CN110872582B - Cold-adapted peroxide reductase and coding gene and application thereof - Google Patents
Cold-adapted peroxide reductase and coding gene and application thereof Download PDFInfo
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- CN110872582B CN110872582B CN201811016358.8A CN201811016358A CN110872582B CN 110872582 B CN110872582 B CN 110872582B CN 201811016358 A CN201811016358 A CN 201811016358A CN 110872582 B CN110872582 B CN 110872582B
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Abstract
The invention discloses a cold-adapted peroxidase, a coding gene and application thereof, and aims to provide a cold-adapted peroxidase reductase, a coding gene and application thereof. The invention firstly uses the Antarctic sea ice microorganism bacillus psychrophilus (A)Psychrobactersp.) has been cloned a peroxiredoxin gene PsPrx, the amino acid residue sequence of which is shown in SEQ ID No. 2. The expression method is to construct a gene containing a peroxiredoxinPsA recombinant expression vector of Prx, and introducing the constructed recombinant expression vector into a host cell (a)Escherichia coli) And inducing to express the peroxide reductase PsPrx gene. The expression product PsPrx has outstanding psychrotrophy, good stability and good capacity of protecting supercoiled DNA from oxidative damage, and can be applied to the related fields of biological medicine, cosmetics, food and the like.
Description
Technical Field
The invention belongs to the fields of biotechnology, medicine, cosmetics, food and the like, and particularly relates to a cold-adapted peroxide reductase, and a coding gene and application thereof.
Background
Peroxoredoxin (Prx) is an important thiol-dependent antioxidant enzyme widely present in eukaryotes and prokaryotes, and can efficiently degrade H 2 O 2 Peroxynitrates and various organic peroxides. Prx in microorganisms is largely classified into 3 types, namely 1-Cys Prx, typical 2-Cys Prx and atypical 2-Cys Prx. The canonical 2-Cys Prx has been shown to be involved in oxidative signaling mechanisms that control apoptosis, differentiation and proliferation, and may be involved in immune responses, and can serve as a biological diagnostic marker for tumors and cancers. In addition, Prx-based antioxidant activity can be used as antioxidant in foodThe agent prolongs shelf life of food. Therefore, the typical 2-Cys Prx has wide application value in the production industries of chemical industry, food, medicine and the like.
The peroxide reductase extracted from animals and plants is easily limited by time, climate and region, and has poor stability, so that the peroxide reductase is difficult to be directly applied to relevant fields such as industrial production and the like. The induction expression by utilizing the genetic engineering bacteria is an effective way for reducing the cost and obtaining the peroxide reductase with natural activity.
The Antarctic environment has the characteristics of strong radiation, high salinity, low temperature and various extreme conditions, and a large amount of oxygen radicals and peroxides are accumulated in the environment. In order to adapt to extreme environments, the antarctic microorganisms evolve a high-efficiency antioxidant enzyme system to eliminate substances such as oxygen radicals and peroxides. Therefore, the Antarctic microorganism is one of important new sources of antioxidant enzyme gene resources.
In view of the above discussion, microorganisms from the Antarctic PolePsychrobactorsp. in vivo cloning Prx to be typical 2-Cys Prx. The gene is expressed in escherichia coli, and the recombinant peroxide reductase PsPrx has the capacity of protecting supercoiled DNA from oxidative damage, and can be applied to the related fields of biological medicines, cosmetics and foods.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the peroxide reductase PsPrx with the characteristics of simple and feasible expression method, easy purification of an expression product and low-temperature catalysis, and the coding gene and the application thereof.
The first object of the present invention is to provide a gene sequence of the cryophilic peroxide reductase gene PsPrx (typically 2-Cys Prx) and an amino acid sequence deduced therefrom. The nucleotide sequence of the psychrotrophic peroxide reductase PsPrx is shown in SEQ ID No.1, and the amino acid sequence of the psychrotrophic peroxide reductase PsPrx is shown in SEQ ID No. 2. The total length of the psychrophilic peroxide reductase PsPrx is 567 bp, and 188 amino acids are coded.
A recombinant expression vector containing the psychrotrophic peroxide reductase PsPrx.
The expression vector is preferably pET-28a (+).
A genetically engineered bacterium containing the recombinant expression vector.
The engineering bacteria are preferablyE.coli BL21 (DE3)。
The medium and conditions for culturing a host cell containing the gene encoding psychrophilic peroxide reductase PsPrx of the present invention may be any medium and conditions for culturing a host. Wherein, when the recombinant host Escherichia coli is cultured, the recombinant host Escherichia coli is cultured at 32-40 ℃ to OD 600 0.4-0.8, adding IPTG into the culture medium until the final concentration is 0.5-1.5 mM, and performing induced fermentation at 25-30 ℃ for 6-10 h to efficiently induce and express the psychrophilic peroxide reductase PsPrx.
The method for optimizing the expression product psychrophilic peroxide reductase PsPrx is preferably Ni column affinity chromatography.
The second purpose of the invention is to provide the PsPrx recombinant protein which can protect the supercoiled DNA from oxidative damage and can be applied to the fields related to biological medicines, cosmetics and foods.
