Recombinant reporter plasmid for detecting human FOXP3 expression of cell line and construction and application of drug screening system of recombinant reporter plasmid
By constructing the recombinant reporter plasmid pGL-FOXP3 prompttor-reporter-FOXP3 enhancer and establishing a drug screening system based on 293 cells, the problem of difficult detection of human FOXP3 expression and screening of regulated drugs in the prior art was solved, and the effect of efficient detection and screening in human cell lines was achieved.
Patent Information
- Application Number
- CN202311221532.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-21
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art is difficult to effectively detect the expression level of human FOXP3 in cell lines, and there is a lack of methods that can effectively screen drugs that regulate FOXP3 expression in human cell lines.
A recombinant reporter plasmid pGL-FOXP3 prompttor-reporter-FOXP3 enhancer was constructed. By introducing human FOXP3 promoter and enhancer sequences into the plasmid, and using Luc or EGFP genes as reporter genes, the FOXP3 expression was effectively detected in human Jurkat cell line. At the same time, a drug screening system based on 293 cells was established, and drugs that affect FOXP3 expression were screened using PMA and TNF-α induction models.
The efficient detection of FOXP3 expression levels in human cell lines and the successful screening of drugs that can regulate FOXP3 expression provides new tools and methods for the study of autoimmune diseases and tumors.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant reporter plasmid pGL-FOXP3 promptor-reporter-FOXP3 enhancer for effectively detecting the expression level of human FOXP3 in a cell line, and the construction and application of a drug screening system thereof. Background Art
[0002] Regulatory T cells (Treg) are a type of T cell subset with immunosuppressive function related to inflammation, autoimmune diseases and tumors, mainly including natural regulatory T cells (nTreg) derived from the thymus and induced regulatory T cells (iTreg) that mature in the periphery, which can regulate the inflammatory immune response of CD4+T cells, CD8+T cells, B cells, natural killer cells, APCs, etc. Forkhead box protein P3 (FOXP3) is a key transcription factor for the development and functional level of Treg cells. Researchers have detected a decrease in FOXP3 levels or functional loss in patients with a variety of autoimmune diseases, and a decrease in its expression level or functional loss can cause severe autoimmune diseases. Therefore, the detection results of FOXP3 expression level and function can reflect the severity of inflammation and autoimmune diseases to a certain extent, and finding drugs that can regulate FOXP3 levels has become an important direction for the treatment of inflammation and autoimmune diseases. In addition, Treg plays a negative regulatory role in the occurrence and development of tumors. Therefore, finding drugs that can reduce the level of FOXP3 in Treg is also of certain significance for the treatment of tumors.
[0003] However, researchers currently use flow cytometry to detect the number of Tregs by means of classification and counting, which requires in vitro separation of lymphocytes or CD4+T cells, which is complicated to operate and expensive instruments and antibodies. In this regard, the reporter gene method has the characteristics of low price and intuitive results, and is more advantageous for FOXP3 expression detection. However, there is currently only genetic engineering technology for FOXP3 detection in mouse cell lines, which fails to effectively detect human FOXP3 expression in cell lines. The traditional mouse cell line FOXP3 detection plasmid only constructs the FOXP3 promoter, which has the significant disadvantage of being cell-specific and active only in primary T cells, but not responding in human cell lines (such as Jurkat, a human CD4+T cell line) (Pierre-YvesMantel, 2006). In addition, human cell lines can more effectively avoid germline differences in disease mechanism and drug screening research, and are more convenient and efficient. Therefore, this patent aims to construct a plasmid that can conveniently detect human FOXP3 in cell lines, and to establish a cell screening system for potential regulatory drugs (FOXP3 expression upregulation or downregulation), which has very important application prospects in basic and clinical research. Summary of the invention
[0004] In order to overcome the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a recombinant reporter plasmid pGL-FOXP3 promptor-reporter-FOXP3 enhancer for detecting the expression level of human FOXP3 in cell lines and a drug screening system thereof.
