CN104877972B - A kind of dual-gene gene-deleted strain of recombinant porcine pseudorabies poison gE/gI and its application - Google Patents
A kind of dual-gene gene-deleted strain of recombinant porcine pseudorabies poison gE/gI and its application Download PDFInfo
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Abstract
It is an object of the invention to provide a kind of porcine pseudorabies virus vaccine; it is made up of antigen and protective agent, wherein antigen includes the attenuated viral strains made of the porcine pseudorabies virus strain that deposit number is CGMCC No.10266 carries out after virulence gene gE and gI missing.Vaccine prepared by the present invention can effectively prevent porcine pseudorabies; and as the porcine pseudorabies virus of antigen it is gene-deleted strain; infected by horizontal transmission in Mice Body continuous passage; it is showed no virulence and returns strong phenomenon; genetic stability; meet porcine pseudorabies virus deletion of vaccine strain avirulence and return strong standard, manufactured vaccine can provide effective immunoprotection, have good commercialized development prospect.
Description
Technical field
The invention belongs to veterinary vaccines preparing technical field, and in particular to a kind of recombinant porcine pseudorabies poison gE/gI is biradical
Because of gene-deleted strain and its application.
Background technology
Porcine pseudorabies virus PRV (Pseudorabies virus) belongs to herpetoviridae a herpesviral subfamilies bubbles
Tobamovirus herpesvirus suis I types, pig are unique natural reservoir (of bird flu viruses) of the virus, cause pig pseudoabies (Pseudorabies,
PR).The disease is in outbreak of epidemic in swinery more, main harm sow group, causes Sow abortion or vertical transmission to be caused just to piglet
Cub's pig mortality, the pig industry to China or even the whole world bring huge economic loss.Having for treatment PR is there is no at present
Medicine is imitated, therefore vaccine inoculation turns into the generation of the control disease and popular major measure.Most popular vaccine master in the world
If PRV Bartha-K61 strain vaccines, but China's pseudorabies pestilence present situation was that the existing vaccine can not be complete in recent years
The new popular PRV of full guard attack, certain economic loss is caused to immune pig farm.Accordingly, it is desirable to provide cri dernier cri is malicious
Vaccine prepared by strain just turns into the study hotspot of porcine pseudorabies virus disease prevention and control field.
The content of the invention
It is an object of the invention to provide a kind of dual-gene gene-deleted strains of recombinant porcine pseudorabies poison gE/gI and its application, i.e., by
The vaccine that the gene-deleted strain of the porcine pseudorabies virus filtered out is prepared as antigen, vaccine of the invention is to currently a popular
The attack of porcine pseudorabies virus can provide good immune protection effectiveness.
Present invention firstly provides a kind of PRV gene-deleted strain, it by deposit number is CGMCC to be
No.10266 porcine pseudorabies virus strain is carried out made of after gE and gI gene delections.
Another aspect of the invention provides application of the above-mentioned PRV gene-deleted strain in vaccine is prepared；
Another aspect of the present invention provides a kind of porcine pseudorabies virus vaccine, is made up of antigen and protective agent, wherein
Antigen includes above-mentioned PRV gene-deleted strain.
Protective agent therein is the viral vaccine protective agent that uses at present, a kind of its specific composition of embodiment be sucrose and
The aqueous solution of gelatin, its mass percent final concentration in vaccine is respectively 20% and 4.8%.
Antibiotic, penicillin, streptomysin final concentration of 200 units/ml are added in above-mentioned vaccine；
Vaccine prepared by the present invention can effectively prevent porcine pseudorabies, and be base as the porcine pseudorabies virus of antigen
Because of gene-deleted strain, infected by horizontal transmission in Mice Body continuous passage, be showed no virulence and return strong phenomenon, genetic stability, meet
Porcine pseudorabies virus deletion of vaccine strain avirulence returns strong standard, and manufactured vaccine can provide effective immunoprotection, tool
There is good commercialized development prospect.
