CN103266125A - Preparation method of diphtheria toxin mutant CRM197 - Google Patents
Preparation method of diphtheria toxin mutant CRM197 Download PDFInfo
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Abstract
The invention relates to a preparation method of a diphtheria toxin mutant CRM197. The preparation method is performed by expressing the recombinant diphtheria toxin mutant CRM197 in escherichia coli and then performing purification, and the purification method comprises the following steps of: performing denaturation and renaturation on an inclusion body, then firstly precipitating with saturated ammonium sulfate, performing ultra-filtration, liquid exchange and concentration, further purifying by utilizing anion exchange chromatography, finally sieving through a molecular sieve, and collecting target protein peaks to get high-purity protein samples. The method has the characteristics of short purification period, simplicity and convenience in operation, high product purity, strong process stability and the like.
Description
Technical field
The present invention relates to genetically engineered and protein purification technology, specifically, relate to expression and the purifying of diphtheria toxin muton CRM 197 in prokaryotic system.
Background technology
Diphtheria toxin (Diphtheria Toxin, DT) be to be about extracellular toxin about 58kD by infecting molecular weight that the genomic diphtheria corynebacterium of phagus beta (Corynebacterium diphtheriae) produces, it is by the eukaryotic peptide chain elongation factor of deactivation II (EF-2), the blocking-up cell protein is synthetic, causes necrocytosis.Toxin can form the A fragment of 193 amino-acid residues and the B fragment of 342 amino-acid residues through trypsin hydrolyzing, A fragment molecular weight is 22kD, be positioned at the toxin N-terminal, it enters in the cell can catalysis ADP-ribosylation, the eukaryotic elongation factor II of deactivation (EF-2); B fragment molecular weight is 38kD, is positioned at C-terminal, by striding film, transposition, receptor binding domain composition.CRM197(cross reacting material197) be a kind of mutant of diphtheria toxin, its 52nd amino acids sports Glu by Gly, causes the diphtheria toxin enzyme active sites to change, thereby can not be to the effect of cell toxigenicity.Though forfeiture enzymic activity and toxicity, CRM197 still preserves consistent with natural diphtheria toxin on antigenicity and immunogenicity.
At present domestic have b type hemophilus influenzae (Hib) combined vaccine (HBOC) with CRM197 as the combined vaccine of carrier, 7 valent pneumococcal conjugate vaccine (PCV, Prevnar), C group meningitis cocci combined vaccine, A+C group meningitis cocci combined vaccine, these combined vaccines are compared with polysaccharide vaccine has advantage, can be the adult, inducing specific immunne response among children and the children below 2 years old, and can continue the long period.And be that the diphtheria vaccine of antigenic component is also in development with CRM197.
It is reported that Nadav etc. have expressed the full gene of CRM197, portion gene fragment and fusion gene in bacterium typhosum strain CVD908-htrA, be used for observing the immunocompetence of rebuild CRM 197.Eliane etc. have realized the expression of CRM197 in cow mycobacteria (rBCG), and discovery rebuild CRM 197 can be induced the generation neutralizing antibody.Recent study finds that (heparin-binding epidermal growth factor HB-EGF) is the special cell-membrane receptor of diphtheria toxin to heparin associative list skin growth factor, increases at some tumour patient expression in vivo, as ovarian cancer patients.Studies have shown that CRM197 has the function in conjunction with HB-EGF equally, can be used as the special inhibitor of HB-EGF, be expected to be applied to tumour patient.Domestic research to CRM197 at present is less, and Zhou Jianhua etc. had once been realized the expression of CRM197 in subtilis.In order to satisfy the domestic market to the demand of CRM197, demand developing that a kind of diphtheria toxin muton CRM 197 efficiently expresses and the method for purifying urgently.
Summary of the invention
The preparation method who the purpose of this invention is to provide diphtheria toxin muton CRM 197.
