CN109134669A - Fusion protein of porcine pseudorabies virus and preparation method thereof, application and vaccine - Google Patents
Fusion protein of porcine pseudorabies virus and preparation method thereof, application and vaccine Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, specifically, providing fusion protein of a kind of porcine pseudorabies virus and preparation method thereof, application and vaccine.The fusion protein of porcine pseudorabies virus provided by the invention includes gB section and gD section, the expression of gB section nucleotide sequence as shown in SEQ ID NO.1, the expression of gD section nucleotide sequence as shown in SEQ ID NO.2.Sequence shown in SEQ ID NO.1 and SEQ ID NO.2 is to select the gene of classical strains and current popular strain as research object, pass through the sequence obtained after comparative analysis, and the sequence is carried out to the optimization and modification of codon, to achieve the purpose that further increase fusion protein antigen broad spectrum activity and improve antigen presentation amount.The present invention also provides the vaccines of the preparation method and application of fusion protein and preparation.
Description
Technical field
The present invention relates to field of biotechnology, in particular to the fusion protein and its system of a kind of porcine pseudorabies virus
Preparation Method, application and vaccine.
Background technique
Porcine pseudorabies (porcine pseudorabies, PR) are by Pseudorabies virus (Pseudorabies
One of bvirus, PRV) the caused, Important Infectious Diseases of serious harm China's pig breeding industry sound development.The virus belongs to bleb poison
The linear dsdna virus of section's A type herpesviral subfamilies, the G+C content of viral DNA are relatively high.It is currently known viral sugared egg
It is white to have 11 kinds, i.e., it must glycoprotein and nonessential glycoprotein.
The susceptible range of pseudoabies is very wide, and newborn piglet is mostly that lethal infection is similar with other susceptible species, more
Die of central nervous system disease.Adult Pig infects the disease multilist and shows respiratory disease, is in mostly subclinical infection, without obvious disease
Shape.After pig infects PRV, the immune system of itself is damaged, immunity degradation, thus is easier to secondary other diseases, such as pig
Pest, porcine reproductive and respiratory syndrome etc..Difficulty is increased to the prevention and treatment of porcine pseudorabies.Currently, the infection of PR and disease incidence are all
It is very high, easily cause huge economic loss.
For the suckling pig within 2 week old, in initial infection it is possible that fever, anorexia, vomiting, diarrhea,
The symptoms such as lassitude；As neural clinical symptom then occurs in the intensification of the state of an illness, such as shake, incoordination, the four limbs that fall down to the ground stroke
It is dynamic；It is often accompanied by epilepsy seizure or jerk, lethargic sleep, last failure death；The death rate may be up to 100%.
The main clinical symptom of pig of somewhat larger about 3-4 week old is same as 2 week old piglets, but compares the disease of 2 week old piglets
Journey is slightly long, and with constipation phenomenon, the death rate is generally up to 40-60%；Even if resistance to mistake, can also occur together have growth retardation, by
The sequelae such as resistance, hemiplegia.
2 pig the autoimmunity more than monthly age enhances, and is in subclinical infection after infection PRV, without apparent clinical symptoms more；Have
Symptom is also relatively slight, transient fever and cough, it is also possible to have vomiting phenomenon.
Farrowing sow is higher to PRV neurological susceptibility, shown as after infection the respiratory systems such as cough, spiritual depressed, appetite stimulator,
Then there is miscarriage symptom, produces the mummification of fetus, produces stillborn foetus and produce weak son, wherein based on stillborn foetus in gastrointestinal symptom.It is produced
Weak pigle is often accompanied by the symptoms such as diarrhea, spasm, ataxia, opisthotonos, dead usually in 24-36h.
Prevention and control to porcine pseudorabies, are puted prevention first both at home and abroad with vaccine immunity, in conjunction with Pseudorabies virus gE antigen and
GE antibody test is eliminated, purifies wild virus infection pig.Currently, the PR vaccine of clinical application both at home and abroad can substantially be divided into following three classes:
First is that the open country by separation is malicious or virulent after formalin-inactivated, add adjuvant emulsion that oil emulsion inactivated vaccine is made；Second is that by separation
Wild poison or the virulent attenuated vaccine through the Attenuation development repeatedly of non-pig source cell or chicken embryo；Third is that utilizing technique for gene engineering
The gene-deleted vaccine of building.Although inactivated vaccine safety, immune efficacy is limited, and cannot play the target of epidemic disease purification.
There are hidden danger for attenuated vaccine safety, and the production cycle is long, at high cost.In addition, porcine pseudorabies virus gene deleted live vaccine, complete
Viral inactivation vaccine is grown on attached cell, and needs animal derived protein, brings risk for vaccine manufacture.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of fusion protein of porcine pseudorabies virus, the fusion protein broad spectrum activity
It is good, there is good immunogenicity, the antibody titer being prepared is high and can prevent the porcine pseudorabies virus of a variety of hypotypes.
The second object of the present invention is to provide a kind of preparation method of above-mentioned fusion protein, which may be implemented
The above-mentioned fusion protein of great expression high quality.
The third object of the present invention is to provide the application of above-mentioned fusion protein.
The fourth object of the present invention is to provide a kind of porcine pseudorabies virus vaccine comprising above-mentioned fusion protein.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of fusion protein of porcine pseudorabies virus, including gB section and gD section；
The expression of gB section nucleotide sequence as shown in SEQ ID NO.1, the gD section is by SEQ ID NO.2 institute
The nucleotide sequence expression shown.
Further, the gB section and the gD section put in order as gB-gD, are connected by Linker；
The Linker has the nucleotide sequence as shown in SEQ ID NO.3.
Further, the fusion protein is the albumen of HEK 293-F cell expression system expression.
A kind of preparation method of above-mentioned fusion protein expresses encoding said fusion protein in mammalian expression systems
Gene.
Further, the gene of encoding said fusion protein is expressed in HEK 293-F cell expression system.
Further, the expression vector of the gene comprising encoding said fusion protein is provided, the expression vector is imported
In HEK 293-F cell, then HEK 293-F cell is screened, obtains stablizing the HEK for expressing the fusion protein
293-F cell strain, the HEK 293-F cell strain express to obtain the fusion protein；
Preferably, the screening includes pressurization screening and monoclonal screening.
