A method for establishing a gli1 deacetylation or pseudoacetylation mutant
By identifying the acetylation sites of the GLI1 protein and constructing GLI1 deacetylated/acylated mutants, the problem of unstable acetylation status of the GLI1 protein was solved, enabling stable expression and functional studies in cells and providing a new disease treatment strategy.
Patent Information
- Application Number
- CN202610391588.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-27
- Publication Date
- 2026-06-19
AI Technical Summary
Existing technologies struggle to maintain stable levels of deacetylation/acylation of GLI1 protein, thus affecting its neuroprotective function.
By identifying the acetylation sites of the GLI1 protein, the GLI1 deacetylated/acylated mutant gene fragment was amplified, ligated to a plasmid vector, and subjected to homologous recombination and plasmid transfection. Protein expression was verified by immunofluorescence and Western blotting, and the GLI1 deacetylated/acylated mutant was constructed.
This study achieved stable maintenance of the deacetylated/acylated state of GLI1 protein within cells, enabling in-depth research into its functional changes and providing new therapeutic strategies for related diseases.
Smart Images
Figure CN122235228A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, and more particularly to a method for establishing a GLI1 deacetylated / acylated mutant. Background Technology
[0002] The acetylation state of a protein affects its transcriptional activity and the switching of downstream signaling networks. Abnormal acetylation levels at specific lysine sites often lead to altered nucleoplasmic distribution, dysregulation of protein stability, or impaired binding to key cofactors, thereby weakening its neuroprotective efficacy under pathological conditions. In existing research, GLI1 is considered one of the important regulatory molecules for neuroprotection, regulating the expression of various genes related to cell proliferation, differentiation, and cell survival. Based on this, deacetylated mutants and acetylated mimic mutants of GLI1 are constructed using site-directed mutagenesis, allowing for the artificial locking of GLI1 protein in a state of sustained activation or sustained inhibition, achieving precise regulation of GLI1 function. These mutants not only provide crucial research tools for elucidating the pathological significance of GLI1 acetylation modification in neurological diseases but also hold promise for restoring or enhancing the neuroprotective function of GLI1 through exogenous intervention, thus playing a direct interventional role in the treatment of related diseases.
[0003] However, GLI1 protein acetylation is affected by a variety of complex physiological mechanisms, making it difficult to maintain its deacetylation / acylation level within cells.
[0004] Based on this, this application proposes a method for establishing GLI1 deacetylated / acylated mutants. Summary of the Invention
[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for establishing GLI1 deacetylated / acylated mutants.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for establishing a GLI1 deacetylated / acylated mutant includes: S1: Identify the acetylation sites of GLI1 protein; S2: Amplify the GLI1 deacetylated / acylated mutant gene fragment; S3: Amplification plasmid vector; S4: The GLI1 deacetylated / acylated gene fragment is linked to the plasmid vector; S5: Immunofluorescence and Western blot (WB) were used to verify the expression of GLI1 deacetylated / acylated mutant proteins.
