CN104017813A - Truncated PCV2-type capsid protein ORF2 (Open Reading Frame 2) virus-like particle and preparation method - Google Patents
Truncated PCV2-type capsid protein ORF2 (Open Reading Frame 2) virus-like particle and preparation method Download PDFInfo
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Abstract
The invention discloses a series of N-terminal truncated PCV2-type ORF2 (Open Reading Frame 2) genes, a virus-like particle of the ORF2 gene and a preparation method of the virus-like particle, a vaccine including the truncated virus-like particle and an application of the vaccine in the prevention of post-weaning multisystemic wasting syndrome infection. The virus-like particle assembled by truncated PCV-ORF2 genes disclosed by the invention has good immunogenicity and protection. Since no denaturing agent is used in the preparation and purification process disclosed by the invention, the activity of the target protein is protected to the greatest extent.
Description
Technical field
The present invention relates to technical field of molecular biology, be specifically related to a kind of PCV2 Cap gene and carrier, bacterial strain and expression method thereof, and the purposes infecting containing the vaccine of this virus-like particle and at prevention pmws (particularly PCV2).
Background technology
Porcine circovirus 2 type PCV2 belongs to PCV-II section, and Circovirus is nonencapsulated strand ring-type DNA virus.This virus is widely current in countries in the world, has caused very large financial loss to world's pig industry.2 type pig circular ring virus comprise again two kinds of hypotype: PCV2a, PCV2b, and PCV2b is the widest at Chinese Epidemic Scope in recent years, proportion maximum, and also the toxicity of PCV2b is stronger.
Porcine circovirus type 2 ORF2 protein is viral capsid protein, and molecular weight is 25kDa, is the main target protein of PCV vaccine.The ORF2 expressing in multiple expression system can be formed on virus-like particle (Virus-Like-Particle, VLP) like morphological structure and natural viral Particle Phase.This virus-like particle is the three-dimensional symmetrical structure of icosahedron.The natural epi-position that it has retained virion, has stronger immunogenicity, can induce the neutralizing antibody for PCV-II.In addition, virus-like particle without viral nucleic acid, without potential carcinogenic danger, has good security.Therefore, VLP vaccine has become the main direction of live vaccine development.Along with Protocols in Molecular Biology is more and more applied to the development of vaccine.The success of virus-like particle package technique also applies to widely modern vaccination research and development and produces.The key of PCV2 vaccine development is efficiently to prepare in a large number VLP particle.
Escherichia expression system is one of gene engineering expression system being most widely used at present.Be easy to cultivate because this expression system has, do not need complex apparatus, safe distinguishing feature, is therefore widely used in bio-pharmaceuticals industry.Also there is before this numerous investigators to attempt utilizing escherichia expression system to carry out the expression of PCV2 ORF2 albumen, and then carry out the exploitation of PCV2 preventative vaccine.But numerous results of study all shows, ORF2 albumen n end contains the nuclear localization signal being made up of 41 amino acid, this signal peptide, due to the inclined to one side preferendum of codon, causes the expression amount of ORF2 albumen in E.coli very low, has lost the possibility of large-scale commercial applications application.
Summary of the invention
The invention provides a kind of N end brachymemma capsid protein PCV2 ORF2 gene of the PCV2 type that meets Chinese Pigs PCV-II feature based on intestinal bacteria selection source, epitope.Based on the technology in this laboratory, by sequence optimisation, PCV2 Cap prokaryotic expression is studied, find N end brachymemma until 27 amino acid whose capsid proteins still can assembling assembly virus-like particle.
The claimed brachymemma capsid protein PCV2 ORF2 gene of the present invention has nucleotide sequence as shown in Seq ID No.1,2,3,4,5,6,7,8,9.
According to a technical scheme of the present invention, the albumen of this genetic expression can be self-assembled into virus-like particle in intestinal bacteria.
