CN106831975B - Application of heat shock transcription factor 1 in regulation and control of expression of 15kDa selenoprotein - Google Patents

Abstract

The invention discloses application of a heat shock transcription factor 1 in regulation and control of expression of 15kDa selenoprotein. The application provided by the invention is specifically the application of the heat shock transcription factor 1 in any one of the following applications: (a1) promoting expression of Sep15 protein in target cells; (a2) promoting transcription of the Sep15 gene in target cells; (a3) the Sep15 promoter activity was enhanced in the target cells. The invention finds that the transcription factor HSF1 can be combined with a Sep15 promoter, regulates the Sep15 gene transcription and promotes the Sep15 protein expression. In the case of heat shock and fever in physiological state, the transcription factor HSF1 can start Sep15 transcription, increase Sep15 expression, thereby participating in the physiological process, helping the protein fold correctly or helping the protein unfold and deliver to other proteins to help the protein fold correctly. Under endoplasmic reticulum stress, HSF1 can also promote Sep15 expression, thereby participating in endoplasmic reticulum protein control and further promoting cell survival. The invention has important significance for preventing and/or treating diseases related to endoplasmic reticulum stress.

Description

Application of heat shock transcription factor 1 in regulation and control of expression of 15kDa selenoprotein

Technical Field

The invention belongs to the technical field of biology, relates to a new application of a heat shock transcription factor 1, and particularly relates to an application of the heat shock transcription factor 1 in regulation and control of expression of 15kDa selenoprotein.

Background

Human heat shock transcription factor 1(HSF1) is expressed in multiple organ tissues and is involved in regulating the heat shock response and inducing the expression of heat shock proteins. It has multiple biological functions, not only can resist apoptosis, protect ischemic myocardial cells, inhibit myocardial fibrosis, participate in growth and development processes, but also can resist inflammatory reaction, and plays a significant role in myocardial cell inflammatory reaction, lung injury and infection, endotoxemia, systemic inflammatory response syndrome and other inflammation-related diseases.

Sep15 is a selenoprotein with a molecular size of 15kDa with a thioredoxin domain at its N-terminus, which predicts that it is a disulfide reductase or disulfide isomerase with redox functions, localized in the lumen of the endoplasmic reticulum in association with glucosyl glycoprotein (UGGT), and involved in endoplasmic reticulum protein mass control.

In many reports, there are many diseases caused by accumulation of misfolded proteins, such as alzheimer's disease, parkinson's disease, and amyotrophic lateral sclerosis, and one of the main causes of disease induction is severe endoplasmic reticulum stress in vivo, which indicates that Sep15 also plays an important role in these diseases, and inducing Sep15 expression may alleviate the progression of these diseases.

At present, no related report that the heat shock transcription factor 1(HSF1) can regulate the expression of the 15kDa selenoprotein exists.

Disclosure of Invention

The invention aims to provide a new application of heat shock transcription factor 1.

The new application of the heat shock transcription factor 1 provided by the invention is specifically the application of the heat shock transcription factor 1 in any one of the following applications:

(a1) promoting expression of Sep15 protein in target cells;

(a2) promoting transcription of the Sep15 gene in target cells;

(a3) enhancing the activity of the Sep15 promoter in the target cell;

(a4) preparing a product having a function of promoting the expression of Sep15 protein in a target cell;

(a5) preparing a product having a function of promoting the transcription of the Sep15 gene in the target cell;

(a6) a product having enhanced Sep15 promoter activity in target cells was prepared.

Of course, the use of a substance capable of promoting the expression of heat shock transcription factor 1 in any of the above (a1) - (a6) is also within the scope of the present invention.

The invention also provides a method for promoting Sep15 protein expression and/or promoting Sep15 gene transcription and/or enhancing Sep15 promoter activity in target cells.

The method for promoting Sep15 protein expression and/or promoting Sep15 gene transcription and/or enhancing Sep15 promoter activity in target cells provided by the invention specifically comprises at least one of the following steps (a) - (c):

(a) increasing the expression level of heat shock transcription factor 1 in the target cell;

(b) heat shock treatment is carried out on the target cells;

(c) and (3) carrying out tunicamycin treatment on the target cells.

In the step (b), the heat shock treatment of the target cells may be performed in a 42-43 ℃ water bath for 1-2h, for example, in a 43 ℃ water bath for 1 h.

In the step (c), the tunicamycin treatment on the target cells may specifically be that the target cells are placed in a tunicamycin solution with a concentration of 100ng-5 μ g/ml (specifically, 100ng/ml or 5 μ g/ml) for 12-24 h.

In the present invention, the GenBank accession number of the amino acid sequence of the Sep15 protein is NP-004252.2 or NP-976086.1 (version 01-DEC-2016). Correspondingly, the Sep15 gene is a gene encoding the Sep15 protein. Further, the nucleotide sequence of the Sep15 gene has GenBank accession No. NM-004261.4 or NM-203341.2 (version 01-DEC-2016).

The Sep15 promoter is the following DNA molecule: at least contains the 1238-1808 th nucleotide sequence of the sequence 1 in the sequence table, and extends from the 1238 th nucleotide sequence of the sequence 1 to the 5' end of the sequence 1 according to the nucleotide sequence of the sequence 1 to obtain any DNA fragment with the length of 571bp to 1808 bp; the DNA molecule has a promoter function.