[0007] Preferably, the identification of GLI1 protein acetylation sites by S1 includes: S11: Transfection: Transfect 10ug of pCMV-EGFP-GLI1(human)-3Myc-Neo plasmid into 293T cells cultured in 10cm dishes; S12: Collect cell protein lysis buffer. After transfection for 24 h, wash the cells with PBS, place the culture dish on ice, and lyse the cells with RIPA protein lysis buffer. Place the cell protein lysis buffer in a 1.5 ml EP tube and incubate at 4 °C for 30 min. S13: Centrifuge the cell protein lysis buffer at 12000 rpm and 4°C for 10 min. Transfer the supernatant to a new 1.5 ml EP tube and discard the precipitate. S14: GLI1 protein enrichment: MYC-tagged magnetic beads were suspended in 1.5 ml of protein lysis supernatant in an EP tube, placed on a rotating rack, and shaken at 4°C for 12 h. S15: GLI1 protein denaturation. Magnetic beads from the S14 protein suspension were adsorbed using a magnetic rack. The protein supernatant was discarded. The magnetic beads were washed three times with 0.5% PBST, and the washing solution was discarded. 40 μl of RIPA protein lysis buffer was added to the magnetic beads, followed by 10 μl of 5×SDS-PAGE protein loading buffer. The mixture was vortexed to obtain a protein suspension. The protein suspension was then placed on a metal bath and heated at 100°C for 10 min to obtain the GLI1 protein solution. S16: Coomassie Brilliant Blue staining. The GLI1 protein solution in S15 was aspirated into one well of a 1.5 mm, 10-well SDS-PAGE protein gel. At the same time, a protein marker well was set up. Electrophoresis was performed at a constant voltage of 120 V for 1 h. After the protein marker was fully separated, the protein gel was removed and immersed in Coomassie Brilliant Blue solution and shaken at room temperature for 4 h. S17: Elute the stained gel, remove the gel, immerse it in Coomassie Brilliant Blue elution buffer, place it on a shaker at room temperature, and change the elution buffer every 30 minutes until the gel is fully eluted. S18: Collect GLI1 protein. After sufficient gel elution, a blue band can be seen at the GLI1 protein site. Cut off the band, about 1 cm², and place the protein gel in a 1.5 ml EP tube. Add enzyme-free water for preservation to obtain the GLI1 protein band. Use this band as the GLI1 protein sample to determine the GLI1 protein acetylation site.
[0008] Preferably, the amplified GLI1 deacetylated / acylated mutant gene fragment of S2 includes: S21: Design primers for GLI1 deacetylation / acylation mutants; S22: Using pCMV-EGFP-GLI1(human)-3Myc-Neo as a template, PCR amplification was performed using the designed primers to obtain GLI1 deacetylated / pseudoacylated mutant gene fragment samples. S23: Perform nucleic acid gel electrophoresis on the amplified GLI1 deacetylated / mimetic acylated mutant gene fragment sample, using DNA Marker as a control, and observe whether the amplified GLI1 deacetylated / mimetic acylated mutant gene fragment is at the corresponding molecular weight after electrophoresis. S24: The amplified GLI1 deacetylated / acylated mutant gene fragment was recovered using a nucleic acid gel recovery kit.
[0009] Preferably, the amplification plasmid vector of S3 comprises: S31: Design vector primers using pCMV-EGFP-GLI1(human)-3Myc-Neo plasmid as a template; S32: Using pCMV-EGFP-3Myc-Neo plasmid as a template, PCR amplification was performed using designed primers to obtain linearized pCMV-EGFP-3Myc-Neo vector samples; S33: Perform nucleic acid gel electrophoresis on the amplified linearized pCMV-EGFP-3Myc-Neo vector sample, using DNA Marker as a control, and observe whether the amplified linearized pCMV-EGFP-3Myc-Neo vector gene fragment is at the corresponding molecular weight after electrophoresis. S34: The amplified linearized pCMV-EGFP-3Myc-Neo vector gene fragment was recovered using a nucleic acid gel recovery kit.
[0010] Preferably, the GLI1 deacetylated / pseudoacylated gene fragment of S4 is linked to a plasmid vector, comprising: S41: Using a seamless cloning kit, the GLI1 deacetylated / pseudoacylated mutant gene fragment obtained in S24 was homologously recombined with the linearized pCMV-EGFP-3Myc-Neo vector gene fragment obtained in S34 to obtain homologous recombinant plasmid products. S42: Transform the homologous recombination plasmid product into competent cells and select positive clones containing the homologous recombination plasmid. S43: Positive clones are identified by colony PCR, and colonies with correct colony PCR results are further cultured to extract plasmids for DNA sequencing. Plasmids with correct sequencing results are GLI1 deacetylated / acylated mutant plasmids.
