CN104152416B - Pseudorabies virus gene delection low virulent strain and its preparation method and application - Google Patents
Pseudorabies virus gene delection low virulent strain and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of Pseudorabies virus gene delection low virulent strain, the gene order lacked includes：Encode the nucleotide sequence of the 1st 404 amino acids of nucleotide sequence and coding Pseudorabies virus gE albumen of the 198th 366 amino acids of Pseudorabies virus gI albumen.The invention also discloses a kind of preparation method and application of Pseudorabies virus gene delection low virulent strain.The Pseudorabies virus gene delection low virulent strain of the present invention, has preferable immune protective effect to Pseudorabies virus, is suitable for the vaccine candidate strain of prevention pseudoabies.
Description
Technical field
The present invention relates to technical field of bioengineering more particularly to a kind of Pseudorabies virus gene delection low virulent strain and its systems
Preparation Method and application.
Background technology
Pseudoabies is by Pseudorabies virus（Pseudorabies virus, PRV）It is caused with fever, very itch, brain ridge
Marrow inflammation is a kind of deadly infectious disease of main feature.PRV can infect a variety of hosts, but pig be the main natural host of the virus,
Storage person and disseminator.The pig of different age group can all infect, and mainly cause pregnant sow miscarriage, stillborn foetus, the mummification of fetus, lactation
Piglet high mortality and boar infertility.PRV belongs to herpetoviridae A type herpesviral subfamilies, and genome is linear double-strand
DNA is about 150kb, is made of the repetitive sequence of long distinct zones (UL), short distinct zones (US) and the both sides US, encodes more than 70 kinds
Albumen.Virion is by nucleocapsid, shell and cyst membrane up of three layers.Nucleocapsid is to wrap up viral genome by 6 kinds of capsid proteins
The icosahedral structure of virus of formation；Shell is made of between nucleocapsid and cyst membrane interlayer 14 kinds of albumen；Cyst membrane is inlayed by 15 kinds of albumen
It is formed in bilayer lipid membrane, wherein 11 kinds of glycoprotein.Virus surface proteins determine the cell tropism of virus and main protection
Property antigen.There is virus virulence and significantly affect in some important viral genes, such as tk, missing can make virus weakening.
Pseudo- mad dog betides the U.S. earliest, and China is found that PRV, the beginning of the sixties in the end of the forties in last century for the first time in cat
Also occurs the viral prevalence in swinery, Pseudorabies virus is still widely current in China so far, seriously threatens China swinery
Health, and cause serious financial consequences.After pig infects PRV, some is in subclinical infection, but long-term carrying is viral；Face
Bed symptom it is resistance to cross pig also can become virus carrier, become the infection sources, which increase PRV prevention difficulty.In order to control
PRV, PRV attenuated live vaccines are widely used since the last century end in China pig farm, such as PRVBartha plants of live vaccines, pseudorabies
Incidence is decreased obviously.But since 2011, it is weak that sow production occur in many large-scale pig farms being immunized using PRV live vaccines
There are the clinical symptoms of the pseudo- mad dog such as nervous symptoms and death in son, stillborn foetus, miscarriage, piglet.Tong Wu, Peng Jinmei et al. also respectively from
It is isolated to PRV street strains in morbid pig, and the street strain detached is confirmed with antibody neutralization test by gene sequencing
Larger variation has occurred compared with vaccine strain and classical poison.This shows occur PRV variants in China pig farm, and uses at present
Commercialized vaccine it is poor to the protecting effect of variant, result in the new line of the pseudo- mad dog incidence in China pig farm.In order to effectively control
The prevalence of PRV variants processed needs to develop highly efficient PRV vaccines.
Invention content
The invention solves the current country PRV variants occurs, and existing commercial PRV vaccines are to variant protecting effect
Difference provides a kind of Pseudorabies virus gene delection low virulent strain, the gene delection there is an urgent need for developing new PRV vaccines
Low virulent strain has preferable immune protective effect to PRV variants, is inoculated with piglet in 2 week old, does not occur PRV clinical symptoms, pacifies
Quan Xinggao is suitable for the vaccine candidate strain of prevention pseudoabies.
In addition, it is also desirable to provide a kind of preparation method and application of above-mentioned Pseudorabies virus gene delection low virulent strain.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a kind of pseudo-rabies viurs attenuated strain strain is provided, which is Pseudorabies virus
Gene delection low virulent strain, the gene order lacked include：Encode Pseudorabies virus gI albumen 198-366 amino acids（SEQ
ID NO.2）Nucleotide sequence and coding Pseudorabies virus gE albumen 1-404 amino acids（SEQ ID NO.3）Core
Nucleotide sequence.
Preferably, the gene order of the low virulent strain missing includes：Pseudorabies virus gI gene 594-1101 bit bases
Nucleotide sequence and Pseudorabies virus gE gene 1-1212 bit bases nucleotide sequence.
