CN107365362B - Method for large-scale production of high-purity porcine circovirus ORF2 protein - Google Patents
Method for large-scale production of high-purity porcine circovirus ORF2 protein Download PDFInfo
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Abstract
The invention belongs to the technical field of vaccine preparation, and particularly relates to a method for producing high-purity porcine circovirus ORF2 protein in a large scale. The method comprises the following steps: adding a small amount of chitosan into the porcine circovirus ORF2 protein culture solution, shaking uniformly, naturally settling, sucking supernatant, clarifying and filtering by using a hollow fiber column, performing ion exchange by using an ion exchange column, desalting eluent by using a G25 chromatographic column, and performing sterilization and filtration to obtain the high-purity porcine circovirus ORF2 protein solution. According to the invention, the preparation of the high-purity porcine circovirus ORF2 protein is realized in large-scale (500L-1000L) high-efficiency production by the processes of chemical reagent sedimentation, clarification filtration, ion exchange, chromatographic column desalination and the like, the recovery rate of the high-purity porcine circovirus ORF2 protein can reach 99.46%, the removal rate of the foreign protein can reach 98.04%, and the content of the effective antigen can reach 93.2%.
Description
Technical Field
The invention belongs to the technical field of vaccine preparation, and particularly relates to a method for producing high-purity porcine circovirus ORF2 protein in a large scale.
Background
Porcine circovirus type 2 (PCV)2) Contains 2 main open reading frames, wherein the viral capsid protein coded by ORF2 gene is a main structural protein, comprises main antigen neutralizing epitopes, has good immunogenicity, and PCV1And PCV2Does not have serological cross reaction, is a good antigen for detecting the antibody level of the virus, and is a good target gene for developing novel vaccines. The recombinant ORF2 protein (28kDa) expressed in vitro by a baculovirus expression system can be self-assembled into viral nucleocapsid-like particles and shows good immunogenicity. The circovirus ORF2 protein has PCV1And PCV2Common immunogenicity is the trend in current vaccines.
At present, the conventional purification method aiming at the circovirus mainly adopts modes of antigen centrifugation, dead-end filtration, membrane-packed concentration and the like, the operation is complex, the cost is high, and the circovirus inactivated vaccine prepared by the conventional production process flow has certain side reaction in clinical application, and is mainly related to the extraantigenic components contained in the vaccine. These ingredients include: residual proteins and residual DNA of cells for virus culture; culture medium residues such as calf serum; residual inactivating agent; preservatives (e.g., thimerosal), and the like. These heterologous components not only cause side effects in the immunized animal, but also greatly reduce the efficacy of the vaccine.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and aims to provide a method for producing high-purity porcine circovirus ORF2 protein in a large scale.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for producing high-purity porcine circovirus ORF2 protein in a large scale is characterized by comprising the following steps:
(1) adding chitosan into porcine circovirus ORF2 protein culture solution, shaking uniformly, naturally settling, and sucking supernatant as protein solution to be clarified;
(2) clarifying and filtering the protein liquid to be clarified obtained in the step (1) by using a hollow fiber column to obtain a porcine circovirus ORF2 protein clarified liquid;
(3) carrying out ion exchange on the porcine circovirus ORF2 protein clear solution obtained in the step (2) by using an ion exchange column, and then eluting to obtain porcine circovirus ORF2 protein purification eluent;
(4) desalting the porcine circovirus ORF2 protein purification eluent obtained in the step (3) by using a G25 chromatographic column, and eluting to obtain a porcine circovirus ORF2 protein purification solution;
(5) and (4) sterilizing and filtering the protein purified liquid obtained in the step (4) to obtain the high-purity porcine circovirus ORF2 protein liquid.
In the scheme, the adding amount of the chitosan in the step (1) is 1-10% of the mass of the porcine circovirus ORF2 protein culture solution, and more preferably, the adding amount of the chitosan is 5%.
In the above embodiment, the pore diameter of the hollow fiber column in step (2) is 0.2 μm to 0.65 μm, and more preferably, the pore diameter of the hollow fiber column is 0.2 μm.