[0011] Preferably, the immunofluorescence and Western blot (WB) verification of GLI1 deacetylated / acylated mutant protein expression in S5 includes: S51: Transfect 2ug of the plasmid with the correct DNA sequencing results from S43 into SH-SY5Y cells (cultured in 6-well plates); S52: 24 hours after plasmid transfection, observe whether the cells have green fluorescence under a fluorescence microscope with 488 nm (blue light) excitation light; S53: 24 h after plasmid transfection, cells were lysed using RIPA protein lysis buffer. The cell protein lysis buffer was placed in a 1.5 ml EP tube and incubated at 4 °C for 30 min. S54: Centrifuge the cell protein lysis buffer at 12000 rpm and 4°C for 10 min. Transfer the protein supernatant to a new 1.5 ml EP tube and discard the precipitate. S55: Add 5×SDS-PAGE protein loading buffer at a volume ratio of protein supernatant: 5×SDS-PAGE protein loading buffer = 4:1, vortex to mix, place on a metal bath, heat at 100℃ for 10 min to obtain protein sample. S56: Perform Western blot (WB) experiments on the protein samples to verify whether the GLI1 deacetylated / acylated mutant plasmid can be successfully expressed as protein.
[0012] The beneficial effects of this invention are as follows: 1. Compared with the traditional artificial control of the acetylation modification state of GLI1 protein, protein acetylation modification is regulated by a variety of biological mechanisms. This invention constructs GLI1 deacetylated / acylated mutants to stably maintain its deacetylated / acylated state.
[0013] 2. The GLI1 deacetylated / acylated mutant of this invention can be transferred into cells / organisms to study changes in protein function, which facilitates a deeper understanding of the role of GLI1 acetylation status in cells / organisms.
[0014] 3. GLI1 is closely related to a variety of diseases. GLI1 deacetylation / acylation mutants can be used in in vitro models to help study the molecular mechanisms of disease development and provide new treatment strategies. Attached Figure Description
[0015] Figure 1 This is a flowchart illustrating a method for establishing a GLI1 deacetylated / pseudoacylated mutant proposed in this invention. Figure 2 Cellular fluorescence image of GLI1 deacetylated / acylated mutant, as presented in this invention, showing a method for establishing GLI1 deacetylated / acylated mutants. Figure 3 This is a schematic diagram of the WB results of the GLI1 deacetylated / mimetic acylated mutant, which is a method for establishing GLI1 deacetylated / mimetic acylated mutants proposed in this invention. Figure 4 This image shows the DNA sequencing results of the mutation sites of the GLI1 deacetylation / mimetic acylation mutant, which is part of the method for establishing GLI1 deacetylation / mimetic acylation mutants proposed in this invention. Detailed Implementation
[0016] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.
[0017] Example 1: A method for establishing a GLI1 deacetylated / acylated mutant, such as... Figure 1 As shown, it includes: S1: Identify the acetylation sites of GLI1 protein; S2: Amplify the GLI1 deacetylated / acylated mutant gene fragment; S3: Amplification plasmid vector; S4: The GLI1 deacetylated / acylated gene fragment is linked to the plasmid vector; S5: Immunofluorescence and Western blot (WB) were used to verify the expression of GLI1 deacetylated / acylated mutant proteins.
[0018] The identification of GLI1 protein acetylation sites in S1 includes the following steps: S11: Transfection: Transfect 10ug of pCMV-EGFP-GLI1(human)-3Myc-Neo plasmid into 293T cells cultured in 10cm dishes; S12: Collect cell protein lysis buffer. After transfection for 24 h, wash the cells with PBS, place the culture dish on ice, and lyse the cells with RIPA protein lysis buffer. Place the cell protein lysis buffer in a 1.5 ml EP tube and incubate at 4 °C for 30 min. S13: Centrifuge the cell protein lysis buffer at 12000 rpm and 4°C for 10 min. Transfer the supernatant to a new 1.5 ml EP tube and discard the precipitate. S14: GLI1 protein enrichment: MYC-tagged magnetic beads were suspended in 1.5 ml of protein lysis supernatant in an EP tube, placed on a rotating rack, and shaken at 4°C for 12 h. S15: GLI1 protein denaturation. Magnetic beads from the S14 protein suspension were adsorbed using a magnetic rack. The protein supernatant was discarded. The magnetic beads were washed three times with 0.5% PBST, and the washing solution was discarded. 40 μl of RIPA protein lysis buffer was added to the magnetic beads, followed by 10 μl of 5×SDS-PAGE protein loading buffer. The mixture was vortexed to obtain a protein suspension. The protein suspension was then placed on a metal bath and heated at 100°C for 10 min to obtain the GLI1 protein solution. S16: Coomassie Brilliant Blue staining. The GLI1 protein solution in S15 was aspirated into one well of a 1.5 mm, 10-well SDS-PAGE protein gel. At the same time, a protein marker well was set up. Electrophoresis was performed at a constant voltage of 120 V for 1 h. After the protein marker was fully separated, the protein gel was removed and immersed in Coomassie Brilliant Blue solution and shaken at room temperature for 4 h. S17: Elute the stained gel, remove the gel, immerse it in Coomassie Brilliant Blue elution buffer, place it on a shaker at room temperature, and change the elution buffer every 30 minutes until the gel is fully eluted. S18: Collect GLI1 protein. After sufficient gel elution, a blue band can be seen at the GLI1 protein site. Cut off the band, about 1 cm², and place the protein gel in a 1.5 ml EP tube. Add enzyme-free water for preservation to obtain the GLI1 protein band. Use this band as the GLI1 protein sample to determine the GLI1 protein acetylation site.