More specifically, in the invention, the Sep15 promoter is any one of the following a1) -a 4):

a1) the nucleotide sequence is a DNA molecule shown by the 1238-1808 th nucleotide of the sequence 1 in the sequence table;

a2) the nucleotide sequence is a DNA molecule shown by the 626-1265 th nucleotide of the sequence 1 in the sequence table;

a3) the nucleotide sequence is a DNA molecule shown as the 626-1808 th nucleotide of the sequence 1 in the sequence table;

a4) the nucleotide sequence is a DNA molecule shown by the 1 st-1808 th nucleotide of the sequence 1 in the sequence table.

In the use or method, the target cell is an animal cell. In the present example, the target cells are specifically HEK293T cells or Neuro 2a cells.

In the present invention, the application or method may be an application or method for non-disease diagnosis and treatment, and may also be an application or method for disease diagnosis and treatment.

The invention finds that the transcription factor HSF1 can be combined with a Sep15 promoter, regulates the Sep15 gene transcription and promotes the Sep15 protein expression. In the case of heat shock and fever in physiological state, the transcription factor HSF1 can start Sep15 transcription, increase Sep15 expression, thereby participating in the physiological process, helping the protein fold correctly or helping the protein unfold and deliver to other proteins to help the protein fold correctly. Under endoplasmic reticulum stress, HSF1 can also promote Sep15 expression, thereby participating in endoplasmic reticulum protein control and further promoting cell survival. The invention has important significance for preventing and/or treating diseases related to endoplasmic reticulum stress. Wherein the diseases related to endoplasmic reticulum stress include Alzheimer disease, Parkinson disease, and muscular dystrophy and lateral sclerosis.

Drawings

FIG. 1 shows the gel electrophoresis of the HEK293T genome DNA extraction.

FIG. 2 is a schematic representation of the corresponding positions of the individual segments on the Sep15 promoter.

FIG. 3 is an agarose gel electrophoresis of human Sep15 promoter region amplified by PCR using human genomic DNA as a template. M: molecular weight standard (DL2000 Marker); pGL4-2055/-248PCR fragment 1; 2 pGL4-2055/-791PCR fragment, 3 pGL4-1430/-248PCR fragment; pGL4-1430/-791PCR fragment 4; pGL4-646/-402PCR fragment 5; pGL4-2055/-1225PCR fragment 6; pGL4-818/-248PCR fragment 7; pGL4-818/-166PCR fragment 8; pGL4-272/+909PCR fragment 9.

FIG. 4 is an agarose gel electrophoresis of PCR products of recombinant plasmid bacterial liquid in the Sep15 promoter region. M: molecular weight standard (DL2000 Marker); 1-4 is pGL4-2055/-248 bacterial liquid PCR band; 5-12 is pGL4-2055/-791 bacterial liquid PCR band, and 13-16 is pGL4-1430/-248 bacterial liquid PCR band; 17-20 is pGL4-272/+909PCR band.

FIG. 5 is an agarose gel electrophoresis of PCR products of recombinant plasmid bacterial liquid in the Sep15 promoter region. M: molecular weight standard (DL2000 Marker); 1-4 is pGL4-818/-248 bacterial liquid PCR band; 5-8 is pGL4-818/-166 bacteria liquid PCR band, 9-11 is pGL4-1430/-791 bacteria liquid PCR band; 12-14 is pGL4-646/-402 bacterial liquid PCR band.

FIG. 6 is an agarose gel electrophoresis of PCR products of recombinant plasmid bacterial liquid in the Sep15 promoter region. M: molecular weight standard (DL2000 Marker); 1-3 is pGL4-2055/-1225 bacterial solution PCR band.

FIG. 7 is the agarose gel electrophoresis of the Sep15 promoter region recombinant plasmid restriction identification. M: molecular weight standard (DL2000 Marker); 1 is pGL4-2055/-248 enzyme cutting product; 2 is pGL4-2055/-791 enzyme-digested product, and 3 is pGL4-1430/-248 enzyme-digested product; 4 is pGL4-1430/-791 enzyme digestion product; 5 is pGL4-646/-402 enzyme cutting product; pGL4-2055/-1225 enzyme digestion product 6; 7 is pGL4-818/-248 enzyme cutting product; 8 is pGL4-818/-166 enzyme cutting product; the 9 is pGL4-272/+909 cleavage product.

FIG. 8 shows the luciferase activity assay of the Sep15 promoter fragments transfected into HEK293T cells with pGL4.10[ LUC2] recombinant. And (4) testing n and t. P < 0.01; p < 0.001.

FIG. 9 shows the luciferase activity assay of the Sep15 promoter fragments after transfection of N2a cells with pGL4.10[ LUC2] recombinant. And (4) testing n and t. P < 0.01; p < 0.001.

FIG. 10 shows the luciferase activity assay after pGL4-818/-248 co-transfected with pcDNA3.1-HSF1 HEK293T cells. -, none; +, and add. And (4) testing n and t. P < 0.001.

FIG. 11 shows the RT-PCR detection of Sep15 mRNA from HEK293T cells overexpressing HSF 1. -, none; +, and add. And (4) testing n and t. P < 0.001.

FIG. 12 shows the protein level of Sep15 detected by the eukaryotic expression vector of over-expressed HSF 1. Wherein, A is the protein level of detecting Sep15 by over-expressing HSF1 eukaryotic expression vector WB; b is Sep15 protein gray value quantitative analysis. -, none; +, and add. And (5) testing n-3. P < 0.01.