[0005] In order to achieve the technical problem of the present invention, the present invention provides the following technical solutions:
[0006] The present invention uses the basic plasmid pGL-basic as a vector, introduces a suitable human FOXP3 promoter and enhancer sequence, and reflects the level of endogenous gene FOXP3 expression. By screening the human FOXP3 gene promoter and enhancer, the -501-+186 region of human FOXP3 is used as a promoter, and the 1800-1922 region is inserted into the pGL-basic plasmid as an enhancer, and Luc and fluorescent protein genes are replaced to obtain a variety of human FOXP3 reporter recombinant plasmids. And verify that it can effectively indicate FOXP3 expression in the human Jurkat cell line. At the same time, 293 cells are used to transfect and induce the human FOXP3 reporter recombinant plasmid to establish a drug screening system.
[0007] The first aspect of the technical solution of the present invention is to provide a Figure 1 The Luc recombinant plasmid used to detect the expression level of human FOXP3 in cell lines is shown.
[0008] The Luc recombinant plasmid for detecting the expression level of human FOXP3 in the cell line includes a human FOXP3 promoter sequence (sequence 1 "Seq_1") and a human FOXP3 enhancer sequence (sequence 2 "Seq_2").
[0009] The steps of constructing the plasmid are as follows: 1) amplifying the target gene; 2) digesting the vector and homologously recombining with multiple target fragments; 3) transforming competent cells with the obtained ligation product; 4) identifying the colonies by PCR after plating and sequencing the extracted plasmid.
[0010] Application of this plasmid: Since the expression level of the FOXP3 reporter gene is controlled by the human FOXP3 promoter and enhancer, this plasmid can reflect the level of endogenous regulation of FOXP3 through the level of the reporter gene, and thus can be used to detect the immune function level of Treg cells in inflammation and autoimmune diseases
[0011] The second aspect of the technical solution of the present invention is to provide the EGFP recombinant plasmid for detecting the expression level of human FOXP3 in cell lines by replacing the reporter gene luciferase with the fluorescent protein EGFP as described in the first aspect, as shown in the attached Figure 2 shown.
[0012] This plasmid replaces the luciferase reporter gene with EGFP, so that the reporter gene expression level can be detected using a fluorescence microscope or a fluorescence detector.
[0013] The present invention only uses EGFP for replacement, but other reporter genes can be used to replace the luciferase gene, such as yellow fluorescent protein (YFP), red fluorescent protein (RFP), DsRedexpress2, mCherry, mOrange, mPlum yellow fluorescent protein (EYFP), cyan fluorescent protein (ECFP), and Sapphire.
[0014] The third aspect of the technical solution of the present invention is to provide a Luc recombinant plasmid that can effectively respond to FOXP3 signals in the human CD4+ cell line Jurkat.
[0015] The specific steps are as follows: Jurkat cells were resuspended in 1640 containing 5% FBS, and then inoculated in a 48-well plate at 100,000 / well. FOXP3-Luc plasmid transfection was performed according to the instructions of Lipofectamin 3000 transfection, and 250ng FOXP3-Luc plasmid was transfected in each well. Specifically, each well was mixed with 0.4μl Lipofectamine 3000 and 10μl Opti-MEM medium, and then mixed with 10μl Opti-MEM medium containing plasmid and 0.4μl P3000 reagent at room temperature for 15 minutes, and then 20μl was added to each well. PMA with a final concentration of 20ng / ml was added to culture for 24h, and the cells were lysed. The protein concentration was measured by G250 method and the luminescence value was detected.
[0016] The fourth aspect of the technical solution of the present invention is to provide a Figure 1 and attached Figure 2 The Luc recombinant plasmid and the EGFP plasmid are used to establish a drug screening system for detecting FOXP3 expression and its application.
[0017] The specific steps are as follows: resuspend 293 cells with DMEM complete medium and inoculate 50,000 cells / well in a 48-well plate. When the cell confluence reaches 70%, transfect FOXP3-Luc plasmid or FOXP3-EGFP and mCherry plasmid according to the Lipofectamin 3000 transfection instructions. Transfect 250ng plasmid per well, specifically: each well is mixed with 0.4μl of Lipofectamine30000 and 10μl of Opti-MEM medium, and then mixed with 10μl of Opti-MEM medium containing plasmid (250ngFOXP3-Luc plasmid or 200ngFOXP3-EGFP+50ngEGFP plasmid) containing 0.4μl P3000 reagent at room temperature for 15 minutes, and then add 20μl to each well. After adding the drug to be screened and culturing for 1-3h, add PMA with a final concentration of 20ng / ml or TNF-α with a final concentration of 1ng / ml and continue culturing for 24h. Lyse cells, measure protein concentration by G250 method and detect luminescence value or observe the results directly by fluorescence microscopy.
