Biological probe for detecting activity of RhoGDIalpha (Rho GDP dissociation inhibitor alpha) protein in living cell

Abstract

The invention belongs to the technical fields of cell biology and molecular biology, and relates to a biological probe designed and prepared according to a basic principle of FRET (fluorescence resonance energy transfer) and capable of detecting the activity of a RhoGDIalpha (Rho GDP dissociation inhibitor alpha) protein in a living cell. The probe is a fusion protein structure, and comprises five parts, i.e. an FRET fluorescent protein pair ECFP and Ypet, a RhoGDIalpha full-length sequence, a substrate structure domain switch II of RhoGDIalpha and an intermediate linker sequence. According to the biological probe, the fusion protein structure of the probe can be independently expressed by a cell after a fusion plasmid is transferred into the living cell, so that a probe function is realized in the cell; the biological probe has the characteristics of being high in specificity, low in cost, free of side effects on the cell and the like.

Description

technical field [0001] The invention belongs to the technical field of cell biology and molecular biology, is designed and prepared based on the principle of fluorescence resonance energy transfer (FRET) and subcloning technology, and detects the activity of RhoGDIα (RhoGDPdissociationinhibitorα) protein in living cells through the change of color and intensity of fluorescent protein Varying biological probes. Background technique [0002] RhoGDIα is a negative regulatory protein of Rho family members, which can participate in the process of cell migration by inhibiting the activity of different members of the Rho family, and plays a vital role in the invasion of cancer cells. At present, methods such as immunohistochemical reaction based on protein antigen-antibody binding, Western Blotting detection, or in situ PCR reaction based on nucleic acid level are generally used to detect intracellular RhoGDIα protein. These methods have certain defects: 1. Cells must be broken or ...

AI Technical Summary

Problems solved by technology

At present, methods such as immunohistochemical reaction based on protein antigen-antibody binding, Western Blotting detection, or in situ PCR reaction based on nucleic acid level are generally used to detect intracellular RhoGDIα protein. These methods have certain defects: 1. Cells must be broken or Pretreatment such as fixation destroys the normal physiological state of cells; 2. It cannot be dynamically observed at the single cell or subcellular level; 3. It can only detect the amount of protein, but cannot detect the activity of protein; 4. High cost

Therefore, there is currently no effective method to detect RhoGDIα activity in living cells

Images