FIG. 13 shows the core promoter activity of Sep15 detected by dual luciferases after heat shock. -, none; +, and add. And (4) testing n and t. P < 0.001.

FIG. 14 shows mRNA levels of Sep15 and Hsp70 detected by RT-PCR after heat shock of HEK293T cells. Wherein, A is the mRNA level of Sep 15; b is the mRNA level of Hsp 70. And (5) testing n-3. P < 0.05; p < 0.01.

FIG. 15 shows Sep15 protein levels detected after heat shock or after HSF1 overexpression and heat shock. Wherein, A is Sep15, HSF1, tag protein MYC, GAPDH protein band; b is Sep15 protein gray value quantitative analysis. -, none; +, and add. And (5) testing n-3. P < 0.05.

FIG. 16 shows the Sep15 core promoter activity detected by over-expression of HSF1 and over-expression of HSF1 and Tm treatment. -, none; +, and add. And (4) testing n and t. P < 0.05; p < 0.01.

FIG. 17 shows mRNA from Sep15 detected by RT-PCR with Tm time gradient processing. And (4) testing n and t. P < 0.001.

FIG. 18 shows the detection of Sep15 protein levels after Tm treatment. Wherein, A is a protein electrophoresis strip of Sep 15; b is Sep15 protein gray value quantitative analysis.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

TABLE 1 Escherichia coli strains used in the following examples

TABLE 2 vector plasmids used in the examples described below

TABLE 3 cell lines used in the following examples

TABLE 4 primers used in the following examples

Bold for protecting bases, italicized underline indicates the region of the cleavage site, and the above primers were synthesized in Shanghai Saiban Bio Inc.

TABLE 5 Primary reagents and kits

TABLE 6 Main solution formulation

TABLE 7 Experimental apparatus

Example 1 cloning of the promoter sequence of the 15kDa selenoprotein Gene and determination of the core region

Extraction of human genome DNA

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. mu.g/. mu.l, the value of A260/A280 was 1.818, and 0.8% agarose gel electrophoresis was performed as shown in FIG. 1. As can be seen, the DNA was degraded in small part, but the overall quality was good and could be used for subsequent experiments.

Secondly, obtaining Sep15 promoter fragments by PCR

The specific primers were used to clone 10 segments of the Sep15 gene promoter, the name and size of the cloned segment and the primers used are shown in Table 8, and the corresponding positions of the cloned segments on the promoter are shown in FIG. 2. And (3) carrying out PCR reaction by using the human genome DNA obtained in the first step as a template, and carrying out 1% agarose gel electrophoresis analysis on the product, wherein the result is shown in figure 3.

TABLE 8 cloning of 10 segments of Sep15 Gene promoter, cloning segment names, sizes and primers used

Note: the specific sequences of the forward and reverse primers are detailed in Table 4.

Third, Sep15 promoter each fragment enzyme digestion

And (3) carrying out enzyme digestion on the pGL4.10[ LUC2] vector and each target fragment obtained in the step two for 3h at 37 ℃ by using corresponding restriction enzymes, wherein the linear vector after enzyme digestion migrates slower than the ring vector without enzyme digestion, and has uniform bands, so that the complete enzyme digestion of the vector and the target fragment can be determined, and the products recovered by enzyme digestion are connected.

Fourth, recombinant bacterium liquid identification of Sep15 promoter activity detection

Transforming the ligation product into Escherichia coli E.coli Top10, selecting 3-5 monoclonals from each plate, respectively inoculating the monoclonals into a liquid culture medium containing aminobenzyl resistance to culture overnight, taking 1 mu L of bacterial liquid as a template, and carrying out PCR identification on the bacterial liquid, wherein the result is shown in figures 4-6, and the monoclonals of the target band which can be amplified are positive clones of the recombinant plasmid with the target gene, and can be used for the next enzyme digestion identification.

Fifth, enzyme digestion identification of recombinant for detecting Sep15 promoter activity

The recombinant plasmid is cut by using a corresponding restriction enzyme at 37 ℃ for 3h, and then 1% agarose gel electrophoresis analysis is carried out, the result is shown in figure 7, and the occurrence of a target band indicates that the restriction enzyme is identified as the successful construction of the target recombinant plasmid.

Sixth, Sep15 promoter activity detection recombinant sequence alignment

The recombinant plasmid is sequenced, and the accuracy of the base sequence of the inserted target fragment is determined to be 100% after the determination result is compared by using a BLAST (BLAST database at NCBI), which indicates that the recombinant plasmid is successfully constructed and can be used for further experiments.

Seventh, Sep15 promoter core region determination

The constructed recombinant plasmids of different segment fragments of the Sep15 promoter and the pRL-TK plasmid are co-transferred into HEK293T and Neuro 2a cells according to the ratio of 19:1 (such as 380 ng: 20ng), and the dual-luciferase activity detection is carried out after transfection for 24h by taking pGL4.10[ LUC2] empty vector as a control, and the specific operation is carried out by a dual-luciferase reporter gene system detection kit instruction.