It is furthermore preferred that the gene order of the low virulent strain missing is nucleotide sequence shown in SEQ ID NO.1.By SEQ
ID NO.1 missing genes sequences it is found that the present invention pseudo-rabies viurs attenuated strain strain, including the 1st of Pseudorabies virus gI genes the to
1213rd to 1740 bit base of 593 bit bases and gE genes, without containing gI genes in Pseudorabies virus genome the 594th to
1101 bit bases, gE genes the 1st to 1212 bit base and gI genes and gE genes between 103bp catenation sequences, i.e., not
The 594th bit base containing gI genes in Pseudorabies virus genome is to the DNA sequence dna between 1212 bit bases of gE genes（It is total
1823bp is lacked, as shown in SEQ ID NO.1）.In 508bp（594th to 1101 bit base）GI gene delection sequences in,
" C " that 1 base, that is, gI genes are the 594th is coding proline（P）Third base, last 3 of remaining 507 base
Base is terminator codon.
In another aspect of this invention, a kind of recombinant vector is provided, which includes Pseudorabies virus gI genes
With the partial sequence of gE genes, wherein do not include following gene order：Encode Pseudorabies virus gI albumen 198-366 bit aminos
The nucleotide sequence of acid and the nucleotide sequence for encoding Pseudorabies virus gE albumen 1-404 amino acids.
Preferably, the recombinant vector also includes the green fluorescence protein gene under CMV promoter control（EGFP gene）,
Recombinant pseudorabies virus for screening-gene missing.
In another aspect of this invention, a kind of preparation method of pseudo-rabies viurs attenuated strain strain, including following step are additionally provided
Suddenly：
(1) above-mentioned recombinant vector is built, which also includes EGFP fluorescent marker genes；
(2) extraction Pseudorabies virus total DNA is contained the recombinant vector cotransfection cells of the total DNA and step (1)
The recombinant pseudorabies virus of EGFP fluorescent markers；
(3) above-mentioned recombinant vector is built, which does not include EGFP fluorescent marker genes；
(4) total DNA for the recombinant pseudorabies virus that extraction step (2) obtains carries the recombination of the total DNA and step (3)
Body cotransfection cells, obtain the recombinant pseudorabies virus not comprising EGFP fluorescent markers, which is pseudo- mad
Dog disease poison low virulent strain.
In another aspect of this invention, a kind of vaccine composition is additionally provided, the composition includes and adjuvant or medicinal
The pseudo-rabies viurs attenuated strain strain of carrier mixing.
The vaccine composition is suitable for collunarium, injection inoculation.
In another aspect of this invention, it is pseudo- in preparation prevention or treatment to additionally provide a kind of above-mentioned pseudo-rabies viurs attenuated strain strain
Application in rabic vaccine.
In another aspect of this invention, it additionally provides and a kind of distinguishing above-mentioned pseudo-rabies viurs attenuated strain strain and street strain infection
Detection kit, including：For the primer pair of missing gene sequence design shown in SEQ ID NO.1.Utilize drawing for design synthesis
Object pair can distinguish the Pseudorabies virus gene delection low virulent strain for identifying the present invention and street strain by pcr amplification reaction.
In another aspect of this invention, it additionally provides and a kind of distinguishing above-mentioned pseudo-rabies viurs attenuated strain strain and street strain infection
Detection kit, including Pseudorabies virus gE protein antibodies.By detecting the gE antibody of infected pigs' body, it can distinguish and identify this
The Pseudorabies virus gene delection low virulent strain of invention and the infection of street strain.
The Pseudorabies virus gene delection low virulent strain of the present invention is inoculated with 11 age in days piglets, clinical symptoms does not occur, safely may be used
It leans on, it can be as the vaccine candidate strain of effective prevention pseudoabies.
