CN105331588A - Preparation process of recombinant spirulina superoxide dismutase (SOD) - Google Patents

Abstract

The invention relates to a preparation process of recombinant spirulina superoxide dismutase (SOD) and belongs to the SOD preparation technology. The preparation process includes: cloning the encoding genes sod of spirulina platensis SOD, building a prokaryotic expression vector pET30a-sod, and expressing recombinant SOD in escherichia coli BL21(DE3); building a fermentation tank fermentation process of recombinant bacteria and a purification process of recombinant protein; measuring the protein concentration and specific activity of recombinant spirulina SOD; analyzing the natural molecular weight of the recombinant spirulina SOD through non-denaturing gel electrophoresis, and using a nitrotetrazolium blue chloride liquid dying method in combination with an inductively coupled plasma optical emission spectrometer to measure metal elements contained by pure enzyme so as to determine the type of the recombinant spirulina SOD; incubating pure enzyme liquid through heat treatment of different temperatures and solutions of different pH, and determining the stability of the pure enzyme. The preparation process has the advantages that the production process of the recombinant spirulina SOD is built, and the defects of residual organic solvent, complex extraction procedure, high product cost, long production cycle, low yield, low purity and poor product safety when the physicochemical technology is used for extracting the SOD are overcome.

Description

A kind of restructuring spirulina superoxide-dismutase preparation technology

Technical field

The present invention relates to a kind of method preparing superoxide-dismutase, especially a kind of method utilizing gene recombination technology to prepare spirulina plalensis superoxide-dismutase.

Background technology

Superoxide-dismutase (SuperoxideDismutase, SOD) be a kind ofly come from the active substance of life entity and antidotal metalloenzyme, its essence is protein, to be found and extract early than 1969 by American scientist McCord and Fridovich from ox red blood corpuscle.In normal metabolic processes, body can produce free radical, if can not remove in time and will produce toxic action to cell, and SOD can catalysis ultra-oxygen anion free radical generation disproportionation reaction, intracellular superoxide radical conversion is made to be oxygen and hydrogen peroxide, thus reaching the object of oxyradical in balanced body, the metal ion combined by SOD is different, can be divided into Fe-SOD, Mn-SOD and Cu/Zn-SOD.Because SOD can balance the oxyradical of body, effective prophylactic activity oxygen, to the toxic action of organism, thus has radioprotective, antitumor and delay the functions such as body aging, in healthcare products, medicine and cosmetic industry, has important using value.

The method of current production SOD has three kinds: the first traditional from animal blood, extracts SOD.Because the various transmissible disease such as mad cow disease, influenza is constantly broken out with popular all over the world, from animal blood, extract SOD there is great potential safety hazard.Ban was promulgated from 1999 in Europe: forbid that animal blood extracts SOD for a long time for the mankind.The second extracts SOD from plant, because in plant materials, SOD content is too low, is difficult to realize industrialization.The third utilizes modern biotechnology producer gene recombination human source SOD, and this is international forefront technology of producing SOD.Gene recombinant human source SOD have without exogenous pollution, virus-free remaining, without immunological rejection, without albumen species variation, without anaphylaxis, purity is high, activity is high, fundamentally solve the mankind use safety problem.The U.S., Japan, some countries of West Europe develop restructuring hSOD medicine for a long time, have entered into II phase, III phase clinical study.In the U.S., the sod gene engineering product output value in 1992 has reached 2,000,000,000 dollars.China has twenties companies to produce SOD at present, is all carry SOD from the animal blood such as ox, pig, annual production 3000 hundred million about U.Siping China Tech biotechnology limited liability company is the producer of domestic first using gene engineering producer gene recombination human source SOD, produces recombination human source SOD vigor 30,000,000,000 U per year.

There is Fe-SOD in spirulina and not containing the SOD of other types, therefore separation and purification Fe-SOD is ideal from spirulina.At present from spirulina, extract SOD and also mainly rely on physiochemical techniques, relate generally to freezing, ultrasonic disruption, ammonium sulfate precipitation, ion exchange chromatography, gel-filtration and the step such as concentrated.This production technique also exists some shortcomings such as organic solvent residual, extraction procedure is complicated, product cost is high, the production cycle is longer, and productive rate is not high, purity is lower, Product Safety is poor.Visible, how to reduce purification step, reduce costs, the SOD obtaining higher specific activity is the key issue of producing SOD.

Summary of the invention

The object of the invention is to overcome weak point of the prior art, a kind of restructuring spirulina superoxide-dismutase preparation technology is provided.

In order to realize object of the present invention, following for employing technical scheme is implemented by we:

A kind of restructuring spirulina superoxide-dismutase, is characterized in that the nucleotide sequence of described restructuring spirulina superoxide dismutase gene sequence and corresponding aminoacid sequence are:

SLEHHHHHH-

A kind of restructuring spirulina superoxide-dismutase preparation technology, is characterized in that: described step of preparation process is as follows:

One, the extraction of spirulina genomic dna

(1), with distilled water, the centrifugal 5min of fresh spirulina plalensis 5000g is washed 3 times altogether;

(2), fresh for 280mg spirulina plalensis being mixed with the quartz sand of a small amount of sterilizing in the mortar of freezing treatment in advance, fully grind;

(3), extraction genomic dna is carried out by the step of plant genome DNA extraction test kit specification sheets;

(4) the genomic dna wash-out, extracted is in 50 μ l elutriant TE;

(5) genomic dna, after wash-out saves backup at-20 DEG C of temperature.

Two, the amplification of sod gene fragment

(1), according to the AY282414 spirulina sod gene order design of amplification primers logged in GenBank: upstream primer SPSODF:5 '-A cATATGgCTTTTGAACTTCCCAG-3 '; Downstream primer SPSODR:5 '-G cTCGAGgCTGGCAGACGCGAGA-3 ', the long 614bp of primer pair expection amplified fragments;

(2), pcr amplification sod gene, with extraction spirulina genomic dna for template, utilize above-mentioned primer pair amplifies sod gene, PCR reaction conditions is: 94 DEG C of denaturation 3min ,-94 DEG C of sex change 45s ,-55 DEG C of annealing 45s, 72 DEG C extend 45s, totally 30 circulations, then, 72 DEG C extend 6min again;

(3), get 5 μ lPCR products and carry out 1.5% agarose gel electrophoresis analysis.

