CN1814753A - Heat-resistant superoxide dismutase and its conding gene and use - Google Patents
Heat-resistant superoxide dismutase and its conding gene and use Download PDFInfo
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Abstract
This invention discloses a heat-resisting super-oxide dismutase, its coding gene and its application, in which, said dismutase is the protein with one of the following amino acid residue sequences: 1, SEQ ID No: 1 in the list, 2, the residue sequences in SEQ ID No: 1 are replaced, deleted or added by 1-10 amino acid residues and having the function of balancing oxygen free redicals in the body, the expression method includes: setting up a recombined expression carrier containing heat-resisting super-oxide dismatase gene to be led into a host cell to be induced to let the dismatase gene to express.
Description
Technical field
The present invention relates to enzyme and encoding gene thereof and application, particularly relate to a kind of heat-resistant superoxide dismutase and encoding gene thereof and its application in heat-resistant superoxide dismutase is produced.
Background technology
(superoxide dismutase SOD) is Fridovich in 1969 and McCord finds and by definite designation to superoxide-dismutase from the ox blood red corpuscle.It is extensively to be present in biological intravital metalloenzyme, can catalysis superoxide anion generation disproportionation reaction, and the biological intravital superoxide ion of single-minded removing, the oxyradical of balance body.SOD mainly is divided into Fe-SOD according to its bonded metal ion, Mn-SOD, four kinds of CuZn-SOD and Ni-SOD.SOD is anti-ageing, radioprotective, and anti-inflammatory suppress tumour and cancer, and the treatment aspect of autoimmune disease has vital role.
At present, the clone of sod gene only is confined to eukaryotes such as people, mouse, yeast and insect, but the SOD poor heat resistance in common source, generally in that be higher than under 50 ℃ of situations will inactivation, the transformation period is short, has limited its application.Up to now, the domestic report that yet there are no relevant heat-resisting sod gene clone.
Aquifex pyrophilus is a kind of extreme thermophile bacteria that derives from the ocean, and it grows in 67-95 ℃ the environment, and its Fe-SOD is first sod gene of being cloned into from extreme thermophile bacteria.This gene has been realized expression in intestinal bacteria, the ratio of reorganization SOD is lived and is 1400U/mg, and enzyme is lived and stablized in the environment of pH 4.0-10.0, has high thermotolerance, and heat resisting temperature can reach 95 ℃.
Summary of the invention
The purpose of this invention is to provide a kind of thermotolerance superoxide-dismutase and encoding gene thereof preferably.
Heat-resistant superoxide dismutase provided by the present invention, name is called Fe-SOD1, is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 1;
2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of oxyradical effect in the balance body.
SEQ ID № in the sequence table: 1 is made up of 211 amino-acid residues.
The encoding gene of above-mentioned heat-resistant superoxide dismutase (Fe-SOD1) has one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
The rigorous condition of described height can be that (or 0.1 * SSC), the solution of 0.1%SDS is hybridized and washed film with 0.1 * SSPE under 65 ℃.
SEQ ID № in the sequence table: 2 by 636 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to the 636th bit base.
Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.
Arbitrary segmental primer is to also within protection scope of the present invention among the amplification Fe-SODl.
Another object of the present invention provides a kind of method of expressing above-mentioned heat-resistant superoxide dismutase.
The method of the above-mentioned heat-resistant superoxide dismutase of expression provided by the present invention, be to make up the recombinant expression vector that contains the heat-resistant superoxide dismutase gene, the recombinant expression vector that makes up is imported host cell, cultivate host cell and make heat-resistant superoxide dismutase genetic expression.
Described host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.
Described intestinal bacteria can be E.coli BL21 (DE3), E.coli BL21 (DE3) plys, BLR (DE3) or B834 etc.
The carrier that sets out that is used to make up described recombinant expression vector can be at expression in escherichia coli expression of exogenous gene carrier, is preferably pET-28a, pET-28b or the pET-28c that can express the His6-Tag structure.
With pET-28a is that the set out recombinant expression vector that contains the heat-resistant superoxide dismutase gene of vector construction is pET28a (+)/Fe-SOD1.
Above-mentioned recombinant expression vector all can make up according to ordinary method.
