CN104877972A - Recombinant porcine pseudorabies virus gE/gI double-gene-deleted strain and application thereof - Google Patents
Recombinant porcine pseudorabies virus gE/gI double-gene-deleted strain and application thereof Download PDFInfo
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Abstract
The invention aims at providing a porcine pseudorabies virus vaccine. The porcine pseudorabies virus vaccine consists of an antigen and a protective agent, wherein the antigen comprises an attenuated virus strain which is prepared by deleting virulence genes gE and gI of a porcine pseudorabies virus strain with a collection number of CGMCC No.10266. The prepared vaccine provided by the invention can be used for effectively preventing porcine pseudorabies; moreover, porcine pseudorabies virus which is taken as the antigen is a gene-deleted strain, and after the porcine pseudorabies virus is subjected to continuous passage in a mouse body by virtue of horizontal transmission infection, no virulence enhancement phenomenon can be caused, and thus the gene-deleted strain is stable in heredity, and can meet the standard that a porcine pseudorabies virus deleted vaccine strain does not have virulence enhancement; and the prepared vaccine can be used for providing effective immune protection, and has very good commercialized development prospects.
Description
Technical field
The invention belongs to veterinary vaccines preparing technical field, be specifically related to a kind of recombinant porcine pseudorabies poison dual-gene gene-deleted strain of gE/gI and application thereof.
Background technology
Porcine pseudorabies virus PRV (Pseudorabies virus) belongs to herpetoviridae a herpesviral subfamilies Vesiculovirus and to be born in the year of pig Simplex Virus Type I, pig is unique natural reservoir (of bird flu viruses) of this virus, cause the pseudoabies (Pseudorabies, PR) of pig.This disease is many in outbreak of epidemic swinery, and main harm sow group, causes Sow abortion or vertical transmission to piglet, cause newborn piglet mortality, brings huge financial loss to the pig industry in China and even the whole world.There is no the active drug for the treatment of PR at present, therefore vaccine inoculation becomes the generation of this disease of control and popular major measure.Most popular vaccine mainly PRV Bartha-K61 strain vaccine in the world, but China's pseudorabies pestilence present situation is the attack that this vaccine existing can not protect new popular PRV completely in recent years, causes certain financial loss to immune pig farm.Therefore, the vaccine providing cri dernier cri strain to prepare is needed just to become the study hotspot of the sick prevention and control field of porcine pseudorabies virus.
Summary of the invention
The object of this invention is to provide a kind of recombinant porcine pseudorabies poison dual-gene gene-deleted strain of gE/gI and application thereof; namely the vaccine prepared as antigen by the gene-deleted strain of the porcine pseudorabies virus filtered out, vaccine of the present invention can provide good immune protection effectiveness to the attack of porcine pseudorabies virus popular at present.
First the present invention provides a kind of PRV (Pseudorabies virus) gene-deleted strain, is to make after gE and gI genetically deficient is carried out in the porcine pseudorabies virus strain being CGMCC No.10266 by deposit number.
Another aspect of the present invention provides above-mentioned PRV (Pseudorabies virus) gene-deleted strain preparing the application in vaccine;
Another aspect of the present invention provides a kind of porcine pseudorabies virus vaccine, is made up of antigen and protective material, and wherein antigen includes above-mentioned PRV (Pseudorabies virus) gene-deleted strain.
Protective material is wherein the virus vaccines protective material used at present, and the concrete composition of its a kind of embodiment is the aqueous solution of sucrose and gelatin, and its mass percent final concentration in vaccine is respectively 20% and 4.8%.
Add microbiotic in above-mentioned vaccine, penicillin, Streptomycin sulphate final concentration are 200 units/ml;
Vaccine prepared by the present invention effectively can prevent porcine pseudorabies; and be gene-deleted strain as the porcine pseudorabies virus of antigen; infected in Mice Body continuous passage by horizontal transmission; be showed no virulence and return strong phenomenon; genetic stability; meet porcine pseudorabies virus deletion of vaccine strain avirulence and return strong standard, the vaccine made can provide effective immunoprotection, has good commercialized development prospect.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, those of ordinary skill in the art, on the basis of technical solution of the present invention, can select the method steps that this area is conventional, and be not limited only to the concrete record of specification sheets embodiment of the present invention.