Three, the clone of sod gene fragment

(1), 45 μ lPCR amplified productions are separated through 1.5% agarose gel electrophoresis, reclaim test kit with sepharose DNA and cut glue recovery goal gene fragment, reclaim product 40 μ l elution buffer EB wash-outs, concrete operations are reclaimed test kit specification sheets by sepharose DNA and are carried out;

(2), the PCR primer of 3 μ l purifying and 1 μ l cloning vector pGEM-TEasyVector are spent the night 4 DEG C of connections;

(3), by 5 μ l connect the heat-shock transformed 100 μ l competence bacillus coli DH 5 alphas of product and coat on the LB agar plate containing 50 μ g/ml penbritins, putting 37 DEG C of incubated overnight;

(4), next day, be inoculated in the LB liquid nutrient medium containing Amp with sterile toothpick picking white colony, 37 DEG C of shaking table overnight incubation;

(5), plasmid is extracted with the little extraction reagent kit of ordinary plasmids.Carry out amplification qualification with the Insert Fragment that SPSODF and SPSODR extracts plasmid for primer pair, with EcoRI, enzyme is carried out to extracted plasmid simultaneously and cut qualification;

(6), the plasmid cutting qualification all correct through PCR, enzyme is checked order;

(7) the Blast software analysis, in sequencing result NCBI.

Four, the structure of sod prokaryotic expression vector

(1), by order-checking correct recombinant plasmid pGEM-T-sod and recombinant plasmid pET30a-apc α (the spirulina plalensis allophycocyanin α subunit gene prokaryotic expression carrier that this laboratory builds) use NdeI and XhoI in 37 DEG C of double digestion 4h respectively, cut glue with sepharose DNA recovery test kit and reclaim sod gene insert and linearizing pET30a carrier respectively;

(2), the SolutionI used in DNALigationKit, sod gene insert is connected with linearizing pET30aVectorDNA;

(3), with connecting in product thermal transition competence e. coli jm109, and be coated on the LB agar plate containing 50 μ g/ml kantlex, 37 DEG C of incubated overnight;

(4), picking list bacterium colony incubated overnight extract plasmid, use primer T7t to institute's upgrading grain order-checking.

Five, the abduction delivering of recombinant plasmid in intestinal bacteria

(1), with the recombinant plasmid pET30a-sod that order-checking is correct transform BL21 (DE3) competent cell, obtain positive expression bacterial strain;

(2), by 100 μ l recombinant strains be seeded in 10ml containing in the LB liquid nutrient medium of 50 μ g/ml kantlex 37 DEG C, 220rpm shaking culture spends the night, and obtains seed liquor;

(3), get described in part steps (2) seed liquor aseptic condition under glycerol adding to 30%, packing-70 DEG C preservation, for subsequent use;

(4), the 1ml seed liquor described in step (2) is inoculated in 100ml containing in 2 × YT liquid nutrient medium of 50 μ g/mL kantlex, 37 DEG C, cultivate 3.5h under 230rpm oscillating condition, take out culturing bottle, sterile sampling 1ml from culture, left blank contrasts;

(5), then in culturing bottle, add the IPTG that final concentration is 0.40mM, regulate culture temperature to 31 DEG C, 230rpm shaking culture 5h;

(6), get induction before and induction after each 0.5ml of bacterium liquid, 4 DEG C, the centrifugal 10min of 9000g, abandon supernatant, precipitation is resuspended in 100 μ lPBS, adds 25 μ l5 × SDS-PAGE sample-loading buffers, after 100 DEG C of thermal treatment 3min, get 10 μ l and carry out 12%SDS-PAGE electrophoretic analysis.

Six, restructuring spirulina SOD ferment tank

(1), the preparation of ferment-seeded

1., get described step 5-(3)-70 DEG C of glycerol stock 100 μ l and be inoculated in 5mlLB (kan ⁺) in substratum, 37 DEG C, 220r/min activated overnight cultivates 18h;

2., 5ml culture is transferred in 150mlTB (kan ⁺) in substratum 37 DEG C, 220r/min incubated overnight 20h, obtains ferment-seeded.

(2), fermentor tank prepares

1., by pH electrode more than aquation 3h in distilled water, the damping fluid of pH6.86 and pH4.0 is then utilized to complete Zero calibration and the slope demarcation of electrode successively;

2., while 1. step carries out by dissolved oxygen electrode, immerse energising polarization more than 6h in distilled water, in rear immersion saturated sodium bisulfite solution, complete Zero calibration;

3., lay down fermentor tank motor, pH electrode, dissolved oxygen electrode are reinstalled in tank the fermentor tank filling 2.25LTB basic medium;

4., by fermentor tank together with feed supplement bottle, mend demijohn, mend alkali bottle, 250ml potassium phosphate buffer and pipeline thereof in 121 DEG C of sterilizing 20min;

5. all circuit and the piping system of fermentor tank, is assembled after sterilizing successively.

(3), ferment

1., open upper computer and air compressor, add 250ml potassium phosphate buffer, 2ml defoamer (Antifoam204SIGMA) and 2mlkan with flame inoculation method successively from inoculation mouth ⁺(50mg/ml);

2., on upper computer be set as follows culture condition: temperature 37 DEG C, air flow 200L/h, pH7.0, rotating speed 500r/min, demarcate 100% value of DO electrode in this case.

3., appropriate regulation air pressure, leave and take 50ml substratum in contrast from material taking mouth.

4., stop stirring and ventilation, aseptic access 150ml seed liquor in tank, adjustment air flow is 200L/h, rotating speed 500r/min, then clicks fermentation and starts, preserve this Batch fermentation record.

5., get 10ml substratum by material taking mouth to contrast as 0h.

6., in fermenting process by regulating air flow and rotating speed to make DO remain on about 30%, temperature is set to automatic control;

7., mend acid solution (2.4MHCl) by automatic dripping and mend alkali lye (4.18M ammoniacal liquor) adjust ph;

8., when fermenting to about 7h, in substratum, carbon source exhausts oxygen dissolving value and rises rapidly, starts to drip feed supplement liquid 400ml continuously in tank; By the rate of addition of adjustment feed supplement liquid, rotating speed and air flow in feed supplement process, make DO remain on about 20%, this process medium speed generally remains on about 700r/min as far as possible;

9., when fermentation, to 12h, from inoculating, mouth is aseptic adds 1.25mlIPTG (0.2g/ml), is 0.4mM, regulates temperature to be 31 DEG C, coinduction 7h to IPTG final concentration;

10., sampling 10ml per hour in fermenting process, for bacterial growth dynamic monitoring; During fermentation ends, reduce air flow and rotating speed, click fermentation ends, preserve data.Fermented liquid is proceeded in aseptic triangular flask, close source of the gas.

