CN101157897A - Schizosaccharomyces pombe engineering strain having soybean MnSOD gene and constructing method thereof - Google Patents
Schizosaccharomyces pombe engineering strain having soybean MnSOD gene and constructing method thereof Download PDFInfo
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Abstract
The invention discloses an engineering bacteria of fission yeast schizosaccharomyces pombe which contains MnSOD genes of beans and a construction method thereof, comprising a special carrier pESPUC which expresses a manganese superoxide dismutase gene MnSOD, and the expression of the recombinant plasmids in the fission yeast schizosaccharomyces pombe at different periods. The invention clones the MnSOD genes of beans and restructures the fission yeast schizosaccharomyces pombe to express the carrier pESPUC-MnSOD. The recombinant plasmids are led into the fission yeast schizosaccharomyces pombe by a lithium acetate method or an electro transformation method to ensure the plasmids can be expressed. The manganese superoxide dismutase MnSOD genes obtained in the invention can get the best condition to express in the fission yeast schizosaccharomyces pombe. At the same time the culture of saccharomyces is used for industrial production, which has the advantages of inexpensive raw material, short growth cycle, large-scaled production and low cost of extraction. The two things combined together has important prospect of industrial application and practical significance.
Description
Technical field
The present invention relates to a kind of schizosaccharomyces pombe engineering bacteria and construction process thereof with soybean MnSOD gene
Background technology
Research to SOD originates in 1938.Britain T.mann in 1938 and D.keilin isolate copper bearing blue green protein the earliest from bovine blood, claimed superoxide dismutase at that time.Nineteen fifty-three isolates albuminoid and claims hepatocuprein from the horse liver, isolate this albumen afterwards again and claim cerebrocuprein from brain.
Mccord in 1969 and Fridovich find that this albumen has catalysis O
2, the function of disproportionation reaction takes place, thus with this enzyme called after superoxide-dismutase (Superoxide Dismutase, SOD, EC1.15.1.1).
(Superoxide dismutase SOD) is a kind of crucial biological enzyme of organism defence oxidative damage to superoxide-dismutase, can catalysis superoxide anion (O
2-) disproportionation reaction takes place, thus the biological intravital superoxide anion of single-minded removing, and the oxyradical of balance body is resisted the toxicity of oxyradical and other oxide compound radical pair cytoplasmic membranes preferably.
In higher plant, SOD is divided three classes according to its prothetic group position bonded different metal ion: MnSOD, FeSOD, CuZnSOD.CuZnSOD is the abundantest class of content in three kinds of superoxide-dismutases in plant, mainly is present in chloroplast(id), kytoplasm and the peroxysome.Each subunit of MnSOD and FeSOD all only contains a metal ion, and MnSOD mainly is present in the plastosome, and FeSOD is present in the chloroplast(id).
The catalytic activity of MnSOD is not subjected to its product H
2O
2Inhibition.Plastosome is the main place that eukaryotic cells carries out aerobic repiration, realizes the function of energy transformation by the electron transport chain of its inner membrance; When biology be in coerce or the pathology condition under, the mitochondrial inner membrane electron transport is obstructed, and will cause the excessive generation of active oxygen, is to cause whole cell to damage to plastosome.Therefore MnSOD eliminates the not key enzyme of receptor 1 activity oxygen injury of superoxide anion, protective wire plastochondria in the cell mitochondrial.
MnSOD is the another kind of SOD except that CuZnSOD in the human body, compare with CuZnSOD to have long, advantage such as molecular weight is little of life-span, and the toxicity of manganese is low than copper iron that its small molecules title complex is good as simulating the MnSOD security.Thereby it is very active about the research of MnSOD in recent years.
The characteristics of MnSOD are in SOD family: biological half-life is longer, and the anti-inflammatory radiation resistance is better than CuZnSOD, and this shows that MnSOD has crucial medical value and potential potential applicability in clinical practice as zymin.
Because MnSOD exists only in the plastosome, content is very little, be difficult to the relatively large MnSOD albumen of separation and purification from natural tissues with traditional biochemical method, therefore adopt the method for molecular cloning to obtain the gene order of MnSOD, and to make MnSOD gene great expression in yeast by transgenic method be the important channel of its function of further investigation.
Yeast is a unicellular eukaryote, and it is fast to have a growth, is easy to genetic manipulation, can translates post-treatment and modification to foreign protein, does not produce characteristics such as toxic products, is considered to express the suitable host of foreign protein.
