CN108611327A - A kind of isolation and purification method of 2 porcine circovirus virus-like particle - Google Patents
A kind of isolation and purification method of 2 porcine circovirus virus-like particle Download PDFInfo
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Abstract
To solve the problems such as step in the isolating and purifying of 2 porcine circovirus virus-like particle is complicated, technique is cumbersome, production cost is high, purification efficiency is low, the present invention provides a kind of isolation and purification methods of 2 porcine circovirus virus-like particle, include the following steps：Step S1 collects recombinant yeast cell and is crushed, obtains the cell pyrolysis liquid of the virus-like particle containing 2 porcine circovirus；Step S2 carries out clarification to cell pyrolysis liquid and obtains virus-like particle first extract；Step S3 carries out ion-exchange chromatography to virus-like particle first extract and obtains virus-like particle slightly pure liquid；Step S4, to virus-like particle, slightly pure liquid carries out that acquisition virus-like particle concentrate is concentrated by ultrafiltration；Step S5 carries out gel filtration chromatography to virus-like particle concentrate, obtains virus-like particle after purification, wherein in the ion-exchange chromatography of step S3, the buffer solution surfactant containing predetermined concentration employed in adsorption process and elution process.
Description
Technical field
The invention belongs to biotechnologies, are related to the isolation and purification method of virus-like particle, and in particular to a boar is justified
The isolation and purification method of 2 virus-like particle of circovirus virus.
Background technology
Porcine circovirus 2 type (Porcine circovirus type 2, PCV2) is to belong to circovirus section
(Circoviridae), the DNA virus of Circovirus (Circovirus), the ring that nucleic acid molecules are sub-thread, head and the tail are closed
Shape structure, no cyst membrane.The not lethal pigs of PCV2 itself, but immunosupress can be caused.PCV2 is postweaning multisystemic failure synthesis
The main pathogen of sign (PMWS) can aggravate the death of sick pig when with other cause of disease mixed infections, be brought to aquaculture huge
Loss.PCV2, which has become, endangers one of main epidemic disease of pig breeding industry, and infection rate is higher in swinery, and vaccine is having for prevention and control PCV2
Effect tool, immunity inoculation can reduce PCV2 to the extent of injury of immune swine and its tissue virus load, also be carried out for pig farm
The purification of PCV2 provides the foundation condition.PCV2 commercialized vaccines currently on the market mainly have totivirus inactivated vaccine and subunit
Two kinds of vaccine：In terms of totivirus inactivated vaccine, the drawbacks of due to viral own characteristic and production technology, often there is virus titer hardly possible
With technical problems such as raisings, and its Effective Antigens purity is relatively low, through often there is different degrees of adverse reaction after immunity inoculation.
Pig circular ring virus subunit vaccine mainly uses eukaryon or prokaryotic expression system high efficient expression Cap protein, Cap eggs
Bai Jing is self-assembly of virus-like particle (Virus-like Particle, VLP), and virus-like particle epidemic disease can be prepared after purified
Seedling.The morphosis of VLP is similar to natural viral height, and immunogenicity is good, and does not contain viral gene, therefore is a kind of reason
The vaccine form thought.Currently, most of commercial PCV2 subunit vaccines use insect baculovirus expression system expression recombinant C ap
Albumen, but the system expression time is long and expensive；And the prokaryotic expression systems such as Escherichia coli cannot carry out recombinant protein
Posttranslational modification, and need to consider endotoxic removal.Yeast cells is expression system more better than prokaryotes, can not only be solved
The certainly endotoxin problem of prokaryotic system, can also be glycosylation modified to the progress of the albumen of expression, and expression is high, is easy to amplify, and
It is more cheap compared to other eukaryotic expression system costs.
For these reasons, the present inventor constructs recombinant expression 2 porcine circovirus virus-like particle (i.e.