(4), bacterial growth dynamically and protein expression effect monitoring

1., with the zeroing of TB substratum, the absorbancy OD of per hour stayed bacterium liquid at 600nm place in fermenting process is measured ₆₀₀;

2., get 10ml bacterium liquid, 4 DEG C, the centrifugal 10min of 9400 × g, abandons supernatant, blots residual liquid with cotton swab, weighs thalline weight in wet base (WCW).

3., target protein expression effect SDS-PAGE analyzes, and before getting induction and each 1.5ml of fermentation ends bacterium liquid, centrifugally abandons supernatant;

4., precipitation is resuspended in 0.8mlPBS, and ultrasonic treatment, cracking condition is: power 400W, and work 4S, stops 6S, altogether about 25 circulations.The centrifugal 10min of lysate 14000 × g, gets 50ul supernatant liquor and carries out SDS-PAGE detection.

(5), tunning is preserved

Abandon supernatant gently after fermented liquid being put 4 DEG C of hold over night, be deposited in 4 DEG C, the centrifugal 20min of 4000g, abandons supernatant, collects bacterium mud, puts-20 DEG C save backup with every part of about 10g.

Seven, the purifying of restructuring spirulina SOD

(1), recombinant bacterium cracking

1., 4 ~ 8g recombinant bacterium precipitation is fully resuspended in 80ml-non denatured binding buffer liquid;

2., connect nanometer high pressure homogenizer air-channel system, open air compressor;

3., with about 100ml distilled water cleaning clarifixator;

4., will treat that cracking bacterial suspension adds in sample introduction bucket, 15000psi pressure cracking bacterium 3 is taken turns to lysate bright;

5. after, by lysate putting 58 DEG C of heating in water bath process 10min, in 9400 × g ,-4 DEG C of centrifugal 30min, 0.45 μm of frit removing cell debris, filtrate can direct upper prop.

(2), column purification

1., get the empty chromatography column 1 of 45ml, fill up with Ni-NTAAgarose resin;

2., pillar is balanced with about 150ml non denatured binding buffer liquid with after the drip washing of about 150ml distilled water;

3., by the bacterial lysate handled well instilled in post by peristaltic pump, if nucleic acid-protein detector reading rises comparatively large in loading process, then collect effluent liquid one and manage;

4., no longer reduce to the nearly background of light absorption value with about 200ml non denatured washing buffer washing pillar;

5., need in process to collect light absorption value rising, peak value, at least each 1 pipe of decrement phase effluent liquid;

6., with low pH elution pillar, start to collect elutriant when light absorption value rises to about 0.080, drop to after below 0.080 to light absorption value and stop collecting;

7. about 200ml distilled water drip washing pillar, is used after purifying;

8., protein purification effect is detected with SDS-PAGE;

Eight, carry out SDS-PAGE, determination of protein concentration, enzyme assay, pure enzyme molecular weight determined, NBT dyeing, metal ion mensuration, thermal stability analysis, ph stability analysis.

Further, the upstream primer SPSODF:5 '-A described in described step one cATATGthe middle underscore part of GCTTTTGAACTTCCCAG-3 ' is NdeI restriction enzyme site; Downstream primer SPSODR:5 '-G cTCGA ggCTGGCAGACGCGAGA-3 ', middle underscore part is XhoI restriction enzyme site.

Further, at the coating of surface uniform before use 200mg/mlIPTG7 μ l, 20mg/mlX-Gal28 μ l on the LB agar plate in described step 4.

Further, described step 6 one (3)-8. described every 400ml of feed supplement liquid contains yeast extract 12.5g, glycerine 240ml.

Further, the non denatured binding buffer liquid described in described step 7: 50mMNaH ₂pO ₄, 0.5MNaCl, 0.1MKCl, 10mM imidazoles, pH7.4; Non denatured washing buffer: 50mMNaH ₂pO ₄, 0.5MNaCl, 0.1MKCl, 20mM imidazoles, pH7.4; Low pH elutriant: 50mMNaH ₂pO ₄, 0.5MNaCl, 0.1MKCl, pH4.5.

Beneficial effect

Take spirulina plalensis as research object, the encoding gene sod of clone's spirulina superoxide-dismutase (SOD), builds prokaryotic expression carrier pET30a-sod, and at e. coli bl21 (DE3) inner expression restructuring SOD; Set up the ferment tank technique of recombinant bacterium and the purifying process of recombinant protein; Measure protein concentration and the specific activity of restructuring spirulina SOD; By the natural molecule amount of native gel electrophoresis analysis restructuring spirulina SOD, NBT liquid (NBT) staining is utilized to measure in conjunction with plasma emission spectrometer the kind that metallic element contained by pure enzyme determines restructuring spirulina SOD; Hatch pure enzyme liquid by heat treatments at different and different pH solution, determine the stability of pure enzyme.By above each step process, finally set up a perfect restructuring spirulina SOD production technique, for the related products such as medicine, foods and cosmetics of subsequent development based on restructuring spirulina SOD lays the foundation.

Accompanying drawing explanation

Fig. 1 is restructuring spirulina superoxide-dismutase technology of preparing route map;

Fig. 2 is the analysis of sod gene PCR amplified production agarose gel electrophoresis;

Fig. 3 is restructuring plasmid pGEM-T-sodNedI/XhoI double digestion electrophorogram;

Fig. 4 is linearized vector pET30a electrophorogram;

Fig. 5 is the open reading frame of pET-30a-sod and the aminoacid sequence of coding thereof;

The expression product electrophorogram of Fig. 6 sod gene in e. coli bl21 (DE3);

Fig. 7 is recombinant bacteria growth curve in fermenting process;

Fig. 8 recombinant bacterium tunning SDS-PAGE electrophorogram;

Fig. 9 is restructuring SOD purified product electrophorogram;

Figure 10 is restructuring SOD native gel electrophoresis NBT dyeing;

The activity change of SOD when Figure 11 is 60 DEG C;

The activity change of SOD when Figure 12 is 65 DEG C;

Figure 13 is the impact of pH value on SOD activity;

Embodiment

By reference to the accompanying drawings the present invention is described further:

As shown in Figure 1, this research with Alkaline lake in Erdos plateau spirulina plalensis for experiment material, by the encoding gene of its SOD of clonal expression, and set up fermentation and the purifying process of extensive restructuring SOD, analyse in depth the biological characteristics of recombination expression product on this basis.

Alkaline lake in Erdos plateau spirulina plalensis, fishes for purifying by Ordos Plateau sand ground alkali lake, cultivates with Zarrouk nutrient solution under room temperature, be so kind as to give by Agricultural University of the Inner Mongol professor Qiao Chen.Competence bacillus coli DH 5 alpha is Biomed Products, and competence e. coli jm109, BL21 (DE3) are TaKaRa Products.