In the above scheme, the hollow fiber column in step (2) has the model number: CFP-2-E-55; the equipment model of the hollow fiber column is VERSAFlux 120.
In the above scheme, the specific operations of clarifying and filtering with the hollow fiber column in the step (2) are as follows: the method comprises the steps of performing circulating sterilization treatment on a hollow fiber column by using a sterile 1mol/LNaOH solution, cleaning the hollow fiber column by using sterile injection water until the pH value is 6.8-7.2, balancing the hollow fiber column by using a sterile 0.01mol/LPBS solution, clarifying and filtering a protein sample to be clarified of porcine circovirus ORF2 protein through the hollow fiber column, and collecting a liquid at the end of permeation to obtain a porcine circovirus ORF2 protein clarified liquid. The process of clarifying and filtering the protein liquid to be clarified by using the hollow fiber column comprises the following steps: firstly permeating 50-70% of protein solution to be clarified, then supplementing 20mmol/L Tris solution with the same volume as the volume of the rest protein solution to be clarified, continuing permeation, after the permeation volume is equal to the volume of the added Tris solution, supplementing 20mmol/L Tris solution with the same volume as the volume of the rest protein solution, washing and filtering for multiple times until residues cannot permeate, and ending the clarification and filtration process.
In the scheme, TMP adopted in the step (2) during clarification and filtration of the hollow fiber column is 2.0-3.0 psi, and the flow rate of the liquid inlet end is controlled at 60-80L/min.
In the above scheme, the ion exchange column in step (3) has the model number: BPG300/500 Column.
In the scheme, the filler of the ion exchange column in the step (3) is Capto SP ImpRes.
In the scheme, the process of ion exchange of the porcine circovirus ORF2 protein clear solution in the step (3) by using an ion exchange column comprises the following steps: washing the ion exchange column with sterile 0.5mol/L NaOH solution for 2 column volumes, then washing with sterile water until the pH is 7.2-7.5, then balancing the ion exchange column with 20mmol/L LTris solution until the conductivity of the ion exchange column before and after the column is consistent (the conductivity of the ion exchange column before and after the column is 6.5-6.6 ms/cm), stabilizing the pH at 7.0, and detecting UV by ultraviolet280The base line is stable; then, the clear solution of the porcine circovirus ORF2 protein obtained in the step (2) is loaded, after the loading is finished, the unbound hetero protein is washed away by using 20mmol/L Tris solution, and the UV is waited280The value is reduced to be lower than 0.100AU, the porcine circovirus ORF2 protein is eluted by high-salt solution, and ultraviolet detection is carried out on the protein280Collecting eluate when the value increases, and waiting for UV280Collection was stopped when the value dropped below 0.100 AU.
In the scheme, the linear flow rate of the clear solution of the porcine circovirus ORF2 protein in the step (3) is 30-100cm/h, the pressure is controlled to be less than 2.50bar, and the loading amount is controlled to be 70-80 mg/ml BSA (the maximum loading amount of the filler is 95mg/ml BSA); the high-salt solution used for elution is 0.5-2 mol/L NaCl solution, and the elution flow rate is controlled to be 30-100 cm/h.
In the above scheme, the model of the G25 gel chromatography column in step (4) is: BPG450/1000 Column.
In the above scheme, the G25 gel chromatography column in step (4) has the following packing: SepHadex G-25.
In the above scheme, the desalting process of the G25 gel chromatography column in step (4) is as follows: washing the G25 gel chromatographic column with sterile 0.5mol/L NaOH solution for 2 column volumes, then washing with sterile water until the pH is 7.2, then balancing the G25 gel chromatographic column with 0.01mol/L PBS solution until the conductivity of the G25 gel chromatographic column before and after the column is consistent (the conductivity of the G25 gel chromatographic column before and after the column is 15-16 ms/cm),the pH is stabilized at 7.0, and ultraviolet detection is carried out280The base line is stable; then, the porcine circovirus ORF2 protein purification eluent obtained in the step (3) is loaded, and after the loading is finished, the elution is carried out until UV is reached280When the value begins to rise, the eluent begins to be collected, and the eluent is stopped being harvested until the first elution peak is finished.
