CN106282132A - The low virulent strain of Pseudorabies virus variant and application thereof - Google Patents
The low virulent strain of Pseudorabies virus variant and application thereof Download PDFInfo
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Abstract
The invention discloses the low virulent strain of a kind of Pseudorabies virus variant, this low virulent strain is the gene delection low virulent strain of Pseudorabies virus variant PRVJS-2012 strain, and the gene order of its disappearance includes: all or part of DNA sequence in gE gene C DS district in PRV JS-2012 variant genome.The low virulent strain that the invention also discloses above-mentioned Pseudorabies virus variant prevents in preparation or treats the application in the vaccine of pseudorabies.The low virulent strain of Pseudorabies virus variant of the present invention; PRV variant and PRV classics virulent strain are all had good immune protective effect, inoculates PRV feminine gender piglet in 2 week old, any clinical ill symptoms does not occur; safety is high, is adapted as the vaccine candidate strain of prevention and control pseudorabies.
Description
Technical field
The present invention relates to biological technical field, particularly relate to low virulent strain and the application thereof of a kind of Pseudorabies virus variant.
Background technology
Pseudorabies be by Pseudorabies virus (Pseudorabies virus, PRV) cause to generate heat, very to itch, encephalomyelitis is
A kind of deadly infectious disease of principal character.PRV can infect multiple host, but the pig natural host that to be this virus main, storage person
And disseminator.The pig of different age group all can infect, and mainly causes in-pig miscarriage, stillborn fetus, mummy tire, suckling pig
High mortality, and boar is sterile.PRV belongs to herpetoviridae A type herpesviral subfamilies, and its genome is wire double-stranded DNA,
It is about 150kb, is made up of the repetitive sequence of long distinct zones (UL), short distinct zones (US) and US both sides, encode hatching egg more than 70
In vain.Virion is by nucleocapsid, shell and cyst membrane up of three layers.Nucleocapsid is by 6 kinds of capsid protein parcel viral genome shapes
The icosahedral structure of virus become；Shell, between nucleocapsid and cyst membrane interlayer, is made up of 14 kinds of albumen；Cyst membrane is inlayed by 15 kinds of albumen
Form in bilayer lipid membrane, wherein glycoprotein 11 kinds.Virus surface proteins determines the cell tropism of virus, is also main protection
Property antigen., such as tk and gI/gE, there is appreciable impact to virus virulence in some important viral genes, its disappearance can make virus
Pathogenicity weakens.
Pseudorabies betides the U.S. the earliest, and China is found that PRV, the beginning of the sixties the end of the forties in last century in cat first
Also occurring in that this viral prevalence in swinery, Pseudorabies virus is still widely current in China so far, and China swinery in serious threat
Health, and cause serious financial consequences.Pig infects after PRV, have in inapparent infection, but carry virus for a long time；Appearance is faced
The resistance to of bed symptom crosses the carrier that pig also can become viral, becomes the source of infection, which increases the difficulty of PRV preventing and treating.In order to control
PRV, China pig farm started the PRV attenuated live vaccines of gene delection to be widely used (such as PRV Bartha from the nineties in last century
Strain live vaccine), pseudorabies sickness rate is decreased obviously, and Some Species pig farm reality has eradicated this virus.But from 2011 with
Coming, many large-scale pig farms using the immunity of PRV live vaccine occur in that sow produces weak son, stillborn fetus, miscarriage, and nerve occurs in piglet
The clinical symptoms of the pseudorabies such as symptom and death.Tong Wu, Peng Jinmei et al. are isolated to PRV street strain the most respectively from morbidity pig,
And confirm that the street strain separated there occurs bigger change compared with vaccine strain and classics poison by gene sequencing with antibody neutralization test
Different.This shows, occurs in that PRV variant in China pig farm, and the protected effect that currently used commercialized vaccine is to variant
Difference, result in the new line of China's pig farm pseudorabies sickness rate.In order to effectively control the popular of PRV variant, need exploitation more
Efficient PRV vaccine.And based on PRV variant, develop new PRV vaccine, can improve for PRV variant
Immune protective effect.
Summary of the invention
The invention solves the problems that the most domestic PRV variant occurs, and existing commercial PRV vaccine be poor to variant protected effect,
Need the technical problem of the new PRV vaccine of exploitation badly, it is provided that the low virulent strain of a kind of Pseudorabies virus variant, this low virulent strain is to PRV
Variant and PRV classics virulent strain all have good immune protective effect, inoculate PRV feminine gender piglet in 2 week old, do not go out
What clinical ill symptoms incumbent, safety is high, is adapted as the vaccine candidate strain of prevention and control pseudorabies.
