CN106543290A - A kind of purification process of 2 type Cap protein of recombinant porcine circovirus - Google Patents
A kind of purification process of 2 type Cap protein of recombinant porcine circovirus Download PDFInfo
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- CN106543290A CN106543290A CN201611120907.7A CN201611120907A CN106543290A CN 106543290 A CN106543290 A CN 106543290A CN 201611120907 A CN201611120907 A CN 201611120907A CN 106543290 A CN106543290 A CN 106543290A
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- porcine circovirus
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 34
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 31
- 238000000746 purification Methods 0.000 title claims abstract description 26
- 241000202347 Porcine circovirus Species 0.000 title claims abstract description 25
- 108010014258 Elastin Proteins 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 101100382437 Porcine circovirus 2 Cap gene Proteins 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 6
- 241000588724 Escherichia coli Species 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 235000018102 proteins Nutrition 0.000 claims description 28
- 241000894006 Bacteria Species 0.000 claims description 21
- 238000005119 centrifugation Methods 0.000 claims description 17
- 238000001556 precipitation Methods 0.000 claims description 15
- 239000000969 carrier Substances 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- 108020004705 Codon Proteins 0.000 claims description 13
- 229920001184 polypeptide Polymers 0.000 claims description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 11
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 11
- 102000016942 Elastin Human genes 0.000 claims description 10
- 241001673669 Porcine circovirus 2 Species 0.000 claims description 10
- 229920002549 elastin Polymers 0.000 claims description 10
- 239000013598 vector Substances 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 7
- 108010077850 Nuclear Localization Signals Proteins 0.000 claims description 6
- 238000005457 optimization Methods 0.000 claims description 6
- 101150044789 Cap gene Proteins 0.000 claims description 5
- 235000001014 amino acid Nutrition 0.000 claims description 5
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 210000001072 colon Anatomy 0.000 claims description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 4
- 230000009466 transformation Effects 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000003780 insertion Methods 0.000 claims description 2
- 230000037431 insertion Effects 0.000 claims description 2
- 230000006920 protein precipitation Effects 0.000 claims description 2
- 238000000844 transformation Methods 0.000 claims description 2
- 238000005267 amalgamation Methods 0.000 claims 1
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- 108020001507 fusion proteins Proteins 0.000 abstract description 10
- 102000037865 fusion proteins Human genes 0.000 abstract description 10
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- 229940031626 subunit vaccine Drugs 0.000 abstract description 4
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 3
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
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- 238000001962 electrophoresis Methods 0.000 description 4
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- 229930027917 kanamycin Natural products 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
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- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229940031551 inactivated vaccine Drugs 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
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- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
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- 241001661006 Pepper cryptic virus 2 Species 0.000 description 1
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- 239000002253 acid Substances 0.000 description 1
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- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 241000933939 bacterium 26 Species 0.000 description 1
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- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000002940 repellent Effects 0.000 description 1
- 239000005871 repellent Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- -1 salt ion Chemical class 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
The present invention relates to a kind of purification process of 2 type Cap protein of recombinant porcine circovirus.Described 2 type Cap protein of recombinant porcine circovirus, by ELP class elastin polypeptide genes and PCV2 ORF2 genomic constitutions.Its preparation method includes expression vector establishment, expressing fusion protein and purification.Compared with the purification process of existing pig circular ring virus vaccine, the 2 type Cap protein purification process of recombinant porcine circovirus of the present invention have the advantages that to prepare simply, easy purification and cheap, large-scale production operation is appropriate to, is that preparation, purification and the application of subsequent recombination pig circular ring virus subunit vaccine provides the foundation.
Description
Technical field
The present invention relates to a kind of purification process of 2 type Cap protein of recombinant porcine circovirus.Belong to veterinary biologicses neck
Domain.
