MAFG transcriptional activation luciferase reporting system based on MYC promoter

Abstract

The invention provides a MAFG transcriptional activation luciferase reporting system based on an MYC promoter, relates to the technical field of biomedicine, and is technically characterized in that the MAFG transcriptional activation luciferase reporting system based on the MYC promoter is provided and comprises a reporter gene plasmid constructed by an MYC promoter truncation body and firefly luciferase, the MYC promoter truncation body comprises a sequence as shown in SEQ ID NO: 01. The invention provides a dual-luciferase report system (Dual-Luciferase report) based on an MYC promoter, and the dual-Luciferase report system (Dual-Luciferase report) is used for detecting the activation transcription capability of a transcription factor MAFG. The luciferase does not need post-translation processing, so that report activity is generated immediately once translation is carried out; and the light product quantum efficiency is high, so that the detection is sensitive and rapid. The method is mainly applied to miRNA target gene verification, transcription factor regulation verification and the like at present. The system can accurately reflect the transcriptional activity of MAFG based on luciferase of an MYC promoter region.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to a MAFG transcriptional activation luciferase reporter system based on a MYC promoter. BACKGROUND

[0002] MAF bZIP transcription factor G (MAFG) is mainly expressed in the nucleus, with a molecular weight of about 18 kD. Human MAFG encodes a basic leucine zipper (bZIP) protein, which is located on human chromosome 17q25 (17th chromosome long arm end q25 band) and belongs to the transcription factor family related to v-maf oncogene. Initially, MAFG forms a heterodimer with Nrf2, binds to the antioxidant response element (ARE) required for Nrf2 signal activation, and participates in antioxidant response. Moreover, MAFG is involved in the differentiation of hematopoietic system, liver and nervous system. For example, in erythropoiesis, MAFG cooperates with transcription factor NF-E2 to regulate the expression of globin gene.

[0003] However, recent studies have shown that MAFG promotes DNA methylation and anti-inflammatory transcriptional programs, and plays an important role in the occurrence and development of cancer. The expression of MAFG is increased in cells and tissues with cholestasis and in human cholangiocarcinoma and hepatocellular carcinoma specimens, and its high expression is associated with tumor progression and shortened survival time. In colon cancer and melanoma, the proto-oncogene (BRAF) variant BRAF (V600E) plays a pro-cancer role by up-regulating MAFG. MAFG promotes the ability of cancer cells to proliferate, migrate, invade and metastasize in prostate cancer by up-regulating MAFG-DT. And in our previous study, MAFG helps cancer cells to escape cell killing by up-regulating super-enhancer-mediated PD-L1 expression. The above studies confirm that MAFG is a pro-cancer gene, but there is currently no effective way to detect the transcriptional activation function of MAFG, so as to further study the specific molecular mechanism of MAFG regulating the expression of tumor-related genes. SUMMARY

[0004] The purpose of the present application is to solve the problem of lack of effective detection of the transcriptional activation function of MAFG in the prior art.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] A MAFG transcriptional activation luciferase reporter system based on a MYC promoter, comprising a reporter gene plasmid constructed by a MYC promoter truncation body and a firefly luciferase, wherein the MYC promoter truncation body comprises the sequence set forth in SEQ ID NO: 01.

[0007] Preferably, the MYC promoter truncation is one of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05.

[0008] The application also provides a MYC promoter-based MAFG transcriptional activation luciferase reporter system use method, using the reporter system described above.

[0009] Preferably, the use method has the following steps:

[0010] S1: combine one of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05 MYC promoter or its truncation with firefly luciferase to construct a reporter gene plasmid;

[0011] S2: co-transfect the reporter gene plasmid with the overexpression MAFG plasmid and the Renilla luciferase internal standard gene plasmid into HEK293T cells, and the ratio of firefly luciferase fluorescence intensity to Renilla fluorescence intensity can be used to judge the transcription intensity of MAFG.