PCV2VLPs kluyveromyces marxianus), and (i.e. for the bacterial strain application of recombination acquisition patent of invention
CN201610806538.0).However, inventor has found in the extraction purification research for carrying out the recombination yeast, in the prior art
The purification process of virus-like particle exists frequently with the precipitation method, gradient centrifugation, anion-exchange chromatography and molecular exclusion chromatography etc.
The problems such as step is complicated, technique is cumbersome, production cost is high, purification efficiency is low.
Invention content
To solve the above problems, the present inventor has carried out the sequence analysis of Porcine circovirus type 2 Cap first, find
In 234 amino acid of Porcine circovirus type 2 Cap, there is the residue number of strong basicity amino acids Arginine, lysine and histidine up to 41,
And the residue number of highly acid amino acid aspartic acid and glutamic acid only 16, the theoretical isoelectric point of PCV2cap albumen is about 10.8,
Therefore in conventional purifying buffer system (pH6-8) surfaces PCV2VLPs be particularly likely that it is positively charged.Therefore, using sun from
Sub- displacement chromatography can preferably isolate PCV2VLPs.
In addition, yeast cells is during high pressure is broken, the electronegative substance such as intracellular polysaccharide largely discharges,
It may be combined by ionization with PCV2VLPs, neutralized the positive charge on the surfaces PCV2VLP, influence cationic exchange
Chromatography purifies the efficiency of PCV2VLP, therefore, during carrying out cation-exchange chromatography, by adding specific surface
Activating agent, while enhancing the ionic strength in buffer system, it will be able to significantly improve the purification efficiency of PCV2VLP.
By analyzing above, to achieve the purpose that establish efficient PCV2VLPs purifying process, inventor is in the present invention
Propose the side of isolating and purifying that specific surfactant is added based on cation-exchange chromatography and in cation-exchange chromatography
Method.Specifically, present invention employs following technical solutions：
The present invention provides a kind of isolation and purification methods of 2 porcine circovirus virus-like particle, are used for from recombination yeast
2 porcine circovirus virus-like particle is isolated and purified in cell pyrolysis liquid, which is characterized in that include the following steps：Step S1 is received
The recombinant yeast cell of collection expression 2 porcine circovirus virus-like particle is simultaneously crushed, and is obtained containing porcine circovirus 2 type virus
The cell pyrolysis liquid of sample particle；Step S2, clarifies cell pyrolysis liquid, obtains virus-like particle first extract；Step S3 is right
Virus-like particle first extract carries out ion-exchange chromatography, obtains virus-like particle slightly pure liquid；Step S4 is slightly pure to virus-like particle
Liquid carries out 3~5 times of ultrafiltration concentration, obtains virus-like particle concentrate；Step S5 carries out gel mistake to virus-like particle concentrate
Chromatographic purifying is filtered, virus-like particle after purification is obtained, wherein in the ion-exchange chromatography of step S3, adsorption process and elution
The used buffer solution surfactant containing predetermined concentration in the process.
The isolation and purification method of 2 porcine circovirus virus-like particle provided by the invention can also have such skill
Art feature, wherein in step S3, buffer solution used by adsorption process is containing 50~150mM NaCl, 80 0.01%Tween
50mM phosphate buffers, the buffer solution employed in elution process is the 50mM phosphorus of NaCl containing 1M, 0.01%Tween 80
Phthalate buffer.
The isolation and purification method of 2 porcine circovirus virus-like particle provided by the invention can also have such skill
Art feature, wherein in step S1, be crushed using mechanical disruption broken, ultrasonic disruption or it is high-pressure homogeneous it is broken in it is any one
Kind or a variety of combinations.
The isolation and purification method of 2 porcine circovirus virus-like particle provided by the invention can also have such skill
Art feature, wherein in step S2, clarification is using full dose filtering, six filtering of mistake or any one or more ultracentrifugal combination.
The isolation and purification method of 2 porcine circovirus virus-like particle provided by the invention can also have such skill
Art feature, wherein it is that 10000g centrifuges 30min that clarification, which uses high speed centrifugation, condition,.
