Method for detecting and / or screening target protein expression cell line based on fluorescence indication

Abstract

The invention relates to a method for detecting and / or screening a target protein expression cell line based on fluorescence indication, and aims to realize rapid detection of specific protein conformation in chassis cells and efficiently screen chassis cells with different functional states, activities and target protein expression levels. The essential difference between the technology and a conventional protein fusion fluorescent label for indicating protein is that the conformation change of the protein is reflected through a cross-linking agent, and the functional state corresponding to the conformation of the protein is reflected based on the change of the fluorescence color of a cross-linking agent skeleton under different protein conformations. The technology provides a convenient, efficient and non-invasive method for the field of biological research, and can be widely applied to the fields of cell activity research, drug screening, protein function research and the like.

Description

Technical Field

[0001] The present invention relates to a method for detecting and / or screening a target protein expressing cell line based on fluorescence indication. Background Art

[0002] Protein expression and structural detection are core issues in biological science research. As the main functional macromolecule in the life process, proteins play various important functions in cells, including signal transduction, metabolic regulation, structural support, etc. Their expression level and structural state directly affect the physiological processes and functional performance of organisms (Nat. Rev. Mol. Cell Biol., 2020, 21, 327-340). Therefore, accurately characterizing the expression level and structural state of proteins is of vital importance for understanding cell biological processes, disease mechanisms and new drug development.

[0003] At present, in protein expression detection, except for a few proteins with self-luminous properties, most of the remaining proteins usually need to be detected by fusing fluorescent expression tags. However, for endogenous proteins that do not have self-luminous properties, it is difficult to accurately indicate their expression and functional status in an in situ environment. Although existing methods such as mass spectrometry can detect protein expression and its conformational state (Proc. Natl. Acad. Sci. USA 2021, 118, e2023360118; J. Biol. Chem. 2017, 292, 1187-1196), these methods usually require cell lysis, thereby destroying the expression and functional state of the protein in the in situ environment. Such methods are difficult to directly detect the expression and functional state of proteins without fluorescent tags in cells, thereby limiting the application of screening and cultivation of target proteins.

[0004] Therefore, a new technology is needed to address the current limitations of protein expression detection. This technology should be able to directly indicate whether the target protein is expressed and characterize its functional status in an in situ environment without labeling or destroying the cell environment. Such innovative technology will provide more efficient and accurate means for cell screening and protein function research, promote the development of the field of cytology, and provide reliable technical support for protein research, biological research, disease treatment and drug development. Summary of the invention

[0005] The present invention provides a method for detecting and / or screening a target protein expression cell line based on fluorescence indication. This technology can realize rapid detection of the expression level and / or structural state of a specific protein in a cell, and can efficiently screen chassis cells of different functional states. By targeting a cross-linking agent with targeting and fluorescence properties to the target protein in the chassis cell for chemical cross-linking reaction, the conformation of the target protein itself and / or the difference in the chemical microenvironment are utilized to cause changes in the cross-linking agent backbone conformation, causing the cross-linking agent to emit fluorescence of different colors, so as to realize accurate and real-time characterization of the protein structure in the chassis cell.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] 1) Design and construct chassis cells of target protein: Transfect the plasmid of target protein into one of the chassis cells such as HEK 293T, HeLa, Escherichia coli, yeast or Bacillus subtilis with lentivirus, culture the cells or bacteria, and use one or more of puromycin, G418, kanamycin and tetracycline to continuously screen the chassis cells transfected with lentivirus for 1 day to one month to obtain chassis cells that can stably express target protein, so as to achieve overexpression of target protein in chassis cells.

