CN113980103A

CN113980103A - mSEB antigen protein purification method

Info

Publication number: CN113980103A
Application number: CN202111356399.3A
Authority: CN
Inventors: 刘冬; 陈克平; 张仁怀; 樊钒; 朱冲; 高婧; 马小洪; 杨茜; 赵丹
Original assignee: Chengdu Olymvax Biopharmaceuticals Inc
Current assignee: Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2022-01-28
Anticipated expiration: 2041-11-16
Also published as: CN113980103B

Abstract

The invention discloses a method for purifying mSEB antigen protein, which solves the technical problems that mSEB protein stock solution obtained by an mSEB protein purification process in the prior art is low in purity, poor in storage stability and capable of causing purity reduction. It comprises the following steps: s1, crushing thalli; s2, clarifying and filtering; s3, SPFF column chromatography; s4, SPHP column chromatography; s5, Phenyl HP column chromatography; s6, G25 column chromatography; s7, Q HP column chromatography; s8, preparing stock solution. The mSEB protein stock solution obtained by the purification method has high purity and good storage stability, and the purity can not be reduced.

Description

mSEB antigen protein purification method

Technical Field

The invention relates to the field of protein purification, in particular to a method for purifying mSEB antigen protein.

Background

Staphylococcus aureus is an important pathogenic bacterium causing hospital infection and community infection, and is difficult to clinically treat due to the development of multiple drug resistance, and a vaccine is urgently needed to be developed to control the infection and the prevalence.

Staphylococcus aureus is a distinct name of "Carmophilus", and is an important pathogenic bacterium causing nosocomial infections and community infections as a representative of gram-positive bacteria. The infection is characterized by acute and suppurative infection, and the local part can cause suppurative infection of skin, soft tissue and the like, and the suppurative infection is not healed for a long time; the whole body can cause serious infection and complication such as acute pneumonia, sepsis, endocarditis, septic arthritis, osteomyelitis and the like, and the death rate is up to 20%. Meanwhile, the exotoxin of staphylococcus aureus can also cause systemic lethal infections such as food poisoning, scald-like skin syndrome and toxic shock syndrome.

With the long-term and wide-spread use of antibiotics, the problem of bacterial resistance is increasingly prominent, and methicillin-resistant Staphylococcus aureus (MRSA), which is a typical representative, has been discovered for the first time since 1961, and is now one of the nosocomial infectious pathogenic bacteria with the highest infection rate in ICU wards, postoperative infections, burns, war wounds and the like all over the world. Meanwhile, the bacterial strain has the difficulty of clinical treatment due to the development of multiple drug resistance because of strong pathogenicity, wide transmission route and easy outbreak and prevalence, and is called as 'first superbacteria'.

MRSA are classified into hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA).

In 2009, reported by the U.S. CDC in the journal of new england medicine, approximately 9 million people are severely infected with MRSA each year in the united states, and approximately 2 million cases are fatal. The conference data of 'new progress of the MRSA hospital infection diagnosis and treatment strategy' in China in 12 months in 2010 shows that the incidence rate of MRSA infection in hospitals in China is about 8%, the average hospitalization time of infected patients in each hospital is prolonged by 14 days, the cost is increased by 6542 yuan, and the direct loss caused by hospital infection in China is over 150 million yuan each year. Meanwhile, due to the abuse of antibiotics, the separation and detection rate of clinical highly drug-resistant staphylococcus aureus, especially MRSA, is rising year by year. The national bacterial drug resistance monitoring report in 2011 in China shows that among 273808 strains of bacteria detected in 129 hospitals in China, the separation amount of gram-positive bacteria of staphylococcus aureus is the first, the MRSA detection rate accounts for 60% of detected staphylococcus aureus, and the wide drug resistance rate exceeds 40%. Based on the severe situation, MRSA has been classified as one of 12 pathogenic microorganisms which may have great influence on the health and health of people in the 21 st century in China. Currently, MRSA is classified as the three most problematic infectious diseases in the world with hepatitis b and AIDS, and is the top. At present, vancomycin is the last line of defense for treating MRSA infection, but with the successive emergence of vancomycin-resistant staphylococcus aureus (VRSA) in 2002, MRSA and VRSA with high drug resistance development are in a global spreading trend, so that clinical staphylococcus aureus infection faces a serious challenge of 'no drug cure'.

The research and development speed of antibiotics can not keep pace with the development speed of bacterial drug resistance, and the safe and effective staphylococcus aureus vaccine can become an effective means for preventing staphylococcus aureus infection. The WHO proposed a ' six-point policy package plan ' for resisting drug-resistant bacteria ' in 2011 and emphasized that the research and development of immune control products such as innovative vaccines and the like are mainly supported in the future. Therefore, the research on immune prevention and treatment of staphylococcus aureus infection is strengthened, and the development of a safe and effective novel staphylococcus aureus vaccine has important practical and strategic significance for effectively controlling wide infection and large-scale outbreak epidemic of staphylococcus aureus, greatly reducing morbidity of infection in a staphylococcus aureus hospital, spreading drug resistance and the like.

Summary of staphylococcus aureus vaccine mSEB antigen proteins:

SEB is superantigen exotoxin, is usually generated by pathogenic staphylococcus aureus, particularly MRSA, and is a main reason for causing human septic shock, systemic inflammatory reaction and food poisoning. SEB is superantigen, is not limited by MHC molecules, and is directly combined with T cell epitope under the condition of not depending on antigen recognition, thereby activating T cells, promoting the T cells to release a large amount of cytokines to cause clinical symptoms such as toxin shock syndrome and the like.

The Blast comparison proves that the SEB has high amino acid sequence conservation in each strain of the staphylococcus aureus. Researches in a mouse model prove that the SEB has better humoral response advantage than other antigens, and the prepared human SEB monoclonal antibody can effectively treat the common toxic shock syndrome of staphylococcus aureus infection. In 2010, the National Institute for Allergy and Infectious Diseases (NIAID) has used wild type SEB to prepare vaccines and has entered human phase I clinical studies.

The mSEB mutant mSEB with three sites (L45R, Y89A and Y94A) is constructed and obtained as one of the antigen components of the recombinant staphylococcus aureus vaccine by the applicant with reference to relevant research documents. In vivo and in vitro experiments prove that: mSEB no longer has toxicity and superantigen activity of wild SEB; animal safety evaluation and immune protection results also show that: mSEB eliminates enterotoxin activity, ensures good safety and retains good immunogenicity. The BALB/c mouse immune challenge protection rate generated by single-component immunity is more than 25%. In addition, no changes in other biological functions were found.

