CN107779464A - A kind of preparation method of human source copper-zinc superoxide dismutase - Google Patents

A kind of preparation method of human source copper-zinc superoxide dismutase Download PDF

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CN107779464A
CN107779464A CN201710756221.5A CN201710756221A CN107779464A CN 107779464 A CN107779464 A CN 107779464A CN 201710756221 A CN201710756221 A CN 201710756221A CN 107779464 A CN107779464 A CN 107779464A
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hsod1
superoxide dismutase
preparation
expression
human source
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朱斌辉
朱艳娟
许仁杰
韩蓉
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SUZHOU DONGQUAN BIOTECHNOLOGY CORP Ltd
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SUZHOU DONGQUAN BIOTECHNOLOGY CORP Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Abstract

The invention discloses a kind of preparation method of human source copper-zinc superoxide dismutase, sequence shown in SEQ ID NO.2 is inserted into initial carrier, builds recombinant vector, expression product is extracted after expanding culture, N His TEV hSOD1 are digested, and purifying obtains the hSOD1 of no label.Method provided by the invention, target gene add His TEV labels according to codon-bias purpose of design gene order, and in N-terminal, both avoid expressing heterologous, turn avoid the security risks of animal or people source；Sequences Design with the addition of His purification tags and label excision TEV recognition sites in method, not only improves purifying, also allows for cutting off His labels to reach the requirement of pharmaceutical protein.The hSOD1 that method expression bacterial strain provided by the invention gives expression to is solubility expression, and adds Cu2+&Zn2+During induction, expression quantity substantially increases, and meets the requirement of large-scale production.

Description

A kind of preparation method of human source copper-zinc superoxide dismutase
Technical field
The invention belongs to superoxide dismutase preparing technical field, and in particular to a kind of human source copper-zinc superoxide disproportionation The preparation method of enzyme.
Background technology
Superoxide dismutase (superoxide dismutase, abbreviation SOD), is that one kind is widely present in organism Metalloenzyme, ultra-oxygen anion free radical can be catalyzed disproportionated reaction occurs, the oxygen radical in balancing machine body.SOD is in anti-spoke Penetrate, anti-aging, anti-inflammatory, suppress tumour and cancer, autoimmune therapeutic etc. show uniqueness function, medical science, food More and more applied in the fields such as product, cosmetics.
Up to the present, from bacterium, protozoan, algae, mould, plant, insect, bird, fish and mammal etc. Isolated SOD in organism.The difference of the metal ion combined according to its activated centre, SOD are broadly divided into three classes：Copper zinc Superoxide dismutase (Cu/Zn-SOD), iron superoxide dismutase (Fe-SOD) and manganese superoxide dismutase (Mn- SOD).Wherein, Fe-SOD is primarily present in the matrix of prokaryotic and eukaryotic, and it is thin that Mn-SOD is primarily present in protokaryon In born of the same parents and a small number of plant cells, and Cu/Zn-SOD is then primarily present in the cytoplasm of eukaryotic cells including humans In.Therefore, the SOD that most suitable human body uses is Cu/Zn-SOD.
Domestic Cu/Zn-SOD research primarily focuses on the extraction and purifying of animals and plants.Wherein animal origin, due to depositing The limited starting material and wholesomeness the problems such as, cause product limits throughput, and the security risks of blood product are big；And Plant origin has different with people itself, the problems such as immunogenicity be present.Therefore, people source is produced using genetic engineering Copper-zinc superoxide dismutase is that solve raw material sources, obtain non-immunogenicity, high activity Cu/Zn-SOD effective way.Mesh It is preceding that the precedent of human source copper-zinc superoxide dismutase (hSOD1) is produced by technique for gene engineering, but still suffer from many and ask Topic, mainly includes：(1) destination protein expression quantity is not high, because heterologous expression system influences destination protein expression quantity；(2) purify Process is complicated, needs by ion exchange, molecular exclusion without purification tag and/or waits a series of chromatography means to carry out Purifying, and zone purification label exists with inclusion bodies, needs to obtain activity by the cumbersome technique such as denaturation, renaturation again Form.
