CN100430479C - Construction of pET-SOD engineering bacterium and method for expression and purification thereof - Google Patents
Construction of pET-SOD engineering bacterium and method for expression and purification thereof Download PDFInfo
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- CN100430479C CN100430479C CNB2005100615026A CN200510061502A CN100430479C CN 100430479 C CN100430479 C CN 100430479C CN B2005100615026 A CNB2005100615026 A CN B2005100615026A CN 200510061502 A CN200510061502 A CN 200510061502A CN 100430479 C CN100430479 C CN 100430479C
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Abstract
The invention discloses a constructing and expressive purifying method of pET-SOD engineering bacterium, which is characterized by the following: designing primer to clone Fe-SOD gene in the CHEN (Nostoc commune strain CHEN); obtaining the recombined product into plasmid pUCm-T; cloning in the escherichia coli DH5alpha; designing primer augmentation Fe-SOD gene; recombining to the prokaryotic expressive carrier pET22b(+); constructing engineering bacterium pET-SOD in the escherichia coli BL21; expressing the purified Fe-SOD with hyperoxide disproportionation enzyme activity and certain antineoplastic ability.
Description
Technical field
The present invention relates to the genetic engineering pharmaceutical field, particularly a kind of pET-SOD engineering bacteria and the structure and expression and purification method that can efficiently express Fe-SOD.
Background technology
Superoxide-dismutase (Superoxide Dismutase is hereinafter to be referred as SOD) is a kind of metal enzyme, according to institute's containing metal prothetic group difference, is divided into Cu, Zn-SOD, Fe-SOD, Mn-SOD and Ni-SOD four classes.Wherein Cu, Zn-SOD mainly are present in the eukaryotic cytoplasm; Mn-SOD is present in eukaryotic plastosome, the bacterium; Fe-SOD only is present in the prokaryotic cell prokaryocyte; Ni-SOD is that recent findings only is present in some only a few protokaryon bacterium.
SOD is as the scavenging agent of free radical, can play skin care, beauty treatment, effect such as antitumor, and the treatment of some autoimmune diseases is helped out.But SOD extracts from natural animal-plant.For solving source restricted problem, the scientific research personnel is doing a lot of work aspect the genetically engineered of SOD both at home and abroad.But the research to SOD biases toward Cu/Zn-SOD, and it is less to the research of prokaryotic organism Fe-SOD, also do not obtain Fe-SOD at present with gene engineering method, along with domestic and international going deep into gradually to Fe-SOD research, the genetically engineered research of Fe-SOD has profound significance, and domestic present gene engineering expression protein content is generally 20%-30%.
Summary of the invention
At the deficiencies in the prior art part, the invention provides pET-SOD engineering bacteria and structure and the expression and purification method of a kind of Fe-SOD of efficiently expressing.
The construction process of pET-SOD engineering bacteria provided by the invention may further comprise the steps:
(1) in the BG11 substratum, cultivates beads algae CHEN (Nostoc commune strain CHEN) 40～50 days, take off a layer culture, adopt SDS-CATB method extracting beads algae CHEN genome;
(2) be template with beads algae CHEN genome,, obtain product and recombinate to plasmid pUCm-T with TD-PCR method amplification Fe-SOD gene, recombinant plasmid called after T-SOD, and in bacillus coli DH 5 alpha, clone;
Two kinds of used primers of TD-PCR are:
SOD-for：5’-GAGGATTTGACACGATGGCATTTG-3’
SOD-orf：5’-CTGACACCTAATTAAGCTTTGGCC-3’；
(3) with T-SOD for touching plate, with PCR method amplification Fe-SOD gene, after Nco I and Xho1 I enzyme point of contact enzyme are cut, recombinate on the expression plasmid pET22b (+) that cuts to same enzyme, sod gene is terminal to link to each other with the gene order of 6 * His label, is built into pET-SOD;
Two kinds of used primers of PCR are:
SODn:5 '-CTAGCCATGGCATTTGTACAGC-3 ' contains Nco I restriction enzyme site
SODx:5 '-CCGCTCGAGAGCTTTGGCCAAGTTTTC-3 ' contains Xho1 I restriction enzyme site;
(4) pET-SOD is transferred to finishes structure in the e. coli bl21.
As a kind of improvement of the present invention: beads algae algae kind is cultivated under aseptic condition.
Improve as another kind of the present invention: beads algae algal species cultivation fate is 45 days.
