CN104404074B - The preparation method of hoof-and-mouth disease virus capsid protein series connection coexpression and virus-like particle - Google Patents
The preparation method of hoof-and-mouth disease virus capsid protein series connection coexpression and virus-like particle Download PDFInfo
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Abstract
The present invention relates to Escherichia coli using the solvable coexpression hoof-and-mouth disease virus capsid protein VP0 (its Gene Fusion as VP4 and VP2) of single plasmid series connection, VP1 and VP3, and the preparation method of hoof-and-mouth disease virus capsid protein virus-like particle.Hoof-and-mouth disease virus capsid protein virus-like particle can be used for the preparation of aftosa vaccine.Method provided by the invention, studied from many aspects coexpression hoof-and-mouth disease virus capsid protein solvable to Escherichia coli, integrated use series connection coexpression and the solvable coexpression hoof-and-mouth disease virus capsid protein VP0 of technology with SUMO labels (it is VP4 and VP2 Gene Fusion), VP1 and VP3, the destination protein finally obtained accounts for about the 20% of bacterial protein, purifies the hoof-and-mouth disease virus capsid protein success assembling assembly virus-like particle of acquisition.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of foot and mouth disease virus capsid protein gene and load
Body, bacterial strain and its expression, and the vaccine containing the virus-like particle and its purposes in prevention infection of foot-and-mouth disease.
Background technology
Aftosa is that illness, the mutation of indivedual foot and mouth disease viruses can pass the artiodactyl as caused by mouth disease virus infection altogether
Dye gives people.Aftosa Symptoms are between coronet, toe, bubble and speck, Some Animals mucous membrane of mouth and nose occur for heel skin
Disk also has same lesion.The infectiousness of aftosa is extremely strong, propagates rapidly, infection and the incidence of disease are very high, can cause mortality, make
Into serious economic loss, a kind of deadly infectious disease is divided into the world.In national capitals such as 50-60 last century, English age, methods
Once large-scale aftosa epidemic situation was broken out.Also there is repeatedly extensive prevalence in China, and turned into the " No.1 to kill of animal husbandry
Hand ".
Foot and mouth disease virus (Foot and Mouth Disease Virus, FMDV) belongs to Picornaviridae, hoof-and-mouth disease
Poison category, the virus have seven serotypes (O, A, C, SAT1 (South Africa 1), SAT2 (South Africa 2), SAT3 (South Africa 3) and Asia1 (Asia
1 type), 65 hypotypes, reacted between various without cross protection, having infected the animal of the aftosa of a serotype can still infect separately
The foot and mouth disease virus of one serotype.FMDV geneome RNAs total length about 8.5kb, it is followed successively by 5 ' UTR, ORF and 3 ' UTR compositions.It is interim
ORF about 6.5kb grow, by L genes, P1 structural protein genes, P2 and P3 nonstructural protein genes and its beginning codon and termination
Codon forms.Foot and mouth disease virus virus coat is symmetrical 20 face body, is made up of capsid protein VP1, VP2, VP3 and VP4.This
A little capsid proteins determine antigenicity, immunity and the serological reaction ability of virus.
The developed country such as Great Britain and America has all eliminated aftosa comprehensively at present.In China, aftosa vaccine is listed in pressure and exempted from
Epidemic disease kind, perform strict vaccine quality standard.Mainly there are three kinds of foot and mouth disease virus hypotypes popular in China：Asia I type, A types,
With it is O-shaped.
At present, prevailing in Chinese market is for O-shaped and traditional inactivated vaccine of Asia I type.Inactivated vaccine etc.
Traditional vaccine has good immunogenicity, is played an important role during preventing and controlling aftosa.But due to going out
Live vaccine exist virus virulence return strong, inactivation of virus not thoroughly, the unsafe factor such as live virus escape, it is remaining in inactivated vaccine
Live virus is possible to that spreading unchecked for foot and mouth disease virus can be caused.
Preferable aftosa vaccine must should safely, effectively while also possess the advantages that inexpensive, easy to spread.Aftosa
For inactivated vaccine in addition to it potential insecurity be present and influence its use, the high cost for preparing of inactivated vaccine limits the vaccine
Promote.Therefore development and production protectiveness and all good foot-and-mouth disease gene engineering vaccine of security, while can prepare as needed
Multivalent virus vaccine, significantly saving production cost turns into the Main way of aftosa vaccine research and development.
The recombinant vaccine based on virus-like particle based on technique for gene engineering Successful utilization in the modern times
The research and development and production of vaccine.But recombinant vaccine generally use eukaryotic expression system.Production cost is higher, is not suitable for big rule
Mould is promoted.
Escherichia expression system is one of gene engineering expression system being most widely used at present.Due to the expression system
System, which has, to be easy to cultivate, it is not necessary to complex device, safe distinguishing feature, therefore it is widely used in bio-pharmaceuticals industry
In.Glycosylation modified is one of main distinction of eukaryotic expression system and prokaryotic expression system, but does not have report to mention a mouthful hoof
The capsid protein of epidemic disease poison, including VP1, VP2, VP3 and VP4, containing any glycosylation site, therefore with two kinds of system expressions
Capsid protein is not had any different at glycosylation modified aspect.The shell of foot and mouth disease virus is by 4 regular group of capsid proteins
The 20 face bodies dressed up.Substantial amounts of research shows, the single expression foot and mouth disease virus coat protein or with not homogeneity in Escherichia coli
The genetic engineering bacterium that grain cotransfection Escherichia coli obtain, the foot and mouth disease virus outer casing protein expression amount of expression are very low and usually
It is insoluble, lose the possibility of large-scale commercial applications application.