[0012] The application also provides an application of a MYC promoter-based MAFG transcriptional activation luciferase reporter system in detecting the activated transcription ability product of transcription factor MAFG, and the reporter system is the reporter system described above.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The application provides a MYC promoter-based dual luciferase reporter assay for detecting the activated transcription ability of transcription factor MAFG. Since luciferase does not require post-translational processing, it immediately produces reporter activity upon translation; and it has a high light product quantum efficiency, so it is sensitive and fast. At present, it is mainly used in miRNA target gene verification, transcription factor regulation verification, etc. Through experiments, we found that MAFG can regulate the expression of MYC at the transcriptional level, and we successfully found the key DNA region of the MYC promoter regulated by MAFG. After constructing a luciferase reporter system by the DNA promoter region, the luciferase based on the MYC promoter region of the system can accurately reflect the transcriptional activity of MAFG. As a transcriptional regulator, MAFG is involved in the regulation of various biological processes in cells, and plays a key role in important diseases such as tumors. Detecting its activity can reflect the state and severity of the disease. The reporter system can detect the transcriptional activation ability of MAFG in cell lines, tumors and other diseases. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Flow chart of the validation experiment of the present application.

[0016] Figure 2 To establish MAFG knockout cell line in RKO cells, differential gene analysis by RNA-seq found that MYC expression decreased.

[0017] Figure 3 To knock out MAFG in RKO cell line, detect the mRNA and protein level expression of MYC by RT-PCR, Western-Blot, immunofluorescence, wherein A: detect the mRNA level expression of MYC in MAFG knockout RKO cell line by RT-PCR. B: detect the protein level expression of MYC in MAFG knockout RKO cell line by Western-Blot. C: detect the expression of MYC in MAFG knockout RKO cell line by immunofluorescence.

[0018] Figure 4 To overexpress MYC and MAFG respectively in RKO cell line, Western-Blot detects the protein expression of MAFG and MYC respectively. RT-PCR detects the mRNA level of MYC in MAFG overexpressing cells. Among them, A: overexpress MYC in RKO cell line, Western-Blot verifies the protein expression of MYC. B: overexpress MAFG in RKO cell line, Western-Blot verifies the protein expression of MAFG. C: RT-PCR detects the mRNA level of MYC in MAFG overexpressing cells.

[0019] Figure 5 To knock out MAFG in HT-29 cell line, RT-PCR and Western-Blot detect the mRNA and protein level of MYC. Western-Blot detects the protein expression of MYC in sg-MAFG tumor tissue. Among them, A: RT-PCR detects the mRNA level of MYC in MAFG knockout HT-29 cell line. B: Western-Blot detects the protein of MYC in MAFG knockout HT-29 cell line. C: Western-Blot detects the protein expression of MYC in MAFG knockout tumor tissue.

[0020] Figure 6 To detect the enrichment of MAFG on the MYC promoter by ChIP-PCR, wherein A: detect the enrichment of MAFG on the MYC promoter by ChIP-PCR. B: quantify the enrichment degree of MAFG on the MYC promoter.

[0021] Figure 7The MYC promoter is combined with firefly luciferase to construct a reporter gene plasmid. The dual luciferase reporter system detects MAFG transcriptional activity, wherein A: schematic diagram of the reporter gene plasmid constructed by combining the MYC promoter with firefly luciferase; B: dual luciferase reporter system for detecting MAFG transcriptional activity.

[0022] Figure 8 The different truncated bodies are combined with firefly luciferase to construct a reporter gene plasmid. The dual luciferase reporter system detects the transcriptional activation of different truncated bodies by MAFG. Among them, A: schematic diagram of the reporter gene plasmid constructed by combining the MYC promoter d1000, d1500, d2100, d2464, d2641 truncated body with firefly luciferase; B: dual luciferase reporter system for detecting the transcriptional activation of MYC promoter d1000 truncated body by MAFG; C: dual luciferase reporter system for detecting the transcriptional activation of MYC promoter d1500 truncated body by MAFG; D: dual luciferase reporter system for detecting the transcriptional activation of MYC promoter d2100 truncated body by MAFG; E: dual luciferase reporter system for detecting the transcriptional activation of MYC promoter d2464 truncated body by MAFG; F: dual luciferase reporter system for detecting the transcriptional activation of MYC promoter d2641 truncated body by MAFG. DETAILED DESCRIPTION

[0023] The application will be further described in detail below in combination with specific embodiments.