Schizosaccharomyces (Schizosaccharomyces) cell ellipse, cylindrical is engaged by vegetative cell, forms ascus.Fermentation capacity is arranged, and representative species is a schizosaccharomyces pombe, separates the earliest from African maize wine, can make the jerusalem artichoke fermentation produce alcohol.Schizosaccharomyces pombe (fission yeast or Schizosaccharomyces pombe) is a kind of simple unicellular organism, and its genome is 14Mb, approximately is 4 times of bacillus coli gene group.This yeast is different from the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) that belongs to ascomycetous fungus, many two primary yeasts that studies show that are at the aspects such as tissue, structure and expression of gene regulation and control of the biosynthesizing of phylogenetic systematics, cell cycle, rRNA and gene and inequality, fission yeast and higher animal but have certain similarity in some aspects, therefore, fission yeast also is the Eukaryotic model animals of a kind of good research.
Summary of the invention
The invention provides a kind of schizosaccharomyces pombe engineering bacteria and construction process thereof with soybean MnSOD gene.
The technical scheme that the present invention takes is: the reorganization schizosaccharomyces pombe expression vector that contains soybean MnSOD gene that will make up imports in the schizosaccharomyces pombe and obtains.
The present invention is inserted into the MnSOD gene at the multiple clone site place of schizosaccharomyces pombe expression vector, this reorganization schizosaccharomyces pombe expression vector pESPUC-MnSOD that makes up is imported in the schizosaccharomyces pombe, obtain reorganization schizosaccharomyces pombe transformant, cultivate the reorganization schizosaccharomyces pombe and make the MnSOD gene obtain expressing.
The present invention schizosaccharomyces pombe expression vector pESPUC-MnSOD that recombinates, this carrier contains Pnmt1 promotor, Pnmt1 terminator sequence, resistant gene is a penbritin, selectable marker gene LEU, and soybean MnSOD gene is cut between site SalI and the BamHI at polyclone enzyme.
Soybean MnSOD gene of the present invention is respectively SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 is described.
Yeast conversion method of the present invention is Lithium Acetate conversion method and electric shock conversion method.
IPTG can induce MnSOD gene great expression in schizosaccharomyces pombe among the present invention.
The present invention has announced the influence of inductor IPTG to the expression of MnSOD albumen in schizosaccharomyces pombe, and IPTG induces yeast expression, and 48 hours MnSOD expressing quantities are the highest cultivating, and expression amount will be higher than and do not add IPTG and cultivate 96 hours proteic expression amounts.Thereby IPTG can induce the great expression of MnSOD gene in schizosaccharomyces pombe as can be known.
The present invention has compared the expression of different lengths MnSOD gene in schizosaccharomyces pombe, has obtained expression vector expression amount and active maximum in yeast that open reading frame sequence makes up.
A kind of construction process that contains the schizosaccharomyces pombe expression vector of manganese superoxide dismutase provided by the present invention.Simultaneously with the expression of different lengths MnSOD gene in schizosaccharomyces pombe.Remedied the deficiency of traditional manganese superoxide dismutase preparation method and prokaryotic expression system, yeast saccharomyces cerevisiae and pichia yeast expression system.In addition, as if the production that invention is applied to the industrialization manganese superoxide dismutase, it is big then to have industrial scale, manganese superoxide dismutase is active high, and enzymatic productivity is strong, and growth cycle is short, the advantage that extraction cost is low has important and practical meanings and wide development prospect.
Description of drawings
Fig. 1 is a schizosaccharomyces pombe expression vector pESPUC collection of illustrative plates.
Fig. 2 is reorganization schizosaccharomyces pombe expression vector pESPUC-MnSOD design of graphics.
Fig. 3 cuts evaluation figure for reorganization schizosaccharomyces pombe expression vector pESPUC-MnSOD enzyme.
Fig. 4 identifies figure for reorganization schizosaccharomyces pombe expression vector pESPUC-MnSOD PCR.
Fig. 5 is reorganization schizosaccharomyces pombe transformant.
Fig. 6 identifies figure for reorganization schizosaccharomyces pombe transformant PCR.
Fig. 7 cuts evaluation figure for reorganization schizosaccharomyces pombe transformant enzyme.
Fig. 8 be contain the MnSOD gene the expression (SDS-PAGE) of recombinant plasmid in schizosaccharomyces pombe (48h-96h).
Fig. 9 be contain the MnSOD gene the expression (Native-PAGE) of recombinant plasmid in schizosaccharomyces pombe (0h-96h).
Figure 10 is same incubation time, and the expression amount of MnSOD different lengths fragment in schizosaccharomyces pombe relatively
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment
Goal gene: MnSOD (deriving from black soya bean), soybean MnSOD gene are respectively full length sequence SEQ ID NO.1, open reading frame sequence SEQ ID NO.2, and mature peptide sequence SEQ ID NO.3 is described.
Structure contain superoxide dismutase gene schizosaccharomyces pombe carrier pESPUC-MnSOD process as shown in Figure 2, concrete steps are as follows:
1) this to test used expression vector be fabric shuttle-type schizosaccharomyces pombe Yeast expression carrier pESPUC, see Fig. 1.CDNA sequence according to MnSOD.With the primer that has restriction enzyme site increase respectively full length sequence, open reading frame sequence and mature peptide sequence.
