CN110092839A - The fusion protein of porcine pseudorabies virus, preparation method, using and comprising porcine pseudorabies virus fusion protein vaccine - Google Patents
The fusion protein of porcine pseudorabies virus, preparation method, using and comprising porcine pseudorabies virus fusion protein vaccine Download PDFInfo
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Abstract
The present invention provides a kind of fusion protein of porcine pseudorabies virus, preparation method, using and comprising porcine pseudorabies virus fusion protein vaccine, it is related to field of biotechnology, the fusion protein of porcine pseudorabies virus provided by the invention includes porcine pseudorabies virus gB albumen section, porcine pseudorabies virus gC albumen section and porcine pseudorabies virus gD albumen section.The fusion protein is to obtain the gene order expressing in series of the gB albumen section of porcine pseudorabies virus, gC albumen section and gD albumen section.The present invention is analyzed by the gene order to gB, gC and gD, it is wherein antigenic good to choose, it connects in easy highly expressed region, obtained fusion protein has antigenicity good, the high advantage of expression quantity, have both that broad spectrum activity is good simultaneously, the antibody titer being prepared is high and the advantages of can preventing the porcine pseudorabies virus of a variety of hypotypes.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to a kind of fusion protein of porcine pseudorabies virus, preparation method,
Using and comprising porcine pseudorabies virus fusion protein vaccine.
Background technique
Pseudorabies virus belongs to herpetoviridae (Herpesviridae), herpesvirus suis category, and virion is circle,
150~180nm of diameter, nucleocapsid diameter are 105~110nm.The outermost layer of virion is virus envelope, it is thin by host
The lipid bilayer structure that born of the same parents are derived.Cyst membrane surface has that be about fibre that 8~10nm is arranged radially prominent.
Pseudoabies (Pseudorabies, PR) is caused by pseudorabies virus (Pseudorabies vires, PRV)
A variety of domestic animals (pig, ox, sheep etc.) and wild animal to generate heat, surprise is itched (except pig) and neurological symptom and encephalomyelitis are
A kind of Important Infectious Diseases of main feature.Pig is the important reservoir host of the virus and the infection sources, mainly pregnant sow is caused to be flowed
Production, stillborn foetus and the mummification of fetus, Infection in Piglets are mainly nervous symptoms, and Adult Pig is then based on subclinical infection.PRV has stronger
Pantropic, neurotropism and latent infection characteristic, peripheral neverous system can long-term latent infection, when latent virus is activated
Become infectious virus, will be fallen ill by the host of latent infection.
Vaccine inoculation is one of the major measure that pseudoabies is even eliminated in prevention, control.Currently, domestic and international clinical application
Pseudorabies vaccine can substantially be divided into following three classes: first is that by the open country poison of separation or virulent after formalin-inactivated, add adjuvant newborn
Oil emulsion inactivated vaccine is made in change；Second is that by the open country poison of separation or virulent through non-pig source cell or chicken embryo, Attenuation is ground repeatedly
The attenuated vaccine of system；Third is that the gene-deleted vaccine constructed using technique for gene engineering.Although inactivated vaccine safety, effect is immunized
Power is limited, and effect is single, and cannot play the target of epidemic disease purification.There are hidden danger, production cycles for attenuated vaccine safety
It is long, it is at high cost.In addition, porcine pseudorabies virus gene deleted live vaccine, inactivated virus vaccine are grown on attached cell,
And animal derived protein is needed, risk is brought 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.
The present invention provides a kind of fusion protein of porcine pseudorabies virus, the fusion protein includes porcine pseudorabies virus gB
Albumen section, porcine pseudorabies virus gC albumen section and porcine pseudorabies virus gD albumen section.
Further, porcine pseudorabies virus gB albumen section nucleotide sequence as shown in SEQ ID NO.1 is expressed,
The expression of porcine pseudorabies virus gC albumen section nucleotide sequence as shown in SEQ ID NO.2, the protein region porcine pseudorabies virus gD
The section expression of the nucleotide sequence as shown in SEQ ID NO.3.