The results are shown in fig. 8 and 9. As can be seen from the figure, the enzyme activities of pGL4-2055/-791, pGL4-2055/-1225, pGL4-646/-402 and pGL4-272/+909 are close to the background value of unloaded pGL4.10[ LUC2], namely, the promoter activity is basically absent, and compared with an empty vector control, the enzyme activities of recombinants of other promoter fragments are obviously increased, which indicates that the several fragments have the promoter activity in both HEK293T and Neuro 2a cells and show the same change trend. Wherein, the structure of pGL4-818/-248 is described as follows: the small fragment between the cleavage sites KpnI and Hind III of the pGL4.10[ LUC2] vector is replaced by a recombinant plasmid of the DNA fragment shown in the 1238-position 1808 of the sequence 1 in the sequence table. The structure of pGL4-1430/-791 is described below: a recombinant plasmid in which a small fragment between the cleavage sites KpnI and Hind III of the pGL4.10[ LUC2] vector is replaced by a DNA fragment shown in the 626-1265 th site of the sequence 1 in the sequence table. The structure of pGL4-1430/-248 is described below: a recombinant plasmid in which a small fragment between the restriction sites KpnI and Hind III of the pGL4.10[ LUC2] vector is replaced by a DNA fragment shown in the 626-1808 th site of the sequence 1 in the sequence table. The structure of pGL4-2055/-248 is described below: the small fragment between the restriction sites KpnI and Hind III of pGL4.10[ LUC2] vector is replaced by a recombinant plasmid of the DNA fragment shown in the 1 st to 1808 th sites of the sequence 1 in the sequence table.

pGL4-2055/-248 containing the longest promoter fragment had a certain promoter activity, but the activity of pGL4-2055/-791 decreased to the background value after deletion of fragment-818/-248, and the activities of other pGL4-646/-402 and pGL4-272/+909 without-818/-248 were all decreased, indicating that-818/-248 plays an important role in the promoter of Sep15 and is the core region.

pGL4-2055/-248 activity is reduced by a plurality of times compared with pGL4-1430/-248 and pGL4-1430/-791 activity, which shows that the-2055/-1430 interval may contain a stronger negative-regulation trans-acting factor binding site, and similarly, pGL4-1430/-248 activity is reduced by about one time compared with pGL4-818/-248 activity, which indicates that-1430/-818 may also contain a negative-regulation trans-acting factor binding site; pGL4-818/-166 was slightly less active than pGL4-818/-248, indicating that-248/-166 may also contain a weaker negatively regulated trans-acting factor binding site.

Example 2 promotion of Sep15 transcription and protein expression by overexpression of HSF1 eukaryotic expression vector

First, detecting Sep15 core promoter activity by overexpression HSF1 eukaryotic expression vector

HEK293T cells were cultured to 80% -90% density, and then cell count was performed to 0.5-2X 10⁵And (2) paving the cells in a 24-well plate, co-transforming the recombinant pGL4-818/-248 and the pcDNA3.1-HSF1 into HEK293T cells with pRL-TK plasmid in a ratio of 1:1 (mass ratio) the next day, transfecting a pcDNA3.1/Myc-His A empty vector serving as a control group for 24h, then cracking the cells, carrying out dual-luciferase activity detection, and specifically operating and detecting a dual-luciferase reporter gene system detection kit instruction.

Wherein, the specific construction process of the pcDNA3.1-HSF1 vector is as follows: using CDS sequence of HSF1 shown in sequence 3 in the artificially synthesized sequence table as a template, adopting primers HSF1-MYC-F and HSF1-MYC-R (the specific sequences are shown in Table 4) to perform PCR amplification, performing double enzyme digestion on the obtained PCR product by adopting EcoRI and HindIII, recovering the enzyme digestion product, and connecting the enzyme digestion product with the large fragment of the pcDNA3.1/Myc-His A vector subjected to the same double enzyme digestion to obtain the recombinant plasmid. The sequencing shows that the recombinant plasmid, which replaces the small segment between the EcoRI and Hind III sites of the restriction enzyme sites of the pcDNA3.1/Myc-His A vector with the DNA segment shown in the 1 st to 1587 th sites of the sequence 3 in the sequence table, is pcDNA3.1-HSF 1. In the pcDNA3.1-HSF1 vector, the 3' end of the HSF1 gene is connected with a coding sequence of a tag protein MYC.

The results are shown in FIG. 10. As can be seen from the figure, after the HSF1 is over-expressed, the activity of the core promoter pGL4-818/-248 of the Sep15 gene is obviously up-regulated and improved by about 2 times, and statistical analysis shows that the groups have obvious difference.

Secondly, detecting the mRNA level of Sep15 by over-expressing HSF1 eukaryotic expression vector

HEK293T cells were cultured to 80% -90% density, and then cell counting was performed, 2-8X 10⁵And (3) paving the cells in a 6-well plate, transfecting HEK293T cells by the recombinant pcDNA3.1-HSF1 the next day, taking a pcDNA3.1/Myc-HisA empty vector as a control group, after transfecting for 24h, cracking the cells, and carrying out real-time fluorescence quantitative PCR (beta-actin gene as an internal reference gene) to detect the mRNA level of Sep 15. See table 4 for specific primer sequences.

The results are shown in FIG. 11. As can be seen from the figure, after HSF1 is over-expressed, the mRNA level of Sep15 can be increased, and the transcription of Sep15 can be promoted, and there are significant differences.