Further, the HEK 293-F cell of the fusion protein is expressed using the screening of Geneticin screening system；
Preferably, expression vector used in the Geneticin screening system is pcDNA3.1, pEE6.4, pEE12.4
Or pGL4.13, preferably pcDNA3.1.
The fusion protein that above-mentioned fusion protein or preparation method are prepared is in following A)-D) at least one of answer
With:
A porcine pseudorabies virus vaccine) is prepared；
B the antibody of porcine pseudorabies virus) is prepared；
C) the kit of preparation detection porcine pseudorabies virus；
D porcine pseudorabies virus diagnostic antigen) is prepared.
A kind of porcine pseudorabies virus vaccine of the fusion protein comprising above-mentioned porcine pseudorabies virus.
Further, the dosage of fusion protein is g/ parts of 70-90 μ in the porcine pseudorabies virus vaccine；
Preferably, the vaccine further includes auxiliary material, and the auxiliary material includes one of vaccine adjuvant, stabilizer and antibiotic
Or it is a variety of；
Preferably, the vaccine adjuvant includes aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white oil assistant
Agent, carbomer, propolis, MF59 adjuvant or ISA201 are, it is preferable to use ISA201.
Compared with prior art, the invention has the benefit that
The fusion protein of porcine pseudorabies virus provided by the invention includes gB section and gD section, and gB section is by SEQ ID
The expression of nucleotide sequence shown in NO.1, the expression of gD section nucleotide sequence as shown in SEQ ID NO.2.SEQ ID NO.1
It is to select the gene of classical strains and current popular strain as research object with sequence shown in SEQ ID NO.2, by right
Than the sequence obtained after analysis, and by the optimization and modification of sequence progress codon, fusion protein is further increased to reach
Antigen broad spectrum activity and the purpose for improving antigen presentation amount.The broad spectrum activity of the fusion protein is good, has good immunogenicity, preparation
Obtained antibody titer is high and can prevent the porcine pseudorabies virus of a variety of hypotypes.
The present invention provides the preparation method of above-mentioned fusion protein, which may be implemented the upper of great expression high quality
Fusion protein is stated, and easy to operate, is suitble to large-scale production.
The present invention provides the porcine pseudorabies virus vaccine comprising above-mentioned fusion protein, and preparation cost is low, less than existing vaccine
The 1% of production cost, while good immune effect.After piglet with 21 age in days of vaccine immunity, blood sampling is surveyed respectively after being immunized 21
Neutralizing antibody, neutralizing antibody on the 21st is not less than 1:1000 as the result is shown, also, attacking malicious vaccine as the result is shown can either be effectively pre-
The attack (JS plants) of anti-classics virulent (SC plants) and current popular strain, protective rate can achieve 100%.The vaccine is better than conventional
Vaccine.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is gB gene PCR amplified production electrophoresis result in the embodiment of the present invention 1, and each swimming lane is respectively as follows: from left to right
Marker DL15000, gB gene PCR amplified production；
Fig. 2 is gD gene PCR amplified production electrophoresis result in the embodiment of the present invention 2, and each swimming lane is respectively as follows: from left to right
Marker DL15000, gD gene PCR amplified production.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
A kind of fusion protein of porcine pseudorabies virus, including gB section and gD section, wherein gB section is by SEQ ID
The expression of nucleotide sequence shown in NO.1, the expression of gD section nucleotide sequence as shown in SEQ ID NO.2.
The important immunogenic components of the main constituents and PRV that gB albumen is constituted as PRV cyst membrane, into machine
Body can be stimulated to generate the neutralizing antibody of complement-dependent, complement dependent/non-dependent after body.
GD albumen is that PRV invasion cell occurs to infect how necessary glycoprotein, gD induction produces as one of required glycoprotein
No matter complement whether there is the neutralization reaction that can carry out PRV to raw neutralizing antibody.
Sequence shown in SEQ ID NO.1 and SEQ ID NO.2 is to select the gene of classical strains and current popular strain
As research object, by the sequence obtained after comparative analysis, and by the optimization and modification of sequence progress codon, to reach
It further increases fusion protein antigen broad spectrum activity and improves the purpose of antigen presentation amount.The broad spectrum activity of the fusion protein is good, has
Good immunogenicity, the antibody titer being prepared is high and can prevent the porcine pseudorabies virus of a variety of hypotypes.
In certain embodiments of the present invention, gB section and the gD section put in order as gB-gD, pass through
Linker connection, wherein Linker has the nucleotide sequence as shown in SEQ ID NO.3.By with Linker by gB and gD
Be attached, both achieved the purpose that Protein reconstitution, in turn avoid gB and gD albumen respectively higher structure formed influence each other.
The nucleotide sequence of encoding fusion protein is as shown in SEQ ID NO.4, the amino acid sequence of fusion protein such as SEQ ID NO.5 institute
Show.
It is preferably carried out in mode at of the invention one, fusion protein is the expression of HEK 293-F cell expression system
Albumen.HEK 293-F cell is mammalian expression systems, uses HEK 293-F cell as host cell, avoids loss
The epitope of the conformation type of the fusion protein.
A kind of preparation method of above-mentioned fusion protein expresses the base of encoding fusion protein in mammalian expression systems
Cause.The above-mentioned fusion protein of great expression high quality may be implemented in the preparation method, and easy to operate, is suitble to scale metaplasia
It produces.Mammalian expression systems for example can be but not to be limited to HEK 293-F cell, HEK 293-E cell, HEK 293-T thin
Born of the same parents, Chinese hamster ovary celI or COS cell.
In the present invention, one is preferably carried out in mode, and expression encodes described melt in HEK 293-F cell expression system
The gene of hop protein.HEK293 is the stable cell lines obtained after adenovirus Ad5 transfection human embryonic kidney cells, and HEK293-F is
There is easy transfection, height to express for the derived cell system of HEK293, Natively glycosylated modification, permission albumen correctly folds and correlation is turned over
The advantages that being modified after translating.
In certain embodiments of the present invention, the index method of the fusion protein of porcine pseudorabies virus includes: offer packet
The expression vector of gene containing encoding fusion protein imports expression vector in HEK 293-F cell, then to HEK 293-F
Cell is screened, and the HEK 293-F cell strain for stablizing expressed fusion protein is obtained, and HEK 293-F cell strain, which is expressed, to be melted
Hop protein.It is preferred that the HEK 293-F cell strain expression quantity of stable expressed fusion protein is not less than 1mg/ml.