The seed selection of embodiment 1:CGMCC No.10266 porcine pseudorabies virus strain
In recent years, the multiple pig farm of China all there occurs pseudoabies, and wherein major part is kind of a pig farm, and has injected pseudo-rabies vaccine before the swinery that falls ill, and infers that the virus infected there occurs variation; Therefore from morbidity swinery, the screening of PRV (Pseudorabies virus) has been carried out.
Get morbidity haslet sample, comprising: heart, liver, lungs, spleen, tonsilla and lymphoglandula etc.Internal organ sample and PBS (0.1M, pH7.2) are made homogenate with V/V1:5, multigelation 3 times, the centrifugal 15min of 3000r/min, get in supernatant and add dual anti-, 1h is made in 37 DEG C of senses, degerming through 0.22 μm of membrane filtration.Get 1ml virus filtrate and be inoculated in the Vero cell growing up to individual layer, blind passage three generations, observation of cell pathology (CPE).To occur that the cell culture fluid of CPE carries out Plaque-purified, the viral packing after purifying be saved to-70 DEG C for subsequent use, and measure viral level.The porcine pseudorabies virus QD strain (chimpanzee agent Porcine herpesvirus Type I) chosen as vaccine development is preserved in China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on March 6th, 2015, deposit number is CGMCC No.10266.
Strain for the present invention's screening carries out PCR detection, blast analysis is carried out after order-checking, although there are at least 2 amino acid whose differences with the corresponding sequence of the porcine pseudorabies virus reported after 2012 in the gE gene that discovery deposit number is the porcine pseudorabies virus of CGMCC No.10266, but sibship is nearer, all be in a relatively independent branch, far away with the strain sibship be separated before.And with the strain be separated recently, there is identical characterization of molecules, namely respectively have the insertion of 1 aspartic acid at gE gene the 48th and 492-496 position, other reports also confirm also this point.And the strain be separated in the past only has individually a position to insert amino acid, the overwhelming majority is insertion not.Therefore the reason place that above-mentioned difference causes existing porcine pseudorabies virus immune effect of vaccine not good just can be inferred.
After this strain does 100 times of dilutions, neutralize with equivalent porcine pseudorabies virus antiserum(antisera), virus all can be neutralized by hyper-immune serum specificity; And with equivalent pig parvoviral, swine influenza virus, Pestivirus suis, porcine circovirus 2 type, porcine reproductive and respiratory syndrome virus, Porcine epidemic diarrhea virus and transmissible gastro-enteritis virus antiserum(antisera) in and group, cell presents obvious cytopathy, and this virus-specific is good as seen.After vaccine immune BALB/C mouse prepared by this strain, the mortality ratio of mouse can be reduced.
Embodiment 2: the structure of the dual-gene gene-deleted strain of recombinant porcine pseudorabies poison gE/gI
From the porcine pseudorabies virus (CGMCC No.10266) be separated, extract the DNA of this strain isolated, adopt engineered method to carry out the disappearance of virulence gene gE and gI gene to this strain isolated, and called after PRV/gE after saving on cell
-/ gI
-, numerous poison, malicious as seed after adding protective material.Embodiment is as follows:
1PCR primer
With reference to PRV whole genome sequence (BK001744), synthesizing series primer voluntarily, being used for amplification is respectively positioned at the left arm fragment (L) that can be used for homologous recombination and the right arm fragment (R) of US7 (gI) gene and US8 (gE) gene both sides (containing part gI and gE gene).Wherein L comprises part US6 gene and part gI gene, and R comprises part gE gene, whole US9 genes and part US2 gene.Design primer amplification EGFP and EGFP eukaryotic expression box in addition simultaneously, and for the identification of the primer of genetically deficient.The size of primer sequence and expection PCR primer is in table 1.