Background technology
Pig circular ring virus (porcine circovirus, PCV) are pig circular ring virus section Circovirus members, PCV point
For two kinds of genotype of PCV1 and PCV2.PCV1 no pathogenicities, and PVC2 is pig postweaning multisystem exhaustion syndrome (PMWS)
Virulence factor.Virus infection rate in China pig farm is high, and the average positive rate of swinery is typically greater than 20%, and what is had is even super
Cross 50%.Sizable economic loss is caused to large-scale pig farm.For the anti-system of the disease mainly relies on vaccine, at present
The PCV2 vaccines of commercialization have PCV2 inactivated virus vaccines, PCV1-PCV2 embedded viruses inactivated vaccine and baculoviruss table
Up to Cap protein inactivated vaccine, although these vaccines are proved to be the mortality rate that can effectively reduce PCV2 infection, due to system
Standby process is loaded down with trivial details, the reason such as purification difficulty is big, with high costs, limits large-scale production and penetration and promotion.
Class elastin polypeptide (ELP) is a kind of sensitive synthetic of with spring function and to external world environmental abnormality
Engineered protein polymer.ELP has good solubility in aqueous, and its structure is mainly by pentapeptide repetitive sequence list
Unit is constituted, i.e. VPGXG stems from the water repellent region of class elastin laminin, and wherein X can be any ammonia in addition to Pro
Base acid.Class elastin polypeptide has a reversible phase transition process, and this process is defined as inverting temperature phase transformation (Inverse
Temperature transition, ITT), even the temperature of surrounding is less than the phase transition temperature (transition
Temperature, Tt) when, ELP is presented high soluble, and structure is the disordered structure state that height stretches.Conversely, working as surrounding ring
When border temperature is higher than the phase transition temperature, aggregation will be disintegrated and be started to aqueous polypeptide chain structure, form gathering rich in ELP
Collection thing, now for state can not be melted.In view of this special nature of ELP, has been widely used for protein purification and plasmid
The isolation technics of DNA.
There is no with colon bacillus (Escherichia coli) (present invention hereinafter referred to as escherichia coli) in the market
System expression produce PCV-2 subunit vaccines report, main cause be Cap genes can not in escherichia coli effective expression,
But remove nuclear localization signal or optimizing codon can be expressed.The present invention is by ELP class elastin polypeptide coded sequences and deletes
Nuclear localization signal (front 39 aminoacid) is simultaneously optimized to the Cap coded sequences of escherichia coli preference codon sequence and carries out weight
Group, is expressed in E. coli system, so as to fusion protein high level expression；Low temperature induction expressing fusion protein is used, with
Just high soluble and the excellent albumen of activity are obtained；ITC purification is carried out with optimal conditions, to reach highest recovery and most
High protein purity.Compared with existing recombinant porcine circovirus vaccine way of purification, with 2 type of recombinant porcine circovirus of the present invention
The way of purification of Cap protein is not only simple, economical, and the fusion protein of purification meets the quality standard of veterinary biologicses, can enter
One step studies the application and production for recombinant subunit vaccine.
The content of the invention
In order to solve the problems, such as prior art, the invention provides a kind of 2 type Cap protein of recombinant porcine circovirus
Purification process.
The 2 type Cap protein of recombinant porcine circovirus of the present invention, by ELP classes elastin polypeptide and porcine circovirus 2 type
Cap protein is constituted.Proceeding to E. coli system expression proves that 2 type Cap protein of recombinant porcine circovirus can Jing after artificial optimization
High efficient expression.Jing ITC purification, can isolate the preferable fusion protein of purity again.
Technical scheme
1. a kind of 2 type Cap protein of recombinant porcine circovirus, it is characterised in that the 2 type Cap protein of recombinant porcine circovirus
It is by ELP class elastin polypeptide genes and PCV2-ORF2 genomic constitutions.