The existing purification process of mSEB protein is to directly capture protein by SPHP chromatography of a bacteria breaking solution so as to achieve the purpose of primary purification of target protein. But this step presents a higher back pressure during process upscaling.

The recombinant mSEB protein is translated and then exists in soluble form in the cytoplasm of E.coli. Due to the existence of 2 cysteines in the sequence, the reducing environment in the cytoplasm of the escherichia coli enables the sulfydryl of the cysteines to exist in an active reducing form, the original mSEB purification process has no special renaturation step, the formation of a disulfide bond is not complete enough, and partial cysteines exist in a free sulfydryl form. During stock storage, these free thiols spontaneously form disulfide bonds, and if cysteines between two protein molecules form disulfide bonds, mSEB dimers or multimers are produced, resulting in a decrease in purity.

The applicant has found that the prior art has at least the following technical problems:

1. the existing purification process of the mSEB protein is to directly capture the protein by SPHP chromatography of a broken bacteria solution, and the situation of high back pressure can occur in the process of process amplification;

2. in the prior art, the mSEB protein stock solution obtained by the mSEB protein purification process has low purity and poor storage stability, and the purity is reduced.

Disclosure of Invention

The invention aims to provide a method for purifying mSEB antigen protein, which aims to solve the technical problems that mSEB protein stock solution obtained by an mSEB protein purification process in the prior art is low in purity, poor in storage stability and prone to purity reduction.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for purifying an mSEB antigenic protein, comprising the steps of:

s1, cell disruption

Dissolving and crushing mSEB thallus to obtain a bacterial liquid A;

s2, clarifying and filtering

Centrifuging the bacterial liquid A in the step S1, and collecting a supernatant; clarifying and filtering the supernatant to obtain a bacterial liquid B;

s3, SPFF column chromatography

Performing primary purification on the bacterial liquid B obtained in the step S2 by filling a GE SPFF filler into a chromatographic column to obtain an SPFF eluent;

s4, SPHP column chromatography

Moderately purifying the SPFF eluent obtained in the step S3 by filling a GE SPHP packing into a chromatographic column to obtain an SPHP eluent;

s5, Phenyl HP column chromatography

E1 dilution of the SPHP eluate with ammonium sulphate solutionReleasing; volume ratio, SPHP eluent: ammonium sulfate solution 1: 0.5 to 5.0; the ammonium sulfate solution is 20mM PB +3M (NH)₄)₂SO₄The pH thereof is 6.0; obtaining an SPHP eluent diluent, and carrying out next purification;

e2, filling a chromatographic column with GE phenyl HP packing, and finely purifying the SPHP eluent diluent to obtain phenyl HP eluent;

s6, G25 column chromatography

Desalting and changing the phenyl HP eluent by adopting a G25 chromatographic column to obtain a G25 chromatographic solution;

s7, Q HP column chromatography

Carrying out chromatography on the G25 chromatographic solution by using a Q HP chromatographic column to obtain a Q HP flow-through solution;

s8 preparation of stock solution

Sterilizing and filtering the Q HP flow-through liquid in a biological safety cabinet or an aseptic isolator to obtain mSEB protein stock solution;

protein renaturation is also included before step S5 by performing SPFF column chromatography renaturation in step S3 or by performing protease cofactor renaturation in step S4.

Further, when PFF column chromatography renaturation is performed in step S3,

step S3 specifically includes:

a chromatographic column: the height of the column is 18-20 cm, the diameter of the column is 200mm, and the volume of the column is 5.5-6.5L;

mobile phase A: 20mM PB (pH6.0);

mobile phase B: 20mM PB +5mM GSH +0.5mM GSSG (pH 6.0);

mobile phase C: 20mM PB +1M NaCl (pH 6.0);

sample loading parameters:

sample loading flow rate: 36-72L/h; ultraviolet detection wavelength: UV280 nm; after the sample loading is finished, the column is balanced by using a mobile phase A;

parameters of complex equilibrium 1:

flow rate: the volume of the column is balanced for 2-3 times at 36-72L/h, and the column renaturation is carried out for 20CV by using the mobile phase B after the base line is leveled;

flow rate: 3.6-7.2L/h, and performing on-column renaturation for 4-20 h; after the next day of renaturation, the column is equilibrated with mobile phase a;

complex equilibrium 2 parameters:

flow rate: 36-72L/h, re-balancing 1-2 column volumes, and starting elution after the base line is leveled;

elution parameters: the flow rate is 36-72L/h; the gradient is: a: 0-50% of C, 10 column volumes;

detecting UV280 absorption spectrum in the elution process, and initially collecting UV value (mAU) more than 600 mAU; ending the collection of the UV value (mAU) less than 700mAU to obtain SPFF eluent;

step S4 specifically includes:

d1, diluting the SPFF eluent obtained in the step S3 by 2-5 times to obtain an SPFF eluent diluent; controlling the conductivity of the SPFF eluent diluent to be 2.0-8.0 ms/cm, and centrifuging the SPFF eluent diluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000-15000 g, centrifuging for 20-30 min at 2-8 ℃, and collecting supernatant;

d2, performing bacteria-reducing filtration on the supernatant by adopting a Cobetter 0.22 mu m filter membrane, and purifying by using a column;

d3, filling a chromatography column with GE SPHP packing, and moderately purifying the SPFF eluent diluent to obtain SPHP eluent.