A second aspect of the present invention, provides the PCV2 ORF2 capsid protein of brachymemma, has the sequence as shown in Seq ID No.10,11,12,13,14,15,16,17,18.Wherein, gene order Seq ID No.1 is corresponding to protein sequence Seq ID No.10; Gene order Seq ID No.2 is corresponding to protein sequence Seq ID No.11; Gene order Seq ID No.3 is corresponding to protein sequence Seq ID No.12; Gene order Seq ID No.4 is corresponding to protein sequence Seq ID No.13; Gene order Seq ID No.5 is corresponding to protein sequence Seq ID No.14; Gene order Seq ID No.6 is corresponding to protein sequence Seq ID No.15; Gene order Seq ID No.7 is corresponding to protein sequence Seq ID No.16; Gene order Seq ID No.8 is corresponding to protein sequence Seq ID No.17; Gene order Seq ID No.9 is corresponding to protein sequence Seq ID No.18.
A third aspect of the present invention, provides a kind of expression vector of the PCV2 ORF2 gene that contains brachymemma of the present invention.
According to a technical scheme of the present invention, described expression vector is the carrier being suitable at expression in escherichia coli.
A fourth aspect of the present invention, provides a kind of cell of expression vector of the PCV2 ORF2 gene containing brachymemma of the present invention.
A fifth aspect of the present invention, provides a kind of method of capsid protein of the PCV2 ORF2 genetic expression of preparing purifying brachymemma, comprises the steps:
(1) the PCV2 ORF2 gene of soluble-expression brachymemma in escherichia expression system,
(2) by having expressed the intestinal bacteria fragmentation of PCV2 ORF2 capsid protein of brachymemma, separate and obtain supernatant liquor,
(3) by the ammonium sulfate fractional separation that contains Repone K, in conjunction with chromatography purifying target protein.
According to a technical scheme of the present invention, described step (3) is first with the 30% ammonium sulfate precipitation remove portion foreign protein that contains Repone K by ammonium sulfate fractional separation, then with the thick target protein of 50% ammonium sulfate precipitation that contains Repone K, finally, above-mentioned purpose albumen is again dissolved in 150mM-2500mM salts solution, and centrifugation obtains target protein.
According to a technical scheme of the present invention, the resuspended supernatant liquor of salts solution, by membrane filtration, is further passed through to chromatography purifying target protein.
A sixth aspect of the present invention, provides a kind of PCV2 ORF2 viruslike particle through the brachymemma of N end, the albumen of the N end brachymemma that wherein this viruslike particle contains PCV2 ORF2 of the present invention genetic expression.
A seventh aspect of the present invention, provides a kind of vaccine for preventing PCV2 to infect, and it comprises: vehicle or carrier for PCV2 virus-like particle and vaccine.
A eighth aspect of the present invention, a kind of method that provides PCV2 of prevention to infect, it comprises holds capsid protein or the virus sample particle vaccines of the PCV2 ORF2 genetic expression of brachymemma to give the pig that need to prevent PCV2 to infect the N of the present invention that contains prevention significant quantity.