Primer following (forward primer restriction enzyme site SalI, reverse primer restriction enzyme site BamHI)
The total length primer
FS 5’ATT?
GTCGAC?ATGGC?CGCGC?GAGCT?CTGT 3’
FA 5’?CG?
GGATCC?GGATC?AAACC?CTGGA?GGCA 3’
The open reading frame sequence primer
CS 5’ATT?
GTCGAC?ATGGC?CGCGC?GAGCT?CTGT 3’
CA 5’?GC?
GGATCC?CTAAG?AGCTC?TCTTT?CTCAT?AC 3’
The mature peptide aligning primer
MS 5’ AA?
GTCGAC?GCTAC?CCGAT?CTGGA?TTIACG 3’
MA 5’ GG?
GAATCC?AGAGC?TCTCT?TTCTC?ATACACT 3’
CDNA sequence with Semen sojae atricolor bud MnSOD is a template, with the MnSOD fragment of the primer amplification different lengths that has restriction enzyme site.
Amplification condition is as follows
1. full length sequence is cloned
10×PCR?buffer 5ul
dNTP 4ul
FS 1ul
FA 1ul
Template 3ul
ddH
2O 36ul
Taq enzyme 0.3ul
The PCR reaction conditions: 94 ℃ of sex change 40s, 57 ℃ of annealing 1min, 72 ℃ are extended 1min10s, and totally 30 circulations are extended 10min after 72 ℃.
2. open reading frame sequence is cloned
10×PCR?buffer 5ul
dNTP 4ul
CS 1ul
CA 1μl
Template 3ul
ddH
2O 36ul
Taq enzyme 0.3ul
The PCR reaction conditions: 94 ℃ of sex change 40s, 52 ℃ of annealing 1min, 72 ℃ are extended 1min10s, and totally 30 circulations are extended 10min after 72 ℃.
3. mature peptide serial response condition:
10×PCR?buffer 5ul
dNTP 4ul
MS 1ul
MA 1ul
Template 3ul
ddH
2O 36ul
Taq enzyme 0.3ul
94 ℃ of sex change 40s, 50 ℃ of annealing 1min, 72 ℃ are extended 1min10s, and totally 30 circulations are extended 10min after 72 ℃.
Electrophoresis reclaims the purpose fragment, cuts with SalI and 37 ℃ of enzymes of BamHI, and 4h, the leakage of electricity swimming is reclaimed.
2) plasmid of the schizosaccharomyces pombe of extraction reorganization simultaneously expression vector, enzyme is cut, and reclaims.
3) carrier and purpose fragment are reclaimed, connect the recombinant plasmid that obtains containing MnSOD, called after pESPUC-MnSOD1001 (full length sequence Yeast expression carrier), pESPUC-MnSOD726 (open reading frame sequence Yeast expression carrier), pESPUC-MnSOD598 (mature peptide sequence Yeast expression carrier).
4) evaluation of recombinant expression plasmid pESPUC-MnSOD.
Enzyme is cut evaluation: cut 3h with SalI and 37 ℃ of enzymes of BamHI
PCR identifies:
10×PCR?buffer 5ul
dNTP 4ul
FS(CS，MS) 1ul
FA(CS，MS) 1ul
Template 3ul
ddH
2O 36ul
Taq enzyme 0.3ul
The full length sequence reaction conditions:
94 ℃ of 40s, 57 ℃ of 1min, 72 ℃ of 1min10s totally 30 circulations are extended 10min after 72 ℃.
The open reading frame sequence reaction conditions:
94 ℃ of 40s, 52 ℃ of 1min, 72 ℃ of 1min10s totally 30 circulations are extended 10min after 72 ℃
Mature peptide serial response condition:
94 ℃ of 40s, 50 ℃ of 1min, 72 ℃ of 1min10s totally 30 circulations are extended 10min after 72 ℃
Enzyme is cut, PCR qualification result such as Fig. 3,4.
Among Fig. 3,1 swimming lane is cut qualification result for the full length sequence enzyme; 2 swimming lanes are that mature peptide sequence enzyme is cut qualification result; 3 swimming lanes are cut qualification result for the open reading frame sequence enzyme; 4 swimming lanes are DL2000
Among Fig. 4,1 swimming lane is DL2000; 2 swimming lanes are mature peptide sequence PCR result;
3 swimming lanes are open reading frame sequence PCR result; 4 swimming lanes are DL2000;
5 swimming lanes are full length sequence PCR result; 6 swimming lanes are DL2000; Vector construction success as seen from the figure.
One, yeast conversion
Plasmid: pESPUC-MnSOD
Host bacterium: schizosaccharomyces pombe S.pombe
A large amount of plasmids that extract recombinant expression vector, utilization AcLi method and electric shocking method change recombinant plasmid in the yeast cell over to.