The isolation and purification method of 2 porcine circovirus virus-like particle provided by the invention can also have such skill
Art feature, wherein in step S3, the medium of ion-exchange chromatography uses strong cation exchange medium, strong cation exchange medium
For any one or more in Capto SP ImpRes, Capto S ImpAct, SP Bestarose FF and POROS HS
Mixing.
The isolation and purification method of 2 porcine circovirus virus-like particle provided by the invention can also have such skill
Art feature, wherein strong cation exchange medium is Capto SP ImpRes.
The isolation and purification method of 2 porcine circovirus virus-like particle provided by the invention can also have such skill
Art feature, wherein in step S4, ultrafiltration concentration uses molecular cut off for the super filter tube centrifugal concentrating of 30kDa.
The isolation and purification method of 2 porcine circovirus virus-like particle provided by the invention can also have such skill
Art feature, wherein the medium of step S5 kinds, gel permeation chromatography uses Sephacryl S-500HR or Sephacryl S-
300HR。
The present invention also provides a kind of 2 porcine circovirus virus-like particles, which is characterized in that using as above any one
The isolation and purification method of 2 porcine circovirus virus-like particle isolates and purifies to obtain from recombinant yeast cell lysate.
Invention effect
According to the isolation and purification method of 2 porcine circovirus virus-like particle provided in this embodiment, as a result of from
Sub- displacement chromatography purifies cell pyrolysis liquid, and is used during ion-exchange chromatography and be added to predetermined concentration surface
The buffer solution of activating agent, for cell sample particle, ion-exchange chromatography has the adsorbance of bigger and higher selectivity,
Therefore it can discharge from viral adsorption sample particle in cell pyrolysis liquid and in elution process, reach and be enriched with from cell pyrolysis liquid
The purpose of virus-like particle.Compared with the conventional precipitation method or gradient centrifugation, this enrichment process is selectively more preferable, intermediate mistake
The product impurity content of journey is relatively low, therefore is more advantageous to the progress of subsequent purification process.
In the present embodiment, virus-like particle is further purified as a result of the filler of gel permeation chromatography,
Therefore the purity of virus-like particle can be made just to can reach 95% or more using primary chromatography after ion-exchange chromatography, met
Demand in practical application.In addition, the step of ultrafiltration concentration is additionally provided with before gel permeation chromatography, therefore volume can also be reduced,
Improve the efficiency of gel chromatography.
Description of the drawings
Fig. 1 is that the sample solution of the different ionic strength in the embodiment of the present invention flows through Capto SP ImpRes ion exchanges
The SDS-PAGE electrophoresis detection results of column chromatography；
Fig. 2 is that the sample solution in the embodiment of the present invention flows through the purifying of Capto SP ImpRes ion exchange columns
280nm test maps；
Fig. 3 be the embodiment of the present invention in antigen slightly pure liquid Sephacryl S-500HR gel permeation chromatography columns when 280nm
Test map；
Fig. 4 is sample before and after the 2 porcine circovirus virus-like particle ion-exchange purification in the embodiment of the present invention
SDS-PAGE electrophoresis detection results；
Fig. 5 is gel filtration chromatography virus-like particle SDS-PAGE electrophoresis detection results in the embodiment of the present invention；
Fig. 6 is the 2 porcine circovirus virus-like particle HPLC testing results purified using the method for the present invention；
Fig. 7 is the electron microscope picture of the 2 porcine circovirus virus-like particle purified using the method for the present invention.
Specific implementation mode
The isolation and purification method of 2 porcine circovirus virus-like particle (hereinafter referred to as virus-like particle) of the present invention
It mainly includes the following steps that：
Step S1 collects the recombinant yeast cell of expression 2 porcine circovirus virus-like particle and is crushed, contained
The cell pyrolysis liquid of virus-like particle；
Step S2, clarifies cell pyrolysis liquid, obtains virus-like particle first extract；
Step S3 carries out ion-exchange chromatography to virus-like particle first extract, obtains virus-like particle slightly pure liquid；
Step S4, to virus-like particle, slightly pure liquid carries out 3~5 times of ultrafiltration concentration, obtains virus-like particle concentrate；
Step S5 carries out gel filtration chromatography to virus-like particle concentrate, obtains virus-like particle after purification.