Further, the porcine pseudorabies virus gB albumen section, porcine pseudorabies virus gC albumen section and pseudorabies
The order of connection of viral gD albumen section is gB-gD-gC.
The present invention also provides the preparation method of the fusion protein of above-mentioned porcine pseudorabies virus, the preparation method includes
The gene for encoding the fusion protein of the porcine pseudorabies virus is expressed in host, obtains the fusion of the porcine pseudorabies virus
Albumen.
Further, the expression vector of the gene comprising the fusion protein for encoding the porcine pseudorabies virus is imported into host
In, it expresses that the gene of the fusion protein of the porcine pseudorabies virus in host, obtains the fusion of the porcine pseudorabies virus
Albumen；
Preferably, the expression vector is plasmid, preferably PMH3。
Further, the host is cell；Preferably CHO-S cell.
Further, the CHO-S cell for importing expression vector is screened and is tamed, obtain melting for porcine pseudorabies virus
Hop protein expression quantity >=1g/L CHO-S cell strain, the CHO-S cell strain express to obtain the fusion of the porcine pseudorabies virus
Albumen；
Preferably, it is screened and is tamed using Geneticin screening system.
It is prepared into the present invention also provides the fusion protein of above-mentioned porcine pseudorabies virus or using above-mentioned preparation method
To porcine pseudorabies virus fusion protein preparation porcine pseudorabies virus vaccine in application.
In addition, the vaccine includes the fusion protein of above-mentioned porcine pseudorabies virus the present invention also provides a kind of vaccine
Or the fusion protein for the porcine pseudorabies virus being prepared using above-mentioned preparation method.
Further, the vaccine further includes adjuvant, the fusion protein of the porcine pseudorabies virus and the volume ratio of adjuvant
For 1:0.5-3.
The fusion protein of porcine pseudorabies virus provided by the invention includes porcine pseudorabies virus gB albumen section, pseudorabies
Viral gC albumen section and porcine pseudorabies virus gD albumen section.The fusion protein is by the protein region gB of porcine pseudorabies virus
The gene order expressing in series of section, gC albumen section and gD albumen section obtains.The present invention passes through the gene sequence to gB, gC and gD
Column are analyzed, and it is wherein antigenic good to choose, and easy highly expressed region is connected, and obtained fusion protein has antigenicity
It is good, the high advantage of expression quantity, while having both that broad spectrum activity is good, the antibody titer being prepared is high and can prevent a variety of hypotypes
The advantages of porcine pseudorabies virus.
The present invention provides the preparation method of above-mentioned fusion protein, will encode the gene order of above-mentioned fusion protein in host
Expression.This method is easy to operate, and universality is wide, easy to accomplish, is suitble to large-scale production and application.
Vaccine provided by the invention containing above-mentioned fusion protein, safety is good, has no adverse reaction, and does not guide and generates other
Antibody, potency is high and stability is good, reduces the production cost of pseudorabies disease vaccine, and protective rate is high.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described reality
Applying example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
It should be understood that
In the present invention, if without particularly illustrating, all embodiments mentioned in this article and preferred implementation method
It can be combined with each other to form new technical solution.
In the present invention, if without particularly illustrating, all technical characteristics and preferred feature mentioned in this article can be with
Intercombination forms new technical solution.
In the present invention, if percentage (%) or part refer to the weight relative to composition without particularly illustrating
Percentage or parts by weight.
In the present invention, if related each component or its preferred ingredient can be combined with each other shape without particularly illustrating
The technical solution of Cheng Xin.
In the present invention, unless otherwise indicated, numberical range " a~b " indicates the contracting of any real combinings between a to b
Sketch form shows that wherein a and b is real number.Such as numberical range " 3~30 " indicate herein all listed " 3~30 " it
Between whole real numbers, " 3~30 " be these combinations of values breviary indicate.
" range " disclosed in this invention can be respectively one or more lower limits and one in the form of lower and upper limit
A or multiple upper limits.