In addition, it is also desirable to provide the application of the low virulent strain of a kind of above-mentioned Pseudorabies virus variant.
In order to solve above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, it is provided that the low virulent strain of a kind of Pseudorabies virus variant, this low virulent strain is Pseudorabies virus
The gene delection low virulent strain of variant PRV JS-2012 strain, the gene order of its disappearance includes: PRV JS-2012 variant gene
All or part of DNA sequence in gE gene C DS district in group.The all DNA in this PRV JS-2012 variant gE gene C DS district
Sequence is as shown in SEQ ID NO.20.
Preferably, the gene order of described disappearance also includes: in PRV JS-2012 variant genome, US2 gene C DS district is complete
Portion or partial dna sequence.The all DNA sequence such as SEQ ID NO.21 in this PRV JS-2012 variant US2 gene C DS district
Shown in.
It is furthermore preferred that the gene order of described disappearance includes: the portion in gE gene C DS district in PRV JS-2012 variant genome
Divide DNA sequence and the partial dna sequence in US2 gene C DS district.Most preferably, the gene order of described disappearance includes: PRV
In JS-2012 variant genome the 437th bit base in gE gene C DS district to US2 gene C DS district the 396th bit base it
Between DNA sequence, consecutive miss 2307bp altogether, the DNA sequence of this consecutive miss is as shown in SEQ ID NO.1.
In another aspect of this invention, additionally providing a kind of vaccine combination, described compositions comprises mixes with adjuvant or pharmaceutical carrier
The low virulent strain of the Pseudorabies virus variant closed.
Described vaccine combination is suitable to inoculate in the way of collunarium or injection.
In another aspect of this invention, the low virulent strain of a kind of above-mentioned Pseudorabies virus variant is additionally provided in preparation prevention or treatment
Application in the vaccine of pseudorabies.
In another aspect of this invention, a kind of low virulent strain distinguishing above-mentioned Pseudorabies virus variant and variant or open country are additionally provided
The detection kit of virus strain infection, it is characterised in that comprise: for the primer pair of described low virulent strain missing gene sequential design.
Utilize the primer pair of design synthesis, by pcr amplification reaction, the Pseudorabies virus variant gene identifying the present invention can be distinguished
Disappearance low virulent strain and variant or street strain.
The low virulent strain of Pseudorabies virus variant of the present invention, is based on PRV variant, by by pseudorabies variant
JS-2012 Vero cell continuous passage under 40 DEG C of high temperature obtains.This low virulent strain has good safety, inoculates 2 week old
, there is not any clinical ill symptoms, can produce gB antibody, but not produce gE antibody in interior PRV feminine gender piglet.This weak poison
After strain immunity 2 week old PRV feminine gender piglets, piglet is highly resistant to variation and the attack of classical pseudorabies poison by force, therefore this weak poison
Strain can be as the vaccine candidate strain of effective prevention and control porcine pseudorabies.
Accompanying drawing explanation
The present invention is further detailed explanation with detailed description of the invention below in conjunction with the accompanying drawings.
Fig. 1 is PRV JS-2012-F50 poison and the parent poison PRV JS-2012 cytopathy on Vero cell of the embodiment of the present invention 1
Become figure；
Fig. 2 is the PRV JS-2012-F120 poison of the embodiment of the present invention 1 early, middle and late phase cytopathy figure on Vero cell；
Fig. 3 is PRV JS-2012-F120 poison and the parent poison PRV JS-2012 plaque on Vero cell of the embodiment of the present invention 1
Aspect graph；
Fig. 4 is the virus genomic gB of PRV JS-2012-F120 of the embodiment of the present invention 1, the PCR qualification result figure of gC, gD gene；
Fig. 5 is the PCR qualification result figure of the PRV JS-2012-F120 virus genomic gI gene of the embodiment of the present invention 1；
Fig. 6 is the PCR qualification result figure of the PRV JS-2012-F120 virus genomic gE gene of the embodiment of the present invention 1；
Fig. 7 is the PCR qualification result figure of the PRV JS-2012-F120 virus genomic US2 gene of the embodiment of the present invention 1；
Fig. 8 is the comparison result first half section figure of the PRV JS-2012-F120gE/US2 gene delection sequence of the embodiment of the present invention 2；
Fig. 9 is the comparison result second half section figure of the PRV JS-2012-F120gE/US2 gene delection sequence of the embodiment of the present invention 2；
Figure 10 is the PRV JS-2012-F120gE/US2 gene delection schematic diagram of the embodiment of the present invention 2；
Figure 11 is PRV JS-2012-F120 virus and the PCR qualification result figure of parent's poison of the embodiment of the present invention 2；
Figure 12 is that the embodiment of the present invention 3 high temperature passes on the dynamic variation diagram of body temperature after different generation virus inoculation feminine gender piglet；
Figure 13 is that the embodiment of the present invention 3 high temperature passes on the survival rate figure of different time points after different generation virus inoculation feminine gender piglet；
Figure 14 is that the embodiment of the present invention 3 high temperature passes on the gB Antibody dynamics of different time points after different generation virus inoculation feminine gender piglet
Variation diagram；
Figure 15 is that the embodiment of the present invention 3 high temperature passes on the gE Antibody dynamics of different time points after different generation virus inoculation feminine gender piglet
Variation diagram；
Figure 16 be the embodiment of the present invention 4 PRV JS-2012-F120 immunity piglet after the different time points Temperature changing figure of counteracting toxic substances；
Figure 17 be the embodiment of the present invention 4 PRV JS-2012-F120 immunity piglet after the different time points survival rate figure of counteracting toxic substances.