The content of the invention
It is an object of the invention to provide a kind of human source copper-zinc superoxide to solve above the deficiencies in the prior art The preparation method of mutase, the expression quantity of destination protein is improved, improve the Rate activity of enzyme, simplify preparation process.
Technical scheme is as follows：
A kind of nucleotide sequence for being used to build human source copper-zinc superoxide dismutase recombinant vector, the sequence are included in people The His-TEV labels of source copper-zinc superoxide dismutase N-terminal addition, the sequence is as shown in SEQ ID NO.2.
A kind of preparation method of human source copper-zinc superoxide dismutase is original by the sequence insertion shown in SEQ ID NO.2 Carrier, recombinant vector is built, extract expression product, N-His-TEV-hSOD1 enzymolysis after expanding culture, purifying obtains no label HSOD1.
Further, the preparation method of described human source copper-zinc superoxide dismutase, the initial carrier can be true Nuclear expression carrier or prokaryotic expression carrier.
Further, the preparation method of described human source copper-zinc superoxide dismutase, the initial carrier can be pET28a。
Further, the preparation method of described human source copper-zinc superoxide dismutase, N-His-TEV-hSOD1 enzymolysis are adopted Use TEV protease.
Further, the preparation method of described human source copper-zinc superoxide dismutase, comprises the following steps：
(1) SEQ ID NO.2 sequence is inserted in initial carrier in a manner of artificial synthesized, to build recombinant vector；
(2) recombinant vector is imported into expression bacterial strain, obtains recombinant bacterial strain；
(3) recombinant bacterial strain is expanded and cultivated；
(4) bacterial cell disruption, centrifugation are obtained into supernatant；
(5) supernatant is purified, obtains the N-His-TEV-hSOD1 of tape label after purification；
(6) N-His-TEV-hSOD1 is digested, obtains cutting off the enzyme liquid of label；
(7) enzyme liquid for cutting off label is purified, obtains the hSOD1 without label after purification.
Further, the preparation method of described human source copper-zinc superoxide dismutase, the middle expression bacterial strain of step (2) is big Enterobacteria, preferably Escherichia coli Codonplus.
Further, the preparation method of described human source copper-zinc superoxide dismutase, recombinant bacterial strain expands in step (3) Incubation is by LB of the recombinant bacterial strain at 25~37 DEG C, containing 10~50 μ g/mL kanamycins and 10~50 μ g/mL chloramphenicol Cultivated in culture medium to OD600Up to 0.4~1.0, final concentration of 0.1-1.0mM IPTG is added, induce 4 at 15~42 DEG C~ 16 hours, thalline is collected by centrifugation；Further, cultivate to OD600Up to 0.4~1.0, add final concentration of 0.1-1.0mM's IPTG, while it is separately added into the Cu within 500 μM2+And Zn2+, inducing temperature is 15~42 DEG C, and induction time is 4~16 small When.
Further, the preparation method of described human source copper-zinc superoxide dismutase, recombinant bacterial strain expands in step (3) Copper ion and zinc ion are also added into culture.
The invention provides a kind of preparation method of human source copper-zinc superoxide dismutase, methods described target gene according to Codon-bias purpose of design gene order, and His-TEV labels are added in N-terminal, obtain objective gene sequence SEQ ID NO.2, both avoid expressing heterologous, turn avoid the security risks of animal or people source；Methods described plasmid construction Target gene is synthesized on plasmid vector by artificial synthesized mode, saves the steps such as digestion, connection, glue reclaim so that Operation be easier and can selectivity removal vector multiple cloning site in each restriction enzyme site gene order, operator and purpose Effable gene order between gene；The starting point of methods described destination protein expression is the initiation codon of target gene Son so that purpose amino acid sequence will not include the amino acid sequence outside design, to avoid its influence to protein structure；Institute State sequences Design in method and with the addition of His purification tags and label excision TEV recognition sites, not only improve purifying, also allow for cutting Except His labels to reach the requirement of pharmaceutical protein.The hSOD1 that methods described expression bacterial strain gives expression to is solubility expression, and Add Cu2+&Zn2+During induction, expression quantity substantially increases, and meets the requirement of large-scale production.