The present invention also provides the Fe-SOD protein expression of pET-SOD engineering bacteria and the method for purifying thereof:
(1) the pET-SOD engineering bacteria adds IPTG and finite concentration Fe in the LB substratum
3+Induced engineering bacteria 4～6 hours, and obtained to express;
(2) after pET-SOD engineering bacteria ultrasonic disruption after inducing and the washing, obtain the SOD inclusion body, it is placed the sex change liquid sex change that contains dodecyl propylhomoserin sodium (hereinafter to be referred as SLS), and cross Ni
2+Affinity column, purified fusion protein.
(3) above purifying acquisition SOD metaprotein places and contains Fe
3+The dissolving damping fluid dialysis renaturation that does not contain SLS.
As a kind of improvement of the present invention: the Fe of adding
3+Be FeCl
3
Improve as another kind of the present invention: the Fe of adding
3+Concentration is 100～300umol/ml substratum.
As a further improvement on the present invention: the Fe of adding
3+Concentration is the 200umol/ml substratum.
Improve as another kind of the present invention: induction time is 5 hours.
A kind of engineering bacteria and construction process thereof that efficiently expresses Fe-SOD of the present invention compared with prior art have the following advantages: (1) clone has obtained the Fe-SOD gene order of the complete 603bp of beads algae, guarantees to express the vigor that obtains Fe-SOD; (2) the Fe-SOD gene order is cloned between the Nco I and Xho1 I site of pET22a (+) plasmid vector, connect the gene order of 6 * His label behind this position, express acquisition Fe-SOD end and contain 6 * His label, make Fe-SOD be able to efficient fast purifying; (3) add Fe in the abduction delivering process
3+, final Fe-SOD obtains to efficiently express, and expression product accounts for whole bacterial protein 78%; (4) utilized gene engineering method to obtain Fe-SOD first, this will solve the industrial source restricted problem of extracting SOD from animals and plants.
Description of drawings
Fig. 1 is the aminoacid sequence of beads algae CHEN (Nostoc commune strain CHEN) Fe-SOD gene order and deduction;
Fig. 2 is the 10%SDS-PAGE electrophoretogram of SOD;
Among Fig. 21,6-Marker; 2,3,4-[Fe
3+]=300uM, 200uM, the SOD albumen of expressing when 100uM induces; Whole bacterial protein when 5-does not induce; SOD behind the 7-purifying;
Fig. 3 is the inhibition test synoptic diagram of Fe-SOD to the SPC-A-1 lung adenocarcinoma cell.
Embodiment
Below the present invention is further described by example:
(1) get 1ml beads algae CHEN (Nostoc commune strain CHEN) culture, with TE damping fluid washed twice, it is standby fully to mill; TE damping fluid wherein: Tris 50mM, EDTA 20mM, pH 8.
Beads algae CHEN (Nostoc commune strain CHEN) is available from the FACHB of the aquatic institute in Chinese Academy of Sciences Wuhan.
(2) the TE damping fluid of adding 567 μ l is mixed thoroughly, and multigelation is 3 times in liquid nitrogen.
(3) Proteinase K of adding 30 μ l 10%SDS and 3 μ l 20mg/ml, mixing, 37 ℃ of insulation 1h.
(4) add the 5mol/L NaCL of 100 μ l, abundant mixing adds the CATB/NaCL mixing of 80 μ l again, at 65 ℃ of insulation 10min down.
CATB/NaCL：2％CTAB、100mmoI/L?Tris.HCI(pH?8.0)、20mmol/L?EDTA、1.4mol/L?NaCl。
(5) add equal-volume chloroform/primary isoamyl alcohol (24: 1), mixing, centrifugal 5min.Supernatant liquor is transferred in another new pipe.
(6) add equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1), mixing, centrifugal 5min is transferred to supernatant in another new pipe.
(7) Virahol of adding 0.6 volume, mixing precipitates (30min) to DNA gently, and centrifugal 5min abandons supernatant, once precipitates with 70% washing with alcohol.Centrifugal 5min.
(8) it is dissolved among the TE of 100 μ l, adds the RNA enzyme,, place 37 ℃ of water-bath 30min until final concentration 50 μ g/ml.
(9) add equal-volume chloroform/primary isoamyl alcohol (24: 1), mixing.Centrifugal 5min.Shift supernatant liquor to new pipe.
(10) add equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1), mixing, centrifugal 5min is transferred to supernatant in another new pipe.