The construction and expression of spirulina sod prokaryotic expression vector

The extraction of spirulina genomic dna

Plant genome DNA is adopted to extract the genomic dna of test kit extraction spirulina.With distilled water, centrifugal for fresh spirulina 5000 × g 5min is washed 3 times altogether.280mg fresh spirulina being mixed with the quartz sand of a small amount of sterilizing in the mortar of freezing treatment in advance, fully grind.All the other step by specifications carry out.Extract genomic dna wash-out in 50 μ l elutriant TE.-20 DEG C save backup.

The amplification of sod gene fragment

According to spirulina sod gene order (AY282414) design of amplification primers logged in GenBank.Upstream primer SPSODF:5 '-A cATATGgCTTTTGAACTTCCCAG-3 ' (underscore part is NdeI restriction enzyme site); Downstream primer SPSODR:5 '-G cTCGAGgCTGGCAGACGCGAGA-3 ', (underscore part is XhoI restriction enzyme site), the long 614bp of primer pair expection amplified fragments.Entrust the synthesis of Invitrogen company.With extraction spirulina genomic dna for template, utilize above-mentioned primer pair amplifies sod gene.PCR reaction conditions is: 94 DEG C of denaturation 3min ,-94 DEG C of sex change 45s, and-55 DEG C of annealing 45s ,-72 DEG C extend 45s, totally 30 circulations, and then, 72 DEG C extend 6min again.Get 5 μ lPCR products and carry out 1.5% agarose gel electrophoresis analysis.

The clone of sod gene fragment

45 μ lPCR amplified productions are separated through 1.5% agarose gel electrophoresis, reclaim test kit with sepharose DNA and cut glue recovery goal gene fragment, reclaim product 40 μ l elution buffer EB wash-outs.Concrete operations are undertaken by test kit specification sheets.The PCR primer of 3 μ l purifying and l μ l cloning vector pGEM-TEasyVector are spent the night 4 DEG C of connections.5 μ l are connected the heat-shock transformed 100 μ l competence bacillus coli DH 5 alphas of product and coats (surface uniform coating 200mg/mlIPTG7 μ l before use on the LB agar plate containing 50 μ g/ml penbritins (Amp), 20mg/mlX-Gal28 μ l), put 37 DEG C of incubated overnight.Next day, be inoculated in the LB liquid nutrient medium containing Amp with sterile toothpick picking white colony, 37 DEG C of shaking table overnight incubation.Plasmid is extracted with the little extraction reagent kit of ordinary plasmids.For primer pair, the Insert Fragment extracting plasmid is identified with SPSODF and SPSODR, with EcoRI, enzyme is carried out to extracted plasmid simultaneously and cut qualification.Cut all correct plasmid of qualification send precious biotechnology (Dalian) company limited to check order by through PCR, enzyme.Blast software analysis in sequencing result NCBI.

The structure of sod prokaryotic expression vector

Use NdeI and XhoI in 37 DEG C of double digestion 4h respectively order-checking correct recombinant plasmid pGEM-T-sod and recombinant plasmid pET30a-apc α, cut glue with sepharose DNA recovery test kit and reclaim sod gene insert and linearizing pET30a carrier respectively.Use the SolutionI in DNALigationKit, sod gene insert is connected with linearizing pET30aVectorDNA.With connecting in product thermal transition competence e. coli jm109, and be coated on the LB agar plate containing 50 μ g/ml kantlex, 37 DEG C of incubated overnight.Picking list bacterium colony incubated overnight also extracts plasmid, uses primer T7t to institute's upgrading grain order-checking.

The abduction delivering of recombinant plasmid in intestinal bacteria

The recombinant plasmid pET30a-sod correct with order-checking transforms BL21 (DE3) competent cell, obtains positive expression bacterial strain.100 μ l recombinant strains are seeded in 10ml containing in the LB liquid nutrient medium of 50 μ g/ml kantlex 37 DEG C, 220rpm shaking culture spends the night, and obtains seed liquor.Get glycerol adding to 30% under Some seeds liquid aseptic condition, packing-70 DEG C preservation.1ml seed liquor is inoculated in 100ml containing in 2 × YT liquid nutrient medium of 50 μ g/mL kantlex.37 DEG C, 230rpm shaking culture 3.5h.Take out culturing bottle, sterile sampling 1ml from culture, left blank contrasts.Then in culturing bottle, add the IPTG that final concentration is 0.40mM, regulate culture temperature to 31 DEG C, 230rpm shaking culture 5h.Get before inducing and the rear each 0.5ml of bacterium liquid of induction, 4 DEG C, the centrifugal 10min of 9000 × g, abandon supernatant, precipitation is resuspended in 100 μ lPBS, adds 25 μ l5 × SDS-PAGE sample-loading buffers, after 100 DEG C of thermal treatment 3min, get 10 μ l and carry out 12%SDS-PAGE electrophoretic analysis.

The foundation of restructuring spirulina SOD ferment tank technique

The preparation of ferment-seeded

Get-70 DEG C of glycerol stock 100ul and be inoculated in 5mlLB (kan ⁺) in substratum, 37 DEG C, 220r/min activated overnight cultivates 18h.5ml culture is transferred in 150mlTB (kan ⁺) in substratum 37 DEG C, 220r/min incubated overnight 20h, obtains ferment-seeded.

Fermentor tank prepares

By pH electrode more than aquation 3h in distilled water, the damping fluid of pH6.86 and pH4.0 is then utilized to complete Zero calibration and the slope demarcation of electrode successively.Dissolved oxygen electrode is immersed energising polarization more than 6h in distilled water simultaneously, in rear immersion saturated sodium bisulfite solution, complete Zero calibration.Lay down fermentor tank motor, pH electrode, dissolved oxygen electrode are reinstalled in tank the fermentor tank filling 2.25LTB basic medium.By fermentor tank together with feed supplement bottle, mend demijohn, mend alkali bottle, 250ml potassium phosphate buffer and pipeline thereof in 121 DEG C of sterilizing 20min.All circuit and the piping system of fermentor tank is assembled successively after sterilizing.