Illustrate the specific implementation mode of the present invention below in conjunction with attached drawing and embodiment.Employed in following each embodiments
Reagent obtained unless otherwise specified from general commercial sources, the experiment condition being not specified with reference to conventional laboratory conditions or is abided by
The condition suggested according to supplier.
Clasmatosis, clarification and the Capto SP Impres ions of 1. 2 porcine circovirus virus-like particle of embodiment are handed over
Change chromatography
The present embodiment mainly comprises the following steps：Porcine circovirus 2 type virus-like is recombinantly expressed with kluyveromyces marxianus
The cell of grain is initial sample, is crushed, is clarified and cation exchange chromatography.Wherein, by repeatedly screening, invention human hair
Now there is using strong cation exchange medium as chromatography media good separating effect；Further, using in strong cation medium
Capto SP ImpRes media there is best separating effect, therefore tested using the medium in the present embodiment.
Specific experiment process is as follows：
1) solution is prepared
The formula of each buffer solution used in the present embodiment is as follows：
Broken bacterium buffer solution：50mM NaH2PO4, 100mM NaCl, 0.01%Tween80, pH6.80
Dilution buffer 1：50mM NaH2PO4, 0.01%Tween80, pH6.80
Dilution buffer 2：50mM NaH2PO4, 100mM NaCl, 0.01%Tween80, pH6.80
Dilution buffer 3：50mM NaH2PO4, 200mM NaCl, 0.01%Tween80, pH6.80
Equilibration buffer 1：50mM NaH2PO4, 50mM NaCl, 0.01%Tween80, pH6.80
Equilibration buffer 2：50mM NaH2PO4, 100mM NaCl, 0.01%Tween80, pH6.80
Equilibration buffer 3：50mM NaH2PO4, 150mM NaCl, 0.01%Tween80, pH6.80
Elution buffer：50mM NaH2PO4, 1M NaCl, 0.01%Tween80, pH6.80
2) preparation of sample
Zymocyte liquid 50mL, 10000g is taken to centrifuge 5min removal culture medium supernatants, precipitation is added deionized water to 50mL, mixes
10000g centrifuges 5min again after closing uniformly, discards supernatant, and precipitation is to express mark of 2 porcine circovirus virus-like particle
This kluyveromyces somatic cells.
The somatic cells that the above process is collected are resuspended with broken bacterium buffer solution to 200mL, high pressure homogenizer 1300bar
2 times broken, 10000g, 4 DEG C of centrifugation 30min collect supernatant, add isometric dilution buffer and are diluted and are split to get cell
Solve liquid.
3) column is filled
It takes the Capto SP Impres12mL after sedimentation to be added in glass chromatography column, and is assembled to AKTA protein purifications
System is rinsed with 50mL deionized waters to balance.
4) ion-exchange chromatography balances
60mL equilibration buffers are taken to balance pillar, A with 2.0mL/min flow velocitys280After baseline is steady, A at this time is demarcated280Value is
Zero.
5) virus-like particle purifies
By cell pyrolysis liquid mixing, takes and A is worked as with 2.0mL/min flow velocity loadings as sample solution after 200mL mixings280It inhales
Light value is collected after rising and flows through liquid.After end of the sample, pillar is rinsed with 2.0mL/min flow velocitys with 60mL equilibration buffers, waits for A280
Baseline steadily stops rinsing afterwards, it is therefore an objective to wash away the foreign protein of non-specific binding.
6) virus-like particle elutes
It is eluted with 2.0mL/min flow velocitys with 50mL eluents, starts to collect eluent after A280 values rise,
Stop collecting after A280 values return zero.