In the scheme, the sample loading linear flow rate of the porcine circovirus ORF2 protein purification eluent desalted by the G25 chromatographic column in the step (4) is 45-100 cm/h, the pressure is less than 2.0bar, the sample loading amount is controlled at 10-30% (v/v) of the column volume, and 0.01mol/L PBS solution is adopted for elution.
The invention has the beneficial effects that:
(1) the invention solves the technical problem that the porcine circovirus ORF2 protein is difficult to realize large-scale production with high purity, realizes the large-scale (500L-1000L) high-efficiency production and preparation of the high-purity porcine circovirus ORF2 protein by the processes of chemical reagent sedimentation, clarification filtration, ion exchange, chromatographic column desalination and the like, and has the advantages that the recovery rate of the porcine circovirus ORF2 protein can reach 99.46%, the removal rate of foreign proteins reaches 98.04%, and the effective antigen content reaches 93.2%.
(2) According to the invention, a chemical reagent is clarified, hollow fiber is clarified and filtered, ion exchange and G25 gel chromatographic column desalination are integrated for 4 steps and applied to the field of porcine circovirus ORF2 protein purification, chitosan is added to increase cell fragment aggregation according to the characteristics of porcine circovirus ORF2 protein, hollow fiber of a specific model/pore size is adopted for clarification, filtration and pretreatment, the purification is further carried out through a Capto SPImpRes ion exchange column, and finally G25 gel chromatographic column desalination is adopted to obtain single pure porcine circovirus ORF2 protein. The antigen is purified by 4 steps, so that impurities are effectively removed, antigen loss is reduced, and the treatment efficiency, the protein recovery rate and the impurity protein removal rate are improved.
(3) The method disclosed by the invention is simple to operate, low in cost and high in treatment efficiency, is suitable for industrial large-scale production, and the purified porcine circovirus ORF2 protein is high in purity, so that the use amount of the antigen is greatly reduced in the later vaccine preparation process, the cost is saved, and the problem of side reaction of the vaccine is fundamentally solved, therefore, the method has a good popularization prospect.
(4) According to the purification process of the porcine circovirus ORF2 protein, the removal rate of the foreign protein is as high as 98.04%, and the content of the effective antigen is as high as 93.2%. The side reaction of the vaccine caused by the hybrid protein is reduced to be lower, the safety of the vaccine is improved, and simultaneously, the good immunogenicity is kept; because the porcine circovirus inactivated vaccine is inoculated to a swinery, the high-quality product greatly reduces the side reaction of the inoculated swinery and brings higher social benefit and economic benefit for farmers.
(5) The method disclosed by the invention can be used for matching with corresponding tank bodies and pipelines under the same experimental conditions, can be used for linearly amplifying the process, can be used for expanding the production scale to 2000L, 3000L and 5000L, and has operability.
Drawings
FIG. 1 is an ion exchange purification profile.
FIG. 2 shows SDS-PAGE results of the whole purification process of porcine circovirus ORF2 protein, wherein 1 is the protein expression sample, 2 is the supernatant of natural sedimentation, 3 is a clarified sample of 0.2 μ M, 4 is a sample purified by an ion exchange column, 5 is a sample diluted 4 times by the ion exchange column, 6 is a sample diluted 8 times by the ion exchange column, 7 is a sample purified by a G25 column, 8 is a sample diluted 3 times by the G25 column, 9 is a blank, and M is Marker.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
In the following examples, the apparatus used is as follows:
the model of the hollow fiber column in the process of clarifying and filtering the hollow fiber column is as follows: CFP-2-E-55.
The model of the hollow fiber column equipment for clarifying and filtering the hollow fiber column is VERSAFlux 120.
The type of the ion exchange column is as follows: BPG300/500 Column.
The ion exchange column packing is: capto SP imprmes.
The model of the desalting gel chromatographic column is as follows: BPG450/1000 Column.
The desalting gel chromatography column packing comprises: SepHadex G-25.
All of the above instruments are available from GE.