Embodiment
The invention will now be further described with reference to specific embodiments, and one of ordinary skill in the art is in the technology of the present invention
On the basis of scheme, method and step commonly used in the art can be selected, is not limited solely to the specific of description of the invention embodiment
Record.
Embodiment 1：The seed selection of CGMCC No.10266 porcine pseudorabies virus strains
In recent years, for the multiple pig farms in China all there occurs pseudoabies, wherein most is kind of a pig farm, and before the swinery that falls ill
Pseudo- rabies vaccine is injected, thus it is speculated that the virus of infection is made a variation；Therefore pseudorabies have been carried out from morbidity swinery
The screening of virus.
Morbidity haslet sample is taken, including：Heart, liver, lungs, spleen, tonsillotome and lymph node etc..By internal organ sample
With PBS (0.1M, pH7.2) with V/V1:5 are made homogenate, multigelation 3 times, 3000r/min centrifugation 15min, take in supernatant and add
Dual anti-, 1h is made in 37 DEG C of senses, degerming through 0.22 μm of membrane filtration.1ml virus filtrates are taken to be inoculated in the Vero cells for growing up to individual layer, it is blind
Pass three generations, observation cytopathy (CPE).The cell culture fluid that will appear from CPE carries out Plaque-purified, virus packing guarantor after purification
Deposit to -70 DEG C it is standby, and determine viral level.Choose the porcine pseudorabies virus QD strain (chimpanzee agents as vaccine development
Porcine herpesvirus Type I) it has been preserved in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on March 6th, 2015
Number China Committee for Culture Collection of Microorganisms's common micro-organisms center of Institute of Microorganism, Academia Sinica, deposit number
For CGMCC No.10266.
The strain screened for the present invention enters performing PCR detection, and blast analyses are carried out after sequencing, it is found that deposit number is
The gE genes of CGMCC No.10266 porcine pseudorabies virus with 2012 after report porcine pseudorabies virus it is relative
Although there is the difference of at least two amino acid in the sequence answered, but affiliation is nearer, be in a relatively independent branch
In, with the strain affiliation that separates before farther out.And the strain with separating recently has identical characterization of molecules, i.e., in gE bases
Respectively there is the insertion of 1 aspartic acid cause the 48th and 492-496 positions, and other reports also confirm that also this point.And it is previously separated
To strain only have position insertion amino acid individually, the overwhelming majority is not inserted into.Therefore above-mentioned difference can speculated just
It is where the reason for causing existing porcine pseudorabies virus immune effect of vaccine bad.
After the strain makees 100 times of dilutions, neutralized with equivalent porcine pseudorabies virus antiserum, virus can be by hyper-immune serum
Specificity neutralizes；And with equivalent pig parvoviral, swine influenza virus, CSFV, porcine circovirus 2 type, pig breeding with breathing
In syndrome virus, Porcine epidemic diarrhea virus and transmissible gastro-enteritis virus antiserum and group, cell present obvious thin
Born of the same parents' lesion, it is seen that the virus-specific is good.After vaccine immune BALB/C mouse prepared by the strain, the death of mouse can be reduced
Rate.
Embodiment 2：The structure of the dual-gene gene-deleted strains of recombinant porcine pseudorabies poison gE/gI
The DNA of the separation strains is extracted from the porcine pseudorabies virus (CGMCC No.10266) of separation, using gene work
The method of journey carries out the missing of virulence gene gE and gI gene to the separation strains, and is named as PRV/ after being saved on cell
gE-/gI-, numerous poison, add after protective agent as seed poison.Embodiment is as follows：
1PCR primers
With reference to PRV whole genome sequences (BK001744), serial primer is voluntarily synthesized, amplification is respectively intended to and is located at US7
(gI) the left arm fragment (L) available for homologous recombination of gene and US8 (gE) genes both sides (gI containing part and gE genes) and the right side
Arm pieces section (R).Wherein L includes part US6 genes and part gI genes, and R includes part gE genes, whole US9 genes and portion
Divide US2 genes.Design primer amplification EGFP and EGFP eukaryotic expression boxes simultaneously in addition, and for identifying drawing for gene delection
Thing.The size of primer sequence and expected PCR primer is shown in Table 1.