7) ion-exchange chromatography cleans
After above-mentioned chromatography process, ion-exchange chromatography filler can be cleaned and be regenerated, to restore filler use
When optimum performance.The detailed process of cleaning and regeneration is as follows：
Pillar is cleaned with 2.0mL/min flow velocitys with 50mL Milli-Q water, then uses 50mL 0.5M NaOH with 1.0mL/
Min flow velocitys are cleaned, and clean pillar with 50mL Milli-Q water with 2.0mL/min flow velocitys again, finally use 20% second of 50mL
Alcohol cleans pillar with 2.0mL/min flow velocitys.
In the present embodiment, tri- groups of cations of A, B, C have been carried out to investigate influence of the different ionic strength to loading and elution
The experiment of displacement chromatography.Elution buffer all same used in three groups of experiments, but dilution buffer and equilibration buffer are not
Together, as follows respectively：
Experimental group A：Dilution buffer 1, equilibration buffer 1；
Experimental group B：Dilution buffer 2, equilibration buffer 2；
Experimental group C：Dilution buffer 3, equilibration buffer 3.
As can be seen that due to containing 50mM NaH in broken bacterium buffer solution2PO4, 100mM NaCl, 0.01%Tween80, and
And dilution buffer is by the supernatant after adding brokenly bacterium in equal volume, therefore in the cell pyrolysis liquid of obtained each experimental group, from
Sub- intensity (NaCl concentration) is identical with corresponding equilibration buffer.That is, the loading ion of each experimental group
Intensity is consistent with corresponding ion balance intensity.
Fig. 1 is that the sample solution of the different ionic strength in the embodiment of the present invention flows through Capto SP ImpRes ion exchanges
The SDS-PAGE electrophoresis detection results of column chromatography.In Fig. 1, the sample of each swimming lane is：Swimming lane 1,2：Experimental group A is flowed through
Liquid；Swimming lane 3：Experimental group A eluents；Swimming lane 4,5：Experimental group B flows through liquid；Swimming lane 6：Experimental group B eluents；Swimming lane 7,8：Experiment
Group C flows through liquid；Swimming lane 9：Experimental group C eluents.
As shown in Figure 1, when using the buffering containing 50~150mM NaCl, 80 0.01%Tween in loading and equilibrium process
When liquid, the virus-like particle protein content flowed through in liquid is less, illustrates that the ionic strength range is conducive to chromatography media to virus
The absorption of sample particle.In addition, the virus-like particle content in eluent is high, illustrate to wash containing 1M NaCl, 0.01%Tween
De- liquid energy is enough fully to elute virus-like particle.
Fig. 2 is that the sample solution in the embodiment of the present invention flows through the purifying of Capto SP ImpRes ion exchange columns
280nm test maps.Test map shown in Figure 2 for experimental group A, wherein abscissa are the chromatography time.
Shown in Fig. 2, when carrying out ion-exchange chromatography using Capto SP ImpRes, eluting peak is narrow and concentrates, and illustrates
Capto SP ImpRes work well to the absorption and desorption of virus-like particle.
2. gel filtration chromatography virus-like particle of embodiment
The present embodiment mainly uses the virus-like particle that ion-exchange chromatography obtains embodiment 1, and slightly pure liquid (strips
Collection liquid in journey) gel permeation chromatography is carried out, improve the purity of virus-like particle.
Sephacryl S-500HR gel filtration chromatography virus-like particles are used in the present embodiment, detailed process is such as
Under：
1) solution is prepared
Solution formula used in the present embodiment is as follows：
Physiological saline：9g NaCl, Milli-Q water is settled to 1L, pH5.95
2) sample ultrafiltration concentration is handled
In the present embodiment, thick pure liquid that ion-exchange chromatography obtains carries out Gel filtration again after being first concentrated by ultrafiltration
Analysis, the process being specifically concentrated by ultrafiltration are：40mL virions slightly pure liquid is taken, is fitted into Millipore 15ml/30KD, is centrifuging
4000rpm centrifuges 30-60min in machine, up to liquid volume to 4mL or so, that is, completes ultrafiltration concentration, obtains virus-like particle
Concentrate.