[0024] The application provides a MAFG transcriptional activation luciferase reporter system based on a MYC promoter, which comprises a reporter gene plasmid constructed by a MYC promoter truncated body and firefly luciferase, and the MYC promoter truncated body comprises the sequence of SEQ ID NO: 01.

[0025] In an embodiment, the MYC promoter truncated body is one of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, and SEQ ID NO: 05.

[0026] Based on the above-mentioned reporter system, the application provides a use method of the MAFG transcriptional activation luciferase reporter system based on the MYC promoter, which comprises the following steps:

[0027] S1: combining one of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, and SEQ ID NO: 05 MYC promoter or its truncated body with firefly luciferase to construct a reporter gene plasmid;

[0028] S2: Co-transfect the reporter plasmid with the overexpression MAFG plasmid, Renilla luciferase internal control plasmid into HEK293T cells, and the ratio of firefly luciferase fluorescence intensity to Renilla fluorescence intensity can determine the transcription intensity of MAFG.

[0029] In addition, based on the above-mentioned reporter system, the application also provides the application of the MYC promoter-based MAFG transcriptional activation luciferase reporter system in detecting the product with the transcription factor MAFG activation transcription ability.

[0030] The above content is described in combination with specific verification experiments as follows:

[0031] Please refer to Figure 1 Firstly, in the present application, MAFG knockout cell lines were constructed in RKO cells, and RNA-seq transcriptome sequencing was performed. Through differential gene analysis, it was found in the present application that MYC was significantly down-regulated, indicating that MAFG might have a regulatory effect on MYC. Figure 2 ) Western Blot, RT-PCR and immunofluorescence results show that the mRNA and protein levels of MYC are significantly reduced after knocking out MAFG. Figure 3 ) In the present application, MYC and MAFG were overexpressed in RKO cells, respectively. Western Blot results prove that overexpression of MYC does not affect the expression of MAFG, but overexpression of MAFG can enhance the protein expression of MYC. Figure 4 ) RT-PCR results also show that overexpression of MAFG can enhance the mRNA level of MYC. Figure 4 ) Therefore, it is reasonable to conclude that MAFG has a positive regulatory effect on MYC.

[0032] To explore whether the regulation of MAFG on MYC is universal, MAFG was knocked out in HT-29 cell lines and the mRNA and protein levels of MYC were detected. Similarly, the expression of MYC in HT-29 cells was also down-regulated due to the knockout of MAFG. Figure 5 ) It is worth mentioning that in the present application, it was found that the protein level of MYC in the MAFG-knockout tumor tissue was also reduced. Figure 5 ) The above results show that the regulation of MAFG on MYC is universal.

[0033] To further understand the regulatory effect of MAFG on MYC, the present application proves that MAFG is enriched on the MYC promoter by ChIP-PCR experiment. Figure 6), indicating that MAFG regulates its transcriptional activity through MYC promoter. Then, the transcriptional activation ability of MAFG was detected based on MYC promoter by using dual luciferase reporter system. First, the MYC promoter (SEQ ID NO: 02) was combined with firefly luciferase to construct a reporter gene plasmid. Subsequently, the overexpression MAFG plasmid and the Renilla luciferase internal standard gene plasmid were co-transfected into HEK293T cells Figure 7 ) MYC promoter was activated, and the firefly luciferase gene was expressed. The transcriptional intensity of MAFG could be judged by the ratio of firefly fluorescence intensity to Renilla fluorescence intensity. The results showed that the transcriptional activity of MAFG based on MYC promoter was significantly enhanced Figure 7

[0034] Next, in order to find the specific position of MYC promoter combined with MAFG, different lengths of truncations were constructed in the application: d1000 (SEQ ID NO: 03), d1500 (SEQ ID NO: 04), d2100 (SEQ ID NO: 05), d2464 (SEQ ID NO: 06), d2641 (SEQ ID NO: 07), and the above truncations were combined with firefly luciferase structure to construct firefly luciferase reporter gene plasmid Figure 8 ), the results showed that the transcriptional activity of MAFG based on truncation d2641 was not enhanced Figure 8 ), suggesting that MAFG binds to SEQ ID NO: 01 sequence in MYC promoter to achieve regulation.