The present invention connects target gene using artificial synthesized mode, during synthesis optimization eliminate more grams of pET-28a carriers Effable gene order in grand site between each restriction enzyme site gene order, operator and target gene etc..
Recombinant vector pET-28a-N-His-TEV-hSOD1 is imported expression bacterial strain by the present invention using calcium chloride transformation In.
In preparation method of the present invention, purifying the N-His-TEV-hSOD1 of tape label method includes affinity chromatography and desalination, Affinity chromatography preferably uses Ni Sepharose chromatographic columns, and desalination preferably uses Sephadex G25 chromatographic columns.The step of purifying Including：
(1) destination protein is slightly carried, is resuspended with disruption buffer (50mM Tris, 500mM NaCl, 5% glycerine, pH8.0) Thalline, afterwards with high-pressure homogeneous crusher machine thalline, supernatant is collected by centrifugation；
(2) affinity chromatography, the Ni first balanced with buffer A (50mM Tris, 500mM NaCl, 5% glycerine, pH8.0) Sepharose chromatographic columns；Afterwards with 4%~60% buffer B (50mM Tris, 500mM NaCl, 5% glycerine, 500mM miaows Azoles, pH 8.0) eluted, collect eluent；
(3) desalination, affinity elution liquid is subjected to desalting processing, first with 4%~60% buffer B (50mM Tris, 500mM NaCl, 5% glycerine, 500mM imidazoles, pH 8.0) balance Desalting chromatographic columns；Afterwards with desalination buffer solution (50mM Tris, 500mM NaCl, 5% glycerine, pH8.0) eluted, eluent is collected, obtains the protein solution of pure tape label.
The method that the present invention purifies the not hSOD1 of tape label includes enzymolysis, affinity chromatography goes label purifying and gel filtration Concentration, affinity chromatography preferably use Ni Sepharose chromatographic columns, and concentration preferably uses the chromatographic columns of Superdex 75.Purifying Step includes：
(1) digest, by the N-His-TEV-hSOD1 of the tape label of purifying protein solution, add TEV protease, 2~8 Digested overnight at DEG C；
(2) affinity chromatography goes label to purify, and first balances Ni Sepharose chromatographic columns with Buffer A, and then loading digests Liquid, and eluted with Buffer A, liquid is spread in collection, is rinsed afterwards with 4%~60%Buffer B affine on chromatographic column His labels, recover chromatographic column affinity；
(3) concentrate, by it is affine flow through liquid carry out concentration, first with gel filtration buffer solution (50mM Tris, pH8.0, 500mM NaCl) the balance chromatographic columns of Superdex 75, then loading is affine flows through liquid, and is eluted with gel filtration buffer solution, receives Collect destination protein peak solution, obtain the protein solution of pure tape label.
The objective gene sequence optimized in the inventive method through codon-bias, can be in E. coli expression strains Expression, and express in the supernatant after the destination protein overwhelming majority that bacterial strain gives expression to is present in bacterial cell disruption.Meanwhile this hair Bright to be induced by adding copper ion and zinc ion simultaneously in recombinant bacterium incubation, expression quantity significantly improves.Furthermore purpose In N-terminal addition His-TEV labels when gene designs so that purifying and the excision of His labels are easy, efficient.It is in addition, of the invention Recombinant vector is built by artificial synthesized mode in method, and selective eliminates gene sequence influential on destination protein Row, and construction method very simple, suitable for industrialization.The method of the present invention is applied to industrially prepare on a large scale HSOD1 with higher specific activity.