In summary, the preventing and treating for aftosa needs, and a kind of with short production cycle there is an urgent need to establish, production cost is low,
The solvable coexpression of hoof-and-mouth disease virus capsid protein series connection of the high Escherichia coli of protein yield and the preparation of virus-like particle
Method, the present invention is therefore.
The content of the invention
The technical problem of first aspect to be solved by this invention, it is to overcome foot and mouth disease virus capsid egg in the prior art
A kind of very low and insoluble situation of expression quantity in Escherichia coli in vain, there is provided solvable table altogether of being connected in Escherichia coli
Up to hoof-and-mouth disease virus capsid protein VP3, VP1 and VP0 (VP4 and VP2 Gene Fusion), the destination protein finally obtained accounts for bacterium
The 20% of the solvable total protein of body.
In order to solve above-mentioned technical problem, the present invention provides a kind of solvable coexpression aftosa capsid protein VP3 that connects,
VP1 and VP0 (VP4 and VP2 Gene Fusion) and virus-like particle process for assembly preparing, comprise the following steps.
(1) the hoof-and-mouth disease virus capsid protein VP0 genes (VP4 and VP2 Gene Fusion) for optimizing codon,
VP1 and VP3 full-length gene is cloned into plasmid pETSUMO respectively, obtains pETSUMO-VP0 respectively, pETSUMO-VP1 and
Tri- plasmids of pETSUMO-VP3, hoof-and-mouth disease virus capsid protein VP0 nucleotide sequences are as shown in Seq ID No.1a, aftosa
Viral capsid proteins VP1 nucleotide sequences are as shown in Seq ID No.1b, hoof-and-mouth disease virus capsid protein VP3 nucleotide sequence
As shown in Seq ID No.1c；
(2) expanded respectively from tri- plasmids of pETSUMO-VP3, pETSUMO-VP1 and pETSUMO-VP0 with PCR method
Go out RBS-SUMO-VP3, RBS-SUMO-VP1 and RBS-SUMO-VP0 DNA fragmentations, be cloned into certain sequence same
PET28b plasmids, obtain recombinant expression plasmid pET-TRI-Asia1-VP310, and wherein RBS refers to ribosome bind site；
(3) the recombinant expression plasmid pET-TRI-Asia1-VP310 is converted into Escherichia coli, obtains genetic engineering bacterium；
(4) genetic engineering bacterium carries out fermented and cultured, solvable mouth of the coexpression with SUMO labels of IPTG induction series connection
Aphtovirus capsid protein VP3, VP1 and VP0 (VP4 and VP2 Gene Fusion)；
(5) supernatant after genetic engineering bacterium bacterial cell disruption is reclaimed, chromatography isolates and purifies the mouth with SUMO labels
Aphtovirus capsid protein VP3, VP1 and VP0 mixture, hoof-and-mouth disease virus capsid protein VP0 amino acid sequence such as Seq ID
Shown in No.2a, hoof-and-mouth disease virus capsid protein VP1 amino acid sequence is as shown in Seq ID No.2b, foot and mouth disease virus capsid
Albumen VP3 amino acid sequence is as shown in Seq ID No.2c；
(6) after digestion removes SUMO labels, the hoof-and-mouth disease virus capsid protein of molecular sieve chromatography chromatography purifying acquisition
VP3, VP1 and VP0 assembling assembly virus-like particle.
Another aspect of the present invention, there is provided one kind can containing claim 1 step (2) the recombinant expression plasmid series connection
Molten coexpression hoof-and-mouth disease virus capsid protein VP0, VP1 and VP3.
Another aspect of the present invention, there is provided one kind is BL21 containing claim 1 step (3) described genetic engineering bacterium
(DE3)/pET-TRI-Asia1-VP310。
Another aspect of the present invention, there is provided one kind is BL21 containing claim 1 step (3) described Escherichia coli
(DE3)。
Another aspect of the present invention, there is provided one kind is solvable containing claim 1 step (4) the genetic engineering bacterium series connection
Hoof-and-mouth disease virus capsid protein VP3, VP1 and VP0 (VP4 and VP2 Gene Fusion) of the coexpression with SUMO labels.
Another aspect of the present invention, there is provided one kind is band containing the albumen isolated and purified claim 1 step (5) described
There are hoof-and-mouth disease the virus capsid protein VP3, VP1 and VP0 (VP4 and VP2 Gene Fusion) of SUMO labels mixture.
Another aspect of the present invention, there is provided one kind removes containing the digestion isolated and purified claim 1 step (6) described
Hoof-and-mouth disease the virus capsid protein VP3, VP1 and VP0 (VP4 and VP2 Gene Fusion) of SUMO labels mixture.