The invention provides and a kind ofly meet the method for the high efficient expression of capsid protein of the PCV2b type of Chinese Pigs PCV-II feature based on selection intestinal bacteria sources, epitope.Based on the technology in this laboratory, by brachymemma N terminal sequence, prokaryotic expression PCV ORF2 albumen is studied, find that the brachymemma of N end still can be assembled into the virus-like particle of diameter 15-20nm up to 27 amino acid whose capsid proteins,
The method of the capsid protein of the PCV2 ORF2 genetic expression of preparing the brachymemma of purifying N end of the present invention, utilizes the method for ammonium sulfate fractional separation precipitation to carry out preliminary purification to target protein.Compared with other intermediate processing, the method is comparatively gentle, not it is generally acknowledged and can damage the structure of protein.In addition, in the time that target protein redissolves, also adopted not containing the redissolution liquid of denaturing agent, this has also further kept the natural radioactivity of the ORF2 albumen of N end brachymemma.By precipitation, redissolve two steps, target protein obtains significant enrichment purifying, and its content that accounts for total protein has risen to 50% left and right from 20%, and in order to carry out, chromatogram is consummate haves laid a good foundation.And, in the process of above-mentioned preliminary purification, not using denaturing agents such as urea, the activity of target protein has obtained maximum protection.In chromatographic purification process, whole technical process is simple, without carrying out the steps such as damping fluid displacement, also without special chromatographic media, and the recovery of two step chromatograms approaches 30%, making becomes possibility with the PCV ORF2 albumen of industrially scalable solubility expression purifying N end brachymemma, and in whole technological process, all do not relate to denaturing agent, purge process and assembling process all can not exert an influence and the neutralizing epitope of break virus sample particle to the conformation of albumen, the virus-like particle of producing like this has the immunogenicity of height, after immunity body, can induce the neutralizing antibody that produces high titre, immune animal is played to good protectiveness effect.
This research with the PCV2 ORF2 albumen of solubility expression N end brachymemma, and on this basis, has been set up the PCV2 vaccine pilot scale production technique of a set of efficient, suitable amplification in intestinal bacteria.Set up the PCV2 VLP vaccine quality research system of a set of N end brachymemma, and the validity of the N in intestinal bacteria source being held to the PCV2 VLP vaccine of brachymemma by experimentation on animals, stability is evaluated.
The explanation of relational language and explanation in the present invention
According to the present invention, term " escherichia expression system " refers to by intestinal bacteria (bacterial strain) and carrier and forms, wherein intestinal bacteria (bacterial strain) derive from available on the market, give an example but be not limited at this: GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3).
According to the present invention, term " carrier " word refers to, and the polynucleotide of certain proteins encoded can be inserted wherein and make albumen obtain a kind of nucleic acid launch vehicle of expressing.Carrier can be by transforming, and transduction or transfection host cell, make its genetic material element carrying in host cell, obtain expression.For instance, carrier comprises: plasmid, phage, coemid etc.
According to the present invention, term " vehicle or carrier for vaccine " refers to and is selected from one or more, includes but not limited to: pH adjusting agent, tensio-active agent, adjuvant, ionic strength toughener.For example, pH adjusting agent is given an example but is not limited to phosphate buffered saline buffer, and tensio-active agent comprises positively charged ion, negatively charged ion or nonionic surface active agent.Give an example but be not limited to: Tween-80.Adjuvant is given an example but is not limited to aluminium hydroxide, Fu Shi Freund's complete adjuvant.Ionic strength toughener is given an example but is not limited to sodium-chlor.
According to the present invention, term " chromatography " includes but not limited to: ion-exchange chromatography (for example cation-exchange chromatography), hydrophobic interaction chromatograph, adsorption chromatography (for example hydroxylapatite chromatography), gel-filtration (gel exclusion) chromatography, affinity chromatography.
According to the present invention, in the method for the PCV2 ORF2 albumen of the N end brachymemma obtaining in the present invention, damping fluid refers to the solution that can maintain within the specific limits pH value stabilization, include but not limited to Tris damping fluid, phosphate buffered saline buffer, HEPES damping fluid, MOPS damping fluid etc.
According to the present invention, during described prokaryotic host cell fragmentation includes but not limited to process by homogenizer fragmentation, clarifixator fragmentation, ultrasonication, grinding, high-pressure extrusion, N,O-Diacetylmuramidase one or multinomial method realize.
According to the present invention, in the method for the PCV2 ORF2 albumen of the N end brachymemma obtaining in the present invention, salt used includes but not limited to it is neutral salt, particularly an alkali metal salt, ammonium salt, hydrochloride, vitriol, vitriol, supercarbonate, phosphoric acid salt or hydrophosphate, particularly NaCl, KCl, NH
4cl, (NH
4)
2sO
4in one or more, preferably NaCl.