Thirdly, detecting protein level of Sep15 by overexpression of HSF1 eukaryotic expression vector

HEK293T cells were cultured to 80% -90% density, and then cell counting was performed, 2-8X 10⁵And (3) paving the cells in a 6-well plate, transfecting HEK293T cells by using a recombinant pcDNA3.1-HSF1 on the next day, taking a pcDNA3.1/Myc-HisA empty vector as a control group, after transfecting for 24h, lysing the cells, quantifying BCA protein, and detecting the protein level of Sep15 by using a weather Blot, wherein the specific operation is carried out according to the kit instructions.

The results are shown in FIG. 12. MYC is a tag protein, and the appearance of a MYC protein band indicates that the HSF1 is successfully overexpressed. The results show that protein expression of Sep15 is significantly promoted after HSF1 is overexpressed.

Example 3 transcriptional and protein level effects of Heat shock on Sep15

First, heat shock detection Sep15 core promoter activity

HEK293T cells were cultured to 80% -90% density, and then cell count was performed to 0.5-2X 10⁵The cells were plated in 24-well plates and the next day the recombinants pGL4-818/-248 were co-transferred with the pRL-TK plasmid at a ratio of 19:1 (e.g.380 ng: 20ng) into HEK293T cells as pGL4.10[ LUC2]]And (3) taking an empty vector as a control, carrying out heat shock for 1h in a water bath at 43 ℃ after transfection is carried out for 24h, recovering for 4h at 37 ℃, then cracking cells to carry out dual-luciferase detection, and specifically operating and detecting a dual-luciferase reporter gene system detection kit instruction.

The results are shown in FIG. 13. As can be seen, the core promoter activity of Sep15 was significantly up-regulated after heat shock compared to cells without heat shock, indicating that heat shock can significantly increase the core promoter activity of Sep 15.

Secondly, RT-PCR detection of mRNA level of Sep15 after heat shock

HEK293T cells were cultured to 80% -90% density, then heat shocked with time gradient (0min, 30min, 1h and 2h) in a water bath at 43 ℃, then RNA extraction and RT-PCR detection were directly performed to Sep15 mRNA level (beta-actin gene as reference gene), and HSP70 was used as a positive control, and the specific sequence of the corresponding primer is detailed in Table 4.

The results are shown in FIG. 14. It can be seen that the heat shock was 1h later to promote Sep15 transcription (A in FIG. 14), HSP70 as a positive control to illustrate the effect of heat shock, and significant HSP up-regulation was seen for 30min (B in FIG. 14).

Third, heat shock and overexpression of protein levels for detection of Sep15

HEK293T cells were cultured to 80% -90% density, and then cell counting was performed, 2-8X 10⁵The cells are paved in a 6-well plate, the recombinant pcDNA3.1-HSF1 is transfected into HEK293T cells the next day, the pcDNA3.1/Myc-HisA empty vector is used as a control group, and after transfection is carried out for 24h, heat shock is carried out in a water bath kettle at the temperature of 43 DEG CAfter 1h, recovery at 37 ℃ for 4h, cell lysis and BCA protein quantification, protein levels of Sep15 were detected by Weathern Blot, see kit instructions for details. MYC is a tag protein, and the appearance of a MYC protein band indicates that the HSF1 is successfully overexpressed.

The results are shown in FIG. 15. As can be seen, in HEK293T cells, heat shock promoted Sep15 protein expression, heat shock and over-expressed HSF1, further promoted Sep15 protein expression.

Example 4 Effect of Tunicamycin (Tm) on the transcriptional and expression Regulation of Sep15

First, Tm processing and detecting Sep15 core promoter activity

HEK293T cells were cultured to 80% -90% density, and then cell count was performed to 0.5-2X 10⁵The cells were plated in 24-well plates, and the HEK293T cells were co-transfected with the recombinant pGL4-818/-248 with pcDNA3.1-HSF1 and pRL-TK plasmid (190 ng: 190 ng: 20ng) the next day, and with pcDNA3.1/Myc-His A vector as a control, Tunicamycin (Tm) treatment was performed for 12h after 24h transfection, wherein the working concentration of Tm was maintained at 100 ng/ml. Then cracking the cells to carry out dual-luciferase detection, and specifically operating and detecting the instructions of the dual-luciferase reporter gene system detection kit.

The results are shown in FIG. 16. As can be seen from the figure, the activity of Sep15 promoter is obviously improved by Tm treatment or over-expression of HSF1, and the activity of Sep15 promoter is enhanced when Tm treatment is carried out and HSF1 is over-expressed.

Secondly, detecting the mRNA level of Sep15 by RT-PCR after Tm time gradient treatment

After HEK293T cells were cultured to 80% -90% density, Tm time gradient (0h, 3h, 6h, 12h, 24h) treatments were performed, wherein the working concentration of Tm was maintained at 5. mu.g/ml. Then RNA extraction and RT-PCR detection are directly carried out to detect the mRNA level of Sep15 (beta-actin gene is used as reference gene), and the specific sequence of the corresponding primer is detailed in Table 4.

As shown in fig. 17, it was found that the Tm treatment significantly increased the mRNA level of Sep15 after 6h, and after 12h, the mRNA level of Sep15 increased twice as much as 0h, and continued increase for 24 h. This indicates that the mRNA expression level of Sep15 continuously increased between 12h and 24h of Tm treatment, promoting continuous transcription of Sep 15.