Therefore, the invention mainly relates to a gene sequence of the psychrotrophic peroxide reductase PsPrx, and more particularly relates to an amino acid sequence of the psychrotrophic peroxide reductase PsPrx, construction of a recombinant expression vector, preparation and purification of recombinant protein, and the PsPrx recombinant protein can protect supercoiled DNA from oxidative damage and can be applied to the related fields of biological medicine, cosmetics and food.
Drawings
FIG. 1 is an SDS-PAGE graph showing expression and purification of the peroxide reductase PsPrx in Acidobacterium.
FIG. 2 shows the recombination of peroxiredoxin PsPrx in FeCl 3 Protection of DNA in catalytic oxidation system.
Detailed Description
The following examples are illustrative of the invention and are not intended to be limiting thereof.
The test methods not specifically mentioned in the following examples can be carried out in accordance with conventional methods or with the instructions of the manufacturers of the products used. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: cloning and sequencing analysis of the peroxidase gene.
Nanji microorganismPsychrobactorANT206 was activated in 2216E liquid medium, cultured to the middle and late stage (about 4 d) of the logarithmic phase, and the total gene DNA of the strain was extracted by combining the CTAB method and the phenol-chloroform extraction method. PCR was performed using degenerate primers with the extracted total DNA as a template.
An upstream primer: 5 '-AMTCAGTGACADTCAG ATGGCGTCT-3'
A downstream primer: 5 '-GSAACTGGKGCATATGTTAGATTTT-3'
The amplification conditions were: denaturation at 94 deg.C for 1 min, annealing at 58.2 deg.C for 1 min, extension at 72 deg.C for 90 s, and circulation for 30 times. Then, agarose gel electrophoresis detection is carried out to find the gene containing the target genePsThe band of Prx is sequenced. Analyzing the sequencing result to obtain a gene with a full-length 567 bp complete reading frame sequence, wherein the nucleotide sequence is shown as SEQ ID No.1, the gene codes a protein consisting of 188 amino acids, and the amino acid sequence is shown as SEQ ID No. 2.
Example 2: expression and purification of peroxidase Gene
And (4) redesigning a primer containing the enzyme cutting site according to the determined full-length sequence of Prx.
An upstream primer: 5' -ATAGGATCCATGGCGTCTATCATCA -3’
A downstream primer: 5' -CGCAAGCTTCGATTTTACCTACTAG -3’
The marked lines are respectivelyBamHI，HindIII restriction enzyme site.
And (3) connecting the Prx gene with a gel recovery product obtained after double enzyme digestion of pET-28a (+) by utilizing T4 ligase in proportion to construct a recombinant expression vector pET-Prx. Transformation of recombinant expression vectors into competent cellsE.coliBL21(DE3) performed positive clone screening and enzyme digestion verification.
And (4) inducing and expressing the screened recombinant strain by IPTG (isopropyl-beta-thiogalactoside). Inoculating the recombinant bacteria into LB culture medium, and culturing at 32-40 deg.C to OD 600 0.4-0.8, adding IPTG to the culture medium to the final concentration of 0.5-1.5 mM, and performing induced fermentation at 25-30 ℃ for 6-10 h. Centrifuging and collectingAnd (3) precipitating the thalli, grinding in an ice bath and carrying out ultrasonic crushing to extract crude protein. And purifying the PsPrx recombinant protein by using Ni column affinity chromatography, and performing SDS-PAGE electrophoretic analysis to finally obtain a single band with the molecular weight of about 25.2 kDa. The results are shown in FIG. 1, M, standard molecular weight protein marker; 1, blank plasmid control; 2, recombining pET-Prx; 3, after purification, PsPrx. The specific embodiment of the Ni column affinity chromatography is as follows: after extracting crude protein, eluting 8-10 column volumes by using 5-20 mM imidazole, eluting 8-10 column volumes by using 40-60 mM imidazole, finally eluting 8-10 column volumes by using 100-500 mM imidazole, and collecting eluent at the peak value to obtain the purified cold-adapted peroxide reductase PsPrx.