According to the present invention, the PCV2 ORF2 protein virion of N end brachymemma obtains as follows: by above-mentioned purity at least the PCV2 ORF2 protein solution of 50%N end brachymemma further separate by for example chromatography, obtain the PCV2 ORF2 protein solution of the N end brachymemma of purifying.The mode of the viruslike particle assembling of the PCV2 ORF2 of N end brachymemma includes but not limited to technology known in the art, for example, and dialysis, ultrafiltration or chromatography etc.
Brief description of the drawings
Below in conjunction with accompanying drawing, the invention will be further described:
Fig. 1 is the PCV2 ORF2 albumen of each brachymemma SDS-PAGE result after purifying.1, through purifying gained PCV2N3C albumen loading of the present invention 2 μ l; 2, through purifying gained PCV2N6C albumen loading of the present invention 2 μ l; 3, through purifying gained PCV2N9C albumen loading of the present invention 2 μ l; 4, through purifying gained PCV2N12C albumen loading of the present invention 2 μ l; 5, through purifying gained PCV2N15C albumen loading of the present invention 2 μ l; 6, through purifying gained PCV2N18C albumen loading of the present invention 2 μ l; 7, through purifying gained PCV2N21C albumen loading of the present invention 2 μ l; 8, through purifying gained PCV2N24C albumen loading of the present invention 2 μ l; 9, through purifying gained PCV2N27C albumen loading of the present invention 2 μ l.
Fig. 2 is PCV2N3C VLPs virus-like particle transmission electron microscope observing (40, the 000 times) result of gained of the present invention.The virus-like particle that in the visual field, visible a large amount of radiuses are 9nm left and right, particle actual size conforms to theoretical size, apparent state uniformity.
Fig. 3 is PCV2N6C VLPs virus-like particle transmission electron microscope observing (40, the 000 times) result of gained of the present invention.The virus-like particle that in the visual field, visible a large amount of radiuses are 9nm left and right, particle actual size conforms to theoretical size, apparent state uniformity.
Fig. 4 is PCV2N9C VLPs virus-like particle transmission electron microscope observing (40, the 000 times) result of gained of the present invention.The virus-like particle that in the visual field, visible a large amount of radiuses are 9nm left and right, particle actual size conforms to theoretical size, apparent state uniformity.
Fig. 5 is PCV2N12C VLPs virus-like particle transmission electron microscope observing (40, the 000 times) result of gained of the present invention.The virus-like particle that in the visual field, visible a large amount of radiuses are 9nm left and right, particle actual size conforms to theoretical size, apparent state uniformity.
Fig. 6 is PCV2N15C VLPs virus-like particle transmission electron microscope observing (40, the 000 times) result of gained of the present invention.The virus-like particle that in the visual field, visible a large amount of radiuses are 9nm left and right, particle actual size conforms to theoretical size, apparent state uniformity.
Fig. 7 is PCV2N18C VLPs virus-like particle transmission electron microscope observing (40, the 000 times) result of gained of the present invention.The virus-like particle that in the visual field, visible a large amount of radiuses are 9nm left and right, particle actual size conforms to theoretical size, apparent state uniformity.
Fig. 8 is PCV2N21C VLPs virus-like particle transmission electron microscope observing (40, the 000 times) result of gained of the present invention.The virus-like particle that in the visual field, visible a large amount of radiuses are 9nm left and right, particle actual size conforms to theoretical size, apparent state uniformity.
Fig. 9 is PCV2N24C VLPs virus-like particle transmission electron microscope observing (40, the 000 times) result of gained of the present invention.The virus-like particle that in the visual field, visible a large amount of radiuses are 9nm left and right, particle actual size conforms to theoretical size, apparent state uniformity.
Figure 10 is PCV2N27C VLPs virus-like particle transmission electron microscope observing (40, the 000 times) result of gained of the present invention.The virus-like particle that in the visual field, visible a large amount of radiuses are 9nm left and right, particle actual size conforms to theoretical size, apparent state uniformity.