[0008] 2) Design of targeted fluorescent indicator crosslinkers:

[0009] (a) the reactive groups of the cross-linking agent are selected from one or more of benzenesulfonyl fluoride group, phenoxysulfonyl fluoride group, N-hydroxysuccinimide group, o-phthalaldehyde group, and diaziridine group, and the reaction rate is 1s-1h and the reaction efficiency is 80%-100%;

[0010] (b) The length of the spacer arm of the cross-linking agent is

[0011] (c) making the crosslinking agent water-soluble by introducing one or more of a hydroxyl group, an amide group, a sulfonic acid group, an amino group, a carboxyl group, and a polyethylene glycol group into the crosslinking agent backbone;

[0012] (d) achieving membrane permeability of the crosslinker by introducing one or more of an amino group, a positively charged group, an amide group, and a polyethylene glycol group into the crosslinker backbone;

[0013] (e) achieving crosslinker backbone targeting by introducing into the crosslinker backbone one or more of an inhibitor of the target protein, an affinity group of the target protein, an antibody of the target protein, and a charge opposite to that of the target protein;

[0014] (f) realizing the fluorescence property of the crosslinker skeleton by introducing one or more of the fluorescent molecules rhodamine, fluorescein, and AlexaFluor series molecules and their derivatives into the crosslinker skeleton;

[0015] 3) Harvesting of chassis cells: scraping cells or digesting with trypsin to obtain cells on the culture dish, and then collecting and washing by centrifugation to obtain chassis cell samples, washing the chassis cells with one or more buffers selected from ammonium bicarbonate buffered saline solution, phosphate buffered saline solution, tris(hydroxymethyl)aminomethane buffered saline solution or 4-hydroxyethylpiperazineethanesulfonic acid buffered saline solution at a pH of 7.0-10.0 to remove impurities such as the culture medium during the chassis cell culture process, and collecting the washed chassis cell samples by centrifugation;

[0016] 4) Preparation of cross-linking solution: prepare the cross-linking agent described in 2) into a solution with a concentration of 0.1 mM-1 M using one or more organic solvents selected from organic alcohols, aprotic polar solvents, organic bases, and organic acids, and add the solution to one or more cell buffer solutions selected from ammonium bicarbonate buffer solution, phosphate buffer solution, 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, or tris(hydroxymethyl)aminomethane buffer solution at a pH of 7.1-10, so that the final concentration of the cross-linking agent is 0.1 mM-100 mM;

[0017] 5) Cross-linking reaction: add the cross-linking solution obtained in 4) to the chassis cell sample collected in 3), the volume ratio of the cross-linking agent solution to the chassis cells is 100:1-1:1, use one or more of a pipette, a dropper, and a mixer to evenly disperse the chassis cells in the cross-linking solution, and perform the cross-linking reaction in one or more of a constant temperature mixer, an oscillator, a shaker, and a test tube rack, the temperature is set at 20-50°C, and the cross-linking reaction time is 1s-1h.

[0018] 6) Harvesting chassis cells that have completed the cross-linking reaction: Collect the cross-linked chassis cell sample by centrifugation, and wash the chassis cells with one or more buffers selected from ammonium bicarbonate buffer solution, phosphate buffer solution, tris buffer solution or 4-hydroxyethylpiperazineethanesulfonic acid buffer solution at a pH of 7.0-10.0 to wash away the unreacted cross-linking agent.

[0019] 7) Protein conformation detection in chassis cells: Monitor the fluorescence signals emitted by the cross-linker backbone in different conformational states through one or more of the following techniques: fluorescence resonance energy transfer, fluorescence activated cell sorting, fluorescence microscopy sorting, and fluorescence live cell imaging sorting, so as to obtain information on the structural state of the target protein of the cross-linker in chassis cells.

[0020] 8) Screening and separation of chassis cells based on the results of conformation detection: By detecting the changes in the fluorescence signal emitted by the crosslinker skeleton through one or more of the following methods: fluorescence resonance energy transfer technology, fluorescence activated cell sorting technology, fluorescence microscopy sorting, and fluorescence live cell imaging sorting, the expression level and structural state of proteins in different chassis cells can be determined. Based on this information, chassis cells in a specific conformational state can be selected to achieve efficient sorting and screening of chassis cells in different functional states.