[0019] The amplified GLI1 deacetylated / acylated mutant gene fragment in S2 includes the following steps: S21: Design primers for GLI1 deacetylation / acylation mutants; sheet GLI1 deacetylation / acylation mutant primers S22: Using pCMV-EGFP-GLI1(human)-3Myc-Neo as a template, PCR amplification was performed using the designed primers to obtain GLI1 deacetylated / pseudoacylated mutant gene fragment samples. sheet GLI1 deacetylation / acylation fragment amplification system sheet GLI1 K180R / Q fragment amplification program sheet GLI1 K518R / Q fragment amplification program S23: Perform nucleic acid gel electrophoresis on the amplified GLI1 deacetylated / mimetic acylated mutant gene fragment sample, using DNA Marker as a control, and observe whether the amplified GLI1 deacetylated / mimetic acylated mutant gene fragment is at the corresponding molecular weight after electrophoresis. S24: The amplified GLI1 deacetylated / acylated mutant gene fragment was recovered using a nucleic acid gel recovery kit.
[0020] The amplification plasmid vector in S3 includes the following steps: S31: Design vector primers using pCMV-EGFP-GLI1(human)-3Myc-Neo plasmid as a template; sheet Vector primers S32: Using pCMV-EGFP-3Myc-Neo plasmid as a template, PCR amplification was performed using designed primers to obtain linearized pCMV-EGFP-3Myc-Neo vector samples; sheet GLI1 K180R / Q and GLI1 K518R / Q vector amplification systems sheet GLI1 K180R / Q and GLI1 K518R / Q vector amplification programs S33: Perform nucleic acid gel electrophoresis on the amplified linearized pCMV-EGFP-3Myc-Neo vector sample, using DNA Marker as a control, and observe whether the amplified linearized pCMV-EGFP-3Myc-Neo vector gene fragment is at the corresponding molecular weight after electrophoresis. S34: The amplified linearized pCMV-EGFP-3Myc-Neo vector gene fragment was recovered using a nucleic acid gel recovery kit.
[0021] The GLI1 deacetylated / pseudoacylated gene fragment in S4 is linked to the plasmid vector, including the following steps: S41: Using a seamless cloning kit, the GLI1 deacetylated / pseudoacylated mutant gene fragment obtained in S24 was homologously recombined with the linearized pCMV-EGFP-3Myc-Neo vector gene fragment obtained in S34 to obtain homologous recombinant plasmid products. sheet Homologous recombination system of GLI1(1-411) and phage-MCS-3×FLAG vector sheet Homologous recombination procedure S42: Transform the homologous recombination plasmid product into competent cells and select positive clones containing the homologous recombination plasmid. S43: Positive clones are identified by colony PCR. Colonies with correct PCR results are further cultured to extract plasmids for DNA sequencing. Plasmids with correct sequencing results are the GLI1 deacetylated / acylated mutant plasmids (see [reference]). Figure 4 ).