In the present invention, one is preferably carried out in mode, and screening includes pressurization screening and monoclonal screening.First with pressurization
Screening is conducive to the cell strain for quickly obtaining expressed fusion protein, then the high table of available fusion protein is screened by monoclonal
Up to pure cell strain.
In one preferred embodiment of the invention, Geneticin screening system screening expressed fusion protein is used
HEK 293-F cell.G Geneticin (G-148) is a kind of aminosugar antibiotics, structure and neomycin, gentamicin, card
That mycin is similar, it is synthesized by influencing 80S ribose body function blocking protein, all toxic to cells such as protokaryon and eukaryons
Property, including bacterium, yeast, plant and mammalian cell, it also include protozoan and worm.When neo gene is integrated into very
Behind the suitable place of nucleus genome, then the sequence that can start neo gene coding is transcribed into mRNA, to obtain resistance product
The high efficient expression of aminoglycoside phosphotransferase makes cell obtain resistance and can train in the selectivity containing G-418 Geneticin
It supports and is grown in base.
In one preferred embodiment of the invention, expression vector used in Geneticin screening system is
PcDNA3.1, pEE6.4, pEE12.4 or pGL4.13, preferably pcDNA3.1.
The fusion protein that above-mentioned fusion protein or preparation method are prepared is in following A)-D) at least one of answer
With:
A porcine pseudorabies virus vaccine) is prepared；
B the antibody of porcine pseudorabies virus) is prepared；
C) the kit of preparation detection porcine pseudorabies virus；
D porcine pseudorabies virus diagnostic antigen) is prepared.
A kind of porcine pseudorabies virus vaccine of the fusion protein comprising above-mentioned porcine pseudorabies virus.The vaccine preparation cost
It is low, less than the 1% of existing production of vaccine cost, while good immune effect.After piglet with 21 age in days of vaccine immunity, it is immunized 21
Neutralizing antibody is surveyed in blood sampling respectively in the future, and neutralizing antibody on the 21st is not less than 1:1000 as the result is shown, also, attacks poison epidemic disease as the result is shown
Seedling can either effectively prevent the attack (JS plants) of classical virulent (SC plants) and current popular strain, and protective rate can achieve 100%.
The vaccine is better than conventional vaccine.
The present invention it is some be preferably carried out in mode, in porcine pseudorabies virus vaccine the dosage of fusion protein be 70-90 μ
G/ parts.
In certain embodiments of the present invention, vaccine further includes auxiliary material, and auxiliary material includes vaccine adjuvant, stabilizer and antibiosis
One of element is a variety of.
In some embodiments of the present invention, vaccine adjuvant includes that aluminium hydroxide gel, Freund's complete adjuvant, Freund are incomplete
Adjuvant, white-oil adjuvant, carbomer, propolis, MF59 adjuvant or ISA201 are, it is preferable to use ISA201.
The technical solution provided to facilitate the understanding of the present invention, below with reference to embodiment to technical solution provided by the invention
It is further described.
The fusion protein sequence of the coding porcine pseudorabies virus of embodiment 1
Select the gene of classical strains (SC plants) and current popular strain (JS plants) as research object from Genebank,
By comparative analysis, ingredient of the Dominant Epitopes as vaccine antigen is selected, according to the partially thermophilic of HEK 293-F cell codon
Property, and by the optimization and modification of sequence progress codon, obtain nucleotide shown in SEQ ID NO.1 and SEQ ID NO.2
Sequence, PCR is enriched with the agarose gel electrophoresis figure of gB sequence as shown in Figure 1, PCR is enriched with the agarose gel electrophoresis figure of gD sequence
As shown in Figure 2.The centre of gB with gD sequence sequence shown in SEQ ID NO.3 connects, and further increases fusion protein to reach
Antigen broad spectrum activity and the purpose for improving antigen presentation amount.
The building of the recombinant vector of 2 expressed fusion protein of embodiment
The sequence of the encoding fusion protein of above-mentioned synthesis is cloned into eukaryon by the insertion of the site I and Hind III Mlu to turn
On transfer body pcDNA3.1.Connection product is obtained after 16 DEG C overnight connection using T4DNA ligase, through E. coli competent
It is coated in the LB plate containing ammonia section penicillin after DH5 α conversion, picking positive bacteria is fallen in containing ammonia section after 37 DEG C of overnight incubations
It is cultivated in the LB culture medium of penicillin, extracts plasmid.Correct recombinant plasmid is obtained by PCR, double digestion and sequence verification.
The pressurization of 3 recombinant cell of embodiment is screened
The HEK 293-F cell that Transfected Recombinant Plasmid to well-grown is suspended entirely carries out cell passage after 72 hours, and
The G418 of 600 μ g/ml is added in culture medium, is forced into motility rate at 10% or so, stops pressurization, is trained with conventional culture medium
It supports, when Cell viability reaches 90% or more, repressurization screening is primary, and same Cell viability carries out next when reaching 90% or more
Step screening.
The monoclonal of 4 recombinant cell of embodiment screens
Positive colony is selected and is detected: in the plate after culture 7 days, the cell of adherent growth being chosen into 96 orifice plates, benefit
After adhere-wall culture base culture 7 days, 100 μ L suspension medium Freestyle HEK 293-F are added and cultivate 2 days, are trained in orifice plate
It supports base and is used for Dot hybridization, wherein 24 orifice plates will be gone to by high-expression clone, using being changed to after the culture of adhere-wall culture base 2 days
Freestyle HEK 293-F culture, culture medium are detected for immunoblotting, finally obtain high expression according to experimental result
Clone strain.The condition of culture of all cells is 37 DEG C, 5%CO2。
3 plants of relatively high cell strains of expression quantity are obtained by screening, are respectively designated as 293F-gB-gD-1,293F-gB-
gD-2、293F-gB-gD-3。
The culture of 5 recombinant cell of embodiment
Cell will be resuspended in Freestyle HEK 293-F culture medium by the HEK 293-F cell of screening, cell connects
Kind density is 0.3 × 106~0.5 × 106VC/ml, inoculating cell are placed in 36~38 DEG C, contain in the cell shaking flask of suitable volumes
5%CO2It is carried out shaking flask culture 48~72 hours in shaking table, continues to pass on amplification cultivation cell.