[0035] The following are the materials and experimental procedures applied in the above embodiments of the application:

[0036] I. Experimental materials and sources

[0037]

[0038]

[0039]

[0040] II. Dual luciferase reporter experiment

[0041] 1. Plasmid construction

[0042] First, the MYC promoter sequence was obtained from the NCBI website, and the cloning primers were designed for PCR amplification, and then inserted into the firefly luciferase (firefly luciferase) reporter gene vector. The insertion site and PCR product were double-digested and ligated, and then transformed into competent cells after ligation by DNA ligase. After resistance screening, the plasmid was extracted and sequenced to ensure successful plasmid construction. ​

[0043] 2. Plasmid transfection

[0044] 1) One day before transfection, HEK 293T cells were seeded in six-well plates according to the grouping, with one duplicate well in each group, and the seeding density was about 70% per well;

[0045] 2) On the day of transfection, the old culture medium in the six-well plates was aspirated and replaced with 2 mL of fresh complete culture medium;

[0046] 3) The corresponding number of 1.5 mL centrifuge tubes was taken, 125 μL of Opti-MEM without antibiotics and serum was added to each tube, and the corresponding volume of DNA plasmid was added according to the DNA plasmid content in each group of the following table, and then mixed gently;

[0047] MYC promoter full-length group cell transfection grouping

[0048]

[0049] Note: ②-④ groups all add 0.1 μg of Renilla luciferase plasmid as internal reference

[0050] MYC promoter truncated group cell transfection grouping

[0051]

[0052] Note: ②-④ groups all add 0.1 μg of Renilla luciferase plasmid as internal reference

[0053] 4) According to the plasmid dosage (μg): Lipo2000 (μL) = 1:1.5, add Lipo2000 in each centrifuge tube, mix gently, then add to the corresponding six-well plate, and continue to culture in the incubator;

[0054] 5) Observe the cell state, and replace the fresh complete culture medium after 6 h;

[0055] 6) Perform dual luciferase detection after 48 h.

[0056] 3. Cell lysis and dual luciferase detection

[0057] 1) When using the dual luciferase detection reagent for the first time, Luciferase Assay Buffer II should be added to Luciferase Assay Substrate to completely dissolve the substrate in the buffer to prepare Luciferase Assay Reagent II (LAR II), which is then aliquoted and stored at -80°C for later use;

[0058] 2) Take out the Stop & Glo Buffer and dissolve at room temperature, dilute 50x Stop & Glo Substrate into 1x Stop & Glo Substrate with the buffer, mix well and place on ice for standby, Stop & Glo Reagent needs to be prepared immediately before use;

[0059] 3) Dissolve the dual-luciferase reporter gene cell lysate at room temperature, remove the culture medium in the six-well plate, wash the cells once with pre-cooled PBS, add 150-200 μL of the melted cell lysate to each well, scrape the cells with a cell scraper and transfer them to a 1.5 mL centrifuge tube, and place it on ice for testing;

[0060] 4) Take out a new 1.5 mL centrifuge tube, add 10 μL of cell lysate, close the cap, and place it in the detector to detect the firefly luciferase fluorescence value and the sea anemone luciferase fluorescence value as a blank control;

[0061] 5) Take 10 μL of cell lysate, add 20 μL of Luciferase Assay Reagent II (LAR II), mix well, and then test the firefly luciferase fluorescence value on the machine;

[0062] 6) Take the centrifuge tube from the detector, add 20 μL of Stop & Glo Reagent to the centrifuge tube, mix well, and then test the sea anemone luciferase fluorescence value on the machine as an internal reference;

[0063] 7) The same sample needs to be tested twice, and after the test is completed, use GraphPad Prism to statistically analyze the ratio of firefly and sea anemone luciferase fluorescence values.