Fermentation

Open upper computer and air compressor, add 250ml potassium phosphate buffer, 2ml defoamer (Antifoam204SIGMA) and 2mlkan with flame inoculation method successively from inoculation mouth ⁺(50mg/ml).Upper computer is set as follows culture condition: temperature 37 DEG C, air flow 200L/h, pH7.0, rotating speed 500r/min, demarcates 100% value of DO electrode in this case.Appropriate regulation air pressure, leaves and takes 50ml substratum in contrast from material taking mouth.Stop stirring and ventilation, aseptic access 150ml seed liquor in tank, adjustment air flow is 200L/h, rotating speed 500r/min, then clicks fermentation and starts, preserve this Batch fermentation record.Get 10ml substratum by material taking mouth to contrast as 0h.By regulating air flow and rotating speed to make DO remain on about 30% in fermenting process, temperature is set to automatic control.By automatic dripping mend acid solution (2.4MH DEG C l) and mend alkali lye (4.18M ammoniacal liquor) adjust ph.When fermenting to about 7h, in substratum, carbon source exhausts oxygen dissolving value and rises rapidly, starts in tank, drip feed supplement liquid 400ml (every 400ml is containing yeast extract 12.5g, glycerine 240ml) continuously.By the rate of addition of adjustment feed supplement liquid, rotating speed and air flow in feed supplement process, make DO remain on about 20%, this process medium speed generally remains on about 700r/min as far as possible.When fermentation is 0.4mM to 12h from the inoculation aseptic 1.25mlIPTG of adding of mouth (0.2g/ml) to final concentration, temperature is regulated to be 31 DEG C, coinduction 7h.Sampling 10ml per hour in fermenting process, for bacterial growth dynamic monitoring.During fermentation ends, reduce air flow and rotating speed, click fermentation ends, preserve data.Fermented liquid is proceeded in aseptic triangular flask, close source of the gas.

Bacterial growth dynamic monitoring

With the zeroing of TB substratum, measure the absorbancy OD of per hour stayed bacterium liquid at 600nm place in fermenting process ₆₀₀; Get 10ml bacterium liquid, 4 DEG C, the centrifugal 10min of 9400 × g, abandons supernatant, blots residual liquid with cotton swab, weighs thalline weight in wet base (WCW).Target protein expression effect SDS-PAGE analyzes, and before getting induction and each 1.5ml of fermentation ends bacterium liquid, centrifugally abandons supernatant.Precipitation is resuspended in 0.8mlPBS, ultrasonic treatment, and cracking condition is: power 400W, and work 4S, stops 6S, about 25 circulations altogether.The centrifugal 10min of lysate 14000 × g, gets 50 μ l supernatant liquor electrophoresis.

Tunning is preserved

Abandon supernatant gently after fermented liquid being put 4 DEG C of hold over night, be deposited in 4 DEG C, the centrifugal 20min of 4000 × g, abandons supernatant, collects bacterium mud, puts-20 DEG C save backup with every part of about 10g.

The purifying of restructuring spirulina SOD

Recombinant bacterium cracking

4 ~ 8g recombinant bacterium precipitation is fully resuspended in 80ml non denatured binding buffer liquid 60mMNaH ₂pO ₄, 0.5MNaCl, 10mM imidazoles, pH7.4) in.Connect nanometer high pressure homogenizer air-channel system, open air compressor.With about 100ml distilled water cleaning clarifixator.To treat that cracking bacterial suspension adds in sample introduction bucket, 15000psi pressure homogeneous bacterium is bright to lysate.After 58 DEG C of heating in water bath process 10min, in 9400 × g ,-4 DEG C of centrifugal 30min, 0.45 μm of frit removing cell debris, filtrate can direct upper prop.

Column purification

Get the empty chromatography column 1 of 45ml, fill up with Ni-NTAAgarose resin.Pillar is balanced with about 150ml non denatured binding buffer liquid with after the drip washing of about 150ml distilled water.The bacterial lysate handled well is instilled in post by peristaltic pump, if nucleic acid-protein detector reading rises comparatively large in loading process, then collects effluent liquid one and manage.With about 200ml non denatured washing buffer (50mMNaH ₂pO ₄, 0.5MNaCl, 20mM imidazoles, pH7.4) wash pillar to the nearly background of light absorption value and no longer reduce.Need in process to collect light absorption value rising, peak value, at least each 1 pipe of decrement phase effluent liquid.With low pH elutriant (50mMNaH ₂pO ₄, 0.5MNaCl, 0.1MKCl, pH4.5.) and wash-out pillar, start to collect elutriant when light absorption value rises to about 0.080, drop to after below 0.080 to light absorption value and stop collecting.With about 200ml distilled water drip washing pillar after purifying.Protein purification effect is detected with SDS-PAGE.

Pure enzyme liquid concentration and determination of activity

Broadford method is adopted to measure pure enzyme liquid concentration.Pyrograllol autoxidation is adopted to measure the activity of enzyme.When enzymic activity is defined as 25 DEG C, the enzyme amount in 1ml reaction solution during every 1min suppression rosette like growth rate 50% is an activity unit.

Native gel electrophoresis

Preparation concentrates the native gel of glue containing 12% separation gel and 0.5%.Concentrated glue damping fluid is 1.5MTris (pH8.8), and separation gel damping fluid is 0.5MTris (pH6.8), does not add SDS in glue.Non denatured electrode buffer (0.025MTris, 0.2M glycine, pH8.3) is added in electrophoresis chamber.Protein sample and sample buffer (0.1MTris-HCl, 10% glycerine, 0.01mg/ml tetrabromophenol sulfonphthalein, pH6.8) equal-volume are mixed, direct loading.Switch on power (upper groove connects negative pole), and 130 volts of constant voltage electrophoresis enter separation gel to sample, and rear regulating voltage 180 volts terminates to electrophoresis.Take out gel, put in ultrapure water and put into microwave oven and boil 7min, totally 2 times.Remove free water with ultrapure water short rinse gel, glue is put into super sensitivity protein electrophorese quick dyeing liquid, put microwave oven and boil 2min, then put decolorization swinging table jog dyeing 10min.Finally glue is immersed in distilled water and cleans, saving result of taking pictures.

The NBT dyeing of restructuring SOD

The native gel electrophoresis of restructuring SOD is separated

Respectively by the SOD of 50 μ l purifying and isopyknic 50mMPBS, 25mMH ₂o ₂solution, 50mMH ₂o ₂solution, chloroform-ethanol (volume ratio 3: 5) mix, and after putting 37 DEG C of incubation 1h, carry out native gel electrophoresis.Scaled off together with its contiguous swimming lane by albumen Marker after electrophoresis, dye with super sensitivity protein electrophorese quick dyeing liquid, all the other gels carry out NBT dyeing.

NBT dyes

By soak in NBT dyeing solution 1 (0.245MmNBT solution), lucifuge places 20min.Then gel is gone to successively NBT dyeing solution 2 (36mMPBS, 28mMTEMED, 0.028mM riboflavin, pH7.8) and in NBT dyeing solution 3 (50mMPBS, 0.1mMEDTA, pH7.8), soak 20min in the sunlight respectively, period needs repeatedly jolting mixing.Finally gel is gone in distilled water saving result of taking pictures after cleaning.