Bioreactor culture step by step is carried out as needed to ferment, and is generally carried out 5~8 times of amplifications, is amplified to designated volume
The expression of Shi Jinhang antigen cooled the temperature to 31~33 DEG C to the 5th day in 36~38 DEG C of cultures, and pH is adjusted to 7.5 ± 0.1, and
To be suitable for that revolving speed is cultivated, the Efficient Feed of 10% starting working volume, every daily test were added on the 4th and the 9th
Concentration of glucose is surveyed, when concentration of glucose is lower than 2.5g/L, supplement glucose to 3~4g/L.When Cell viability is lower than 80%
When, harvest supernatant is required antigen.
By optimizing condition of culture, feed supplement time and the feed supplement amount of the cell strain, it is finally obtained 1.0~1.2mg/ml's
GB-gD protein expression.
6 porcine pseudorabies virus vaccine of embodiment
Go cell fragment by expression it is quantitative after, be prepared by mixing into vaccine with ISA201 adjuvant.Wherein water phase and oily mutually cream
Change ratio is 1:1 mass ratio, first mutually imports oily in beaker, mixes slowly, be slowly added to water phase, add rear 10000r/min and cut
Cut emulsification 5min, terminate emulsification before 1% thimerosal solution is added in an amount, make its final concentration of 0.01%, be sufficiently stirred mixed
It is even.Sterile quantitative separating, seals, and obtains porcine pseudorabies virus subunit vaccine, sets 2~8 DEG C of preservations.
After piglet with 21 age in days of vaccine immunity, takes a blood sample respectively after being immunized 21 and survey neutralizing antibody, 21 days as the result is shown
Neutralizing antibody is not less than 1:1000, also, attacks poison the vaccine can either effectively prevent classical virulent (SC plants) and work as the result is shown
The attack of preceding prevalence strain (JS plants), protective rate can achieve 100%.All immune swines are increased without body temperature and abnormal clinical disease
The generation of shape.
Comparative example 1
With 21 age in days piglets of classical virulent (SC plants) and (JS plants) of current popular strain attack health, compare as the result is shown
Pig occur body temperature increase 41 degree or more and with nervous symptoms, lethargic sleep, toot the facing for porcine pseudorabies virus such as cry, vomit, have loose bowels
Bed symptom.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Tian Kang Biological Co., Ltd.
<120>fusion protein of porcine pseudorabies virus and preparation method thereof, application and vaccine
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 2745
<212> DNA
<213>artificial sequence
<400> 1
atgcccgccg gcggcggcct gtggcgcggc ccccgcggcc accgccccgg ccaccacggc 60
ggcgccggcc tgggccgcct gtggcccgcc ccccaccacg ccgccgccgc ccgcggcgcc 120
gtggccctgg ccctgctgct gctggccctg gccgccaccc ccacctgcgg cgccgccgcc 180
gtgacccgcg ccgccagcgc cagccccgcc cccggcaccg gcgccacccc cgacggcttc 240
agcgccgagg agagcctgga ggagatcgac ggcgccgtga gccccggccc cagcgacgcc 300
cccgacggcg agtacggcga cctggacgcc cgcaccgccg tgcgcgccgc cgccaccgag 360
cgcgaccgct tctacgtgtg cccccccccc agcggcagca ccgtggtgcg cctggagccc 420
gagcaggcct gccccgagta cagccagggc cgcaacttca ccgagggcat cgccgtgctg 480
ttcaaggaga acatcgcccc ccacaagttc aaggcccaca tctactacaa gaacgtgatc 540
gtgaccaccg tgtggagcgg cagcacctac gccgccatca ccaaccgctt caccgaccgc 600
gtgcccgtgc ccgtgcagga gatcaccgac gtgatcgacc gccgcggcaa gtgcgtgagc 660
aaggccgagt acgtgcgcaa caaccacaag gtgaccgcct tcgaccgcga cgagaacccc 720
gtggaggtgg acctgcgccc cagccgcctg aacgccctgg gcacccgcgg ctggcacacc 780
accaacgaca cctacaccaa gatcggcgcc gccggcttct accacaccgg caccagcgtg 840
aactgcatcg tggaggaggt ggaggcccgc agcgtgtacc cctacgacag cttcgccctg 900
agcaccggcg acatcgtgta catgagcccc ttctacggcc tgcgcgaggg cgcccacggc 960
gagcacatcg gctacgcccc cggccgcttc cagcaggtgg agcactacta ccccatcgac 1020
ctggacagcc gcctgcgcgc cagcgagagc gtgacccgca acttcctgcg caccccccac 1080
ttcaccgtgg cctgggactg ggcccccaag acccgccgcg tgtgcagcct ggccaagtgg 1140
cgcgaggccg aggagatgat ccgcgacgag acccgcgacg gcagcttccg cttcaccagc 1200
cgcgccctgg gcgccagctt cgtgagcgac gtgacccagc tggacctgca gcgcgtgcac 1260
ctgggcgact gcgtgctgcg cgaggccagc gaggccatcg acgccatcta ccgccgccgc 1320
tacaacaaca cccacgtgct ggccggcgac aagcccgagg tgtacctggc ccgcggcggc 1380