Further, when the protease cofactor restoration is performed in step S4,

step S3 specifically includes:

spf column chromatography for primary purification:

buffer solution:

SPFF equilibrium liquid: PB with the pH value of 6.0-6.5 and the concentration of 20-50 mM;

SPFF eluent: 20mM PB +1M NaCl, pH 6.0;

sample loading flow rate: 36-72L/h;

and (3) re-balancing: the column was again equilibrated with equilibration buffer, equilibration flow rate: 36-72L/h, equilibrium volume: 5-8 CV until the UV value and the conductance are stable;

and (3) elution: the elution flow rate is 36-72L/h, the elution gradient is 0-50% B, and the CV is 10;

collection criteria: starting to collect peaks when UV280nm is 200-600 mAU, and stopping collecting when UV280nm is 700-200 mAU to obtain SPFF eluent;

step S4 specifically includes:

d1, diluting the SPFF eluent obtained in the step S4 by 2-5 times to obtain an SPFF eluent diluent; controlling the conductivity of the SPFF eluent diluent to be 2.0-8.0 ms/cm, and adding CuSO with the final concentration of 1-50 mu mM₄Stirring the solution at a rotating speed of 50-100 RPM, and keeping the temperature of 2-8 ℃ for 4-20 h;

d2, centrifuging the SPFF eluent diluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000-15000 g, centrifuging for 20-30 min at 2-8 ℃, and collecting supernatant;

d3, performing bacteria-reducing filtration on the supernatant by adopting a Cobetter 0.22 mu m filter membrane, and purifying by using a column;

d4, filling a chromatography column with GE SPHP packing, moderately purifying the dilution liquid of the SPFF eluent, when the SPHP column chromatography is moderately purified,

buffer solution:

SPHP equilibrium liquid: PB with a pH of 6.0-6.5 and a concentration of 20-50 mM;

SPHP eluent: 20mM PB +1M NaCl, pH 6.0-6.5;

sample loading flow rate: 12-24L/h;

and (3) re-balancing: re-balancing the chromatographic column by using a balancing buffer solution at the flow rate of 12-24L/h; equilibrium volume: 2-5 CV until the UV value and the conductance are stable;

and (3) elution: the elution flow rate is 12-24L/h, the elution gradient is 0-50% B, and the CV is 10;

collection criteria: and starting to collect peaks when the UV280nm is 200-600 mAU, and stopping collecting when the UV280nm is 1000-200 mAU to obtain SPHP eluent.

Further, in step S1, the cell disruption specifically includes:

a1, dissolving mSEB thallus, and according to the mass-volume ratio, the mSEB thallus: cell lysis solution 1: 15-25, and dissolving the mSEB thalli at the temperature not more than 20 ℃;

a2, crushing mSEB thallus, and crushing for 2-4 times by using a homogenizer at the temperature of no more than 20 ℃ and the pressure of 700-800 bar after the mSEB thallus is dissolved to obtain a bacterium liquid A.

Further, in the step A1, the cell lysis solution is 20 to 50mM PB and has a pH of 6.0 to 6.5.

Further, in step S2, the clarifying and filtering specifically includes:

b1, centrifuging the bacterial liquid A in the step S1 by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000-15000 g, centrifuging for 20-30 min at 2-8 ℃, and collecting supernatant;

b2, clarifying and filtering the supernatant collected in the step B1 by using a Cobetter 0.6-0.8 μm or Pall PDH4 deep filter plate.

Furthermore, when Phenyl HP column chromatography is used for fine purification in the step S5,

a chromatographic column: the height of the column is 9-11 cm, the diameter of the column is 140mm, and the volume of the column is 1-2L;

buffer solution:

phenyl HP equilibrium liquid: 20 to 50mM PB +0.25 to 2.5M (NH)₄)₂SO₄The pH value is 6.0-6.5;

phenyl HP eluent: PB with a pH of 6.0-6.5 and a concentration of 20-50 mM;

sample loading flow rate: 12-24L/h;

and (3) re-balancing: the column was again equilibrated with equilibration buffer, equilibration flow rate: 12-24L/h, equilibrium volume: 3-5 CV until UV value, pH value and electric conductivity are stable;

and (3) elution: elution flow rate: 12-24L/h, elution gradient: 0-100% B,10 CV;

collecting samples: collecting the eluent, wherein the collection is started when the UV280nm is 500-1500 mAU, and the collection is stopped when the UV280nm is 1000-200 mAU, so as to obtain phenyl HP eluent.

Furthermore, in the step S6, when desalting and liquid changing are carried out by G25 column chromatography,

a chromatographic column: the height of the column is about 28-30 cm, the diameter of the column is 300mm, and the volume of the column is 20-22L;

balance liquid: 10-50 mML-His +0.9% -1.5% NaCl, and the pH value is 6.0-6.5;

sample loading flow rate: 50-80L/h, and RT is 15 min;

the sample loading amount is less than or equal to 30 percent of the volume of the column bed;

detecting the absorption of the eluent UV280, starting collection when the UV280nm is more than or equal to 50mAU, and stopping collection when the UV280nm is less than or equal to 50mAU to obtain G25 chromatographic solution; if the chromatography is carried out in several times, the eluates are combined.

Furthermore, in the step S7, when the Q HP column chromatography is used for chromatography,

balance liquid: 10-50 mM L-His +0.9% -1.5% NaCl, the pH value is 6.0-6.5;

sample loading flow rate: 12-24L/h;

collecting flow-through liquid, and collecting standard: when the UV280nm is more than or equal to 50mAU, the collection is started, and when the UV280nm is less than or equal to 50mAU, the collection is stopped, and Q HP flow-through liquid is obtained.

Further, in the step S8, a 0.22 μm filter is used for the sterile filtration.

Based on the technical scheme, the embodiment of the invention can at least produce the following technical effects:

the mSEB protein stock solution obtained by the mSEB antigen protein purification method provided by the invention has high purity and good storage stability, and the purity can not be reduced, and the method has the following advantages:

(1) according to the invention, a clarification filtration step is added, so that fine particles, a large amount of nucleic acid and other impurities in the bacterial liquid A are removed, the clarity of the bacterial liquid B is improved, the high column pressure of subsequent chromatographic packing is reduced, and the service life of the packing is prolonged;

(2) the invention adds SPFF column chromatography (SPFF cation chromatography) as a primary purification step to quickly capture the target protein, so that the target protein is concentrated and stabilized from the bacteria-breaking supernatant with complex components; the SPHP column chromatography (SPHP cation chromatography) is set as a moderate purification step to remove a large amount of impurities contained in the mSEB thallus that have not been removed in the SPFF step, such as HCPs, nucleic acids, enzymes and possibly aggregates; and, in the primary purification or moderate purification step, a protein renaturation step is added, under the influence of an oxidation environment, 2 cysteines of the mSEB can be oxidized, active sulfydryl is changed into an inert disulfide bond, the formation of the disulfide bond is realized, the stability of the protein is increased, and dimers and polymers formed by mismatching the disulfide bond of the mSEB protein during renaturation are removed during SPHP column chromatography and Phenyl HP column chromatography, so that the purity and the stability of the stock solution are improved;

(4) according to the invention, Phenyl HP column chromatography (PHP hydrophobic chromatography) is set as a fine purification step, the gradient elution peak-collecting standard is optimized, and trace impurities are removed, so that all indexes of the target protein meet the quality standard requirements;

(5) according to the invention, G25 chromatography is adopted for buffer solution replacement, Q HP chromatography is adopted for effective removal of endotoxin, the steps are effectively linked, and finally a sample is sterilized and filtered by a 0.22-micron filter membrane and then subpackaged to obtain a protein stock solution for storage.