Detailed description of the invention
In the following example, the experimental technique of unreceipted actual conditions, the most routinely condition, as " fine works molecular biology is real
Test guide " (F.M. Ao Sibai, R.E. James Kingston, J.G. Sai Deman etc. edits, and Ma Xuejun, Su Yuelong translates. north
Capital: Science Press, 2004) method described in is carried out.
Owing to the Pseudorabies virus variation strain of popular can not be provided preferably immunity to protect by current PRV (Pseudorabies virus) vaccine
Protect effect, it is therefore necessary to develop PRV new generation vaccine.This laboratory is separated to a strain Pseudorabies virus variation virulent strain for 2012
PRV JS-2012.By this variation virulent strain Vero cell, under 40 DEG C of hot conditionss, the weak poison of strain that continuous passage obtains
Strain PRV JS-2012-F120.The cause of this low virulent strain not only virulence is weak and gE gene has disappearance, and this gene delection is consecutive miss,
Deletion sites to before 397 bit bases in US2 gene C DS district, amounts to and lacks after the 436th bit base in gE gene C DS district
Lose 2307 bases.This low virulent strain has preferable safety, inoculates PRV feminine gender piglet in 2 week old, do not occur any not
Good clinical symptoms, and by after these low virulent strain immunity 2 week old PRV feminine gender piglets, be highly resistant to variation and classical pseudorabies is strong
The attack of poison.Low virulent strain the most of the present invention can be as the vaccine candidate strain of prevention and control porcine pseudorabies.
In the following embodiment of the present invention, the experiment material of use is as follows:
Virus and cell: Vero cell (American Type Culture Collection committee of Chinese Academy of Sciences cell bank, preserving number is: GN010),
(this laboratory preserves in Pseudorabies virus JS-2012 strain；Publish an article and see: Tong Wu, Zhang Qingzhan, Zheng Hao, Liu Fei, Jiang Yifeng,
Dan Tongling, Zhou Yanjun, Tong Guangzhi. the separation of Pseudorabies virus and qualification in immunity sequela piglet. China's epizootiology
Report, 2013,21 (3): 1-7).
Other reagent: hyclone, pancreatin, DMEN, Life technologies Products；DNA extraction kit, sky
Root biochemical technology (Beijing) company limited；2 × GC Buffer II, dNTP Mix (2.5mmol/L each), LA-Taq,
DL-15000, DL-2000DNA Marker is purchased from TakaRa；Cell is cultivated consumptive material and is matched bio tech ltd purchased from Shanghai hundred.
In an embodiment of the present invention, the cultural method of Vero cell is as follows:
Vero cell in the T25 Tissue Culture Flask of the DMEM culture medium containing 10%FBS adherent cover with monolayer after, discard cell training
Supporting culture medium in bottle, and wash once with PBS, the pancreatin with 0.25%, by under cell dissociation, adds the new DMEM containing 10%FBS
Culture medium, divides with the ratio of 1:6 and plants in cell bottle or Tissue Culture Plate.