ttcgtggtgg ccttccgccc cctgatcagc aacgagctgg cccagctgta cgcccgcgag 1440
ctggagcgcc tgggcctggc cggcgtggtg ggccccgcca gccccgccgc cgcccgccgc 1500
gcccgccgca gccccggccc cgccggcacc cccgagcccc ccgccgtgaa cggcaccggc 1560
cacctgcgca tcaccaccgg cagcgccgag ttcgcccgcc tgcagttcac ctacgaccac 1620
atccaggccc acgtgaacga catgctgagc cgcatcgccg ccgcctggtg cgagctgcag 1680
aacaaggacc gcaccctgtg gggcgagatg agccgcctga accccagcgc cgtggccacc 1740
gccgccctgg gccagcgcgt gagcgcccgc atgctgggcg acgtgatggc catcagccgc 1800
tgcgtggagg tgcgcggcgg cgtgtacgtg cagaacagca tgcgcgtgcc cggcgagcgc 1860
ggcacctgct acagccgccc cctggtgacc ttcgagcaca acggcaccgg cgtgatcgag 1920
ggccagctgg gcgacgacaa cgagctgctg atcagccgcg acctgatcga gccctgcacc 1980
ggcaaccacc gccgctactt caagctgggc ggcggctacg tgtactacga ggactacagc 2040
tacgtgcgca tggtggaggt gcccgagacc atcagcaccc gcgtgaccct gaacctgacc 2100
ctgctggagg accgcgagtt cctgcccctg gaggtgtaca cccgcgagga gctggccgac 2160
accggcctgc tggactacag cgagatccag cgccgcaacc agctgcacgc cctgaagttc 2220
tacgacatcg accgcgtggt gaaggtggac cacaacgtgg tgctgctgcg cggcatcgcc 2280
aacttcttcc agggcctggg cgacgtgggc gccgccgtgg gcaaggtggt gctgggcgcc 2340
accggcgccg tgatcagcgc cgtgggcggc atggtgagct tcctgagcaa ccccttcggc 2400
gccctggcca tcggcctgct ggtgctggcc ggcctggtgg ccgccttcct ggcctaccgc 2460
cacatcagcc gcctgcgccg caaccccatg aaggccctgt accccgtgac caccaaggcc 2520
ctgaaggagg acggcgtgga ggaggacgac gtggacgagg ccaagctgga ccaggcccgc 2580
gacatgatcc gctacatgag catcgtgagc gccctggagc agcaggagca caaggcccgc 2640
aagaagaaca gcggccccgc cctgctggcc agccgcgtgg gcgccatggc cacccgccgc 2700
cgccactacc agcgcctgga gaacgaggac cccgacgccc cctaa 2745
<210> 2
<211> 1209
<212> DNA
<213>artificial sequence
<400> 2
atgctgctgg ccgccctgct ggccgccctg gtggcccgca ccaccctggg cgccgacgtg 60
gacgccgtgc ccgcccccac cttccccccc cccgcctacc cctacaccga gagctggcag 120
ctgaccctga ccaccgtgcc cagccccttc gtgggccccg ccgacgtgta ccacacccgc 180
cccctggagg acccctgcgg cgtggtggcc ctgatcagcg acccccaggt ggaccgcctg 240
ctgaacgagg ccgtggccca ccgccgcccc acctaccgcg cccacgtggc ctggtaccgc 300
atcgccgacg gctgcgccca cctgctgtac ttcatcgagt acgccgactg cgacccccgc 360
cagatcttcg gccgctgccg ccgccgcacc acccccatgt ggtggacccc cagcgccgac 420
tacatgttcc ccaccgagga cgagctgggc ctgctgatgg tggcccccgg ccgcttcaac 480
gagggccagt accgccgcct ggtgagcgtg gacggcgtga acatcctgac cgacttcatg 540
gtggccctgc ccgagggcca ggagtgcccc ttcgcccgcg tggaccagca ccgcacctac 600
aagttcggcg cctgctggag cgacgacagc ttcaagcgcg gcgtggacgt gatgcgcttc 660
ctgaccccct tctaccagca gcccccccac cgcgaggtgg tgaactactg gtaccgcaag 720
aacggccgca ccctgccccg cgcctacgcc gccgccaccc cctacgccat cgaccccgcc 780
cgccccagcg ccggcagccc ccgcccccgc ccccgccccc gcccccgccc ccgccccaag 840
cccgagcccg cccccgccac ccccgccccc cccggccgcc tgcccgagcc cgccacccgc 900
gaccacgccg ccggcggccg ccccaccccc cgcccccccc gccccgagac cccccaccgc 960
cccttcgccc cccccgccgt ggtgcccagc ggctggcccc agcccgccga gcccttcccc 1020
ccccgcacca ccgccgcccc cggcgtgagc cgccaccgca gcgtgatcgt gggcaccggc 1080
accgccatgg gcgccctgct ggtgggcgtg tgcgtgtaca tcttcttccg cctgcgcggc 1140
gccaagggct accgcctgct gggcggcccc gccgacgccg acgagctgaa ggcccagccc 1200
ggcccctaa 1209
<210> 3
<211> 15
<212> DNA
<213>artificial sequence
<400> 3
cctcccagcc ccagc 15
<210> 4
<211> 3963
<212> DNA
<213>artificial sequence
<400> 4
atgcccgccg gcggcggcct gtggcgcggc ccccgcggcc accgccccgg ccaccacggc 60
ggcgccggcc tgggccgcct gtggcccgcc ccccaccacg ccgccgccgc ccgcggcgcc 120
gtggccctgg ccctgctgct gctggccctg gccgccaccc ccacctgcgg cgccgccgcc 180
gtgacccgcg ccgccagcgc cagccccgcc cccggcaccg gcgccacccc cgacggcttc 240
agcgccgagg agagcctgga ggagatcgac ggcgccgtga gccccggccc cagcgacgcc 300
cccgacggcg agtacggcga cctggacgcc cgcaccgccg tgcgcgccgc cgccaccgag 360
cgcgaccgct tctacgtgtg cccccccccc agcggcagca ccgtggtgcg cctggagccc 420
gagcaggcct gccccgagta cagccagggc cgcaacttca ccgagggcat cgccgtgctg 480
ttcaaggaga acatcgcccc ccacaagttc aaggcccaca tctactacaa gaacgtgatc 540
gtgaccaccg tgtggagcgg cagcacctac gccgccatca ccaaccgctt caccgaccgc 600
gtgcccgtgc ccgtgcagga gatcaccgac gtgatcgacc gccgcggcaa gtgcgtgagc 660
aaggccgagt acgtgcgcaa caaccacaag gtgaccgcct tcgaccgcga cgagaacccc 720
gtggaggtgg acctgcgccc cagccgcctg aacgccctgg gcacccgcgg ctggcacacc 780
accaacgaca cctacaccaa gatcggcgcc gccggcttct accacaccgg caccagcgtg 840
aactgcatcg tggaggaggt ggaggcccgc agcgtgtacc cctacgacag cttcgccctg 900
agcaccggcg acatcgtgta catgagcccc ttctacggcc tgcgcgaggg cgcccacggc 960