Another aspect of the invention is the hoof-and-mouth disease virus capsid protein (VP0 (VP4 prepared using the method for the invention
With VP2 Gene Fusion), VP1 and VP3) composition virus-like particle.
Another aspect of the invention is the vaccine prepared using the method for the invention for preventing infection of foot-and-mouth disease, and it is wrapped
Include：It is foregoing to assemble obtained foot and mouth disease virus sample particle, and vaccine excipient or carrier.Another aspect of the invention is use
The genetic engineering bacterium of the method for the invention structure, its feature is, the genetic engineering bacterium is connected solvable coexpression aftosa
Viral capsid proteins VP3 genes, VP1 genes and VP0 genes (for VP4 and VP2 Gene Fusion)；VP0 nucleotide sequences are such as
Shown in SeqID No.1a；VP1 gene nucleotide series are as shown in SeqID No.1b；VP3 gene nucleotide series such as SeqID
Shown in No.1c.
The present invention provides a kind of solvable coexpression of the series connection based on Escherichia coli in Chinese popular foot and mouth disease virus
Capsid protein high efficiency method.Technology based on this laboratory, from many aspects to the solvable coexpression aftosa of Escherichia coli
Viral capsid proteins are studied, integrated use series connection coexpression and the solvable coexpression hoof-and-mouth disease of technology with SUMO labels
Virus capsid protein VP3, VP1, VP0 (VP4 and VP2 Gene Fusion), the destination protein finally obtained accounts for bacterial protein
About 20%.Then hoof-and-mouth disease virus capsid protein VP3, VP1 and VP0 with SUMO labels are obtained by chromatography separation,
After digestion removes SUMO labels, hoof-and-mouth disease virus capsid protein VP3, VP1 and VP0 that chromatography isolates and purifies acquisition are assembled into
Virus-like particle.
Chromatography separation method mild condition, do not obtained using denaturant, the activity of destination protein such as urea
Maximum protection, is not damaged to the structure of protein, it ensure that the aftosa capsid protein that purifying obtains is protected
Natural activity has been held, natural binding activity is at utmost maintained between each aftosa capsid protein.Moreover, in chromatogram purification
During, whole technological process is simple, without carrying out the steps such as buffer exchange, it is not necessary to using complicated technologies such as ultracentrifugations,
And the recovery of two step chromatograms is close to 30% so that with industrially scalable solubility expression purify hoof-and-mouth disease virus capsid protein into
To be possible, and in whole technical process, denaturant is not directed to, purge process and assembling process will not be to the structures of albumen
Neutralizing epitope as having an impact and destroying virus-like particle, the virus-like particle so produced have the immunogenicity of height,
The neutralizing antibody for producing high titre can be induced after immune body, good protectiveness effect is played to immune animal.
The technical scheme is that the solvable coexpression hoof-and-mouth disease virus capsid protein VP3 that connected in Escherichia coli,
VP1, VP0 (VP4 and VP2 Gene Fusion), and on this basis, establish a set of aftosa vaccine efficiently, suitably amplified
Scale up test technique.A set of aftosa vaccine quality research system is established, and by zoopery to Escherichia coli
The validity of aftosa vaccine, stability are evaluated.
The explanation of relational language and explanation in the present invention
According to the present invention, term " escherichia expression system " refers to be made up of with carrier Escherichia coli (bacterial strain), wherein
Escherichia coli (bacterial strain) are illustrated but are not limited to from available on the market herein：BL21 (DE3), B834 (DE3), BLR
(DE3), JM109, XL1Blue, ER2566, Rosetta, GI698.It is preferred that BL21 (DE3).
According to the present invention, the word of term " carrier " one refers to and can be inserted the polynucleotide of certain encoding proteins simultaneously
Albumen is set to obtain a kind of nucleic acid delivery vehicle of expression.Carrier can take it by conversion, transduction or transfection host cell
The inhereditary material element of band is expressed in host cell.For example, carrier includes：Plasmid；Bacteriophage；Coemid etc.
Deng.
According to the present invention, term " series connection coexpression " word refers to multiple genes to be inserted into same carrier table altogether
Reach.Coexpression of connecting order includes but is not limited to VP3, the various possibility between VP1, VP0 (for VP4 and VP2 Gene Fusion)
Various possible combinations between combination and VP1, VP2, VP3, VP4, such as can be VP1-VP3-VP0 series sequence,
VP3-VP0-VP1 series sequence, VP1-VP0-VP3 series sequence, VP3-VP1-VP2-VP4 series sequence, VP1-
The series sequence of VP2-VP3-VP4 series sequence etc., preferably VP3-VP1-VP0.
According to the present invention, term " vaccine excipient or carrier " refers to, selected from one or more, include but is not limited to：pH
Conditioning agent, surfactant, adjuvant, ionic strength reinforcing agent.For example, pH adjusting agent citing but it is not limited to phosphate buffer,
Surfactant includes cation, anion or nonionic surface active agent.Illustrate but be not limited to:Tween-80.Adjuvant
Illustrate but be not limited to aluminium hydroxide, Fu Shi Freund's complete adjuvants.Ionic strength reinforcing agent is illustrated but is not limited to sodium chloride.