2. the preparation method of the 2 type Cap protein of recombinant porcine circovirus described in claim 1, it is characterised in that the method
Comprise the following steps：
(1) synthesize the sequence SEQ ID No.1 insertion pET-30a carriers of ELP classes elastin polypeptide coding, merged
Expression vector pET-ELP；
(2) synthesize without nuclear localization signal and be optimized to the PCV-2Cap coded sequence SEQ ID of e. coli codon
No.2, inserts pET-ELP carriers, obtains recombinant vector pELP-Cap；
(3) recombinant vector is proceeded to into BLR escherichia coli, obtains recombinant bacterium and be named as colon bacillus
Beijing Chaoyang was delivered in (Escherichia coli) pELP-Cap BLR (DE3) strain, this plant of bacterium on November 11st, 2016
The common micro- life of No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of institute of area North Star West Road 1
Thing center CGMCC No.13417；This plant of bacterium is expressed with IPTG induced proteins at 20 DEG C；
(4) the full bacterium after room temperature 12000g centrifugal breaking, goes precipitation to take supernatant；
(5) the addition NaCl to final concentration of 2M in supernatant；
(6) in 26 DEG C, 12000g protein precipitation by centrifugation；
(7) with the albumen of the resuspended dissolution precipitation of 4 DEG C of PBS；
(8) albumen of resuspended dissolving is centrifuged in 4 DEG C, 12000g, goes precipitation to take supernatant.
3. the preparation method of the 2 type Cap protein of recombinant porcine circovirus as described in right 2, it is characterised in that：Step (1)
It is：Escherichia coli preference codon sequence of the ELP coded sequences for artificial optimization；Step (2) is：PCV-2Cap coded sequences are deleted
Except 39 aminoacid before nuclear localization signal, and artificial optimization is into e. coli codon sequence；Step (3) is：Carry out in low temperature
The expression of carrying Cap gene of porcine circovirus type 2；Step (5) is：Under appropriate salt ion and temperature conditionss, circulated using ELP phase transformations
Characteristic, to recombinate carrying Cap gene of porcine circovirus type 2 carry out purification.
Specific implementation step
Biological material source：
1.pET-30a carriers：Buy from Novagen companies of the U.S., our company's laboratory is preserved.
2.DH5a escherichia coli：Buy from BD Biosciences Clontech companies of the U.S., our company's laboratory is protected
Deposit.
3.BLR escherichia coli：Buy from Novagen companies of the U.S., our company's laboratory is preserved.
Concrete operation step is as follows：
1.pET-ELP vector constructions
(1) ELP class elastin polypeptide coded sequences (SEQ ID No.1) are synthesized, 5' ends introduce Nde I restriction enzyme sites,
3' ends introduce Sac I restriction enzyme sites, send Nanjing Jin Ruisi bio tech ltd to synthesize sequence, are cloned in pUC57 plasmids
In carrier, SEQ ID No.1 are as shown：
(2) pUC57-ELP carriers are digested with restriction enzyme Nde I and Sac I, after agarose gel electrophoresiies are separated, use
AXYGEN DNA gel QIAquick Gel Extraction Kits (healthy and free from worry Biology Science Co., Ltd) reclaim ELP sequences, carry with identical enzyme action pET-30a
Body connects, and obtains fusion expression vector pET-ELP (Fig. 1).
(3) after by 39 amino acid deletions before PCV2-Cap coded sequences, with JAVA Codon Adaption Tool (texts
Offer：Grote A,Hiller K,Scheer M,Münch RB,Hempel DC,Jahn D.JCat:a novel tool to
adapt codon usage of a target gene to its potential expression host.Nucleic
Acids Research, 2005,1:33) escherichia coli preference codon sequence (SEQ ID No.2) will be optimized to, 5' ends introduce
HindIII restriction enzyme sites, 3' ends introduce XhoI restriction enzyme sites, send Nanjing Jin Ruisi bio tech ltd to synthesize sequence,
It is cloned in pUC57 carriers, obtains pUC57-Cap carriers.SEQ ID No.2 are as shown：
(4) Cap coded sequences are cut from pUC57-Cap carriers with restriction enzyme HindIII and XhoI, inserts pET-ELP
Carrier corresponding site, obtains recombinant vector pELP-Cap (Fig. 1).