Description of the drawings
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is the pBIE-GFP plasmid construction route maps of the embodiment of the present invention 1；
Fig. 2 is I double digestion qualification result figure of the pXEGFP-C3 plasmids Nhe I that the embodiment of the present invention 1 has been transformed and Xho；
Fig. 3 is the PCR result figures that the embodiment of the present invention 1 or so recombinates arm；
Fig. 4 is the pBIE-GFP plasmids Sac I and Nhe I, Nhe I and Xba I, I and of Xba of the embodiment of the present invention 1
Xho I carries out double digestion qualification result figure respectively；
Fig. 5 is that the different transfection reagent of the transfer vector that builds of the embodiment of the present invention 1 transfects after BHK-21 cells for 24 hours
Fluorescence microscope result figure；
Fig. 6 be 2PRV JS-2012- △ gI/gE-EGFP poison of the embodiment of the present invention in purification process different generations glimmering
Result figure under light microscope；
Fig. 7 is that 2PRV JS-2012- △ gI/gE-EGFP poison differences of the embodiment of the present invention connect plaque form under toxic dose；
Fig. 8 is the PCR mirror of 2PRV JS-2012- △ gI/gE-EGFP poison gBup/gBdown primers of the embodiment of the present invention
Determine result figure；
Fig. 9 is the PCR mirror of 2PRV JS-2012- △ gI/gE-EGFP poison gEup/gEdown primers of the embodiment of the present invention
Determine result figure；
Figure 10 is the PCR identification knots of 2PRV JS-2012- △ gI/gE-EGFP poison gIU/gID primers of the embodiment of the present invention
Fruit is schemed；
Figure 11 is the PCR identification knots of 2PRV JS-2012- △ gI/gE-EGFP poison gEU/gED primers of the embodiment of the present invention
Fruit is schemed；
Figure 12 is the PCR identification knots of 2PRV JS-2012- △ gI/gE-EGFP poison gIU/gED primers of the embodiment of the present invention
Fruit is schemed；
Figure 13 is the structure route map of 3pBIE plasmids of the embodiment of the present invention；
Figure 14 be after 3PRV JS-2012- △ gI/gE poison inoculating cells of the embodiment of the present invention under fluorescence and natural light
Lesion figure；
Figure 15 is the PCR qualification result figures of 3PRV JS-2012- △ gI/gE poison gIU/gED primers of the embodiment of the present invention；
Figure 16 is body temperature after 4PRV JS-2012- △ gI/gE vaccine virus of the embodiment of the present invention and its parent's poison inoculation piglet
Variation diagram；
Figure 17 be 4PRV JS-2012- △ gI/gE vaccine virus of the embodiment of the present invention and its parent's poison inoculation piglet after it is dead
Die result statistical chart.
Specific implementation mode
In the following example, test method without specific conditions, usually routinely condition, such as《Fine works molecular biosciences
Learn experiment guide》(F.M. Ao Sibai, R.E. James Kingstons, the chief editors such as J.G. Sai Deman, Ma Xuejun, Su Yuelong's translates the Beijing：Section
Learn publishing house, 2004) described in method carry out.
Since existing porcine pseudorabies virus vaccine cannot provide preferably currently a popular Pseudorabies virus variation strain
Immune protective effect, it is therefore necessary to develop PRV new generation vaccines.The present invention is by by Pseudorabies virus velogen strain PRV JS-
2012 gI genes and the portion gene of gE genes are lacked, and are obtained virulence and are caused weak Pseudorabies virus gene-deleted vaccine
PRV JS-2012-△gI/gE.The vaccine has preferable safety to piglet.It is expected to mark attenuated vaccine as genetic engineering
Applied to the prevention to Pseudorabies virus variant.
In following embodiments of the present invention, the experiment material used is as follows：
Virus and cell:(American Cell collection ATCC, preserving number are BHK-21 cells：CCL-10), Vero cells
(the American Type Culture Collection committee of Chinese Academy of Sciences cell bank, preserving number are：GN010), Pseudorabies virus JS-2012 plants (these
Laboratory preserves；It publishes an article and sees：Tong Wu, Zhang Qingzhan, Zheng Hao, Liu Fei, Jiang Yifeng, Dan Tongling, Zhou Yanjun, virgin light will are immune
The separation of Pseudorabies virus and identification China zoonosis journal, 2013,21 (3) in morbidity piglet afterwards:1-7).
Plasmid and bacterial strain：PEGFP-N1, pEGFP-C3 and pBluescript SK (+) have purchased from Shanghai fundamental tone biotechnology
Limit company；Bacillus coli DH 5 alpha competence；Purchased from Beijing Tiangeng.
Other reagents:MEM and DMEN, Invitrogen Products；AseI, SacI, XhoI, ScaI are connected with T4DNA
Enzyme, NEB Products；Fugene HD transfection reagents, promega Products；DNA fragmentation quick QIAquick Gel Extraction Kit in a small amount,
Omega companies；2 × GC Buffer II, dNTP Mix (2.5mmol/L each), LA-Taq, DL-15000, DL-2000DNA
Marker is purchased from TakaRa；Other chemical reagent are that import or domestic analysis are pure.
In an embodiment of the present invention, the cultural method of BHK-21 cells is as follows：
BHK-21 cells in the T25 Tissue Culture Flasks of the MEM culture mediums containing 10%FBS it is adherent cover with single layer after, discard
Culture medium in Tissue Culture Flask, and being washed once with PBS under cell dissociation, will be added with 0.25% pancreatin and new contain 10%FBS
MEM culture mediums, with 1:8 ratio point kind is in cell bottle or tissue culture plate.
In an embodiment of the present invention, the cultural method of Vero cells is as follows：
Vero cells in the T25 Tissue Culture Flasks of the DMEM culture mediums containing 10%FBS it is adherent cover with single layer after, discard
Culture medium in Tissue Culture Flask, and being washed once with PBS under cell dissociation, will be added with 0.25% pancreatin and new contain 10%FBS
DMEM culture mediums, with 1:6 ratio point kind is in cell bottle or tissue culture plate.