According to the present invention, term " chromatography " includes but is not limited to：Ion-exchange chromatography (such as cation exchange color
Spectrum), hydrophobic interaction chromatograph, adsorption chromatography (such as hydroxylapatite chromatography), gel filtration (gel exclusion) chromatography,
Affinity chromatography, molecular sieve chromatography.
According to the present invention, in hoof-and-mouth disease virus capsid protein VP3, VP1, VP0 (VP4 and the VP2 gene that the present invention obtains
Fusion) method in, buffer solution refers to the solution that can maintain pH stable within the specific limits, include but is not limited to, Tris delays
Fliud flushing, phosphate buffer, HEPES buffer solution, MOPS buffer solutions etc..
According to the present invention, the prokaryotic host cell is broken include but is not limited to by homogenizer is broken, homogeneous crusher machine,
One or more method in ultrasonication, grinding, high-pressure extrusion, bacteriolyze ferment treatment is realized.
According to the present invention, in hoof-and-mouth disease virus capsid protein VP3, VP1, VP0 (VP4 and the VP2 gene that the present invention obtains
Fusion) albumen method in, salt used includes but is not limited to be neutral salt, particularly alkali metal salt, ammonium salt, hydrochloride, sulphur
Hydrochlorate, sulfate, bicarbonate, phosphate or hydrophosphate, particularly NaCl, KCl, NH4Cl、(NH4)2SO4In one kind or
It is several.It is preferred that NaCl.
According to the present invention, hoof-and-mouth disease virus capsid protein virus-like particle is obtained as below：Digestion is removed into SUMO labels
Hoof-and-mouth disease virus capsid protein VP3, VP1, VP0 (VP4 and VP2 Gene Fusion) protein solution enter for example, by chromatography
One step separates, aftosa the capsid protein VP3, VP1, VP0 (VP4 and VP2 Gene Fusion) that are purified protein solution.Mouthful
The mode of aphtovirus capsid protein virus-like particle assembling includes but is not limited to techniques known in the art, for example, dialysis, ultrafiltration
Or chromatography etc..
According to the present invention, (VP4 and VP2 gene melt hoof-and-mouth disease the virus capsid protein VP3, VP1, VP0 that the present invention obtains
Close) include from the highly similar different hoof-and-mouth disease serotypes of protein sequence, hypotype, the capsid protein VP0 of mutant strain
(for VP4 and VP2 Gene Fusion), VP1 and VP3, and the protein fragments of these albumen.
Brief description of the drawings
The invention will be further described below in conjunction with the accompanying drawings：
Fig. 1 is aftosa capsid protein VP3, VP1 and VP0 (VP4 and the VP2 gene of the invention with SUMO tag expressions
Fusion) SDS-PAGE results.M:Molecular weight Marker；1：Full bacterium before induction；2：The supernatant after full bacterium is cracked after induction；As a result
It has been shown that, hoof-and-mouth disease virus capsid protein solvable coexpression in a manner of band SUMO labels in Escherichia coli, and destination protein is about
Account for 20% or so of the solvable total protein of thalline.
Fig. 2 is that the hoof-and-mouth disease virus capsid protein with SUMO labels passes through the SDS-PAGE results of affinity chromatography after purification.
M:Molecular weight Marker；1, purify hoof-and-mouth disease virus capsid protein loading 2 μ l of the gained with SUMO labels through the present invention；2, through this
Hoof-and-mouth disease virus capsid protein loading 3 μ l of the invention purifying gained with SUMO labels.As a result show, purified band SUMO marks
The aftosa capsid protein loading purity of label has reached more than 70%.
Fig. 3 is the aftosa capsid protein with SUMO labels, and molecular sieve chromatography purifies after digestion removes SUMO labels
SDS-PAGE results.1, purify the μ l of gained hoof-and-mouth disease virus capsid protein loading 1 through the present invention；2, purify gained mouth through the present invention
The μ l of aphtovirus capsid protein loading 2.As a result show, purified hoof-and-mouth disease virus capsid protein purity reached 90% with
On.
Fig. 4 is the hoof-and-mouth disease virus capsid protein virus-like particle transmission electron microscope observing result of present invention gained.In the visual field
It can be seen that the virus-like particle that a large amount of uniform radius are 15nm or so, particle actual size are consistent with theoretical size, apparent state is equal
It is even consistent.
Fig. 5 is the dynamic light scattering observed result of the hoof-and-mouth disease virus capsid protein virus-like particle of present invention gained.Knot
Fruit shows that the hydrated molecule kinetics radius of hoof-and-mouth disease virus capsid protein virus-like particle are 23.37nm, and particle assembles percentage
Than being about 100%.
Fig. 6 is total for different phase serum after the hoof-and-mouth disease virus capsid protein virus-like particle Mice Inoculated of present invention gained
Antibody titer.Arrow show immunization time in figure.After just January is exempted from, total anti-titre has obvious rising, by once
After booster immunization, the titre of antibody can reach 107High level.
Sequence table explanation：
Seq ID No.1a are the nucleotides of aftosa capsid protein VP0 genes (VP4 and VP2 Gene Fusion) of the present invention
Sequence.
Seq ID No.1b are the nucleotide sequence of aftosa capsid protein VP1 genes of the present invention.
Seq ID No.1c are the nucleotide sequence of aftosa capsid protein VP3 genes of the present invention.