Determination of Metals in pure enzyme liquid

ICPE-9000 plasma emission spectrometer is utilized to measure the metallic element such as iron, manganese, copper, zinc, chromium, cadmium, lead, aluminium in pure enzyme liquid.Test basis is " drinking water standard method of inspection metal index " (GB/T5750.6-2006).Utilize the standard solution preparation standard seriess such as iron, manganese, copper, zinc, chromium, cadmium, lead, aluminium, conditioning instrumentation is to optimum Working, and bioassay standard series, drawing standard curve, calculates regression equation.Then pure enzyme liquid is imported instrument, measure each metallic element.

Temperature is on the impact of enzyme liquid activity

By certain batch of pure enzyme liquid respectively at 60 DEG C of heat treated 15min, 30min, 1h, 2h, 3h; At 65 DEG C of heat treated 15min, 30min, 1h.Put ice bath cooling after in the centrifugal 10min of 14000 × g.Get centrifugal after enzyme liquid supernatant, with Broadford method measure protein concentration, measure specific activity with pyrograllol autoxidation, and compare with untreated enzyme liquid activity.

PH environment is on the impact of enzyme liquid activity

After certain batch of pure enzyme liquid is done 1: 5 dilution with the buffered soln of pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH6.5, pH7.0, pH7.5, pH8.0, pH8.5, pH9.0, pH9.5 and pH10.0 respectively, put 25 DEG C and hatch at least 20min.The activity of ferment treatment liquid is measured with pyrograllol autoxidation.

The construction and expression of pET30a-sod expression plasmid

The clone of sod gene

As shown in Figure 2, go out a specific band with Auele Specific Primer to SPSODF/SPSODR Successful amplification from extracted spirulina plalensis DNA, this band is between 750bp and 500bp, and its size is consistent with expection amplification gene clip size.

Recombinant plasmid constructed by the PCR primer of purifying is connected with pGEM-TEasyVector cuts qualification through pcr amplification and EcoRI enzyme, and the fragment of the 600bp that all confirms to have an appointment successfully is inserted in cloning vector.Check order to this Insert Fragment and carry out Blast analysis to obtained sequence, result shows that in institute's cloned gene sequence and GenBank, spirulina plalensis sod gene order (accession number: AY282414) is completely the same.

The structure of pET30a-sod recombinant plasmid

As shown in Figure 3, Figure 4, utilize NdeI and XhoI double digestion recombinant plasmid pGEM-T-sod and pET30a-apc α respectively, obtain length and be about the Insert Fragment of 614bp and the pET30a linearized vector of 5076bp respectively.This Insert Fragment is connected with linearized vector and transformed competence colibacillus cell JM109.Extracted plasmid order-checking is shown that Insert Fragment is successfully connected with carrier, illustrates that pET30a-sod expression plasmid successfully builds.Be illustrated in figure 5 the open reading frame of plasmid pET30a-sod and the aminoacid sequence of coding thereof.

The expression of restructuring SOD

As shown in Figure 6, the e. coli bl21 (DE3) proceeding to recombinant expression plasmid pET30a-sod is the IPTG5h induction of 0.40mM through final concentration, and successful expression molecular weight is about the recombinant protein of 22kDa.

The high density fermentation result of recombinant protein

It is relatively slow that fermentation starts front 3h bacterial growth, and when fermenting to 3h, OD600 is 4.31, and bacterium weight in wet base is 13g/L.After 3h, bacterium starts ramp, and during to 6h, OD600 reaches 20.45, and bacterium weight in wet base is 22g/L.After 7h, bacterial growth is slack-off, starts feed supplement.After this, bacterium is restoration ecosystem again, and enters mild vegetative period.When fermentation is to 18h, OD600 reaches 33.2, and bacterium weight in wet base reaches 38g/L.After this in 2h, bacterial growth is slack-off, and OD600 and bacterium weight in wet base decline all to some extent, terminates fermentation during 20h.As shown in Figure 7, the growth curve of bacterium in fermenting process.As shown in Figure 8, SDS-PAGE analyzes display, and when IPTG starts to induce, without protein expression in bacterial lysate supernatant, after IPTG induction, successful expression molecular weight is the recombinant protein of 22kDa.

The purifying of restructuring SOD

As shown in Figure 9, recombinant bacterium lysate, after specific binding resin Ni-NTAAgarose mono-takes turns purifying, obtains the target protein SOD that composition is single, and in crude protein washings after purifying, target protein amount is little.

The concentration of restructuring SOD and determination of activity

The restructuring SOD of purifying measures through Broadford, and its protein concentration is for being 3mg/ml.It is 10517U/ml that pyrograllot autoxidation records its enzymic activity, and specific activity is 3506U/mg.

Restructuring SOD native gel electrophoresis and NBT coloration result

As shown in Figure 10, native gel electrophoresis analysis shows that the natural molecule amount of restructuring SOD is 38kDa, and prompting restructuring SOD may exist with dimeric form.By SOD and PBS, H ₂o ₂solution, chloroform-ethanol mixed solution etc. carry out native gel electrophoresis after hatching respectively again, and carry out NBT dyeing to gel.Result display H ₂o ₂solution partly can suppress the activity of SOD, and chloroform-ethanol mixed solution can suppress the activity of SOD completely, and contrasts the activity that PBS can not suppress SOD.Experimental result shows that the standby SOD of this institute system is Fe-SOD.

Metal element content in pure enzyme liquid

ICPE-9000 plasma emission spectrometer is utilized to record the middle metallic element contained amount of pure enzyme liquid (protein concentration is 0.95mg/ml) in table 1.By calculating, learn that each pure enzyme dimer is about chelating 1.35 iron atoms.

Metal element content in the pure enzyme liquid of table 1.

The thermostability of restructuring SOD

As shown in figure 11, the pure enzyme liquid of certain batch of purifying is through 60 DEG C of heating, and along with the prolongation of heat-up time, the specific activity of SOD is in slow reduction.Through 60 DEG C of thermal treatment 180min, the specific activity of SOD is reduced to 2343U/mg from 3380U/mg, and its activity is reduced to original 69.3%.As shown in figure 12, restructuring SOD is active under 65 DEG C of conditions to be reduced comparatively fast, and through 65 DEG C of heat treated 60min, its activity is reduced to original 42.7%.