[0064] 4、Cell culture

[0065] The RKO, HT29, and HEK293T cells used in the experiment were cultured in complete high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and were placed in a 37°C constant temperature incubator with a carbon dioxide concentration of 5%. The cell processing process was completed on a clean bench, and the whole process was protected and sterilely operated. The cell morphology and cleanliness were observed regularly.

[0066] 5、Chromatin immunoprecipitation (CHIP)

[0067] (1) Sample collection and ultrasonic process

[0068] 1) Place PBS in an ice box for ice bath, take out the SDS Lysis Buffer in the kit and place it at room temperature, and gently shake the SDS inside.

[0069] 2) Take the dish, discard the medium, wash the cells with PBS twice. Discard the PBS, prepare 1% formaldehyde (10 mL pre-cooled PBS + 270 μL 37% formaldehyde), add 10 mL 1% formaldehyde to the dish, incubate on a shaker at 40 rpm for 10 min at room temperature to cross-link the target protein and its associated DNA fragments.

[0070] 3) Continue to add 1.1 mL Glycine Solution (10x), mix gently and place on a shaker at room temperature at 40 rpm for 6 min.

[0071] 4) Prepare fresh PBS containing 1 mM PMSF (10 mL pre-cooled PBS + 100 μL 100 mM PMSF), discard the 1% formaldehyde and Glycine Solution (10x) added in the previous two steps, try to ensure that there is no residual liquid in the dish.

[0072] 5) Add 4 mL PBS containing 1 mM PMSF, wash the cells, discard the liquid after washing, try to ensure that there is no residual liquid. This step is performed twice to achieve the purpose of thorough washing.

[0073] 6) Add 1 mL PBS containing the above pre-cooled PBS in the dish, carefully scrape the cells with a cell scraper on an ice box, collect the cell liquid in a 1.5 mL EP tube.

[0074] 7) Set the centrifuge parameters to 4°C, 1000 g for 5 min, discard the supernatant in the EP tube.

[0075] 8) Prepare SDS Lysis Buffer containing 1 mM PMSF (300 μL SDS Lysis Buffer + 3 μL 100 mM PMSF), add to the EP tube, repeatedly blow the cell pellet. Place on ice for lysis for 10 min.

[0076] 9) Use ultrasonic instrument to ultrasonic cell suspension, set parameters: power 50W, set to 30% of maximum power, 15 times of ultrasonic, 10s each time, 10s interval between each ultrasonic.

[0077] (2) Chromatin immunoprecipitation

[0078] 1) After ultrasonic, place the sample at 4°C, 14000 g centrifuge for 15 min, transfer the supernatant to a new 2 mL EP tube and place on ice.

[0079] 2) Prepare CHIP Dilution Buffer with 1 mM PMSF (1.8 mL CHIP Dilution Buffer + 18 μL 100 mM PMSF), add to the EP tube above and mix by pipetting.

[0080] 3) Take 40 μL of the sample as Input and store at -20 °C. Add 100 μL of Protein A+G Agarose / Salmon Sperm DNA to the remaining sample, mix gently by inverting and rotate at 4 °C for 2 h.

[0081] 4) Remove the sample and centrifuge at 1000 g for 5 min at 4 °C. Transfer the supernatant to a new EP tube and divide it into two aliquots, add 2 μg of the protein of interest and 2 μg of the corresponding species IgG to each and rotate at 4 °C overnight.

[0082] 5) The next day, remove the sample and add 60 μL of Protein A+G Agarose / Salmon Sperm DNA to each, mix gently by inverting and rotate at 4 °C for 2 h.

[0083] 6) Centrifuge at 1000 g for 5 min at 4 °C, discard the supernatant, being careful not to aspirate the pellet. Add Low Salt Immune Complex Wash Buffer, pipette the pellet up and down, rotate at 4 °C for 5 min, then centrifuge at 1000 g for 3 min at 4 °C, discard the supernatant.

[0084] 7) Add High Salt Immune Complex Wash Buffer, pipette the pellet up and down, rotate at 4 °C for 5 min, then centrifuge at 1000 g for 3 min at 4 °C, discard the supernatant.

[0085] 8) Add LiCI Immune Complex Wash Buffer, pipette the pellet up and down, rotate at 4 °C for 5 min, then centrifuge at 1000 g for 3 min at 4 °C, discard the supernatant.