1, AcLi conversion method
YPD substratum (PH=5.3): 1% yeast extract, 2% Tryptones, 2% glucose, 2% agar.
TE?Buffer：10mM?Tris·Cl，1mM?EDTA(PH＝8.0)。
0.2M LiAc:0.21g LiAc is dissolved in the 100ml distilled water.
70%PEG4000:35g PEG4000 is dissolved in the 50ml distilled water.
1) spends the night at 5mlYPD inoculation of medium S.pombe.
2) 170rpm, 30 ℃ of incubated overnight.To OD
600=1.0.
3) 2500rpm, 5min washes primary sedimentation with 5ml TE buffer.
4) be suspended in 0.6ml TE Buffer, mix with 0.5ml cell suspending liquid and 0.5ml 0.2M LiAc.
5) getting the above-mentioned mixed solution of 10ul plasmid DNA and 0.1ml mixes.30 ℃ are incubated 30 fens.
6) add 0.1ml 70%PEG4000,30 ℃ of insulation 1h.
7) add the 2ml sterilized water, after the mixing, 2500rpm 5min.
8) cell precipitation is suspended in 0.5ml water, gets 0.1ml suspension and be applied on the EMM selection flat board.
9) 30 ℃ are incubated 3-4 days.
2, electric shocking method
The YCD substratum: yeast soaks powder 10g, glucose 20g, and caseinhydrolysate 2g, sorbyl alcohol 218.6g is dissolved in the 1L distilled water
1.2M sorbyl alcohol: the 218.604g sorbyl alcohol is dissolved in the 1L distilled water.
1) preparation of electric shock yeast cell
(1) spends the night at the YCD of 500ml inoculation of medium s.pombe.
(2) 250rpm, 30 ℃ of incubated overnight, to concentration be 1*10
7Cell/ml (OD
600=0.7).
(3) cell is placed in the ice-water bath 15 minutes, and it is stopped growing.
(4) gently cell is poured in the 250ml centrifuge tube of two bacterium of going out into 3000g, 5min, 4 ℃, sedimentation cell.
(5) abandon supernatant, have the centrifuge tube of cell precipitation to place on ice collection.
(6) add the went out sorbyl alcohol of precooling on ice of bacterium of 50ml in each pipe, the suspension cell precipitation adds sorbyl alcohol then to 250ml.3000g, 5min, 4 ℃ of sedimentation cells are abandoned supernatant.
(7) repeat 6.
(8) add 20ml the suspend precipitation and transferring in the freezing 50ml centrifuge tube of the ice-cold sorbyl alcohol of bacterium of going out, 3000g, 5min, 4 ℃ of sedimentation cells are abandoned supernatant.
(9) with the 0.5ml sterilization, the sorbyl alcohol of the 1.2M of precooling suspends and precipitates, and making the final cell volume is 1.3ml, and cell concn is 1*10
9Cell/ml is put into cell on ice, is used for electric shock as quickly as possible.
2) electric shock
(1) the plasmid DNA sample (1ug) of drawing pre-inversion places pipe on ice in the 1.5ml centrifuge tube.
(2) add the 200ul competent cell in each DNA sample, mix.
(3) micropulser is made as " ShS ".
(4) the DNA cell sample is changed over to 0.2cm by in the electric shock cup of precooling, (beat the pipe suspension at the end gently, put into the electric shock pond) electric shock.
(5) cup is taken out, added ice-cold 0.8ml 1.2M sorbyl alcohol immediately, the cell with dilution is transferred in the pipe of the bacterium of going out then.
(6) inspection and record pulse parameter, the time should be controlled at 5 milliseconds, and magneticstrength can accurately be calculated by the distance (cm) of present voltage (kv)/cup.
(7) at room temperature, will manage and place 40～60min, make electric shock cell incubation growth in comprising the micro-substratum of 1.2M sorbyl alcohol..30 ℃, 60-96h incubated cell.
Fig. 5 is the recombination yeast transformant that filters out
1. mature peptide sequence transformant; 2.CDS sequence transformant; 3. full length sequence transformant; 4.pESPUC empty carrier transformant
Two, the evaluation of recombination yeast transformant
1, the extraction of yeast plasmid
Yeast extracts damping fluid: 2%Triton X-100,1%SDS, 100mM NaCl, 10mM Tris-Cl (PH=8), 1mMEDTA.
1) the single bacterium colony in the picking EMM solid medium in the EMM liquid nutrient medium 30 ℃ cultivated 1 day.
Collect the yeast thalline, 13000rpm, 1min, 4 ℃ are centrifugal.
2) abandon supernatant, with thalline vortex on residual liquid.