In order to realize the object of the invention, the preparation method of a kind of diphtheria toxin muton CRM 197 of the present invention comprises that diphtheria toxin muton CRM 197 is in the step of expression in escherichia coli and purifying; Wherein, the step of protein purification comprises:
1) adopts intestinal bacteria with the recombinant protein c RM197 of inclusion body formal representation, after inclusion body process sex change and the renaturation, obtain containing the renaturation solution of target protein;
2) renaturation solution is behind ammonium sulfate precipitation, and the centrifuging and taking supernatant separates and concentrates by ultrafiltration, obtains ultrafiltration and concentration liquid;
3) above-mentioned ultrafiltration and concentration liquid is crossed ion exchange column and carry out purifying, obtain containing the classification component of target protein CRM197;
4) above-mentioned classification component is passed through molecular sieve system, obtain containing the target protein liquid of diphtheria toxin muton CRM 197.
Aforesaid preparation method, step 1) is specially: add 500mL sex change liquid in 5g target protein CRM197 inclusion body, behind the stirring and dissolving 60min, the centrifugal 10min of 12000rpm, collect supernatant in dialysis tubing, place 10L TE damping fluid current system, stir renaturation 24-72h in 4 ℃; Wherein, described sex change liquid contains 8mol/L urea, 1mL beta-mercaptoethanol, Tris-HCl50mmol/L, pH8.0.Sex change, refolding method also can adopt protein denaturation, renaturation method in common.
Aforesaid preparation method, step 2) carries out the ammonium sulfate concentrations that ammonium sulfate precipitation uses in and be 20-40%, preferred 20%.
Aforesaid preparation method, step 2) ultrafiltration is specially in: be that the ultra-filtration membrane of 10kD carries out balance with the TE damping fluid to ultra-fine filter and aperture earlier, the maintenance inlet and outlet pressure is respectively 1.3bar and 0.3bar, begins to dialyse to change liquid, remove the salt ion in the supernatant liquor, collect ultrafiltration and see through liquid; According to the method described above, again ultrafiltration is seen through liquid and concentrate, be concentrated into 1/10th of original volume, namely get ultrafiltration and concentration liquid.
Aforesaid preparation method, step 2) supernatant liquor of collecting in separates by ultrafiltration and concentrates, can disposablely carry out ultrafiltration and concentration, also can first ultrafiltration removal salt ion and foreign protein, carry out ultrafiltration and concentration again.
Aforesaid preparation method, step 3) is specially: earlier with TE damping fluid balance (the DEAE-Sepharose FF chromatography column of 1.6cm * 15cm), then ultrafiltration and concentration liquid upper prop is carried out purifying, carry out Balance Treatment with 3 column volumes of TE damping fluid flushing, to remove most of impurity, carry out wash-out with the anion-exchange chromatography post of the TE damping fluid that contains 0.5-1.5mol/L NaCl after to above-mentioned processing then, obtain containing the classification component of target protein CRM197.Wherein, the chromatography media of the employed anion-exchange chromatography post of anion-exchange chromatography is selected from sepharose (Sepharose) class, and aglucon is selected from diethyl aminoethyl (DEAE).Employed anion-exchange chromatography medium is selected DEAE-Sepharose FF chromatography media for use.
Aforesaid preparation method, the type of elution in the step 3) adopts the continuous gradient wash-out.
Aforesaid preparation method, step 4) is specially: earlier with TE damping fluid balance molecular sieve Sephacryl S-100, will contain the protein solution upper prop of above-mentioned classification component, when first peak occurring, begin to collect eluted protein liquid, namely get the target protein liquid that contains diphtheria toxin muton CRM 197.
Aforesaid preparation method, step 2), the TE buffer concentration that uses in step 3) and the step 4) is 10mmol/L-50mmol/L, pH value 7.0-8.0.
Aforesaid preparation method, diphtheria toxin muton CRM 197 in the step of expression in escherichia coli is: accession number is the gene order of the diphtheria toxin muton CRM 197 of CQ967258 in according to GenBank, carry out codon optimized, synthetic gene; Designing primer simultaneously, is template with synthetic gene, carries out pcr amplification; Amplified production is building up in the expression vector after checking order accurately, changes over to and carries out abduction delivering in the intestinal bacteria.
Wherein, primer sequence is:
Forward primer: 5 '-AGATCTATGGGTGCTGACGACGTTGTT-3 ',
Reverse primer: 5 '-CTCGAGATTAAGATTTGATTTCGA-3 '.
