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A kind of site-directed mutagenesis Escherichia coli dna photorepair enzyme and its construction method

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 poor stability and low antioxidant capacity, and achieve stable activity and antioxidant effects Improved effect

Active Publication Date: 2017-06-06
ANHUI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
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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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  • A kind of site-directed mutagenesis Escherichia coli dna photorepair enzyme and its construction method
  • A kind of site-directed mutagenesis Escherichia coli dna photorepair enzyme and its construction method
  • A kind of site-directed mutagenesis Escherichia coli dna photorepair enzyme and its construction method

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Embodiment 1

[0033] 1. Obtain the photorepair enzyme gene phr of WT Escherichia coli

[0034] Primers were designed through the known Escherichia coli (Escherichia coli) photorepair enzyme (cyclobutane pyrimidinedimer photolyase, CPDase, EC4.1.99.3) (WT) gene, and the target fragment was amplified by PCR using E. coli genomic DNA as a template ( Such as figure 1 ). The phr amplification primers and PCR conditions were as follows:

[0035] phrF(NdeI): 5′-CTCCATATGACTACCCATCTGGTCTG-3′

[0036] phrR(XhoI): 5'-GTGCTCGAGTTTCCCCCTTCCGCGCC-3'

[0037] 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.

[0038] 2. Construction of recombinant vector pET22b-phr

[0039] The amplified gene fragment and plasmid pET22b were digested with NdeI and XhoI, ligated at 16°C for 20 hours, and transformed into E.coli DH5α. Positive transformants were screened out to obtain recombinant plasmid pET22b-phr. After do...

Embodiment 2

[0049] Example 2: A377N activity experiment.

[0050] 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.coli CPDase types can be divided into oxidation type, free radical type and reduction type. Among the three types of E.coli CPDase, only the reduced E.coli CPDase has photorepair activity, and the specific absorption wavelength is about 360nm visible light; while the oxidized and free radical types of E.coli CPDase have no photorepair enzyme activity. The oxidized E.coli CPDase 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.coli CPDase specifically absorbs visible light w...

Embodiment 3

[0054] Example 3: Antioxidative ability of A377N.

[0055] In the air, the oxidation process of DNA photorepair enzyme in Escherichia coli is transformed from the free radical type E.coli CPDase to the oxidized E.coli CPDase, that is, the free radical type E.coli CPDase is continuously reduced, and the oxidized E.coli CPDase is continuously increased . The oxidized E.coli CPDase 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.coli CPDase 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 e...

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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

[0001] Technical field: [0002] 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. [0003] Background technique: [0004] 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 mid-band ultraviolet on the induction of keratinocyte apoptosis and calcium signaling [J]. Chinese Pharma...

Claims

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Application Information

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Patent Type & Authority Patents(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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