Construction method of Grx-roGFP/Gpx3-roGFP gene overexpression MDCK monoclonal cell line

Abstract

The invention discloses a construction method of a Grx-roGFP / Gpx3-roGFP gene overexpression MDCK (Madin Darby Canine Kidney) monoclonal cell line. The construction method comprises the following steps: step 1, firstly, cloning target genes Grx-roGFP and Gpx-roGFP to a lentiviral vector pLV-puro; 2, a lentivirus vector pLV-W is further used for packaging into lentivirus to infect MDCK cells, a W gene overexpression stable cell strain is screened through puromycin, and therefore the cell line capable of dynamically observing the redox dynamic state in kidney cell mitochondria in real time is constructed, and an in-situ model is provided for research on reactive oxygen lesion caused by toxicants, drugs and metabolites in the kidney; according to the present invention, GSH and H2O2 capable of accurately reflecting the redox dynamic state in mitochondria are selected, and glutaredoxin (Grx) capable of specifically detecting GSH is fused with glutathione peroxidase Gpx3 and roGFP capable of specifically detecting H2O2; glutathione and H2O2 in cells can be dynamically detected in real time by observing Grx-roGFP and GPX3-roGFP, so that the change of an oxidation-reduction state in a cell body is speculated, and the pathogenesis of active oxygen is disclosed.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a Grx-roGFP / Gpx3-roGFP gene overexpressed MDCK monoclonal cell line. Background technique [0002] This cell line is a mitochondrial-targeted Grx-roGFP and Gpx3-roGFP Martin Darby dog ​​kidney cell (MDCK) stably transfected cell line. As we all know, reactive oxygen species (Reactive Oxygen Species, ROS) is a kind of oxygen-containing substances with lively chemical properties and strong oxidizing ability, which cause many harms to the body, and the occurrence and development of many diseases are closely related to ROS. Therefore, exploring the changes in ROS levels is of great significance for in-depth exploration of drugs or poisons or their own internal environment. [0003] Mitochondria is an important place for the generation of intracellular reactive oxygen species, and its fine structure and complex functions make it the initial target organelle for ...

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.