Seq ID No.2a are ammonia corresponding to aftosa capsid protein VP0 genes (VP4 and VP2 Gene Fusion) of the present invention
Base acid sequence.
Seq ID No.2b are amino acid sequence corresponding to aftosa capsid protein VP1 genes of the present invention.
Seq ID No.2c are amino acid sequence corresponding to aftosa capsid protein VP3 genes of the present invention.
Embodiment
Such scheme is described further below in conjunction with specific embodiment.It should be understood that these embodiments are to be used to illustrate
The present invention and be not limited to limit the scope of the present invention.The implementation condition used in embodiment can be done according to the condition of specific producer
Further adjustment, unreceipted implementation condition is usually the condition in normal experiment.
Material and reagent：It is related to being synthesized by Shanghai Bo Ya Bioisystech Co., Ltd for gene chemical synthesis in the present invention,
PETSUMO plasmids are purchased from Invitrogen companies, and 50L fermentation tanks are purchased from Shanghai Bao Xing biotech firms, and other reagents and medicine are
Ordinary commercial products.PET28b plasmids are purchased from Novagen companies, and e. coli bl21 (DE3) is purchased from New England
Biolabs。
Embodiment 1：Hoof-and-mouth disease virus capsid protein VP3, VP1, VP0 with sequence 1 (1a, 1b, 1c) be (VP4 and VP2's
Gene Fusion) the solvable coexpression of series connection
1.1 are used as the preparation of hoof-and-mouth disease virus capsid protein VP3, VP1, the VP0 genetic fragment of template
Foot and mouth disease virus capsid protein gene total length (VP3, VP1, VP0) by e. coli codon optimization is by Suzhou
Jin Wei intelligence bio tech ltd synthesizes.Synthesized genetic fragment total length is respectively 909 bp (VP0), 657 bp (VP3),
627 bp(VP1).Herein aftosa of the present invention is prepared on the basis of artificial synthesized foot and mouth disease virus capsid protein gene fragment
The template of viral capsid proteins.
The structure of 1.2 band SUMO label foot and mouth disease virus capsid protein genes (VP3, VP1, VP0) series connection coexpression vectors
Distinguished with foot and mouth disease virus capsid protein gene total length (VP3, VP1, VP0) genetic fragment synthesized by previous step
It is used as the template of PCR reactions.Using Asia1-VP0F as forward primer, using Asia1-VP0R as reverse primer, using VP0 as PCR moulds
Plate amplification obtains hoof-and-mouth disease virus capsid protein VP0 genes；It is reverse using Asia1-VP1R using Asia1-VP1F as forward primer
Primer, expanded using VP1 as pcr template and obtain hoof-and-mouth disease virus capsid protein VP1 genes；Using Asia1-VP3F as forward primer,
Using Asia1-VP3R as reverse primer, expanded using VP3 as pcr template and obtain hoof-and-mouth disease virus capsid protein VP3 genes.Forward direction is drawn
5 ' ends of thing introduce restriction enzyme BamH I sites and protectiveness base, and wherein BamHI site sequences are GGATCC；Reversely
5 ' ends of primer introduce restriction enzyme Not I sites, two terminator codons and protectiveness base, wherein Not I sites
Sequence is GCGGCCGC.Enter performing PCR reaction according to following condition in PCR instrument：
Obtained DNA fragmentation is expanded, the fragment obtained after BamH I/Not I digestions, digestion identical with process
PETSUMO carriers connect, and respectively obtain the positive colony pETSUMO-VP3 of insertion foot and mouth disease virus capsid protein gene,
PETSUMO-VP1 and pETSUMO-VP0.The plasmid connected is transformed into the DH5a competent cells prepared with Calcium Chloride Method
In, when waiting monoclonal bacterium colony high-visible, in picking monoclonal to the LB fluid nutrient mediums containing kanamycins, 37 DEG C 230 turns/
Minute, cultivate 12 hours overnight, extract plasmid.
With Infusion-F：5 '-CAATTCCCCTCTAGAAATA-3 ' are forward primer and Infusion-R：5’-
CAGCCAACTCAGCTTCCTT-3 ' is reverse primer, is made respectively with pETSUMO-VP3, pETSUMO-VP1 and pETSUMO-VP0
For pcr template, amplification obtains the aftosa for the SUMO labels with ribosome bind site for being suitable for In-fusion clone technologies
Viral capsid proteins RBS-SUMO-VP3, RBS-SUMO-VP1 and RBS-SUMO-VP0 DNA fragmentation.By above-mentioned fragment with In-
Fusion clone technologies, same pET28b carriers are cloned into order, obtain positive colony pET-TRI-Asia1-VP310.
The plasmid connected is transformed into the DH5a competent cells prepared with Calcium Chloride Method, when waiting monoclonal bacterium colony high-visible,
In picking monoclonal to the LB fluid nutrient mediums containing kanamycins, 37 DEG C 230 revs/min, cultivate 12 hours overnight, extract matter
Grain.