3) Sephacryl S-500 HR chromatograph column equilibration
Pillar is rinsed with 0.5mL/min flow velocitys with 200mL Milli-Q water, uses 500mL physiological saline after the completion of rinsing again
Pillar is balanced with 0.5mL/min flow velocitys, it is zero that light absorption value at 280nm at this time is demarcated after A280 baselines are steady.
4) virus-like particle purifies
4mL virus-like particle concentrate loadings are taken, is eluted with the flow velocity of 0.5mL/min with physiological saline, works as extinction
Value A280 values occur collecting eluent after rising, and stop collecting after A280 values tend to be steady.Collected eluent is height
The 2 porcine circovirus virus-like particle solution of purity.
5) Sephacryl S-500 HR chromatographic columns clean
After above-mentioned chromatography process, the filler of gel permeation chromatography can be cleaned and be regenerated, made with restoring filler
The optimum performance of used time.The detailed process of cleaning and regeneration is as follows：
Pillar is cleaned with 0.5mL/min flow velocitys with 300mL Milli-Q water, then use 200mL 0.5M NaOH with
0.5mL/min flow velocitys are cleaned, then clean pillar with 300mL Milli-Q water with 0.5mL/min flow velocitys, finally use 200mL
20% ethyl alcohol rinses pillar with 0.5mL/min flow velocitys.
Fig. 3 be the embodiment of the present invention in antigen slightly pure liquid Sephacryl S-500HR gel permeation chromatography columns when 280nm
Test map.In Fig. 3, abscissa is the chromatography time.
As shown in figure 3, when carrying out gel permeation chromatography using Sephacryl S-500HR, eluting peak is narrow and concentrates, and
There are apparent multiple impurity peaks in other times section, illustrates that Sephacryl S-500HR conciliate the absorption of virus-like particle
Suction works well, and to the good separation of impurity.
The ion-exchange chromatography of 3. 2 porcine circovirus virus-like particle of embodiment and gel filtration chromatography sample
Detection
The present embodiment main process be the sample during the isolating and purifying of embodiment 1 and embodiment 2 is detected and
Analysis.
1) virus first, obtained using SDS-PAGE electrophoresis detection Capto SP Impres cation exchange chromatographies
Sample particulate samples (i.e. thick pure liquid sample).
Fig. 4 is sample before and after the 2 porcine circovirus virus-like particle ion-exchange purification in the embodiment of the present invention
SDS-PAGE electrophoresis detection results.In Fig. 4, the sample of each swimming lane is：Swimming lane 1：Albumen Marker；Swimming lane 2：Ion exchange layer
Analyse sample before purification；Swimming lane 3-4：Ion-exchange chromatography flows through liquid；Swimming lane 5：Ion-exchange chromatography cleaning solution；Swimming lane 6：Ion
The virus-like particle of displacement chromatography purifying.
As shown in figure 4, can be by porcine circovirus 2 type virus-like using Capto SP Impres ion-exchange chromatographies
Grain is separated from yeast cells lysate, and purity reaches 50% or more.
2) the virus-like particle purity that then, gel filtration chromatography step obtains uses SDS-PAGE electrophoresis and HPLC
Method detects.Wherein, the chromatographic column that HPLC analytic approach is selected is TSKgel G4000SWXL (7.8 × 300mm, 5 μm), for flowing
It is mutually 50mM NaH2PO4,50mM Na2HPO4,100mM Na2SO4, pH6.75, flow velocity 0.6ml/min, Detection wavelength
280nm。
Fig. 5 is gel filtration chromatography virus-like particle SDS-PAGE electrophoresis detection results in the embodiment of the present invention.Fig. 5
In, the sample of each swimming lane is respectively：Swimming lane 1：Albumen Marker；Swimming lane 2：Gel filtration chromatography virus-like particle.
Fig. 6 is the 2 porcine circovirus virus-like particle HPLC testing results purified using the method for the present invention.
As shown in Figure 5 and Figure 6, the 2 porcine circovirus virus-like particle obtained after gel filtration chromatography step
Purity is up to 95% or more.