[0021] The method of the present invention has the following advantages:

[0022] 1) Rapid detection of specific protein expression levels: This technology can quickly detect the expression levels of specific proteins in chassis cells, greatly shortening the research process and time;

[0023] 2) Efficient screening of chassis cells with different functional states: By analyzing the different color fluorescence signals emitted by the cross-linking agent, chassis cells with different functional states can be efficiently screened, thereby distinguishing the structural state of intracellular proteins;

[0024] 3) Convenient and non-invasive: This technology uses a non-invasive method, which will not cause additional impact on the chassis cells, maintain the original state of the cells, and ensure the reliability and repeatability of the research results;

[0025] 4) Provide accurate intracellular information: The application of quantitative analysis of fluorescence signals and high-resolution fluorescence microscopy enables this technology to provide accurate structural information of target proteins within cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of a method for detecting and / or screening target protein-expressing cell lines based on fluorescence indication.

[0027] Figure 2 This is a confocal image of the target protein in the cross-linked chassis. Only the cross-linked target protein has fluorescent properties. DETAILED DESCRIPTION

[0028] The present invention provides the following embodiments to illustrate the present invention, but does not limit the protection scope of the present invention. Although the present invention is described in detail with reference to the embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0029] Synthesis of targetable fluorescent cross-linkers:

[0030]

[0031] Synthesis of compound 3:

[0032] Compound 1 (2.93 g, 8 mmol, rhodamine 110) was dissolved in 80 ml DMSO, and compound 2 (4.2 g, 8 mmol, reference: Hang Gao, et al., Anal. Chem. 2022, 94, 21, 7551–7558) was dissolved in 20 ml DMSO and added dropwise to the solution. Stir at room temperature for 5 min and purify by semi-preparative reverse phase HPLC (Hanbang Technology, model: NS4000, A: volume concentration 0.1% TFA / H2O, B: ACN, linear gradient method set to: B: 20%-50%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring). Freeze-dried at 30 ° C for 24 h to obtain 1.6 g of the target product, which was a light yellow viscous oil, with a yield of 38.1%, LCMS [M+H] + = 740.23.

[0033] Synthesis of compound 5:

[0034] Dissolve compound 3 (1.6 g, 2.16 mmol) in 50 ml DMSO, dissolve compound 4 (865 mg, 4 mmol, Bis-PEG2-NHS ester) in 10 ml DMSO, and drop into the solution. Stir at room temperature for 5 min, and purify by semi-preparative reverse phase high performance liquid chromatography (Hanbang Technology, model: NS4000, A: volume concentration 0.1% TFA / H2O, B: ACN, linear gradient method set to: B: 30%-60%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring). Freeze-dry at 30 ° C for 24 h to obtain 720 mg of the target product, which is a light yellow viscous oil, with a yield of 45%, LCMS [M+H] + = 1025.32.

[0035] Synthesis of compound 7:

[0036] Dissolve compound 5 (720 mg, 0.7 mmol) in 10 ml DMSO, and dissolve compound 6 (143 mg, 0.7 mmol) in 1.5 ml DMSO, and drip into the solution. Stir at room temperature for 5 min, and purify by semi-preparative reverse phase high performance liquid chromatography (Hanbang Technology, model: NS4000, A: volume concentration 0.1% TFA / H2O, B: ACN, linear gradient method set to: B: 30%-70%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring). Freeze-dry at 30 ° C for 24 h to obtain 216 mg of the target product, which is a light yellow viscous oil, with a yield of 30%, LCMS [M+H] + = 1113.33.