[0018] In order to achieve the purpose of the present invention, the present invention relates to the use of a recombinant reporter plasmid for detecting the expression level of human FOXP3 in a PMA-stimulated 293 cell model with elevated FOXP3 expression and a TNF-α-stimulated 293 cell model with reduced FOXP3 expression, and in screening drugs that affect the up-regulation or down-regulation of FOXP3 expression.
[0019] In this study, phorbol ester (PMA) and human TNF-α were used as modeling agents to construct a 293 cell model induced by PMA or TNF-α and transfected with FOXP3-Luc, which was applied to the screening of the effects of drugs such as EIE-2, MFA-1, yellow flower or MTX on the expression of human FOXP3. The results showed that when PMA was used as a stimulator, the luminescence value of luciferase reflecting the expression of human FOXP3 in 293 cells increased. When TNF-α was used as a stimulator, the luminescence value of luciferase reflecting the expression of FOXP3 in 293 cells decreased. Among them, compounds EIE-2, MFA-1, and MTX can reduce the expression of FOXP3 in 293 cells when stimulated by PMA, and at the same time, EIE-2 can increase the expression of FOXP3 in 293 cells when stimulated by TNF-α. Further, 293 cells transfected with FOXP3-Luc or FOXP3-EGFP were used to detect the luminescence or take photos with a fluorescence microscope, which confirmed that EIE-2 has the effect of bidirectionally regulating the expression of FOXP3. In summary, based on the established screening system, a compound EIE-2 that can bidirectionally regulate the balance of FOXP3 expression, as well as compounds MFA-1 and MTX that can reduce FOXP3 expression were screened, thus providing candidate drugs for the research of autoimmune diseases and tumor drugs.
[0020] Beneficial technical effects
[0021] The advantages of the present invention are: (1) the present invention is the first to construct a recombinant reporter plasmid that can effectively detect the expression of human FOXP3 in cell lines, and can be effectively expressed in human cell lines; (2) the recombinant reporter plasmid for detecting the expression of human FOXP3 of the present invention can detect the expression of FOXP3 by a variety of detection methods; (3) the present invention uses the recombinant reporter plasmid for detecting the expression of human FOXP3 to construct a 293 cell FOXP3 increase or decrease model, which can effectively screen candidate drugs that up-regulate or down-regulate FOXP3 expression. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 .pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid map
[0023] Figure 2 .pGL-FOXP3 promptor-EGFP-FOXP3 enhancer plasmid map
[0024] Figure 3 .The colony image of pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid after transformation into competent cells
[0025] Figure 4.The colony image of pGL-FOXP3 promptor-EGFP-FOXP3 enhancer plasmid after transformation into competent cells
[0026] Figure 5 .Validation of the effectiveness of pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid transfection into 293 cells
[0027] Figure 6 .Validation of the effectiveness of pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid transfection into Jurkat cells
[0028] Figure 7 .Drug screening of 293 cells transfected with pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid
[0029] Figure 8 .pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid transfection PMA stimulation 293 cell drug screening diagram
[0030] Fig. 9 .Figure 2: EIE-2 effect of 293 cells stimulated by PMA after transfection of pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid
[0031] Fig.10 .EIE-2 effect of pGL-FOXP3 promptor-EGFP-FOXP3 enhancer plasmid transfected with PMA in 293 cells
[0032] Fig.11 .EIE-2 effect of TNF-α on 293 cells transfected with pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid
[0033] Fig.12 . EIE-2 effect of TNF-α on 293 cells transfected with pGL-FOXP3 promptor-EGFP-FOXP3 enhancer plasmid
[0034] Fig.13 .Structure diagram of the 3-arylbenzofuran derivative EIE-2 compound in this laboratory DETAILED DESCRIPTION