3) add the 0.2ml yeast and extract damping fluid, 0.2ml phenol/chloroform/primary isoamyl alcohol (25: 24: 1) and 0.3g granulated glass sphere, vortex 5min.13000rpm，8min。
4) supernatant is got 0.2ml and put into a new pipe.Add 1/10 3M NaAc and isopyknic Virahol, static 3min
5)13000rpm，10min。
6) wash once 13000rpm, 5min with 1ml 70% ethanol.
7) at air drying 10min.
8) be resuspended in 20ul ddH
2Among the O (containing 10%RNase).
9) at 37 ℃ of incubation 30min.
10) be stored in-20 ℃.
2, PCR and enzyme are cut the evaluation transformant
PCR identifies:
10×PCR?buffer 5ul
dNTP 4ul
Sense 1ul
Antisense 1ul
Template 3ul
ddH
2O 36ul
Taq enzyme 0.3ul
The full length sequence reaction conditions:
94 ℃ of 40s, 57 ℃ of 1min, 72 ℃ of 1min10s totally 30 circulations are extended 10min after 72 ℃.
The open reading frame sequence reaction conditions:
94 ℃ of 40s, 52 ℃ of 1min, 72 ℃ of 1min10s totally 30 circulations are extended 10min after 72 ℃.
Mature peptide serial response condition:
94 ℃ of 40s, 50 ℃ of 1min, 72 ℃ of 1min10s totally 30 circulations are extended 10min after 72 ℃.
Enzyme is cut evaluation: cut 3h with SalI and 37 ℃ of enzymes of BamHI
Transformant PCR, enzyme cut and identify as Fig. 67.
Among Fig. 7, swimming lane 1:DL2000; Swimming lane 2: mature peptide sequence transformant PCR qualification result
Swimming lane 3:DL2000; Swimming lane 4: open reading frame sequence transformant PCR qualification result
Among Fig. 7, swimming lane 1: mature peptide sequence transformant enzyme is cut qualification result; Swimming lane 2:DL2000;
Swimming lane 3: full length sequence transformant enzyme is cut qualification result; Swimming lane 4: open reading frame sequence transformant enzyme is cut qualification result; Swimming lane 5:DL2000;
The expression of embodiment 3:MnSOD gene in schizosaccharomyces pombe
One, containing the recombinant plasmid zymic induces:
1, the extraction of Yeast protein (being used for SDS-PAGE)
1) yeast cell that will contain the yeast cell of recombinant plasmid and contain empty carrier is inoculated into 30 ℃ of incubated overnight in the EMM liquid nutrient medium of 20ml, taking-up 10ml adds IPTG and induces, and cultivates with adding the continuation of IPTG inducing culture simultaneously, does not add IPTG and adds IPTG and cultivate 0h respectively, 24h, 48h, 72h, 96h and 0h, 24h, 48h, 72h, sampling.
2) collect thalline, 12000rpm, 3min, 4 ℃, abandon supernatant, add the 50mM Tris-Cl (PH=7.5) of 1.5ml, 10mmol/L sodiumazide, the precipitation that suspends, 12000rpm, 5min, 4 ℃ of centrifugation cells.
3) abandon supernatant, use 30ul ESB damping fluid suspension cell again.
4) 100 ℃ of insulation 3min make after the proteolytic enzyme inactivation, sample are deposited in-20 ℃.
5) granulated glass sphere of adding 0.3g diameter 0.2mm, thermal agitation mixing 2min.
6) add 150ul ESB damping fluid, 100 ℃ of insulation 1min are put in vibration a little.
7) sample carries out the SDS-PAGE electrophoresis.
2, the extraction of Yeast protein (being used for native-PAGE)
Collect thalline, 5000rpm, 5min, with aseptic washing once, the centrifugal thalline that gets off adds 500ul granulated glass sphere and the broken damping fluid of 500ul, place ice bath, vibration broken (1min vibration, 1min ice bath) 10 times, 12000rpm, 15min, suct clearly, centrifugal again, suct again clearly, be shell-broken liquid.Add isopyknic sample loading buffer, mixing carries out the native-PAGE electrophoresis.
Two, MnSOD gene expression product in schizosaccharomyces pombe detects
1, SDS-PAGE electrophoresis: the discontinuous polyacrylamide gel slab-electrophoresis that adopts the non-system of dissociating.