gagcacatcg gctacgcccc cggccgcttc cagcaggtgg agcactacta ccccatcgac 1020
ctggacagcc gcctgcgcgc cagcgagagc gtgacccgca acttcctgcg caccccccac 1080
ttcaccgtgg cctgggactg ggcccccaag acccgccgcg tgtgcagcct ggccaagtgg 1140
cgcgaggccg aggagatgat ccgcgacgag acccgcgacg gcagcttccg cttcaccagc 1200
cgcgccctgg gcgccagctt cgtgagcgac gtgacccagc tggacctgca gcgcgtgcac 1260
ctgggcgact gcgtgctgcg cgaggccagc gaggccatcg acgccatcta ccgccgccgc 1320
tacaacaaca cccacgtgct ggccggcgac aagcccgagg tgtacctggc ccgcggcggc 1380
ttcgtggtgg ccttccgccc cctgatcagc aacgagctgg cccagctgta cgcccgcgag 1440
ctggagcgcc tgggcctggc cggcgtggtg ggccccgcca gccccgccgc cgcccgccgc 1500
gcccgccgca gccccggccc cgccggcacc cccgagcccc ccgccgtgaa cggcaccggc 1560
cacctgcgca tcaccaccgg cagcgccgag ttcgcccgcc tgcagttcac ctacgaccac 1620
atccaggccc acgtgaacga catgctgagc cgcatcgccg ccgcctggtg cgagctgcag 1680
aacaaggacc gcaccctgtg gggcgagatg agccgcctga accccagcgc cgtggccacc 1740
gccgccctgg gccagcgcgt gagcgcccgc atgctgggcg acgtgatggc catcagccgc 1800
tgcgtggagg tgcgcggcgg cgtgtacgtg cagaacagca tgcgcgtgcc cggcgagcgc 1860
ggcacctgct acagccgccc cctggtgacc ttcgagcaca acggcaccgg cgtgatcgag 1920
ggccagctgg gcgacgacaa cgagctgctg atcagccgcg acctgatcga gccctgcacc 1980
ggcaaccacc gccgctactt caagctgggc ggcggctacg tgtactacga ggactacagc 2040
tacgtgcgca tggtggaggt gcccgagacc atcagcaccc gcgtgaccct gaacctgacc 2100
ctgctggagg accgcgagtt cctgcccctg gaggtgtaca cccgcgagga gctggccgac 2160
accggcctgc tggactacag cgagatccag cgccgcaacc agctgcacgc cctgaagttc 2220
tacgacatcg accgcgtggt gaaggtggac cacaacgtgg tgctgctgcg cggcatcgcc 2280
aacttcttcc agggcctggg cgacgtgggc gccgccgtgg gcaaggtggt gctgggcgcc 2340
accggcgccg tgatcagcgc cgtgggcggc atggtgagct tcctgagcaa ccccttcggc 2400
gccctggcca tcggcctgct ggtgctggcc ggcctggtgg ccgccttcct ggcctaccgc 2460
cacatcagcc gcctgcgccg caaccccatg aaggccctgt accccgtgac caccaaggcc 2520
ctgaaggagg acggcgtgga ggaggacgac gtggacgagg ccaagctgga ccaggcccgc 2580
gacatgatcc gctacatgag catcgtgagc gccctggagc agcaggagca caaggcccgc 2640
aagaagaaca gcggccccgc cctgctggcc agccgcgtgg gcgccatggc cacccgccgc 2700
cgccactacc agcgcctgga gaacgaggac cccgacgccc cccctcccag ccccagcctg 2760
ctggccgccc tgctggccgc cctggtggcc cgcaccaccc tgggcgccga cgtggacgcc 2820
gtgcccgccc ccaccttccc cccccccgcc tacccctaca ccgagagctg gcagctgacc 2880
ctgaccaccg tgcccagccc cttcgtgggc cccgccgacg tgtaccacac ccgccccctg 2940
gaggacccct gcggcgtggt ggccctgatc agcgaccccc aggtggaccg cctgctgaac 3000
gaggccgtgg cccaccgccg ccccacctac cgcgcccacg tggcctggta ccgcatcgcc 3060
gacggctgcg cccacctgct gtacttcatc gagtacgccg actgcgaccc ccgccagatc 3120
ttcggccgct gccgccgccg caccaccccc atgtggtgga cccccagcgc cgactacatg 3180
ttccccaccg aggacgagct gggcctgctg atggtggccc ccggccgctt caacgagggc 3240
cagtaccgcc gcctggtgag cgtggacggc gtgaacatcc tgaccgactt catggtggcc 3300
ctgcccgagg gccaggagtg ccccttcgcc cgcgtggacc agcaccgcac ctacaagttc 3360
ggcgcctgct ggagcgacga cagcttcaag cgcggcgtgg acgtgatgcg cttcctgacc 3420
cccttctacc agcagccccc ccaccgcgag gtggtgaact actggtaccg caagaacggc 3480
cgcaccctgc cccgcgccta cgccgccgcc accccctacg ccatcgaccc cgcccgcccc 3540
agcgccggca gcccccgccc ccgcccccgc ccccgccccc gcccccgccc caagcccgag 3600
cccgcccccg ccacccccgc cccccccggc cgcctgcccg agcccgccac ccgcgaccac 3660
gccgccggcg gccgccccac cccccgcccc ccccgccccg agacccccca ccgccccttc 3720
gccccccccg ccgtggtgcc cagcggctgg ccccagcccg ccgagccctt ccccccccgc 3780
accaccgccg cccccggcgt gagccgccac cgcagcgtga tcgtgggcac cggcaccgcc 3840
atgggcgccc tgctggtggg cgtgtgcgtg tacatcttct tccgcctgcg cggcgccaag 3900
ggctaccgcc tgctgggcgg ccccgccgac gccgacgagc tgaaggccca gcccggcccc 3960
taa 3963
<210> 5
<211> 1320
<212> PRT
<213>artificial sequence
<400> 5
Met Pro Ala Gly Gly Gly Leu Trp Arg Gly Pro Arg Gly His Arg Pro
1 5 10 15
Gly His His Gly Gly Ala Gly Leu Gly Arg Leu Trp Pro Ala Pro His
20 25 30
His Ala Ala Ala Ala Arg Gly Ala Val Ala Leu Ala Leu Leu Leu Leu
35 40 45
Ala Leu Ala Ala Thr Pro Thr Cys Gly Ala Ala Ala Val Thr Arg Ala
50 55 60
Ala Ser Ala Ser Pro Ala Pro Gly Thr Gly Ala Thr Pro Asp Gly Phe
65 70 75 80
Ser Ala Glu Glu Ser Leu Glu Glu Ile Asp Gly Ala Val Ser Pro Gly
85 90 95
Pro Ser Asp Ala Pro Asp Gly Glu Tyr Gly Asp Leu Asp Ala Arg Thr
100 105 110
Ala Val Arg Ala Ala Ala Thr Glu Arg Asp Arg Phe Tyr Val Cys Pro
115 120 125
Pro Pro Ser Gly Ser Thr Val Val Arg Leu Glu Pro Glu Gln Ala Cys
130 135 140
Pro Glu Tyr Ser Gln Gly Arg Asn Phe Thr Glu Gly Ile Ala Val Leu
145 150 155 160