In the present invention, unless otherwise indicated, it is each reaction or operating procedure can sequentially carry out, can also in sequence into
Row.Preferably, reaction method herein is that sequence carries out.
Unless otherwise indicated, profession used herein and meaning phase known to scientific term and one skilled in the art
Together.In addition, any method similar to or equal to what is recorded or material can also be applied in the present invention.
The present invention provides a kind of fusion protein of porcine pseudorabies virus, the fusion protein includes porcine pseudorabies virus gB
Albumen section, porcine pseudorabies virus gC albumen section and porcine pseudorabies virus gD albumen section.
The fusion protein is by the gene of the gB albumen section of porcine pseudorabies virus, gC albumen section and gD albumen section
Sequence expressing in series obtains.The present invention is analyzed by the gene order to gB, gC and gD, and it is wherein antigenic good to choose, easily
Highly expressed region is connected, and obtained fusion protein has antigenic good, the high advantage of expression quantity, while having both broad spectrum activity
Good, the antibody titer being prepared is high and the advantages of can preventing the porcine pseudorabies virus of a variety of hypotypes.
In some preferred embodiments, porcine pseudorabies virus gB albumen section core as shown in SEQ IDNO.1
Nucleotide sequence expression, the expression of porcine pseudorabies virus gC albumen section nucleotide sequence as shown in SEQ ID NO.2, pseudorabies
The expression of viral gD albumen section nucleotide sequence as shown in SEQ ID NO.3.
Sequence shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 is to select classical strains and current stream
The gene of row strain is as research object, and by the sequence obtained after comparative analysis, to reach, to further increase fusion protein anti-
Former broad spectrum activity and the purpose for improving antigen presentation amount.
To porcine pseudorabies virus gB albumen section, porcine pseudorabies virus gC albumen section and the protein region porcine pseudorabies virus gD
Section the order of connection without limitation, as long as being linked together by concatenated mode.In some preferred embodiments,
The company of the porcine pseudorabies virus gB albumen section, porcine pseudorabies virus gC albumen section and porcine pseudorabies virus gD albumen section
Connecing sequence is gB-gD-gC.
The present invention also provides the preparation method of the fusion protein of above-mentioned porcine pseudorabies virus, the preparation method includes
The gene for encoding the fusion protein of the porcine pseudorabies virus is expressed in host, obtains the fusion of the porcine pseudorabies virus
Albumen.
This method is easy to operate, and universality is wide, easy to accomplish, is suitble to large-scale production and application.
In some preferred embodiments, by the table of the gene comprising the fusion protein for encoding the porcine pseudorabies virus
It up in vector introduction host, expresses that the gene of the fusion protein of the porcine pseudorabies virus in host, it is pseudo- to obtain the pig
The fusion protein of rabies viruses.
Preferably, the expression vector is plasmid, preferably PMH3。
In some preferred embodiments, the host is cell；Preferably CHO-S cell.
The typical but non-limiting method for importing expression vector in host is cell transfecting method, specifically, is prepared high
Concentration endotoxin-free plasmid, with the method for liposome transfection by eukaryotic expression plasmids into host cell strain.It transfects previous
It, is inoculated on culture plate with suitable cell density, when cell fusion degree reaches 80%, according to Lipofectamine
The specification of 3000 transfection reagents carries out the operation of expression vector transfection cell.
In some preferred embodiments, the CHO-S cell for importing expression vector is screened and is tamed, obtain pig
Fusion protein expression >=1g/L CHO-S cell strain of Pseudorabies virus, the CHO-S cell strain express to obtain the pig puppet
The fusion protein of rabies viruses；
Preferably, it is screened and is tamed using Geneticin screening system.
In a specific embodiment, after cell growth two days adherent, liquid pressurization is changed, is trained with the selection containing G418
Support the stable transfected cells strain that base screening is integrated into fusion secreting, expressing gB-gD-gC.All clones are chosen to culture plate
In, it sets in incubator and cultivates, it is long to 80% bottom hole is paved with wait clone, take supernatant to pass through SDS-PAGE electrophoresis and Western blot
The high cell strain of method screening expression quantity, which leaves, to be continued to screen.Stablized and merged after repeating the general plate colony screening of low-density
The highest positive CHO-S monoclonal cell strain of expressing quantity, stability and high efficiency express the CHO-S Dan Ke of gB-gD-gC fusion protein
Grand cell strain.