[0035] The following examples further illustrate the present invention, but the present invention is not limited to these examples, which are entirely illustrative and are only used to specifically describe the present invention. The inventor provides a recombinant reporter plasmid for detecting human FOXP3 expression, comprising a resistance gene, a human FOXP3 promoter gene, a reporter gene, and a human FOXP3 enhancer gene. The plasmid of the present invention is transfected into human cells, and the expression of the reporter gene is promoted by activating the human FOXP3 promoter and under the synergistic effect of the human FOXP3 enhancer. The reporter gene can be a luciferase that is currently used more. By adding a substrate of luciferase, the luminescence value is detected using a luminometer to judge the expression and change of FOXP3 in the cell. Due to the problems of transfection efficiency differences, plasmid copy differences, and cell number effects of the single transfection plasmid expressing luciferase, the present invention is used to correct experimental errors by quantitatively detecting the protein content used to measure the luminescence value. In addition, by replacing the luciferase reporter gene with a green fluorescent protein gene and co-transfecting with a red fluorescent protein plasmid, the convenience of directly observing the experimental results under a fluorescence microscope is achieved. Moreover, previous studies have shown that the FOXP3 promoter has cell specificity and can only show activity in primary T cells, but not in the human cell line Jurkat. The human FOXP3-reporter plasmid of the present invention can show activity in human Jurkat cells, which is more conducive to in vitro drug screening and mechanism research. The present invention stimulates 293 cells transfected with human FOXP3-reporter gene plasmids with PMA and TNF-α, respectively, and constructs a 293 cell FOXP3 increase or decrease cell model, providing a tool for drug screening.
[0036] Example 1: Synthesis of pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid
[0037] The steps for plasmid synthesis are as follows:
[0038] 1) Extraction of human genomic DNA: Lyse normal human cells at 55°C overnight using lysis buffer, add an equal volume of phenol:chloroform:isoamyl alcohol mixture, centrifuge to obtain the upper aqueous phase, and transfer to a clean EP tube. Precipitate the genomic DNA with an equal volume of isopropanol, wash with pre-cooled 70% ethanol, dry, dissolve in water or TE and store for later use.
[0039] 2) PCR technology used primers of the promoter and enhancer of the human FOXP3 gene and human genomic DNA as a template to amplify the human FOXP3 promoter and enhancer fragments, specifically: high temperature pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, renaturation at 55-60°C for 45 s, extension at 72°C for 1 min, 30 cycles, reaction at 72°C for 10 min, and constant cooling at 4°C.
[0040] 3) The amplified human FOXP3 promoter and enhancer fragments were purified using a PCR purification kit.
[0041] 4) The purified human FOXP3 promoter and enhancer fragments were digested with restriction endonucleases of the pGL-basic plasmid.
[0042] 5) Construct a ligation system in proportion by adding 80 ng of the digested human FOXP3 promoter or enhancer fragment, 20 ng of the linearized pGL-basic plasmid, 1 μl of 10x ligation buffer, 1 U of T4 DNA ligase and water to make up to 10 μl, and ligate at 20°C for 2 h.
[0043] 6) Gently mix 5 μl of the ligated product with 50 μl of E. coli, place on ice for 30 min, heat shock at 42°C for 90 s, place on ice for 90 s, add 500 μl of SOC medium, shake on a shaker for 40 min, concentrate and inoculate on LB medium containing Amp resistance, select single clones, expand the bacteria to extract plasmids and sequence to verify. The pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid was obtained. See the attached picture of the colony growth on the plate. Figure 3 .
[0044] Example 2: Synthesis of pGL-FOXP3 promptor-EGFP-FOXP3 enhancer plasmid
[0045] The steps for plasmid synthesis are as follows:
[0046] 1) PCR technology uses EGFP primers and EGFP plasmid as a template to amplify the EGFP fragment, specifically: high temperature pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, renaturation at 55-60°C for 45 s, extension at 72°C for 1 min, 30 cycles, reaction at 72°C for 10 min, and constant cooling at 4°C.
[0047] 2) Purify the amplified EGFP fragment using a PCR purification kit.
[0048] 3) The purified EGFP fragment was digested with restriction endonuclease of pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid.
