Site-directed mutagenesis escherichia coli DNA photolyase and construction method thereof

A technology of Escherichia coli and photorepair enzymes, applied in recombinant DNA technology, botany 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 effects Improved effect

Active Publication Date: 2015-11-25
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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  • Site-directed mutagenesis escherichia coli DNA photolyase and construction method thereof
  • Site-directed mutagenesis escherichia coli DNA photolyase and construction method thereof
  • Site-directed mutagenesis escherichia coli DNA photolyase and construction method thereof

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Embodiment 1

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

[0029] Primers were designed through the known Escherichia coli (Escherichia coli) photorepair enzyme (cyclobutanepyrimidinedimerphotolyase, CPDase, EC4.1.99.3) (WT) gene, using E.coli genomic DNA as a template, the target fragment was amplified by PCR (such as figure 1 ). The phr amplification primers and PCR conditions were as follows:

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

[0031] phrR(XhoI): 5'-GTGCTCGAGTTTCCCCCTTCCGCGCC-3'

[0032] Pre-denature at 95°C for 5 minutes; cycle 30 times at 94°C for 30s, 60°C for 30s, and 72°C for 2 minutes; fully extend at 72°C for 10 minutes.

[0033] 2. Construction of 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. Positive transformants were screened out to obtain recombinant plasmid pET22b-phr. After double-enzyme di...

Embodiment 2

[0044] Example 2: A377N activity experiment.

[0045] Escherichia coli DNA photorepair enzyme contains two coenzymes, which are methenyltetrahydrofolate (MTHF) and flavin adenine dinucleotide (FAD). MTHF can enhance the activity of photorepair enzymes. While FAD enables the enzyme to have photorepair activity, there are three forms of FAD, oxidized state, free radical state and reduced state. So E.coliCPDase type can be divided into oxidation type, free radical type and reduction type. Among the three types of E.coliCPDase, only the reduced E.coliCPDase has photorepair activity, and the specific absorption wavelength is about 360nm visible light; while the oxidized and free radical types of E.coliCPDase 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 type E.coliCPDase specifically absorbs visible light with a w...

Embodiment 3

[0049] Example 3: Antioxidative ability of A377N.

[0050] In the air, the oxidation process of Escherichia coli DNA photorepair enzyme changed from free radical E.coliCPDase to oxidized E.coliCPDase, that is, the free radical E.coliCPDase decreased continuously, while the oxidized E.coliCPDase increased continuously. 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 type E.coliCPDase specifically absorbs visible light with a wavelength of about 580nm. Therefore, the photorepair enzyme oxidation process includes two characteristics, one is that the absorbance of visible light with a wavelength of about 580nm gradually decreases until it becomes stable; the other is that the absorbance of visible light with a wavelength of about 450nm gradually increases until it becomes stable. Store the purified DNA photorepair enzyme at 4°C. At regular intervals, d...

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Abstract

The invention discloses a site-directed mutagenesis escherichia coli DNA photolyase and a construction method thereof. The photolyase has an amino acid sequence shown by SEQ ID NO:1. The method comprises the following steps: obtaining a DNA photolyase gene from the existing WT escherichia coli, designing a mutation primer to obtain a mutated gene fragment, and connecting the mutated gene fragment with a pET22b plasmid to construct a recombinant expression plasmid pET22b-A377N; and converting the recombinant expression plasmid into an escherichia coli competent cell to construct a genetic engineering bacterium BL21(DE3)/pET22b-A377N for mutant enzyme expression. The site-directed mutagenesis escherichia coli DNA photolyase obtains better expression under a 1mM ITPG condition at 18 DEG C. The expressed photolyase is more stable in activity, and the antioxidation effect of the photolyase is significantly improved.

Description

Technical field: [0001] The invention relates to an Escherichia coli photorepair enzyme and a construction method thereof, in particular to an Escherichia coli DNA photorepair 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 the normal replication and transcription of DNA, and causes changes in the signaling pathways in skin and tissue cells, resulting in immune suppression, skin inflammation, accumulation of a large amount of CPD in cells and even skin cancer ( Li Peibo, Guan Yongyuan, He Hua, Qiu Qinying et al. Effects of medium-band ultraviolet on the induction of keratinocyte apoptosis and calcium signaling [J]. Chinese Pharmacology Bulle...

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 文斌徐蕾王鹏朱国萍田常青曹正宇黄士平
Owner ANHUI NORMAL UNIV
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