The structure of embodiment 1PRV JS-2012gI/gE Deleted Transfers pBIE-GFP
By PCR method, 121632 to 123031 bit bases and 124854 in PRV JS-2012 genomes are amplified
To two bar segments of 126372 bit bases, it is used as the left and right recombination arm of homologous recombination.Meanwhile by endonuclease digestion, from
CMV promoter-EGFP gene segment is cut out in pEGFP-C3 plasmids, and SV40polyA signal sequences are cut out from pEGFP-N1 plasmids
Column-slice section, and left recombination arm-CMV promoter-EGFP gene-SV40polyA signal sequences-right recombination arm is sequentially assembled in
In pBluescript SK (+) carrier, recombinant transfer vector pBIE-GFP is obtained.Specific building process is as follows（Referring to Fig. 1）：
1.1 design of primers
According to JS-2012 plants of whole genome sequences of PRV, with 2 couples of primer PRELF/ of Oligo6.0 Software for Design
PRELR and PRERF/PRERR (being shown in Table 1), is used for expand homologous recombination left arm and right arm, respectively two before and after left recombination arm
End plus I restriction enzyme site of Sac I and Ase have added II restriction enzyme site of EcoR V and Afl in the front end of right recombination arm, and rear end adds
I restriction enzyme sites of Xho.Left recombination arm（PRELF/PRELR）Length be 1416bp, nucleotide sequence such as SEQ ID NO.4 institutes
Show, right recombination arm（PRERF/PRERR）Length be 1543bp, nucleotide sequence is as shown in SEQ ID NO.5.
Table 1 expands the primer of homologous recombination arm
The transformation of 1.2pEGFP-C3
In order to delete the unwanted restriction enzyme sites of pEGFP-C3, using I digestion pEGFP-C3 of Sca I and Sma, recycling is big
Segment, and large fragment is connected with T4DNA ligases and is cyclized, conversion chooses bacterium and extracts plasmid, by plasmid Nhe I and Xho I
Double digestion is identified that the plasmid being transformed is because I restriction enzyme sites of Xho are deleted, so an only band, the plasmid not being transformed
Two band of 3.9kb and 750bp will be cut into.The results are shown in Figure 2, obtains new plasmid pXEGFP-C3.In fig. 2,
" M " refers to DNA Marker DL2000, and " 1 " refers to plasmid 1, and " 2 " refer to plasmid 2, and " 3 " refer to plasmid 3, and " 4 " refer to pEGFP-C3, and " M " refers to
DNA Marker DL15000。
1.3 homologous recombination arms expand
PRELF/PRELR and PRERF/PRERR two is utilized respectively to primer, using the full-length genome of PRVJS-2012 as template
PCR operations are carried out, reaction system is 50 μ L：2×GCBuffer Ⅱ25ul；dNTP Mix(2.5mmol/Leach)4ul；Upstream
Primer 1ul (10pmol/ul)；Downstream primer 1ul (10pmol/ul)；LA-Taq1ul；ddH2O15ul；Genomic DNA 3ul.Instead
The condition is answered to be:94℃5min；94 DEG C of 30s, 69 DEG C of 30s, 72 DEG C of 2min are recycled 35 times；Finally extend 10min in 72 DEG C.It is placed in
Electrophoresis on 1% agarose amplifies the left recombination arm pieces section of about 1400bp and the right recombination arm pieces section of 1500bp, and recycles two
Bar segment（Fig. 3）.In figure 3, " 1 " refers to the left arm amplified, and " 2 " refer to positive control, and " 3 " refer to negative control, and " M " refers to
DNAMarkerDL2000, " 4 " negative control, " 5 " refer to positive control, and " 6 " refer to the right arm amplified.
The structure of 1.4pBLA-CMV-EGFP carriers
By the left homologous recombination arm pieces section of recycling with I double digestion of Sac I and Ase, and recycle the segment of about 1400bp.It is double
Digestion system is（50ul）：NEBuffer25ul, BSA0.5ul, Sac I 1ul, Ase I 1ul, DNA fragmentation 2ug add ddH2O is extremely
50ul.37 DEG C of digestions are stayed overnight.
The segment of 1300bp or so will be recycled after pXEGFP-C3 I double digestions of Ase I and Xba.Double digestion system is
（50ul）：NEBuffer25ul, BSA0.5ul, Ase I 1ul, Xba I 1ul, pXEGFP-C32ug, add ddH2O to 50ul.It sets
It is stayed overnight in 37 DEG C of digestions.
By pBluescript SK (+) to recycle the segment of 2900bp or so after I double digestion of Sac I and Xba.Double digestion
System is（50ul）：NEBuffer45ul, BSA0.5ul, Sac I 1ul, Xba I 1ul, pBluescript SK (+) 2ug, adds
ddH2O to 50ul.37 DEG C of digestions are stayed overnight.
Three above recycling segment T4DNA ligases are connected, and convert bacillus coli DH 5 alpha competence, by plasmid
Extraction, PCR identifications and sequencing analysis filter out positive recombinant plasmid, and are named as pBLA-CMV-EGFP.