Brief description of the drawings
Fig. 1 is the physical map of recombinant vector pET-28a-N-His-TEV-hSOD1 described in embodiment 1；
Fig. 2 is the electrophoretogram that recombination bacillus coli produces hSOD1 screenings in embodiment 2.Wherein, M is protein molecular weight Standard；Swimming lane 1 is full cell；Swimming lane 2 is broken full cell；Swimming lane 3 is broken supernatant；Swimming lane 4 is by Ni Sepharose Sample after purification；A, B, C, D are the 4 restructuring single bacterium colonies selected
Fig. 3 is the electrophoretogram of induction production hSOD1 in embodiment 3, wherein (a) is IPTG induction productions hSOD1 electrophoresis Figure；
(a) in, M is protein molecular weight standard；Swimming lane 1 is full cell；Swimming lane；2 be broken full cell；Swimming lane 3 is Broken supernatant；Swimming lane 4 is by Ni Sepharose samples after purification；
(b) it is IPTG and Cu2+&Zn2+Induction production hSOD1 electrophoretogram, in (b), M is protein molecular weight standard； Swimming lane 1 is full cell；Swimming lane 2 is broken supernatant；Swimming lane 3 is by Ni Sepharose samples after purification；
Fig. 4 is the electrophoretogram in induction and purge process in embodiment 5, wherein, M is protein molecular weight standard；Swimming lane 1 To contrast empty carrier；Swimming lane 2 is non-inducible vectors；Swimming lane 3 precipitates for inducible vectors；Swimming lane 4 is inducible vectors supernatant；Swimming lane 5 For pET-28a-N-His-TEV-hSOD1 after enriching and purifying；Swimming lane 6 is hSOD1 after TEV digestions；
Fig. 5 is the mass spectrogram of pET-28a-N-His-TEV-hSOD1 and hSOD1 in embodiment 5.
Embodiment：
Embodiment 1
Experimental method used, is conventional method unless otherwise specified in embodiment of the present invention.
Material used, reagent etc., unless otherwise specified, are commercially obtained in embodiment of the present invention.
Nucleotide fragments used comprising hSOD1 gene coded sequences of the invention can be expanded by PCR, be artificial synthesized The methods of obtain.For artificial synthesized target gene, those skilled in the art are known by the software analysis codon such as Emboss The technology such as preferences, purpose of design gene order.Different organisms have certain preference to the codon encoded with monoamino-acid Property, by the analysis of codon usage bias, the expression quantity that can improve target gene is designed for these optimal codons. The present invention preferably designs the artificial synthesized nucleotide fragments for including hSOD1 gene coded sequences by codon-bias.
Initial carrier used in the present invention can be prokaryotic expression carrier and carrier for expression of eukaryon.Prokaryotic expression system, which has, to be lost Pass background understands, expression quantity is high, expression product isolate and purify it is relatively easy and the advantages of suitable for industrialized production.Preferably, The initial carrier is prokaryotic expression carrier, such as pET serial carriers, pQE serial carriers, pGEX serial carriers, pMAL series Carrier etc..In prokaryotic expression carrier, the present invention preferably pET serial carriers, particularly pET-28a.
The present invention using in incubation addition copper, zinc ion improve hSOD1 expression quantity.Because of Cu2+&Zn2+It is The important component of hSOD1 active sites, and it is closely related with hSOD1 whether to form correct foldable structure, and The specific incorrect integration that peptide chain can not correctly be folded is the main reason for causing inclusion body to be formed.Therefore induced except reducing Cultivation temperature during expression, adds Cu in the medium2+&Zn2+Reaching makes the purpose of hSOD1 solubility expressions, to improve table Up to amount.
The method of superoxide dismutase Enzyme activity assay is as follows in embodiment of the present invention：
Superoxide dismutase enzyme activity is defined as, and suppresses enzyme amount required during mouse thymus cells speed 50% at 25 DEG C For an enzyme activity unit (U).
It is formulated as follows reagent：
A liquid：The 0.1mol/L trishydroxymethylaminomethanes (Tris) of pH 8.20-hydrochloric acid buffer solution (includes 1mmol/L ED TA·2Na).1.2114g Tris and 37.2mg EDTA2Na is weighed to be dissolved in 62.4mL 0.1mol/L hydrochloric acid solutions, 100mL is settled to distilled water.
B liquid：4.5mmol/L pyrogallol hydrochloric acid solutions.Weigh pyrogallol (A.R) 56.7mg and be dissolved in a small amount of 10mmol/L Hydrochloric acid solution, and it is settled to 100mL.