[0049] 4) Construct a ligation system according to the proportions of 80 ng of the digested EGFP fragment, 20 ng of the linearized pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid, 1 μl of 10x ligation buffer, 1 U of T4 DNA ligase and water to make up to 10 μl, and ligate at 20°C for 2 h.
[0050] 5) Gently mix 5 μl of the ligated product with 50 μl of E. coli, place on ice for 30 min, heat shock at 42°C for 90 s, place on ice for 90 s, add 500 μl of SOC medium, shake on a shaker for 40 min, concentrate and inoculate on LB medium containing Amp resistance, select single clones, expand the bacteria to extract plasmids and sequence to verify. The pGL-FOXP3 promptor-EGFP-FOXP3 enhancer plasmid was obtained. See the attached picture of the colony growth on the plate. Figure 4 .
[0051] Pharmacological application experiments:
[0052] Experimental Example 1: Effective response of pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid in 293 cell line and Jurkat cell line
[0053] Experimental methods:
[0054] 1) The bacteria successfully transformed with pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid (FOXP3-Luc plasmid for short) were amplified, and the endotoxin-free plasmid was extracted using the Tiangen plasmid extraction kit. The concentration of the extracted plasmid was measured using the double-stranded DNA concentration measurement module of the microplate reader. If necessary, the plasmid was concentrated and stored in TE buffer.
[0055] 2) After the human embryonic kidney epithelial cells 293 were revived and subcultured for 3 generations, the cells were resuspended in DMEM complete medium and inoculated in a 48-well plate at 50,000 / well. When the cell confluence reached 70%, FOXP3-Luc plasmid transfection was performed according to the Lipofectamin 3000 transfection instructions, and 250ng FOXP3-Luc plasmid was transfected per well. Specifically, each well was mixed with 0.4μl Lipofectamine3000 and 10μl Opti-MEM medium containing plasmid and 10μl Opti-MEM medium containing 0.4μl P3000 reagent at room temperature for 15min, and then 20μl was added to each well. PMA with a final concentration of 20ng / ml was added and cultured for 24h, and the cells were lysed. The protein concentration was measured by G250 method and the luminescence value was detected.
[0056] 3) After resuspending Jurkat cells in 1640 containing 5% FBS, 100,000 cells were inoculated in a 48-well plate. FOXP3-Luc plasmid transfection was performed according to the instructions of Lipofectamin3000 transfection. 250 ng FOXP3-Luc plasmid was transfected in each well. Specifically, each well was mixed with 0.4 μl of Lipofectamine 3000 and 10 μl of Opti-MEM medium, and then mixed with 10 μl of Opti-MEM medium containing plasmid and 0.4 μl P3000 reagent at room temperature for 15 minutes, and then 20 μl was added to each well. PMA with a final concentration of 20 ng / ml was added and cultured for 24 hours. The cells were lysed, and the protein concentration was measured by G250 method and the luminescence value was detected.
[0057] 4) The steps for measuring the luminescence value are as follows: Dissolve the luciferase substrate with buffer in advance, store it in a -80℃ refrigerator away from light. Before the experiment, take out the luciferase mixture from the -80℃ refrigerator in advance, thaw it in a room temperature water bath, and equilibrate it to room temperature. Dilute the 5x PLB solution with deionized water to 1x PLB and equilibrate it to room temperature. Pipette the 293 cells in the well plate and transfer them to a 2ml EP tube. Centrifuge at 2000rpm for 5min, discard the supernatant, wash once with PBS, and add an appropriate amount of 1x PLB solution. Alternatively, wash the cells directly in the well plate with PBS and lyse them with an appropriate amount of 1x PLB solution. Vortex or puff with a gun for 15s, freeze at -80℃ for 5min, shake on a shaker at room temperature for 15min, centrifuge at 12000rpm for 3min, and take the supernatant for luminescence detection. Mix 20ul of lysis supernatant with 100ul of luciferase detection solution, and test it on the machine. The machine is set to react for 2s and read the value for 10s. Data processing T test was used for comparison between two groups, one-way ANOVA test was used for comparison between multiple groups, and Dunnett's post hoc test was used for post hoc comparison. GraphPad Prism 9 software was used for data analysis and plotting.
