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Grx-roGFP Gpx3-roGFP gene overexpression 16HBE monoclonal cell line model

A gene overexpression, 16HBE technology, applied in animal cells, genetic engineering, respiratory/lung cells, etc., can solve the problems of real-time dynamic observation, poor specificity, irreversible cytotoxicity and non-living detection

Pending Publication Date: 2022-05-24
HEBEI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescein probes such as 2',7'-dichlorodihydrofluorescein (2',7'-dihydrodichlorofluore-scein (H2DCF) can produce fluorescence when oxidized by H2O2, but in addition to H2O2, H2DCF can also be oxidized by metal ions, Oxidation by oxidase and cytochrome c, poor specificity
The ESR method requires high sample preparation, and can only measure the average value of sample cell ROS, which is complex and expensive
Other methods such as chemical dye 3,3'-diaminobenzidine (DAB) and nitrotetrazolium blue chloride (NBT) can react with active oxygen to form precipitates, which are Traditional ROS in situ detection method, but this application is affected by factors such as substrate absorption, cell penetration, tissue fixation, etc.
Moreover, both fluorescent reagents and dyes have shortcomings such as cytotoxicity, irreversibility, and non-living detection, and cannot perform real-time dynamic observation of the development of ROS in cells.
[0007] To sum up, there are still many deficiencies and limitations in the current research methods of reactive oxygen species, so it is imperative to develop a method that is efficient, non-toxic and capable of real-time dynamic monitoring of ROS levels

Method used

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Embodiment Construction

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0025] In the embodiment of the present invention, a method for constructing a 16HBE monoclonal cell line model with Grx-roGFP / Gpx3-roGFP gene overexpression, the construction method steps are as follows:

[0026] Step 1: First clone the target genes Grx-roGFP and Gpx-roGFP into the lentiviral vector pLV-puro;

[0027] The specific steps of the first step are as follows:

[0028] S1: First, select the double-enzyme digested gene fragments Grx-roGFP and Gpx-roGF...

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Abstract

The invention discloses a construction method of a Grx-roGFP / Gpx3-roGFP gene overexpression 16HBE monoclonal cell line model. The construction method comprises the following steps: step 1, firstly, cloning target genes Grx-roGFP and Gpx-roGFP to a lentiviral vector pLV-puro; and 2, further packaging the lentivirus by using a lentiviral vector pLV-W, infecting 16HBE cells with the lentivirus, screening Grx-roGFP and Gpx-roGFP gene overexpression stable cell strains through puromycin, thereby constructing a cell line capable of dynamically observing the redox dynamic state in bronchial epithelial cell mitochondria in real time, and specifically detecting glutaredoxin (Glutaredoxin, Gpx-roGFP and Gpx-roGFP) of GSH. Grx is fused with glutathione peroxidase Gpx3 and roGFP for specifically detecting H2O2, and glutathione and H2O2 in cells can be dynamically detected in real time by observing the Grx-roGFP and the GPX3-roGFP, so that the change of the oxidation-reduction state in the cells can be speculated, the pathogenesis of active oxygen is disclosed, and a new foundation is laid for research on bacterial and virus infection and tobacco exposure. And lung lesions caused by dust and smoke, weather and air quality in some workplaces are provided with an in-situ model.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a model of a 16HBE monoclonal cell line overexpressing a Grx-roGFP / Gpx3-roGFP gene. Background technique [0002] This cell line is a mitochondria-targeted Grx-roGFP and Gpx3-roGFP bronchial epithelial cell (16HBE) stably transduced cell line. As we all know, reactive oxygen species (Reactive Oxygen Species, ROS) are a class of oxygen-containing substances with active chemical properties and strong oxidative ability, which cause many harm to the body. The occurrence and development of many diseases are closely related to reactive oxygen species. Therefore, exploring the changes of ROS levels is of great significance for in-depth study of drugs or poisons or their own internal environment. [0003] Mitochondria are important sites for the generation of intracellular reactive oxygen species, and their fine structure and complex functions make them the initial...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66C12N15/65C12N15/62C12N5/10C12R1/91
CPCC12N15/86C12N15/65C12N9/0004C12N9/0065C12N5/0688C12Y120/04001C12Y111/01009C12N2740/15043C12N2800/107C12N2510/00C07K2319/60
Inventor 李筱楠王娜温然
Owner HEBEI MEDICAL UNIVERSITY
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