The ph stability of restructuring SOD

As shown in figure 13, by the activity measuring enzyme after the buffered soln process of different pH value of the SOD after purifying, SOD is more stable in pH4.0 to pH9.5 scope in result display, and within the scope of this, activity change is little.When pH value reaches 10, the activity of enzyme slightly declined.

Claims (7)

1. a restructuring spirulina superoxide-dismutase, is characterized in that the nucleotide sequence of described restructuring spirulina superoxide dismutase gene sequence and corresponding aminoacid sequence are:

2. recombinate a spirulina superoxide-dismutase preparation technology, it is characterized in that: described step of preparation process is as follows:

One, the extraction of spirulina genomic dna

(1) with distilled water, the centrifugal 5min of fresh spirulina plalensis 5000g is washed 3 times altogether;

(2) fresh for 280mg spirulina plalensis being mixed with the quartz sand of a small amount of sterilizing in the mortar of freezing treatment in advance, fully grind;

(3) extraction genomic dna is carried out by the step of plant genome DNA extraction test kit specification sheets;

(4) the genomic dna wash-out extracted is in 50 μ l elutriant TE;

(5) genomic dna after wash-out saves backup at-20 DEG C of temperature.

Two, the amplification of sod gene fragment

(1) according to the AY282414 spirulina sod gene order design of amplification primers logged in GenBank: upstream primer SPSODF:5 '-ACATATGGCTTTTGAACTTCCCAG-3 '; Downstream primer SPSODR:5 '-GCTCGAGGCTGGCAGACGCGAGA-3 ', the long 614bp of primer pair expection amplified fragments;

(2) pcr amplification sod gene, with extraction spirulina genomic dna for template, utilize above-mentioned primer pair amplifies sod gene, PCR reaction conditions is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 45s, 55 DEG C of annealing 45s, 72 DEG C extend 45s, totally 30 circulations, then, 72 DEG C extend 6min again;

(3) get 5 μ lPCR products and carry out 1.5% agarose gel electrophoresis analysis.

Three, the clone of sod gene fragment

(1) 45 μ lPCR amplified productions are separated through 1.5% agarose gel electrophoresis, reclaim test kit with sepharose DNA and cut glue recovery goal gene fragment, reclaim product 40 μ l elution buffer EB wash-outs, concrete operations are reclaimed test kit specification sheets by sepharose DNA and are carried out;

(2) PCR primer of 3 μ l purifying and 1 μ l cloning vector pGEM-TEasyVector are spent the night 4 DEG C of connections;

(3) 5 μ l connected the heat-shock transformed 100 μ l competence bacillus coli DH 5 alphas of product and coat on the LB agar plate containing 50 μ g/ml penbritins, putting 37 DEG C of incubated overnight;

(4) next day, be inoculated in the LB liquid nutrient medium containing Amp with sterile toothpick picking white colony, 37 DEG C of shaking table overnight incubation;

(5) plasmid is extracted with the little extraction reagent kit of ordinary plasmids.Carry out amplification qualification with the Insert Fragment that SPSODF and SPSODR extracts plasmid for primer pair, with EcoRI, enzyme is carried out to extracted plasmid simultaneously and cut qualification;

(6) plasmid cutting qualification all correct through PCR, enzyme is checked order;

(7) the Blast software analysis in sequencing result NCBI.

Four, the structure of sod prokaryotic expression vector

(1) be that the spirulina plalensis allophycocyanin α subunit gene prokaryotic expression carrier that this laboratory builds uses NdeI and XhoI in 37 DEG C of double digestion 4h respectively by order-checking correct recombinant plasmid pGEM-T-sod and recombinant plasmid pET30a-apc α, reclaim test kit with sepharose DNA and cut glue and reclaim sod gene insert and linearizing pET30a carrier respectively;

(2) use the SolutionI in DNALigationKit, sod gene insert is connected with linearizing pET30aVectorDNA;

(3) with connecting in product thermal transition competence e. coli jm109, and be coated on the LB agar plate containing 50 μ g/ml kantlex, 37 DEG C of incubated overnight;

(4) picking list bacterium colony incubated overnight extract plasmid, uses primer T7t to institute's upgrading grain order-checking.

Five, the abduction delivering of recombinant plasmid in intestinal bacteria

(1) transform BL21 (DE3) competent cell with the recombinant plasmid pET30a-sod that order-checking is correct, obtain positive expression bacterial strain;

(2) 100 μ l recombinant strains are seeded in 10ml containing in the LB liquid nutrient medium of 50 μ g/ml kantlex 37 DEG C, 220rpm shaking culture spends the night, and obtains seed liquor;

(3) glycerol adding to 30% under the seed liquor aseptic condition described in part steps (2) is got, packing-70 DEG C preservation, for subsequent use;

(4) the 1ml seed liquor described in step (2) is inoculated in 100ml containing in 2 × YT liquid nutrient medium of 50 μ g/mL kantlex, 37 DEG C, cultivate 3.5h under 230rpm oscillating condition, take out culturing bottle, sterile sampling 1ml from culture, left blank contrasts;

(5) then in culturing bottle, add the IPTG that final concentration is 0.40mmol/l, regulate culture temperature to 31 DEG C, 230rpm shaking culture 5h;

(6) get before induction and the rear each 0.5ml of bacterium liquid of induction, 4 DEG C, the centrifugal 10min of 9000g, abandon supernatant, precipitation is resuspended in 100 μ lPBS, adds 25 μ l5 × SDS-PAGE sample-loading buffers, after 100 DEG C of thermal treatment 3min, get 10 μ l and carry out 12%SDS-PAGE electrophoretic analysis.

Six, restructuring spirulina SOD ferment tank

(1) preparation of ferment-seeded

1. getting described step 5-(3)-70 DEG C of glycerol stock 100 μ 1 is inoculated in 5mlLB (kan+) substratum, 37 DEG C, and 220r/min activated overnight cultivates 18h;

2. 5ml culture to be transferred in 150mlTB (kan+) substratum 37 DEG C, 220r/min incubated overnight 20h, obtain ferment-seeded.

(2) fermentor tank prepares

1. by pH electrode more than aquation 3h in distilled water, the damping fluid of pH6.86 and pH4.0 is then utilized to complete Zero calibration and the slope demarcation of electrode successively;

2. dissolved oxygen electrode is immersed energising polarization more than 6h in distilled water while 1. step carries out, in rear immersion saturated sodium bisulfite solution, complete Zero calibration;

3. lay down fermentor tank motor, pH electrode, dissolved oxygen electrode are reinstalled in tank the fermentor tank filling 2.25LTB basic medium;

4. by fermentor tank together with feed supplement bottle, mend demijohn, mend alkali bottle, 250ml potassium phosphate buffer and pipeline thereof in 121 DEG C of sterilizing 20min;

5. all circuit and the piping system of fermentor tank is assembled after sterilizing successively.

