CN108018266A - A kind of marine source superoxide dismutase and its encoding gene and application - Google Patents
A kind of marine source superoxide dismutase and its encoding gene and application Download PDFInfo
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- CN108018266A CN108018266A CN201810105875.6A CN201810105875A CN108018266A CN 108018266 A CN108018266 A CN 108018266A CN 201810105875 A CN201810105875 A CN 201810105875A CN 108018266 A CN108018266 A CN 108018266A
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- sequence
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- superoxide dismutase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
Abstract
The invention discloses a kind of marine source superoxide dismutase and its encoding gene and application.The present invention provides a kind of protein, is following (a1) or (a2)：(a1) protein being made of the amino acid sequence shown in sequence in sequence table 1；(a2) amino acid sequence of sequence 1 is passed through to substitution and/or missing and/or addition and the protein as derived from sequence 1 with identical function of one or several amino acid residues.The present invention is by being sequenced south west Indian Ocean sediment sample member genome, it was found that and obtain Io SOD1, and successfully realize expression of the gene in Escherichia coli, expression is simple and practicable, expression product easy purification, stability is good, than living up to 1315U/mg, and the enzyme is with good stability, Io SOD1 of the invention are by with wide prospects for commercial application.
Description
Technical field
The present invention relates to a kind of marine source superoxide dismutase and its encoding gene and application.
Background technology
Oxygen is of crucial importance for vital movement, but simultaneously because the participation of oxygen, cell also inevitably produce oxygen certainly
By base (i.e. usually said active oxygen).Active oxygen can be caused a series of harmful by oxidativestress damage cellular macromolecule
Biochemical reaction, causes the inactivation of Protein Damage, lipid peroxidation, DNA mutation and enzyme.Early in 1969, McCord and
Fridovich is found that oxygen radical in a kind of erythrocuprein energy scavenger-cell body, and is named as superoxide dismutase
Enzyme (SOD).Research shows SOD enzymes by the way that oxygen radical is converted into H2O2, H2O2It is converted into again by catalase and oxidizing ferment
Water, so as to achieve the purpose that oxygen radical in scavenger-cell, protection cell.Oxygen is being protected cells from just because of SOD enzymes certainly
By the important function played in the murder by poisoning of base, SOD enzymes are before medicine, food and agriculture field embody boundless application
Scape.The production of SOD mainly includes natural extraction and microbial fermentation production two ways.Particularly with molecule clone technology
Development, its advantage is increasingly embodied using genetically engineered bacteria production SOD.
Ocean is covered with the area of earth surface 70%.Microorganism spreads all over ocean, or even survives in 11000 meters of deep-sea, pressure
Power 100Mpa, temperature are higher than 100 extreme marine environment.Estimate that educable microorganism only accounts for the 1% of microorganism total amount at present.
Therefore, substantial amounts of undeveloped microorganism and enzyme resource are contained in marine environment.By combine microorganism member genome and
High-flux sequence can develop a large amount of unknown novel enzyme resources contained in marine environment to a greater extent.
The content of the invention
The object of the present invention is to provide a kind of marine source superoxide dismutase and its encoding gene and application.
The present invention provides a kind of protein (being named as Io-SOD1 albumen), is any in following (a1)-(a5)：
(a1) protein being made of the amino acid sequence shown in sequence in sequence table 1；
(a2) amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and there is the protein as derived from sequence 1 of identical function；
(a3) fusion protein of (a1) or (a2) is contained；
(a4) fusion protein that small peptide of the connection containing label obtains in the end of (a1) or (a2)；
(a5) fusion protein that connection label obtains in the end of (a1) or (a2).
In order to make protein in (a1) easy to purifying and detection, amino acid sequence that can be in as sequence table shown in sequence 1
Arrange the amino terminal or the upper label as shown in Table 1 of carboxyl terminal connection of the protein of composition.