(11) Virahol of adding 0.6 volume, mixing precipitates (30min) to DNA gently, and centrifugal 5min abandons supernatant, precipitates centrifugal 5min once with 70% washing with alcohol.
(12) supernatant discarded precipitates air-dryly, and the TE buffered soln dissolving DNA with 20 μ l adds the two volumes dehydrated alcohol then, puts into 4 ℃ of refrigerators precipitation 30min. and finally is dissolved in 20 μ l TE.
(1) under aseptic condition, beads algae CHEN algae kind 2ml is inserted in the 1000mlBG11 substratum, in 25 ℃, 2000XL intensity of illumination illumination every day 10 hours, and shake bottle once.Cultivate after 45 days, take off a layer culture, adopt the SDS-CATB method extracting beads algae genome of Kim.
(2) live species Fe-SOD gene order in the inquiry Genbank database, their homology of blastn compare of analysis, predict its trend of evolution, and design primer SOD-for (5 '-GAGGATTTGACACGATGGCATTTG-3 ') and SOD-orf (5 '-CTGACACCTAATTAAGCTTTGGCC-3 ') according to the Fe-SOD gene order of the highest species of homology (Spirulina platensis, Nostoc linckia etc.).
(3) be primer with SOD-for and SOD-orf, it is template that above extracting obtains genome, TD-PCR amplification Fe-SOD gene, and the TD-PCR program is as follows:
Fs: 95 ℃ of 3min,
Subordinate phase: 95 ℃ of 55s
55℃ 60s
72℃ 40s
Subordinate phase circulation 20 times, every second step of two-wheeled round-robin is reduced by 1 ℃, and extremely last circulation is 45 ℃;
Phase III: 95 ℃ of 55s
46℃ 60s
72℃ 40s
Phase III circulation 16 times, last circulation final step keeps 72 ℃ of 10min;
Recovery PCR product is also recombinated to pUCm-T, recombinant plasmid called after T-SOD, and transformed competence colibacillus E.coli.DH5 α, the method for cutting by PCR and enzyme filters out positive colony, and correct positive colony is determined in order-checking at last.
The pUCm-T plasmid is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
(4) with T-SOD being template, is primer with SODn and SODx, amplification Fe-SOD gene,
SODn:5 '-CTAGCCATGGCATTTG TACAGC-3 ' contains Nco I restriction enzyme site,
SODx:5 '-CCGCTCGAGAGCTTTGGCCAAGTTTTC-3 ' contains Xho1 I restriction enzyme site,
The PCR program is:
Fs: 94 ℃ of 5min,
Subordinate phase: 94 ℃ of 50s
50℃ 60s
72℃ 60s
Subordinate phase circulation 40 times, last circulation final step keeps 72 ℃ of 10min;
Reclaim the PCR product, cut with Nco I and Xho1 I enzyme, and reorganization is to pET22b (+) plasmid of cutting through same enzyme, sod gene is terminal to link to each other with the gene order of 6 * His label, recombinant plasmid called after pET-SOD, and transformed competence colibacillus E.coli.BL21, the method for cutting by PCR and enzyme filters out positive colony, and correct positive colony is determined in order-checking at last.
PET22b (+) plasmid is available from Invitrogen company.
Efficiently expressing of embodiment 3, pET-SOD engineering bacteria:
(1) 1L contains in the LB substratum of 50 μ g/ml Amp, and 37 ℃, 0.5mM IPTG, 200uMFeCl
3, induced engineering bacteria 5 hours.
(2) the centrifugal 10min of 5000g collects thalline, is dissolved in 50ml TE;
TE：20mM?Tris-HCl，10mM?EDTA，pH?8.0。
(3) in 200W ultrasonic disruption bacterium liquid 10min, cleer and peaceful precipitation in the centrifugal 30min collection of 12000g is carried out the 10%SDS-PAGE electrophoresis respectively.
Electrophoresis result shows in top condition: 37 ℃, 0.5mM IPTG, 200 μ M FeCl
3, induced engineering bacteria 5 hours, engineering bacteria obtains to efficiently express, the about 28kD of expressing protein molecular weight, it accounts for 78% (Fig. 2, swimming lane 3) of whole bacterial protein, and all with the inclusion body formal representation.
Embodiment 4:
As embodiment 3 steps, wherein FeCl
3Amount be 300uM.