Table 1：The primer used in this research
The structure of 2gE and gI gene transfer vectors
Using PRV QD pnca genes groups as template, using primer gEILF/GEILR and gEIRF/GEIRR, position is amplified respectively
Sequence (gE containing part and gI genes) in gE and gI both sides recombinates the one of arm gEIL, gEIR, wherein gEIL, gEIR as left and right
End is respectively provided with a loxP site.Enter performing PCR and digestion identification to it after being cloned into pMD19-T, identify correct rear survey
Sequence, double digestion is carried out using Hind III and Pst I to correct positive colony T-gEIL is sequenced, the fragment is cloned into together
On the pBluescript SK carriers of sample digestion processing, using Spe I and Xba I respectively to the recombinant plasmid after identifying correctly
Double digestion is carried out with the recombinant plasmid vector for being connected with right side homology arm, the linearisation restructuring matter containing left side homology arm is separately recovered
Grain and right side homology arm, the two is attached.PCR and digestion are identified.Send sequencing after identification is correct, sequencing result is correct
Be named as pSKgEILR.
Using pCDNA3.1-EGFP plasmids as template, EGFP reading frames are expanded using primer EorfF/EorfR, are connected into eucaryon
Expression vector pVAX1, the eukaryotic expression containing EGFP is amplified using primer cassetteF/cassetteF after identifying correctly
Box, pMD19-T carriers are connected into, pMDEV is named as after identification is correct.
Double digestion is carried out to pMDEV using Pst I and Spe I, eukaryotic expression box is reclaimed, is connected into same double digestion
PSKgEILR, correct rear as gE and gI gene transfer vectors are identified, are named as pSKgE-EGFP.
The structure of 3gE and gI gene delection strain virus and purifying rescue
3.1 transfection
Recombinant virus rescue is carried out in six porocyte culture plates, is transfected when Vero cells cover with 90%.Take PRV
The μ g of QD pnca genes group 3, after being mixed with 1 μ g transferring plasmids pSKgE-EGFP, cotransfection according to a conventional method, method detailed referring to
LipofectamineTM2000 specifications.Set the only control group containing transferring plasmid simultaneously.
3.2 identification
PRV QD pnca genes groups are with after pSKgE-EGFP cotransfections 48h, observing transfectional cell lesion formational situation and fluorescence
Protein expression situation.In the generation of blind passage two after transfectional cell cracking, still there are cytopathy and green fluorescence, tentatively judge that recombinant virus is saved
Rescue success.Recombinant virus is identified through plaque purification and PCR, is named as PRV/gE-/gI-/EGFP。
The rejecting of 3.3 gene-deleted strain reporter genes
Reporter gene EGFP is rejected using the site-specific recombination of Cre-loxP System-mediateds.10 μ g are taken through Cre
The DNA that ferment treatment is crossed, using calcium phosphate procedure transfected Vero cells, put 37 DEG C of CO22-3d is cultivated in incubator, treats that cell produces
Virus liquid is harvested during raw 80% lesion.Virus liquid multigelation is taken into supernatant afterwards three times, being inoculated in 24 orifice plates by 2uL/ holes grows up to
The Vero cells of individual layer, low melting-point agarose is spread when Jian begins and cytopathy occurs, picking does not have fluorescence under fluorescence microscope
Plaque.Purified virus so repeatedly, until all virus plaques, without the prominent light of green, it is PRV/ to name the gene-deleted strain
gE-/gI-。
3.4PRV/gE-/gI-Identification
Extract PRV/gE-/gI-Genomic DNA, enter performing PCR with primer gEIF/gEIR and primer gBF/gBR and expand, identification
Whether the viral gene interior segments of rescue lack, if parent plant, PRV/gE-/gI-/ EGFP gene group DNA is as control.
