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

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...

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 si