Electrophoresis result shows: target protein accounts for 68% (Fig. 2, swimming lane 2) of whole bacterial protein.
Embodiment 5:
As embodiment 3 steps, wherein FeCl
3Amount be 100uM.
Electrophoresis result shows: target protein accounts for 69% (Fig. 2, swimming lane 4) of whole bacterial protein.
Embodiment 6:
As embodiment 3 steps, wherein do not add IPTG and FeCl
3
Electrophoresis result shows: target protein accounts for 0% (Fig. 2, swimming lane 5) of whole bacterial protein.
The purifying of embodiment 7, pET-SOD engineering bacteria:
(1) behind the 1L engineering bacteria abduction delivering 5h, ultrasonic disruption obtains all SOD inclusion body precipitations, uses 50ml inclusion body lysate I and 50ml inclusion body lysate II washed twice respectively,
Inclusion body lysate I:1mM EDTA, 5% glycerine, 0.5%Triton X-100; Inclusion body lysate II: inclusion body lysate I adds 2mol/L urea.
(2) in 50ml 50mmol/L Tris-Hcl, 0.3%SLS, the dissolving of 0.1mmol/L dithiothreitol (DTT) damping fluid is spent the night.
(3) this sex change liquid is crossed 5mlNi
2+Resin column, flow velocity is adjusted into 0.4ml/min.(albumen of 0～500mM) elution buffer elution of bound, every part of 3ml is in charge of collection, monitoring A with the imidazole concentration gradient of 5 times of column volumes
280Protein-contg elutriant collection tube is carried out the 10%SDS-PAGE electrophoresis.
Electrophoresis result shows that the target protein behind the purifying is more single, does not contain other foreign protein (Fig. 2, swimming lane 7).
Under 4 ℃, above purifying acquisition SOD metaprotein places and contains 0.04mM FeCl
3, do not contain the dissolving damping fluid dialysis renaturation 72h of SLS.Obtain the pure product of albumen with ddH
2O is in 4 ℃ of 24h that dialyse down ,-80 ℃ of freeze-drying, liquid nitrogen preservation.
The enzyme activity of embodiment 8, purifying protein:
(1) with Marklund[9] pyrogallol autoxidation method measure the enzyme activity of purifying protein:
SOD behind the purifying is made into finite concentration with pH7.8PBS, the about 0.07OD/min of control pyrogallol autoxidation speed, inhibiting rate about 50%.
The result shows, when protein concentration is 0.8mg/ml, recombinant protein has the SOD activity, and its vigor is 1100u/mg.
Embodiment 9:
The SPC-A-1 lung adenocarcinoma cell is inoculated 90 μ l (5 * 10
5Individual cells/ml), 37 ℃, cultivates 24h in carbonic acid gas (5%) incubator in 96 orifice plates.Add 10 μ l SOD to finite concentration (1 μ M, 2 μ M, 5 μ M, 10 μ M), control group adds PBS, and mixing continues to cultivate 48h.Press shown in the CCK-8 test kit [10-11], every hole adds CCK-8 reagent 10 μ l, and mixing continues to cultivate 1h, and 450nm measures down and absorbs absorbancy, carries out cell counting.
Result such as Fig. 3, Fig. 3 show that after 48 hours, the absorbancy under the PBS control group 450nm is 1.692, and the absorbancy of 1uM SOD control group is 1.095, and its inhibiting rate is 35.3%, and the absorbancy of 10uM SOD control group is 0.734, and its inhibiting rate reaches 56.6%, IC
50Be 5.20 μ M.
SOD has the ability of certain inhibition SPC-A-1 lung adenocarcinoma cell growth behind the purifying.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (8)
1, a kind of construction process of pET-SOD engineering bacteria, its feature may further comprise the steps successively:
(1) in the BG11 substratum, cultivates beads algae CHEN (Nostoc commune strain CHEN) 40～50 days, take off a layer culture, adopt SDS-CATB method extracting beads algae CHEN genome;
(2) be template with beads algae CHEN genome,, obtain product and recombinate to plasmid pUCm-T with TD-PCR method amplification Fe-SOD gene, recombinant plasmid called after T-SOD, and in bacillus coli DH 5 alpha, clone;
Two kinds of used primers of TD-PCR are:
SOD-for：5’-GAGGATTTGACACGATGGCATTTG-3’
SOD-orf：5’-CTGACACCTAATTAAGCTTTGGCC-3’；
(3) with T-SOD for touching plate, with PCR method amplification Fe-SOD gene, after Nco I and Xhol I enzyme point of contact enzyme are cut, recombinate on the expression plasmid pET22b (+) that cuts to same enzyme, sod gene is terminal to link to each other with the gene order of 6 * His label, is built into pET-SOD;
Two kinds of used primers of PCR are:
SODn:5 '-CTAGCCATGGCATTTGTACAGC-3 ' contains Nco I restriction enzyme site
SODx:5 '-CCGCTCGAGAGCTTTGGCCAAGTTTTC-3 ' contains the XholI restriction enzyme site;
(4) pET-SOD is transferred to finishes structure in the e. coli bl21.