Embodiment 1PRV JS-2012 high temperature passes on the structure of low virulent strain
Based on pseudorabies variant PRV JS-2012 strain, this PRV JS-2012 strain is passed through on Vero cell 40 DEG C of companies
Continuous high temperature passes on, it is thus achieved that pass on strain PRV JS-2012-F120.PRV JS-2012-F120 strain is bred on Vero cell,
Its virus titer is up to 108.5TCID50/ 0.1mL, and the cytopathic morphology that caused on Vero cell of this poison and plaque shape
State differs markedly from its parent poison PRV JS-2012.Verify through PCR, gE and the US2 base of PRV JS-2012-F120 strain
Because all there being disappearance.
1 method
1.1 design of primers
According to the whole genome sequence of PRV JS-2012 strain, by Oligo 6.0 software design a plurality of primer (being shown in Table 1),
GBU/gBD, gCU/gCD, gDU/gDD, gIU/gID, gEU/gED, US2U/US2D are used for expanding Pseudorabies virus gB, gC, gD,
The part of gI, gE, US2 gene or full length sequence.
Table 1 primer sequence
The high temperature of 1.2PRV JS-2012 virus passes on
37 DEG C, the CO of 5%2Condition of culture under, Vero cell is at 25cm2Tissue Culture Flask in cultivate to monolayer, inoculation
Containing 106Individual TCID50PRV JS-2012 virus liquid 5ml.Vero cell after connecing poison is put to 40 DEG C, the CO of 5%2Training
Cultivate under the conditions of Yanging, after about 24h, after the cytopathy of 80%-90%, virus is put to-20 DEG C of freeze thawing once, then by disease
Venom goes to 10000g in the EP pipe of 1.5ml and is centrifuged 2min, takes in the EP pipe of the clean 1.5ml of supernatant device, by this disease
Poison is designated as PRV JS-2012-F1.
PRV JS-2012-F1 is made 1:50 dilution with the DMEM containing 2%FBS, takes 5ml virus dilution inoculation T25 cell bottle
In Vero cell monolayer, put to 40 DEG C, the CO of 5%2Condition of culture under cultivate, after the cytopathy of 80%-90%,
Put to-20 DEG C of freeze thawing once, then virus liquid is gone to 10000g in the EP pipe of 1.5ml be centrifuged 2min, take supernatant and be sub-packed in 1.5ml
EP pipe in, this virus is designated as PRV JS-2012-F2.Refer again to said method and carry out continuous passage, until reaching 120
In generation, the virus in the 120th generation is designated as PRV JS-2012-F120.Succeeding generations often reaches 30n-3 (n is 1,2,3,4) for time
By continuous 3 generation Plaque Clone purification (process is as follows) passaged virus.
By passaged virus with DMEM dilute, be seeded on the Vero cell monolayer in 60mm Tissue Culture Dish, 37 DEG C, 5%
CO2Condition of culture under hatch 1h after, abandon viral adsorption liquid, and wash once with PBS, add containing 1% agar and 2%FBS
Cover layer, room temperature is put to agar solidification, is put by cell to 37 DEG C, the CO of 5%2Condition of culture under cultivate, 2 to 3 days
After, the plaque that picking is single is placed in 0.5mL DMEM.After 1 freeze thawing, dilute plaque liquid with DMEM, inoculate 60mm
Vero cell monolayer in Tissue Culture Dish carries out second and takes turns plaque purification.Third round plaque liquid is dilute with the DMEM containing 2%FBS
Release to 5mL, the Vero cell monolayer in inoculation T25 cell bottle, put to 40 DEG C, the CO of 5%2Condition of culture under continue to pass on.
The PCR of 1.3PRV JS-2012-F120 virus identifies, poison valency measures and plaque morphologic observation
The DNA of PRV JS-2012-F120 is extracted by DNA extraction kit, with gBU/gBD, gCU/gCD, gDU/gDD,
GIU/gID, gEU/gED, US2U/US2D six carries out PCR qualification respectively to primer.PCR reaction system is 20 μ L:2 × GC
BufferⅡ10ul；dNTP Mix(2.5mmol/L each)2ul；Forward primer 0.5ul (10pmol/ul)；Downstream is drawn
Thing 0.5ul (10pmol/ul)；LA-Taq 0.5ul；ddH2O 4.5ul；The DNA 2ul of virus.Reaction condition is: 94 DEG C
Denaturation 5min；94 DEG C of degeneration 30s, 69 DEG C of annealing 30s, 72 DEG C extend 2.5min, circulate 35 times；Last in 72 DEG C of extensions
10min.PCR primer, with the sepharose electrophoresis of 1%, is observed in gel imaging instrument.