[0037] Synthesis of compound 9:

[0038] Compound 7 (216 mg, 0.19 mmol) was dissolved in 5 ml DMSO, and compound 6 (39 mg, 0.19 mmol) was dissolved in 1 ml DMSO, and then added dropwise to the solution. Stirred at room temperature for 5 min, and purified by semi-preparative reverse phase high performance liquid chromatography (Hanbang Technology, model: NS4000, A: volume concentration 0.1% TFA / H2O, B: ACN, linear gradient method set to: B: 30%-70%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring). Freeze dried at 30 ° C for 24 h to obtain 110.2 mg of the target product, which was a light yellow viscous oil, with a yield of 51%, LCMS [M+H] + = 1201.35. The structure of the compound was confirmed to be consistent with the expected structure by nuclear magnetic resonance technology, mass spectrometry, and liquid chromatography.

[0039] Synthesis of compound 10:

[0040] Compound 7 (216 mg, 0.19 mmol) was dissolved in 5 ml DMSO, and compound 8 (42 mg, 0.19 mmol) was dissolved in 1 ml DMSO, and then dropped into the solution. Stir at room temperature for 5 min, and purify by semi-preparative reverse phase high performance liquid chromatography (Hanbang Technology, model: NS4000, A: volume concentration 0.1% TFA / H2O, B: ACN, linear gradient method set to: B: 30%-70%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring). Freeze-dried at 30 ° C for 24 h, 105.8 mg of the target product was obtained, the properties were light yellow viscous oil, yield: 49%, LCMS [M+H] + = 1217.32. The structure of the compound was confirmed to be consistent with the expected structure by nuclear magnetic resonance technology, mass spectrometry, and liquid chromatography.

[0041] Synthesis of cross-linker 1:

[0042] Compound 9 (110.2 mg, 0.09 mmol) was dissolved in 3 ml PBS, and ubquitin antibody (0.2 mmol) was added to the solution. The reaction was carried out at room temperature for 90 min, and semi-preparative reverse phase high performance liquid chromatography (Hanbang Technology, model: NS4000, A: volume concentration 0.1% TFA / H2O, B: ACN, linear gradient method set to: B: 50%-70%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring) was used for purification. The target product was obtained by freeze drying at 30°C for 24 h.

[0043] Synthesis of cross-linker 2:

[0044] Compound 10 (105.8 mg, 0.09 mmol) was dissolved in 3 ml PBS, and SIRT2 antibody (0.2 mmol) was added to the solution. The reaction was carried out at room temperature for 90 min, and semi-preparative reverse phase high performance liquid chromatography (Hanbang Technology, model: NS4000, A: volume concentration 0.1% TFA / H2O, B: ACN, linear gradient method set to: B: 50%-70%, 30 min, flow rate of 50 mL / min, 200 nm UV wavelength monitoring) was used for purification. The target product was obtained by freeze drying at 30°C for 24 h.

[0045] Synthesis of cross-linker 3:

[0046] Compound 10 (105.8 mg, 0.09 mmol) was dissolved in 3 ml PBS, and FGL1 antibody (0.2 mmol) was added to the solution. The mixture was reacted at room temperature for 90 min, and then purified by semi-preparative reverse phase HPLC (Hanbang Technology, model: NS4000, A: volume concentration 0.1% TFA / H2O, B: ACN, linear gradient method set to: B: 50%-70%, 30 min, flow rate 50 mL / min, 200 nm UV wavelength monitoring). The mixture was freeze-dried at 30°C for 24 h to obtain the target product.