Above-mentioned pET-TRI-Asia1-VP310 plasmids are transformed into the competence Escherichia coli that 40 μ l are prepared with Calcium Chloride Method
BL21 (DE3), the solid LB media of kalamycin resistance is coated on, 37 DEG C of quiescent culture 10-12 hours are clear to single bacterium colony
It is distinguishable.Picking single bacterium drops down onto the test tube of the LB liquid medium of the kalamycin resistance containing 4ml, 37 DEG C of 230 revs/min of shaken cultivations 12
Hour, 1mL bacterium solutions are therefrom taken in -80 DEG C of lyophilized preservations.
The series connection coexpression of 1.3 hoof-and-mouth disease virus capsid proteins (VP3, VP1, VP0)
The strain Escherichia coli with recombinant plasmid pET-TRI-Asia1-VP310 is taken out from -80 DEG C, that is mould for access card
The 50ml LB fluid nutrient mediums of plain resistance, 250rpm, 37 DEG C, after cultivating about 12 hours, in 1L LB fluid nutrient mediums of transferring,
37 DEG C of great expressions, wait OD600After value reaches 0.8,0.4mM IPTG is added, inducible protein expression, 20 DEG C overnight.
Key instrument, 50 liters of fermentation tanks of Shanghai Bao Xing biotech firms
Fermentation tank pH electrodes are corrected, 30 liters of culture mediums is prepared and loads fermentation tank, 121 DEG C sterilize 30 minutes, correction dissolved oxygen electricity
Pole, with before not ventilated after sterilizing for zero point, as 100% during initial mixing speed 100rpm before not being inoculated with after ventilation during fermenting.
Second day by 10 bottles of seed liquors, 5 liters of access fermentation tanks, 30 DEG C of temperature, pH value 7.0, adjust manually mixing speed and
Throughput, dissolved oxygen is maintained more than 40%.
Flow feeding, 50% glucose and 30% casein hydrolysate are pressed into Solute mass ratio 2：1 ratio mixing.
Flow acceleration is as follows：
100% speed is 25ml/min
First hour：5% speed；
Second hour：10% speed；
3rd hour：20% speed；
4th hour：40% speed；
60% speed after 5th hour.
Culture to bacteria concentration reaches OD600Cultivation temperature is down to 25 DEG C of addition 16gIPTG Fiber differentiations when about 10 or so
12 hours.Final concentration is about 45 or so (OD600) under tank, thalline about 3.0kg is collected by centrifugation.
LB culture medium prescriptions are following (1 liter)：Peptone 10g, dusty yeast 5g, NaCl 10g.
Embodiment 2：It is prepared by the affinity chromatography purifying of the hoof-and-mouth disease virus capsid protein (VP3, VP1, VP0) with SUMO labels
Bacterium is resuspended in the ratio that 10mL lysates (20mM Tris pH of buffer 7.2,300mM NaCl) are corresponded to by 1g thalline
Body, homogenizer is used with 700bar pressure breakings thalline 4 times.JA-14 rotary heads 13500rpm (28000g), 35min is centrifuged, is left and taken
Supernatant, detected by 15%SDS- polyacrylamide gel electrophoresises, now the foot and mouth disease virus capsid with SUMO labels in supernatant
The purity of albumen (VP3, VP1, VP0) is about 20%.Supernatant is with after 0.45 μm of aperture membrane filtration, with HisTrap FF pillars
(GE Healthcare Life Sciences) does chromatogram purification.
Instrument system：The AKTA purifying preparative liquids of GE Healthcare original Amershan Pharmacia companies production
Phase chromatographic system.
Chromatography media：Ni Sepharose.
Column volume：5ml.
Buffer solution：20mM phosphate buffers pH8.0,0.2M NaCl；
Eluent：20mM phosphate buffers pH8.0,0.2M NaCl+300mM imidazoles.
Flow velocity：5 ml/min.
Detector wavelength：280nm.
Sample is coli somatic supernatants through be homogenized crusher machine after of the 3L after 0.45 μm of aperture membrane filtration.
Elution program is：After penetrating, buffer solution elution foreign protein, foot and mouth disease virus clothing of the elution with SUMO labels
Glutelin (VP3, VP1, VP0) product, hoof-and-mouth disease virus capsid protein (VP3, VP1, VP0) sample with SUMO labels is obtained altogether
900mL。
The μ l of sample 20 of the present embodiment method of learning from else's experience purifying, add the μ l of 5 × Loading Buffer 5 and mix, in 80 DEG C of water
Take 1 μ l and 2 μ l in 10%SDS- polyacrylamide gels with 240V electrophoresis 40min after bath 10min.Then with coomassie
Bright blue dyeing shows electrophoretic band, and electrophoresis result is shown in Fig. 2.From electrophoresis result, destination protein concentration is about 2mg/ml, SDS-
It is about 70% that PAGE, which examines dye purity,.
Embodiment 3：Remove the molecular sieve chromatography purifying of the hoof-and-mouth disease virus capsid protein (VP3, VP1, VP0) of SUMO labels
Collect embodiment 2 and obtain hoof-and-mouth disease virus capsid protein (VP3, VP1, the VP0) sample of acquisition with SUMO labels altogether
The molecular sieve chromatography purifying of next step is carried out after 12 hours in 4 ° of digestions.
Instrument system：The AKTA purifying preparative liquids of GE Healthcare original Amershan Pharmacia companies production
Phase chromatographic system.