The structure of 1.5pBSV40-RA
After the right homologous recombination arm pieces section of recycling I double digestion of Ecor V and Xho, the segment of 1500bp is recycled.It is double
Digestion system is（50ul）：NEBuffer45ul, BSA0.5ul, Ecor V 1ul, Xho I 1ul, DNA fragmentation 2ug add ddH2O
To 50ul.37 DEG C of digestions are stayed overnight.
By pBluescript SK (+) with I double digestion of Ecor V and Xho after, the segment of recycling 2900bp or so.Double enzymes
The system of cutting is（50ul）：NEBuffer45ul,BSA0.5ul,Ecor Ⅴ1ul,Xho Ⅰ1ul,pBluescript SK(+)
2ug adds ddH2O to 50ul.37 DEG C are placed in, overnight.
The respective segments of above two step recycling T4DNA ligases are connected into simultaneously transformed competence colibacillus bacillus coli DH 5 alpha, warp
It crosses plasmid extraction, PCR identifications and sequencing analysis and filters out positive recombinant plasmid, and be named as pBRA.
Again by after pBRA I double digestions of Afl II and Xba, the segment of 4400bp or so is recycled.Double digestion system is
（50ul）：NEBuffer45ul, BSA0.5ul, Afl II 1ul, Xba I 1ul, pBRA2ug, add ddH2O to 50ul.It is placed in 37
DEG C, overnight.
With I double digestion pEGFP-N1 of Afl II and Xba, and the segment for recycling 220bp or so.Double digestion system is
（50ul）：NEBuffer45ul, BSA0.5ul, Afl II 1ul, Xba I 1ul, pEGFP-N12ug, add ddH2O to 50ul.It sets
In 37 DEG C, overnight.
Above-mentioned Afl II is connected with two segments that I digestions of Xba are recycled with T4DNA ligases, and converts Escherichia coli
DH5 α competence filters out positive recombinant plasmid by plasmid extraction, PCR identifications and sequencing analysis, and is named as
pBSV40-RA。
The structure of 1.6PRV JS-2012gI/gE Deleted Transfers
With I double digestion pBLA-CMV-EGFP of Xba I and Xho, the segment of recycling 5600bp or so；With Xba I and Xho I
Double digestion pBSV40-RA, the segment of recycling 1700bp or so.Double digestion system is（50ul）：NEBuffer45ul,
BSA0.5ul, Xho I 1ul, Xba I 1ul, DNA2ug, add ddH2O to 50ul.37 DEG C are placed in, overnight.
The segment of 5600bp or so is connected with the segment of 1700bp or so with T4DNA ligases and converts Escherichia coli
DH5 α competence filters out positive recombinant plasmid by plasmid extraction, PCR identifications and sequencing analysis, positive recombinant plasmid is used
Sac I and Nhe I, Nhe I and Xba I, Xba I and Xho I carry out double digestion respectively, are as a result consistent with expection.Positive weight
Group plasmid can two bands of 5.4kb and 2kb be cut by Sac I and Nhe I, can by Nhe I and Xba I, cut into 6.6kb and
Two bands of 750bp can cut into two bands of 5.6kb and 1.8kb by Xba I and Xho I（See Fig. 4）.By the positive restructuring
Plasmid is named as pBIE-GFP, as PRV JS-2012gI/gE Deleted Transfers.In Fig. 4, left side " M " refers to DNA
Marker DL2000, " 1-4 " refer to pBIE-GFP clone's 1-4 I digestions of Sac I and Nhe, and " 5-8 " refers to pBIE-GFP clones 1-4
With I digestion of Nhe I and Xba, " 10-12 " refers to pBIE-GFP clone's 1-4 I digestions of Xba I and Xho, and right side " M " refers to DNA
MarkerDL15000.In the PRV JS-2012gI/gE Deleted Transfers that success is built, " left recombination arm+CMV+EGFP
The sequence of the right recombination arms of+SV40+ " is as shown in SEQ ID NO.6.
The activity verification of 1.7pBIE-GFP marker gene
With reference to Fugene HD transfection reagent specifications, pBIE-GFP transfections are grown in 6 orifice plates with Fugene HD
BHK-21 cells.24 to 48 hours after transfection, in fluorescence microscopy microscopic observation, the results are shown in Figure 5, transfectional cell high level table
Up to GFP.
The structure of embodiment 2PRVJS-2012- △ gI/gE-EGFP strains
With PRVJS-2012 infection cells and total DNA is extracted, by total DNA and pBIE-GFP cotransfection cells, obtains weight
Group virus.Plaque-purified, the recombinant virus JS-2012- △ for obtaining gI and gE gene delections and being marked containing EGFP are taken turns by 6
gI/gE-EGFP。
2.1 design of primers
According to PRVJS-2012 plants of whole genome sequence, with 2 couples of primer gIU/gID of Oligo6.0 Software for Design with
GEU/gED is used for the full length sequence of the gI and gE to expand PRV JS-2012.The common detection primer gBup/gBdown of PRV,
And the identification primer gEup/gEdown for distinguishing wild poison and vaccine virus is preserved by this laboratory.Each primer sequence is shown in Table 2.