[0058] Experimental results:
[0059] (1) Verification of the effectiveness of FOXP3-Luc plasmid transfection into 293 cells: Figure 5The results showed that compared with the blank control group (NC), the expression of luciferase in the cell lysate increased significantly after 293 cells were transfected with FOXP3-Luc plasmid, indicating that the design of FOXP3-Luc plasmid was effective; Phorbol ester PMA is a proteinase C (PKC) activator. Studies have reported that PKC can activate two downstream transcription factors, AP-1 and NFAT-1. When designing the FOXP3 promoter and enhancer sequence, AP-1 and or NFAT-1 binding sequences are included. Therefore, PMA can promote the expression of FOXP3. The experimental results showed that PMA 20ng / ml stimulation can significantly promote the increase of luciferase indicating FOXP3 expression, indicating that FOXP3-Luc plasmid can effectively respond to PMA stimulation and increase the activity of FOXP3 promoter and enhancer.
[0060] (2) Verification of the effectiveness of FOXP3-Luc plasmid transfection into Jurkat cells: Figure 6 The results showed that compared with the blank control group (NC), the expression of luciferase in the cell lysate increased significantly after Jurkat cells were transfected with FOXP3-Luc plasmid, indicating that FOXP3-Luc plasmid can effectively verify the expression of FOXP3 in Jurkat cells. The experimental results showed that PMA 20ng / ml stimulation can significantly promote the increase of luciferase indicating FOXP3 expression in Jurkat cells, indicating that FOXP3-Luc plasmid can effectively respond to PMA stimulation in Jurkat cells and increase the activity of FOXP3 promoter and enhancer, indicating that FOXP3-Luc plasmid can be successfully transfected and expressed in Jurkat cells, and can be used in the study of human CD4+T cell-related FOXP3 expression signaling pathways and drug screening.
[0061] Experimental Example 2: Screening of candidate drugs for regulating human FOXP3 expression by transfecting 293 cells
[0062] Experimental methods:
[0063] 1) Resuspend 293 cells in DMEM complete medium and inoculate 50,000 cells / well in a 48-well plate. When the cell confluence reaches 70%, transfect FOXP3-Luc plasmid according to the Lipofectamine 3000 transfection instructions, and transfect 250ng FOXP3-Luc plasmid per well. Specifically, each well is mixed with 0.4μl Lipofectamine 3000 and 10μl Opti-MEM medium, and then mixed with 10μl Opti-MEM medium containing plasmid and 0.4μl P3000 reagent at room temperature for 15 minutes, and then 20μl is added to each well. After adding MFA-1 (final concentration 10uM), EIE-2 (final concentration 10uM), goldenrod (final concentration 10uM), and methotrexate (MTX, final concentration 5uM) and culturing for 2h, PMA with a final concentration of 20ng / ml was added and cultured for 24h or directly cultured for 24h, the cells were lysed, and the protein concentration was measured by the G250 method and the luminescence value was detected.
[0064] Experimental results:
[0065] (1) Application of FOXP3-Luc in drug screening: The testing laboratory has conducted experiments on the effects of three drugs and the clinically available drug methotrexate on FOXP3 expression in 293 cells. The results are shown in the attached figure. Figure 7 As shown, MFA-1 and EIE-2 can inhibit the expression of FOXP3 in 293 cells, and the positive drug MTX can also inhibit the expression of FOXP3 in 293 cells, and it is statistically significant, but goldenrod can slightly reduce the expression of FOXP3, but there is no statistical difference.
[0066] (2) Application of FOXP3-Luc in PMA-stimulated 293 cells: The above drugs were tested for their effects on FOXP3 expression in PMA-stimulated 293 cells. Figure 8 The results showed that MFA-1, EIE-2, and MTX could reduce the increased expression of FOXP3 in 293 cells stimulated by PMA, while A. chrysanthemi had a slight increasing trend, but no statistical difference was observed.