Concentrated glue is the gel of PH=6.8 Tris-Cl damping fluid 5%
Separation gel is the gel of PH=8.8 Tris-Cl damping fluid 12%
Electrode buffer is the Tris-Gly damping fluid of PH=8.3
2, Native-PAGE electrophoresis:
Concentrated glue is the gel of PH=6.7Tris-Cl damping fluid 2.5%
Separation gel is the gel of PH=8.9Tris-Cl damping fluid 15%
Electrode buffer is the Tris-Gly damping fluid of PH=8.3
3, the NBT method is surveyed active
Draw the 30ul sample, put into 10ml transparent glass small beaker, add 3ml NBT reaction solution, mixing, in 28 ℃ of SOD photochmeical reaction chambers, 2 * 15W fluorescent lamp illumination 20min measures absorbancy at the 560nm place, makes blank with the NBT reaction solution, establish two repetitions for every group, whole mensuration process is all carried out under lucifuge or low light condition except that photochmeical reaction, is decided to be an enzyme activity unit with the 50% required enzyme amount that suppresses NBT photochemical reduction speed of reaction.Formula is as follows:
Sample enzyme activity (U/ml)=(OD
Contrast-OD
Sample)/(1/2 OD
Contrast) * extension rate
Fig. 8 be contain the MnSOD gene the expression (SDS-PAGE) of recombinant plasmid in schizosaccharomyces pombe (48h-96h).
Fig. 8 a is the expression (48h-96h) of expression vector in yeast of MnSOD mature peptide sequence construct.
Swimming lane 1:96h; Swimming lane 2:72h; Swimming lane 3:48h; Swimming lane 4: low molecular weight protein (LMWP) standard
The expression (48h-96h) of expression vector in yeast that Fig. 8 b makes up for the MnSOD open reading frame sequence.
Swimming lane 1: low molecular weight protein (LMWP) standard; Swimming lane 2:48h; Swimming lane 3:72h; Swimming lane 4:96h
The expression (48h-96h) of expression vector in yeast that Fig. 8 c makes up for the MnSOD full length sequence.
Swimming lane 1: low molecular weight protein (LMWP) standard; Swimming lane 2:48h; Swimming lane 3:72h; Swimming lane 4:96h
Can find out that the MnSOD expressing quantity increases in time and increases.
Fig. 9 be contain the MnSOD gene the expression (Native-PAGE) of recombinant plasmid in schizosaccharomyces pombe (0h-96h)
(it is the IPTG abduction delivering that the swimming lane that indicates i among Fig. 9 is represented this swimming lane albumen)
Fig. 9 a be MnSOD mature peptide sequence construct the expression (Native-PAGE) of expression vector in yeast (0h-96h).
Swimming lane 1:0h; Swimming lane 2:24h; Swimming lane 3:48h; Swimming lane 4:72h; Swimming lane 5:96h; Swimming lane 6:i24h; Swimming lane 7:i48h; Swimming lane 8:i72h
The expression (Native-PAGE) of expression vector in yeast that Fig. 9 b makes up for the MnSOD open reading frame sequence (0h-96h).
Swimming lane 1:0h; Swimming lane 2:24h; Swimming lane 3:48h; Swimming lane 4:72h; Swimming lane 5:96h; Swimming lane 6:H
2O
2Handle; Swimming lane 7: chloroform/ethanol is handled; Swimming lane 8:i24h; Swimming lane 9:i48h; Swimming lane 10:i96h
The expression (Native-PAGE) of expression vector in yeast that Fig. 9 c makes up for the MnSOD full length sequence (0h-96h).
Swimming lane 1:0h; Swimming lane 2:24h; Swimming lane 3:48h; Swimming lane 4:72h; Swimming lane 5:96h;
Swimming lane 6:i24h; Swimming lane 7:i48h; Swimming lane 8:i72h
As shown in Figure 9, add active maximum that IPTG inductive bacterial strain 48h produces.Do not add active maximum that IPTG inductive bacterial strain 72h produces.And IPTG induces the activity of 48h generation than not inducing the activity and the expression amount of 72h generation much bigger with IPTG.
Get 3ul bacterium liquid NBT method and survey activity, the activity change rule as mentioned above.
Figure 10 is same incubation time, and the expression amount of MnSOD different lengths fragment in schizosaccharomyces pombe relatively
Swimming lane 1: empty carrier contrast; Swimming lane 2: the expression of mature peptide sequence in yeast; Swimming lane 3: the expression of open reading frame sequence in yeast; Swimming lane 4: the expression of full length sequence in yeast.
As seen from the figure, secondly the expression amount maximum of expression vector in schizosaccharomyces pombe that open reading frame sequence makes up be mature peptide sequence and full length sequence.
The yeast carrier for expression of eukaryon that is made up by the above-mentioned MnSOD of reaching a conclusion albumen has obtained a large amount of expression in schizosaccharomyces pombe, MnSOD open reading frame sequence expressing quantity maximum wherein, mature peptide sequence expressing quantity secondly, be full length sequence once more, and expression amount can obviously increase under IPTG inductive situation, so open reading frame sequence IPTG abduction delivering in schizosaccharomyces pombe of clone soybean MnSOD gene can obviously improve the proteic expression amount of MnSOD, provides fundamental basis for further carrying out suitability for industrialized production.