Table 2PRV detection primers
The extraction of 2.2PRV JS-2012 genomes
By the BHK-21 cells of JS-2012 plants of inoculation fusion single layers of PRV, wait for that the cell of 70%-80% all generates lesion,
3/4ths culture solutions are discarded, with cell scraper by under cell scraper, and cell liquid are moved into 50ml centrifuge tubes, 1500rmp centrifugations
5min abandons supernatant, and sedimentation cell is resuspended with TNE solution.Total DNA is extracted from infection cell with phenol-chloroform method, and is dissolved in TE
In, it is saved backup in -30 DEG C.
2.3 cotransfection
The BHK-21 cells being grown in 35mm culture dishes when density reaches 70%~85%, with reference to specification, are used
Fugene HD transfection reagents will be in DNA the and pBIE-GFP cotransfections to BHK-21 cells of 2.2 extractions.Cell after transfection is set
In 37 DEG C of CO2It is cultivated in incubator, occurs cytopathy after 36 hours, harvest supernatant.
The purifying of 2.4PRV JS-2012- △ gI/gE-EGFP
By the virus of receipts after dilution, the Vero cells of inoculation fusion single layer.1h after infection sucks infection liquid, in cell
Upper berth adds the MEM culture mediums containing 1% agarose and 2%FBS.After room temperature solidification, inoculating cell is moved into CO2It is cultivated in incubator.
After 60h, inoculating cell is placed in fluorescence microscopy under the microscope, marks and have beside picking the plaque of cell expression green fluorescence,
It is placed in DMEM.Plaque liquid through freeze thawing is inoculated with Vero cells, progress is Plaque-purified, and picking has the sky that green fluorescence is expressed
Spot carries out next round purifying.Plaque-purified by 6 wheels, the virus inoculation vero cells of acquisition, the plaque of generation expresses EGFP
（See Fig. 6, Fig. 7）.This shows to obtain pure recombinant virus PRV JS-2012- △ gI/gE-EGFP.
The PCR of 2.5PRV JS-2012- △ gI/gE-EGFP is identified
With the DNA of DNA extraction kit extraction PRV JS-2012- △ gI/gE-EGFP, with gIU/gID, gEU/gED,
GBup/gBdown, gEup/gEdown and gIU/gED five carries out PCR identifications respectively to primer.PCR reaction systems are 20 μ L：2
×GC Buffer Ⅱ10ul；dNTP Mix(2.5mmol/L each)2ul；Sense primer 0.5ul (10pmol/ul)；Downstream
Primer 0.5ul (10pmol/ul)；LA-Taq0.5ul；ddH2O4.5ul；PRVJS-2012- △ gI/gE-EGFP poison
DNA2ul.Primer gIU/gID, gEU/gED and gIU/gED reaction condition is:94 DEG C of pre-degeneration 5min；94 DEG C denaturation 30s, 69 DEG C
Anneal 30s, and 72 DEG C of extension 2.5min are recycled 35 times；Finally extend 10min in 72 DEG C.Primer gBup/gBdown reaction conditions
For:94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 68 DEG C of annealing 30s, 72 DEG C extend 45s, recycle 35 times；Finally in 72 DEG C of extensions
10min.Primer gEup/gEdown reaction conditions are:94 DEG C/5min, 94 DEG C/30s, 64 DEG C/30s, 72 DEG C/30s, cycle 35
It is secondary；Finally extend 10min in 72 DEG C.PCR product is observed in gel imager with 1% agarose electrophoresis.PCR the results show that
The gBup/gBdown amplifications of PRV JS-2012- △ gI/gE-EGFP are positive（Fig. 8）；gEup/gEdown、gIU/gID、gEU/
The amplification of gED is negative（Fig. 9, Figure 10, Figure 11）；Though gIU/gED expands shaping from PRV JS-2012- △ gI/gE-EGFP
Band, but less than parent poison PRV JS-2012's（Figure 12）.These results indicate that recombinant virus PRV JS-2012- △ gI/gE-
EGFP can not expand gI and gE genetic fragments, be successfully realized the missing of gI/gE genes." M " refers to DNA in Fig. 8-12
Marker DL2000, " 1 " refer to PRV JS-2012- △ gI/gE-EGFP Plaque Clones 1, and " 2 " refer to PRV JS-2012- △ gI/
GE-EGFP Plaque Clones 2, " 3 " refer to PRV JS-2012, and " 4 " refer to negative control, and subsequent " M " refers to DNA Marker in Figure 12
DL15000。
The deletion of embodiment 3PRVJS-2012- △ gI/gE-EGFP virus signature genes
With reference to the thinking of embodiment 1, I restriction enzyme sites of end Ase of left recombination arm are changed into Xba I.By with Sac I
It connects left recombination arm being filled in the pBRA plasmids built in 1.5 with I digestions of Xba and forms new homologous recombination transfer vector
pBIE（See Figure 13）.With PRV JS-2012- △ gI/gE-EGFP infection cells and total DNA is extracted, by total DNA and pBIE corotation
Cell is contaminated, recombinant virus is obtained.It is Plaque-purified by 3 wheels, obtain the recombinant virus PRV JS- marked without EGFP
2012-△gI/gE。
3.1 primer
Since experiment needs I restriction enzyme sites of end Ase by the left recombination arm in embodiment 1 to change Xba I into, need
A downstream primer is redesigned, changes I restriction enzyme sites of Ase into Xba I.Primer PRELR-X sequences are：5’
TTCTAGAAACTAGGGTCCACGACGCGCAGGCTG3’(SEQ ID NO.20).Left recombination arm（PRELF/PRELR-X）Sequence
Row are as shown in SEQ ID NO.7.