Table 1: the primer used in this research
The structure of 2gE and gI gene transfer vector
With PRV QD pnca gene group for template, utilize primer gEILF/GEILR and gEIRF/GEIRR, amplify the sequence (containing part gE and gI gene) that is positioned at gE and gI both sides respectively as left and right restructuring arm gEIL, gEIR, wherein one end of gEIL, gEIR is respectively with a loxP site.After being cloned into pMD19-T, PCR is carried out to it and enzyme cuts qualification, order-checking after qualification is correct, the positive colony T-gEIL correct to order-checking adopts Hind III and Pst I to carry out double digestion, this fragment being cloned into same enzyme cuts on the pBluescript SK carrier of process, Spe I and Xba I is adopted to carry out double digestion to this recombinant plasmid and the recombinant plasmid vector being connected with right side homology arm respectively after qualification is correct, reclaim the linearizing recombinant plasmid containing left side homology arm and right side homology arm respectively, the two is connected.PCR and enzyme are cut and are identified.Order-checking is sent, the called after pSKgEILR that sequencing result is correct after qualification is correct.
With pCDNA3.1-EGFP plasmid for template, adopt primer EorfF/EorfR amplification EGFP reading frame, be connected into eukaryotic expression vector pVAX1, after qualification is correct, employing primer cassetteF/cassetteF amplifies the eukaryotic expression box containing EGFP, be connected into pMD19-T carrier, called after pMDEV after qualification is correct.
Adopt Pst I and Spe I to carry out double digestion to pMDEV, reclaim eukaryotic expression box, be connected into the pSKgEILR of same double digestion, after qualification is correct, be gE and gI gene transfer vector, called after pSKgE-EGFP.
The structure of 3gE and gI genetically deficient strain virus and purifying rescue
3.1 transfection
Recombinant virus rescue is carried out on six porocyte culture plates, when Vero cell covers with 90%, carry out transfection.Get PRV QD pnca gene group 3 μ g, after mixing with 1 μ g transferring plasmid pSKgE-EGFP, cotransfection according to a conventional method, method detailed is see Lipofectamine
tM2000 specification sheetss.Establish only containing the control group of transferring plasmid simultaneously.
3.2 qualification
After PRV QD pnca gene group and pSKgE-EGFP cotransfection 48h, observe transfectional cell pathology formational situation and fluorescent protein expression situation.In blind passage two generation after transfectional cell cracking, still have cytopathy and green fluorescence, tentatively judge that recombinant virus is saved successfully.Recombinant virus is identified through plaque purification and PCR, called after PRV/gE
-/ gI
-/ EGFP.
The rejecting of 3.3 gene-deleted strain reporter genes
The site-specific recombination of Cre-loxP System-mediated is utilized to reject reporter gene EGFP.Get the DNA that 10 μ g cross through Cre ferment treatment, adopt calcium phosphate procedure transfected Vero cells, put 37 DEG C of CO
2cultivate 2-3d in incubator, when cell produces 80% pathology, gather in the crops virus liquid.Get supernatant by after virus liquid multigelation three times, be inoculated in by 2uL/ hole the Vero cell that 24 orifice plates grow up to individual layer, begin to spread low melting-point agarose when there is cytopathy at Jian, under fluorescent microscope, picking does not have the plaque of fluorescence.Purified virus so repeatedly, until all virus plaques all to be dashed forward light without green, names this gene-deleted strain to be PRV/gE
-/ gI
-.
3.4PRV/gE
-/ gI
-qualification
Extract PRV/gE
-/ gI
-genomic dna, carries out pcr amplification with primer gEIF/gEIR and primer gBF/gBR, and whether the virogene interior segments of qualification rescue lacks, if parent plant, PRV/gE
-/ gI
-/ EGFP genomic dna in contrast.
The genetic stability of 4 recombinant viruses detects
What screening obtained is first for PRV/gE
-/ gI
-recombinant virus continuous passage on Vero cell, in every 5 generations, extract cells infected STb gene, and the PCR carrying out lack part gene detects.