Figure 11 is PCV2N3C VLPs, PCV2N6C VLPs, PCV2N9C VLPs, PCV2N12C VLPs, PCV2N15C VLPs, PCV2N18C VLPs, PCV2N21C VLPs, PCV2N24C VLPs, the total antibody titers of different steps serum after PCV2N27C VLPs inoculation rabbit.Just exempting from after three weeks, total anti-titre has obvious rising, and after a booster immunization, the titre of antibody can reach 10
7high level.
Note: PCV2-N3C represents the virus-like particle of 3 expression of amino acids of N end brachymemma; PCV-N6C represents the virus-like particle of 6 expression of amino acids of N end brachymemma; By that analogy, PCV-N9C, PCV-N12C, PCV-N15C, PCV-N18C, PCV-N21C, PCV-N24C, PCV-N27C, represent respectively 9,12,15,18,21,24,27 expression of amino acids of N end brachymemma virus-like particle.
Embodiment
Below in conjunction with specific embodiment, such scheme is described further.Should be understood that these embodiment are not limited to limit the scope of the invention for the present invention is described.The implementation condition adopting in embodiment can be done further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in normal experiment.
Material and reagent: in the present invention, relate to synthetic being synthesized by Shanghai Bo Ya Bioisystech Co., Ltd of gene, pET carrier is purchased from Novagen company, and 50L fermentor tank is purchased from Shanghai Bao Xing biotech firm, and other reagent and medicine are common commercially available prod.
Embodiment 1: the protein expression with the PCV2 ORF2 gene of the N end brachymemma of sequence 1-9
1.1 are used as the preparation of the PCV2 ORF2 gene fragment of template
The PCV2 ORF2 full length gene of optimizing through e. coli codon is synthetic by Shanghai Bo Ya Bioisystech Co., Ltd.The gene fragment total length of synthesized is 702bp.On the basis of the PCV2 of this synthetic ORF2 gene fragment, prepare the template of the PCV2 ORF2 gene of N end of the present invention brachymemma.
The structure of the nonfusion expression vector of the PCV2 ORF2 gene of 1.2N end brachymemma
Be used as the template of PCR reaction with the PCV2 ORF2 full-length gene fragment of previous step synthesized.Respectively with PCV2-N3F, PCV2-N6F, PCV2-N9F, PCV2-N12F, PCV2-N15F, PCV2-N18F, PCV2-N21F, PCV2-N24F and PCV2-N27F are forward primer, taking PCV2-R as reverse primer, amplification obtains the PCV2 ORF2 gene of N end brachymemma.5 ' end of forward primer is introduced restriction enzyme Nde I site and protectiveness base, and wherein Nde I site sequence is CATATG, and ATG is the initiator codon in intestinal bacteria system; 5 ' end of reverse primer is introduced restriction enzyme Xho I site and protectiveness base, and wherein Xho I site sequence is CTCGAG.