Cultivation contains the substratum and the culture condition of the host cell of heat-resistant superoxide dismutase encoding gene of the present invention, all can be substratum and the culture condition of cultivating the host that sets out.Wherein, add inductor IPTG when cultivating described recombination bacillus coli host, add IPTG concentration be 0.1-1mM, be preferably 0.75mM, inducing temperature is 30-37 ℃, induction time is 3-5 hour.
The described method that the expression product heat-resistant superoxide dismutase is carried out purifying is preferably Ni post affinity chromatography.
The present invention is cloned into Fe-SOD1 from the plasmid library of Tengchong hot spring environment DNA, and make it obtain to express, expression method is simple, the easy purifying of expression product, good stability can reach 2100U/mg than work, and this enzyme has outstanding thermotolerance and good resistance to acids and bases, its prothetic group institute chelated metal ion is analyzed, proved that this enzyme is Fe-SODs.Fe-SOD1 of the present invention will have wide prospect in industrial application.
Description of drawings
Fig. 1 is the SDS-PAGE collection of illustrative plates of expressing protein
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The clone of embodiment 1, Fe-SOD1 gene
One, slightly the carrying of DNA in the environmental sample
With reference to the yellow power of Wang Xiaobo, the separation and purification of DNA and library construction in the environmental sample, 2001, the second phase, the microorganism journal, in method extract DNA in Yunnan Tengchong hot spring (80 ℃, the pH 7.0) environmental sample, concrete steps are as follows:
1) takes by weighing 5g (dry weight) environmental sample, after fully grinding, place centrifuge tube, add 13.5mL DNA extraction buffer and (contain 100mmol/L Tris-HCL pH8.0,100mmol/L EDTA, 100mmol/L sodium phosphate buffer pH8.0,1.5mol/L NaCL, 1%CTAB) in the rearmounted liquid nitrogen of mixing, take out extremely thawing of insulation in 60 ℃ of water-baths then, freeze thawing three times.
2) behind adding 50 μ L Proteinase Ks (20mg/mL) and the 15mL 10%SDS mixing, 60 ℃ of insulation 2-3min, every 15-20min voltage regulator tube mixing several times, the centrifugal 10min of 7000g/min room temperature then, collect supernatant liquor, with with quadrat method with twice of washing of precipitate (in precipitation, adding 5mL water), merges the supernatant liquor of three collections, with twice of isopyknic phenol/chloroform (1: 1) extracting.
3) add 1g pickling cross-linked polyvinylpyrrolidone (PVPP), 37 ℃ of insulation 30min, via hole diameter is that the membrane filtration of 0.45um is removed PVPP, the Virahol that in filtrate, adds 0.6 times of volume again, after room temperature is placed 30min, 12000g/min, 4 ℃ of centrifugal 15min will precipitate with being dissolved in 1.5mL TE (pH 8.0) after 70% washing with alcohol.
4) behind the adding 0.1g anhydrous acetic acid ammonium mixing, 8000g/min, 4 ℃ of centrifugal 30min, get supernatant liquor, the Virahol that adds 1 times of volume, behind the ice bath 10min, 12000g/min, 4 ℃ of centrifugal 30min will precipitate with being dissolved in 0.5mL TE (containing 20 μ g/mL RNase A) after 70% washing with alcohol, obtain the DNA in the environmental sample.
Two, the structure of environmental sample DNA plasmid library
Building process may further comprise the steps:
1) DNA that adopts the LMP gel that step 1 is slightly carried carries out purifying and recovery;
2) with restriction enzyme Pst I the DNA of purifying is carried out enzyme and cut, separate enzyme with 1% agarose gel electrophoresis and cut product;
3) reclaiming length with DEAE Mierocrystalline cellulose filter paper is the endonuclease bamhi of 3-8kb, these endonuclease bamhis are connected with the carrier pUC 19 that cuts through Pst I enzyme with handling through the alkaline phosphatase dephosphorylation, to connect product Calcium Chloride Method transformed into escherichia coli D H5 α, be integrated with the intestinal bacteria bacterium colony of recombinant plasmid then with blue hickie method screening, obtain the plasmid library of environmental sample DNA.
