CN110872582B - A cold-adapted peroxide reductase and its encoding gene and application - Google Patents

Abstract

The invention discloses a cold-adapting peroxidase, its encoding gene and application, and aims to provide a cold-adapting peroxidase and its encoding gene and application. The present invention firstly clones the peroxide reductase gene PsPrx from the Antarctic sea ice microorganism Psychrobacter sp., and the amino acid residue sequence of the peroxide reductase is shown in SEQ ID No.2. The expression method is to construct a recombinant expression vector containing the peroxide reductase gene Ps Prx, and introduce the constructed recombinant expression vector into a host cell ( Escherichia coli ), and induce the expression of the peroxide reductase PsPrx gene. The expression product PsPrx of the present invention has outstanding cold adaptability, good stability, and good ability to protect supercoiled DNA from oxidative damage, and can be applied to related fields such as biomedicine, cosmetics and food.

Description

A cold-adapted peroxide reductase and its encoding gene and application

technical field

The invention belongs to the fields of biotechnology, medicine, cosmetics, food and the like, and particularly relates to a cold-adapting peroxide reductase and its encoding gene and application.

Background technique

Peroxiredoxin (Prx) is a class of important thiol-dependent antioxidant enzymes that widely exist in eukaryotes and prokaryotes. They can efficiently degrade H ₂ O ₂ , peroxynitrate and a variety of Organic peroxides. Prx in microorganisms is mainly divided into three categories, namely 1-Cys Prx, typical 2-Cys Prx and atypical 2-Cys Prx. Typical 2-Cys Prx has been shown to be involved in oxidative signaling mechanisms that control cell apoptosis, differentiation and proliferation, and may be involved in immune responses, and can be used as biomarkers for tumor and cancer diagnosis. In addition, based on the antioxidant capacity of Prx, it can also be used as a food antioxidant to extend the shelf life of food. Therefore, typical 2-Cys Prx has a wide range of application value in chemical, food, pharmaceutical and other production industries.

Peroxidoreductases extracted from animals and plants are easily limited by seasons, climates and regions, and have poor stability, making it difficult to directly apply to related fields such as industrial production. The use of genetically engineered bacteria to induce expression is an effective way to reduce costs and obtain peroxidoreductases with natural activity.

The Antarctic environment is characterized by various extreme conditions such as strong radiation, high salinity and low temperature, and a large number of oxygen free radicals and peroxides are accumulated in the environment. In order to adapt to the extreme environment, Antarctic microorganisms have evolved a high-efficiency antioxidant enzyme system to scavenge oxygen free radicals and peroxides. Therefore, Antarctic microorganisms are one of the important new sources of antioxidant enzyme gene resources.

In view of the above discussion, Prx was cloned from Antarctic microorganism Psychrobactor sp. as a typical 2-CysPrx. The gene is expressed in Escherichia coli, and the recombinant peroxide reductase PsPrx has the ability to protect supercoiled DNA from oxidative damage, and can be used in biomedicine, cosmetics and food-related fields.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a peroxidase reductase PsPrx and its encoding gene and application which are simple and easy to express, the expression product is easy to purify, and has low-temperature catalytic properties.

The first object of the present invention is to provide a gene sequence of a cold-adapted peroxide reductase gene PsPrx (typical 2-CysPrx) and an amino acid sequence deduced therefrom. The nucleotide sequence of the cold-adapted peroxide reductase PsPrx of the present invention is shown in SEQ ID No.1, and its amino acid sequence is shown in SEQ ID No.2. The cold-adapted superoxide reductase PsPrx is 567 bp in length and encodes 188 amino acids.

The recombinant expression vector containing the above-mentioned cold-adapted peroxide reductase PsPrx.

The expression vector is preferably pET-28a(+).

A genetically engineered bacterium containing the above-mentioned recombinant expression vector.

The engineering bacteria is preferably E. coli BL21 (DE3).