Mouse thymus cells rate determination sequentially adds A liquid 2.35mL at 25 DEG C in 10mL colorimetric cylinders, distillation Water 2.00mL, B liquid 0.15mL.Add B liquid to mix and be poured into cuvette immediately, determine respectively under 325nm wavelength conditions just Light absorption value, the difference between the two are mouse thymus cells speed Δ A during the beginning and after 1min325(min-1)。
SOD enzyme liquids suppress mouse thymus cells rate determination makes suppression neighbour benzene three by a certain amount of enzyme liquid of above-mentioned steps addition Phenol autoxidation speed is about 1/2 Δ A325(min-1)。
The vigor (U/mL) of superoxide dismutase in solution to be measured is calculated according to equation below：
In above-mentioned formula, Δ A325For mouse thymus cells speed；ΔA’325Suppress pyrogallol from oxygen for enzyme liquid to be measured Change speed；V is enzyme liquid volume to be measured, unit mL；4.5 be reaction system cumulative volume, unit mL；D is extension rate.It is real Apply example 1：Recombinant expression carrier pET28a-N-His-TEV-hSOD1 structure
1) acquisition of target gene, with hSOD1 amino acid sequence (SEQ ID NO.1) for template, according to Escherichia coli Codon-bias (as shown in table 1), codon corresponding to express amino acid sequence SEQ ID NO.1 is converted into large intestine bar The high codon of bacterium frequency of use, and gene order GC percentage is adjusted, the design of gene order is completed, and in N ends His-TEV labels are added, obtain objective gene sequence SEQ ID NO.2.
The codon preference of table 1
Note：Parenthetic numeral represents the frequency for occurring corresponding codon in every 1000 codons behind codon
2) structure of recombinant expression carrier, by artificial synthesized mode, the DNA fragmentation of step 1) is directly synthesized At initial carrier pET28a multiple cloning sites, pET28a is fixed on carrier, by being deprotected, being coupled, capping, aoxidizing Directly target gene is singly connected in order of sequence etc. a series of chemical synthesis processes more grams of pET28a carriers Long Weidianchu, builds recombinant vector pET28a-N-His-TEV-hSOD1, and its physical map is as shown in Figure 1.Confirmed through sequencing, The sequence shown in SEQ ID NO.2 is connected at initial carrier pET28a multiple cloning sites.
Embodiment 2：The structure of recombination bacillus coli
1) preparation of competent escherichia coli cell
Use CaCl2Chemical method prepares competent escherichia coli cell, and step includes：Take the Escherichia coli bacterium that 1ml has been activated Liquid is seeded in 100ml LB culture mediums；37 DEG C of shaken cultivations are to OD600During=0.35-0.5；Centrifuged and received with 4000g at 4 DEG C Collect thalline；Add the 0.1MCaCl of 40ml precoolings2Cell, ice bath 10 minutes is resuspended；Bacterium is collected by centrifugation with 4000g at 4 DEG C Body；Add the 0.1M CaCl of 2ml precoolings2Cell is resuspended；Add the glycerine of final concentration 15%~20%；It is distributed into every μ L of pipe 50； And 4 DEG C preserve 24 hours after go in -80 DEG C of refrigerators and freeze.
2) recombinant vector pET28a-N-His-TEV-hSOD1 converts competent escherichia coli cell
By the μ L of recombinant vector pET28a-N-His-TEV-hSOD1 2 (content is no more than 50ng) and E. coli competent The μ L of cell 200 are mixed；Ice bath 30 minutes, 42 DEG C of heat shocks 90 seconds, subsequent ice bath 3~5 minutes；Often pipe adds 1mL LB culture mediums, 37 DEG C of cultures 45-60 minutes (≤220rpm), make bacterium restore normal growth state；Then above-mentioned bacterium solution is shaken up and be coated on LB flat boards containing antibiotic (kanamycins and chloramphenicol), 37 DEG C of inversion overnight incubations.
3) hSOD1 expression screening is recombinated
Picking single bacterium colony is inoculated into LB fluid nutrient mediums of the 5mL containing antibiotic from flat board, 37 DEG C of culture 4.5h conducts Seed liquor.Take 0.9ml seeds to be transferred in LB fluid nutrient mediums of the 2ml containing antibiotic, and add 0.3mM IPTG, 37 DEG C lure Lead 2 hours.Thalline is collected by centrifugation and carries out SDS-PAGE detections, analyzes hSOD1 expression.Remaining seed liquor is added eventually The glycerine packing of concentration 15%~20%, 4 DEG C preserve 24 hours after go in -80 DEG C of refrigerators and freeze.