The genetic stability detection of 4 recombinant viruses
The primary PRV/gE obtained will be screened-/gI-Recombinant virus continuous passage on Vero cells, every 5 generation extraction infection
Cell STb gene, carry out the PCR detections of lack part gene.
5PRV/gE-/gI-Seed culture of viruses safety testing
With PBS by 10 times of antigen diluent, 100 grams of intramuscular inoculation mouse four, every 0.2mL, observe 14 days, its react or
Dead must not exceed two.
6PRV/gE-/gI-Immune efficacy detection
The BALB/C mice of 6 week old is randomly divided into 3 groups, every group 5 and weighed, vaccinates a plant Bartha- respectively
K61, gene-deleted strain PRV/gE-/gI-And DMEM.The μ L DMEM of every injection of control group 100, other two groups of hindlimb muscles injections
104TCID50The vaccine of viral dosage.The clinical symptoms of daily observation mouse, whether there is One's spirits are drooping, apocleisis, itch, shake after immune
Quiver, 21d weighs again, and in 14d, 21d tail vein blood, serum is separated, with the coated ELISA of PRV QD strain totivirus
Plate detects antibody level of serum.21d attacks poison with PRV QD strains after immune, with 104TCID50Each test group of dose inoculation and control
Group, injected using hindlimb muscle, Continuous Observation 14d after virus inoculation.
7 results
Using PRV QD strain virus as template, gE and gI gene transfer vectors are successfully obtained；It is then that it is same with PRV genomes
Source recombinates, and successfully saves and is purified into gE the and gI gene delection strain virus containing EGFP marker gene；Using Cre recombinases
After removing EGFP marker gene, gE the and gI gene delection strain virus for not containing EGFP marker gene is successfully saved and is purified into
(PRV/gE-/gI-)。
The PRV/gE obtained will be screened-/gI-The continuous passage on Vero cells of primary recombinant virus, every 5 generation extraction infection
Cell STb gene, the PCR testing results of lack part gene show that the size of gene is the size after deletion fragment, show this
Recombinant virus stability is good.
Seed culture of viruses safety testing result shows that the vaccine on mouse is safe, no mad dog specific symptom of puppet, does not influence
Grow.Porcine pseudorabies virus gene-deleted vaccine (the PRV/gE that the present invention transforms-/gI-) it is safe, it can be used for
Prepare vaccine.
21d, Bartha-K61, PRV/gE after immune-/gI-Group and DMEM control group mice average weight gains be respectively 4.4g,
4.1g, 4.5g, weightening difference is little, illustrates weightening unrestraint effect of the gene-deleted strain to mouse.Do not examined yet during 14d after immune anti-
Body, PRV/gE during 21d-/gI-Group antibody level is significantly raised, and other groups are still without significant change.Show PRV/gE-/gI-Missing
Strain can induce mouse and produce obvious immune response.After immune 21d poison, PRV/gE are attacked with PRV QD strains-/gI-Group protective rate is obvious
Higher than Bartha-K61 groups, protective rate is respectively 100% and 20%.
The preparation and application of the porcine pseudorabies virus gene-deleted vaccine of embodiment 3
1 material
1.1 seed culture of viruses
Vaccine porcine pseudorabies virus is manufactured, deposit number is：CGMCC No.10266.
1.2 experimental animal
BALB/C small white mouses, purchased from Shandong University's Experimental Animal Center.
1.3 seedling Other Instruments, reagent
There is provided by Shandong Sinder Technology Co., Ltd..