5PRV/gE
-/ gI
-seed culture of viruses safety testing
With PBS by antigen diluent 10 times, intramuscular inoculation 100 grams of mouse four, every 0.2mL, observes 14 days, its reaction or death must not more than two.
6PRV/gE
-/ gI
-immune efficacy detect
The BALB/C mice in 6 week age is divided into 3 groups at random, often organizes 5 and weigh, respectively vaccinate strain Bartha-K61, gene-deleted strain PRV/gE
-/ gI
-and DMEM.Control group often only injects 100 μ LDMEM, other two groups of hindlimb muscles injections 10
4tCID
50the vaccine of virus dosage.Observe the clinical symptom of mouse after immunity every day, with or without One's spirits are drooping, apocleisis, itch, tremble, 21d weighs again, and in 14d, 21d tail vein blood, separation of serum, detects antibody level of serum with the elisa plate of PRV QD strain totivirus bag quilt.After immunity, 21d PRV QD strain attacks poison, with 10
4tCID
50the each test group of dose inoculation and control group, adopt hindlimb muscle injection, Continuous Observation 14d after virus inoculation.
7 results
With PRV QD strain virus for template, successfully obtain gE and gI gene transfer vector; Subsequently by itself and PRV genome homologous recombination, successfully save and be purified into gE and the gI genetically deficient strain virus containing EGFP marker gene; After adopting Cre recombinase to remove EGFP marker gene, successfully save and be purified into gE and the gI genetically deficient strain virus (PRV/gE not containing EGFP marker gene
-/ gI
-).
By the PRV/gE that screening obtains
-/ gI
-just for recombinant virus continuous passage on Vero cell, in every 5 generations, extract cells infected STb gene, the PCR detected result of lack part gene show the size of gene be deletion fragment after size, show this recombinant virus good stability.
Seed culture of viruses safety testing result shows that this vaccine on mouse is safe, without pseudo-rabies specific symptom, does not affect and grows.Porcine pseudorabies virus gene-deleted vaccine (the PRV/gE of the present invention's transformation
-/ gI
-) be safe, can be used for preparing vaccine.
21d, Bartha-K61, PRV/gE after immunity
-/ gI
-group and DMEM control group mice average weight gain are respectively 4.4g, 4.1g, 4.5g, and weightening finish difference is little, and the weightening finish unrestraint effect of gene-deleted strain to mouse is described.Antibody is examined not yet, PRV/gE during 21d during 14d after immunity
-/ gI
-group antibody horizontal obviously raises, and other groups still do not have considerable change.Show PRV/gE
-/ gI
-gene-deleted strain can produce obvious immune response by inducing mouse.Poison is attacked, PRV/gE with PRV QD strain after immunity 21d
-/ gI
-group protection ratio is apparently higher than Bartha-K61 group, and protection ratio is respectively 100% and 20%.
The preparations and applicatio of embodiment 3 porcine pseudorabies virus gene-deleted vaccine
1 material
1.1 seed culture of viruses
Manufacture vaccine porcine pseudorabies virus, deposit number is: CGMCC No.10266.
1.2 experimental animal
BALB/C small white mouse, 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 cell is cultivated according to a conventional method, with 5-10PFU virus infected cell, gathers in the crops virus, puts-80 DEG C of multigelations three times, measure the TCID of virus when cytopathy (CPE) reaches about 90%
50.According to the malicious valency of results virus liquid, carry out suitable dilution, the virus liquid after dilution and protectant volume ratio are 1:1.5, fully mix, and wherein sucrose final concentration is 20%, and gelatin final concentration is 4.8%.Every bottled 2.5mL, carries out freeze-drying in juxtaposition Freeze Drying Equipment.
2.2 inspection after construction
2.2.1 proterties
Observe the appearance color of vaccine, proterties, depart from situation and dissolving situation after adding diluent with bottle wall.
2.2.2 steriling test
Test by version " Chinese veterinary pharmacopoeia " annex method in 2010.
2.2.3 mycoplasma inspection
Test by version " Chinese veterinary pharmacopoeia " annex method in 2010.