Phe Lys Glu Asn Ile Ala Pro His Lys Phe Lys Ala His Ile Tyr Tyr
165 170 175
Lys Asn Val Ile Val Thr Thr Val Trp Ser Gly Ser Thr Tyr Ala Ala
180 185 190
Ile Thr Asn Arg Phe Thr Asp Arg Val Pro Val Pro Val Gln Glu Ile
195 200 205
Thr Asp Val Ile Asp Arg Arg Gly Lys Cys Val Ser Lys Ala Glu Tyr
210 215 220
Val Arg Asn Asn His Lys Val Thr Ala Phe Asp Arg Asp Glu Asn Pro
225 230 235 240
Val Glu Val Asp Leu Arg Pro Ser Arg Leu Asn Ala Leu Gly Thr Arg
245 250 255
Gly Trp His Thr Thr Asn Asp Thr Tyr Thr Lys Ile Gly Ala Ala Gly
260 265 270
Phe Tyr His Thr Gly Thr Ser Val Asn Cys Ile Val Glu Glu Val Glu
275 280 285
Ala Arg Ser Val Tyr Pro Tyr Asp Ser Phe Ala Leu Ser Thr Gly Asp
290 295 300
Ile Val Tyr Met Ser Pro Phe Tyr Gly Leu Arg Glu Gly Ala His Gly
305 310 315 320
Glu His Ile Gly Tyr Ala Pro Gly Arg Phe Gln Gln Val Glu His Tyr
325 330 335
Tyr Pro Ile Asp Leu Asp Ser Arg Leu Arg Ala Ser Glu Ser Val Thr
340 345 350
Arg Asn Phe Leu Arg Thr Pro His Phe Thr Val Ala Trp Asp Trp Ala
355 360 365
Pro Lys Thr Arg Arg Val Cys Ser Leu Ala Lys Trp Arg Glu Ala Glu
370 375 380
Glu Met Ile Arg Asp Glu Thr Arg Asp Gly Ser Phe Arg Phe Thr Ser
385 390 395 400
Arg Ala Leu Gly Ala Ser Phe Val Ser Asp Val Thr Gln Leu Asp Leu
405 410 415
Gln Arg Val His Leu Gly Asp Cys Val Leu Arg Glu Ala Ser Glu Ala
420 425 430
Ile Asp Ala Ile Tyr Arg Arg Arg Tyr Asn Asn Thr His Val Leu Ala
435 440 445
Gly Asp Lys Pro Glu Val Tyr Leu Ala Arg Gly Gly Phe Val Val Ala
450 455 460
Phe Arg Pro Leu Ile Ser Asn Glu Leu Ala Gln Leu Tyr Ala Arg Glu
465 470 475 480
Leu Glu Arg Leu Gly Leu Ala Gly Val Val Gly Pro Ala Ser Pro Ala
485 490 495
Ala Ala Arg Arg Ala Arg Arg Ser Pro Gly Pro Ala Gly Thr Pro Glu
500 505 510
Pro Pro Ala Val Asn Gly Thr Gly His Leu Arg Ile Thr Thr Gly Ser
515 520 525
Ala Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asp His Ile Gln Ala His
530 535 540
Val Asn Asp Met Leu Ser Arg Ile Ala Ala Ala Trp Cys Glu Leu Gln
545 550 555 560
Asn Lys Asp Arg Thr Leu Trp Gly Glu Met Ser Arg Leu Asn Pro Ser
565 570 575
Ala Val Ala Thr Ala Ala Leu Gly Gln Arg Val Ser Ala Arg Met Leu
580 585 590
Gly Asp Val Met Ala Ile Ser Arg Cys Val Glu Val Arg Gly Gly Val
595 600 605
Tyr Val Gln Asn Ser Met Arg Val Pro Gly Glu Arg Gly Thr Cys Tyr
610 615 620
Ser Arg Pro Leu Val Thr Phe Glu His Asn Gly Thr Gly Val Ile Glu
625 630 635 640
Gly Gln Leu Gly Asp Asp Asn Glu Leu Leu Ile Ser Arg Asp Leu Ile
645 650 655
Glu Pro Cys Thr Gly Asn His Arg Arg Tyr Phe Lys Leu Gly Gly Gly
660 665 670
Tyr Val Tyr Tyr Glu Asp Tyr Ser Tyr Val Arg Met Val Glu Val Pro
675 680 685
Glu Thr Ile Ser Thr Arg Val Thr Leu Asn Leu Thr Leu Leu Glu Asp
690 695 700
Arg Glu Phe Leu Pro Leu Glu Val Tyr Thr Arg Glu Glu Leu Ala Asp
705 710 715 720
Thr Gly Leu Leu Asp Tyr Ser Glu Ile Gln Arg Arg Asn Gln Leu His
725 730 735
Ala Leu Lys Phe Tyr Asp Ile Asp Arg Val Val Lys Val Asp His Asn
740 745 750
Val Val Leu Leu Arg Gly Ile Ala Asn Phe Phe Gln Gly Leu Gly Asp
755 760 765
Val Gly Ala Ala Val Gly Lys Val Val Leu Gly Ala Thr Gly Ala Val
770 775 780
Ile Ser Ala Val Gly Gly Met Val Ser Phe Leu Ser Asn Pro Phe Gly
785 790 795 800
Ala Leu Ala Ile Gly Leu Leu Val Leu Ala Gly Leu Val Ala Ala Phe
805 810 815
Leu Ala Tyr Arg His Ile Ser Arg Leu Arg Arg Asn Pro Met Lys Ala
820 825 830
Leu Tyr Pro Val Thr Thr Lys Ala Leu Lys Glu Asp Gly Val Glu Glu
835 840 845
Asp Asp Val Asp Glu Ala Lys Leu Asp Gln Ala Arg Asp Met Ile Arg
850 855 860
Tyr Met Ser Ile Val Ser Ala Leu Glu Gln Gln Glu His Lys Ala Arg
865 870 875 880
Lys Lys Asn Ser Gly Pro Ala Leu Leu Ala Ser Arg Val Gly Ala Met
885 890 895
Ala Thr Arg Arg Arg His Tyr Gln Arg Leu Glu Asn Glu Asp Pro Asp
900 905 910
Ala Pro Pro Pro Ser Pro Ser Leu Leu Ala Ala Leu Leu Ala Ala Leu
915 920 925
Val Ala Arg Thr Thr Leu Gly Ala Asp Val Asp Ala Val Pro Ala Pro
930 935 940
Thr Phe Pro Pro Pro Ala Tyr Pro Tyr Thr Glu Ser Trp Gln Leu Thr
945 950 955 960
Leu Thr Thr Val Pro Ser Pro Phe Val Gly Pro Ala Asp Val Tyr His
965 970 975
Thr Arg Pro Leu Glu Asp Pro Cys Gly Val Val Ala Leu Ile Ser Asp
980 985 990
Pro Gln Val Asp Arg Leu Leu Asn Glu Ala Val Ala His Arg Arg Pro
995 1000 1005
Thr Tyr Arg Ala His Val Ala Trp Tyr Arg Ile Ala Asp Gly Cys
1010 1015 1020
Ala His Leu Leu Tyr Phe Ile Glu Tyr Ala Asp Cys Asp Pro Arg
1025 1030 1035
Gln Ile Phe Gly Arg Cys Arg Arg Arg Thr Thr Pro Met Trp Trp
1040 1045 1050
Thr Pro Ser Ala Asp Tyr Met Phe Pro Thr Glu Asp Glu Leu Gly
1055 1060 1065