[0047] The ubquitin sequence is:

[0048] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG

[0049] The SIRT2 sequence is:

[0050] MEGVPADLRAKQSVVCTGSFYEEYDTLETMRNLVVLDAGSEAVWRALELNLVDCKYKSHPHPEFNLVVPRGRSLGKGLDFIYKMKGDSPFRGKFAVYAGVADTQPLEKGIDPNSKGQKPFVTDYRRKHTLQDNIVFNLPNVSFYETAK DLTKWVDIQHCFKGHAEFSFSYRRGEVNFEYGDYHVVGTAEVSLANAKNDTWTGERKVLTTHFNVIDFLENKLQTDKRDEAPYQGVPFIFELIDGSSVHRNIYIWDTAGGSSVAEAQGESSSDPSMKGPDASDEDSQQESGKQETSLD

[0051] The FGL1 sequence is:

[0052] MVPPLLLLSGLALWLQLCSLFSLHSTLPDSQVCQDLQTRKKRLHVCADARFYHLPLSAQQAEGKTIYFPDLHATPKDITQIGLQNGIRVWVHKGAHVVPNRVCEKFIHHSTNYLQKVAEYFQSGKGQSESTEHLLKASSLNDPLPKCKRLVVPDDIFGVEGFHRGEYLFSVPGPNELEFPRNPMSLGAADWPKFLSHEHH

[0053] Example 1

[0054] 1) Construction of chassis cells expressing the target protein: The ubquitin gene was transfected into HEK 293T cells through the NcmDH5-α vector (reference: Mingen Lin, et al., J Clin Invest. 2023, 133, e164528), and the cells were cultured. Puromycin was used to continuously screen the chassis cells transfected with lentivirus (reference: MingenLin, et al., J Clin Invest. 2023, 133, e164528), and the chassis cells stably expressing the target protein were cultured to a density of 80%-90%.

[0055] 2) Design of targeted fluorescent indicator crosslinker 1: The reactive group of the crosslinker is selected as benzenesulfonyl fluoride group, the reaction time is 1 min, and the reaction efficiency is 50%; the length of the spacer arm of the crosslinker is The water solubility of the crosslinker is achieved by introducing polyethylene glycol groups into the crosslinker skeleton, and the membrane permeability of the crosslinker is achieved by introducing amide groups; the targeting of the crosslinker skeleton is achieved by introducing antibodies of target proteins into the crosslinker skeleton, and the fluorescent molecule rhodamine is introduced to achieve the fluorescent properties of the crosslinker skeleton; the preparation process and structure of crosslinker 1 are shown in the section "Synthesis of targetable crosslinkers with fluorescent properties";

[0056] 3) Harvesting chassis cells: gently place the scraper of the cell scraper on the cell culture dish, and then scrape along the surface to scrape the chassis cell sample, so as to collect the chassis cell sample by centrifugation, wash the chassis cells with 10mM phosphate buffered saline solution with a pH of 7.4 to remove impurities such as the culture medium during the chassis cell culture process, and collect the washed chassis cell sample by centrifugation;

[0057] 4) Preparation of cross-linking solution: The cross-linking agent described in 2) is prepared into a 1 M solution using a non-protonic polar solvent dimethyl sulfoxide, and added to a 10 mM phosphate buffered saline solution at a pH of 7.4, so that the final concentration of the cross-linking agent is 100 mM;

[0058] 5) Cross-linking reaction: add the cross-linking solution obtained in 4) to the chassis cell sample collected in 3), the volume ratio of the cross-linking agent solution to the chassis cells is 10:1, blow the chassis cells with a pipette to evenly disperse them in the cross-linking solution, and carry out the cross-linking reaction in a constant temperature mixer at 37°C for 1 min.

[0059] 6) Harvesting chassis cells that have completed the cross-linking reaction: Collect the cross-linked chassis cell samples by centrifugation, and wash the chassis cells with 10 mM phosphate buffered saline solution with a pH of 7.4 to wash away the unreacted cross-linking agent.

[0060] 7) Protein conformation detection in chassis cells: Use fluorescence live cell imaging sorting technology to monitor the fluorescence signals emitted by the cross-linker backbone in different conformational states (reference: Daniel Schraivogel et al., High-speed fluorescence image–enabled cell sorting.