The reagents used were as follows: 1000L of a porcine circovirus culture solution provided by the company Ministry of production;
the porcine circovirus type 2 ELISA antibody detection kit is provided by the diagnostic reagent part of the company.
The protein obtained by purification is used for detecting the protein content by an SDS-PAGE method, and the ORF2 protein concentration is determined by a double antibody sandwich ELISA method.
The linear flow velocity formula v is 30-100cm/h pi r2In ml/min, r represents the radius of the column.
Example 1
First, antigen clarification pretreatment
Adding sterile chitosan into porcine circovirus ORF2 protein culture solution according to the proportion of 1 wt%, 5 wt% and 10 wt% respectively, shaking uniformly, naturally settling at 4 ℃, and sucking supernatant as a protein sample to be clarified.
TABLE 1 evaluation of Effect of porcine circovirus ORF2 protein clarification pretreatment
| Sample (I) | Protein concentration (ug/ml) | Removal rate of hetero-protein | Turbidity (FTU) | Protein recovery |
| Protein expression profiles | 11214 | - | 2000 | - |
| 1% chitosan | 5612 | 85.01％ | 1000 | 99.8％ |
| 5% chitosan | 4321 | 92.12％ | 300 | 99.1％ |
| 10% chitosan | 3123 | 93.50％ | 50 | 91％ |
With A280The method detects the protein concentration and the double-antibody sandwich ELISA method detects the effective target protein content. The results are shown in Table 1. The results in table 1 show that the addition of chitosan can obviously increase the aggregation of macromolecules such as cell debris and the like, increase the clarity of protein samples, and lay a foundation for later-stage antigen clarification. The recovery rate of protein after 5 percent of chitosan treatment reaches up to 99.1 percent, the removal rate of foreign protein is 92.12 percent, and the 5 percent of chitosan is more suitable for the clarification pretreatment of antigen by integrating several parameters of the removal rate, turbidity and protein recovery rate of the foreign protein.
Example 2
Second, hollow fiber column clarification process
1 System Pre-processing
1.1 respectively installing hollow fiber columns of 0.2 μm, 0.45 μm and 0.65 μm into a hollow fiber column control device, connecting corresponding pipelines, and circularly infiltrating the hollow fiber columns with sterile water for injection for 10min after assembly.
1.2 System integrity detection
Pressure maintenance measures the integrity of the system.
1.3 processing of the System
Cleaning and sterilizing: carrying out circulating sterilization treatment on the system for 30min by using a sterile 0.5mol/L NaOH solution, then cleaning the system by using sterile water for injection, and washing off the residual alkali solution until the pH value is 7.0;
1.4 detection of Water flux
The water flux of the hollow fiber column at the corresponding temperature was calculated by permeation with sterile water for injection at a prescribed pump speed.
1.5 hollow fiber column Balancing
The hollow fiber column was equilibrated with sterile 0.01mol/L PBS solution for 5 min.
2 clarification filtration Process of sample to be clarified
2.1 taking 200L of virus liquid of pure suspension SF-9 cell culture, adding 5 percent chitosan, shaking uniformly, settling, and sucking supernatant as a protein sample to be clarified;
2.2, respectively enabling the protein sample to be clarified to pass through a hollow fiber column with the diameter of 0.2-0.65 mu m, controlling the pump speed at 40-50% (the flow rate of a liquid inlet end is 60-80L/min) after the sample circulates in the fiber column for 5min, opening a permeation end, maintaining the TMP (transmembrane pressure) at 1.5-2.0 psi, and collecting liquid at the permeation end, wherein the circulating flow rate is 78L/min; and continuously replenishing 20mmol/L Tris solution at the flow rate of 2L/min when the volume of the liquid at the permeation end to be collected is 170L, continuously permeating, replenishing 20mmol/L Tris solution with the same volume as the rest sample after the volume of the sample to be permeated is equal to the volume of the added Tris solution, washing and filtering for three times, and finishing the clarification process until the residue cannot permeate, wherein 280L of the liquid collected at the permeation end is the porcine circovirus ORF2 protein clarified liquid.