The present invention is the method for the reorganization diphtheria toxin muton CRM 197 being carried out purifying behind expression in escherichia coli to it, purification process comprises the steps: after inclusion body sex change, the renaturation, earlier through saturated ammonium sulphate, after ultrafiltration is changed liquid and is concentrated then, utilize anion-exchange chromatography to carry out purifying, cross molecular sieve at last, collect the target protein peak, can obtain highly purified protein sample.Present method has short, easy and simple to handle, characteristics such as product purity is high, technology stability is strong of purifying cycle.
For the recombinant protein c RM197 that adopts intestinal bacteria with the inclusion body formal representation; method provided by the invention is suitable for mass-producing and produces CRM197 fast; have that technology is simple, cost is low, purpose product yield height and pilot process be convenient to characteristics such as control, in addition the increase security of products.
Description of drawings
Fig. 1 is for utilizing the CRM197 mutant purity collection of illustrative plates behind the SDS-PAGE electrophoretic analysis purifying in the embodiment of the invention 2; Wherein, M is albumen Marker, and 1 was the sample behind the anion column, and 2 was the sample behind the molecular sieve.
Fig. 2 is the Western hybridization qualification result of recombinant protein c RM197 and positive control in this embodiment of the invention 3; Wherein, 1 is the recombinant protein c RM197 behind the purifying, 2 positive contrasts.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
Be the gene order of the diphtheria toxin muton CRM 197 of CQ967258 according to accession number among the GenBank, carry out codon optimized, synthetic gene, sequence is shown in SEQ ID No.1.
According to the gene of synthetic, design pcr amplification primer:
Forward primer: 5 '-AGATCTATGGGTGCTGACGACGTTGTT-3 '
Reverse primer: 5 '-CTCGAGATTAAGATTTGATTTCGA-3 '
Be template with synthetic gene, carry out pcr amplification.
The PCR reaction system is:
The PCR reaction conditions is: after 95 ℃ of initial sex change 5min, and according to 95 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 2min, carry out 30 circulations, and last 72 ℃ are extended 7min, to guarantee the complete of extension.
Amplified production is after checking order accurately, with the synthetic CRM197 gene fragment of Bgl II/Xho I double digestion, be connected with the pBAD-DEST49 carrier of cutting processing through same enzyme, 42 ℃ of heat shocks are transformed in the bacillus coli DH 5 alpha competent cell, screening positive clone, carry out enzyme and cut, the leakage of electricity swimming is identified, obtains expression vector pBAD-DEST49/CRM197.The positive expression carrier changes in the e. coli bl21, and picking transformant incubated overnight is then in 1:100(v:v) ratio transfer in the LB substratum that contains 0.1% ammonia benzyl resistance, 37 ℃, the 180r/min shaking table is cultured to OD
600Be 0.6 o'clock, in 1:100(v:v) ratio to add final concentration be 37 ℃ of abduction delivering 5h of pectinose of 20%.Centrifugal, collect thalline, smudge cells separates obtaining inclusion body.
The purifying of embodiment 2 diphtheria toxin muton CRM 197 albumen
May further comprise the steps:
1, adopts intestinal bacteria with the recombinant protein c RM197 of inclusion body formal representation, after inclusion body process sex change and the renaturation, obtain containing the renaturation solution of target protein.Be specially: add 500mL sex change liquid in 5g target protein CRM197 inclusion body, behind the stirring and dissolving 60min, the centrifugal 10min of 12000rpm collects supernatant in dialysis tubing, places 10L TE damping fluid current system, stirs renaturation 24-72h in 4 ℃; Wherein, described sex change liquid contains 8mol/L urea, 1mL beta-mercaptoethanol, Tris-HCl50mmol/L, pH8.0.
2, renaturation solution is behind 20% ammonium sulfate precipitation, and the centrifuging and taking supernatant separates and concentrates by ultrafiltration, and changes liquid with the TE damping fluid, obtains ultrafiltration and concentration liquid.Be specially: be that the ultra-filtration membrane of 10kD carries out balance with the TE damping fluid to ultra-fine filter and aperture earlier, keep inlet and outlet pressure to be respectively 1.3bar and 0.3bar, begin to dialyse and change liquid, remove the salt ion in the supernatant liquor, collect ultrafiltration through liquid; According to the method described above, again ultrafiltration is seen through liquid and concentrate, be concentrated into 1/10th of original volume.