[0061] Science 2022,375,315-320.), thereby obtaining information on the structural state of the target protein of the crosslinker in the chassis cells. The red fluorescent signal indicates that the target protein is in a compact structural state (the protein structure is similar to a sphere at this time), while the green fluorescent signal indicates that the target protein is in an extended structural state (the protein structure is similar to a line at this time). The absence of fluorescent signal indicates that the crosslinker has not undergone crosslinking reaction with the target protein. Figure 2 shown.

[0062] 8) Screen and separate chassis cells based on the results of conformation detection: Detect the fluorescence signal emitted by the crosslinker backbone through fluorescent live cell imaging sorting technology to determine the expression level and structural state of proteins in different chassis cells. The stronger the fluorescence signal, the higher the expression level of the target protein. See the attached experimental process. Figure 1 . Through the fluorescent live cell imaging sorting technology, the chassis cells emitting red fluorescent signals are sorted into the collection pool, and the ones emitting green fluorescent signals are discarded, thereby isolating the chassis cells with the target protein in a compact structure state. Next, through the fluorescent live cell imaging sorting technology, the chassis cells emitting strong red fluorescent signals are sorted into the collection pool (the red fluorescent signal reaches 10 2 The cells with weak red fluorescence signals were discarded, thereby isolating the chassis cells with high expression of the target protein with a compact structure. The number of cells obtained in this step is 10% of the total number of initial cells.

[0063] 9) Application: Collect and select chassis cells that emit red fluorescence signals. The target protein of the chassis cells is in a compact structural state. By adding the interacting protein of the target protein, when the target protein interacts with the interacting protein, the conformation of the target protein becomes more compact (from a loose spherical structure to a solid spherical structure), so that the cross-linking agent skeleton emits a stronger red fluorescence signal (the fluorescence signal reaches 10 3 The above), fluorescence resonance energy transfer analysis again can screen out the target proteins that interact with each other for protein function research and analysis.

[0064] Example 2

[0065] 1) Construction of chassis cells expressing the target protein: The SIRT2 gene was transfected into HEK 293T cells through the NcmDH5-α vector (reference: Mingen Lin, et al., J ClinInvest.2023,133,e164528), and the cells were cultured. Puromycin was used to continuously screen the chassis cells transfected with lentivirus (reference: Mingen Lin, et al., J ClinInvest.2023,133,e164528), and the chassis cells stably expressing the target protein were cultured to a density of 80%-90%.

[0066] 2) Design of targeted fluorescent indicator crosslinker 2: The reactive groups of the crosslinker are selected as benzenesulfonyl fluoride group and benzenesulfonyl chloride group, the reaction time is 5 min, and the reaction efficiency is 40%; the length of the spacer arm of the crosslinker is The water solubility of the crosslinker is achieved by introducing polyethylene glycol groups into the crosslinker skeleton, and the membrane permeability of the crosslinker is achieved by introducing amide groups; the targeting of the crosslinker skeleton is achieved by introducing antibodies of target proteins into the crosslinker skeleton, and the fluorescent molecule rhodamine is introduced to achieve the fluorescent properties of the crosslinker skeleton; the preparation process and structure of crosslinker 2 are shown in the section "Synthesis of targetable crosslinkers with fluorescent properties";

[0067] 3) Harvesting chassis cells: gently place the scraper of the cell scraper on the cell culture dish, and then scrape along the surface to scrape the chassis cell sample, so as to collect the chassis cell sample by centrifugation, wash the chassis cells with a 10mM 4-hydroxyethylpiperazineethanesulfonic acid buffer solution with a pH of 7.0 to remove impurities such as the culture medium during the chassis cell culture process, and collect the washed chassis cell sample by centrifugation;

[0068] 4) Preparation of cross-linking solution: prepare the cross-linking agent described in 2) into a solution with a concentration of 300 mM with deionized water, and add the solution to a 10 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer solution with a pH of 7.0, so that the final concentration of the cross-linking agent is 30 mM;

[0069] 5) Cross-linking reaction: add the cross-linking solution obtained in 4) to the chassis cell sample collected in 3), the volume ratio of the cross-linking agent solution to the chassis cells is 8:1, blow the chassis cells with a pipette to evenly disperse them in the cross-linking solution, and carry out the cross-linking reaction in a constant temperature mixer at 25°C for 5 minutes.