[0086] 9) Add TE Buffer, pipette the pellet up and down, rotate at 4 °C for 5 min, then centrifuge at 1000 g for 3 min at 4 °C, discard the supernatant. Repeat this process twice.

[0087] (3) PCR amplification of the gene of interest

[0088] 1) Prepare fresh Elution Buffer (10 mL CHIP Dilution Buffer + 0.1 g SDS + 0.084 g NaHCO3)

[0089] 2) Add 250 μL of Elution Buffer to the last cell pellet, vortex, rotate at room temperature for 5 min, then centrifuge at 1000 g for 3 min.

[0090] 3) Transfer the supernatant to a new 1.5 mL EP tube, add 250 μL of Elution Buffer, vortex, rotate at room temperature for 5 min, then centrifuge at 1000 g for 3 min.

[0091] 4) Mix the supernatant and the supernatant of the previous step, a total of 500 μL of supernatant.

[0092] 5) Add 20 μL of 5M Nacl to the supernatant, mix and heat at 65°C for 4h to release the crosslinking of protein and DNA. Add 2 μL of 5M Nacl to the Input sample, mix and heat at 65°C for 4h.

[0093] 6) Continue to add 10 μL of 0.5M EDTA, 20 μL of 1M Tris, and 1 μL of 20 mg / mL proteinase K to the sample, mix by blowing, and place in a 45°C water bath for 1h.

[0094] 7) Similarly, continue to add 0.77 μL of 0.5M EDTA, 1.54 μL of 1M Tris, and 0.1 μL of 20 mg / mL proteinase K to the Input sample, mix by blowing, and place in a 45°C water bath for 1h.

[0095] 8) Purify the sample with a DNA purification kit, perform PCR detection, separate the PCR product by agarose gel electrophoresis, and compare the differences.

[0096] 6. Real-time fluorescent quantitative PCR

[0097] (1) Extract RNA

[0098] Trizol method was used to extract sample RNA. First, the cells were taken out into an enzyme-free EP tube, and after the supernatant was discarded, 1 mL of Trizol lysis solution was added. The cell pellet was repeatedly blown by a pipette gun, and was placed at room temperature for 5 min. Then 200 μL of chloroform was poured in, and the EP tube was vigorously shaken on a vortex shaker for 15 s. After the liquid in the EP tube turned light pink, it was placed for 3 min. Finally, it was centrifuged at 4°C, 12000 rpm for 15 min, and the sample was divided into three layers (the uppermost layer was the RNA layer, the middle layer was the DNA layer, and the bottom layer was the organic protein layer). The RNA layer was transferred to another enzyme-free EP tube, 500 μL of isopropanol was poured in, and the mixture was inverted several times. It was placed at room temperature for 10 min, and then was centrifuged at 4°C, 12000 rpm for 10 min. A white RNA precipitate was visible at the bottom of the tube. The supernatant was removed, and the precipitate was retained. Pre-cooled 75% ethanol was added to wash the RNA precipitate. The centrifuge was set to 7500 rpm for 5 min, and the RNA precipitate was washed with 75% ethanol twice. Finally, the supernatant was removed as much as possible, and the sample was dried at room temperature for 20 min. 30 μL of DEPC water was added and blown by a gun head. The RNA sample was placed in a 55°C oven for 10 min to dissolve, and finally was stored at -80°C.

[0099] (2) Reverse transcription

[0100] The RNA sample was subjected to reverse transcription. An enzyme-free octuple tube was placed in an ice box, and 4 μL of 4×gDNAwiper Mix, 1 μg of RNA, and RNase free H2O were added to make up the system to 16 μL. Then it was placed in a 42°C water bath for 2 min. After the water bath, 5×Hiscript III 4 μL was added, and finally it was placed in a PCR instrument. The parameters were set as follows: 37°C, 15 min, 85°C, 5 s, 4°C cycle. After the reverse transcription was completed, 80 μL of ddH2O was added to dilute the product.