TABLE 2 evaluation of porcine circovirus ORF2 protein clarification filtration Effect
| Sample (I) | Protein concentration (ug/ml) | Removal rate of hetero-protein | ORF2 protein concentration (ug/ml) | Protein recovery |
| Protein expression profiles | 11214 | - | 9.83 | - |
| 0.2um | 5612 | 94.98％ | 4.62 | 97.00％ |
| 0.45um | 4321 | 96.73％ | 4.3 | 90.49％ |
| 0.65um | 4123 | 97.62％ | 4.2 | 85.45％ |
With A280The method detects the protein concentration and the double-antibody sandwich ELISA method detects the effective target protein content. The results of the experiment (see table 2). The results in table 2 show that the removal rate of the foreign protein after the hollow fiber clarification filtration is higher than 94%, the recovery rate of the protein is as high as 97%, and almost no loss is caused. Compared with conventional centrifugation and dead-end filtration, the open flow channel structure of the hollow fiber membrane is more conducive to the permeation and removal of foreign proteins. In actual operation, the hollow fiber column with the aperture ratio of 0.2um to 0.65um has higher treatment efficiency and higher protein recovery rate. By combining the removal rate of the hybrid protein, the recovery rate of the protein and the operation efficiency, the effect of clarifying the protein of the porcine circovirus ORF2 by adopting a hollow fiber column with the aperture of 0.2um is better.
EXAMPLE 3 ion exchange column purification Process
1 System Pre-processing
1.1 packing the ion exchange packing Capto SP Impres, uniformly pounding the mixture into a BPG300/500column, and measuring the column effect after the ion exchange column is assembled.
1.2 treating 2 Column Volumes (CV) of the molecular sieve gel chromatographic column with sterile 0.5mol/L NaOH, washing with sterile injection water to pH7.5, balancing the ion exchange column with 20mmol/L Tris solution until the front and back of the column are consistent (the conductivity before and after the column is 6.5-6.6 ms/cm), stabilizing pH at 7.0, and performing ultraviolet UV280The baseline was stable.
2 purification Process
And (3) loading the protein clarified liquid, setting the linear flow rate of loading to be 30-100cm/h, controlling the pressure to be less than 2.50bar, and controlling the loading amount to be 70-80 mg/ml BSA (the maximum loading amount of the filler is 95mg/ml BSA). After all 280L of protein clarified liquid sample was loaded on the ion exchange column, the unbound hetero-proteins were washed with 20mmol/L Tris. To be UV cured280The value is reduced to be lower than 0.100AU, and the porcine circovirus ORF2 protein is eluted by using high-salt solution of 0.5mol/L NaCl, 1mol/L NaCl and 2mol/L NaCl respectively. UV to ultraviolet280Collecting eluted sample when the value is increased, and waiting for UV280When the value is reduced to be lower than 0.100AU, the protein collection is stopped, and the elution is continued until all the antigen liquid flows out of the column. The collected elution sample is 30-35L of porcine circovirus ORF2 protein purification eluent.
TABLE 3 evaluation of the purification Effect of porcine circovirus ORF2 protein
| Sample (I) | Protein concentration (ug/ml) | Removal rate of hetero-protein | Cap protein concentration (ug/ml) | Protein recovery |
| Protein expression profiles | 11214 | - | 9.83 | - |
| 0.5mol/L NaCl | 3012 | 95.30％ | 293 | 89.00％ |
| 1mol/L NaCl | 3321 | 94.20％ | 345 | 97.12％ |
| 2mol/L NaCl | 3392 | 93.90％ | 357 | 98.20％ |
With A280The method detects the protein concentration and the double-antibody sandwich ELISA method detects the effective target protein content, and the experimental results are shown in Table 3. The results in table 3 show that different salt ion concentrations have a significant effect on the recovery of protein. The elution process of 1mol/L NaCl and 2mol/L NaCl has more charges, is more beneficial to the recovery of target protein, and has the protein recovery rate as high as 97%. Comprehensively considering the removal rate of the hybrid protein and the recovery rate of the protein, the ion exchange purification has better elution effect by adopting 2mol/L NaCl.