The sequence of 1 label of table
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Protein in above-mentioned (a2) can be artificial synthesized, also can first synthesize its encoding gene, then carries out biological expression and obtain.
The present invention also protection encodes the gene (Io-SOD1 genes) of the Io-SOD1 albumen.
The gene is any DNA molecular in following (b1)-(b4)：
(b1) DNA molecular of the code area as shown in sequence 2 in sequence table；
(b2) DNA molecular in sequence table shown in sequence 2；
(b3) the DNA sequence dna hybridization limited under strict conditions with (b1) or (b2) and encoding superoxide dismutase
DNA molecular；
(b4) there is more than 90% homology and encoding superoxide discrimination with (b1) or (b2) or (b3) DNA sequence dna limited
Change the DNA molecular of enzyme.
Above-mentioned stringent condition can be miscellaneous in DNA or RNA with 0.1 × SSPE (or 0.1xSSC), the solution of 0.1%SDS
Hand over and hybridize in experiment at 65 DEG C and wash film.
The present invention also protects the recombinant expression carrier containing Io-SOD1 genes, expression cassette, transgenic cell line or restructuring
Bacterium.
The recombinant expression carrier is concretely in multiple cloning sites (such as the NdeI and HindIII of pET28a (+) carrier
Between restriction enzyme site) the obtained recombinant plasmid of the double chain DNA molecule shown in the sequence 2 of insetion sequence table from 5 ' 1-612, ends.
The present invention also protects application of the Io-SOD1 albumen in as superoxide dismutase.
The present invention also protects a kind of recombinant bacterium, will be obtained in Io-SOD1 channel genes host strains.
The Io-SOD1 genes can import host strain by the recombinant expression carrier containing Io-SOD1 genes and be recombinated
Bacterium.
The recombinant expression carrier is concretely in multiple cloning sites (such as the NdeI and HindIII of pET28a (+) carrier
Between restriction enzyme site) the obtained recombinant plasmid of the double chain DNA molecule shown in the sequence 2 of insetion sequence table from 5 ' 1-612, ends.
The host strain can be Escherichia coli, concretely e. coli bl21 (DE3).
The present invention also protects a kind of preparation method of superoxide dismutase, includes the following steps：The recombinant bacterium is cultivated,
Superoxide dismutase is obtained from recombinant bacterium.
The present invention also protects the Io-SOD1 albumen or Io-SOD1 genes or the recombinant bacterium or the method to exist
Prepare the application in superoxide dismutase product.
The present invention had found and obtains Io-SOD1 by the way that south west Indian Ocean sediment sample member genome is sequenced, and
Successfully realize expression of the gene in Escherichia coli, expression is simple and practicable, and expression product easy purification, stability is good, than
Living up to 1315U/mg, and the enzyme is with good stability, and Io-SOD1 of the invention is by before with wide commercial Application
Scape.
Brief description of the drawings
Fig. 1 expresses SDS-PAGE collection of illustrative plates for Io-SOD1.
Fig. 2 is the thermal stability analysis of Io-SOD1 at different temperatures.
Fig. 3 is the stability analyses of Io-SOD1 at room temperature.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiments, is respectively provided with three repeated experiments, as a result makes even
Average.
The acquisition of embodiment 1, Io-SOD1 and its encoding gene
STb gene is extracted from from the deposit of the Indian Ocean, lots of genes group sequencing analysis are carried out to it, are therefrom found
A kind of protein, as shown in the sequence 1 of sequence table, is named as Io-SOD1.The encoding gene of Io-SOD1 is named as Io-
SOD1, as shown in the sequence 2 of sequence table.
The expression and purifying of embodiment 2, Io-SOD1
1st, pET28a (+) carrier is substituted using the double chain DNA molecule shown in the sequence 2 from 5 ' 1-612, ends of sequence table
Small fragment between (Novagen companies) NdeI and HindIII restriction enzyme sites, obtains recombinant expression carrier pET28a (+)/Io-
SOD1 (sequence verification).