Chromatography media：HiLoad 26/60 Superdex 200.
Column volume：2.6 × 60 cm, 318 ml.
Eluent：20mM phosphate buffers pH8.0,0.2M NaCl.
Flow velocity：5 ml/min.
Detector wavelength：280nm.
Sample is hoof-and-mouth disease virus capsid protein (VP3, VP1, VP0) protein solution that 900mL removes SUMO labels.
Elution program is：After penetrating, 500mL elution products, obtain altogether hoof-and-mouth disease virus capsid protein (VP3,
VP1, VP0) purification of samples about 50mL.
The μ l of aftosa capsid protein (VP3, VP1, VP0) sample 20 of the present embodiment method of learning from else's experience purifying, addition 5 ×
The μ l of Loading Buffer 5 are mixed, and take 1 μ l and 2 μ l to be coagulated in 10%SDS- polyacrylamides respectively after 80 DEG C of water-bath 10min
With 240V electrophoresis 40min in glue.Then dyed with Coomassie brilliant blue and show electrophoretic band, electrophoresis result is shown in Fig. 3.By electrophoresis
As a result understand, destination protein concentration is about 1.5mg/ml, and SDS-PAGE examines dye purity and is more than 90%.
Embodiment 4：The morphologic detection and its immunogenicity determining mouth hoof of hoof-and-mouth disease virus capsid protein virus-like particle
Epidemic disease virus capsid protein virus-like particle transmission electron microscope observing
Instrument is FEI transmission electron microscopes, 42,000 times of multiplication factor.Hoof-and-mouth disease virus capsid protein virus-like particle is with soon
Quickly cooling freeze techniques, which are fixed on the copper mesh of spray charcoal, to be observed.As a result such as Fig. 4, it is seen that a large amount of radiuses of gained sample of embodiment 4 are about
It is uniform in size for 15 nm or so virus-like particle, it is rendered as hollow form.
Hoof-and-mouth disease virus capsid protein virion dynamic light scattering is observed
Instrument is the DynaPro MS/X types dynamic light scattering of Protein Solutions companies of U.S. production (containing temperature
Spend controller), the use of algorithm is Regulation algorithms.Sample is the gained sample of embodiment 3.Sample is through 0.22 μm of membrane filtration
After measure.Measurement result is shown in Fig. 5.As a result the hydrated molecule dynamics half of hoof-and-mouth disease virus capsid protein virion is shown
Footpath is 23.37nm.
The immune protective evaluation of animal is immunized in the hoof-and-mouth disease virus capsid protein virus sample particle vaccines of embodiment 5
Mouse：Regular grade, female, 6~8 week old.Hoof-and-mouth disease virus capsid protein virus-like particle prepared by embodiment 3
Just exempt to mix with equivalent Freund's complete adjuvant, booster immunization is then mixed with equivalent freund 's incomplete adjuvant and prepared, and side is immunized
Formula is intramuscular injection, and initial immunity dosage is 100ug/, and hereafter each respectively at 4,10 weeks to strengthen once, booster immunization dosage is
50ug/ is only.From after immune, peripheric venous blood is extracted weekly, separates serum, is preserved to be checked.Measurement result is shown in Fig. 6.
The foregoing examples are merely illustrative of the technical concept and features of the invention, its object is to allow the person skilled in the art to be
Present disclosure can be understood and implemented according to this, it is not intended to limit the scope of the present invention.It is all smart according to the present invention
The equivalent transformation or modification that refreshing essence is done, should all be included within the scope of the present invention.
Claims (6)
- The preparation method of solvable coexpression 1. hoof-and-mouth disease virus capsid protein is connected, it is characterised in that comprise the following steps：(1) the hoof-and-mouth disease virus capsid protein VP0 genes for optimizing codon, VP1 and VP3 full-length gene are cloned respectively Enter plasmid pETSUMO, obtain tri- plasmids of pETSUMO-VP0, pETSUMO-VP1 and pETSUMO-VP3, wherein VP0 bases respectively Because VP4 and VP2 Gene Fusion forms, its nucleotide sequence is as shown in Seq ID No.1a, the nucleotide sequence of VP1 genes As shown in Seq ID No.1b, the nucleotide sequence of VP3 genes is as shown in Seq ID No.1c；(2) amplified respectively from tri- plasmids of pETSUMO-VP3, pETSUMO-VP1 and pETSUMO-VP0 with PCR method RBS-SUMO-VP3, RBS-SUMO-VP1 and RBS-SUMO-VP0DNA fragment, it is cloned into by VP3, VP1 and VP0 order same PET28b plasmids, obtain recombinant expression plasmid pET-TRI-Asia1-VP310, and wherein RBS refers to ribosome bind site；(3) the recombinant expression plasmid pET-TRI-Asia1-VP310 is converted into Escherichia coli, obtains genetic engineering bacterium；(4) genetic engineering bacterium carries out fermented and cultured, solvable aftosa of the coexpression with SUMO labels of IPTG induction series connection Viral capsid proteins VP3, VP1 and VP0；(5) supernatant after genetic engineering bacterium bacterial cell disruption is reclaimed, affinity chromatography isolates and purifies the mouth with SUMO labels Aphtovirus capsid protein VP3, VP1 and VP0 protein mixture；(6) after digestion removes SUMO labels, the purifying of molecular sieve chromatography chromatography obtains hoof-and-mouth disease virus capsid protein VP3, VP1 With VP0 protein mixtures, and assembling assembly virus-like particle.