[0101] (3) Real-time fluorescent quantitative PCR

[0102] System: 2×SYBR Green 5 μL, 0.2 μL of upstream and downstream primers, 2.6 μL of ddH2O, 2 μL of cDNA. They were sequentially added to a 96-well plate, and were shaken well after centrifugation. Then they were loaded into the machine.

[0103] 7. Immunofluorescence staining

[0104] Place round glass in 24-well plate, inoculate cells to make the confluence 60%-70%. When the cells are well attached and spread, use PBS to flush 3 times, then fix with 4% paraformaldehyde for 20 min, and then wash with PBS for 3 times. Then use 0.5% Triton X-100 to treat the cells for 5 min. Prepare 1% BSA blocking buffer and block for 1 h at room temperature. After removing the blocking buffer, directly add the primary antibody and incubate at 4°C for 24 h. Remove the primary antibody and flush with PBS, then add the corresponding species of fluorescently labeled secondary antibody, avoid light for 2 h, and finally remove the secondary antibody and flush with PBS. Finally, use Hoechst to re-stain the nucleus, avoid light for 30 min, discard the Hoechst staining solution and wash with PBS for 3 times. Use antifade reagent to mount the slide, and finally place it under an inverted fluorescence microscope to select typical fields and take pictures.

[0105] 8. Western blotting

[0106] (1) Cell protein extraction

[0107] Discard the culture medium in the culture dish, trypsinize the cells, and then add them to a sterile 1.5 mL EP tube. Set the centrifuge at 1500 rpm for 5 min, end the centrifugation, take out the EP tube, remove the supernatant, add 1 mL PBS and blow the cell pellet evenly, centrifuge at 1500 rpm for 5 min in the centrifuge. Discard the supernatant and add an appropriate amount of protein lysis buffer RIPA and protease inhibitor PMSF, and repeatedly blow the cell pellet. If the extracted protein is a nuclear protein, it needs to be ultrasonicated on ice using an ultrasonic instrument. If the extracted protein exists in the cell membrane or cytoplasm, it does not need to be ultrasonicated. After the ultrasonication is completed, place it in an ice box and shake at 200 rpm for 45 min, then centrifuge at 12000 rpm for 15 min, take out the EP tube, transfer the supernatant to a new EP tube, add 5x protein loading buffer and mix well, then place the EP tube in a 105°C metal bath for 5 min to denature the protein. At this point, the protein sample preparation is complete.

[0108] (2) Electrophoresis

[0109] Firstly, prepare the Tris-glycine electrophoresis buffer solution, the system is as follows: Tris 3.01 g, glycine 18.8 g, SDS 1 g, add ddH2O to 1 L, store at room temperature. Then prepare SDS-PAGE gel, prepare the separation gel by adding appropriate amount of ddH2O, 30% polyacrylamide, Tris-Hcl buffer (pH 8.8), 10% AP, 10% SDS and TEMED. Mix the above liquids, add to the glass plate for gel preparation, add 1 mL of isopropanol to make the liquid surface flat. Place at a constant temperature for 30 to 60 min, after the separation gel is completely hardened, remove the isopropanol on the surface, then wash with water twice, and then wipe off the water with a clean absorbent paper.

[0110] Then prepare the concentrated gel, add appropriate amount of ddH2O, 30% polyacrylamide, Tris-Hcl buffer (pH 6.8), 10% AP, 10% SDS and TEMED in turn. Mix the above liquids again, add to the glass plate for gel preparation until the liquid overflows, insert a clean comb, wait for about 30 min at room temperature until the concentrated gel solidifies. Finally, place the gel preparation plate in the electrophoresis tank and pour in an appropriate amount of electrophoresis buffer solution. Pull out the comb, add the extracted protein sample and protein Maker in order in the comb holes, connect the power supply of the electrophoresis tank, adjust the parameters to 80 V, when the sample runs to the boundary line between the concentrated gel and the separation gel, the voltage can be increased for further electrophoresis, until the protein sample runs to the bottom of the gel and the protein Maker separates from each other, stop electrophoresis.

