RCAN1.4 promoter fragment for cell hypoxia and HIF1alpha protein activity indicator and application of RCAN1.4 promoter fragment

Abstract

The invention discloses an RCAN1.4 promoter fragment for detecting a cell hypoxia state and HIF1alpha protein activity and application of the RCAN1.4 promoter fragment, and relates to the technical field of gene engineering. According to the invention, a dual-luciferase reporter gene detection system is adopted, an RCAN1.4-315-15 promoter fragment is inserted into a blank vector pGL3-basic with a firefly luciferase reporter gene, a recombinant plasmid RCAN1.4-315-15-LUC for expressing firefly luciferase is constructed, the activation condition of the RCAN1.4-315-15-LUC promoter fragment is judged by detecting the activity of the RCAN1.4-315-15-LUC luciferase, and the activity of the RCAN1.4-315-15-LUC promoter fragment is judged by detecting the activity of the firefly luciferase reporter gene. Further, the hypoxia state of the cells and the activity of the HIF1alpha protein are judged. The RCAN1.4-315-15 promoter fragment provided by the invention can be activated by the HIF1 alpha protein, has high sensitivity to the HIF1 alpha protein, and can be used as a cell hypoxia and HIF1 alpha protein activity indicator.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an RCAN1.4-315-15 promoter fragment used as an indicator of cell hypoxia and HIF1α protein activity and an application thereof. Background technique [0002] The RCAN1 gene is located in the Down Syndrome Critical Region (DSCR), including 7 exons and 6 introns, of which the first 4 exons are selective exons, and different expressions can be Four different protein isoforms are produced, the most important of which are RCAN1.1 encoded by exon 1 and RCAN1.4 encoded by exon 4. The RCAN1 gene has two transcription start sites, which are located in front of exon 1 and exon 4, and the 168 amino acids encoded by exons 5, 6, and 7 are highly conserved regions, which are expressed in all RCAN1 subtypes , and contains a calcineurin-binding domain. Studies have shown that RCAN1 has a dual regulatory effect on the signal transduction pathway of calcineurin, and partic...

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the WB operation steps are relatively cumbersome and complicated. When the cells are lysed, a sufficient amount of protease inhibitors and phosphatase inhibitors that are still within the validity period should be added to the lysate in time to slow down the degradation of the protein. Lysis, centrifugation to immunoprecipitation, etc. need to be kept at 0-4°C throughout the operation, and the reaction takes a long time, usually four hours to overnight incubation, long-term exposure to 4°C even in the presence of protease inhibitors. susceptible to protein degradation

In addition, HIF1α protein is very easy to degrade rapidly under normoxic conditions, and the operation process must be skilled and fast, otherwise the results will be affected

Furthermore, compared with other common protein antibodies of the same specification, HIF1α antibodies currently on the market have disadvantages such as high price, unstable activity, and short storage time, which have a certain impact on the accuracy of the results.

[0005] Generally speaking, this kind of method is time-consuming and laborious to detect, the result is unstable and the detection cost is high

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