2nd, it is (complete to import e. coli bl21 (DE3) by the recombinant expression carrier pET28a (+) for obtaining step 1/Io-SOD1
Shi Jin Bioisystech Co., Ltd, article No.：CD601-01), recombinant bacterium is obtained.
3rd, the recombinant bacterium for obtaining step 2 is seeded in the LB fluid nutrient mediums of 50ml kanamycins containing 50ug/mL, and 37
DEG C, 200rpm shake cultures to bacterium solution OD600nm=0.6, IPTG is added in bacterium solution and makes its concentration in system be 1mM, 16
DEG C, 200rpm inductions 4 it is small when, 12000rpm is centrifuged after induction, is collected bacterial sediment and is carried out SDS-PAGE detections (result is such as
The swimming lane 1 of Fig. 1).
4th, with PBS (135mMNaCl, 2.7mMKCl, 1.5mM KH2PO4, 8mM K2HPO4, pH 7.2) be resuspended step 3 receive
The bacterial sediment of collection, ultrasound (150W, crushes 10s, is spaced 10s, 20 circulations), collects full cell pyrolysis liquid after broken, will be complete
Cell pyrolysis liquid 12000rpm centrifuges 10min, collects supernatant and precipitates respectively.Supernatant is subjected to SDS-PAGE detections (knot
The swimming lane 2 of fruit such as Fig. 1), while precipitation is also subjected to SDS-PAGE detections (swimming lane 3 of result such as Fig. 1) testing result display warp
IPTG is induced, and the albumen that intracellular expression molecular size range is about 25kD, is consistent with the theoretical molecular of the mono- subunits of SOD, shows
The Io-SOD1 correctly expressed in aforementioned manners.
5th, by the operating method of SNBC 3S NTA Resin (Shanghai Sheng Neng lottery industries bio tech ltd) non denatured
Under the conditions of in the supernatant that can be obtained to step 4 expression product with His-Tag labels purify, obtain after purification
Io-SOD1 protein solutions, protein concentration 0.12mg/mL, the swimming lane 4-6 through SDS-PAGE testing results such as Fig. 1.With facing benzene three
Phenol Autoxidation Method (measure of superoxide dismutase (SOD) activity in GB/T 5009.171-2003 health foods) measures Io-
SOD enzyme activity in SOD1 protein solutions, its unit of activity are about 198U/mL nutrient solutions, are 1315U/mg than work.
The definition of SOD unit of activity：Required SOD amounts are one when suppressing mouse thymus cells speed 50% at 25 DEG C
Unit of activity.
Embodiment 3, Io-SOD1 stability analyses
1st, the Io-SOD1 protein solutions for preparing embodiment 2 respectively under the conditions of 50 DEG C and 60 DEG C stand 3 it is small when, process
It is middle using face benzenetriol Autoxidation Method (in GB/T 5009.171-2003 health foods superoxide dismutase (SOD) activity
Measure) measure Io-SOD1 protein solutions in remaining enzymatic activity (SOD enzyme activity is set to 100% before heat treatment, after heat treatment
The percentage that enzyme activity accounts for the enzyme activity of before processing is remaining enzyme activity), the results are shown in Figure 2.The result shows that Io-SOD1 is at 50 DEG C
Under the conditions of handle 3 hours, still retain more than 90% enzyme activity, more than 80% can still be retained by being handled 30 minutes under the conditions of 60 DEG C
Enzyme activity.
2nd, Io-SOD1 protein solutions prepared by embodiment 2 are placed into half a year under room temperature (25 DEG C), during using facing benzene
Enzymatic activity in triphenol Autoxidation Method measure Io-SOD1 protein solutions.The results are shown in Figure 3.The result shows that Io-SOD1 room temperatures
Lower preservation is up to half a year, can still maintain more than 60% enzyme activity
The above results show that Io-SOD1 is with good stability.