Three, the clone of Fe-SOD1 gene
The positive colony that step 2 is obtained carries out random sequencing, and utilizing the BlastX program of Genebank to carry out sequence alignment, the result shows that one of them positive colony H314 contains complete superoxide-dismutase mature protein coding sequence and upstream portion regulating and controlling sequence.According to this sequencing result design primer, primer sequence is (band underscore base is the restriction enzyme enzyme recognition site):
The SODF:(upstream primer) 5 '-CG
GGATCCATGCCAGTGCATAAGTTA-3 '
The SODR:(downstream primer) 5 '-CC
AAGCTTTTATTTCACAAAATCCTT-3 '
Genomic dna with above-mentioned positive colony H314 is a template, the encoding sequence of this potential of pcr amplification SOD maturation protein, pcr amplification product is checked order, sequencing result shows that it has the nucleotide sequence of sequence 2 in the sequence table, by 636 based compositions, encoding sequence is that coding has the protein of the amino acid residue sequence of sequence 1 in the sequence table from 5 ' end the 1st to the 636th bit base, and this proteic theoretical molecular is 24kD.With this dna fragmentation called after Fe-SOD1.
The expression of embodiment 2, Fe-SOD1 and metal ion analysis
One, the expression of Fe-SOD1
The Fe-SOD1 that embodiment 1 is obtained is cloned among the carrier pET28a (+) (Novagen company), obtains containing the recombinant expression vector of Fe-SOD1, with its called after pET28a (+)/Fe-SOD1, again with recombinant expression vector pET28a (+)/Fe-SOD1 CaCl
2Method transformed into escherichia coli BL21 (DE3), with blue hickie method screening recon, recon is inoculated in the test tube that contains the LB liquid nutrient medium 37 ℃ shaking culture 12-24 hour, be inoculated in the 500mL triangular flask that 50mL LB liquid nutrient medium is housed by 1% inoculum size again, at 30 ℃, 0.75mM IPTG induces down expression (is contrast with what do not add IPTG), take a sample behind the 4h, collect supernatant liquor after centrifugal respectively and through the precipitation of ultrasonication, and carry out SDS-PAGE and detect, the result as shown in Figure 1, swimming lane 1 is the protein standard molecular weight, swimming lane 2 is not for adding IPTG inductive bacterial cell disruption supernatant liquor, swimming lane 3 is through IPTG inductive bacterial cell disruption supernatant liquor, swimming lane 4 is the Aquifex pyrophilus Fe-SOD of purifying, induce through IPTG, born of the same parents have expressed the albumen that the molecular weight size is about 28kD outward, conform to the theoretical molecular of the single subunit of SOD, show the Fe-SOD1 that has obtained correct expression with aforesaid method, and its Partial Protein still keeps polymeric structure under the sex change condition, with the similar performance of Aquifex pyrophilusFe-SOD.The expression product that has the His-Tag label by the working method of SNBC 3S NTA Resin (Shanghai give birth to can lottery industry bio tech ltd) in can be to supernatant liquor under non-sex change condition carries out extracting.Measure the activity of the Fe-SOD1 that expresses with facing benzenetriol autoxidation method, its unit of activity is about the 324U/mL nutrient solution, is 2100U/mg than living, and measuring expressing quantity with Folin-phenol method is the 0.16mg/mL fermented liquid.
Two, the metal ion analysis of Fe-SOD1 prothetic group
1, Fe-SOD1 being broken away from son handles, and carry out Fe, the reconstruction of Mn SOD is to analyze its ion chelating characteristic, method is: break away from the son processing with 8M urea to implementing 2 Fe-SOD1 that obtain, it is lost activity, and then use Fe respectively, the Mn metal ion is rebuild it, the result shows that the Fe-SOD of reconstruction recovers original in 1890U/mg alive, and the Mn-SOD that rebuilds significantly is reduced to 45U/mg than living, and shows that SOD1 is Fe-SODs.
2, analyze the kind and the content of Fe-SOD1 protein protomer institute chelated metal ions with atomic absorption spectrum (AAnalyst 100, Parkin Elmer), the result shows that Fe-SOD1 prothetic group institute chelated metal ion is an iron ion.