Fig. 2 is the screening that recombination bacillus coli produces hSOD1.As illustrated, M is protein molecular weight standard；Swimming lane 1 For full cell；Swimming lane 2 is broken full cell；Swimming lane 3 is broken supernatant；Swimming lane 4 is by Ni Sepharose samples after purification Product；A, B, C, D are the 4 restructuring single bacterium colonies selected.It can be seen that hSOD1 has expression in recombinant bacterial strain, and it is solubility expression.
Embodiment 3：Recombinant bacterial strain produces hSOD1
1) recombinant bacterial strain induces production hSOD1 through IPTG
Glycerine preserves the μ L of bacterium solution 20 in pipe and is inoculated into LB fluid nutrient mediums of the 200ml containing antibiotic in Example 2, 37 DEG C, 140rpm is cultivated to OD600Up to 0.4~0.6.LB liquid of the 1000ml containing antibiotic is transferred to by 2.5% inoculum concentration to train Support in base.When 37 DEG C of shaken cultivations to OD600During up to 0.4~1.0, IPTG to final concentration of 0.3mM is added, 30 DEG C of inductions 4 are small When.Thalline is collected by centrifugation.SDS-PAGE detections are carried out, analyze hSOD1 expression and enzyme activity implementations.
(a) is IPTG induction productions hSOD1 in Fig. 3.As illustrated, M is protein molecular weight standard；Swimming lane 1 is complete thin Born of the same parents；Swimming lane；2 be broken full cell；Swimming lane 3 is broken supernatant；Swimming lane 4 is by Ni Sepharose samples after purification.
2) recombinant bacterial strain is through IPTG and Cu2+&Zn2+Induction production hSOD1
Glycerine preserves the μ L of bacterium solution 20 in pipe and is inoculated into LB fluid nutrient mediums of the 200ml containing antibiotic in Example 2, 37 DEG C, 140rpm is cultivated to OD600Up to 0.4~0.6.LB liquid of the 1000ml containing antibiotic is transferred to by 2.5% inoculum concentration to train Support in base.When 37 DEG C of shaken cultivations to OD600During up to 0.4~1.0, addition IPTG to final concentration of 0.3mM, while addition 0~ 500μM Cu2+&Zn2+, 30 DEG C induce 4 hours.Thalline is collected by centrifugation.SDS-PAGE detections are carried out, analyze hSOD1 expression feelings Condition and enzyme activity implementations.
(b) is IPTG and Cu in Fig. 32+&Zn2+Induction production hSOD1.As illustrated, M is protein molecular weight standard； Swimming lane 1 is full cell；Swimming lane 2 is broken supernatant；Swimming lane 3 is by Ni Sepharose samples after purification.
As shown in figure 3, Cu is added simultaneously2+&Zn2+Induction hSOD1 expression quantity is substantially better than than individually being induced with IPTG 's.
The enzymatic activity contrast situation of the method for embodiment 3, as a result as shown in table 2.
Table 2
Cu2+&Zn2+Concentration (μM) Rate activity (U/mg)
0 7371
50 11075
150 18912
200 26775
300 35463
500 23902
Embodiment 4：Tape label N-His-TEV-hSOD1 purifying
(1) destination protein is slightly carried, is resuspended with disruption buffer (50mM Tris, 500mM NaCl, 5% glycerine, pH8.0) Thalline, afterwards with high-pressure homogeneous crusher machine thalline, supernatant is collected by centrifugation；
(2) affinity chromatography, the Ni first balanced with buffer A (50mM Tris, 500mM NaCl, 5% glycerine, pH8.0) Sepharose chromatographic columns；Afterwards with 4%~60% buffer B (50mM Tris, 500mM NaCl, 5% glycerine, 500mM miaows Azoles, pH 8.0) eluted, collect eluent；
(3) desalination, affinity elution liquid is subjected to desalting processing, first with 4%~60% buffer B (50mM Tris, 500mM NaCl, 5% glycerine, 500mM imidazoles, pH 8.0) balance Desalting chromatographic columns；Afterwards with desalination buffer solution (50mM Tris, 500mM NaCl, 5% glycerine, pH8.0) eluted, eluent is collected, obtains the protein solution of pure tape label.Embodiment 5： Not tape label hSOD1 purifying
(1) digest, by the N-His-TEV-hSOD1 of the tape label of purifying protein solution, add TEV protease, 4 DEG C Lower enzymolysis is overnight；
(2) affinity chromatography goes label to purify, and first balances Ni Sepharose chromatographic columns with Buffer A, and then loading digests Liquid, and eluted with Buffer A, liquid is spread in collection, is rinsed afterwards with 4%~60%Buffer B affine on chromatographic column His labels, recover chromatographic column affinity；
(3) concentrate, by it is affine flow through liquid carry out concentration, first with gel filtration buffer solution (50mM Tris, pH8.0, 500mM NaCl) the balance chromatographic columns of Superdex 75, then loading is affine flows through liquid, and is eluted with gel filtration buffer solution, receives Collect destination protein peak solution, obtain the protein solution of pure tape label.