2, the construction process of a kind of pET-SOD engineering bacteria according to claim 1 is characterized in that: the beads algae algae kind in the described step (1) is cultivated under aseptic condition.
3, the construction process of a kind of pET-SOD engineering bacteria according to claim 1 is characterized in that: the beads algae algal species cultivation fate in the described step (1) is 45 days.
4, a kind of Fe-SOD protein expression of the pET-SOD engineering bacteria as each described construction process gained in the claim 1 to 3 and the method for purifying, its feature may further comprise the steps successively:
(1) the pET-SOD engineering bacteria adds IPTG and Fe in the LB substratum
3+Induced engineering bacteria 4～6 hours, and obtained to express;
(2) after pET-SOD engineering bacteria ultrasonic disruption after inducing and the washing, obtain the SOD inclusion body, it is placed the sex change liquid sex change that contains dodecyl propylhomoserin sodium, and cross Ni
2+Affinity column, purified fusion protein;
(3) above purifying acquisition SOD metaprotein places and contains Fe
3+The dissolving damping fluid dialysis renaturation that does not contain dodecyl propylhomoserin sodium.
5, the Fe-SOD protein expression of a kind of pET-SOD engineering bacteria according to claim 4 and the method for purifying is characterized in that: the Fe that adds in the described step (1)
3+Be FeCl
3
6, the Fe-SOD protein expression of a kind of pET-SOD engineering bacteria according to claim 4 and the method for purifying is characterized in that: the Fe that adds in the described step (1)
3+Concentration is 100～300umol/ml substratum.
7, the Fe-SOD protein expression of a kind of pET-SOD engineering bacteria according to claim 4 and the method for purifying is characterized in that: the Fe that adds in the described step (1)
3+Concentration is the 200umol/ml substratum.
8, the Fe-SOD protein expression of a kind of pET-SOD engineering bacteria according to claim 4 and the method for purifying is characterized in that: induction time is 5 hours in the described step (1).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101148679B (en) * | 2007-09-10 | 2011-06-15 | 重庆大学 | Extraneous sources IPTG and O2 concentration simultaneously regulating and controlling expression carrier and construction method thereof |
| CN101818166B (en) * | 2010-05-12 | 2013-06-19 | 西南民族大学 | Yak copper zinc superoxide dismutase recombinant expression protein |
| CN105331588A (en) * | 2015-02-02 | 2016-02-17 | 鄂尔多斯市疾病预防控制中心 | Preparation process of recombinant spirulina superoxide dismutase (SOD) |
| CN106318940A (en) * | 2016-08-18 | 2017-01-11 | 河南佰柯生物科技有限公司 | High-temperature-resistant SOD protein, and cloning and purifying method thereof |
| CN111254125B (en) * | 2020-04-30 | 2020-08-25 | 中国农业大学 | Superoxide dismutase and preparation method thereof, superoxide dismutase oral liquid and solid preparation |
Citations (1)
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| CN1263158A (en) * | 1999-02-11 | 2000-08-16 | 吴光耀 | Recombinant superoxide dismutase gene sequence and preparation method of its engineering bacterium and product |
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| CN1263158A (en) * | 1999-02-11 | 2000-08-16 | 吴光耀 | Recombinant superoxide dismutase gene sequence and preparation method of its engineering bacterium and product |
Non-Patent Citations (3)
| Title |
|---|
| CN,A,1263158 2000.08.16 |
| 钝顶螺旋藻Fe－SOD基因的克隆及序列分析. 郭建军等.浙江大学学报（理学版），第31卷第6期. 2004 |
| 钝顶螺旋藻Fe－SOD基因的克隆及序列分析. 郭建军等.浙江大学学报（理学版），第31卷第6期. 2004 * |
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