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Application of heat shock transcription factor 1 in regulating expression of 15kDa selenoprotein

A technology for transcription factor and protein expression, which is applied to the new use of heat shock transcription factor 1, the application field of heat shock transcription factor 1 in regulating the expression of 15kDa selenoprotein

Active Publication Date: 2017-06-13
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no related report that heat shock transcription factor 1 (HSF1) can regulate the expression of 15kDa selenoprotein

Method used

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  • Application of heat shock transcription factor 1 in regulating expression of 15kDa selenoprotein
  • Application of heat shock transcription factor 1 in regulating expression of 15kDa selenoprotein
  • Application of heat shock transcription factor 1 in regulating expression of 15kDa selenoprotein

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Experimental program
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Effect test

Embodiment 1、15

[0071] The cloning of embodiment 1,15kDa selenoprotein gene promoter sequence and the determination of core region

[0072] 1. Extraction of Human Genomic DNA

[0073] Genomic DNA was extracted from HEK293T cells by using a DNA extraction kit, the concentration of DNA detected by a nucleic acid protein detector was 0.450 μg / μl, and the value of A260 / A280 was 1.818, and 0.8% agarose gel electrophoresis was carried out as figure 1 shown. It can be seen from the figure that although a small part of the DNA is degraded, the overall quality is good and can be used for subsequent experiments.

[0074] 2. Obtaining fragments of Sep15 promoter by PCR

[0075] Use specific primers to clone 10 segments of the Sep15 gene promoter respectively. The names, sizes and primers of the cloned segments are shown in Table 8. The corresponding positions of the cloned segments on the promoter are shown in Table 8. figure 2 shown. Using the human genomic DNA obtained in step 1 as a template, PC...

Embodiment 2

[0093] Example 2, the promoting effect of overexpressing HSF1 eukaryotic expression vector on the transcription and protein expression of Sep15

[0094] 1. Detection of Sep15 core promoter activity by overexpressing HSF1 eukaryotic expression vector

[0095] After the HEK293T cells were cultured to 80%-90% density, the cells were counted, and 0.5-2×105 cells were plated in a 24-well plate, and the next day, the recombinant pGL4-818 / -248 and pcDNA3.1-HSF1 were co-transfected into HEK293T cells with the pRL-TK plasmid at a ratio of 1:1 (mass ratio), and pcDNA3. 1 / Myc-His A empty vector was used as the control group. After 24 hours of transfection, the cells were lysed and the dual luciferase activity was detected.

[0096] Wherein, the specific construction process of the pcDNA3.1-HSF1 vector is as follows: using the CDS sequence of HSF1 shown in sequence 3 in the artificially synthesized sequence listing as a template, using primers HSF1-MYC-F and HSF1-MYC-R (see Table 4) PCR ...

Embodiment 3

[0104] Example 3. Effect of heat shock on the transcription and protein level of Sep15

[0105] 1. Heat shock detection of Sep15 core promoter activity

[0106] After the HEK293T cells were cultured to 80%-90% density, the cells were counted, and 0.5-2×10 5 Cells were plated in a 24-well plate, and the next day, the recombinant pGL4-818 / -248 and pRL-TK plasmids were co-transfected into HEK293T cells at a ratio of 19:1 (such as 380ng:20ng) to pGL4.10 [LUC2] The empty vector was used as the control. After 24 hours of transfection, heat shock was performed in a water bath at 43°C for 1 hour, and then recovered at 37°C for 4 hours. After that, the cells were lysed for dual-luciferase detection. box instructions.

[0107] The result is as Figure 13 shown. It can be seen from the figure that after heat shock, compared with cells without heat shock, the core promoter activity of Sep15 was significantly up-regulated, indicating that heat shock can significantly increase the core ...

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Abstract

The invention discloses an application of a heat shock transcription factor 1 in regulating expression of a 15kDa selenoprotein. The application provided by the invention is specifically any one of the applications of the heat shock transcription factor 1: (a1) promoting expression of a Sep15 protein in a target cell; (a2) promoting transcription of a Sep15 gene; and (a3) enhancing the activity of a Sep15 promoter in the target cell. According to the application disclosed by the invention, a transcription factor HSF1 can be combined with the Sep15 promoter to adjust transcription of the Sep15 gene and accelerate expression of the Sep15 protein. Under a heat shock and fever of a physiological status, the transcription factor HSF1 can start transcription of Sep15 and improve expression of Sep 15 so as to participate the physiological process and help the protein being corrected folded or help the protein being unfolded and delivered to other proteins to help the proteins to be folded correctly. Under endoplasmic reticulum stress, the HSF1 also can promote expression of Sep 15 to participate protein quality control of endoplasmic reticulum so as to further promote cell survival. The application disclosed by the invention is of important significance in preventing and / or treating endoplasmic reticulum stress related diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a new application of heat shock transcription factor 1, in particular to an application of heat shock transcription factor 1 in regulating the expression of 15kDa selenoprotein. Background technique [0002] Human heat shock transcription factor 1 (HSF1) is expressed in multiple organs and tissues, and is involved in regulating the heat shock response and inducing the expression of heat shock proteins. It has a variety of biological functions, not only anti-apoptosis, protection of ischemic cardiomyocytes, inhibition of myocardial fibrosis, participation in growth and development, but also anti-inflammatory response, in myocardial cell inflammatory response, lung injury and infection, internal It plays a pivotal role in toxemia, systemic inflammatory response syndrome and other inflammation-related diseases. [0003] Sep15 is a selenoprotein with a molecular size of 15 kDa and a thior...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47A61P25/28A61P25/16A61P21/00
CPCC07K14/4702
Inventor 田静黄燕妹邹琛刘琼倪嘉缵
Owner SHENZHEN UNIV
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