| Title | Primer (5 '～3 ') |
| PCV2-N3F: | GGAATTCCATATGCGTCGTCGTTACCGTCGT |
| PCV2-N6F | GGAATTCCATATGTACCGTCGTCGTCGTCAC |
| PCV2-N9F: | GGAATTCCATATGCGTCGTCACCGTCCGCGT |
| PCV2-N12F: | GGAATTCCATATGCGTCCGCGTTCTCACCTG |
| PCV2-N15F: | GGAATTCCATATGTCTCACCTGGGTCAGATC |
| PCV2-N18F: | GGAATTCCATATGGGTCAGATCCTGCGTCGT |
| PCV2-N21F: | GGAATTCCATATGCTGCGTCGTCGTCCGTGG |
| PCV2-N24F: | GGAATTCCATATGCGTCCGTGGCTGCTGCAC |
| PCV2-N27F: | GGAATTCCATATGCTGCTGCACCCGCGTCAC |
| PCV2-R | CCGCTCGAGTTACGGGTTCAGCGGCGGGTC |
Note: PCV2-N3F represents the forward clone primer of 3 amino acid whose gene order Seq No.1 of N end brachymemma; PCV2-N6F represents the forward clone primer of 6 amino acid whose gene order Seq No.2 of N end brachymemma; By that analogy, PCV2-N9F, PCV2-N12F, PCV2-N15F, PCV2-N18F, PCV2-N21F, PCV2-N24F, PCV2-N27F, represent respectively 9,12,15,18,21,24,27 amino acid whose gene order Seq No.3 of N end brachymemma, 4,5,6,7,8,9 forward clone primer.Carry out PCR reaction at PCR instrument according to following condition:
Amplification obtains the special DNA fragmentation of 0.7kb left and right size, and the fragment obtaining after Nde I/Xho I enzyme is cut digestion connects with the pET carrier of cutting through identical enzyme, obtains the positive colony pET-PCV2 ORF2 of the PCV2 ORF2 gene that inserts the brachymemma of N end.The plasmid connecting is transformed into and uses CaCl
2in the DH5a competent cell of preparation, coat on the solid LB substratum of kalamycin resistance, while waiting mono-clonal bacterium colony high-visible, picking mono-clonal is to the LB liquid nutrient medium that contains kantlex, 37 DEG C 230 revs/min, cultivate and spend the night for 12 hours, extract plasmid.
Above-mentioned pET-PCV2 ORF2 plasmid is transformed into the competence e. coli bl21 (DE3) that 40 μ l are prepared with Calcium Chloride Method by 1.3, coat on the solid LB substratum of kalamycin resistance, within 10-12 hour, extremely single bacterium colony is clear and legible in 37 DEG C of standing cultivations.Picking list bacterium colony is to the test tube containing the liquid LB substratum of 4ml kalamycin resistance, and 37 DEG C of 230 revs/min of shaking culture 12 hours, therefrom get 1mL bacterium liquid and preserve in-80 DEG C of freeze-drying.
The great expression of the PCV2 ORF2 albumen of 1.4N end brachymemma
From-80 DEG C, take out the intestinal bacteria bacterial classification with recombinant plasmid pET-PCV2 ORF2, the 50ml LB liquid nutrient medium of access kalamycin resistance, 230rpm, 37 DEG C, cultivate after approximately 12 hours, to transfer in 1L LB liquid nutrient medium, 37 DEG C of great expression, wait OD
600after value reaches 0.6, add the IPTG of 0.1mM, inducible protein is expressed, and 20 DEG C are spent the night.
Proofread and correct 50 liters of fermentor tank pH electrode, prepare 30 liters of substratum and pack fermentor tank into, 121 DEG C of sterilizings 30 minutes, proofread and correct dissolved oxygen electrode, taking after sterilizing before ventilation as zero point, when fermenting after ventilation before inoculation when initial stirring velocity 100rpm as 100%.
Second day by 5 liters of access fermentor tanks of 10 bottles of seed liquor, 30 DEG C of temperature, and pH value 7.0, manual regulation stirring velocity and air flow, maintain dissolved oxygen more than 40%.
Flow feeding, mixes 50% glucose and 30% casein hydrolysate in the ratio of solute mass ratio 2:1.
Flow acceleration is as follows:
100% speed is 25ml/min
First hour: 5% speed;
Second hour: 10% speed;
The 3rd hour: 20% speed;
The 4th hour: 40% speed;
60% speed after the 5th hour.
Be cultured to bacteria concentration and reach OD
600when about 10 left and right, culture temperature is down to 25 DEG C and adds 4gIPTG inducing culture 12 hours.Final concentration is approximately 40 left and right (OD
600) lower tank, the about 2.5kg of centrifugal collection thalline.
LB culture medium prescription following (1 liter): peptone 10g, yeast powder 5g, NaCl10g.