2. the expression and purification of 2 type Cap protein of recombinant porcine circovirus
(1) recombiant plasmid pELP-Cap is proceeded to into BLR escherichia coli, obtains recombinant bacterium and be named as escherichia coli pELP-
Section of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China was delivered in Cap BLR (DE3) strain, this plant of bacterium on November 11st, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology of institute CGMCC No.13417；Picking
Single bacterium colony inoculation kanamycin (50 μ g/ml) the LB culture medium of recombinant bacterium, 37 DEG C, 220r/min overnight incubations；By 1:100 ratios
Inoculation kanamycin 2 × YT culture fluid (Tryptone 16g, Yeast Extract 10g, NaCl 5g, pH7.2), 37 DEG C,
220r/min is cultivated to OD600=0.6, add 0.2mM IPTG, 37 DEG C of abduction delivering 6h；4 DEG C, 5000g centrifugation 10min, precipitation
Thalline PBS (NaCl 7.9g, KCl 0.2g, Na2HPO41.44g, KH2PO47.4) 0.24g, pH suspend, and ultrasound wave is abundant
Cracking (200W, 2s stop 3s, 20min)；4 DEG C, 12000g centrifugation 10min, take supernatant and add NaCl to be respectively 2M to final concentration,
5min is incubated in 20 DEG C, 22 DEG C, 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C of water-baths respectively, the 12000g centrifugations 10min at each same temperature
Precipitation is collected, SDS-PAGE electrophoretic analysiss after PBS is resuspended, are carried out.As a result show：Recombinant bacterium pELP-Cap (BLR) can express expection
The fusion protein of 67kDa, after 26 DEG C of incubations can obtain maximum recovery, reach 92% (Fig. 2).
(2) take cracking after thalline, 4 DEG C, 12000g centrifugation 10min, take supernatant add NaCl to final concentration be respectively 1,2,
3M, in 26 DEG C of water-bath incubation 5min, same temperature 12000g centrifugation 10min collects precipitation, redissolves precipitation with 4 DEG C of PBS, equally
Temperature 12000g centrifugation 10min goes precipitation, and taking supernatant carries out SDS-PAGE electrophoretic analysiss.As a result show：In the 2M NaCl response rate
Most preferably, 92% is reached, purity reaches 93% (Fig. 3).
Description of the drawings
Fig. 1 is pELP-Cap fusion expression vector structural representations.PT7 is escherichia coli T7 promoteres；ELP is class elasticity
Protein polypeptide coded sequence；Cap is carrying Cap gene of porcine circovirus type 2 coded sequence.
Fig. 2 is that purified fusion protein is analyzed in the SDS-PAGE of different salt ionic concentrations.M is albumen Marker；1 is cracking
The full bacterium of recombinant bacterium；2-4 is to crack the centrifugation after recombinant bacterium 26 DEG C of supernatant of centrifugation is acted on 1M, 2M, 3M NaCl respectively.
Fig. 3 is purified fusion protein SDS-PAGE analyses at different temperatures.M is albumen Marker；1 is cracking restructuring
The full bacterium of bacterium；2～7 for cracking thalline centrifugation supernatant in the final concentration of 2M of NaCl, respectively at 20,22,24,26,28,30 DEG C
ITC results.
The present invention relates to biomaterial resource information
Colon bacillus (Escherichia coli) pELP-Cap strain recombinant bacteriums, abbreviation escherichia coli pELP-Cap
BLR (DE3) strain, this plant of bacterium deliver the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences on November 11st, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology CGMCC No.13417.
Other biomaterials used in the present invention, are the material of commercialization, except the Products shown purchased from foreign countries
Outward, other are purchased from domestic biological reagent company.
The positive effect of the present invention
The present invention relates to a kind of preparation of 2 type Cap protein of recombinant porcine circovirus and purification process.Described Recombinant Swine circle
2 type Cap protein of circovirus virus, by ELP class elastin polypeptide genes and PCV2-ORF2 genomic constitutions.Its preparation method includes table
Up to vector construction, expressing fusion protein and purification.Compared with the purification process of existing pig circular ring virus vaccine, the weight of the present invention
Group carrying Cap gene of porcine circovirus type 2 purification process has the advantages that to prepare simple, easy purification and cheap, is appropriate to greatly
Large-scale production is operated, and is that preparation, purification and the application of subsequent recombination pig circular ring virus subunit vaccine provides the foundation.