EXAMPLE 4G 25 chromatography column desalination Process
Washing the G25 gel chromatographic column with sterile 0.5mol/L NaOH solution for 2 column volumes, then washing with sterile water until the pH is 7.2, then balancing the G25 gel chromatographic column with 0.01mol/L PBS solution until the conductivity of the G25 gel chromatographic column before and after the column is consistent (the conductivity of the G25 gel chromatographic column before and after the column is 15-16 ms/cm), the pH is stabilized at 7.0, and detecting UV by ultraviolet280The base line is stable; then, loading the porcine circovirus ORF2 protein purification eluent obtained in the step (3) into a G25 chromatographic column for desalting by 15% of column volume, setting the linear flow rate of the loaded sample to be 45-100 cm/h and the pressure to be less than 2.0bar, after the loading is finished, eluting by using 0.01mol/L PBS solution, and waiting for UV280And when the value begins to rise, collecting the eluent, stopping collecting the eluent until the first elution peak is finished, and obtaining a porcine circovirus ORF2 protein purified sample after elution. The purpose of desalting the G25 column is to replace the high salt eluent with a low salt buffer to facilitate protein stabilization.
The optimal purification process determined according to the embodiments 1 to 4 comprises the following specific process flows:
1, pretreatment: after the protein culture solution of pure suspension culture is added with 5% chitosan for sedimentation, the supernatant is sucked.
2, clarification: and treating the clarified protein sample by a 0.2-micron hollow fiber column to obtain the clarified protein sample.
3, purification: and purifying the clarified protein sample by an ion exchange column, and eluting by 2mol/L NaCl to obtain porcine circovirus ORF2 protein eluent.
4, desalting: desalting the purified protein elution sample by a G25 chromatographic column to obtain a porcine circovirus ORF2 protein purified sample.
5, detection: after sampling, the protein concentration and the ORF2 protein content were examined.
Tests of 3 batches of samples were run against this process and data collected and compared.
1. Clarification: adding 5% chitosan into the pure suspension culture protein culture solution, shaking, settling, sucking supernatant 300L, and treating with 0.2 μm hollow fiber column to obtain clarified protein sample 400L.
2. And (3) purification: purifying the clarified protein sample 400L by an ion exchange column to obtain a protein elution sample 40L.
3. Desalting: desalting the protein eluted sample by a G25 chromatographic column to obtain a protein purified sample.
4. And (3) detection: after sampling, the protein concentration and the ORF2 protein content were examined.
1. Clarification: adding 5% chitosan into the pure suspension culture protein culture solution, shaking, settling, sucking 400L of supernatant, and treating with 0.45 μm hollow fiber column to obtain 500L of clarified protein sample.
2. And (3) purification: purifying the clarified protein sample 500L by ion exchange column to obtain protein elution sample 43L.
3. Desalting: desalting the protein eluted sample by a G25 chromatographic column to obtain a protein purified sample.
4. And (3) detection: after sampling, the protein concentration and the ORF2 protein content were examined.
1. Clarification: adding 5% chitosan into the protein culture solution for pure suspension culture, shaking uniformly, settling, sucking 500L of supernatant, and treating by a 0.65 μm hollow fiber column to obtain 600L of clarified sample.
2. And (3) purification: the clarified protein sample was purified by ion exchange column to obtain 50L of protein eluate.
3. Desalting: desalting the protein eluted sample by a G25 chromatographic column to obtain a protein purified sample.
4. And (3) detection: after sampling, the protein concentration and the ORF2 protein content were examined.
With A280The method detects the protein concentration and the double-antibody sandwich ELISA method detects the effective target protein content. The results are shown in Table 4.