As shown in figure 4, according to SDS-PAGE electrophoresis showeds pET28a-N-His-TEV-SOD1 in Bacillus coli expression strain There is obvious solubility expression, can be enriched with very well by Ni affinity columns.TEV digestions experiment shows that pET28a-N-His-TEV-SOD1 can It is removed label and forms hSOD1.As shown in figure 5, the people source for showing Bacillus coli expression according to LC-MS data recombinates SOD1 eggs White molecular weight is consistent with theoretical molecular, is destination protein.
Sequence table
<110>Suzhou Dongquan Biotechnology Co., Ltd.
<120>A kind of preparation method of human source copper-zinc superoxide dismutase
<130> 1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 154
<212> PRT
<213>Amino acid sequence (hSOD1)
<400> 1
Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln
1 5 10 15
Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val
20 25 30
Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val
35 40 45
His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His
50 55 60
Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg
65 70 75 80
His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala
85 90 95
Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys
100 105 110
Ile Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly
115 120 125
Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg
130 135 140
Leu Ala Cys Gly Val Ile Gly Ile Ala Gln
145 150
<210> 2
<211> 515
<212> DNA
<213>Artificial sequence (N-His-TEV-hSOD1)
<400> 2
ccatgcatca tcatcatcat cacgaaaact tgtatttcca gggcatggca accaaagcag 60
tttgtgttct gaaaggtgat ggtccggttc agggtattat taattttgaa cagaaggaaa 120
gcaacggtcc tggggtagca ggttaaagtt ttaaaggtct gaccgaaggt ctgcatggtt 180
ttcatgttca tgaatttggt cagcgcaggt gataataccg caggttgtac ccgcatttta 240
atccgctgag ccgtaaacat ggtggtccga aagatgaaga acgtcatgtt ggtgatctgg 300
gtaatgttac cgcagataaa gatggtgttg cagatgttag cattgaagat agcgttatta 360
gcctgagcgg tgatcattgt attattggtc gtaccctggt ccaaaaccgg aaagcagatg 420
atctgggtaa aggtggtaat gaagaaagca tgttcatgaa taatgcaggt agccgtctgg 480
catgtggtgt tattggtatt gcacagtaac tcgag 515

Claims (10)

  1. A kind of 1. nucleotide sequence for being used to build human source copper-zinc superoxide dismutase recombinant vector, it is characterised in that the sequence Row are included in human source copper-zinc superoxide dismutase N-terminal addition His-TEV labels, and the sequence is as shown in SEQ ID NO.2.
  2. 2. a kind of preparation method of human source copper-zinc superoxide dismutase, it is characterised in that by the sequence shown in SEQ ID NO.2 Row insert initial carrier by artificial synthesized mode, build recombinant vector, and expression product, N-His- are extracted after expanding culture TEV-hSOD1 is digested, and purifying obtains the hSOD1 of no label.
  3. 3. the preparation method of human source copper-zinc superoxide dismutase according to claim 2, it is characterised in that described original Carrier is carrier for expression of eukaryon or prokaryotic expression carrier.
  4. 4. the preparation method of human source copper-zinc superoxide dismutase according to claim 3, it is characterised in that described original Carrier is pET28a.