It is prepared into the present invention also provides the fusion protein of above-mentioned porcine pseudorabies virus or using above-mentioned preparation method
To porcine pseudorabies virus fusion protein preparation porcine pseudorabies virus vaccine in application.
In addition, the vaccine includes the fusion protein of above-mentioned porcine pseudorabies virus the present invention also provides a kind of vaccine
Or the fusion protein for the porcine pseudorabies virus being prepared using above-mentioned preparation method.
The vaccine safety is good, has no adverse reaction, and does not guide and generates other antibody, and potency is high and stability is good, reduces
The production costs of pseudorabies disease vaccines, protective rate are high.
In some preferred embodiments, the vaccine further includes adjuvant, the fusion protein of the porcine pseudorabies virus
Volume ratio with adjuvant is 1:0.5-3, such as can be, but be not limited to 1:0.5,1:1,1:2 or 1:3.
Wherein, typical adjuvant include aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white-oil adjuvant,
MF59 adjuvant or ISA201 are, it is preferable to use ISA201.
In order to help to further understand the present invention, technical solution of the present invention is carried out now in conjunction with preferred embodiment detailed
Explanation.
The building of 1 eukaryon expression plasmid of embodiment
1, PCR amplification gB, gD, gC protein gene sequence overall length is connected gB, gD, gC sequence using gibson connection method
Get up, building gB-gD-gC expresses frame, and gB-gD-gC expresses frame both ends and distinguishes primer EcoRI and XhoI restriction enzyme site.
2, EcoRI and XhoI enzyme cuts gB-gD-gC expression frame from carrier, is connected to EcoRI and XhoI digestion
In carrier for expression of eukaryon PMH3.For the ease of protein purification, can in sequence table sequence shown in SEQ ID No.3 carboxyl
End connects upper Poly-His label, constructs recombinant eukaryon expression vector PMH3-gB-gD-gC-his.
3, double digestion is identified, label needs well the 1.5ml EP used to manage, and sample-adding: 20ul reactant is carried out according to the following table
System, is placed in 37 DEG C of thermostat water baths, water-bath 2h.Double digestion system sample is subjected to agarose gel electrophoresis, checks insertion piece
Whether Duan great little is correct, selects Insert Fragment correctly to clone and sequencing company is sent to be sequenced.
1 reaction system of table
2 cell culture of embodiment
1, cell recovery: taking out cryopreservation tube from liquid nitrogen container, is immediately placed in 37 DEG C of thermostat water baths, shakes gently, make
Its fast melt.Cell suspension in cryopreservation tube is drawn in centrifuge tube, 1000rpm/min is centrifuged 5min, discards supernatant, and is added suitable
It measures fresh culture and cell is resuspended, be put into 37 DEG C, 5%CO2It is cultivated in incubator.
2, cell passes on: Chinese hamster ovary celI is incubated in adhere-wall culture base, and 37 DEG C, 5%CO2It is cultivated in incubator.Cell is long extremely
When degrees of fusion is up to 80% or more, culture medium is sucked out, is washed 2 times with PBS, the digestion of 0.25% trypsin solution of 1ml is added, it is light to shake
Cell bottle waits for that pancreatin infiltrates bottle wall, and part pancreatin is sucked out, continues to digest with remaining a small amount of pancreatin, and as under inverted microscope
Observation removes remaining trypsin solution to the cell rounding hypsokinesis that brightens, the fresh culture containing serum is added and terminates digestion, and uses suction pipe
Featheriness bottle wall all falls off to cell, discards part cell liquid, and suction pipe is slightly blow molded into individual cells suspension, adds appropriate training
Base is supported, is put into and continues to cultivate in incubator.Suspension cell then takes in growth logarithmic phase cell, counts, and observes cell state, presses
After being calculated according to required concentration and volume, with certain quantity of fresh culture medium gravity treatment, piping and druming, which mixes, is placed on 100rpm/min, and 37
It is cultivated in DEG C constant-temperature table.
