which is characterized in that its deposit number is
CGMCC No:16290.
3. the porcine pseudorabies virus of six gene delection of any of claims 1 or 2 is preparing the application in pseudorabies vaccines.
4. a kind of pseudorabies disease vaccine, which is characterized in that it includes the pig puppet of six gene delection of any of claims 1 or 2
Rabies viruses and stabilizer.
5. a kind of preparation method of pseudorabies disease vaccine as claimed in claim 4, which is characterized in that it includes following step
It is rapid:
Step (a): the porcine pseudorabies virus of six gene delection described in claim 1 is inoculated with suitable cell, harvests virus liquid；
Step (b): the virus liquid of harvest is mixed with stabilizer.
6. preparation method according to claim 5, which is characterized in that the stabilizer includes: oligosaccharide, amino acid, bright
Glue and protein zymolyte.
7. preparation method according to claim 6, which is characterized in that the oligosaccharide is the combination of sucrose and trehalose；
Preferably, amino acid is sodium glutamate；
Preferably, protein zymolyte is lactoalbumin hydrolysate.
8. preparation method according to claim 6, which is characterized in that by mass percentage, the stabilizer contains:
5%-10% sucrose, 1%-5% trehalose, 1%-5% sodium glutamate and 2%-10% lactoalbumin hydrolysate, 0.5%~1%
Gelatin, surplus are water.
9. according to the described in any item preparation methods of claim 5-8, which is characterized in that in step (a), the virus liquid with
Stabilizer is mixed according to the ratio of 1:1-1:10.
10. according to the described in any item preparation methods of claim 5-8, which is characterized in that the suitable cell is that BHK21 is thin
Born of the same parents, ST cell, 293T cell, PK15 cell, Vero cell or MDBK cell, preferably BHK21 cell.
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| CN112501133A (en) * | 2020-12-01 | 2021-03-16 | 山东信得科技股份有限公司 | Pseudorabies virus QD strain three-gene deletion weakening strain |
| CN114657151A (en) * | 2022-02-25 | 2022-06-24 | 广东海大畜牧兽医研究院有限公司 | Porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain and construction method and application thereof |
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| CN112501133A (en) * | 2020-12-01 | 2021-03-16 | 山东信得科技股份有限公司 | Pseudorabies virus QD strain three-gene deletion weakening strain |
| CN114657151A (en) * | 2022-02-25 | 2022-06-24 | 广东海大畜牧兽医研究院有限公司 | Porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain and construction method and application thereof |
| CN114657151B (en) * | 2022-02-25 | 2024-03-12 | 广东海大畜牧兽医研究院有限公司 | Porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain, construction method and application thereof |
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