Embodiment 2: the acquisition of the Porcine circovirus type 2 ORF2 protein of the N end brachymemma of purity approximately 70%
Press the resuspended thalline of ratio of the corresponding 10mL lysate of 1g thalline (20mMTris pH of buffer 7.2,300mMNaCl), adopt clarifixator with 700bar pressure breaking thalline 4 times.JA-14 rotary head 13500rpm (28000g), centrifugal 40min, leaves and takes supernatant, detects by 15%SDS-polyacrylamide gel electrophoresis, and now in supernatant, the purity of PCV2ORF2 is about 20%.Adopt ammonium sulfate fractional separation precipitation, just remove part foreign protein by the 30% saturated thiamines precipitation that contains Repone K, then with the 50% thiamines precipitation target protein that contains Repone K.With isopyknic 10mM phosphoric acid buffer pH7.0,10mM DTT, the resuspended precipitation of 300mMNaCl, stirs 30min.JA-14 rotary head (Beckman J25 supercentrifuge), 13500rpm (30000g), centrifugal 20min, the centrifugal supernatant that obtains, uses 0.22 μ m aperture membrane filtration sample, carries out next step chromatogram purification with this sample.Get 20 μ L and filter rear sample, add 5X Loading Buffer4 μ L.Mix, after 100 DEG C of water-bath 10min, get 10 μ L in 10%SDS-polyacrylamide gel with 240V voltage electrophoresis 35min.Show electrophoretic band with Coomassie brilliant blue dyeing subsequently, electrophoresis result is shown in Fig. 1.The PCV2 ORF2 albumen of analyzing the brachymemma of known N end by SDS-PAGE is after the step by precipitation, redissolution, and target protein has obtained purifying and enrichment, and purity has reached approximately 50% left and right.
The chromatogram purification of the PCV2 ORF2 of embodiment 3:N end brachymemma
The cation-exchange chromatography purifying of the PCV2 ORF2 of N end brachymemma
Instrument system: the AKTA purification type liquid chromatographic system that the former Amershan Pharmacia of GE Healthcare company produces.
Chromatography media: Butyl Sepharose4Fast Flow.
Column volume: 5.5cm × 20cm.
Damping fluid: 20mM phosphoric acid buffer pH8.0,0.2M NaCl;
20mM phosphoric acid buffer pH8.0,2M NaCl.
Flow velocity: 25ml/min.
Detector wavelength: 280nm.
Sample is the PCV2 ORF2 protein solution that 3L purity is about the brachymemma of 70%N end.
Elution program is: after penetrating, and 1500mMNaCl wash-out foreign protein, 200mM NaCl wash-out target protein, collects 1000mMNaCl eluted product, obtains altogether the PCV2 ORF2 purification of samples 900mL of N end brachymemma.
The SP purifying of the PCV2 ORF2 of N end brachymemma
Instrument system: the AKTA purification type liquid chromatographic system that the former Amershan Pharmacia of GE Healthcare company produces.
Chromatography media: SP (purchased from Bio-RAD).
Column volume: 5.5cm × 20cm.
Damping fluid: 20mM phosphoric acid buffer pH8.0,0.2M NaCl;
20mM phosphoric acid buffer pH8.0,2M NaCl.
Flow velocity: 20ml/min.
Detector wavelength: 280nm.
Sample is: Butyl Sepharose4Fast Flow200mM NaCl eluted product.
Elution program is: first collect the sample penetrating, and 500mMNaCl wash-out foreign protein, 1000mMNaCl wash-out target protein, collects 1000mMNaCl eluted product, obtains altogether the PCV2 ORF2 purification of samples 800mL of N end brachymemma.