Sequence table
＜110〉Jilin Agriculture University
＜120〉a kind of schizosaccharomyces pombe engineering bacteria and construction process thereof with soybean MnSOD gene
<130>wangpiwu200704-06
<160>3
<170>PatentIn?version?3.2
<210>1
<211>1200
<212>DNA
＜213〉black soya bean
<400>1
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ctgctgcacc?accagaagca?ccaccagact?tacatcacca?acttcaacaa?ggccctcgag 300
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tctggatggg?tgtggcttgg?tctggacaaa?gagttgaaga?ggcttgtagt?tgaaaccact 600
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tctgaaagtg?ctacttgata?tagctcttga?gatgacagat?ttgcagcacg?gtaagagatg 840
tggaataaaa?tgatgtgatt?tgatgtaata?aataacacta?actattttta?tgtagttgta 900
ctgaagaacc?tcgagcattg?tatggctgca?tttctttgta?ctaagttccc?tcttgtctta 960
tgggattttt?ctgatgctct?gttaagatac?tattcgtttc?cagtgatgta?tatcttttgc 1020
ctccagggtt?tgatcccgat?cgtaatctct?agaggatccc?cgggtacgga?gctcgaattc 1080
gtaatcatgg?tcatagctgt?ttcctggtgc?gaaattgtaa?tcggctccac?aattccacac 1140
aaccaacgaa?gccgggaagc?ataaaggtgt?aaggcctggg?gggtgcccta?atgaagtgag 1200
<210>2
<211>1200
<212>DNA
＜213〉black soya bean
<400>2
cgggatcctt?ctagagatta?tagatctgat?ggccgcgcga?gctctgttga?ccagaaaaac 60
cctagccacc?gtgctccgca?acgacgcgaa?gcccataatc?ggagttggca?taacagcagc 120
ggctactcat?tcacgcgggt?tgcacgtgta?cacgctaccc?gatctggatt?acgactatgg 180
cgcactggag?ccagccatca?gcggcgacat?catgcagctg?caccaccaga?agcaccacca 240
gacttacatc?accaactaca?acaaggccct?cgagcagctc?caagacgcca?tcgccaagaa 300
agattcctcc?gccgtcgtta?agctccaggg?cgccatcaag?ttcaacggcg?gaggtcatgt 360
caaccattct?attttctgga?aaaatctagc?tcctgttcgt?gaaggaggtg?gtgaaccacc 420
caagggttca?ctgggatggg?ctattgacac?acattttggt?tcttttgaag?cattaataca 480
aaaagttaac?gcagaaggtg?ctgcactaca?ggggtctgga?tgggtgtggc?ttggtctgga 540
caaagagttg?aagaggcttg?tagttgaaac?cactgccaac?caggacccac?tggttactaa 600
gggaccaaat?ttggttccat?tgattggtat?tgatgtttgg?gagcatgcgt?actacttaca 660
gtacaagaat?gttagaccag?actatcttaa?gaacatttgg?aaagttatta?attggaaata 720
tgccagtgaa?gtgtatgaga?aagagagctc?ttagcgatcg?ccatcgtcga?cctgcaggca 780
tgcaagcttg?gcactggccg?tcgttttaca?acgtcgtgac?tgggaaaacc?ctggcgttac 840
caacttaatc?gccttgcagc?acatccccct?ttcgccagct?ggcgtaatag?cgaagagccc 900
gcaccgatcg?cccttcccaa?cagttgcgca?gcctgaatgg?cgaatggcgc?ctgatgcggt 960
attttctcct?tacgcatctg?tgcggtattt?cacaccgcat?atggtgcact?ctcagtacaa 1020
tctgctctga?tgccgcatag?tttaagccag?cccgacaccc?gccacacccg?ctgacgcgcc 1080
ctggacggct?tgttctgcct?ccccggcatc?cgcttacaag?acaagctgtg?tacgccctcc 1140
ggagcttgca?tgtgtccaaa?aggttttcca?ccgtctactt?acccggaacc?ggccggagac 1200
<210>3
<211>583
<212>DNA
＜213〉black soya bean
<400>3
gcgtctctgt?gagcagccat?cagcggtgaa?tcatgcagct?gcaccaccag?aagcaccacc 60
agacttacat?caccaacttc?aacaaggccc?tcgagcagct?ccaagacgcc?gtcgccaaga 120
aagattcctc?cgccgtcgtt?aagctccagg?gcgccatcaa?gttcaacggc?ggaggtcacg 180
tcaaccattc?tattttctgg?aaaaatctag?ctcctgttcg?tgaaggaggt?ggtgaaccac 240
ccaagggttc?actgggatgg?gccattgaca?cacattttgg?ctcttttgaa?gcattagtac 300
aaaaagttaa?tgcagaaggt?gctgcactac?aggggtctgg?atgggtgtgg?cttggtctgg 360
acaaagagtt?gaagaggctt?gtagttgaaa?ccactgccaa?ccaggaccca?ctggttacta 420
agggaccaaa?tttggttcca?ttgcttggta?ttgatgtttg?ggagcatgcg?tactacttac 480
agtacaagaa?tgttagacca?gactatctga?agaacatttg?gaaagttatt?aattggaaac 540
atgccagtga?agtgtatgag?aaagagagct?ctggatcccc?aat 583
Claims (5)
1. the schizosaccharomyces pombe engineering bacteria with soybean MnSOD gene is to obtain in the reorganization schizosaccharomyces pombe expression vector importing schizosaccharomyces pombe that contains soybean MnSOD gene that will make up.
