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Cold-adapted peroxiredoxin as well as coding gene and application thereof

A technology of peroxide and reductase, which is applied in the fields of cosmetics, food, biotechnology and medicine, and can solve problems such as poor stability

Active Publication Date: 2020-03-10
HARBIN INST OF TECH AT WEIHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Peroxide reductase extracted from animals and plants is easily restricted by season, climate and region, and has poor stability, so it is difficult to be directly applied to related fields such as industrial production

Method used

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  • Cold-adapted peroxiredoxin as well as coding gene and application thereof
  • Cold-adapted peroxiredoxin as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Cloning and sequencing analysis of peroxide reductase gene.

[0021] Antarctic microbes Psychrobactor sp.ANT206 was activated in 2216E liquid medium, cultured to the middle and late logarithmic growth phase (about 4d), and the total gene DNA of the strain was extracted by combining CTAB method and phenol-chloroform extraction method. Using the extracted total DNA as a template, PCR was performed using degenerate primers.

[0022] Upstream primer: 5’- AMTCAGTGACADTCAG ATGGCGTCT -3’

[0023] Downstream primer: 5'- GSAACTGGKGCATATGTTAGATTTT -3'

[0024] The amplification conditions were: denaturation at 94°C for 1 min, annealing at 58.2°C for 1 min, extension at 72°C for 90 s, and 30 cycles. Then detect the gene of interest by agarose gel electrophoresis PS The band of Prx was sequenced. The sequencing results were analyzed to obtain a gene with a complete reading frame sequence of 567 bp in full length. The nucleotide sequence is shown in SEQIDNo.1, which...

Embodiment 2

[0025] Embodiment 2: Expression and purification of peroxide reductase gene

[0026] According to the determined full-length sequence of Prx, primers containing restriction sites were redesigned.

[0027] Upstream primer: 5'-ATA GGATCC ATGGCGTCTATCATCA -3'

[0028] Downstream primer: 5'-CGC AAGCTT CGATTTTACCTACTAG -3'

[0029] The lines are Bam Hi, Hin dIII restriction site.

[0030] The Prx gene and pET-28a (+) double-enzyme-digested gel recovery product were connected in proportion using T4 ligase to construct the recombinant expression vector pET-Prx. Transform recombinant expression vectors into competent cells E. coli In BL21 (DE3), positive clone screening and enzyme digestion verification were carried out.

[0031] The recombinant strain obtained by screening was induced to express by IPTG. Inoculate the recombinant bacteria into LB medium and cultivate to OD at 32-40°C 600 0.4-0.8, add IPTG to the medium to a final concentration of 0.5-1.5 mM, and induc...

Embodiment 3

[0032] Example 3: FeCl 3 Catalytic Oxidative Supercoiled DNA Protection Assay

[0033] 15 μL reaction contains 15-18 mM FeCl 3 , 15-18 mM DTT and a certain amount of purified PsPrx were placed in a water bath at 20-30 °C for 2-3 h, then 750-1250 ng of pUC19 supercoiled DNA was added, and placed in a water bath at 20-30 °C for 2-3 h. After the reaction, the degradation results were detected by agarose gel electrophoresis. The results showed that the cold-adapted peroxide reductase PsPrx can protect supercoiled DNA from metal-catalyzed oxidation system damage. The result is as figure 2 As shown, 1, pUC19 plasmid; 2, pUC19 plasmid+FeCl; 3, pUC19 plasmid+DTT; 4, pUC19 plasmid+FeCl 3 +DTT+BSA; 5, pUC19 plasmid+FeCl 3 +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-adapte...

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Abstract

The invention discloses a cold-adapted peroxidase as well as a coding gene and application thereof, and aims to provide the cold-adapted peroxiredoxin as well as the coding gene and application thereof. According to the invention, peroxiredoxin gene PsPrx is firstly cloned from Antarctic sea ice microorganism psychrobacter sp., and the amino acid residue sequence of the peroxiredoxin is shown in SEQ ID No.2. An expression method of the peroxiredoxin comprises the steps of constructing a recombinant expression vector containing the peroxiredoxin gene PsPrx, introducing the constructed recombinant expression vector into a host cell escherichia coli, and performing induction to realize the peroxiredoxin gene PsPrx gene expression. The expression product PsPrx provided by the invention has outstanding cold adaptability, better stability, and good ability to protect supercoiled DNA against oxidative damage, and can be applied to the related fields such as biomedicine, cosmetics and food.

Description

technical field [0001] The invention belongs to the fields of biotechnology, medicine, cosmetics, food and the like, and specifically relates to a cold-adapted peroxide reductase and its coding gene and application. Background technique [0002] Peroxiredoxin (Prx) is an important class of thiol-dependent antioxidant enzymes widely present in eukaryotes and prokaryotes, which can efficiently degrade H 2 o 2 , peroxynitrate and various organic peroxides. Prx in microorganisms is mainly divided into three types, namely 1-Cys Prx, typical 2-Cys Prx and atypical 2-Cys Prx. Typical 2-Cys Prx has been shown to be related to the oxidative signaling mechanism controlling apoptosis, differentiation and proliferation, and may be involved in the immune response, which can be used as a biodiagnostic marker for tumors and cancers. 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, the typical 2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0065C12Y111/01015
Inventor 侯艳华王全富王一帆王亚彤徐一凤
Owner HARBIN INST OF TECH AT WEIHAI
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