(3) ferment

1. open upper computer and air compressor, add 250ml potassium phosphate buffer, 2ml defoamer (Antifoam204SIGMA) and 2mlkan+ (50mg/ml) with flame inoculation method successively from inoculation mouth;

2. on upper computer, be set as follows culture condition: temperature 37 DEG C, air flow 200L/h, pH7.0, rotating speed 500r/min, demarcate 100% value of DO electrode in this case.

3. appropriate regulation air pressure, leaves and takes 50ml substratum in contrast from material taking mouth.

4. stop stirring and ventilation, aseptic access 150ml seed liquor in tank, adjustment air flow is 200L/h, rotating speed 500r/min, then clicks fermentation and starts, preserve this Batch fermentation record.

5. get 10ml substratum by material taking mouth to contrast as 0h.

6. make DO remain on about 30% by adjustment air flow and rotating speed in fermenting process, temperature is set to automatic control;

7. mend acid solution (2.4MHCl) by automatic dripping and mend alkali lye (4.18M ammoniacal liquor) adjust ph;

8. when fermenting to about 7h, in substratum, carbon source exhausts oxygen dissolving value and rises rapidly, starts to drip feed supplement liquid 400ml continuously in tank; By the rate of addition of adjustment feed supplement liquid, rotating speed and air flow in feed supplement process, make DO remain on about 20%, this process medium speed generally remains on about 700r/min as far as possible;

9. when fermentation, to 12h, from inoculating, mouth is aseptic adds 1.25mlIPTG (0.2g/ml), is 0.4mM, regulates temperature to be 31 DEG C, coinduction 7h to IPTG final concentration;

10. sampling 10ml per hour in fermenting process, the dynamic and protein expression level monitoring for bacterial growth; During fermentation ends, reduce air flow and rotating speed, click fermentation ends, preserve data.Fermented liquid is proceeded in aseptic triangular flask, close source of the gas.

(4) bacterial growth dynamically and protein expression effect monitoring

1. with the zeroing of TB substratum, the absorbancy OD600 of per hour stayed bacterium liquid at 600nm place in fermenting process is measured;

2. get 10ml bacterium liquid, 4 DEG C, the centrifugal 10min of 9400 × g, abandons supernatant, blots residual liquid with cotton swab, weighs thalline weight in wet base (WCW).

3. target protein expression effect SDS-PAGE analyzes, and before getting induction and each 1.5ml of fermentation ends bacterium liquid, centrifugally abandons supernatant;

4. precipitation is resuspended in 0.8mlPBS, ultrasonic treatment, and cracking condition is: power 400W, and work 4S, stops 6S, about 25 circulations altogether.The centrifugal 10min of lysate 14000 × g, gets 50ul supernatant liquor and carries out SDS-PAGE detection;

5. precipitation is resuspended in 1ml distilled water, gets 50ul electrophoresis.

(5) tunning is preserved

Abandon supernatant gently after fermented liquid being put 4 DEG C of hold over night, be deposited in 4 DEG C, the centrifugal 20min of 4000g, abandons supernatant, collects bacterium mud, puts-20 DEG C save backup with every part of about 10g.

Seven, the purifying of restructuring spirulina SOD

(1) recombinant bacterium cracking

1. 4 ~ 8g recombinant bacterium precipitation is fully resuspended in 80ml non denatured binding buffer liquid;

2. connect nanometer high pressure homogenizer air-channel system, open air compressor;

3. with about 100ml distilled water cleaning clarifixator;

4. will treat that cracking bacterial suspension adds in sample introduction bucket, 15000psi pressure cracking bacterium 3 is taken turns to lysate bright;

5. after lysate being put 58 DEG C of heating in water bath process 10min, in 9400 × g ,-4 DEG C of centrifugal 30min, 0.45 μm of frit removing cell debris, filtrate can direct upper prop.

(2) column purification

1. get the empty chromatography column 1 of 45ml, fill up with Ni-NTAAgarose resin;

2. pillar is balanced with about 150ml non denatured binding buffer liquid with after the drip washing of about 150ml distilled water;

3. the bacterial lysate handled well is instilled in post by peristaltic pump, if nucleic acid-protein detector reading rises comparatively large in loading process, then collect effluent liquid one and manage;

4. no longer reduce to the nearly background of light absorption value with about 200ml non denatured washing buffer washing pillar;

5. need in process to collect light absorption value rising, peak value, at least each 1 pipe of decrement phase effluent liquid;

6. with low pH elution pillar, start to collect elutriant when light absorption value rises to about 0.080, drop to after below 0.080 to light absorption value and stop collecting;

7. about 200ml distilled water drip washing pillar is used after purifying;

8. protein purification effect is detected with SDS-PAGE.

3. one restructuring spirulina superoxide-dismutase preparation technology according to claim 2, is characterized in that: inductor IPTG optimal expression condition added in (5), (6), (7) in described step 5 is induce 5h with the IPTG of 0.40mmol/l.

4. one restructuring spirulina superoxide-dismutase preparation technology according to claim 2, is characterized in that: the upstream primer SPSODF:5 '-A described in described step one cATATGthe middle underscore part of GCTTTTGAACTTCCCAG-3 ' is NdeI restriction enzyme site; Downstream primer SPSODR:5 '-G cTCGAGgCTGGCAGACGCGAGA-3 ', middle underscore part is XhoI restriction enzyme site.

5. one restructuring spirulina superoxide-dismutase preparation technology according to claim 2, is characterized in that: at surface uniform coating 200mg/mlIPTG7 μ l, 20mg/mlX-Gal28 μ l before use on the LB agar plate in described step 4.

6. one restructuring spirulina superoxide-dismutase preparation technology according to claim 2, is characterized in that: in described step 6, (3)-8. described every 400ml of feed supplement liquid contains yeast extract 12.5g, glycerine 240ml.

7. one restructuring spirulina superoxide-dismutase preparation technology according to claim 1, is characterized in that: the non denatured binding buffer liquid described in described step 7: 50mMNaH ₂pO ₄, 0.5MNaCl, 0.1MKCl, 10mM imidazoles, pH7.4; Non denatured washing buffer: 50mMNaH ₂pO ₄, 0.5MNaCl, 0.1MKCl, 20mM imidazoles, pH7.4; Low pH elutriant: 50mMNaH ₂pO ₄, 0.5MNaCl, 0.1MKCl, pH4.5.