[0022] The immunofluorescence and Western blot (WB) verification of GLI1 deacetylated / acylated mutant protein expression in S5 includes the following steps: S51: Transfect 2ug of the plasmid with the correct DNA sequencing results from S43 into SH-SY5Y cells (cultured in 6-well plates); S52: 24 hours after plasmid transfection, observe the cells under a fluorescence microscope using 488 nm (blue light) excitation light to see if there is green fluorescence (see [reference]). Figure 2 ); S53: 24 h after plasmid transfection, cells were lysed using RIPA protein lysis buffer. The cell protein lysis buffer was placed in a 1.5 ml EP tube and incubated at 4 °C for 30 min. S54: Centrifuge the cell protein lysis buffer at 12000 rpm and 4°C for 10 min. Transfer the protein supernatant to a new 1.5 ml EP tube and discard the precipitate. S55: Add 5×SDS-PAGE protein loading buffer at a volume ratio of protein supernatant: 5×SDS-PAGE protein loading buffer = 4:1, vortex to mix, place on a metal bath, heat at 100℃ for 10 min to obtain protein sample. S56: Perform Western blotting on the protein samples to verify whether the GLI1 deacetylated / acylated mutant plasmid can be successfully expressed as protein (see [reference]). Figure 3 ).
[0023] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for establishing a GLI1 deacetylated or pseudoacylated mutant, characterized in that, Includes the following steps: S1: Identify acetylation sites of GLI1 protein; S2: Amplify the GLI1 deacetylated or pseudoacylated mutant gene fragment; S3: Amplification plasmid vector; S4: GLI1 deacetylated or pseudoacylated gene fragments are linked to plasmid vectors; S5: Immunofluorescence and Western blotting were used to verify the expression of GLI1 deacetylated or acylated mutant proteins.
2. The method for establishing a GLI1 deacetylated or pseudoacylated mutant according to claim 1, characterized in that, The identification of GLI1 protein acetylation sites by S1 includes: S11: Transfection: Transfect 10ug of pCMV-EGFP-GLI1-3Myc-Neo plasmid into 293T cells cultured in 10cm dishes; S12: Collect cell protein lysis buffer. After transfection for 24 h, wash the cells with PBS, place the culture dish on ice, and lyse the cells with RIPA protein lysis buffer. Place the cell protein lysis buffer in a 1.5 ml EP tube and incubate at 4 °C for 30 min. S13: Centrifuge the cell protein lysis buffer at 12000 rpm and 4°C for 10 min. Transfer the supernatant to a new 1.5 ml EP tube and discard the precipitate. S14: GLI1 protein enrichment: MYC-tagged magnetic beads were suspended in 1.5 ml of protein lysis supernatant in an EP tube, placed on a rotating rack, and shaken at 4°C for 12 h. S15: GLI1 protein denaturation. Magnetic beads from the S14 protein suspension were adsorbed using a magnetic rack. The protein supernatant was discarded. The magnetic beads were washed three times with 0.5% PBST, and the washing solution was discarded. 40 μl of RIPA protein lysis buffer was added to the magnetic beads, followed by 10 μl of 5×SDS-PAGE protein loading buffer. The mixture was vortexed to obtain a protein suspension. The protein suspension was then placed on a metal bath and heated at 100°C for 10 min to obtain the GLI1 protein solution. S16: Coomassie Brilliant Blue staining. The GLI1 protein solution in S15 was aspirated into one well of a 1.5 mm, 10-well SDS-PAGE protein gel. At the same time, a protein marker well was set up. Electrophoresis was performed at a constant voltage of 120 V for 1 h. After the protein marker was fully separated, the protein gel was removed and immersed in Coomassie Brilliant Blue solution and shaken at room temperature for 4 h. S17: Elute the stained gel, remove the gel, immerse it in Coomassie Brilliant Blue elution buffer, place it on a shaker at room temperature, and change the elution buffer every 30 minutes until the gel is fully eluted. S18: Collect GLI1 protein. After sufficient gel elution, a blue band can be seen at the GLI1 protein site. Cut off the band, about 1 cm², and place the protein gel in a 1.5 ml EP tube. Add enzyme-free water for preservation to obtain the GLI1 protein band. Use this band as the GLI1 protein sample to determine the GLI1 protein acetylation site.