3 protein purification of embodiment
1, cell culture fluid is collected, 8000g is centrifuged 30min, takes supernatant, crosses 0.8um filter membrane, and loading reserves 90ul sample
4 × SDS Buffer of 30ul is added, is detected for SDS-PAGE.
2, it column equilibration: with ultrapure 2~3 column volume of water balance, excludes ethyl alcohol and saves liquid, then use column equilibration liquid (20mM
NaH2PO4, 500nM NaCl) and 2~3 column volumes of balance, 5ml/min.
3, loading: 5ml prepacked column, 1ml/min carry out loading, stay column time 5min, collect stream liquid leakage, 90ul sample is taken to add
Enter 4 × SDS Buffer of 30ul, is detected for SDS-PAGE.
4, it washs: with 4% eluent (20mM NaH2PO4, 500mM, 20nM imidazole) and wash column, flow velocity 2ml/
Min rinses the weaker foreign protein of the albumen and binding ability of unbonded upper prop well, until OD280nm baseline is steady.
5, it elutes: with 50% eluent (20mM NaH2PO4, 500mM, 250nM imidazole) and elution destination protein is extremely
Baseline washes flat, flow velocity 2ml/min, 10ml/ pipe collection, collects the 4 × SDS for taking 90ul sample that 30ul is added after sample mixing
Buffer is detected for SDS-PAGE.
6, it washs: with 100% eluent (20mM NaH2PO4, 500mM, 500nM imidazole), 4ml/min rinses 2
~3 column volumes, until baseline wash it is flat.2~3 column volumes of ultrapure water balance.Liquid, which is saved, with 20% ethyl alcohol balances 2~3 cylinders
It is saved after product.
7, liquid is changed in dialysis: the imidazole elution containing destination protein being poured into bag filter, with 1 × PBS dialysis at least 10
Times, take 90ul to keep sample detection.
8, in Biohazard Safety Equipment, 0.22um vacuum filter, filtered protein solution sample storage aseptic filtration: are crossed
In -80 DEG C of refrigerators.
9, protein concentration protein concentration and purity testing: is measured using BCA method；Purity, purity are detected using HPLC method
It is attained by 90% or more.
4 vaccine preparation of embodiment and safety verification, efficacy test
1, vaccine preparation
The porcine pseudorabies virus gB-gD-gC albumen of appropriate expressing cho cell is added to (volume in ISA206VG adjuvant
Than 50:50), make final concentration of protein 100ug/ml, emulsify, quality inspection qualification is placed on 4 DEG C of preservations.
2, safety verification
With 3~4 week old piglet 10 (PRV antigen-antibody is negative), it is randomly divided into 2 groups, every group 5, wherein one group of conduct
Control group, another group, as the immune group musculi colli above-mentioned vaccine 2ml of injection, is observed 14, and note seedling is local without severe reaction, and
It is all strong living for qualification.As a result it see the table below, the results showed that vaccine is qualified, and injection site, whole body and body temperature are showed no exception.
2 safety verification result of table
3, efficacy test
The susceptible piglet 30 of 3~4 week old health (PRV antigen-antibody is negative) is screened, is randomly divided into 3 groups, every group 10,
In one group as a control group, remaining two groups be used as immune group, respectively be immunized expressing cho cell fusion protein preparation vaccine and
Commercial seedling Reearch in Pseudorabies virus gE Gene lacks live vaccine (Bathar-K61 plants), and 28 days after being immunized, together with control pig, each collunarium is connect
Kind classical virulent (SC plants) and current popular strain (JS plants), are observed 14.Compare pig should at least 4 hairs disease, and at least 2 are dead
It dies, immune swine should at least 4 head protections.