[0111] (3) Transferring film

[0112] Firstly, prepare the film transfer buffer solution, the system is as follows: Tris 3.03 g, glycine 14.4 g, dd H2O 800 mL, methanol 200 mL, store at 4°C. Then use wet transfer method, pour pre-cooled film transfer solution into a square iron pan, place the sandwich clamp for film transfer into the film transfer solution, and put two pieces of filter paper on it. Take out the gel plate after electrophoresis, cut out the gel inside and place it on the clamp and filter paper, take a PVDF film, first soak it in methanol for polarization, then place it in ddH2O for depolarization, and finally place the film flat on the gel block. Close the sandwich clamp, place it in the film transfer instrument, set the parameters to 300 mA and appropriate time (determined according to the size of the molecule). Since the film transfer process will generate heat, causing the volatilization of methanol, some ice blocks need to be placed beside the film transfer instrument to cool down.

[0113] (4) Blocking

[0114] Prepare 5% skimmed milk blocking solution, the system is as follows: 5 g of skimmed milk powder, 100 mL of TBS film washing solution.

[0115] Then mix evenly on a shaker. Then take out the transferred membrane and seal it with the sealing liquid for 2h at room temperature.

[0116] (5) Incubate the first antibody

[0117] Rinse the membrane with TBST washing solution, then add the corresponding first antibody of the target protein in an antibody incubation box, dilute the first antibody with 5% skimmed milk sealing liquid (1:2000), and incubate the first antibody on a shaker at room temperature for 2h.

[0118] Place the membrane in a shaker at 4°C for overnight incubation.

[0119] (6) Incubate the second antibody

[0120] Take out the membrane being incubated, rinse it with TBST washing solution, add the corresponding second antibody of the species, dilute the second antibody with 5% skimmed milk sealing liquid (1:2000), and incubate the second antibody on a shaker at room temperature for 2h.

[0121] (7) Development

[0122] After the incubation of the second antibody is completed, rinse the membrane with TBST washing solution, place the membrane on a shaker for 20min,

[0123] Rinse three times. Mix ECL luminescent developing A and B liquids in a ratio of 1:1, prepare them immediately before use, drop them evenly on the membrane, develop them using a chemiluminescence instrument, and the development time and number can be determined according to the differences of different protein antibodies.

[0124] 9. Statistical analysis

[0125] All data are subjected to t-test and variance analysis, and if the p value is less than 0.05, the difference has statistical significance. Use Graphpad to analyze the difference, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001.

[0126] In summary, the MAFG transcriptional activation luciferase reporter system based on the MYC promoter provided in the application improves the specific detection ability of MAFG transcriptional activation activity through the application of the MYC promoter. And by detecting the luciferase activity, the transcriptional activation activity of MAFG can be accurately quantified. At the same time, the reporter system can be used to study the transcriptional activation function of MAFG under different cell lines and different physiological and pathological conditions, and has a wide application prospect.

Claims

1. A MAFG transcriptional activation luciferase reporter system based on MYC promoter, characterized in that: The report gene plasmid is constructed by combining MYC promoter truncation body and luciferase, and the MYC promoter truncation body comprises a sequence described in SEQ ID NO:

01.

2. The MAFG transcriptional activation luciferase reporter system based on MYC promoter according to claim 1, characterized in that: The MYC promoter truncation body is one of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04 and SEQ ID NO:

05.

3. A method for using the MAFG transcriptional activation luciferase reporter system based on the MYC promoter, characterized in that: The report system is used in claim 1 or 2.

4. The method of using a MYC promoter-based MAFG transcriptional activation luciferase reporter system according to claim 3, wherein: The steps of the use method are as follows: S1: one of the MYC promoters of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04 and SEQ ID NO: 05 or a truncation body thereof is combined with luciferase to construct a report gene plasmid; S2: the report gene plasmid is co-transfected with a MAFG overexpression plasmid and a renilla luciferase internal reference gene plasmid in HEK293T cells, and the transcription intensity of MAFG can be judged by the ratio of the luciferase fluorescence intensity to the renilla fluorescence intensity.

5. The use of the MAFG transcriptional activation luciferase reporter system based on the MYC promoter in the product for detecting the transcriptional activation ability of the transcription factor MAFG, characterized in that: The report system is the report system in claim 1 or 2.

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