[0067] Experimental Example 3: FOXP3-Luc and FOXP3-EGFP detection of the effect of EIE-2 on FOXP3 expression in 293 cells under different stimulation conditions
[0068] Experimental methods:
[0069] 1) The bacteria successfully transformed with pGL-FOXP3 promptor-Luc-FOXP3 enhancer plasmid (FOXP3-Luc plasmid for short), pGL-FOXP3 promptor-EGFP-FOXP3 enhancer plasmid (FOXP3-EGFP plasmid for short) and mCherry plasmid were amplified, and the endotoxin-free plasmids were extracted using the Tiangen plasmid extraction kit. The concentration of the extracted plasmids was measured using the double-stranded DNA concentration measurement module of the microplate reader. If necessary, the plasmids were concentrated and stored in TE buffer.
[0070] 2) Resuspend 293 cells with DMEM complete medium and inoculate 50,000 cells / well in a 48-well plate. When the cell confluence reaches 70%, transfect FOXP3-Luc plasmid or FOXP3-EGFP and mCherry plasmid according to the Lipofectamin 3000 transfection instructions. Transfect 250ng of plasmid per well, specifically: each well is mixed with 0.4μl of Lipofectamine 3000 and 10μl of Opti-MEM medium containing plasmid (250ngFOXP3-Luc plasmid or 200ngFOXP3-EGFP+50ngEGFP plasmid) and 10μl of Opti-MEM medium containing 0.4μl P3000 reagent at room temperature for 15 minutes, and then add 20μl to each well. After adding EIE-2 (final concentration 2.5uM, 5uM, 10uM) and MTX (final concentration 5uM) for 2h, PMA with a final concentration of 20ng / ml or TNF-α with a final concentration of 1ng / ml was added and cultured for another 24h. The cells were lysed, and the protein concentration was measured by G250 method and the luminescence value was detected or the results were observed directly by fluorescence microscopy.
[0071] Experimental results:
[0072] (1) Application of FOXP3-Luc and FOXP3-EGFP in PMA-stimulated 293 cells: To examine the dose-dependent effect of EIE-2 on FOXP3 expression in PMA-stimulated 293 cells. Fig. 9 and attached Fig.10 The results showed that EIE-2 and MTX could dose-dependently reduce the increase in FOXP3 luciferase and fluorescent protein expressions in 293 cells under PMA stimulation.
[0073] (2) Application of FOXP3-Luc and FOXP3-EGFP in TNF-α-stimulated 293 cells: TNF-α is a major inflammatory factor in the serum of patients with autoimmune diseases, and TNF-α can inhibit FOXP3 expression in autoimmune diseases. Therefore, TNF-α was used to stimulate 293 cells to construct a cell model with reduced FOXP3, simulating the inflammatory environment of reduced immune tolerance in autoimmune diseases, so as to find suitable drugs to improve immune tolerance in autoimmune diseases. Fig.11 and attached Fig.12 The results showed that EIE-2 could dose-dependently increase the expression of FOXP3 luciferase and fluorescent protein in 293 cells under TNF-α stimulation, while MTX further reduced the expression of FOXP3 in 293 cells.
[0074] In summary, the results of the present invention show that the pGL-FOXP3 promptor-reporter-FOXP3 enhancer plasmid can be effectively applied to the study of FOXP3 expression and drug screening in human Jurkat and 293 cell lines. By stimulating 293 cells with PMA or TNF-α under different conditions, a cell model with increased or decreased FOXP3 expression in 293 cells was constructed, which was applied to the screening and evaluation of drugs that regulate FOXP3 expression, and provided biological tool support for subsequent animal experiments and clinical trials of candidate drugs. Among them, the candidate compound 3-arylbenzofuran derivative EIE-2 (structure as shown in the attached figure) was selected. Fig.13 ) can maintain the balance of FOXP3 in both directions. In the active stage of rheumatoid arthritis, it has the effect of increasing the expression of FOXP3, and in the inactive stage, it has the effect of reducing the expression of FOXP3. Therefore, EIE-2 can be used as a candidate drug for the treatment of Treg immune balance regulation in autoimmune diseases for subsequent exploration and research. In the study of tumor diseases, the incidence and severity of tumors are related to the enhanced inhibitory function of Treg cells, and inhibiting the expression of FOXP3, which is mainly involved in its inhibitory function, is considered to be an effective treatment. The inventors found in the experiment that MFA-1 in this laboratory and MTX used clinically can effectively reduce the expression of FOXP3. MTX can reduce the expression of FOXP3 under a variety of stimuli, confirming the clinical application of MTX in the treatment of malignant tumors.