TABLE 4 porcine circovirus ORF2 protein purification comparative experiment results
| Sample (I) | Protein concentration (ug/ml) | Removal rate of hetero-protein | ORF2 protein concentration (ug/ml) | Protein recovery |
| Protein expression profiles | 11214 | - | 9.83 | - |
| |
3142 | 91.30％ | 364 | 98.10 |
| Test | ||||
| 2 | 2456 | 94.20％ | 398 | 91.00 |
| Test | ||||
| 3 | 2598 | 96.00％ | 429 | 87.00％ |
The results show that the removal rate of the foreign protein of the antigen prepared by the process after three batches of purification can reach 96.0 percent at most, and the recovery rate of the protein can reach 98.1 percent. The purification process is stable, the protein concentration can be improved, and the difference between finished products in the later period is reduced. Comparative experiments also show that clarification filtration with 0.2 μm hollow fiber and ion exchange purification are the best process combination and the advantages are more obvious in large-scale production and are also the most protected.
It is apparent that the above embodiments are only examples for clearly illustrating and do not limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are therefore intended to be included within the scope of the invention as claimed.
Claims (4)
1. A method for mass production of highly pure porcine circovirus ORF2 protein, comprising
The following steps:
(1) adding chitosan into porcine circovirus ORF2 protein culture solution, shaking uniformly, naturally settling, and sucking supernatant as protein solution to be clarified;
(2) clarifying and filtering the protein liquid to be clarified obtained in the step (1) by using a hollow fiber column to obtain a porcine circovirus ORF2 protein clarified liquid; the aperture of the hollow fiber column is 0.2-0.65 μm; the specific operation process of clarifying and filtering the protein liquid to be clarified by utilizing the hollow fiber column comprises the following steps: firstly permeating 50-70% of protein solution to be clarified, then supplementing 20mmol/L Tris solution with the same volume as the volume of the rest protein solution to be clarified, continuing permeation, supplementing 20mmol/L Tris solution with the same volume as the volume of the rest protein solution after the permeated volume is equal to the volume of the added Tris solution, washing and filtering for multiple times until residues cannot permeate, and finishing the clarifying and filtering process;
(3) carrying out ion exchange on the porcine circovirus ORF2 protein clear solution obtained in the step (2) by using an ion exchange column, and then eluting to obtain porcine circovirus ORF2 protein purification eluent; the filler of the ion exchange column is Capto SP ImpRes; when the ion exchange column is used for carrying out ion exchange, the sample loading linear flow rate of the porcine circovirus ORF2 protein clear solution is 30-100cm/h, the pressure is controlled to be less than 2.50bar, and the sample loading amount is controlled to be 70-80 mg/ml BSA; the elution solution is 0.5-2 mol/L NaCl solution, and the elution flow rate is 30-100 cm/h;
(4) desalting the porcine circovirus ORF2 protein purification eluent obtained in the step (3) by using a G25 chromatographic column, and eluting to obtain a porcine circovirus ORF2 protein purification solution; the packing of the G25 gel chromatographic column is as follows: SepHadex G-25; when the G25 chromatographic column is used for desalting, the sample loading linear flow rate of the porcine circovirus ORF2 protein purification eluent is 45-100 cm/h, the pressure is less than 2.0bar, the sample loading amount is controlled to be 10-30% of the column volume, and the eluent used for elution is 0.01mol/L PBS solution;
(5) and (4) sterilizing and filtering the protein purified liquid obtained in the step (4) to obtain the high-purity porcine circovirus ORF2 protein liquid.
2. The method for mass production of highly pure porcine circovirus ORF2 protein according to claim 1, which
Is characterized in that the addition amount of the chitosan in the step (1) is 1-10% of the mass of the porcine circovirus ORF2 protein culture solution.
3. The method for mass production of highly pure porcine circovirus ORF2 protein according to claim 1,
the method is characterized in that TMP adopted in the clarification and filtration of the hollow fiber column in the step (2) is 2.0-3.0 psi, and the flow rate of a liquid inlet end is controlled to be 60-80L/min.
4. The method for mass production of highly pure porcine circovirus ORF2 protein according to claim 1,
the method is characterized in that the hollow fiber column in the step (2) has the following model: CFP-2-E-55; the type of the ion exchange column in the step (3) is as follows: BPG300/500 Column; the model of the G25 gel chromatographic column in the step (4) is as follows: BPG450/1000 Column.
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