Third, protein level of Sep15 after Tm time gradient treatment

HEK293T cells were plated in 6-well plates and cultured to 70-80% density, followed by Tm time gradient (0h, 3h, 6h, 12h, 24h) with working Tm at 5. mu.g/ml. And (3) collecting cells at different time points, extracting protein, carrying out BCA protein quantification, and finally detecting the Sep15 protein concentration by Western Blot.

The results are shown in FIG. 18. As can be seen, the protein expression of Sep15 is promoted by Tm treatment for 3h, and the protein level of Sep15 gradually increases with time, which is consistent with the mRNA level trend of Sep15 after previous Tm time gradient treatment. The fact that the endoplasmic reticulum stress inducer of Tm promotes HSF1 to enter the nucleus and is combined with a specific site of a Sep15 promoter, so that the transcription of Sep15 is enhanced, the massive expression of Sep15 protein is promoted, the endoplasmic reticulum stress inducer participates in the control of endoplasmic reticulum protein, and the correct folding of misfolded protein is assisted or the unfolding of the misfolded protein is assisted to be delivered to other proteins for correct folding is facilitated.

In combination with the above examples, the results of the dual luciferase reporter system (DLR) show that the 5' -818/-248 of Sep15 gene plays an important role in promoter activity, the-2055/-1430 region may contain a strong negative-regulatory trans-acting factor, the-1430/-818 region may also contain a negative-regulatory trans-acting factor, and the 248/-166 region may also contain a weak negative-regulatory trans-acting factor. Whether the HSF1 is over-expressed, heat shock or tunicamycin treatment is adopted, the promoter activity of Sep15 can be improved, Sep15 transcription is promoted, and then protein of Sep15 is massively expressed, and the Sep15 expression is regulated to participate in various biological events by inducing HSF1 to enter a nucleus, combining with a Sep15 promoter sequence to start transcription. Sep15 is a selenoprotein with a thioredoxin domain at its N-terminus, which is predictive of being a disulfide reductase or disulfide isomerase, redox, localized in the lumen of the endoplasmic reticulum in association with glucosyl glycoprotein (UGGT), and involved in endoplasmic reticulum protein control. The endoplasmic reticulum stress is induced by an endoplasmic reticulum stress inducer Tm, and in the process, Sep15 expression is up-regulated, which indicates that Sep15 plays an important role in the process. In many reports, there are many diseases caused by accumulation of misfolded proteins, such as alzheimer's disease, parkinson's disease, and amyotrophic lateral sclerosis, and one of the main causes of disease induction is severe endoplasmic reticulum stress in vivo, which indicates that Sep15 also plays an important role in these diseases, and inducing Sep15 expression may alleviate the progression of these diseases.

110 Shenzhen university

Application of 120 heat shock transcription factor 1 in regulation and control of expression of 15kDa selenoprotein

160 3

170 PatentIn version 3.5

210 1

211 1808

212 DNA

213 (Homo sapiens)

400 1

aactctcgct ctccatgctg agctcttcca aatgccaaag tgccctatta atccgaaagt 60

acctattatc tgtgcttttt tctggcctgt ctttcacaac ttcacaccaa ctttttgtac 120

tttagacccc ctcatccatt aaagcacctg ttaacccttt aaggtattta accatatggt 180

tagctttccg cataatccta cttttccttt aacaaacaca aacttcacta cataattccc 240

cttcagcgtt ccctcctctt cgaaattcac atcaacactg ccgtgaagca acatacatag 300

gcatccctat tctcaagccc caattcatca gtttagcgcc actaaagaag aatttaaatc 360

tccggaccct catttccgta ctccgatgaa gctttgcact cgcgttcccg cactacccgg 420

ccagcctccc tcagctggca attacaaggt cctttgctgc ccggagcaca agagtgcaac 480

tcgctacagt gttcacatgt tgctctccgt catcccagca tctcctccca tttctgcgca 540

cctatccctt catgagcccc caaaagaatt cgtatccaga tgcccatagc tcaaattaga 600

atacccctca tctgacattg ccagggcttc ctgtacgact tccaagagaa cacgcgatgc 660

taagcgcttc tgtctccgga gaccagcaac acacgctccc cactgagttc ggtcttctct 720

ctcgctctct ctccgcggaa tgcctacttc ggggctaccc tttggctagg gaagcccaac 780

cttgcaacct ctgccccgtg acctcgcgag cccagcacgc cgctcaagct taattcaacc 840

cattcttgga gctcgtgctc tcgctgcccc ttgttcccgg ttctctttga aaccgctttc 900

agcccccgag ccagacccca ggttagatga acgcccactt gctccggcgg cagcgcctcc 960

tcggcccctt ccacagcact cagcccgggg cagttcagtt cctcacctag cttcgagcgg 1020

gactcctcca gtttctggat ctggttttcc aagaagagca tcgccaccgc gaaggccacc 1080

accgccagca gctgcaactt gggcggcatc ataatcctga ggagccccat gaaacccgct 1140

gctcgggatc aagacatacc gcggggggcg gggagcggga ccccggaggc gagagagacg 1200

ccgggaggag cccgccgagc cgggagcgag gcaaagagag agcgacagcg agagaaccac 1260

cgcagcgacc ccccttctcg cctctccagt ccccctcccc caggcgctat caccaacgcc 1320

gctggtgtcc gcttctcctc agccgactaa acggccgccc cctccaaaag cccgcgcagc 1380

aaacagcgcg aagcggctgc cgggctctcg cctccctcta ctgcctcagt ccccgacccc 1440

cgcgcccgcc tgcttgcccg cctccctctc ttccttcctt ccctccctcc tttgctcgcg 1500

gccgcgcgcc cctccaccgg cgctgcggct gcgccgccct catccccgcc cccgcccatc 1560

cccctccgcc accacgcgca cgcatccgcg tacgctgctc ggcaaccaga agccccgccc 1620

ctccccccgt tcgggtccgc ttattggacg gaagagcccc tgcgcttcgc tcttggttcg 1680

gtttacggtt ggtttcctct gccacagagc tcaatcaaga cgacattcaa ttgggtaaac 1740

ttggagaaga aggcggggct aaaactggcg aaggcgtggc ttcttggctg cttgacgaag 1800

tgtcgtga 1808

210 2

211 529

212 PRT

213 (Homo sapiens)