[0070] 6) Harvesting chassis cells that have completed the cross-linking reaction: Collect the chassis cell samples after cross-linking by centrifugation, and wash the chassis cells with 10 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer solution with a pH of 7.0 to wash away the unreacted cross-linking agent.

[0071] 7) Protein conformation detection in chassis cells: Use fluorescence resonance energy transfer technology to monitor the fluorescence signals emitted by the cross-linker backbone in different structural states (reference: Erika Cecon et al., SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET. Cell Chem. Biol. 2022, 29, 74-83), so as to obtain the structural state information of the target protein of the cross-linker in chassis cells. The red fluorescence signal indicates that the target protein is in a compact structural state (the protein structure is similar to a sphere at this time), while the green fluorescence signal indicates that the target protein is in an extended structural state (the protein structure is similar to a line at this time), and the absence of a fluorescent signal indicates that the cross-linker has not undergone a cross-linking reaction with the target protein.

[0072] 8) Screen and separate chassis cells based on the results of conformation detection: The fluorescence signal emitted by the cross-linker backbone is detected by fluorescence resonance energy transfer technology, which can determine the expression level and structural state of proteins in different chassis cells. The stronger the fluorescence signal, the higher the expression level of the target protein. Through fluorescence resonance energy transfer technology, chassis cells that emit red fluorescence signals are sorted into a collection pool, and those that emit green fluorescence signals are discarded, thereby isolating chassis cells with target proteins in a compact structural state. Next, chassis cells that emit strong red fluorescence signals are sorted into a collection pool (red fluorescence signals reach 10 2 The cells with weak red fluorescence signals were discarded, thereby isolating the chassis cells with high expression of the target protein in a compact structure. The number of cells obtained in this step was 13% of the total number of initial cells.

[0073] 9) Application: Collect and select chassis cells that emit red fluorescence signals. The target protein of the chassis cells is in a compact structural state. By adding the interacting protein of the target protein, when the target protein interacts with the interacting protein, the conformation of the target protein becomes more compact (from a loose spherical structure to a solid spherical structure), so that the cross-linking agent skeleton emits a stronger red fluorescence signal (the fluorescence signal reaches 10 3 The above), fluorescence resonance energy transfer analysis again can screen out the target proteins that interact with each other for protein function research and analysis.

[0074] Example 3

[0075] 1) Construction of chassis cells expressing target proteins: The FGL1 gene was transfected into HEK 293T cells via NcmDH5-α vector (reference: Mingen Lin, et al., J Clin Invest.

[0076] 2023,133,e164528), cell culture was performed, and puromycin was used to continuously screen the lentiviral transfected chassis cells (reference: Mingen Lin, et al., J Clin Invest.

[0077] 2023,133,e164528), and culture the chassis cells stably expressing the target protein to 80%-90% density.

[0078] 2) Design of targeted fluorescent indicator crosslinker 3: The reactive groups of the crosslinker are selected as benzenesulfonyl fluoride group and benzenesulfonyl chloride group, the reaction time is 5 min, and the reaction efficiency is 55%; the length of the spacer arm of the crosslinker is The water solubility of the crosslinker is achieved by introducing polyethylene glycol groups into the crosslinker skeleton, and the membrane permeability of the crosslinker is achieved by introducing amide groups; the targeting of the crosslinker skeleton is achieved by introducing antibodies of target proteins into the crosslinker skeleton, and the fluorescent molecule rhodamine is introduced to achieve the fluorescent properties of the crosslinker skeleton; the preparation process and structure of crosslinker 3 are shown in the section "Synthesis of targetable crosslinkers with fluorescent properties";