The culture medium and culture conditions for culturing the host cells containing the gene encoding the cold-adapted peroxide reductase PsPrx of the present invention can be both the culture medium and the culture conditions for culturing the host. Wherein, when culturing the recombinant host Escherichia coli, it is cultured at 32-40 °C to an OD ₆₀₀ of 0.4-0.8, IPTG is added to the medium to a final concentration of 0.5-1.5 mM, and fermentation is induced at 25-30 °C for 6-10 h, the expression of cold-adapted superoxide reductase PsPrx can be efficiently induced.

The method for optimizing the expression product, the cold-adapted peroxide reductase PsPrx, is preferably Ni-column affinity chromatography.

The second object of the present invention is to provide that the above-mentioned PsPrx recombinant protein can protect supercoiled DNA from oxidative damage, and can be applied to the fields of biomedicine, cosmetics and food.

Therefore, the present invention mainly relates to the gene sequence of a cold-adapted peroxide reductase PsPrx, more specifically, to the amino acid sequence of the cold-adapted peroxide reductase PsPrx, the construction of recombinant expression vector, the preparation and purification of recombinant protein, and the present invention also The PsPrx recombinant protein can protect the supercoiled DNA from oxidative damage, and can be applied to the fields of biomedicine, cosmetics and food.

Description of drawings

Figure 1 is an SDS-PAGE chart of the expression and purification of the peroxidoreductase PsPrx in Psychrophilic Bacillus.

Figure 2 is an analysis diagram of DNA protection by recombinant peroxide reductase PsPrx in FeCl ₃ catalytic oxidation system.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the present invention.

The test methods that are not specifically indicated in the following examples can be carried out according to conventional methods, or according to the instructions of the used product manufacturers. The materials, reagents, etc. used in the following examples can be obtained through commercial channels unless otherwise specified.

Example 1: Cloning and sequencing analysis of peroxidoreductase gene.

The Antarctic microorganism Psychrobactor sp. ANT206 was activated in 2216E liquid medium and cultivated to the middle and late logarithmic growth phase (about 4 days), and the total gene DNA of the strain was extracted by combining CTAB method and phenol-chloroform extraction method. PCR was performed using degenerate primers using the extracted total DNA as a template.

Upstream primer: 5'-AMTCAGTGACADTCAG ATGGCGTCT-3'

Downstream primer: 5'-GSAACTGGKGCATATGTTAGATTTT-3'

The amplification conditions were as follows: denaturation at 94 °C for 1 min, annealing at 58.2 °C for 1 min, and extension at 72 °C for 90 s, with 30 cycles. Then, the band containing the target gene Ps Prx was detected by agarose gel electrophoresis and sequenced. Analyzing the sequencing results, a gene with a full-length reading frame of 567 bp was obtained. The nucleotide sequence is shown in SEQ ID No. 1, which encodes a protein composed of 188 amino acids, and the amino acid sequence is shown in SEQ ID No. 2.

Example 2: Expression and purification of peroxidoreductase gene

The primers containing the restriction sites were redesigned according to the determined full-length sequence of Prx.

Upstream primer: 5'-ATA GGATCC ATGGCGTCTATCATCA -3'

Downstream primer: 5'-CGC AAGCTT CGATTTTACCTACTAG -3'

The underlined points are Bam HI and Hin dIII restriction sites, respectively.

The Prx gene and pET-28a(+) double-enzyme digested gel recovery products were ligated by T4 ligase in proportion to construct the recombinant expression vector pET-Prx. The recombinant expression vector was transformed into competent cells E.coli BL21 (DE3), and positive clones were screened and verified by enzyme digestion.