400 2

Met Asp Leu Pro Val Gly Pro Gly Ala Ala Gly Pro Ser Asn Val Pro

1 5 10 15

Ala Phe Leu Thr Lys Leu Trp Thr Leu Val Ser Asp Pro Asp Thr Asp

20 25 30

Ala Leu Ile Cys Trp Ser Pro Ser Gly Asn Ser Phe His Val Phe Asp

35 40 45

Gln Gly Gln Phe Ala Lys Glu Val Leu Pro Lys Tyr Phe Lys His Asn

50 55 60

Asn Met Ala Ser Phe Val Arg Gln Leu Asn Met Tyr Gly Phe Arg Lys

65 70 75 80

Val Val His Ile Glu Gln Gly Gly Leu Val Lys Pro Glu Arg Asp Asp

85 90 95

Thr Glu Phe Gln His Pro Cys Phe Leu Arg Gly Gln Glu Gln Leu Leu

100 105 110

Glu Asn Ile Lys Arg Lys Val Thr Ser Val Ser Thr Leu Lys Ser Glu

115 120 125

Asp Ile Lys Ile Arg Gln Asp Ser Val Thr Lys Leu Leu Thr Asp Val

130 135 140

Gln Leu Met Lys Gly Lys Gln Glu Cys Met Asp Ser Lys Leu Leu Ala

145 150 155 160

Met Lys His Glu Asn Glu Ala Leu Trp Arg Glu Val Ala Ser Leu Arg

165 170 175

Gln Lys His Ala Gln Gln Gln Lys Val Val Asn Lys Leu Ile Gln Phe

180 185 190

Leu Ile Ser Leu Val Gln Ser Asn Arg Ile Leu Gly Val Lys Arg Lys

195 200 205

Ile Pro Leu Met Leu Asn Asp Ser Gly Ser Ala His Ser Met Pro Lys

210 215 220

Tyr Ser Arg Gln Phe Ser Leu Glu His Val His Gly Ser Gly Pro Tyr

225 230 235 240

Ser Ala Pro Ser Pro Ala Tyr Ser Ser Ser Ser Leu Tyr Ala Pro Asp

245 250 255

Ala Val Ala Ser Ser Gly Pro Ile Ile Ser Asp Ile Thr Glu Leu Ala

260 265 270

Pro Ala Ser Pro Met Ala Ser Pro Gly Gly Ser Ile Asp Glu Arg Pro

275 280 285

Leu Ser Ser Ser Pro Leu Val Arg Val Lys Glu Glu Pro Pro Ser Pro

290 295 300

Pro Gln Ser Pro Arg Val Glu Glu Ala Ser Pro Gly Arg Pro Ser Ser

305 310 315 320

Val Asp Thr Leu Leu Ser Pro Thr Ala Leu Ile Asp Ser Ile Leu Arg

325 330 335

Glu Ser Glu Pro Ala Pro Ala Ser Val Thr Ala Leu Thr Asp Ala Arg

340 345 350

Gly His Thr Asp Thr Glu Gly Arg Pro Pro Ser Pro Pro Pro Thr Ser

355 360 365

Thr Pro Glu Lys Cys Leu Ser Val Ala Cys Leu Asp Lys Asn Glu Leu

370 375 380

Ser Asp His Leu Asp Ala Met Asp Ser Asn Leu Asp Asn Leu Gln Thr

385 390 395 400

Met Leu Ser Ser His Gly Phe Ser Val Asp Thr Ser Ala Leu Leu Asp

405 410 415

Leu Phe Ser Pro Ser Val Thr Val Pro Asp Met Ser Leu Pro Asp Leu

420 425 430

Asp Ser Ser Leu Ala Ser Ile Gln Glu Leu Leu Ser Pro Gln Glu Pro

435 440 445

Pro Arg Pro Pro Glu Ala Glu Asn Ser Ser Pro Asp Ser Gly Lys Gln

450 455 460

Leu Val His Tyr Thr Ala Gln Pro Leu Phe Leu Leu Asp Pro Gly Ser

465 470 475 480

Val Asp Thr Gly Ser Asn Asp Leu Pro Val Leu Phe Glu Leu Gly Glu

485 490 495

Gly Ser Tyr Phe Ser Glu Gly Asp Gly Phe Ala Glu Asp Pro Thr Ile

500 505 510

Ser Leu Leu Thr Gly Ser Glu Pro Pro Lys Ala Lys Asp Pro Thr Val

515 520 525

Ser

210 3

211 1590

212 DNA

213 (Homo sapiens)