Claims
1. A recombinant reporter plasmid pGL-FOXP3promptor-reporter-FOXP3 enhancer for detecting the expression level of human FOXP3 in cell lines, characterized in that: They are respectively composed of resistance gene, human FOXP3 promoter, reporter gene and human FOXP3 enhancer.
2. The recombinant reporter plasmid pGL-FOXP3promptor-reporter-FOXP3 enhancer for detecting the expression level of human FOXP3 in cell lines according to claim 1, characterized in that: The resistance gene contained is the ampicillin resistance gene.
3. The recombinant reporter plasmid pGL-FOXP3promptor-reporter-FOXP3 enhancer for detecting the expression level of human FOXP3 in cell lines according to claim 1, characterized in that: The human FOXP3 promoter is the human FOXP3 gene Eukaryotic promoter database (EPD) -501-+186 base region, and the sequence is shown in sequence 1 "Seq_1".
4. The recombinant reporter plasmid pGL-FOXP3promptor-reporter-FOXP3 enhancer for detecting the expression level of human FOXP3 in cell lines according to claim 1, characterized in that: The human FOXP3 enhancer is the 1800-1922 base region of the human FOXP3 gene, and the sequence is shown in sequence 2 "Seq_2".
5. The recombinant reporter plasmid pGL-FOXP3promptor-reporter-FOXP3 enhancer for detecting the expression level of human FOXP3 in cell lines according to claim 1, characterized in that: The reporter gene can be luciferase or fluorescent protein: green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), DsRedexpress2, mCherry, mOrange, mPlum yellow fluorescent protein (EYFP), cyan fluorescent protein (ECFP), and Sapphire.
6. A method for constructing a recombinant reporter plasmid pGL-FOXP3promptor-reporter-FOXP3 enhancer for the expression level of human FOXP3 in a cell line according to any one of claims 1 to 3, characterized in that: 1) Amplify the target gene; 2) Cut the vector and homologously recombine with multiple target fragments; 3) Transform competent cells with the obtained ligation products; 4) After plating, the colonies were identified by PCR and the extracted plasmids were sequenced.
7. A method for constructing a recombinant reporter plasmid pGL-FOXP3promptor-reporter-FOXP3 enhancer for the expression level of human FOXP3 in a cell line according to any one of claims 1 to 3, characterized in that: The expression level of the reporter gene is jointly controlled by the human FOXP3 promoter and enhancer. The level of the reporter gene reflects the level of endogenous regulation of FOXP3 and can be used to detect the immune function level of Treg cells in inflammation and autoimmune diseases.
8. The recombinant reporter plasmid pGL-FOXP3 promptor-reporter-FOXP3 enhancer of the cell line human FOXP3 expression level described in any one of claims 1 to 3 can be effectively used for the study of expression in the cell line human CD4+T cell Jurkat cells.
9. A method for detecting the expression level of human FOXP3 and constructing a drug screening system using the recombinant plasmid pGL-FOXP3 promptor-reporter-FOXP3 enhancer, which is a reporter of the expression level of human FOXP3 in a cell line according to any one of claims 1 to 3, characterized in that: Using the human embryonic kidney epithelial cell family 293 cells as screening cells, a 293 cell model with elevated FOXP3 was constructed by phorbol methyl ester (PMA) stimulation, which was used to screen drugs that reduce FOXP3 expression; a 293 cell model with reduced FOXP3 was constructed by human TNF-α stimulation, which was used to screen drugs that increase FOXP3 expression.
10. The method for constructing a detection and drug screening system according to claim 9, characterized in that: The method comprises the following steps: 1) resuspending 293 cells with DMEM complete medium and seeding them in a well plate; 2) When the cell confluence reaches 60-80%, transfect the FOXP3-reporter plasmid according to the Lipofectamin 3000 transfection instructions; after adding the drug to be screened, place the cells in a cell culture incubator for 1-3 hours, and then add PMA or TNF-α to stimulate for 12-36 hours; 3) Measure the protein concentration by G250 method and detect the luminescence value or take photos directly under a fluorescence microscope.