Escherichia coli DNA photolyase and construction method thereof

A technology of Escherichia coli and photorepair enzyme, which is applied in recombinant DNA technology, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of low antioxidant capacity and poor stability, and achieve stable activity and antioxidant effect. improved effect

Active Publication Date: 2015-11-18
ANHUI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing DNA photorepair enzymes are mainly used in animal models and human experiments, showing that they play an important role in repairing gene damage and preventing the biological effects induced by gene damage, but the stability of existing DNA photorepair enzymes is relatively low. Poor, low antioxidant capacity

Method used

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  • Escherichia coli DNA photolyase and construction method thereof
  • Escherichia coli DNA photolyase and construction method thereof
  • Escherichia coli DNA photolyase and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Obtain the phr of WT E. coli photorepair enzyme gene

[0029] Design primers by known Escherichiacoli (cyclobutanepyrimidinedimer, CPDase, EC4.1.99.3) (WT) gene, and use E.coli genomic DNA as a template to amplify the target fragment (such as figure 1 ). The phr amplification primers and PCR conditions are:

[0030] phrF(NdeI): 5′-CTCCATATGACTACCCATCTGGTCTG-3′

[0031] phrR(XhoI): 5′-GTGCTCGAGTTTCCCCTTCCGCGCC-3′

[0032] Pre-denaturation at 95°C for 5 minutes; 30 cycles at 94°C for 30s, 60°C for 30s, 72°C for 2 minutes; Full extension at 72°C for 10 minutes.

[0033] 2. Construct the recombinant vector pET22b-phr

[0034] The amplified gene fragment and plasmid pET22b were digested with NdeI and XhoI, ligated at 16°C for 20h, and transformed into E.coliDH5α. The positive transformants were screened out and the recombinant plasmid pET22b-phr was obtained. After the double enzyme digestion and identification, it was sent to Nanjing GenScript Biotech Co., Ltd. for sequencing. T...

Embodiment 2

[0044] Example 2: A377S activity test.

[0045] The E. coli DNA photorepair enzyme contains two coenzymes, namely methine tetrahydrofolate (MTHF) and flavin adenine dinucleotide (FAD). MTHF has the effect of enhancing the activity of light repair enzymes. While FAD makes enzymes have photoremediation activity, FAD has three existing forms, oxidation state, free radical state and reduction state. Therefore, the types of E.coliCPDase can be divided into oxidized type, free radical type and reduced type. Among the three types of E. coli CPDase, only the reduced E. coli CPDase has photorepair activity, with a specific absorption wavelength of about 360 nm of visible light; while the oxidized and free radical types of E. coli CPDase have no photorepair enzyme activity. The oxidized E.coliCPDase specifically absorbs visible light with a wavelength of about 450nm, and does not absorb visible light with a wavelength above 550nm. The free radical E.coliCPDase specifically absorbs visib...

Embodiment 3

[0049] Example 3: Antioxidant ability of A377S.

[0050] In the air, the oxidation process of E. coli DNA photorepair enzyme changes from free radical E.coliCPDase to oxidized E.coliCPDase, that is, the free radical E.coliCPDase keeps decreasing, and the oxidized E.coliCPDase keeps increasing. The oxidized E.coliCPDase specifically absorbs visible light with a wavelength of about 450nm, and does not absorb visible light with a wavelength above 550nm. The free radical E.coliCPDase specifically absorbs visible light with a wavelength of about 580nm. Therefore, the photoremediation enzyme oxidation process includes two features. One feature is that the absorbance of visible light with a wavelength of about 580nm gradually decreases until it stabilizes; the other feature is that the absorbance of visible light with a wavelength of about 450nm gradually rises until it stabilizes. Store the purified DNA photorepair enzyme at 4°C. At regular intervals, draw 800μl of purified enzyme so...

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Abstract

The invention discloses escherichia coli DNA photolyase and a construction method thereof. The enzyme has the amino acid sequence shown in SEQ ID NO: 1 (in the description). The construction method comprises the following steps: obtaining a DNA photolyase gene from the conventional WT escherichia coli; designing a mutation primer to obtain a mutational gene segment; connecting the mutational gene segment with pET22b plasmid; constructing recombinant expression plasmid pET22b-A377S; converting the recombinant expression plasmid into a competent cell of escherichia coli; constructing genetically engineered bacterium BL21 (DE3) / pET22b-A377S for mutant enzyme expression. According to the invention, better expression is achieved under the conditions of 18 DEG C and 1 mM ITPG; the expressed photolyase is more stable in activity, and the antioxidant effect of the enzyme is remarkably improved.

Description

Technical field: [0001] The invention relates to an Escherichia coli light repair enzyme and a construction method thereof, in particular to an Escherichia coli DNA light repair enzyme and a construction method thereof. Background technique: [0002] Due to the pollution and destruction caused by human activities, the ozone layer in the atmosphere has become thinner, and the intensity of ultraviolet light in the sun has increased, causing more and more damage to humans, especially the DNA inside cells. Ultraviolet light destroys the structure of DNA, prevents normal replication and transcription of DNA, and causes changes in signal pathways in skin and tissue cells, resulting in immune suppression, skin inflammation, accumulation of large amounts of CPD in cells and even skin cancer ( Li Peibo, Guan Yongyuan, He Hua, Qiu Qinying et al. The effect of mid-wavelength ultraviolet on the induction of keratinocyte apoptosis and calcium signal[J]. Chinese Pharmacological Bulletin, 2004,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21
CPCC12N9/88C12Y401/99013
Inventor 徐蕾文斌王鹏朱国萍吕培培曹正宇黄士平
Owner ANHUI NORMAL UNIV
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