Drawings

FIG. 1 is an electrophoretogram of a stock mSEB protein solution obtained in comparative example 1;

FIG. 2 is an electrophoretogram of a stock mSEB protein solution obtained in example 1;

FIG. 3 is an electrophoretogram of an intermediate process sample of comparative example 1;

FIG. 4 is an electrophoretogram of a sample of the intermediate process of example 1.

Detailed Description

Example 1:

the invention provides a method for purifying mSEB antigen protein, which comprises the following steps:

s1, cell disruption

A1, dissolving mSEB thallus, and according to the mass-volume ratio, the mSEB thallus: cell lysis solution 1: 20, dissolving the mSEB thalli at the temperature not higher than 20 ℃; the thallus dissolving solution is PB with the pH value of 6.0 and 20 mM;

a2, crushing mSEB thallus, and crushing 3 times by using a homogenizer at the temperature of no more than 20 ℃ and the pressure of 750bar after the mSEB thallus is dissolved to obtain a bacterium liquid A; sampling 1 ml/branch of supernatant of the bacterial liquid A and multiplying 20 branches to obtain a sample I;

s2, clarifying and filtering

B1, centrifuging the bacterial liquid A in the step S1 by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000g, centrifuging for 20min at 2-8 ℃, and collecting supernatant;

b2, clarifying and filtering the supernatant collected in the step B1 by using Cobetter 0.6-0.8 mu m to obtain a bacterial liquid B; detecting the comprehensive turbidity value before and after filtration, and recording turbidity data; sampling the bacterial liquid B into 1 ml/count multiplied by 20 counts to obtain a sample II;

s3, SPFF column chromatography

And (3) primarily purifying the bacterial liquid B obtained in the step (S2) by adopting a GE SPFF filler filled chromatographic column, wherein when the SPFF column chromatography is used for primary purification:

a chromatographic column: the height of the column was about 19cm, the diameter of the column was 200mm, and the volume of the column was about 6.0L.

Buffer solution:

SPFF equilibrium liquid: PB, pH6.0, 20 mM;

SPFF eluent: 20mM PB +1M NaCl, pH 6.0;

sample loading flow rate: 36-72L/h;

and (3) re-balancing: the column was again equilibrated with equilibration buffer, equilibration flow rate: 54L/h, equilibrium volume: 6CV until UV value and stable conductance;

and (3) elution: the elution flow rate is 54L/h, the elution gradient is 0-50% B, and the CV is 10;

collection criteria: starting to collect peaks when UV280nm equals 400mAU and stopping collecting when UV280nm equals 300mAU, resulting in SPFF eluent;

recording the volume of the eluted SPFF liquid, and sampling 1 ml/count multiplied by 20 counts of the SPFF eluent to obtain a sample;

regeneration: regeneration was performed by washing the column 1-2CV with 20mM PB +1M NaCl (pH 6.0);

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

and (3) storage: preserving the chromatographic column by using 0.01M NaOH or 20% ethanol solution;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S4 by 3 times to obtain an SPFF eluent diluent; controlling the conductivity of the SPFF eluent diluent to be 5.0ms/cm, and adding the final concentrationCuSO at 25. mu.mM₄Stirring the solution at the rotating speed of 75RPM, and keeping the temperature at 5 ℃ for 12 h;

d2, centrifuging the SPFF eluent diluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000g, centrifuging for 20min at 2-8 ℃, and collecting supernatant;

a chromatographic column: the height of the column is about 19cm, the diameter of the column is 200mm, and the volume of the column is about 6.0L;

buffer solution:

SPHP equilibrium liquid: PB, pH6.0, 20 mM;

SPHP eluent: 20mM PB +1M NaCl, pH 6.0;

sample loading flow rate: 18L/h;

and (3) re-balancing: re-equilibrating the chromatographic column with an equilibration buffer solution at the flow rate of 18L/h; equilibrium volume: 3CV until UV value and stable conductance;

and (3) elution: the elution flow rate is 18L/h, the elution gradient is 0-50% B, and the CV is 10;

collection criteria: starting to collect peaks when UV280nm is 400mAU, and stopping collecting when UV280nm is 800mAU to obtain SPHP eluent;

recording the volume of the SPHP eluent, and sampling 1 ml/branch multiplied by 20 branches of the SPHP eluent to obtain a sample;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s5, Phenyl HP column chromatography

E1, diluting the SPHP eluent with an ammonium sulfate solution; volume ratio, SPHP eluent: ammonium sulfate solution 1: 3.0; the ammonium sulfate solution is 20mM PB +3M (NH)₄)₂SO₄The pH thereof is 6.0; obtaining the diluted solution of the SPHP eluent, and carrying out the next stepPurifying;

e2, filling a chromatography column with GE Phenyl HP packing, finely purifying the diluted solution of the SPHP eluent, when finely purifying Phenyl HP column chromatography,

a chromatographic column: the height of the column is about 10cm, the diameter of the column is 140mm, and the volume of the column is about 1.5L;

buffer solution:

phenyl HP equilibrium liquid: 20mM PB +1.5M (NH)₄)₂SO₄The pH thereof is 6.0;

phenyl HP eluent: PB, pH6.0, 20 mM;

sample loading flow rate: 18L/h;

and (3) re-balancing: the column was again equilibrated with equilibration buffer, equilibration flow rate: 18L/h, equilibrium volume: 4CV until UV value, pH and conductivity are stable;

and (3) elution: elution flow rate: 18L/h, elution gradient: 0-100% B,10 CV;

collecting samples: collecting eluate, starting with UV280nm (1000 mAU), and stopping collection when UV280nm (200 mAU) to obtain phenyl HP eluate;

recording the volume of phenyl HP eluent, wherein the sampling of phenyl HP eluent is 1 ml/branch multiplied by 20, and is sample;

regeneration: regeneration was performed by washing the column 1-2CV with 20mM PB (pH 6.0);

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s6, G25 column chromatography