Collect 1000mM NaCl wash-out target protein, obtain the PCV2 ORF2 sample 800mL of the N end brachymemma of purifying.The PCV2 ORF2 sample 20 μ L of the N end brachymemma of the present embodiment method of learning from else's experience purifying, add 5X Loading Buffer5 μ L to mix, after 80 DEG C of water-bath 10min, get 10 μ l in 10%SDS-polyacrylamide gel with 240V voltage electrophoresis 40min.Show electrophoretic band with Coomassie brilliant blue dyeing subsequently, electrophoresis result is shown in Fig. 1.From electrophoresis result, target protein concentration is about 0.7mg/ml, and SDS-PAGE examines and dyes purity and be greater than 96%.
Morphologic detection and the immunogenicity determining thereof of the PCV2 ORF2 VLP of embodiment 4:N end brachymemma
The PCV2 ORF2 virus-like particle transmission electron microscope observing of N end brachymemma
Instrument is the 200kV transmission electron microscope that NEC company produces, and magnification is 40,000 times.The PCV2 virus-like particle of N end brachymemma, through 5% phospho-wolframic acid pH7.0 negative staining, is fixed on the copper mesh that sprays charcoal and observes.Result is respectively as shown in Fig. 2-10, and the visible a large amount of radiuses of embodiment 4 gained sample are the virus-like particle of 9nm left and right, and size evenly, is rendered as hollow form.
The immune protective evaluation of the PCV2 VLPs vaccine immunity animal of embodiment 5 N end brachymemmas
Rabbit: regular grade, female, 6～8 week age.The prepared N of embodiment 2 or 3 holds the PCV2ORF2 virus-like particle of brachymemma just to exempt to mix with equivalent Freund's complete adjuvant, booster immunization mixes and is prepared with equivalent freund 's incomplete adjuvant, immunization ways is intramuscular injection, initial immunity dosage is 100ug/, after this respectively at the 1st, 2 weeks each reinforcements once, booster immunization dosage is 50ug/.After immunity, extract weekly peripheric venous blood, separation of serum, preserves to be checked.
Above-mentioned example is only explanation technical conceive of the present invention and feature, and its object is to allow person skilled in the art can understand content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalent transformations that spirit is done according to the present invention or modification, within all should being encompassed in protection scope of the present invention.
Claims (10)
1. the PCV2 ORF2 gene of a series of N end brachymemma, its nucleotide sequence after optimizing is as shown in Seq ID No.1,2,3,4,5,6,7,8,9.
2. the PCV2 ORF2 gene of brachymemma according to claim 1, wherein the albumen of this genetic expression can be self-assembled into virus-like particle in intestinal bacteria.
3. the PCV2 ORF2 capsid protein of nine kinds of brachymemmas, has the sequence as shown in Seq ID No.10,11,12,13,14,15,16,17,18.
4. contain the expression vector of gene described in claim 1.
5. expression vector according to claim 4, described expression vector is the carrier being suitable at expression in escherichia coli.
6. contain the cell of claim 4 expression vector.
7. a method of preparing the capsid protein of purifying brachymemma PCV2 ORF2 genetic expression, comprises the steps:
(1) the PCV2-ORF2 gene of soluble-expression brachymemma in escherichia expression system,
(2) by having expressed the intestinal bacteria fragmentation of PCV2 ORF2 capsid protein of brachymemma, separate and obtain supernatant liquor,
(3) by the ammonium sulfate fractional separation that contains Repone K, in conjunction with chromatography purifying target protein.
8. nine kinds of PCV2 ORF2 viruslike particles through brachymemma, the albumen that wherein this viruslike particle contains claim 1 or 2 genetic expressions or the albumen that comprises claim 3.
9. the vaccine for preventing PCV2 to infect, it comprises: the PCV2ORF2 virus-like particle of (1) claim 8, and vehicle or carrier for vaccine.
10. a method of preventing PCV2 to infect, it comprises the pig that the vaccine of the claim 9 containing the prevention PCV2 ORF2 capsid protein of claim 1-2 any one of significant quantity or the vaccine of the virus-like particle of claim 8 or prevention significant quantity is given to prevent PCV2 infection.
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