The structure of 3.2 transfer vector pBIE
New recombination left arm is amplified with primer PRELF/PRELR-X.The pBRA plasmids that will be built in the left arm and 1.5
Double digestion is carried out with Sac I and Xba I respectively, respective large fragment is separately recovered, two segments are connected with T4DNA ligases,
And bacillus coli DH 5 alpha competence is converted, positive recombinant plasmid is filtered out by plasmid extraction, PCR identifications and sequencing analysis, and
It is named as pBIE.
The deletion of 3.3PRV JS-2012- △ gI/gE-EGFP virus signature genes
The full-length genome of method extraction PRV JS-2012- △ gI/gE-EGFP viruses in reference 2.2, refers again to 2.3
Method is obtained in the full-length genome of PRV JS-2012- △ gI/gE-EGFP and pBIE cotransfections to cell by plaque purification
Recombinant virus without EGFP green fluorescent labels, as shown in figure 14, A is the photo under fluorescence in fig. 14, and B is same regards
Photo of the open country under natural light shows the EGFP marker gene of PRV JS-2012- △ gI/gE-EGFP viruses by fluorescence results
It deletes successfully.And the recombinant virus is named as PRV JS-2012- △ gI/gE.
The PCR of 3.4PRV JS-2012- △ gI/gE is identified
Since PRV JS-2012- △ gI/gE viruses are deleted on the basis of PRV JS-2012- △ gI/gE-EGFP viruses
CMV-EGFP-SV40 sequences（Size is about 1500bp）, with reference to 2.5 method PCR identifications, PCR results are carried out with gIU/gED
The segment that display PRV JS-2012- △ gI/gE viruses are expanded is 1000bp or so, hence it is evident that is less than PRV JS-2012- △ gI/
The segment that gE-EGFP viruses are expanded（2500bp or so）, it is consistent with expected results（Figure 15）, therefore PRV JS-2012- △ gI/
GE poison is built successfully." M " refers to DNA Marker DL2000 in Figure 15, and " 1 " refers to PRV JS-2012- △ gI/gE poison, and " 2 " refer to PRV
JS-2012- △ gI/gE-EGFP poison, " 3 " refer to PRV JS-2012 poison, and " 4 " refer to negative control, and subsequent " M " refers to DNA Marker
DL15000。
The titer determination of 3.5PRV JS-2012- △ gI/gE
PRV JS-2012- △ gI/gE are inoculated with Vero cells, when there is CPE in 80% cell, harvest virus.With reference to text
It offers（Yin Zhen etc., animal virology, Science Press, 1997）, PRV JS-2012- △ are measured with tissue culturing plates with 96 hole method
The titre of gI/gE.After PRV JS-2012- △ gI/gE are serially diluted with 10 times of the DMEM works containing 2%FBS, by diluted virus
The Vero cell monolayers being inoculated in 96 porocyte culture plates.8 holes are inoculated with per dilution, if the control of 8 holes is (with the DMEM containing 2%FBS
Inoculation), it sets in 37 DEG C 5% of carbon dioxide incubator and cultivates, to after being inoculated with the 4th day, there is the hole count of CPE in record for observation daily,
TCID is calculated according to Reed-Muench methods50.The results show that titre of the PRV JS-2012- △ gI/gE poison on Vero cells is
105.5TCID50/ml。
The safety testing of 11 age in days piglets of embodiment 4PRV JS-2012- △ gI/gE couple
Recombination deficient virus JS-2012- △ gI/gE are inoculated with 11 age in days piglets, are not led to that piglet falls ill, are had preferable
Safety.The piglet of inoculation JS-2012- △ gI/gE only generates PRV gB antibody, without generating PRV gE antibody, can be used as and exempt from
Epidemic disease marker vaccine Candidate Strain.
4.1 experiment piglets
Piglet purchase is tested in Shaoxing, Zhejiang Province city Shengzhou Large-scale pig farm, 11 age in days sodium selenites, pseudo- mad dog
Viral gB and gE antibody is feminine gender, and Pseudorabies virus（PRV）, reproductive and respiratory syndrome virus (PRRSV), swine fever virus (CSFV) and
The Pathogen test of porcine circovirus 2 type (PCV2) is feminine gender.