3 piglet immunological of table attacks malicious (SC plants), and body temperature reacts afterwards
4 piglet immunological of table attacks malicious (SC plants), and result counts afterwards
Illustrated by the experimental data of table 3 and table 4, after the porcine pseudorabies vaccine immunity piglet of preparation, is inoculated with classical strong
(SC plant) attacks of poison, 100% (5/5) can be provided for piglet and is protected, suitable with commercial seedling protective rate, and is compareed after piglet attacks poison
100% morbidity and 3/5 death, show good immunoprotection.
5 piglet immunological of table attacks malicious (JS plants), and body temperature reacts afterwards
6 piglet immunological of table attacks malicious (JS plants), and result counts afterwards
Illustrated by the experimental data of table 5 and table 6, after the porcine pseudorabies vaccine immunity piglet of preparation, is inoculated with current stream
(JS plants) of row strain attacks can provide 100% (5/5) protection for piglet, be better than commercial seedling protective rate (2/5), and compare piglet
It attacks after poison 100% morbidity and 3/5 is dead, show good immunoprotection.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
SEQUENCE LISTING
<110>Tian Kang Biological Co., Ltd.
<120>fusion protein of porcine pseudorabies virus, preparation method, the fusion protein using and comprising porcine pseudorabies virus
Vaccine
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 2745
<212> DNA
<213> Pseudorabies virus
<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> 1464
<212> DNA
<213> Pseudorabies virus
<400> 2
atggcctcgc tcgcgcgtgc gatgctcgcg ctgctggcgc tctacacggc ggccatcgcc 60
gcggcgccgt cgtccacgac ggcgctcggc acgacgccca acgggggcgg gggcggcaac 120
agcagcgcgg gcgagctctc gccctcgccg ccctcgacgc ccgagcccgt ctcggggacg 180
acgggggccg cggcctccac gcccgccgcc gtctcgacgc cccgggtccc gccgccctcg 240
gtctcgcgcc ggaagcccca gcggaacggc aacaggacgc gcgtccacgg cgacaaggcc 300
acctcgcacg ggcgcaagcg catcgtgtgc cgcgagcggc tgttctcggc gagggtgggg 360
gacgcggtca gcttcgggtg cgccgtcgtc ccgcgcgccg gggagacctt cgaggtccgc 420
ttctgccgcc gcgggcgctt ccgctcgccc gacgccgacc ccgagtactt tgacgagccc 480
ccgcgcccgg agctcccgcg ggagcggctc ctcttcagct ccgccaacgc ctccctcgcc 540
cacgcggacg cgctcgcctc cgccgtcgtc gtcgagggcg agcgcgcgac cgtcgccaac 600
gtctcgggcg aggtgtccgt gcgcgtggcc gcggcggacg ccgagaccga gggcgtctac 660
acgtggcgcg tgctgtccgc caacggcacc gaggtccgca gcgccaacgt ctcgctcgtc 720
ctgtaccacc agcccgagtt cggcctgagc gcgccgcccg tcctcttcgg cgagcccttc 780
cgggcggtgt gcgtcgtccg cgactactac ccgcggcgca gcgtgcgcct gcgctggttc 840
gcggacgagc acccggtgga cgccgccttc gtgaccaaca gcaccgtggc cgacgagctc 900
gggcgccgca cgcgcgtctc cgtggtgaac gtgacgcgcg cggacgtccc gggcctcgcg 960
gccgcggacg acgcggacgc gctcgcgccg agcctgcgct gcgaggccgt gtggtaccgc 1020
gacagcgtgg cctcgcagcg cttctccgag gccctgcgcc cccacgtcta ccacccggcg 1080
gcggtctcgg tgcgcttcgt cgagggcttc gccgtctgcg acggcctctg cgtgcccccg 1140
gaggcgcgcc tcgcctggtc cgaccacgcc gccgacaccg tctaccacct cggcgcctgc 1200
gccgagcacc ccggcctgct caacgtgcgg agcgcccgcc cgctgtcgga cctcgacggg 1260
cccgtcgact acacctgccg cctcgagggc atgccctcgc agctgcccat cttcgaggac 1320
acgcagcgct acgacgcctc ccccacgtcc gtgagctggc ccgtcgtgac cagcatgatc 1380
accgtcatcg ccggcatcgc catcctagcc atcgtgctgg tcatcatggc gacgtgcgtc 1440
tactaccgcc ggtccgcgct gtga 1464
<210> 3
<211> 1209
<212> DNA
<213> Pseudorabies virus
<400> 3
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
Claims (10)
1. a kind of fusion protein of porcine pseudorabies virus, which is characterized in that the fusion protein includes porcine pseudorabies virus gB egg
White area section, porcine pseudorabies virus gC albumen section and porcine pseudorabies virus gD albumen section.