400 3

atggatctgc ccgtgggccc cggcgcggcg gggcccagca acgtcccggc cttcctgacc 60

aagctgtgga ccctcgtgag cgacccggac accgacgcgc tcatctgctg gagcccgagc 120

gggaacagct tccacgtgtt cgaccagggc cagtttgcca aggaggtgct gcccaagtac 180

ttcaagcaca acaacatggc cagcttcgtg cggcagctca acatgtatgg cttccggaaa 240

gtggtccaca tcgagcaggg cggcctggtc aagccagaga gagacgacac ggagttccag 300

cacccatgct tcctgcgtgg ccaggagcag ctccttgaga acatcaagag gaaagtgacc 360

agtgtgtcca ccctgaagag tgaagacata aagatccgcc aggacagcgt caccaagctg 420

ctgacggacg tgcagctgat gaaggggaag caggagtgca tggactccaa gctcctggcc 480

atgaagcatg agaatgaggc tctgtggcgg gaggtggcca gccttcggca gaagcatgcc 540

cagcaacaga aagtcgtcaa caagctcatt cagttcctga tctcactggt gcagtcaaac 600

cggatcctgg gggtgaagag aaagatcccc ctgatgctga acgacagtgg ctcagcacat 660

tccatgccca agtatagccg gcagttctcc ctggagcacg tccacggctc gggcccctac 720

tcggccccct ccccagccta cagcagctcc agcctctacg cccctgatgc tgtggccagc 780

tctggaccca tcatctccga catcaccgag ctggctcctg ccagccccat ggcctccccc 840

ggcgggagca tagacgagag gcccctatcc agcagccccc tggtgcgtgt caaggaggag 900

ccccccagcc cgcctcagag cccccgggta gaggaggcga gtcccgggcg cccatcttcc 960

gtggacaccc tcttgtcccc gaccgccctc attgactcca tcctgcggga gagtgaacct 1020

gcccccgcct ccgtcacagc cctcacggac gccaggggcc acacggacac cgagggccgg 1080

cctccctccc ccccgcccac ctccacccct gaaaagtgcc tcagcgtagc ctgcctggac 1140

aagaatgagc tcagtgacca cttggatgct atggactcca acctggataa cctgcagacc 1200

atgctgagca gccacggctt cagcgtggac accagtgccc tgctggacct gttcagcccc 1260

tcggtgaccg tgcccgacat gagcctgcct gaccttgaca gcagcctggc cagtatccaa 1320

gagctcctgt ctccccagga gccccccagg cctcccgagg cagagaacag cagcccggat 1380

tcagggaagc agctggtgca ctacacagcg cagccgctgt tcctgctgga ccccggctcc 1440

gtggacaccg ggagcaacga cctgccggtg ctgtttgagc tgggagaggg ctcctacttc 1500

tccgaagggg acggcttcgc cgaggacccc accatctccc tgctgacagg ctcggagcct 1560

cccaaagcca aggaccccac tgtctcctag 1590

Claims (3)

1. Use of heat shock transcription factor 1 in any one of:

(a1) Preparing a product having a function of promoting the expression of Sep15 protein in a target cell;

(a2) Preparing a product having a function of promoting the transcription of the Sep15 gene in the target cell;

(a3) Preparing a product having enhanced Sep15 promoter activity in target cells;

wherein, the heat shock transcription factor 1 is HSFl which is encoded by a nucleotide sequence in an overexpression sequence table 3 in HEK293T cell;

the GenBank accession number of the amino acid sequence of the Sep15 protein is NP-004252.2 or NP-976086.1; the Sep15 gene is a gene encoding the Sep15 protein;

the Sep15 promoter is the following DNA molecule: at least contains the 1238-1808 th nucleotide sequence of the sequence 1 in the sequence table, and extends from the 1238 th nucleotide sequence of the sequence 1 to the 5' end of the sequence 1 according to the nucleotide sequence of the sequence 1 to obtain any DNA fragment with the length of 571bp to 1808 bp; the DNA molecule has a promoter function; the nucleotide sequence of the DNA molecule is a DNA molecule shown as the 1238-th-1808-bit nucleotide of the sequence 1 in the sequence table.

2. The application of the substance capable of promoting the expression of the heat shock transcription factor 1 in any one of the following substances:

(a1) Preparing a product having a function of promoting the transcription of the Sep15 gene in the target cell;

(a2) Preparing a product having enhanced Sep15 promoter activity in target cells;

wherein, the heat shock transcription factor 1 is HSFl which is encoded by a nucleotide sequence in an overexpression sequence table 3 in HEK293T cell;

the GenBank accession number of the amino acid sequence of the Sep15 protein is NP-004252.2 or NP-976086.1; the Sep15 gene is a gene encoding the Sep15 protein;

the Sep15 promoter is the following DNA molecule: at least contains the 1238-1808 th nucleotide sequence of the sequence 1 in the sequence table, and extends from the 1238 th nucleotide sequence of the sequence 1 to the 5' end of the sequence 1 according to the nucleotide sequence of the sequence 1 to obtain any DNA fragment with the length of 571bp to 1808 bp; the DNA molecule has a promoter function; the nucleotide sequence of the DNA molecule is a DNA molecule shown as the 1238-th-1808-bit nucleotide of the sequence 1 in the sequence table.

3. Use according to claim 2, characterized in that: the GenBank accession number of the nucleotide sequence of the Sep15 gene is NM _004261.4 or NM _ 203341.2.

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