Leu Leu Met Val Ala Pro Gly Arg Phe Asn Glu Gly Gln Tyr Arg
1070 1075 1080
Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu Thr Asp Phe Met
1085 1090 1095
Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala Arg Val Asp
1100 1105 1110
Gln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser Asp Asp Ser
1115 1120 1125
Phe Lys Arg Gly Val Asp Val Met Arg Phe Leu Thr Pro Phe Tyr
1130 1135 1140
Gln Gln Pro Pro His Arg Glu Val Val Asn Tyr Trp Tyr Arg Lys
1145 1150 1155
Asn Gly Arg Thr Leu Pro Arg Ala Tyr Ala Ala Ala Thr Pro Tyr
1160 1165 1170
Ala Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro Arg
1175 1180 1185
Pro Arg Pro Arg Pro Arg Pro Arg Pro Lys Pro Glu Pro Ala Pro
1190 1195 1200
Ala Thr Pro Ala Pro Pro Gly Arg Leu Pro Glu Pro Ala Thr Arg
1205 1210 1215
Asp His Ala Ala Gly Gly Arg Pro Thr Pro Arg Pro Pro Arg Pro
1220 1225 1230
Glu Thr Pro His Arg Pro Phe Ala Pro Pro Ala Val Val Pro Ser
1235 1240 1245
Gly Trp Pro Gln Pro Ala Glu Pro Phe Pro Pro Arg Thr Thr Ala
1250 1255 1260
Ala Pro Gly Val Ser Arg His Arg Ser Val Ile Val Gly Thr Gly
1265 1270 1275
Thr Ala Met Gly Ala Leu Leu Val Gly Val Cys Val Tyr Ile Phe
1280 1285 1290
Phe Arg Leu Arg Gly Ala Lys Gly Tyr Arg Leu Leu Gly Gly Pro
1295 1300 1305
Ala Asp Ala Asp Glu Leu Lys Ala Gln Pro Gly Pro
1310 1315 1320
Claims (10)
1. a kind of fusion protein of porcine pseudorabies virus, which is characterized in that including gB section and gD section；
The expression of gB section nucleotide sequence as shown in SEQ ID NO.1, the gD section is as shown in SEQ ID NO.2
Nucleotide sequence expression.
2. fusion protein according to claim 1, which is characterized in that the gB section and the gD section put in order
For gB-gD, connected by Linker；
The Linker has the nucleotide sequence as shown in SEQ ID NO.3.
3. fusion protein according to claim 1, which is characterized in that the fusion protein is HEK293-F cell expression system
The albumen of system expression.
4. the preparation method of any one of the claim 1-3 fusion protein, which is characterized in that in mammalian expression systems
In, express the gene of encoding said fusion protein.
5. the preparation method according to claim 4, which is characterized in that express and compile in HEK 293-F cell expression system
The gene of the code fusion protein.
6. the preparation method according to claim 4, which is characterized in that provide the gene comprising encoding said fusion protein
The expression vector is imported in HEK 293-F cell, is then screened to HEK 293-F cell by expression vector, is obtained steady
Surely the HEK 293-F cell strain of the fusion protein is expressed, the HEK 293-F cell strain expresses to obtain the fusion protein；
Preferably, the screening includes pressurization screening and monoclonal screening.
7. preparation method according to claim 6, which is characterized in that using described in the screening expression of Geneticin screening system
The HEK 293-F cell of fusion protein；
Preferably, expression vector used in the Geneticin screening system be pcDNA3.1, pEE6.4, pEE12.4 or
PGL4.13, preferably pcDNA3.1.
8. the described in any item fusion proteins of claim 1-3 or the described in any item preparation methods of claim 4-7 are prepared into
The fusion protein arrived is in following A)-D) at least one of application:
A porcine pseudorabies virus vaccine) is prepared；
B the antibody of porcine pseudorabies virus) is prepared；
C) the kit of preparation detection porcine pseudorabies virus；
D porcine pseudorabies virus diagnostic antigen) is prepared.
9. a kind of porcine pseudorabies virus vaccine of the fusion protein comprising any one of the claim 1-3 porcine pseudorabies virus.
10. porcine pseudorabies virus vaccine according to claim 9, which is characterized in that in the porcine pseudorabies virus vaccine
The dosage of fusion protein is g/ parts of 70-90 μ；
Preferably, the vaccine further includes auxiliary material, and the auxiliary material includes one of vaccine adjuvant, stabilizer and antibiotic or more
Kind；
Preferably, the vaccine adjuvant includes aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white-oil adjuvant, card
Wave nurse, propolis, MF59 adjuvant or ISA201 are, it is preferable to use ISA201.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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