2.2.4 exogenous virus inspection
Test by version " Chinese veterinary pharmacopoeia " annex method in 2010.
2.2.5 safety verification
By the BALB/C mice 10 in 6 week age, intramuscular injection 0.2ml (containing 10 plumage parts) vaccine, observes 21, all should be good for and live, without any local or systemic adverse reactions.
2.2.6 efficacy test
Following method is appointed and is selected one.
2.2.6.1 cellular assay is used
By plumage part that label indicates, vaccine 10%DMEM is diluted to l plumage part/0.2ml, remakes 10 times of serial dilutions, get 10
-2, 10
-3, 10
-4, 10
-5four extent of dilution, inoculate the good Vero cell of growth conditions respectively, each extent of dilution inoculates 8 holes, every hole 0.2m1, separately gets 8 holes for inoculating 10%DMEM in contrast.Hatch CO for 37 DEG C
2after carrying out hatching 3 in incubator, observe pathology situation, calculate TCID by Reed-Muench method
50, viral level answers>=10
4.0tCID
50/ 0.2ml.
2.2.6.2 check with mouse
Get BALB/C mice 10 in 6 week age, every intramuscular injection vaccine 1 plumage part.After 21 days, together with 10 control mice, hindlimb muscle injection 10
4.0tCID
50/ only, observe 14.Control group should at least 8 death or occur pseudo-rabies specific symptom, and immune group should at least 8 protections.
2.2.7 residual moisture measures
Undertaken by version " Chinese veterinary pharmacopoeia " annex method in 2010.
2.2.8 vacuum tightness measures
Undertaken by version " Chinese veterinary pharmacopoeia " annex method in 2010.
2.2.9 vaccine result of use is checked
Choose 15 30 age in days piglets before test, be divided into 3 groups at random, often organize 5 and weigh, respectively vaccinate strain Bartha-K61, gene-deleted strain PRV/gE
-/ gI
-vaccine and DMEM.Control group every injection 2mL DMEM, other two groups of hindlimb muscle injections 10
5.0tCID
50the vaccine of virus dosage.Within 4th week, carry out attacking poison after immunity, intramuscular injection 10
7.0tCID
50pRV QD strain strain virus, observes clinical symptom, calculates protection ratio situation.
3 results
3.1 viral levels measure
Porcine pseudorabies virus content is 10
6.5tCID
50/ 0.1ml.
3.2 inspection after construction results
3.2.1 proterties
Micro-yellow spongy loosens agglomerate, and during dandle, sample is easy to depart from bottle wall.Dissolve all rapidly after adding diluent.
3.2.2 steriling test
Vaccine random sampling 10 bottles, recovers commercial weight with 10%DMEM respectively, tests respectively for every bottle by version " Chinese veterinary pharmacopoeia " annex method in 2010.All without bacterium, mould-growth.
3.2.3 mycoplasma inspection
Vaccine random sampling 5 bottles, recovers commercial weight with 10%DMEM respectively and mixes, testing by version " Chinese veterinary pharmacopoeia " annex method in 2010.Vaccine all grows without mycoplasma.
3.2.4 exogenous virus inspection
Extract cell toxicant genome, carry out the PCR qualification of exogenous virus, result is feminine gender, shows that vaccine exogenous virus is up to the standards.
3.2.5 safety verification
Vaccine samples 3 bottles, and do suitably dilution after recovering commercial weight with 10%DMEM respectively, each intramuscular injection 0.2ml vaccine, observes 21.Result shows, and Mice Inoculated is all without any untoward reaction, and 10/10 is strong alive.
3.2.6 efficacy test
3.2.6.1 cellular assay is used
By plumage part that label indicates, vaccine 10%DMEM is diluted to l plumage part/0.2ml, remakes 10 times of serial dilutions, after inoculating cell, calculate TCID according to Reed-Muench method
50, result shows that every plumage part is 10
4.5tCID
50.Control mice is all without the specific clinical symptoms of pseudo-rabies.