2. the fusion protein of porcine pseudorabies virus according to claim 1, which is characterized in that the porcine pseudorabies virus gB
The expression of albumen section nucleotide sequence as shown in SEQ ID NO.1, porcine pseudorabies virus gC albumen section is by SEQ ID NO.2
Shown in nucleotide sequence expression, porcine pseudorabies virus gD albumen section nucleotide sequence as shown in SEQ ID NO.3 expression.
3. the fusion protein of porcine pseudorabies virus according to claim 1, which is characterized in that the porcine pseudorabies virus gB
The order of connection of albumen section, porcine pseudorabies virus gC albumen section and porcine pseudorabies virus gD albumen section is gB-gD-gC.
4. the preparation method of the fusion protein of porcine pseudorabies virus as described in any one of claims 1-3, which is characterized in that institute
Stating preparation method includes expressing the gene for the fusion protein for encoding the porcine pseudorabies virus in host, and it is pseudo- to obtain the pig
The fusion protein of rabies viruses.
5. the preparation method according to claim 4, which is characterized in that by the fusion comprising encoding the porcine pseudorabies virus
The expression vector of the gene of albumen imports in host, makes the gene of the fusion protein of porcine pseudorabies virus table in host
It reaches, obtains the fusion protein of the porcine pseudorabies virus；
Preferably, the expression vector is plasmid, preferably PMH3。
6. the preparation method according to claim 4, which is characterized in that the host is cell；Preferably CHO-S cell.
7. according to the described in any item preparation methods of claim 4-6, which is characterized in that thin to the CHO-S for importing expression vector
Born of the same parents screen and tame, and obtain fusion protein expression >=1g/L CHO-S cell strain of porcine pseudorabies virus, the CHO-
S cell strain expresses to obtain the fusion protein of the porcine pseudorabies virus；
Preferably, it is screened and is tamed using Geneticin screening system.
8. any one of fusion protein or application claim 4-7 of porcine pseudorabies virus as described in any one of claims 1-3
Application of the fusion protein for the porcine pseudorabies virus that the preparation method is prepared in preparation porcine pseudorabies virus vaccine.
9. a kind of vaccine, which is characterized in that the vaccine includes melting for the described in any item porcine pseudorabies virus of claim 1-3
The fusion protein of hop protein or the porcine pseudorabies virus being prepared using the described in any item preparation methods of claim 4-7.
10. vaccine according to claim 9, which is characterized in that the vaccine further includes adjuvant, the porcine pseudorabies virus
Fusion protein and adjuvant volume ratio be 1:0.5-3.
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| CN201910392417.XA CN110092839A (en) | 2019-05-10 | 2019-05-10 | The fusion protein of porcine pseudorabies virus, preparation method, using and comprising porcine pseudorabies virus fusion protein vaccine |
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| CN201910392417.XA CN110092839A (en) | 2019-05-10 | 2019-05-10 | The fusion protein of porcine pseudorabies virus, preparation method, using and comprising porcine pseudorabies virus fusion protein vaccine |
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| CN110981968A (en) * | 2019-12-17 | 2020-04-10 | 天康生物(上海)有限公司 | Fusion protein containing rabies virus G protein, preparation method, application and vaccine thereof |
| CN114146172A (en) * | 2021-12-07 | 2022-03-08 | 重庆市动物疫病预防控制中心 | Nano antibody vaccine capable of preventing porcine pseudorabies virus infection, preparation method and application |
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