<110>Ke Xinyang bio tech ltd in Shenzhen
<120>A kind of marine source superoxide dismutase and its encoding gene and application
<160> 2
<210> 1
<211> 204
<212> PRT
<213>Artificial sequence
<220>
<223>
<400> 1
Met Pro Thr His Thr Leu Pro Asp Leu Pro Tyr Asp His Glu Ala Leu
1 5 10 15
Ala Pro His Ile Asp Ala Arg Thr Met Glu Ile His His Gly Lys His
20 25 30
His Gln Gly Tyr Val Asn Asn Leu Asn Ala Ala Leu Glu Gly His Pro
35 40 45
Glu Leu Gln Ala Lys Ser Val Glu Glu Leu Val Ala Gly Ile Asp Ala
50 55 60
Val Pro Glu Ala Ile Arg Thr Ala Val Arg Asn Asn Gly Gly Gly His
65 70 75 80
Ala Asn His Ser Leu Phe Trp Leu Ser Met Ser Pro Gln Gly Gly Gly
85 90 95
Ala Pro Asp Gly Ala Leu Ala Thr Ala Leu Ala Asn Thr Phe Gly Ser
100 105 110
Phe Gly Asp Phe Lys Glu Gln Leu Thr Ala Ala Ser Leu Ala Arg Phe
115 120 125
Gly Ser Gly Trp Gly Trp Leu Val Val Thr Ser Gly Gly Glu Leu Gly
130 135 140
Val Tyr Ser Thr Ala Asn Gln Asp Asn Pro Tyr Met Gln Gly Asp Val
145 150 155 160
Pro Ile Leu Gly Val Asp Val Trp Glu His Ala Tyr Tyr Leu Asn Tyr
165 170 175
Gln Asn Arg Arg Pro Asp Tyr Leu Ala Ala Trp Trp Ser Val Val Asp
180 185 190
Trp Asp Glu Val Ala Arg Arg Phe Asp Ala Thr Arg
195 200
<210> 2
<211> 615
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
atgccgactc acaccctgcc ggacctcccg tacgatcatg aagcgctggc accacacatc 60
gatgctcgca ctatggaaat ccaccacggc aaacaccacc agggttacgt gaacaacctg 120
aatgctgcac tggaaggtca cccggaactc caagctaaat ctgtggaaga actggttgca 180
ggtatcgacg cggttccgga agctattcgt accgcagtac gtaacaacgg cggtggtcat 240
gcaaatcaca gcctgttttg gctgtccatg tcgccgcagg gtggcggtgc accggacggc 300
gcactggcaa ctgcgctggc aaacactttc ggtagctttg gtgacttcaa agaacagctg 360
acggcggctt ccctggctcg ttttggctct ggttggggtt ggctggttgt tacctctggc 420
ggcgaactgg gcgtgtactc caccgcgaac caagataatc catacatgca aggtgatgta 480
ccgattctgg gcgtggacgt gtgggagcac gcgtactacc tgaactacca gaaccgtcgt 540
ccggactatc tggcggcatg gtggtccgtt gttgattggg atgaagttgc gcgccgtttc 600
gatgcgaccc gttaa 615
Claims (8)
1. a kind of protein, is any in following (a1)-(a5)：
(a1) protein being made of the amino acid sequence shown in sequence in sequence table 1；
(a2) by the amino acid sequence of sequence 1 by the substitution and/or missing and/or addition of one or several amino acid residues and
With identical function as derived from sequence 1 protein；
(a3) fusion protein of (a1) or (a2) is contained；
(a4) fusion protein that small peptide of the connection containing label obtains in the end of (a1) or (a2)；
(a5) fusion protein that connection label obtains in the end of (a1) or (a2).
2. encode the gene of protein described in claim 1.