3.2.6.2 check with mouse
Get BALB/C mice 10 in 6 week age, every intramuscular injection vaccine 1 plumage part.After 21 days, together with 10 control mice, hindlimb muscle injection 10
4.0tCID
50/ only, observe 14.Result: control group 10 death or occur pseudo-rabies specific symptom.
3.2.7 residual moisture measures
Vaccine samples 4 bottles and tests with boulton process.Product test sample residual moisture content 2.0% ~ 2.7%, all≤4%.Illustrate that vaccine residual moisture measures inspection all qualified.
3.2.8 vacuum tightness measures
Vaccine is tested with vacuum leak detector respectively.Product test sample is all in purple glow.Illustrate that vaccine vacuum tightness measures inspection all qualified.
3.2.9 vaccine result of use
To 30 age in days piglets immune vaccine strain Bartha-K61, gene-deleted strain PRV/gE respectively
-/ gI
-vaccine and DMEM, carry out attacking poison after immunity, attack malicious result and show, PRV/gE on the 4th week
-/ gI
-group protection ratio is apparently higher than Bartha-K61 group, and protection ratio is respectively 100% and 20%.Show PRV/gE of the present invention
-/ gI
-vaccine immune effect clinically is obviously better than present widely used Bartha-K61 vaccine.And Detection results shows, the immune effect of the porcine pseudorabies virus (CGMCC No.10266) that PRV/TK-/gE-vaccine uses the present invention is significantly better than other vaccine (p < 0.05); Prove the specificity had as the porcine pseudorabies virus (CGMCC No.10266) of starting strain in heredity.
Claims (9)
1. a pseudorabies disease vaccine, is characterized in that, described vaccine is made up of antigen and protective material, the attenuated viral strains made after the virus strain that wherein it is CGMCC No.10266 that antigen includes by deposit number carries out virulence gene disappearance.
2. pseudorabies disease vaccine as claimed in claim 1, it is characterized in that, described virulence gene is gE and gI gene.
3. pseudorabies disease vaccine as claimed in claim 1, it is characterized in that, described protective material is virus vaccines protective material.
4. pseudorabies disease vaccine as claimed in claim 3, it is characterized in that, described protective material is the aqueous solution of sucrose and gelatin.
5. pseudorabies disease vaccine as claimed in claim 4, it is characterized in that, described sucrose and the gelatin mass percent final concentration in vaccine is respectively 20% and 4.8%.
6. pseudorabies disease vaccine as claimed in claim 1, is characterized in that, add microbiotic in described vaccine.
7. pseudorabies disease vaccine as claimed in claim 6, it is characterized in that, described microbiotic is penicillin and Streptomycin sulphate.
8. pseudorabies disease vaccine as claimed in claim 7, it is characterized in that, described penicillin and the final concentration of Streptomycin sulphate are respectively 200 units/ml.
9. the preparation method of pseudorabies disease vaccine according to claim 1, is adopt engineered method to be lacked by genomic for porcine pseudorabies virus virulence gene, expands the virus liquid that poison obtains after cell is saved; Add that protective material makes.
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| CN109609468A (en) * | 2018-12-10 | 2019-04-12 | 四川华神兽用生物制品有限公司 | A kind of porcine pseudorabies virus of six gene delection, pseudorabies disease vaccine and preparation method |
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| CN110684746A (en) * | 2019-09-26 | 2020-01-14 | 北京市动物疫病预防控制中心 | Preparation method and detection method of freeze-dried Newcastle disease virus nucleic acid standard substance |
| 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 |
| CN113249341A (en) * | 2021-02-03 | 2021-08-13 | 铜仁职业技术学院 | Porcine pseudorabies virus double-gene deletion strain |
| CN112773892A (en) * | 2021-02-09 | 2021-05-11 | 铜仁职业技术学院 | Porcine pseudorabies virus low virulent strain freeze-dried vaccine |
| CN113502275A (en) * | 2021-06-21 | 2021-10-15 | 江西正邦科技股份有限公司 | Porcine pseudorabies virus strain and application thereof |
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