3) PCV2 virus-like particle (the i.e. gels that the purifying of the present invention obtains finally, are had detected using transmission electron microscope observing
The virus-like particle sample obtained after filtration chromatography step).Film copper mesh is supported to adsorb using carbon, after 3% phosphotungstic acid negative staining,
Using the formational situation of transmission electron microscope observation viruslike particle.
Fig. 7 is the electron microscope picture of the 2 porcine circovirus virus-like particle purified using the method for the present invention.
As shown in fig. 7, the circovurus type 2 Cap protein obtained using the purification process of the present invention is with virus-like particle shape
Formula exists, and illustrates it with good immunogenicity and reactionogenicity.
Embodiment effect
According to the isolation and purification method of 2 porcine circovirus virus-like particle provided in this embodiment, as a result of
Capto SP Impres carry out cation-exchange chromatography as the chromatographic stuffing of ion-exchange chromatography, the filler with it is similar
Other fillers compare with bigger adsorbance and higher selectivity, therefore can from cell pyrolysis liquid viral adsorption sample
Grain simultaneously discharges in elution process, achievees the purpose that the enriching virus sample particle from cell pyrolysis liquid.Meanwhile the cation exchanges
The adsorption process of chromatography uses the 50mM phosphate buffers containing 50~150mM NaCl, 80 0.01%Tween, strips
Journey uses the 50mM phosphate buffers of NaCl containing 1M, 0.01%Tween 80, these buffer solutions are added to debita spissitudo
Surfactant, therefore the purification efficiency that the ionic strength in buffer system can be enhanced, significantly improve PCV2 VLP.
Compared with the conventional precipitation method or gradient centrifugation, cation-exchange chromatography that the present embodiment uses this be enriched with
Journey is selectively more preferable, and the product impurity content of pilot process is relatively low, therefore is more advantageous to the progress of subsequent purification process.
In the present embodiment, the filler as a result of Sephacryl S-500 HR as gel permeation chromatography, therefore energy
The residual impurity after ion-exchange chromatography is enough removed, virus-like particle is further purified, the pure of virus-like particle is made
Degree just can reach 95% or more after ion-exchange chromatography using primary chromatography, substantially meet the demand in practical application.Separately
Outside, the step of being additionally provided with ultrafiltration concentration before gel permeation chromatography, therefore volume can also be reduced, improve the efficiency of gel chromatography.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. a kind of isolation and purification method of 2 porcine circovirus virus-like particle, for dividing from recombinant yeast cell lysate
From purifying porcine circovirus 2 type virus-like particle, which is characterized in that include the following steps：
Step S1 collects the recombinant yeast cell of expression 2 porcine circovirus virus-like particle and is crushed, obtains and justify containing pig
The cell pyrolysis liquid of 2 virus-like particle of circovirus virus；
Step S2 clarifies the cell pyrolysis liquid, obtains virus-like particle first extract；
Step S3 carries out ion-exchange chromatography to the virus-like particle first extract, obtains virus-like particle slightly pure liquid；
Step S4, to the virus-like particle, slightly pure liquid carries out 3~5 times of ultrafiltration concentration, obtains virus-like particle concentrate；
Step S5 carries out gel filtration chromatography to the virus-like particle concentrate, obtains virus-like particle after purification,
Wherein, in the ion-exchange chromatography of step S3, the buffer solution employed in adsorption process and elution process contains
The surfactant of predetermined concentration.
2. the isolation and purification method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that：
Wherein, in step S3, buffer solution used by the adsorption process is containing 50~150mM NaCl, 80 0.01%Tween
50mM phosphate buffers, the buffer solution employed in the elution process is NaCl containing 1M, 0.01%Tween 80
50mM phosphate buffers.
3. the isolation and purification method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that：
Wherein, in step S1, the broken broken, ultrasonic disruption using mechanical disruption or it is high-pressure homogeneous it is broken in it is any one
Kind or a variety of combinations.