The recombinant strains obtained by screening were induced to express by IPTG. The recombinant bacteria were inoculated into LB medium, cultured at 32-40 °C until OD ₆₀₀ was 0.4-0.8, IPTG was added to the medium to a final concentration of 0.5-1.5 mM, and fermentation was induced at 25-30 °C for 6-10 h. After centrifugation, the cell pellet was collected, ground in an ice bath and sonicated to extract crude protein. The PsPrx recombinant protein was purified by Ni-column affinity chromatography, and analyzed by SDS-PAGE electrophoresis. Finally, a single target band was obtained, and its molecular weight was about 25.2 kDa. The results are shown in Figure 1, M, standard molecular weight protein marker; 1, blank plasmid control; 2, recombinant pET-Prx; 3, PsPrx after purification. The specific embodiment of Ni column affinity chromatography is as follows: after extracting crude protein onto Ni column, use 5-20 mM imidazole to elute 8-10 column volumes, 40-60 mM imidazole to elute 8-10 column volumes, and finally use 5-20 mM imidazole to elute for 8-10 column volumes. 100-500 mM imidazole is eluted for 8-10 column volumes, and the eluate at the peak is collected to obtain PsPrx, which is a suitable cold peroxide reductase after purification.

Example ₃ : FeCl3-catalyzed oxidation of supercoiled DNA protection assay

The 15 μL reaction system contains 15-18 mM FeCl ₃ , 15-18 mM DTT and a certain amount of purified PsPrx in a water bath at 20-30 °C for 2-3 h, followed by adding 750-1250 ng pUC19 supercoiled DNA, 20- Water bath at 30 °C for 2-3 h. After the reaction, the degradation results were detected by agarose gel electrophoresis. The results show that the cold-adapted peroxidoreductase PsPrx can protect supercoiled DNA from damage by metal-catalyzed oxidation systems. The results are shown in Figure 2, 1, pUC19 plasmid; 2, pUC19 plasmid+FeCl; ₃ , pUC19 plasmid+DTT; 4, pUC19 plasmid+FeCl3+DTT+BSA; ₅ , pUC19 plasmid+FeCl3+DTT+PsPrx ( 5 μg/mL); 6, pUC19 plasmid+FeCl ₃ +DTT+PsPrx (10 μg/mL); 7, pUC19 plasmid+FeCl ₃ +DTT+PsPrx (15 μg/mL); 8, pUC19 plasmid+FeCl ₃ + DTT+PsPrx (20 μg/mL); NF, nicked plasmid DNA; SF, supercoiled plasmid DNA. This experiment proves that the cold-adapted peroxide reductase PsPrx has a good ability to protect supercoiled DNA from oxidative damage, and can be used in related fields such as biomedicine, cosmetics and food.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, and combinations made without departing from the spirit and principle of the present invention , simplification, all should be equivalent replacement modes, and are all included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Harbin Institute of Technology (Weihai)

<120> A cold-adapted peroxide reductase and its encoding gene and application

<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 cold-adapting peroxide reductase PsPrx, characterized in that its amino acid sequence is shown in SEQ ID No.2. 2. A gene encoding the cold-adapted peroxide reductase PsPrx of claim 1. 3. The gene encoding cold-adapted peroxide reductase PsPrx according to claim 2, characterized in that its nucleotide sequence is shown in SEQ ID No.1. 4. A recombinant expression vector containing the gene of claim 2 or 3. 5. A genetically engineered bacterium containing the recombinant expression vector of claim 4. 6. The genetically engineered bacterium according to claim 5, wherein the engineered bacterium is Escherichia coli E. coliBL21 (DE3). 7. a method for inducing expression of the cold-adapted peroxide reductase PsPrx according to claim 1, characterized in that, comprising the following steps: The recombinant bacteria transformed into the gene encoding the cold-adapted peroxide reductase PsPrx were cultured at 32-40° C. to an OD600 of 0.4-0.8, and IPTG was added to the medium to a final concentration of 0.5-1.5 mM, 25- The cold-adapted peroxide reductase PsPrx can be induced to express by inducing fermentation at 30 °C for 6-10 h. 8. The application of the cold-adapted peroxide reductase PsPrx of claim 1 in protecting supercoiled DNA from oxidative damage, the application being an application for non-disease diagnosis and treatment purposes.

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