Example 3: FeCl 3 Catalytic oxidation supercoiled DNA protection assay
15 μ L of the reaction system contained 15-18 mM FeCl 3 15-18 mM DTT and a certain amount of purified PsPrx are subjected to water bath at 20-30 ℃ for 2-3 h, and then 750-1250 ng of pUC19 supercoiled DNA are added and the water bath is carried out at 20-30 ℃ for 2-3 h. The degradation results were detected by agarose gel electrophoresis after the reaction. The results indicate that the psychrotrophic peroxide reductase PsPrx is able to protect supercoiled DNA from damage by a metal-catalyzed oxidation system. The results are shown in FIG. 2, plasmid 1, pUC 19; 2, pUC19 plasmid + FeCl; 3, pUC19 plasmid + DTT; 4, pUC19 plasmid + FeCl 3 + DTT + BSA; 5, pUC19 plasmid + FeCl 3 + DTT + PsPrx (5. mu.g/mL); 6, pUC19 plasmid + FeCl 3 + DTT + PsPrx (10. mu.g/mL); 7, pUC19 plasmid + FeCl 3 + DTT + PsPrx (15. mu.g/mL); 8, pUC19 plasmid + FeCl 3 + DTT + PsPrx (20. mu.g/mL); NF, nicked plasmid DNA; SF, supercoiled plasmid DNA. The experiment proves that the psychrotrophic peroxide reductase PsPrx has good capability of protecting supercoiled DNA from oxidative damage, and can be applied to related fields of biological medicine, cosmetics, food and the like.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
SEQUENCE LISTING
<110> Harbin Industrial university (Weihai)
<120> cold-adapted peroxide reductase and coding gene and application thereof
<130> 11
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 567
<212> DNA
<213> Artificial Sequence
<220>
<223> Psychrobactor sp . ANT206
<400> 1
atggcgtcta tcatcaatca agaaatccca gaattttcaa cacaagcgtt ttacaacggt 60
gaattcaaaa ccatcacctc tgaagacgta aaaggcaagt ggtcgatttt catgttttac 120
ccacacgact ttacctttgt ttgcccaact gaacttgaag acatggcaga ccattacgaa 180
gagctaaaaa agttaggcgt agaagtgtac tcagtatcaa cggatacgca tttcacccat 240
aaagcatggc atgattcttc agaagcaatc tccaaaattc agtacccaat gattggtgat 300
cctactggtc gtatcacgcg tggctttaac attatgattg aagaagagca ccaagctgag 360
cgcggtacat tcttagtaga tcctgatggc tttattcaag ttgctgagat tcatgacctc 420
ggtattggcc gtagtgcaaa agatatgctg cgtaaagtaa aagcggcaca gtatgttcgt 480
gaaaacgacg gcgaagtttg cccagccgct tgggaagagg gtcaagagac tttaaaacca 540
agtcttgatc tagtaggtaa aatctaa 567
<210> 2
<211> 188
<212> PRT
<213> Artificial Sequence
<220>
<223> Psychrobactor sp . ANT206
<400> 2
Met Ala Ser Ile Ile Asn Gln Glu Ile Pro Glu Phe Ser Thr Gln Ala
1 5 10 15
Phe Tyr Asn Gly Glu Phe Lys Thr Ile Thr Ser Glu Asp Val Lys Gly
20 25 30
Lys Trp Ser Ile Phe Met Phe Tyr Pro His Asp Phe Thr Phe Val Cys
35 40 45
Pro Thr Glu Leu Glu Asp Met Ala Asp His Tyr Glu Glu Leu Lys Lys
50 55 60
Leu Gly Val Glu Val Tyr Ser Val Ser Thr Asp Thr His Phe Thr His
65 70 75 80
Lys Ala Trp His Asp Ser Ser Glu Ala Ile Ser Lys Ile Gln Tyr Pro
85 90 95
Met Ile Gly Asp Pro Thr Gly Arg Ile Thr Arg Gly Phe Asn Ile Met
100 105 110
Ile Glu Glu Glu His Gln Ala Glu Arg Gly Thr Phe Leu Val Asp Pro
115 120 125
Asp Gly Phe Ile Gln Val Ala Glu Ile His Asp Leu Gly Ile Gly Arg
130 135 140
Ser Ala Lys Asp Met Leu Arg Lys Val Lys Ala Ala Gln Tyr Val Arg
145 150 155 160
Glu Asn Asp Gly Glu Val Cys Pro Ala Ala Trp Glu Glu Gly Gln Glu
165 170 175
Thr Leu Lys Pro Ser Leu Asp Leu Val Gly Lys Ile
180 185
Claims (8)
1. A psychrotrophic peroxide reductase PsPrx is characterized in that the amino acid sequence of the psychrotrophic peroxide reductase PsPrx is shown as SEQ ID No. 2.
2. A gene encoding the psychrophilic peroxide reductase PsPrx of claim 1.
3. The gene encoding the cryophilic peroxide reductase PsPrx according to claim 2, which has the nucleotide sequence shown in SEQ ID No. 1.
4. A recombinant expression vector containing the gene of claim 2 or 3.
5. A genetically engineered bacterium comprising the recombinant expression vector of claim 4.
6. The genetically engineered bacterium of claim 5, wherein the engineered bacterium is Escherichia coli E.coli BL21(DE 3).
7. A method for inducible expression of the psychrophilic peroxide reductase PsPrx of claim 1, comprising the steps of:
culturing the recombinant strain with the gene encoding the psychrotrophic peroxide reductase PsPrx at 32-40 ℃ until OD600 is 0.4-0.8, adding IPTG into a culture medium until the final concentration is 0.5-1.5 mM, and performing induced fermentation for 6-10 h at 25-30 ℃, thus obtaining the psychrotrophic peroxide reductase PsPrx through induced expression.
8. Use of the psychrophilic peroxide reductase PsPrx of claim 1 for protecting supercoiled DNA from oxidative damage for non-disease diagnostic and therapeutic purposes.
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