2 methods
2.1 seedling processes
Vero cells are cultivated according to a conventional method, with 5-10PFU virus infected cells, when cytopathy (CPE) is left up to 90%
Virus is harvested when right, puts -80 DEG C of multigelations three times, determines the TCID of virus50.According to the malicious valency of harvest virus liquid, fitted
When dilution, virus liquid after dilution is 1 with protectant volume ratio:1.5, it is sufficiently mixed, wherein sucrose final concentration of 20%,
Gelatin final concentration of 4.8%.Per bottled 2.5mL, freezed in juxtaposition freeze dryer.
2.2 product inspection
2.2.1 character
Appearance color, character and the bottle wall for observing vaccine depart from situation and dissolve situation after adding dilution.
2.2.2 steriling test
By version in 2010《Chinese veterinary pharmacopoeia》Annex method is tested.
2.2.3 mycoplasma is examined
By version in 2010《Chinese veterinary pharmacopoeia》Annex method is tested.
2.2.4 exogenous virus is examined
By version in 2010《Chinese veterinary pharmacopoeia》Annex method is tested.
2.2.5 safety verification
With the BALB/C mice 10 of 6 week old, intramuscular injection 0.2ml (containing 10 plumage parts) vaccine, observe 21, should all be good for
It is living, without any locally or systemically adverse reaction.
2.2.6 efficacy test
Following method, which is appointed, selects one.
2.2.6.1 cellular assay is used
The plumage part indicated by label, is diluted to l plumages part/0.2ml with 10%DMEM by vaccine, remakes 10 times and be serially diluted,
Take 10-2、10-3、10-4、10-5Four dilution factors, are inoculated with the good Vero cells of growth conditions respectively, and each dilution factor is inoculated with 8
Hole, per hole 0.2m1,8 holes are separately taken to be used as control for inoculation 10%DMEM.37 DEG C of incubation CO2Carried out in incubator after being incubated 3,
Lesion situation is observed, TCID is calculated by Reed-Muench methods50, viral level should >=104.0TCID50/0.2ml。
2.2.6.2 examined with mouse
Take 6 week old BALB/C mice 10, every plumage part of intramuscular injection vaccine 1.After 21 days, together with 10 control mices,
Hindlimb muscle injection 104.0TCID50/ only, observe 14.Control group at least 8 death or pseudo- mad dog specific symptom should occur,
Immune group should at least 8 protections.
2.2.7 residual moisture determines
By version in 2010《Chinese veterinary pharmacopoeia》Annex method is carried out.
2.2.8 vacuum determines
By version in 2010《Chinese veterinary pharmacopoeia》Annex method is carried out.
2.2.9 vaccine using effect is examined
15 first 30 age in days piglets are chosen before experiment, 3 groups, every group 5 is randomly divided into and weighs, vaccinate strain respectively
Bartha-K61, gene-deleted strain PRV/gE-/gI-Vaccine and DMEM.Every injection 2mL DMEM of control group, other two groups of hindlimb muscles
Injection 105.0TCID50The vaccine of viral dosage.Carry out within 4th week attacking poison, intramuscular injection 10 after immune7.0TCID50PRV QD strain strains
Virus, clinical symptoms are observed, calculate protective rate situation.
3 results
3.1 viral levels determine
Porcine pseudorabies virus content is 106.5TCID50/0.1ml。
3.2 product inspection results
3.2.1 character
Slightly yellow Sponge Porosity agglomerate, when rocking up and down, sample is easy to depart from bottle wall.It is fast after addition dilution
Instant solution.
3.2.2 steriling test
10 bottles of vaccine grab sample, recover commercial weight with 10%DMEM respectively, every bottle is pressed version in 2010 respectively《Chinese veterinary drug
Allusion quotation》Annex method is tested.Without bacterium, fungus growth.
3.2.3 mycoplasma is examined
5 bottles of vaccine grab sample, recover commercial weight with 10%DMEM respectively and mix, by version in 2010《Chinese veterinary pharmacopoeia》
Annex method is tested.Vaccine grows without mycoplasma.
3.2.4 exogenous virus is examined
Cell toxicant genome is extracted, carries out the PCR identifications of exogenous virus, result is feminine gender, shows vaccine exogenous virus
It is qualified to examine.