By PRV JS-2012-F120 virus inoculation Vero cell, when CPE occurs in 80% cell, results virus.Reference literature (Yin
Shake etc., animal virology, Science Press, 1997), the titre of this virus is measured by 96 hole tissue culturing plate methods.By this disease
After poison makees 10 times of serial dilutions with the DMEM containing 2%FBS, by thin for the Vero monolayer on the virus inoculation 96 porocyte culture plate of dilution
Born of the same parents.Every dilution factor inoculates 8 holes, if 8 hole comparisons (inoculating with the DMEM containing 2%FBS), puts in the CO2 gas incubator of 37 DEG C 5%
Cultivating, observe every day to inoculation the 4th day, there is the hole count of CPE in record, calculates TCID according to Reed-Muench method50。
PRV JS-2012-F120 virus is seeded on the Vero cell of monolayer with suitable poison amount, at 37 DEG C, the CO of 5%2
Condition of culture under hatch 1h, by the agar that mixes, (agar of 2% mixes with 2*MEM culture medium 1:1, adds overall
The hyclone of long-pending 2%) it is added to Vero cell, room temperature is put to agar solidification, is put by cell to 37 DEG C, the CO of 5%2Training
Cultivate under the conditions of Yanging, after 2 to 3 days, dye with 5% crystal violet, observe plaque form.
2 results
The high temperature of 2.1PRV JS-2012 virus passes on
Along with the high temperature of virus passes on, to when reaching for 50 generation, it is thin that PRV JS-2012-F50 virus is formed on Vero cell
Born of the same parents' pathological changes is entirely different with the cytopathy of parental virus PRV JS-2012.Parental virus infects and cell can be caused to form syncytium,
Assembling occurs in infection cell, and agglomerating comes off；PRV JS-2012-F50 infection then presents individual cells and becomes round, comes off, and does not goes out
(result is shown in Fig. 1, and wherein A is parent poison PRV JS-2012 cytopathy figure on Vero cell, and B is in existing infection cell gathering
PRV JS-2012-F50 poison cytopathy figure on Vero cell).50 instead of after until during 120 generation, each generation pass on disease
The cytopathy that poison is formed on Vero cell is completely the same with the cytopathy of PRV JS-2012-F50 virus, and Fig. 2 is PRV
The early stage that JS-2012-F120 virus being on Vero cell, mid-term and advanced lesions figure, in Fig. 2, A is that B is mid-term, C in early days
For late period.
Plaque form shows: as it is shown on figure 3, PRV JS-2012-F120 virus and the plaque shape of parental virus PRV JS-2012
State is entirely different, and the plaque of parental virus is rounded (Fig. 3 A), and the plaque of PRV JS-2012-F120 virus is fusiformis (figure
B)。
2.2PRV the PCR of JS-2012-F120 virus identifies and poison valency measures
PRV JS-2012-F120 breeds on Vero cell, through TCID50Measuring, its virus titer is up to 108.5TCID50/0.1
mL.PCR qualification result shows, the result of gBU/gBD, gCU/gCD, gDU/gDD, gIU/gID primer is all positive,
Illustrate that these genes all lack, see Fig. 4, Fig. 5.Fig. 4 A, B, C are respectively the full genome of gB, gC, gD and expand
Increasing result, wherein numeral numbering 1-3 is the different clones of PRV JS-2012-F120 poison, and 4 are positive control, and 5 are
Negative control.Fig. 5 is the full genome amplification of gI, and wherein 1-10 is the different clones of PRV JS-2012-F120 poison,
11 is positive control, and 12 is negative control.
The result of gEU/gED, US2U/US2D primer is negative, and illustrates that disappearance occurs in this two gene, sees Fig. 6, Fig. 7.Fig. 6
For the full genome amplification of gE, wherein 1-20 is the different clones of PRV JS-2012-F120 poison, and 21 is positive control,
22 is negative control.Fig. 7 is the full genome amplification of US2, and wherein 1-6 is the difference of PRV JS-2012-F120 poison
Clone, 7 is positive control, and 8 is negative control.
Embodiment 2 PRV JS-2012-F120 virus genomic deletion sequence analysis
Through PCR checking, embodiment 1 finds that gE, the US2 gene of PRV JS-2012-F120 virus exists disappearance, this enforcement
For this two gene design, 1 pair of primer (gE330up/US2down) expands parent poison PRV JS-2012 and biography to example respectively
GE to the us2 gene (see SEQ ID NO.14, SEQ ID NO.15) of generation poison PRV JS-2012-F120, finds through order-checking,
DNA for passing on poison relatively parent's poison, between 396 bit bases of 437 bit bases in gE gene C DS district to US2 gene C DS district
Sequentially continuous lacks, and amounts to 2307 bases of disappearance.1 pair of primer (DF120up/DF120down) is devised with reference to this disappearance
For distinguishing PRV JS-2012-F120 poison and parent poison PRV JS-2012.