3, above-mentioned ultrafiltration and concentration liquid is crossed the anion-exchange chromatography post and carry out purifying, obtain containing the classification component of target protein CRM197.Be specially: (1.6cm * 15cm), then ultrafiltration and concentration liquid upper prop is carried out purifying obtains containing the classification component of target protein CRM197 with TE damping fluid balance DEAE-Sepharose FF chromatography column earlier.Wherein, the chromatography media of the employed anion-exchange chromatography post of anion-exchange chromatography is selected from sepharose (Sepharose) class, and aglucon is selected from diethyl aminoethyl (DEAE).Employed anion-exchange chromatography medium is selected DEAE-Sepharose FF chromatography media for use.
4, with level pad the anion-exchange chromatography post is carried out balance, wash 3 column volumes, to remove most of impurity.
5, carry out wash-out with the anion-exchange chromatography post of the level pad that contains 1mol/L NaCl after to above-mentioned processing, collect eluted protein liquid.The purity of target protein can reach more than 80% in the protein liquid of gained.
6, earlier with TE damping fluid balance molecular sieve Sephacryl S-100, with above-mentioned protein solution upper prop, when first peak occurring, begin to collect eluted protein liquid, namely get the target protein liquid that contains diphtheria toxin muton CRM 197.The purity of target protein CRM197 can reach more than 95% in the gained protein liquid.
Fig. 1 is the CRM197 mutant purity collection of illustrative plates that utilizes behind the SDS-PAGE electrophoretic analysis purifying of the present invention; 1 was the sample behind the anion column, and 2 was the sample behind the molecular sieve.
The Western of embodiment 3 diphtheria toxin muton CRM 197 albumen detects
1, the separation gel of preparation 12% and 5% concentrated glue, sample carries out the SDS-PAGE electrophoresis.
2, electrotransfer: with separation gel, 1 and pvdf membrane in transfering buffering liquid the balance 15-20min of gel phase with size, 4 thick filter paper are cut into and gel phase with size, wetting in transfering buffering liquid; According to the order of filter paper, gel, pvdf membrane, filter paper, with the half-dried glue electrotransfer of Bio-Rad instrument, albumen is transferred on the pvdf membrane from the negative electrode to the anode, constant current 120mA, 90-100min finish electricity to be changeed.
3, sealing: pvdf membrane is put into confining liquid (1%BSA is dissolved in PBST), put shaker platform, the room temperature sealing is spent the night.
4, discard confining liquid, (horse lgG 1:2000), puts shaker platform, room temperature incubation 1h to add the diphtheria flocculoreaction toxinicide national standard product that dilute with confining liquid.
5, discard primary antibodie, PBST washes film 3 times, each 10-15min.
6, the anti-horse IgG of the rabbit antibody that adds the HRP mark that dilutes with confining liquid is in room temperature shaker platform incubation lh.
7, discard two and resist, wash film 3 times with PBST, each 10-15min.
8, add substrate DAB colour developing, use the purified water termination reaction in the 10min.
9, blot residual liquid on the film with filter paper, take pictures, saving result.Western hybridization detected result shows, is that the 58kD place produces specific band at molecular weight.
The toxicity test of embodiment 4 diphtheria toxin muton CRM 197 albumen
Experiment purpose: whether detect toxic reaction symptom
Respectively the cavy about body weight 250g is carried out subcutaneous injection 25ng diphtheria toxin or 250 μ gCRM197.Diphtheria toxin positive control treated animal is all dead in the 48h of injection back; And CRM197 test group animal all survives, and injection site is reactionless, and spirit is good, food is normal, tracing observation 30d, and body weight is stable to rise.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the preparation method of diphtheria toxin muton CRM 197 comprises that diphtheria toxin muton CRM 197 in the step of expression in escherichia coli and purifying, is characterized in that, the step of protein purification comprises:
1) adopts intestinal bacteria with the recombinant protein c RM197 of inclusion body formal representation, after inclusion body process sex change and the renaturation, obtain containing the renaturation solution of target protein;
2) renaturation solution is behind ammonium sulfate precipitation, and the centrifuging and taking supernatant separates and concentrates by ultrafiltration, obtains ultrafiltration and concentration liquid;
3) above-mentioned ultrafiltration and concentration liquid is crossed ion exchange column and carry out purifying, obtain containing the classification component of target protein CRM197;
4) above-mentioned classification component is passed through molecular sieve system, obtain containing the target protein liquid of diphtheria toxin muton CRM 197.