Embodiment
The present embodiment is not construed as limiting to the technical scheme that the present invention is claimed to further illustrate the present invention.
Embodiment 1
--- the preparation of 2 type Cap protein of recombinant porcine circovirus
Biological material source：
1.pET-30a carriers：Buy from Novagen companies of the U.S., our company's laboratory is preserved.
2.DH5a escherichia coli：Buy from BD Biosciences Clontech companies of the U.S., our company's laboratory is protected
Deposit.
3.BLR escherichia coli：Buy from Novagen companies of the U.S., our company's laboratory is preserved.
Concrete operation step is as follows：
1.pET-ELP vector constructions
(1) ELP class elastin polypeptide coded sequences (SEQ ID No.1) are synthesized, 5' ends introduce Nde I restriction enzyme sites,
3' ends introduce Sac I restriction enzyme sites, send Nanjing Jin Ruisi bio tech ltd to synthesize sequence, are cloned in pUC57 plasmids
In carrier, SEQ ID No.1 are as shown：
(2) pUC57-ELP carriers are digested with restriction enzyme Nde I and Sac I, after agarose gel electrophoresiies are separated, use
AXYGEN DNA gel QIAquick Gel Extraction Kits (healthy and free from worry Biology Science Co., Ltd) reclaim ELP sequences, carry with identical enzyme action pET-30a
Body connects, and obtains fusion expression vector pET-ELP (Fig. 1).
(3) after by 39 amino acid deletions before PCV2-Cap coded sequences, with JAVA Codon Adaption Tool (texts
Offer：Grote A,Hiller K,Scheer M,Münch RB,Hempel DC,Jahn D.JCat:a novel tool to
adapt codon usage of a target gene to its potential expression host.Nucleic
Acids Research, 2005,1:33) escherichia coli preference codon sequence (SEQ ID No.2) will be optimized to, 5' ends introduce
HindIII restriction enzyme sites, 3' ends introduce XhoI restriction enzyme sites, send Nanjing Jin Ruisi bio tech ltd to synthesize sequence,
It is cloned in pUC57 carriers, obtains pUC57-Cap carriers.SEQ ID No.2 are as shown：
(4) Cap coded sequences are cut from pUC57-Cap carriers with restriction enzyme HindIII and XhoI, inserts pET-ELP
Carrier corresponding site, obtains recombinant vector, and the carrier is named as recombinant vector pELP-Cap (Fig. 1).
2. the expression and purification of 2 type Cap protein of recombinant porcine circovirus
(1) recombiant plasmid pELP-Cap is proceeded to into BLR escherichia coli, obtains recombinant bacterium and be named as escherichia coli pELP-
Cap BLR (DE3) strain, and the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is delivered on November 11st, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology CGMCC No.13417；Picking is recombinated
Single colony inoculation kanamycin (50 μ g/ml) the LB culture medium of bacterium, 37 DEG C, 220r/min overnight incubations；By 1:100 ratios are inoculated with
Kanamycin 2 × YT culture fluid (Tryptone 16g, Yeast Extract 10g, NaCl 5g, pH7.2), 37 DEG C, 220r/
Min is cultivated to OD600=0.6, add 0.2mM IPTG, 37 DEG C of abduction delivering 6h；4 DEG C, 5000g centrifugation 10min, precipitate thalline
With PBS (NaCl 7.9g, KCl0.2g, Na2HPO41.44g, KH2PO4 0.24g, pH7.4) suspend, ultrasound wave is fully cracked
(200W, 2s stop 3s, 20min)；4 DEG C, 12000g centrifugation 10min, take supernatant and add NaCl to be respectively 2M to final concentration, respectively
5min is incubated in 20 DEG C, 22 DEG C, 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C of water-baths, 12000g centrifugations 10min is collected at each same temperature
Precipitation, carries out SDS-PAGE electrophoretic analysiss after PBS is resuspended.As a result show：Recombinant bacterium pELP-Cap (BLR) can express expection
The fusion protein of 67kDa, after 26 DEG C of incubations can obtain maximum recovery, reach 92% (Fig. 2).