3, NaN
3But specificity ground suppresses Fe/Mn SODs, and the SOD of other type is not influenced, thereby when the enzyme of measuring Fe-SOD1 is lived, adds the inhibitor NaN of different concns in reaction solution
3With NaF to measure it to the influence that enzyme is lived, measurement result shows NaN
3503nhibiting concentration to Fe-SOD1 is 37mM, meets the characteristic of Fe/Mn SODs.
4, H
2O
2Can suppress the activity of Fe SODs by the Fe ion in SOD active centre, and MnSODs is not influenced.Fe-SOD1 is dissolved in respectively contains 0.24mM and 1.2mM H
2O
2Phosphate buffered saline buffer (pH 7.0) in, 25 ℃ of down insulations, add catalase and measure its residual enzyme vigor after handling different time.Measurement result shows that Fe-SOD1 is to N
2O
2Sensitivity is containing 1.2mM H
2O
2Phosphate buffered saline buffer in its enzyme half inhibition time of living be 82min, meet the character of Fe SODs.
The above-mentioned Fe-SOD1 of experimental results show that is Fe SODs.
Embodiment 3, Fe-SOD1 and zymologic property analysis thereof
1, under 90 ℃ and 100 ℃, the Fe-SOD1 that embodiment 2 is expressed handles respectively and analyzed its thermotolerance in 1-3 hour, the result handles enzyme almost not loss alive in 2 hours down at 90 ℃, handles 60 minutes alive still remaining on more than 60% of enzyme down at 100 ℃, shows that Fe-SOD1 has higher thermotolerance.
2, be to handle the ph stability of measuring Fe-SOD1 in three hours in the buffering system of 2.2-12 in the pH value, the enzyme of result Fe-SOD1 in the pH4.0-11.0 scope is lived stable, shows that this enzyme has good resistance to acids and bases.
3, (chromatography column model: Superdex 200HR 10/30, chromatographic system: AKT FPLC), measurement result shows that the molecular weight of Fe-SOD1 under native state is 64kDa, infers that it is a dimer to measure the molecular weight of Fe-SOD1 with gel chromatography.
Sequence table
<160>2
<210>1
<211>211
<212>PRT
＜213〉the unknown
<220>
<223>
<400>1
Met?Pro?Val?His?Lys?Leu?Glu?Pro?Lys?Asn?His?Leu?Lys?Pro?Ser?Asn
1 5 10 15
Leu?Asn?Gly?Ile?Ser?Asn?Glu?Gln?Ile?Glu?Pro?His?Phe?Glu?Ala?His
20 25 30
Tyr?Lys?Gly?Tyr?Val?Thr?Lys?Tyr?Asn?Glu?Ile?Gln?Glu?Lys?Leu?Ala
35 40 45
Asp?Leu?Asn?Phe?Ser?Asp?Arg?Ser?Lys?Ala?Asn?Gln?Asn?Tyr?Ser?Glu
50 55 60
Tyr?Arg?Glu?Leu?Lys?Val?Glu?Glu?Thr?Phe?Asn?Tyr?Met?Gly?Val?Val
65 70 75 80
Leu?His?Glu?Leu?Tyr?Phe?Gly?His?Leu?Gly?Ala?Lys?Gly?Glu?Pro?Ser
85 90 95
Glu?Ala?Phe?Lys?Lys?Lys?Val?Glu?Glu?Asp?Phe?Gly?Ser?Trp?Asp?Ala
100 105 110
Cys?Ile?Gln?Glu?Ile?Lys?Ala?Ala?Gly?Met?Ala?Phe?Arg?Gly?Trp?Ala
115 120 125
Ile?Leu?Gly?Leu?Asp?Ile?Phe?Ser?Gly?Arg?Leu?Val?Val?Asn?Gly?Leu
130 135 140
Asp?Ala?His?Asn?Val?Tyr?Asn?Tyr?Thr?Gly?Leu?Ile?Pro?Leu?Ile?Val
145 150 155 160
Leu?Asp?Thr?Tyr?Glu?His?Ala?Tyr?Tyr?Val?Asp?Gln?Lys?Asn?Lys?Arg
165 170 175
Pro?Pro?Tyr?Ile?Asp?Ala?Phe?Leu?Gln?Asn?Leu?Asn?Trp?Glu?Val?Ile
180 185 190
Asn?Glu?Arg?Phe?Glu?Lys?Ala?Met?Lys?Ala?Tyr?Glu?Thr?Leu?Lys?Asp
195 200 205
Phe?Val?Lys
210
<210>2
<211>636
<212>DNA
＜213〉the unknown
<220>
<223>
<400>2
atgccagtgc?ataagttaga?gccaaagaac?catcttaagc?cttcaaacct?caatggcatt 60
tccaacgagc?agattgagcc?acactttgaa?gctcattata?agggctatgt?gactaaatac 120
aacgagattc?aggaaaagct?cgcagacctt?aacttctctg?acagaagcaa?agcaaaccaa 180
aactactctg?agtataggga?gctaaaggtg?gaggagactt?tcaattacat?gggtgttgtt 240
ctacacgagc?tttattttgg?tcatcttggt?gcaaagggag?agccttcaga?ggctttcaaa 300
aagaaagtag?aagaagactt?tggctcttgg?gatgcctgta?ttcaggagat?aaaggcagca 360