3. gene as claimed in claim 2, it is characterised in that：The gene is any DNA in following (b1)-(b4)
Molecule：
(b1) DNA molecular of the code area as shown in sequence 2 in sequence table；
(b2) DNA molecular in sequence table shown in sequence 2；
(b3) DNA sequence dna limited under strict conditions with (b1) or (b2) hybridizes and the DNA of encoding superoxide dismutase divides
Son；
(b4) there is more than 90% homology and encoding superoxide dismutase with (b1) or (b2) or (b3) DNA sequence dna limited
DNA molecular.
4. recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium containing gene described in Claims 2 or 3.
5. application of the protein described in claim 1 in as superoxide dismutase.
6. a kind of recombinant bacterium, is that will be obtained in the channel genes host strain described in Claims 2 or 3.
7. a kind of preparation method of superoxide dismutase, includes the following steps：The recombinant bacterium described in claim 6 is cultivated, from
Superoxide dismutase is obtained in recombinant bacterium.
8. the protein described in claim 1, or, the gene described in Claims 2 or 3, or, the restructuring described in claim 6
Bacterium, or, the method described in claim 7, the application in superoxide dismutase product is prepared.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810105875.6A CN108018266B (en) | 2018-02-02 | 2018-02-02 | Marine-derived superoxide dismutase and coding gene and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810105875.6A CN108018266B (en) | 2018-02-02 | 2018-02-02 | Marine-derived superoxide dismutase and coding gene and application thereof |
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| Publication Number | Publication Date |
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| CN108018266A true CN108018266A (en) | 2018-05-11 |
| CN108018266B CN108018266B (en) | 2020-11-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810105875.6A Active CN108018266B (en) | 2018-02-02 | 2018-02-02 | Marine-derived superoxide dismutase and coding gene and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115011493A (en) * | 2022-06-14 | 2022-09-06 | 深圳中科欣扬生物科技有限公司 | Saccharomyces cerevisiae strain for separating and producing SOD (superoxide dismutase) from hot spring soil in Quzhuomu region in Tibet and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814753A (en) * | 2005-02-01 | 2006-08-09 | 中国科学院微生物研究所 | Heat-resistant superoxide dismutase and its conding gene and use |
| CN102010466A (en) * | 2010-11-10 | 2011-04-13 | 中国农业科学院作物科学研究所 | Plant resistance associated protein MYB, as well as coding gene and application thereof |
| CN102757487A (en) * | 2011-04-27 | 2012-10-31 | 中国农业大学 | Plant dwarfing related protein GA2ox, and encoding gene and application thereof |
| US9832939B2 (en) * | 2010-01-21 | 2017-12-05 | Austin Russell | Systems and methods for water harvesting and recycling |
-
2018
- 2018-02-02 CN CN201810105875.6A patent/CN108018266B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814753A (en) * | 2005-02-01 | 2006-08-09 | 中国科学院微生物研究所 | Heat-resistant superoxide dismutase and its conding gene and use |
| US9832939B2 (en) * | 2010-01-21 | 2017-12-05 | Austin Russell | Systems and methods for water harvesting and recycling |
| CN102010466A (en) * | 2010-11-10 | 2011-04-13 | 中国农业科学院作物科学研究所 | Plant resistance associated protein MYB, as well as coding gene and application thereof |
| CN102757487A (en) * | 2011-04-27 | 2012-10-31 | 中国农业大学 | Plant dwarfing related protein GA2ox, and encoding gene and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115011493A (en) * | 2022-06-14 | 2022-09-06 | 深圳中科欣扬生物科技有限公司 | Saccharomyces cerevisiae strain for separating and producing SOD (superoxide dismutase) from hot spring soil in Quzhuomu region in Tibet and application thereof |
| CN115011493B (en) * | 2022-06-14 | 2023-07-18 | 深圳中科欣扬生物科技有限公司 | Saccharomyces cerevisiae strain for producing SOD by separating hot spring soil in Qu Zhuomu-Tibet region and application thereof |
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| Publication number | Publication date |
|---|---|
| CN108018266B (en) | 2020-11-03 |
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