4. the isolation and purification method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that：
Wherein, in step S2, the clarification is using full dose filtering, six filtering of mistake or any one or more ultracentrifugal connection
With.
5. the isolation and purification method of 2 porcine circovirus virus-like particle according to claim 3, it is characterised in that：
Wherein, it is that 10000g centrifuges 30min that the clarification, which uses the high speed centrifugation, condition,.
6. the isolation and purification method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that：
Wherein, in step S3, the medium of the ion-exchange chromatography uses strong cation exchange medium,
The strong cation exchange medium be Capto SP ImpRes, Capto S ImpAct, SP Bestarose FF and
The mixing of any one or more in POROS HS.
7. the isolation and purification method of 2 porcine circovirus virus-like particle according to claim 6, it is characterised in that：
Wherein, the strong cation exchange medium is Capto SP ImpRes.
8. the isolation and purification method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that：
Wherein, in step S4, the ultrafiltration concentration uses molecular cut off for the super filter tube centrifugal concentrating of 30kDa.
9. the isolation and purification method of 2 porcine circovirus virus-like particle according to claim 1, it is characterised in that：
Wherein, the medium of step S5 kinds, the gel permeation chromatography uses Sephacryl S-500HR or Sephacryl S-
300HR。
10. a kind of 2 porcine circovirus virus-like particle, which is characterized in that using according to any one of claims 1 to 9
The isolation and purification method of 2 porcine circovirus virus-like particle isolates and purifies to obtain from recombinant yeast cell lysate.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113637054A (en) * | 2021-08-30 | 2021-11-12 | 雪莱(武汉)生物医药技术有限公司 | Purification method and application of recombinant Sendai virus-like particles |
| CN114085829A (en) * | 2021-11-18 | 2022-02-25 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Efficient enrichment method for viruses in environmental medium |
| CN116768986A (en) * | 2022-12-07 | 2023-09-19 | 华北制药金坦生物技术股份有限公司 | Novel chromatographic purification method for virus-like particles |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365362A (en) * | 2017-07-04 | 2017-11-21 | 武汉科前生物股份有限公司 | A kind of method for mass producing high-purity pig circular ring virus ORF2 albumen |
| CN107384878A (en) * | 2017-09-04 | 2017-11-24 | 肇庆大华农生物药品有限公司 | A kind of method of consummate pig circular ring virus |
-
2018
- 2018-07-03 CN CN201810712312.3A patent/CN108611327B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365362A (en) * | 2017-07-04 | 2017-11-21 | 武汉科前生物股份有限公司 | A kind of method for mass producing high-purity pig circular ring virus ORF2 albumen |
| CN107384878A (en) * | 2017-09-04 | 2017-11-24 | 肇庆大华农生物药品有限公司 | A kind of method of consummate pig circular ring virus |
Non-Patent Citations (1)
| Title |
|---|
| MINDAUGAS ZAVECKAS ET.AL.,: "Purification of recombinant virus-like particles of porcine circovirustype 2 capsid protein using ion-exchange monolith chromatography", 《JOURNAL OF CHROMATOGRAPHY B》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113637054A (en) * | 2021-08-30 | 2021-11-12 | 雪莱(武汉)生物医药技术有限公司 | Purification method and application of recombinant Sendai virus-like particles |
| CN113637054B (en) * | 2021-08-30 | 2023-10-03 | 雪莱(武汉)生物医药技术有限公司 | Purification method and application of recombinant Sendai virus-like particles |
| CN114085829A (en) * | 2021-11-18 | 2022-02-25 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Efficient enrichment method for viruses in environmental medium |
| CN114085829B (en) * | 2021-11-18 | 2023-08-11 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Efficient enrichment method for viruses in environmental medium |
| CN116768986A (en) * | 2022-12-07 | 2023-09-19 | 华北制药金坦生物技术股份有限公司 | Novel chromatographic purification method for virus-like particles |
| CN116768986B (en) * | 2022-12-07 | 2024-03-12 | 华北制药金坦生物技术股份有限公司 | Novel chromatographic purification method for virus-like particles |
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