Desalting and liquid changing are carried out on phenyl HP eluent by adopting a G25 chromatographic column, when desalting and liquid changing are carried out by adopting a G25 chromatographic column,

a chromatographic column: the height of the column is about 29cm, the diameter of the column is 300mm, and the volume of the column is about 20L;

balance liquid: 10mM L-His +0.9% NaCl, pH 6.0;

sample loading flow rate: 50-80L/h;

detecting the absorption of the eluent UV280, starting collection when the UV280nm is 50mAU, and stopping collection when the UV280nm is less than or equal to 50mAU (if fractional chromatography is adopted, combining eluents for each time) to obtain G25 chromatographic solution;

mixing the eluates, and sampling at a rate of 1ml per branch multiplied by 20 to obtain a sample;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s7, Q HP column chromatography

Performing chromatography on the G25 chromatographic solution by using a Q HP chromatographic column,

balance liquid: 10mM L-His +0.9% NaCl, pH 6.0;

sample loading flow rate: 18L/h;

collecting flow-through liquid, and collecting standard: collecting when UV280nm is 50mAU, stopping collecting when UV280nm is 40mAU, and obtaining Q HP flow-through liquid;

sampling Q HP flow-through liquid by 1 ml/branch multiplied by 20 branch and is sample;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s8 preparation of stock solution

And (4) performing sterile filtration on the Q HP flow-through solution in a biological safety cabinet or a sterile isolator by adopting a 0.22 mu m filter to obtain a mSEB protein stock solution.

Example 2:

s1, cell disruption

A1, dissolving mSEB thallus, and according to the mass-volume ratio, the mSEB thallus: cell lysis solution 1: 25, dissolving the mSEB thalli at the temperature not higher than 20 ℃; the thallus dissolving solution is PB with the pH value of 6.5 and the concentration of 50 mM;

a2, crushing mSEB thallus, and crushing for 2 times at a temperature not more than 20 ℃ and a pressure of 800bar by using a homogenizer after the mSEB thallus is dissolved to obtain a bacterium liquid A; sampling 1 ml/branch of supernatant of the bacterial liquid A and multiplying 20 branches to obtain a sample I;

s2, clarifying and filtering

B1, centrifuging the bacterial liquid A in the step S1 by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 15000g, centrifuging for 30min at 2 ℃, and collecting supernatant;

s3, SPFF column chromatography

Buffer solution:

SPFF equilibrium liquid: PB, pH6.5, 50 mM;

SPFF eluent: 20mM PB +1M NaCl, pH 6.5;

sample loading flow rate: 72L/h;

and (3) re-balancing: the column was again equilibrated with equilibration buffer, equilibration flow rate: 72L/h, equilibrium volume: 8CV until UV value and stable conductance;

and (3) elution: the elution flow rate is 72L/h, the elution gradient is 0-50% B, and the CV is 10;

collection criteria: starting to collect peaks when UV280nm equals 600mAU and stopping collecting when UV280nm equals 300mAU to obtain SPFF eluent;

regeneration: regeneration was performed by flushing the column 1-2CV with 50mM PB +1M NaCl (pH 6.5);

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S4 by 5 times to obtain an SPFF eluent diluent; controlling the conductivity of the SPFF eluent diluent to be 8.0ms/cm, and adding CuSO with the final concentration of 50 mu mM₄Stirring the solution at the rotating speed of 100RPM and preserving the heat at the temperature of 8 ℃ for 4 hours;

d2, centrifuging the SPFF eluent diluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 15000g, centrifuging for 30min at 2-8 ℃, and collecting supernatant;

buffer solution:

SPHP equilibrium liquid: PB, pH6.5, 50 mM;

SPHP eluent: 20mM PB +1M NaCl, pH 6.5;

sample loading flow rate: 24L/h;

and (3) re-balancing: re-balancing the chromatographic column by using an equilibrium buffer solution at the flow rate of 24L/h; equilibrium volume: 5CV until UV value and stable conductance;

and (3) elution: the elution flow rate is 24L/h, the elution gradient is 0-50% B, and the CV is 10;

collection criteria: starting to collect peaks when UV280nm is 600mAU, stopping collecting when UV280nm is 200mAU, obtaining SPHP eluent;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s5, Phenyl HP column chromatography

E1, diluting the SPHP eluent with an ammonium sulfate solution; volume ratio, SPHP eluent: sulfurAmmonium salt solution ═ 1: 5.0; the ammonium sulfate solution is 20mM PB +3M (NH)₄)₂SO₄Obtaining the diluted solution of the SPHP eluent with the pH value of 6.0, and carrying out next purification;

buffer solution:

phenyl HP equilibrium liquid: 50mM PB +2.5M (NH)₄)₂SO₄The pH is 6.5;

phenyl HP eluent: PB, pH6.5, 50 mM;

sample loading flow rate: 24L/h;

and (3) re-balancing: the column was again equilibrated with equilibration buffer, equilibration flow rate: 24L/h, equilibrium volume: 5CV until UV value, pH and conductivity are stable;

and (3) elution: elution flow rate: 24L/h, elution gradient: 0-100% B,10 CV;

collecting samples: collecting eluent, starting to collect the eluent with UV280nm equal to 1500mAU, and stopping collecting the eluent with UV280nm equal to 500mAU to obtain phenyl HP eluent;

regeneration: regeneration was performed by flushing the column with 1-2CV of 50mM PB (pH 6.5);

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s6, G25 column chromatography

balance liquid: 50mM L-His + 1.5% NaCl, pH 6.5;

sample loading flow rate: 50-80L/h;

detecting the absorption of the eluent UV280, starting collection when the UV280nm is 50mAU, and stopping collection when the UV280nm is 50mAU (if fractional chromatography is adopted, combining eluents for each time) to obtain G25 chromatographic solution;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s7, Q HP column chromatography

balance liquid: 50mM L-His + 1.5% NaCl, pH 6.5;

sample loading flow rate: 12-24L/h;

collecting flow-through liquid, and collecting standard: collecting when UV280nm equals 60mAU, and stopping collecting when UV280nm equals 50mAU to obtain Q HP flow-through;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s8 preparation of stock solution