- 2. according to the method for claim 1, it is characterised in that step (3) described Escherichia coli are BL21 (DE3).
- 3. according to the method for claim 1, it is characterised in that step (3) described genetic engineering bacterium be BL21 (DE3)/ pET-TRI-Asia1-VP310。
- 4. hoof-and-mouth disease virus capsid protein VP0, VP1 and VP3 composition are prepared by claim 1-3 any one methods describeds Virus-like particle, the VP0 albumen be VP4 and VP2 gene fusion expression product, VP0 amino acid sequences such as SeqID Shown in No.2a, VP1 amino acid sequences are as shown in SeqID No.2b, and VP3 amino acid sequences are as shown in SeqID No.2c.
- 5. a kind of vaccine for being used to prevent infection of foot-and-mouth disease, it includes：The foot and mouth disease virus sample particle of claim 4, and vaccine With excipient or carrier.
- A kind of 6. genetic engineering bacterium for building to obtain by claim 1-3 any one methods describeds, it is characterised in that the base Because of the solvable coexpression hoof-and-mouth disease virus capsid protein VP3 genes of engineering bacteria series connection, VP1 genes and VP0 genes；The VP0 genes For VP4 and VP2 Gene Fusion, its nucleotide sequence is as shown in SeqID No.1a；VP1 gene nucleotide series such as SeqID Shown in No.1b；VP3 gene nucleotide series are as shown in SeqID No.1c.
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| CN105821011B (en) * | 2015-01-07 | 2020-10-30 | 普莱柯生物工程股份有限公司 | Vaccine composition for resisting O-type foot-and-mouth disease and preparation and application thereof |
| CN111944024B (en) * | 2015-04-17 | 2022-08-09 | 普莱柯生物工程股份有限公司 | Foot-and-mouth disease virus-like particle, preparation method, vaccine composition and application |
| CN104873967A (en) * | 2015-04-30 | 2015-09-02 | 中国农业科学院特产研究所 | O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof |
| CN106540248B (en) * | 2015-09-21 | 2020-11-17 | 普莱柯生物工程股份有限公司 | Vaccine composition for resisting foot-and-mouth disease and preparation method and application thereof |
| CN106479986B (en) * | 2016-10-31 | 2019-07-02 | 中国农业科学院兰州兽医研究所 | A kind of O-shaped foot and mouth disease virus sample particle and its preparation method and application |
| CN106636016B (en) * | 2017-02-24 | 2020-03-17 | 天津大学 | Method for inducing auxiliary virus-like particle self-assembly through positive and negative charges and application |
| CN108624609B (en) * | 2017-03-24 | 2020-04-07 | 斯澳生物科技（苏州）有限公司 | Nucleic acid constructs and methods for making coxsackievirus type a16 virus-like particles |
| CN107119058B (en) * | 2017-05-10 | 2020-12-08 | 斯澳生物科技（苏州）有限公司 | MS2 capsid protein fusion expression method and application thereof |
| CN107261135B (en) * | 2017-07-07 | 2020-11-10 | 中国农业科学院兰州兽医研究所 | Application of gold nanoparticles as adjuvant in preparation of virus-like particle vaccine |
| CN110777160B (en) * | 2018-07-31 | 2023-06-16 | 普莱柯生物工程股份有限公司 | Preparation method of foot-and-mouth disease virus-like particle antigen, foot-and-mouth disease virus-like particle antigen prepared by same and application thereof |
| CN109187967B (en) * | 2018-09-19 | 2021-10-01 | 郑州大学 | Duplex rapid detection card for detecting and distinguishing O-type and A-type foot-and-mouth disease viruses and preparation method thereof |
| CN110951757A (en) * | 2019-11-25 | 2020-04-03 | 中国农业科学院兰州兽医研究所 | Prokaryotic soluble expression method of foot-and-mouth disease virus VP3 gene of south Africa type 2 |
| CN110981946A (en) * | 2019-12-30 | 2020-04-10 | 华派生物工程集团有限公司 | Solution for large-scale production of foot-and-mouth disease virus-like particle antigen and purification and assembly method |
| US20230149529A1 (en) * | 2020-04-29 | 2023-05-18 | Pulike Biological Engineering, Inc. | Foot-and-mouth disease virus-like particle antigen, vaccine composition, preparation method, and use thereof |
| CN111773383B (en) * | 2020-07-03 | 2021-02-02 | 中国农业科学院兰州兽医研究所 | O-type foot-and-mouth disease subunit vaccine and preparation method and application thereof |
| CN113956335B (en) * | 2020-07-21 | 2024-04-12 | 普莱柯生物工程股份有限公司 | Preparation method of O-type foot-and-mouth disease virus-like particle antigen, prepared O-type foot-and-mouth disease virus-like particle antigen and application thereof |
| CN112076313B (en) * | 2020-09-24 | 2021-08-06 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease subunit vaccine and preparation method and application thereof |
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