1 material and method
1.1 design of primers
According to the whole genome sequence of PRV JS-2012 strain, with Oligo 6.0 software for gE and us2 gene design 1 pair of primer
GE330up/US2down, gE330up:5 ' GCGCCGTGCTGGCCGGGATCTGGAC 3 ' (SEQ ID NO.16)；US2down:
5 ' GCCCCGCTCTGTTCTTCCTCCACCC 3 ' (SEQ ID NO.17), are used for expanding PRV JS-2012 and PRV JS-2012
-F120 passes on the sequence of the gE to US2 of poison.
The extraction of 1.2PRV JS-2012-F120 genome
PRV JS-2012-F120 strain inoculation is merged the BHK-21 cell of monolayer, treats that the cell of 70%-80% all produces pathological changes,
Discarding 3/4ths culture fluid, scrape with cell and scraped by cell, and Cell sap moves into 50ml centrifuge tube, 1500rmp is centrifuged 5min,
Abandon supernatant, with TNE solution, sedimentation cell is resuspended.From infection cell, extract STb gene with phenol-chloroform method, and be dissolved in TE,
Save backup in-30 DEG C.
1.3PCR amplification and order-checking
The DNA extracted in 1.2 is carried out PCR amplification as template.PCR reaction system is 20 μ L:2 × GC Buffer II 10ul；
dNTP Mix(2.5mmol/L each)2ul；gE330up 0.5ul(10pmol/ul)；US2down 0.5ul(10pmol/ul)；
LA-Taq 0.5ul；ddH2O 4.5ul；The DNA 2ul of virus.Reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of degeneration
30s, 69 DEG C of annealing 30s, 72 DEG C extend 2.5min, circulate 35 times；Finally extend 10min in 72 DEG C.PCR primer is with 1%
Sepharose electrophoresis, observes in gel imaging instrument.
Reclaim the purpose fragment of PRV JS-2012-F120 amplification, clone with pMD-18T, after transformed competence colibacillus cell, sieve
Positive bacteria is selected to drop into row gene sequencing.
1.4 pass on gene-deleted strain identifies PCR method
According to PRV JS-2012-F120 absent region and both sides sequence, design has synthesized 1 couple of primer: DF120up/DF120down,
DF120up:5'-GGCCGGGATCTGGACGTT-3'(SEQ ID NO.18)；DF120down:5'-CGGGTCACG
ATCTGGGCAT-3'(SEQ ID NO.19).With PRV JS-2012-F120 and PRV JS-2012 genome as template, ginseng
PCR amplification is carried out according to the test method in 1.3.
2. result
The deletion sequence analysis of 2.1PRV JS-2012-F120
Reclaim test kit with glue and reclaim the PRV JS-2012-F120 virus band of primer gE330up/US2down amplification, warp
Connecting, choose positive bacteria and drop into row order-checking after conversion, sequencing result shows: gE gene C DS of PRV JS-2012-F120 virus
437th bit base in district all lacks to the 396th bit base in US2 gene C DS district, lacks 2307bp altogether, see Fig. 8,9,
10.Fig. 8 is the comparison result first half section figure of PRV JS-2012-F120gE/US2 gene delection sequence, and Fig. 9 is PRV
The comparison result second half section figure of JS-2012-F120gE/US2 gene delection sequence.Figure 10 A, B are PRV JS-2012-F120
GE/US2 gene delection schematic diagram, wherein the red area of Figure 10 B is the position of 2307bp consecutive miss.
2.2 identify PCR result
The PCR result of primer DF120up/DF120down shows, PRV JS-2012 virus amplification has gone out the purpose sheet of 2728bp
Section, is consistent with expected results；And the purpose fragment of PRV JS-2012-F120 virus is about 421bp, two fragments just differ
2307bp, is consistent with expected results, sees Figure 11.This shows, primer DF120up/DF120down detects PRV JS-2012-F120,
Only amplifying the band of specific 421bp, this can distinguish PRV JS-2012-F120 and other PRV strain.In fig. 11,1
For the purpose fragment of PRV JS-2012-F120 poison, 2 is the purpose fragment of PRV JS-2012 poison, and 3 is negative control, the left side
M is 2000DNA maker, and the M on the right is 15000DNA maker.