Example 3:

s1, cell disruption

A1, dissolving mSEB thallus, and according to the mass-volume ratio, the mSEB thallus: cell lysis solution 1: 15, dissolving the mSEB thalli at the temperature not higher than 20 ℃; the thallus dissolving solution is PB with pH of 6.2 and 35 mM;

a2, crushing mSEB thallus, and crushing 4 times by using a homogenizer at the temperature of no more than 20 ℃ and the pressure of 700bar after the mSEB thallus is dissolved to obtain a bacterial liquid A; sampling 1 ml/branch of supernatant of the bacterial liquid A and multiplying 20 branches to obtain a sample I;

s2, clarifying and filtering

B1, centrifuging the bacterial liquid A in the step S1 by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 13500g, centrifuging at 2 ℃ for 25min, and collecting supernatant;

b2, clarifying and filtering the supernatant collected in the step B1 by using a Pall PDH4 deep filter plate to obtain a bacterial liquid B; detecting the comprehensive turbidity value before and after filtration, and recording turbidity data; sampling the bacterial liquid B into 1 ml/count multiplied by 20 counts to obtain a sample II;

s3, SPFF column chromatography

Buffer solution:

SPFF equilibrium liquid: PB, pH6.2, 35 mM;

SPFF eluent: 20mM PB +1M NaCl, pH 6.2;

sample loading flow rate: 36L/h;

and (3) re-balancing: the column was again equilibrated with equilibration buffer, equilibration flow rate: 36L/h, equilibrium volume: 5CV until UV value and stable conductance;

and (3) elution: the elution flow rate is 36L/h, the elution gradient is 0-50% B, and the CV is 10;

collection criteria: starting to collect peaks when UV280nm equals 200mAU and stopping collecting when UV280nm equals 700mAU, resulting in SPFF eluent;

regeneration: regeneration was performed by washing the column 1-2CV with 35mM PB +1M NaCl (pH 6.2);

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S4 by 2 times to obtain an SPFF eluent diluent; controlling the electric conductivity of the eluent diluent of SPFF to be 2.0ms/cm, and adding CuSO with the final concentration of 1 mu mM₄Stirring the solution at the rotating speed of 50RPM and preserving the heat at the temperature of 2 ℃ for 20 hours;

d2, centrifuging the SPFF eluent diluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12500g, centrifugating at 2 deg.C for 25min, and collecting supernatant;

buffer solution:

SPHP equilibrium liquid: PB, pH6.2, 35 mM;

SPHP eluent: 20mM PB +1M NaCl, pH 6.2;

sample loading flow rate: 12L/h;

and (3) re-balancing: re-balancing the chromatographic column by using an equilibrium buffer solution at the flow rate of 12L/h; equilibrium volume: 2CV until UV value and stable conductance;

and (3) elution: the elution flow rate is 12L/h, the elution gradient is 0-50% B, and the CV is 10;

collection criteria: starting to collect peaks when UV280nm is 200mAU, stopping collection when UV280nm is 1000mAU, resulting in SPHP eluate;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s5, Phenyl HP column chromatography

E1, diluting the SPHP eluent with an ammonium sulfate solution; volume ratio, SPHP eluent: ammonium sulfate solution 1: 0.5; the ammonium sulfate solution is 20mM PB +3M (NH)₄)₂SO₄The pH thereof is 6.0; obtaining an SPHP eluent diluent, and carrying out next purification;

buffer solution:

phenyl HP equilibrium liquid: 35mM PB +1.5M (NH)₄)₂SO₄The pH thereof is 6.2;

phenyl HP eluent: PB, pH6.2, 35 mM;

sample loading flow rate: 12L/h;

and (3) re-balancing: the column was again equilibrated with equilibration buffer, equilibration flow rate: 12L/h, equilibrium volume: 3CV until UV value, pH and conductivity are stable;

and (3) elution: elution flow rate: 12L/h, elution gradient: 0-100% B,10 CV;

collecting samples: collecting eluate, starting with UV280nm (500 mAU), and stopping collection when UV280nm (1000 mAU) to obtain phenyl HP eluate;

regeneration: regeneration was performed by washing the column 1-2CV with 35mM PB (pH 6.2);

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s6, G25 column chromatography

balance liquid: 35mM L-His + 1.2% NaCl, pH 6.2;

sample loading flow rate: 50-80L/h;

detecting the absorption of the eluent UV280, starting collection when the UV280nm is 55mAU, and stopping collection when the UV280nm is 45mAU (combining eluents for each time if fractional chromatography is performed), thus obtaining G25 chromatographic solution;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s7, Q HP column chromatography

balance liquid: 35mM L-His + 1.2% NaCl, pH 6.2;

sample loading flow rate: 12L/h;

collecting flow-through liquid, and collecting standard: collecting when UV280nm equals 55mAU, and stopping collecting when UV280nm equals 45mAU to obtain Q HP flow-through;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s8 preparation of stock solution

Example 4:

this example differs from example 1 in that:

s3, SPFF column chromatography

mobile phase A: 20mM PB (pH6.0);

mobile phase B: 20mM PB +5mM GSH +0.5mM GSSG (pH 6.0);

mobile phase C: 20mM PB +1M NaCl (pH 6.0);

sample loading parameters:

sample loading flow rate: 54L/h; ultraviolet detection wavelength: UV280 nm; after the sample loading is finished, the column is balanced by using a mobile phase A;

parameters of complex equilibrium 1:

flow rate: 54L/h, 3 column volumes are subjected to rebalancing, and after the base line is leveled, the column renaturation is carried out by 20CV by using the mobile phase B;

flow rate: 5.4L/h, and the column renaturation is carried out for 12h overnight; after the next day of renaturation, the column is equilibrated with mobile phase a;

complex equilibrium 2 parameters:

flow rate: 54L/h, repeatedly balancing 2 column volumes, and starting elution after the base line is leveled;

elution parameters: the flow rate is 54L/h; the gradient is: a: 0-50% of C, 10 column volumes;

detecting a UV280 absorption spectrum in the elution process, and initially collecting a UV value (mAU) of 620 mAU; ending the collection of the UV value (mAU) 680mAU to obtain SPFF eluent;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 by 3 times to obtain an SPFF eluent diluent; controlling the conductivity of the SPFF eluent diluent to be 5.0ms/cm, centrifuging the SPFF eluent diluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000g, centrifuging for 20min at 5 ℃, and collecting supernatant;

d3, filling a chromatography column with GE SPHP packing, moderately purifying the dilution liquid of the SPFF eluent, when the SPHP column chromatography is moderately purified,

buffer solution:

SPHP equilibrium liquid: PB, pH6.0, 20 mM;

SPHP eluent: 20mM PB +1M NaCl, pH 6.0;

sample loading flow rate: 18L/h;

collection criteria: starting to collect peaks when UV280nm equals 400mAU, stopping collecting when UV280nm equals 200mAU, obtaining SPHP eluent;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

and (3) storage: the column was preserved using 0.01M NaOH or 20% ethanol solution.

