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Recombinant plasmid for screening nrf2 activator and its construction method and use

A recombinant plasmid and activator technology, 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: 2020-01-17
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-term (2) The detection sensitivity is poor and needs to be further improved; (3) The above-mentioned reporter gene adopts transient transfection, and the transfection efficiency of the most commonly used HaCaT cells is very low (transfection efficiency is about 20%), resulting in inconsistent results. Stablize

Method used

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  • Recombinant plasmid for screening nrf2 activator and its construction method and use
  • Recombinant plasmid for screening nrf2 activator and its construction method and use
  • Recombinant plasmid for screening nrf2 activator and its construction method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 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 Cell Research, Chinese Academy of Sciences, Shanghai).

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

[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: ...

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 present invention relates to the field of biomedicine, in particular to a recombinant plasmid for screening Nrf2 activators and its construction method and application, wherein the recombinant plasmid comprises one or more than two antioxidant response elements connected in series, basic transcription elements, secreted Luciferase gene, secreted alkaline phosphatase gene. Compared with the prior art, the present invention uses secreted luciferase as a reporter gene, which has high detection sensitivity and does not need to lyse cells when detecting luciferase gene activity, thus realizing high-throughput screening and comparison of antioxidants, as well as long-term effects The detection of antioxidants provides an efficient, fast and simple method for screening antioxidants or detecting antioxidant activity, which can be applied to the screening of natural medicines and synthetic compounds with antioxidant effects.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant plasmid used for screening Nrf2 activators and a construction method and application thereof. Background technique [0002] The human body will produce reactive oxygen species (ROS) when exposed to various environmental factors, such as ultraviolet radiation. As a second messenger, ROS induce inflammatory response, cell membrane lipid peroxidation, protein and DNA oxidative damage, directly or indirectly destroy the physiological functions of intracellular proteins, lipids, nucleic acids and other macromolecular substances, and then induce cellular oxidative stress. damage or apoptosis, which further leads to the formation of tumors, etc. 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 stress pathway. It can initiate ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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