2. the construction process with schizosaccharomyces pombe engineering bacteria of soybean MnSOD gene as claimed in claim 1, its construction process is the multiple clone site place that the MnSOD gene is inserted into the schizosaccharomyces pombe expression vector, this reorganization schizosaccharomyces pombe expression vector pESPUC-MnSOD that makes up is imported in the schizosaccharomyces pombe, obtain reorganization schizosaccharomyces pombe transformant, cultivate the reorganization schizosaccharomyces pombe and make the MnSOD gene obtain expressing.
3. the construction process with schizosaccharomyces pombe engineering bacteria of soybean MnSOD gene as claimed in claim 2, it is characterized in that: the reorganization schizosaccharomyces pombe expression vector pESPUC-MnSOD of structure, this carrier contains Pnmt1 promotor, Pnmt1 terminator sequence, resistant gene is a penbritin, selectable marker gene LEU, soybean MnSOD gene is cut between site SalI and the BamHI at polyclone enzyme.
4. the construction process with schizosaccharomyces pombe engineering bacteria of soybean MnSOD gene as claimed in claim 1 is characterized in that: soybean MnSOD gene is respectively SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 is described.
5. the construction process with schizosaccharomyces pombe engineering bacteria of soybean MnSOD gene as claimed in claim 2 is characterized in that: the yeast conversion method is Lithium Acetate conversion method and electric shock conversion method.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591622B (en) * | 2009-06-30 | 2011-01-19 | 重庆大学 | Melittin gene fission yeast engineering bacteria and construction method and application thereof |
| CN102226155A (en) * | 2011-05-12 | 2011-10-26 | 江南大学 | Screening and application of yeast with high ethanol yield and low fusel oil yield in Chinese Maotai-flavor liquor production |
| CN102766585A (en) * | 2011-05-12 | 2012-11-07 | 江南大学 | Screening and application of yeast CGMCC 4740 for high production of ethanol and low production of fusel oil in production of Chinese Maotai-flavor liquor |
| CN104450768A (en) * | 2014-11-18 | 2015-03-25 | 昆明理工大学 | Shuttle vector of targeted yeast mitochondria and application of shuttle vector |
| CN105274014A (en) * | 2015-11-17 | 2016-01-27 | 江南大学 | Engineering bacterium of saccharomyces cerevisiae for efficiently expressing superoxide dismutase of macrobrachium rosenbergii |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2004254640B2 (en) * | 2003-07-02 | 2010-08-26 | Bp Corporation North America Inc. | Glucanases, nucleic acids encoding them and methods for making and using them |
| CN1995377A (en) * | 2006-12-25 | 2007-07-11 | 西南大学 | Catechin-O-methyltransferase regulator molecule sieve model and its uses |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591622B (en) * | 2009-06-30 | 2011-01-19 | 重庆大学 | Melittin gene fission yeast engineering bacteria and construction method and application thereof |
| CN102226155A (en) * | 2011-05-12 | 2011-10-26 | 江南大学 | Screening and application of yeast with high ethanol yield and low fusel oil yield in Chinese Maotai-flavor liquor production |
| CN102226155B (en) * | 2011-05-12 | 2012-08-22 | 江南大学 | Screening and application of yeast with high ethanol yield and low fusel oil yield in Chinese Maotai-flavor liquor production |
| CN102766585A (en) * | 2011-05-12 | 2012-11-07 | 江南大学 | Screening and application of yeast CGMCC 4740 for high production of ethanol and low production of fusel oil in production of Chinese Maotai-flavor liquor |
| CN104450768A (en) * | 2014-11-18 | 2015-03-25 | 昆明理工大学 | Shuttle vector of targeted yeast mitochondria and application of shuttle vector |
| CN104450768B (en) * | 2014-11-18 | 2018-04-24 | 昆明理工大学 | A kind of shuttle vector for targeting yeast mitochondrial and its application |
| CN105274014A (en) * | 2015-11-17 | 2016-01-27 | 江南大学 | Engineering bacterium of saccharomyces cerevisiae for efficiently expressing superoxide dismutase of macrobrachium rosenbergii |
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|---|---|
| CN101157897B (en) | 2011-04-27 |
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