3.2.5 safety verification
Vaccine samples 3 bottles, makees appropriate dilution after recovering commercial weight with 10%DMEM respectively, each intramuscular injection 0.2ml vaccines, sees
Examine 21.As a result show, Mice Inoculated is without any adverse reaction, and 10/10 is good for work.
3.2.6 efficacy test
3.2.6.1 cellular assay is used
The plumage part indicated by label, is diluted to l plumages part/0.2ml with 10%DMEM by vaccine, remakes 10 times and be serially diluted,
After inoculating cell TCID is calculated according to Reed-Muench methods50, the results showed that every plumage part is 104.5TCID50.The equal nothing of control mice
The specific clinical symptoms of pseudo- mad dog.
3.2.6.2 examined with mouse
Take 6 week old BALB/C mice 10, every plumage part of intramuscular injection vaccine 1.After 21 days, together with 10 control mices,
Hindlimb muscle injection 104.0TCID50/ only, observe 14.As a result：There is pseudo- mad dog specific symptom in 10 death of control group.
3.2.7 residual moisture determines
Vaccine samples 4 bottles and tested with boulton process.Product test sample residual moisture content 2.0%~
2.7% ,≤4%.It is qualified to illustrate that vaccine residual moisture measure is examined.
3.2.8 vacuum determines
Vaccine is tested with vacuum leak detector respectively.Product test sample is in purple glow.Illustrate vaccine vacuum
It is qualified that measure is examined.
3.2.9 vaccine using effect
To 30 age in days piglets difference immune vaccine strain Bartha-K61, gene-deleted strain PRV/gE-/gI-Vaccine and DMEM, it is immunized
Carry out attacking poison within the 4th week afterwards, attack malicious result and show, PRV/gE-/gI-Group protective rate divides apparently higher than Bartha-K61 groups, protective rate
Wei 100% and 20%.Show the PRV/gE of the present invention-/gI-The immune effect of vaccine clinically is substantially better than now extensively
The Bartha-K61 vaccines used.Moreover, Detection results show, the pseudorabies that PRV/TK-/gE- vaccines are used the present invention
The immune effect of virus (CGMCC No.10266) is significantly better than other vaccines (p ＜ 0.05)；Prove as starting strain
Porcine pseudorabies virus (CGMCC No.10266) has specificity genetically.
Claims (7)
1. a kind of pseudorabies disease vaccine, it is characterised in that described vaccine is made up of antigen and protective agent, wherein antigen bag
Attenuated viral strains made of containing carrying out after virulence gene missing by the Strain that deposit number is CGMCC No.10266；It is described
Virulence gene be gE and gI genes.
2. pseudorabies disease vaccine as claimed in claim 1, it is characterised in that described protective agent is protected for viral vaccine
Agent.
3. pseudorabies disease vaccine as claimed in claim 2, it is characterised in that described protective agent is the water of sucrose and gelatin
Solution.
4. pseudorabies disease vaccine as claimed in claim 3, it is characterised in that the matter of described sucrose and gelatin in vaccine
It is respectively 20% and 4.8% to measure percentage final concentration.
5. pseudorabies disease vaccine as claimed in claim 1, it is characterised in that add antibiotic in described vaccine.
6. pseudorabies disease vaccine as claimed in claim 5, it is characterised in that described antibiotic is penicillin and strepto-
Element.
7. pseudorabies disease vaccine as claimed in claim 6, it is characterised in that described penicillin and the final concentration of streptomysin
Respectively 200 units/ml.
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| CN106834236A (en) * | 2016-02-23 | 2017-06-13 | 南京农业大学 | PRV variant TK, gE and gI gene delection strain and its application |
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| 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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| gl/gE双基因缺失伪狂犬病病毒变异株的构建和免疫效力评价;王春花;《中国优秀硕士学位论文全文数据库农业科技辑》;20141115(第11期);D050-62 * |
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