Otherwise, the mSEB protein stock solution was obtained as in example 1.

Example 5:

this example differs from example 1 in that:

s3, SPFF column chromatography

a chromatographic column: the height of the column is about 19cm, the diameter of the column is 200mm, and the volume of the column is 6.0L;

mobile phase A: 20mM PB (pH6.0);

mobile phase B: 20mM PB +5mM GSH +0.5mM GSSG (pH 6.0);

mobile phase C: 20mM PB +1M NaCl (pH 6.0);

sample loading parameters:

sample loading flow rate: 72L/h; ultraviolet detection wavelength: UV280 nm; after the sample loading is finished, the column is balanced by using a mobile phase A;

parameters of complex equilibrium 1:

flow rate: 72L/h, carrying out repeated balance for 2-3 column volumes, and carrying out on-column renaturation by using a mobile phase B for 20CV after a base line is leveled;

flow rate: 7.2L/h, and keeping the column renaturation overnight for 4 h; after the next day of renaturation, the column is equilibrated with mobile phase a;

complex equilibrium 2 parameters:

flow rate: 72L/h, re-balancing 2 column volumes, and starting elution after the base line is leveled;

elution parameters: the flow rate is 72L/h; the gradient is: a: 0-50% of C, 10 column volumes;

detecting UV280 absorption spectrum in the elution process, and initially collecting UV value (mAU) more than 600 mAU; ending the collection of the UV value (mAU) less than 700mAU to obtain SPFF eluent; recording the volume of the eluted SPFF liquid, and sampling 1 ml/count multiplied by 20 counts of the SPFF eluent to obtain a sample;

regeneration: regeneration was performed by flushing the column 1-2CV with 50mM PB +1M NaCl (pH 6.5); 10

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 by 5 times to obtain an SPFF eluent diluent; controlling the conductivity of the SPFF eluent diluent to be 8.0ms/cm, and centrifuging the SPFF eluent diluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 15000g, centrifuging for 30min at 2 ℃, and collecting supernatant;

buffer solution:

SPHP equilibrium liquid: PB, pH6.5, 50 mM;

SPHP eluent: 20mM PB +1M NaCl, pH 6.5;

sample loading flow rate: 24L/h; and (3) re-balancing: re-balancing the chromatographic column by using an equilibrium buffer solution at the flow rate of 24L/h; equilibrium volume: 5CV until UV value and stable conductance;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

Otherwise, the mSEB protein stock solution was obtained as in example 1.

Example 6:

this example differs from example 1 in that:

s3, SPFF column chromatography

mobile phase A: 20mM PB (pH6.0);

mobile phase B: 20mM PB +5mM GSH +0.5mM GSSG (pH 6.0);

mobile phase C: 20mM PB +1M NaCl (pH 6.0);

sample loading parameters:

sample loading flow rate: 36L/h; ultraviolet detection wavelength: UV280 nm; after the sample loading is finished, the column is balanced by using a mobile phase A;

parameters of complex equilibrium 1:

flow rate: the volume of 2 columns is rebalanced at 36L/h, and after the base line is leveled, the column renaturation is carried out by 20CV by using the mobile phase B;

flow rate: 3.6L/h, and keeping the column renaturation overnight for 20 h; after the next day of renaturation, the column is equilibrated with mobile phase a;

complex equilibrium 2 parameters:

flow rate: at 36L/h, re-balancing 1 column volume, and starting elution after the base line is leveled;

elution parameters: the flow rate is 36L/h; the gradient is: a: 0-50% of C, 10 column volumes;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 by 2 times to obtain an SPFF eluent diluent; controlling the conductivity of the SPFF eluent diluent to be 2.0ms/cm, centrifuging the SPFF eluent diluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000g, centrifuging for 30min at 8 ℃, and collecting supernatant;

buffer solution:

SPHP equilibrium liquid: PB, pH6.2, 35 mM;

SPHP eluent: 20mM PB +1M NaCl, pH 6.2;

sample loading flow rate: 12L/h;

CIP: flushing the chromatographic column 1-2CV with 1M NaOH solution for CIP; 5

Otherwise, the mSEB protein stock solution was obtained as in example 1.

Second, comparative example

Comparative example 1:

the difference from example 1 is: s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 by 2-5 times to obtain an SPFF eluent diluent; controlling the conductivity of the SPFF eluent diluent to be 2.0-8.0 ms/cm (without adding CuSO)₄Solution);

the rest of the same procedure as in example 1 gave a mSEB protein stock solution.

Third, Experimental example

1. The mSEB protein stock solutions obtained in examples 1 to 6 and comparative example 1 and the samples of the intermediate process were compared in terms of electrophoretic purity and examined by Image Lab analysis software of BioRad, as shown in FIGS. 1 to 4 and Table 1 below.

TABLE 1 comparison of mSEB protein purity of examples and comparative examples (unheated)

Example 1

Example 2

Example 3

Example 4

Example 5

Example 6

Comparative example 1

Sample 2

40.0％

42.1％

44.0％

43.5％

41.9％

40.9％

43.8％

Sample

3

95.3％

95.6％

85.4％

95.2％

94.7％

93.1％

89.6％

Sample iv

97.9％

98.2％

99.8％

97.9％

98.2％

97.2％

94.5％

Sample (v)

100.0％

98.7％

100.0％

99.7％

99.9％

98.9％

98.4％

Sample (c)

100.0％

99.8％

100.0％

99.7％

97.2％

Sample (c)

100.0％

99.8％

100.0％

99.9％

98.3％

mSEB protein stock solution

100.0％

97.4％

As can be seen from fig. 1-4 and table 1, after the mSEB is renatured, the sample sampled after the SPHP column chromatography can meet the 95% purity requirement, i.e., the sample can meet the quality standard, while the comparative example 1 can not, the comparative example 1 can reach 97% after Phenyl HP, but the highest purity of the embodiment of the invention can reach 100%, the purity of the mSEB protein stock solution obtained in the embodiments 1 to 6 can reach 100.0%, the purity standard of the stock solution can be met, and the purity is much higher than that of the stock solution in the comparative example 1; in addition, in comparative example 1, the data of the sample iv, sample iv and the mSEB protein stock solution are not repeated, because comparative example 1 is not renatured, the sample in the process is not repeated, and the purity is not good because aggregates are generated without renaturation.