(2) take cracking after thalline, 4 DEG C, 12000g centrifugation 10min, take supernatant add NaCl to final concentration be respectively 1,2,
3M, in 26 DEG C of water-bath incubation 5min, same temperature 12000g centrifugation 10min collects precipitation, redissolves precipitation with 4 DEG C of PBS, equally
Temperature 12000g centrifugation 10min goes precipitation, and taking supernatant carries out SDS-PAGE electrophoretic analysiss.As a result show：In the 2M NaCl response rate
Most preferably, 92% is reached, purity reaches 93% (Fig. 3).
Sequence table
<110>In believe in promise biotechnology Taizhou company limited
<120>A kind of purification process of 2 type Cap protein of recombinant porcine circovirus
<130>
<160>2
<170> Patentin version 3.5
<210> 1
<211> 1679
<212> DNA
<213>Artificial sequence
<223>Description to artificial sequence：ELP class elastin polypeptide coded sequences
<400> 1
CATATGGGCC ACGGCGTGGG TGTTCCGGGC GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC 60
GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT GTTGGTGTGC CGGGTGTTGG TGTACCAGGT 120
GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC 180
GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT 240
GTTGGTGTGC CGGGTGTTGG TGTACCAGGT GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT 300
GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC 360
GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT GTTGGTGTGC CGGGTGTTGG TGTACCAGGT 420
GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC 480
GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT 540
GTTGGTGTGC CGGGTGTTGG TGTACCAGGT GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT 600
GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC 660
GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT GTTGGTGTGC CGGGTGTTGG TGTACCAGGT 720
GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC 780
GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT 840
GTTGGTGTGC CGGGTGTTGG TGTACCAGGT GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT 900
GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC 960
GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT GTTGGTGTGC CGGGTGTTGG TGTACCAGGT 1020
GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC 1080
GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT 1140
GTTGGTGTGC CGGGTGTTGG TGTACCAGGT GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT 1200
GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC 1260
GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT GTTGGTGTGC CGGGTGTTGG TGTACCAGGT 1320
GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC 1380
GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT 1440
GTTGGTGTGC CGGGTGTTGG TGTACCAGGT GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT 1500
GGCGGTGTGC CGGGCGTGGG TGTTCCGGGC GTGGGTGTTC CGGGTGGCGG TGTGCCGGGC 1560
GCAGGTGTTC CTGGTGTAGG TGTGCCGGGT GTTGGTGTGC CGGGTGTTGG TGTACCAGGT 1620
GGCGGTGTTC CGGGTGCAGG CGTTCCGGGT GGCGGTGTGC CGGGCGGGCT GGTGAGCTC 1679
<210> 2
<211> 598
<212> DNA
<213>Artificial sequence
<223>Description to artificial sequence：The escherichia coli preference codon sequence of optimization
<400> 2
TGTACACAAC AACGGTATCT TCAACACCCG TCTGTCTCGT ACCATCGGTT ACACCGTTAA 60
AAAAACCACC GTTCGTACCC CGTCTTGGAA CGTTGACATG ATGCGTTTCA ACATCAACGA 120
CTTCCTGCCG CCGGGTGGTG GTTCTAACCC GCTGACCGTT CCGTTCGAAT ACTACCGTAT 180
CCGTAAAGTT AAAGTTGAAT TCTGGCCGTG CTCTCCGATC ACCCAGGGTG ACCGTGGTGT 240
TGGTTCTACC GCTGTTATCC TGGACGACAA CTTCGTTACC AAAGCTAACG CTCTGACCTA 300
CGACCCGTAC GTTAACTACT CTTCTCGTCA CACCATCACC CAGCCGTTCT CTTACCACTC 360
TCGTTACTTC ACCCCGAAAC CGGTTCTGGA CCGTACCATC GACTACTTCC AGCCGAACAA 420
CAAACGTAAC CAGCTGTGGC TGCGTCTGCA GACCACCGGT AACGTTGACC ACGTTGGTCT 480
GGGTACCGCT TTCGAAAACT CTATCTACGA CCAGGACTAC AACATCCGTA TCACCATGTA 540
CGTTCAGTTC CGTGAATTCA ACCTGAAAGA CCCGCCGCTG AACCCGAAAT AATCTAGA 598
2
Claims (3)
1. 2 type Cap protein of a kind of recombinant porcine circovirus, it is characterised in that the 2 type Cap protein of recombinant porcine circovirus be by
ELP class elastin polypeptide genes and PCV2-ORF2 genomic constitutions.
