Recombinant plasmid for screening Nrf2 activating agent and construction method and application thereof

A technology of recombinant plasmids and activators, applied in the field of biomedicine, can solve the problems of difficult long-term detection, complicated operation steps, unstable results, etc., and achieve the effects of long-term activity monitoring, simplified operation steps, and convenient and fast detection

Active Publication Date: 2017-01-04
广州市皮肤病防治所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still a series of problems in these reporter systems: (1) Various enzymes such as firefly luciferase are used as reporter genes. Subsequent detection of their activity requires lysed cells to be detected. The operation steps are complicated and inconvenient, and it is difficult to achieve long

Method used

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  • Recombinant plasmid for screening Nrf2 activating agent and construction method and application thereof
  • Recombinant plasmid for screening Nrf2 activating agent and construction method and application thereof
  • Recombinant plasmid for screening Nrf2 activating agent and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Construction method of recombinant plasmid

[0037] (1) Experimental materials

[0038] Plasmids, strains and cells HaCaT cells, secreted luciferase vector, Escherichia coli plasmid (purchased from Institute of Cells, Chinese Academy of Sciences, Shanghai).

[0039] Reagents: Secrete-Pair Dual Luminescence Assay Kit (purchased from Genecopoeai); lipofec3000 liposomes (purchased from Invitrogen).

[0040] Equipment: Infinite F200Pro multifunctional microplate reader (purchased from TECAN Company), UV lamp tube (purchased from Philips Company).

[0041] (2) Experimental steps

[0042]In this example, the secreted luciferase (Gaussia Luciferase, Gluc) derived from Gaussia princeps was used as the reporter gene, and the Nrf2 with easy detection, high sensitivity and high stability was constructed by screening the antioxidant response element (ARE). The reporting system for signaling pathway activation, the specific steps are as follows:

[0043] (1) The cD...

Embodiment 2

[0058] Embodiment 2: Identify the reactivity of the recombinant plasmid of embodiment 1 in cells with known Nrf2 activators

[0059] (1) HaCaT cells were cultured in DMEM medium supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin and 10% fetal bovine serum at 37°C in an atmosphere containing 5% CO2, using Life Technologies (Carlsbad, CA, USA) Lipofectamine 2000 reagent purchased was used for the recombinant plasmid transfected cells prepared in Example 1 according to the recommended process;

[0060] (2) After 24 hours of transfection, replace the fresh medium containing SF (10uM) and tBHQ (20uM), and continue to cultivate for 24 hours;

[0061] (3) Take 100 ul of the medium supernatant for luciferase activity detection. The assay of each reporter activity used the Secrete-Pair Dual Luminescence Assay Kit of Genecopoeai Company and adopted the Infinite F200Pro multifunctional microplate reader of TECAN Company, and determined luciferase (Gluc) and alkaline phosphata...

Embodiment 3

[0063] Embodiment 3: Use the recombinant plasmid of embodiment 1 to detect the reactivity of known Nrf2 activators with different action times and the application of screening antioxidants

[0064] 1. Recombinant plasmids are used to detect the reactivity of known Nrf2 activators with different action times

[0065] (1) HaCaT cells were cultured in DMEM medium supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin and 10% fetal bovine serum at 37°C in an atmosphere containing 5% CO2, using Life Technologies The Lipofectamine 2000 reagent purchased by the company (Carlsbad, CA, USA) was used in the p8XARE-ML-Gluc-AP-Puro plasmid transfected cells of Example 1 according to the recommended procedure;

[0066] (2) After 24 hours of transfection, fresh medium containing SF (10uM) and tBHQ (20uM) was replaced, and then 100ul medium supernatant was taken at 0, 6, and 24h for the detection of luciferase (Gluc) activity.

[0067] (3) The determination of the reactivity of known...

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Abstract

The invention relates to the field of biological medicine, in particular to recombinant plasmid for screening an Nrf2 activating agent and a construction method and application thereof. The recombinant plasmid comprises one or two or above series antioxidant reaction elements, basal transcription elements, secretion-type luciferase genes and secretion-type alkaline phosphatase genes. Compared with the prior art, as secretion-type luciferase is used as reporter genes, the detection sensitivity is high, cells do not need to be pyrolysed when the activity of the luciferase genes is detected, high-throughput screening and comparing and detection of the long-term effect of an antioxidant are achieved, and an efficient, rapid, simple and convenient method is provided for screening the antioxidant or detecting the activity of the antioxidant, and the recombinant plasmid can be applied to screening of natural medicine with the antioxidation effect and artificially-synthesized compounds.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant plasmid for screening Nrf2 activators and its construction method and application. Background technique [0002] The human body will produce reactive oxygen species (reactive oxygen species, ROS) when it is exposed to various environmental factors such as ultraviolet radiation. As a second messenger, ROS induces inflammatory response, cell membrane lipid peroxidation, protein and DNA oxidative damage, and directly or indirectly destroys the physiological functions of intracellular proteins, lipids, nucleic acids and other macromolecular substances, thereby inducing cellular oxidative stress. damage or apoptosis, further leading to the formation of tumors and the like. When exposed to ROS, the body itself can induce a series of protective proteins, among which the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is the most important anti-oxidative str...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12Q1/66C12Q1/02
CPCC12N15/66C12N15/85C12N2800/107C12Q1/02C12Q1/66
Inventor 朱慧兰李华平
Owner 广州市皮肤病防治所
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