2. preparation method according to claim 1, it is characterized in that, step 1) is specially: add 500mL sex change liquid in 5g target protein CRM197 inclusion body, behind the stirring and dissolving 60min, the centrifugal 10min of 12000rpm, collect supernatant in dialysis tubing, place 10L TE damping fluid current system, stir renaturation 24-72h in 4 ℃;
Wherein, described sex change liquid contains 8mol/L urea, 1mL beta-mercaptoethanol, Tris-HCl50mmol/L, pH8.0.
3. preparation method according to claim 1 is characterized in that step 2) in carry out the ammonium sulfate concentrations that ammonium sulfate precipitation uses and be 20-40%.
4. preparation method according to claim 1, it is characterized in that, step 2) ultrafiltration is specially in: be that the ultra-filtration membrane of 10kD carries out balance with the TE damping fluid to ultra-fine filter and aperture earlier, keep inlet and outlet pressure to be respectively 1.3bar and 0.3bar, begin to dialyse and change liquid, remove the salt ion in the supernatant liquor, collect ultrafiltration and see through liquid; According to the method described above, again ultrafiltration is seen through liquid and concentrate, be concentrated into 1/10th of original volume, namely get ultrafiltration and concentration liquid.
5. preparation method according to claim 1, it is characterized in that, step 3) is specially: earlier with TE damping fluid balance DEAE-Sepharose FF chromatography column, then ultrafiltration and concentration liquid upper prop is carried out purifying, carry out Balance Treatment with 3 column volumes of TE damping fluid flushing, to remove most of impurity, carry out wash-out with the chromatography column of the TE damping fluid that contains 0.5-1.5mol/L NaCl after to above-mentioned processing then, obtain containing the classification component of target protein CRM197.
6. preparation method according to claim 5 is characterized in that, the type of elution in the step 3) adopts the continuous gradient wash-out.
7. preparation method according to claim 1, it is characterized in that, step 4) is specially: earlier with TE damping fluid balance molecular sieve Sephacryl S-100, with above-mentioned classification component upper prop, when first peak occurring, begin to collect eluted protein liquid, namely get the target protein liquid that contains diphtheria toxin muton CRM 197.
8. according to each described preparation method of claim 4-7, it is characterized in that step 2), the TE buffer concentration that uses in step 3) and the step 4) is 10mmol/L-50mmol/L, pH value 7.0-8.0.
9. according to each described preparation method of claim 1-7, it is characterized in that, diphtheria toxin muton CRM 197 in the step of expression in escherichia coli is: accession number is the gene order of the diphtheria toxin muton CRM 197 of CQ967258 in according to GenBank, carry out codon optimized, synthetic gene; Designing primer simultaneously, is template with synthetic gene, carries out pcr amplification; Amplified production is building up in the expression vector after checking order accurately, changes over to and carries out abduction delivering in the intestinal bacteria.
10. preparation method according to claim 9 is characterized in that, the primer sequence of design is:
Forward primer: 5 '-AGATCTATGGGTGCTGACGACGTTGTT-3 ',
Reverse primer: 5 '-CTCGAGATTAAGATTTGATTTCGA-3 '.
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| CN106119320A (en) * | 2016-06-29 | 2016-11-16 | 山东省生物制品研究所 | A kind of removal expresses, because of protokaryon inclusion body, the polymeric method that refolding strategy produces |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016079755A1 (en) * | 2014-11-20 | 2016-05-26 | Biological E Limited | Codon optimized polynucleotide for high level expression of crm197 |
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| JP7042305B2 (en) | 2014-11-20 | 2022-03-25 | バイオロジカル イー リミテッド | Codon-optimized polynucleotide for high-level expression of CRM197 |
| CN106119320A (en) * | 2016-06-29 | 2016-11-16 | 山东省生物制品研究所 | A kind of removal expresses, because of protokaryon inclusion body, the polymeric method that refolding strategy produces |
| CN107163111A (en) * | 2017-06-15 | 2017-09-15 | 华兰生物工程股份有限公司 | The method for purifying diphtheria toxin |
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Application publication date: 20130828 |