4.2 zooperies are grouped and attack poison
12 piglets of purchase, are randomly divided into 3 groups：J groups, E groups and D groups.J groups 5, number：J1、J2、J3、J4、
Strong 2 milliliters of the poison of J5, equal collunarium inoculation PRVJS-2012（Containing 2 × 105A TCID50）.E groups 5, number：E1, E2, E3, E4 and
GI/gE2 milliliters of poison of E5, equal collunarium inoculation PRV JS-2012- △ (contain 2 × 105A TCID50).D groups 2, number：D1 and D2,
Equal collunarium is inoculated with DMEM2 milliliters.
4.3 clinicing symptom observations and body temperature measurement
After inoculation, 9 points or so of every morning, the clinical symptoms of animal house observation experiment pig are gone, is examined daily within first 7 days after attacking poison
Body temperature measurement, one fixed clinical thermometer of every pig are carried out to each experiment pig when clinical symptoms.It is opened within second day after attacking poison
Begin, J groups body temperature all increases and all at 41 degree or more（Figure 16）.It attacks after poison the 4th day, J2, J4 and J5 pig are dead；J1 is on the point of with J3
Si ﹑ four limbs are in shape of striking.It attacks after poison the 5th day, J1 and J3 pigs are dead.E groups and the body temperature and clinical symptoms of D group pigs are normal（Figure
17）.It attacks after poison the 17th day, the experiment pig of E groups and D groups is cutd open and is killed, the internal organs of each head pig of visual inspection.The heart of slaughter pig, liver, spleen,
Lung, kidney and lymph node are normal, do not occur lesion.This shows that JS-2012 plants have stronger virulence, can cause 11 age in days piglets
100% is dead, and the gene-deleted strain JS-2012- △ gI/gE that the present invention is built are in then weak malicious feature, are lost to 11 age in days piglets
Pathogenicity.
4.4 blood sampling detection antibody
The experiment pig blood of 1d, 3d, 5d, 7d and 17d after poison are attacked before poison and are attacked in acquisition, and detach serum.With IDEXX's
Pseudorabies virus gB, gE antibody assay kit are respectively detected all serum.As a result 3 and table 4 are see the table below, due to J groups
(PRV JS-2012 inoculation groups) attacks complete dead in 5d after poison, the antibody of anti-PRV gB and gE is not detected.And E groups (PRV JS-
2012- △ gI/gE inoculation groups) attack poison after 7d start to generate anti-PRV gB antibody, there is gB antibody, but anti-PRV gE after 17d
Antibody is not detected always.Then PRV gB and gE antibody are feminine gender to control group D groups.This shows recombination deficient strain PRV JS-
2012- △ gI/gE can induce Pigs Inoculated and generate anti-gB antibody, but due to the gE gene delections of PRV JS-2012- △ gI/gE, no
It can induce the antibody for generating anti-gE.JS-2012- △ gI/gE can be developed into a kind of immune labeled epidemic disease of PRV gene delections by this
Seedling is used for the prevention and control and elimination of pig farm PRV.
After table 3PRV JS-2012- △ gI/gE vaccine virus and its parent's poison inoculation piglet gB antibody levels are detected with ELISA
Variation
Note：In table 3, numerical value ＜ 0.6 is antibody positive, and 0.6 ＜ numerical value ＜ 0.7 is that antibody is suspicious, and 0.7 antibody of numerical value ＞ is
It is negative.
After table 4PRV JS-2012- △ gI/gE vaccine virus and its parent's poison inoculation piglet gE antibody levels are detected with ELISA
Variation
Note：In table 4, numerical value ＜ 0.6 is antibody positive, and 0.6 ＜ numerical value ＜ 0.7 is that antibody is suspicious, and 0.7 antibody of numerical value ＞ is
It is negative.
Embodiments of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but can not
Therefore it is interpreted as the limitation to the scope of the claims of the present invention.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection model of the present invention
It encloses.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (3)
1. a kind of pseudo-rabies viurs attenuated strain strain, which is characterized in that the low virulent strain is the gene delection of JS-2012 plants of Pseudorabies virus
Low virulent strain, the gene order of missing are the nucleotides sequence of JS-2012 plants of gI gene 594-1101 bit bases of Pseudorabies virus
The nucleotide sequence of row and JS-2012 plants of gE gene 1-1212 bit bases of Pseudorabies virus, the gene order of the missing
For nucleotide sequence shown in SEQ ID NO.1.
2. application of the pseudo-rabies viurs attenuated strain strain described in claim 1 in preparing the vaccine for preventing pseudoabies.
3. a kind of distinguishing the detection kit that pseudo-rabies viurs attenuated strain strain is infected with street strain described in claim 1, feature exists
In, including：For the primer pair of missing gene sequence design shown in SEQ ID NO.1, the primer pair is gEup/gEdown,
Its nucleotide sequence is as shown in SEQ ID NO.18,19.
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