The pathogenic experiment to 14 age in days piglets of the high temperature passaged virus of embodiment 3 PRV JS-2012 difference generation
Choose PRV JS-2012-F5 in embodiment 1, PRV JS-2012-F50, PRV JS-2012-F91, PRV JS-2012-F120,
The PRV feminine gender piglet of 14 ages in days, the weak effect of cause of checking virus is inoculated by collunarium.Found that PRV JS-2012-F5 poison energy
100% lethal test pig；PRV JS-2012-F50 poison can make pig 100% fall ill, but fatality rate is 40%；PRV JS-2012-F91
Poison can make pig 80% fall ill, but pig can not be made lethal；PRV JS-2012-F120 is the safest to 14 age in days piglets.Attacked by analysis
Antibody in serum after poison, result shows: the most dead all test pig all can detect the antibody of gB gene after the 7th day,
And until experiment in the 21st day terminates gE antibody and is all negative, PRV JS-2012-F50, PRV JS-2012-F91 is described, PRV
JS-2012-F120 virus gE gene all has disappearance.
1 material and method
1.1 test pig
The negative piglet of 14 ages in days is chosen in this experiment, and (porcine pseudorabies, cause of disease and antibody are feminine gender；Pig blue-ear disease, swine fever,
The antibody of porcine circovirus 2 type is also feminine gender) 20
1.2 zoopery packet and counteracting toxic substances
20 negative piglets in 1.1 are randomly divided into 4 groups, often group 5.1st group of 5 test pig collunarium inoculation PRV respectively
JS-2012-F5 virus 106Individual TCID502ml；2nd group of 5 test pig collunarium inoculation PRV JS-2012-F50 virus 10 respectively6Individual
TCID502ml；3rd group of 5 test pig collunarium inoculation PRV JS-2012-F91 virus 10 respectively6Individual TCID502ml；4th group 5
Test pig collunarium inoculation PRV JS-2012-F120 virus 10 respectively6Individual TCID502ml.After counteracting toxic substances, every day detects each group by rectum
The body temperature of test pig, and separation serum of taking a blood sample, observe clinical symptoms and death condition, and make a record.
1.3PRV detection of specific antibody
To specifications, with Pseudorabies virus gB, gE antibody assay kit of IDEXX respectively to the 1.2 Virus monitory PRV collected
GB and gE antibody.
2 results
PRV JS-2012-F5 group the 2nd day body temperature after counteracting toxic substances is all had a fever to more than 41 DEG C, within after counteracting toxic substances the 4th day, occurs dead, and the 5th
It is the most dead.PRV JS-2012-F50 group the 2nd day body temperature after counteracting toxic substances is all had a fever to more than 40.5 DEG C, after counteracting toxic substances the 6th day and
8 days each dead 1 pigs.PRV JS-2012-F91 group has 4 temperature of pig body to have a fever to more than 40.5 DEG C for the 2nd day after counteracting toxic substances, and wherein 3
It is normal that head pig continues temperature recovery after 2 to 3 days, and 1 pig fever continues 7 days, separately have 1 whole experimentation of pig is showed no any
Clinical response.PRV JS-2012-F120 group test pig is showed no any clinical symptoms in whole experimentation.Result is shown in Figure 12,
Figure 13.This shows, along with viral passages number of times increases, virus virulence constantly declines, the viral PRV JS-2012-F120 in 120 generations
Two week old piglets are not had pathogenic, be a strain low virulent strain.
Antibody test result shows, PRV JS-2012-F5 inoculates piglet, and in the 5d that it is survived, gB antibody and gE antibody are
Negative；And PRV JS-2012-F50, PRV JS-2012-F91, PRV JS-2012-F120 tri-groups inoculation survival piglet, inoculation
Rear 7d gB is positive, and arrive inoculate after the 21st day, gE antibody is still negative (see Figure 14, Figure 15).PRV wild virus infection pig,
Within after infection the 7th day, produce gB antibody, gE antibody after 2 weeks, then can be detected.In this experiment, the survival pig more than 1 week after inoculation,
All produce gB antibody, and the experiment pig of all survivals, the most do not produce gE antibody during by 3 weeks, show PRV JS-2012-F50, PRV
All there is gE gene delection in JS-2012-F91, PRV JS-2012-F120.The gE gene order of PRV JS-2012-F120 detects also
Confirming, there is disappearance in its gE gene, this is consistent with results of serological detection.