3. The method for establishing a GLI1 deacetylated or pseudoacylated mutant according to claim 1, characterized in that, The amplified GLI1 deacetylated or pseudoacylated mutant gene fragment of S2 includes: S21: Design primers for GLI1 deacetylation or pseudoacylation mutants; S22: Using pCMV-EGFP-GLI1-3Myc-Neo as a template, PCR amplification was performed using the designed primers to obtain GLI1 deacetylated or pseudoacylated mutant gene fragment samples. S23: Perform nucleic acid gel electrophoresis on the amplified GLI1 deacetylated or pseudoacylated mutant gene fragment sample, using DNA Marker as a control, and observe whether the amplified GLI1 deacetylated or pseudoacylated mutant gene fragment is at the corresponding molecular weight after electrophoresis. S24: Use a nucleic acid gel recovery kit to recover the amplified GLI1 deacetylated or pseudoacylated mutant gene fragment.
4. The method for establishing a GLI1 deacetylated or pseudoacylated mutant according to claim 1, characterized in that, The amplification plasmid vector of S3 includes: S31: Design vector primers using pCMV-EGFP-GLI1-3Myc-Neo plasmid as a template; S32: Using pCMV-EGFP-3Myc-Neo plasmid as a template, PCR amplification was performed using designed primers to obtain linearized pCMV-EGFP-3Myc-Neo vector samples; S33: Perform nucleic acid gel electrophoresis on the amplified linearized pCMV-EGFP-3Myc-Neo vector sample, using DNA Marker as a control, and observe whether the amplified linearized pCMV-EGFP-3Myc-Neo vector gene fragment is at the corresponding molecular weight after electrophoresis. S34: The amplified linearized pCMV-EGFP-3Myc-Neo vector gene fragment was recovered using a nucleic acid gel recovery kit.
5. A method for establishing a GLI1 deacetylated or pseudoacylated mutant according to claim 1, characterized in that, The GLI1 deacetylated or pseudoacylated gene fragment of S4 is linked to the plasmid vector, including: S41: Using a seamless cloning kit, the deacetylated or pseudoacylated mutant gene fragment of GLI1 obtained in S24 was homologously recombined with the linearized pCMV-EGFP-3Myc-Neo vector gene fragment obtained in S34 to obtain homologous recombinant plasmid products. S42: Transform the homologous recombination plasmid product into competent cells and select positive clones containing the homologous recombination plasmid. S43: Positive clones are identified by colony PCR, and colonies with correct colony PCR results are further cultured to extract plasmids for DNA sequencing. Plasmids with correct sequencing results are GLI1 deacetylated or pseudoacylated mutant plasmids.
6. A method for establishing a GLI1 deacetylated or pseudoacylated mutant according to claim 1, characterized in that, The immunofluorescence and Western blotting verification of GLI1 deacetylated or pseudoacylated mutant protein expression by S5 includes: S51: Transfect 2 μg of plasmid with the correct DNA sequencing results from S43 into a 6-well plate of SH-SY5Y cells for culture. S52: 24 hours after plasmid transfection, observe whether the cells have green fluorescence under a fluorescence microscope using 488 nm blue light excitation. S53: 24 h after plasmid transfection, cells were lysed using RIPA protein lysis buffer. The cell protein lysis buffer was placed in a 1.5 ml EP tube and incubated at 4 °C for 30 min. S54: Centrifuge the cell protein lysis buffer at 12000 rpm and 4°C for 10 min. Transfer the protein supernatant to a new 1.5 ml EP tube and discard the precipitate. S55: Add 5×SDS-PAGE protein loading buffer at a volume ratio of protein supernatant: 5×SDS-PAGE protein loading buffer = 4:1, vortex to mix, place on a metal bath, heat at 100℃ for 10 min to obtain protein sample. S56: Perform Western blot (WB) experiments on the protein samples to verify whether the GLI1 deacetylated or pseudoacylated mutant plasmids can be successfully expressed as proteins.