ggtatggctt?ttagaggatg?ggctattctt?ggacttgata?tattctctgg?caggcttgtg 420
gtaaacggtc?ttgacgctca?caatgtttac?aactacacag?ggcttatacc?tctcatagtc 480
cttgacactt?acgaacacgc?ctactatgtg?gaccaaaaga?acaagagacc?tccttacatt 540
gatgccttcc?tccagaacct?aaactgggaa?gttataaacg?aaaggtttga?aaaggctatg 600
aaggcttatg?aaaccctcaa?ggattttgtg?aaataa 636
Claims (10)
1, a kind of heat-resistant superoxide dismutase is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 1;
2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of oxyradical effect in the balance body.
2, the gene of the described heat-resistant superoxide dismutase of coding claim 1.
3, gene according to claim 3, it is characterized in that: it has one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
4, contain arbitrary segmental primer in claim 2 or 3 described expression carrier, transgenic cell line, host bacterium and amplification claim 2 or the 3 described genes.
5, the expression method of the described heat-resistant superoxide dismutase of a kind of claim 1, be to make up the recombinant expression vector that contains the heat-resistant superoxide dismutase gene, the recombinant expression vector that makes up is imported host cell, cultivate host cell and make heat-resistant superoxide dismutase genetic expression.
6, the expression method of heat-resistant superoxide dismutase according to claim 5 is characterized in that: being used to make up the described carrier that sets out that contains the recombinant expression vector of heat-resistant superoxide dismutase gene is pET-28a, pET-28b or pET-28c.
7, the expression method of heat-resistant superoxide dismutase according to claim 6 is characterized in that: described recombinant expression vector is pET28a (+)/Fe-SOD1.
8, the expression method of heat-resistant superoxide dismutase according to claim 5 is characterized in that: described host is E.coli BL21 (DE3), E.coli BL21 (DE3) plys, BLR (DE3) or B834.
9, the expression method of heat-resistant superoxide dismutase according to claim 8, it is characterized in that: add inductor IPTG when cultivating described recombination bacillus coli host, add IPTG concentration be 0.1-1mM, inducing temperature is 30-37 ℃, induction time is 3-5 hour.
10, according to the expression method of the arbitrary described heat-resistant superoxide dismutase of claim 5-9, it is characterized in that: the described method that the expression product heat-resistant superoxide dismutase is carried out purifying is a Ni post affinity chromatography.
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| CN108018266A (en) * | 2018-02-02 | 2018-05-11 | 深圳中科欣扬生物科技有限公司 | A kind of marine source superoxide dismutase and its encoding gene and application |
| CN108440035A (en) * | 2018-05-25 | 2018-08-24 | 福建农林大学 | A kind of compost method reducing nitrous oxide and ammonia emission |
| CN113321742A (en) * | 2021-06-04 | 2021-08-31 | 江苏大学 | Recombinant superoxide dismutase and construction method and application thereof |
| CN116042551B (en) * | 2022-12-13 | 2024-01-12 | 广州美神生物科技有限公司 | High-activity superoxide dismutase, preparation method thereof and application thereof in preparation of antioxidant products |
| CN116042551A (en) * | 2022-12-13 | 2023-05-02 | 广州美神生物科技有限公司 | High-activity superoxide dismutase, preparation method thereof and application thereof in preparation of antioxidant products |
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