The immune protective of 14 age in days piglets is tested by embodiment 4 PRV JS-2012-F120 virus
PRV JS-2012-F120 is inoculated 14 age in days piglets, by variation virulent strain PRV JS-2012 and classical virulent strain PRV after 28 days
SC attacks, and can obtain the protection of 100%.Therefore PRV JS-2012-F120 poison can carry out prevention and control pig pseudo-as vaccine candidate strain
Rabies.
1 material and method
1.1 experiment piglets
The negative piglet of 14 ages in days is chosen in this experiment, and (porcine pseudorabies, cause of disease and antibody are feminine gender；Pig blue-ear disease, swine fever,
The antibody of porcine circovirus 2 type is also feminine gender) 20
1.2 zoopery packet and counteracting toxic substances
By 20 piglets in 1.1, being randomly divided into 4 groups, often group the 5: 1st group and the 2nd group, totally 10 equal collunariums of pig connect
Plant PRV JS-2012-F120 virus 105Individual TCID502ml；3rd group and the 4th group, totally 10 pig equal collunariums inoculations are without serum
DMEM 2ml.After inoculating 28 days, the 1st group and the 3rd group, totally 10 pig equal collunariums inoculation PRV JS-2012 poison 10 by force5Individual TCID502ml；
2nd group and the 4th group, totally 10 pig equal collunariums inoculation PRV SC poison 10 by force5Individual TCID502ml.After counteracting toxic substances, every day is detected by rectum
Each body temperature organizing test pig, observes clinical symptoms and death condition, and makes a record.
2 results
1st group and the 2nd group after attacking strong poison in whole experimentation test pig be showed no any clinical symptoms.Attacking PRV for 3rd group
The 2nd day body temperature after JS-2012 poison by force is all had a fever to more than 41 DEG C, death within after counteracting toxic substances the 4th day, occurs, and the 7th day the most dead.4th
The group the 3rd day body temperature after attacking PRV SC poison by force is all had a fever to more than 41 DEG C, the 6th day dead 2 after counteracting toxic substances, and the 7th day and the 9th day each
Dead 1.Result is shown in Figure 16, Figure 17.This shows, with 105TCID50PRV JS-2012-F120 inoculates piglet, can effectively support
The attack of the different strong poison of resistance and classics poison by force, provides piglet and protects completely.
In view of PRV JS-2012-F120 is to piglet no pathogenicity, it is provided that good immune protective effect, and after inoculation piglet not
Produce gE antibody, novel PRV vaccine can be developed as vaccine candidate strain.
Embodiment described above only have expressed embodiments of the present invention, and it describes more concrete and in detail, but can not therefore and
It is interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for the person of ordinary skill of the art, do not taking off
On the premise of present inventive concept, it is also possible to make some deformation and improvement, these broadly fall into protection scope of the present invention.Therefore,
The protection domain of patent of the present invention should be as the criterion with claims.
Claims (8)
1. the low virulent strain of a Pseudorabies virus variant, it is characterised in that this low virulent strain is Pseudorabies virus variant PRV
The gene delection low virulent strain of JS-2012 strain, the gene order of its disappearance includes: gE gene in PRV JS-2012 variant genome
All or part of DNA sequence in CDS district.
Low virulent strain the most according to claim 1, it is characterised in that the gene order of described disappearance also includes: PRV JS-2012
All or part of DNA sequence in US2 gene C DS district in variant genome.
Low virulent strain the most according to claim 2, it is characterised in that the gene order of described disappearance includes: PRV JS-2012
The partial dna sequence in gE gene C DS district and the partial dna sequence in US2 gene C DS district in variant genome.
Low virulent strain the most according to claim 3, it is characterised in that the gene order of described disappearance includes: PRV JS-2012
In variant genome, the 437th bit base in gE gene C DS district is to the DNA between the 396th bit base in US2 gene C DS district
Sequence, the DNA sequence of this disappearance is as shown in SEQ ID NO.1.
5. a vaccine combination, it is characterised in that described compositions comprises the claim 1 mixed with adjuvant or pharmaceutical carrier
Low virulent strain to the Pseudorabies virus variant according to any one of 4.
Vaccine combination the most according to claim 5, it is characterised in that described vaccine combination is suitable to collunarium or injection
Mode inoculate.
7. the low virulent strain of the Pseudorabies virus variant according to any one of Claims 1-4 is preparation prevention or treatment pseudorabies
The sick application in vaccine.
8. distinguish the low virulent strain of Pseudorabies virus variant according to any one of Claims 1-4 and variant or street strain for one kind
The detection kit infected, it is characterised in that comprise: for the primer pair of described low virulent strain missing gene sequential design.
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