2. the preparation method of the 2 type Cap protein of recombinant porcine circovirus described in claim 1, it is characterised in that the method includes
Following steps：
(1) synthesize the sequence SEQ ID No.1 insertion pET-30a carriers of ELP classes elastin polypeptide coding, obtain amalgamation and expression
Carrier pET-ELP；
(2) synthesize without nuclear localization signal and be optimized to the PCV-2Cap coded sequence SEQ ID No.2 of e. coli codon, insert
Enter pET-ELP carriers, obtain recombinant vector pELP-Cap；
(3) recombinant vector is proceeded to into BLR escherichia coli, the bacterial strain is named as colon bacillus (Escherichia coli)
PELP-Cap BLR (DE3) strain, this plant of bacterium deliver Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on November 11st, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center of Institute of Microorganism, Academia Sinica CGMCC
No.13417；This plant of bacterium is expressed with IPTG induced proteins at 20 DEG C；
(4) the full bacterium after room temperature 12000g centrifugal breaking, goes precipitation to take supernatant；
(5) the addition NaCl to final concentration of 2M in supernatant；
(6) in 26 DEG C, 12000g protein precipitation by centrifugation；
(7) with the albumen of the resuspended dissolution precipitation of 4 DEG C of PBS；
(8) albumen of resuspended dissolving is centrifuged in 4 DEG C, 12000g, goes precipitation to take supernatant.
3. the preparation method of the 2 type Cap protein of recombinant porcine circovirus as described in right 2, it is characterised in that：Step (1) is：
Escherichia coli preference codon sequence of the ELP coded sequences for artificial optimization；Step (2) is：PCV-2Cap coded sequences are deleted
39 aminoacid before nuclear localization signal, and artificial optimization is into e. coli codon sequence；Step (3) is：Pig is carried out in low temperature
The expression of circovurus type 2 Cap protein；Step (5) is：Under appropriate salt ion and temperature conditionss, circulated using ELP phase transformations
Characteristic, to recombinating, carrying Cap gene of porcine circovirus type 2 carries out purification.
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| CN107022026A (en) * | 2017-06-12 | 2017-08-08 | 扬州大学 | A kind of purification process of chicken yolk antibody |
| CN108264541A (en) * | 2018-01-12 | 2018-07-10 | 温州大学 | A kind of method of high efficient expression dog circovirus Cap protein |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
| CN109425738A (en) * | 2017-09-01 | 2019-03-05 | 洛阳普莱柯万泰生物技术有限公司 | 3 type antibody assay kit of pig circular ring virus and its application |
| CN111087452A (en) * | 2019-12-18 | 2020-05-01 | 中国农业科学院兰州兽医研究所 | Prokaryotic soluble expression method of porcine circovirus 2b subtype Cap protein |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107022026A (en) * | 2017-06-12 | 2017-08-08 | 扬州大学 | A kind of purification process of chicken yolk antibody |
| CN109425738A (en) * | 2017-09-01 | 2019-03-05 | 洛阳普莱柯万泰生物技术有限公司 | 3 type antibody assay kit of pig circular ring virus and its application |
| CN108264541A (en) * | 2018-01-12 | 2018-07-10 | 温州大学 | A kind of method of high efficient expression dog circovirus Cap protein |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
| CN111087452A (en) * | 2019-12-18 | 2020-05-01 | 中国农业科学院兰州兽医研究所 | Prokaryotic soluble expression method of porcine circovirus 2b subtype Cap protein |
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