2. The mSEB protein stock solutions obtained in examples 1 to 6 and comparative example 1 were stored at ultralow temperatures of-60 to-80 ℃ for 12 months, and purity measurements were performed after 1 month, 3 months, 6 months, 9 months and 12 months, respectively, and the results are shown in Table 2 below.

TABLE 2 comparison of mSEB protein stock stability

Time

Example 1

Example 2

Example 3

Example 4

Example 5

Example 6

Comparative example 1

0 month

100％

97.4％

1 month

100％

96.4％

3 month

99.7％

99.9％

99.3％

99.5％

99.6％

99.8％

96.0％

6 month

99.4％

99.7％

99.4％

99.5％

99.6％

94.5％

9 month

99.5％

99.7％

99.6％

99.2％

99.5％

99.6％

93.7％

12 month

99.3％

99.5％

99.2％

99.5％

Not determined

As can be seen from Table 2, the mSEB protein stock solutions obtained in examples 1-6 can meet the requirement of no significant reduction in purity for 12 months; while comparative example 1 was already below the quality standard for 6 months, stability was maintained for only 3 months. Since the test time is already lower than the standard at 6 months and 9 months, the test is not needed for 12 months, and the product is not qualified. The mSEB protein stock solutions obtained in examples 1-6 did not produce aggregates, and were of a purity close to zero. The method has good renaturation effect, can well solve the problem of mSEB polymers, and improves the purity of the mSEB protein stock solution and the storage stability of the mSEB protein stock solution.

Claims

1. A method for purifying mSEB antigen protein, which is characterized by comprising the following steps:

s1, cell disruption

Dissolving and crushing mSEB thallus to obtain a bacterial liquid A;

s2, clarifying and filtering

s3, SPFF column chromatography

s4, SPHP column chromatography

s5, Phenyl HP column chromatography

E1, diluting the SPHP eluent with an ammonium sulfate solution; volume ratio, SPHP eluent: ammonium sulfate solution = 1: 0.5 to 5.0; the ammonium sulfate solution is 20mM PB +3M (NH)₄）₂SO₄The pH thereof is 6.0; obtaining an SPHP eluent diluent, and carrying out next purification;

s6, G25 column chromatography

s7, Q HP column chromatography

s8 preparation of stock solution

2. The method for purifying mSEB antigen protein according to claim 1, wherein when PFF column chromatography renaturation is performed in step S3,

step S3 specifically includes:

mobile phase A: 20mM PB (pH6.0);

mobile phase B: 20mM PB +5mM GSH +0.5mM GSSG (pH 6.0);

mobile phase C: 20mM PB +1M NaCl (pH 6.0);

sample loading parameters:

parameters of complex equilibrium 1:

complex equilibrium 2 parameters:

elution parameters: the flow rate is 36-72L/h; the gradient is: a: c =0-50%, 10 column volumes;

step S4 specifically includes:

3. The method for purifying mSEB antigen protein according to claim 1, wherein when the protease cofactor renaturation is performed in step S4,

step S3 specifically includes:

spf column chromatography for primary purification:

buffer solution:

SPFF eluent: 20mM PB +1M NaCl, pH 6.0;

sample loading flow rate: 36-72L/h;

collection criteria: starting to collect peaks when UV280nm = 200-600 mAU, and stopping collecting when UV280nm = 700-200 mAU to obtain SPFF eluent;

step S4 specifically includes:

d1, diluting the SPFF eluent obtained in the step S4 by 2-5 times to obtain an SPFF eluent diluent; controlling dilution of SPFF eluentsThe conductivity is 2.0 to 8.0ms/cm, CuSO with the final concentration of 1 to 50 mu mM is added₄Stirring the solution at a rotating speed of 50-100 RPM, and keeping the temperature of 2-8 ℃ for 4-20 h;

buffer solution:

SPHP eluent: 20mM PB +1M NaCl, pH 6.0-6.5;

sample loading flow rate: 12-24L/h;

collection criteria: the peak starts to collect when UV280nm = 200-600 mAU, and stops collecting when UV280nm = 1000-200 mAU, and SPHP eluent is obtained.

4. The method for purifying the mSEB antigen protein according to claim 1, wherein in the step S1, the thallus disruption specifically comprises:

a1, dissolving mSEB thallus, and according to the mass-volume ratio, the mSEB thallus: cell lysis solution = 1: 15-25, and dissolving the mSEB thalli at the temperature not more than 20 ℃;

5. The method for purifying mSEB antigen protein according to claim 4, wherein in the step A1, the cell lysis solution is 20-50mM PB and has a pH of 6.0-6.5.

6. The method for purifying mSEB antigen protein according to claim 1, wherein in step S2, the clarification filtration is specifically:

7. The method for purifying mSEB antigen protein according to claim 1, wherein in the step S5, when Phenyl HP column chromatography is used for fine purification,

buffer solution:

phenyl HP equilibrium liquid: 20 to 50mM PB +0.25 to 2.5M (NH)₄）₂SO₄The pH value is 6.0-6.5;

phenyl HP eluent: PB with a pH of 6.0-6.5 and a concentration of 20-50 mM;

sample loading flow rate: 12-24L/h;

and (3) elution: elution flow rate: 12-24L/h, elution gradient: 0-100% B,10 CV;

collecting samples: collecting eluent, starting to collect the eluent with UV280nm = 500-1500 mAU, and stopping collecting the eluent with UV280nm = 1000-200 mAU to obtain phenyl HP eluent.

8. The method for purifying mSEB antigen protein according to claim 1, wherein in the step S6, when G25 column chromatography is used for desalting and liquid changing,

balance liquid: 10-50 mML-His +0.9% -1.5% NaCl, and the pH value is 6.0-6.5;

sample loading flow rate: 50-80L/h, and RT is 15 min;

9. The method for purifying mSEB antigen protein according to claim 1, wherein in the step S7, when the Q HP column chromatography is used for chromatography,

balance liquid: 10-50 mM L-His +0.9% -1.5% NaCl, the pH value is 6.0-6.5;

sample loading flow rate: 12-24L/h;

10. The method for purifying the mSEB antigen protein according to any one of claims 1 to 9, wherein in the step S8, a 0.22 μm filter is used for sterile filtration.