  5. 5. the preparation method of human source copper-zinc superoxide dismutase according to claim 2, it is characterised in that N-His- TEV-hSOD1 enzymolysis uses TEV protease.
  6. 6. the preparation method of human source copper-zinc superoxide dismutase according to claim 2, it is characterised in that including following Step：
    （1）SEQ ID NO.2 sequence is inserted in initial carrier in a manner of artificial synthesized, to build recombinant vector；
    （2）Recombinant vector is imported into expression bacterial strain, obtains recombinant bacterial strain；
    （3）Recombinant bacterial strain is expanded and cultivated；
    （4）By bacterial cell disruption, centrifugation obtains supernatant；
    （5）Supernatant is purified, obtains the N-His-TEV-hSOD1 of tape label after purification；
    （6）N-His-TEV-hSOD1 is digested, obtains cutting off the enzyme liquid of label；
    （7）The enzyme liquid for cutting off label is purified, obtains the hSOD1 without label after purification.
  7. 7. the preparation method of human source copper-zinc superoxide dismutase according to claim 6, it is characterised in that step（2） Middle expression bacterial strain is Escherichia coli, preferably Escherichia coli Codonplus.
  8. 8. the preparation method of human source copper-zinc superoxide dismutase according to claim 6, it is characterised in that step（3） Middle recombinant bacterial strain, which expands in culture, is also added into copper ion and zinc ion.
  9. 9. the preparation method of human source copper-zinc superoxide dismutase according to claim 6, it is characterised in that step（3） It is at 25 ~ 37 DEG C, containing 10 ~ 50 μ g/mL kanamycins and 10 ~ 50 μ g/mL by recombinant bacterial strain that middle recombinant bacterial strain, which expands incubation, Cultivated in the LB culture mediums of chloramphenicol to OD600Up to 0.4 ~ 1.0, final concentration of 0.1-1.0mM IPTG is added, 15 ~ Induced 4 ~ 16 hours at 42 DEG C, thalline is collected by centrifugation.
  10. 10. the preparation method of human source copper-zinc superoxide dismutase according to claim 9, it is characterised in that culture is extremely OD600Up to 0.4 ~ 1.0, final concentration of 0.1-1.0mM IPTG is added, while be separately added into the Cu within 500 μM2+And Zn2 +, inducing temperature is 15 ~ 42 DEG C, and induction time is 4 ~ 16 hours.
CN201710756221.5A 2017-08-29 2017-08-29 A kind of preparation method of human source copper-zinc superoxide dismutase Pending CN107779464A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480445A (en) * 2022-01-26 2022-05-13 西南交通大学 Preparation and application of humanized superoxide dismutase hSOD1 mutant
CN114983900A (en) * 2022-06-22 2022-09-02 杭州水芭莎生物医药科技有限公司 Preparation method of emulsion with antioxidant and repairing effects

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101955918A (en) * 2009-07-17 2011-01-26 华东理工大学 Method for producing recombinant high-activity manganese superoxide mutase
CN102465134A (en) * 2010-11-16 2012-05-23 杭州纽龙生物科技有限公司 Method for preparing recombinant anthropogenic Cu/Zn superoxide dismutase
CN103374579A (en) * 2012-04-13 2013-10-30 上海医药工业研究院 Plectasin as well as gene and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101955918A (en) * 2009-07-17 2011-01-26 华东理工大学 Method for producing recombinant high-activity manganese superoxide mutase
CN102465134A (en) * 2010-11-16 2012-05-23 杭州纽龙生物科技有限公司 Method for preparing recombinant anthropogenic Cu/Zn superoxide dismutase
CN103374579A (en) * 2012-04-13 2013-10-30 上海医药工业研究院 Plectasin as well as gene and preparation method thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480445A (en) * 2022-01-26 2022-05-13 西南交通大学 Preparation and application of humanized superoxide dismutase hSOD1 mutant
CN114983900A (en) * 2022-06-22 2022-09-02 杭州水芭莎生物医药科技有限公司 Preparation method of emulsion with antioxidant and repairing effects
CN114983900B (en) * 2022-06-22 2023-09-15 杭州水芭莎生物医药科技有限公司 Preparation method of emulsion with antioxidant and repairing effects

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