[0079] 3) Harvesting chassis cells: gently place the scraper of the cell scraper on the cell culture dish, and then scrape along the surface to scrape the chassis cell sample, so as to collect the chassis cell sample by centrifugation, wash the chassis cells with a 10mM 4-hydroxyethylpiperazineethanesulfonic acid buffer solution with a pH of 7.0 to remove impurities such as the culture medium during the chassis cell culture process, and collect the washed chassis cell sample by centrifugation;

[0080] 4) Preparation of cross-linking solution: prepare the cross-linking agent described in 2) into a solution with a concentration of 500 mM using deionized water, and add the solution to a 10 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer solution with a pH of 7.0, so that the final concentration of the cross-linking agent is 50 mM;

[0081] 5) Cross-linking reaction: add the cross-linking solution obtained in 4) to the chassis cell sample collected in 3), the volume ratio of the cross-linking agent solution to the chassis cells is 5:1, blow the chassis cells with a pipette to evenly disperse them in the cross-linking solution, and carry out the cross-linking reaction in a constant temperature mixer at 37°C for 5 minutes.

[0082] 6) Harvesting chassis cells that have completed the cross-linking reaction: Collect the chassis cell samples after cross-linking by centrifugation, and wash the chassis cells with 10 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer solution with a pH of 7.0 to wash away the unreacted cross-linking agent.

[0083] 7) Protein conformation detection in chassis cells: Use fluorescence resonance energy transfer technology to monitor the fluorescence signals emitted by the cross-linker backbone in different structural states (reference: Erika Cecon et al., SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET. Cell Chem. Biol. 2022, 29, 74-83), so as to obtain the structural state information of the target protein of the cross-linker in chassis cells. The red fluorescence signal indicates that the target protein is in a compact structural state (the protein structure is similar to a sphere at this time), while the green fluorescence signal indicates that the target protein is in an extended structural state (the protein structure is similar to a line at this time), and the absence of a fluorescent signal indicates that the cross-linker has not undergone a cross-linking reaction with the target protein.

[0084] 8) Screen and separate chassis cells based on the results of conformation detection: The fluorescence signal emitted by the cross-linker backbone is detected by fluorescence resonance energy transfer technology, which can determine the expression level and structural state of proteins in different chassis cells. The stronger the fluorescence signal, the higher the expression level of the target protein. Through fluorescence resonance energy transfer technology, chassis cells that emit red fluorescence signals are sorted into a collection pool, and those that emit green fluorescence signals are discarded, thereby isolating chassis cells with target proteins in a compact structural state. Next, chassis cells that emit strong red fluorescence signals are sorted into a collection pool (red fluorescence signals reach 10 2 The cells with weak red fluorescence signals were discarded, thereby isolating the chassis cells with high expression of the target protein in a compact structure. The number of cells obtained in this step was 14% of the total number of initial cells.

[0085] 9) Application: Collect and select chassis cells that emit strong red fluorescence signals. The target protein of the chassis cells is in an extended structural state, which is related to the formation of T cell dysfunction. By adding therapeutic target drugs, the conformation of the target protein can be compacted (from a loose spherical structure to a solid spherical structure), so that the cross-linker skeleton emits a stronger red fluorescence signal (fluorescence signal reaches 10 3 The above), and then the fluorescence resonance energy transfer technology analysis can screen out drugs with therapeutic effects, thereby achieving the purpose of drug screening analysis.

Claims

1. A method for detecting and / or screening a target protein-expressing cell line based on fluorescence indication, characterized in that: By targeting the target protein in chassis cells with cross-linking agents with targeting and fluorescent properties for chemical cross-linking reaction, the differences in the target protein's own conformation and / or chemical microenvironment are used to cause changes in the cross-linker backbone conformation, resulting in the cross-linker emitting fluorescence of different colors, which can be used to quickly detect and / or efficiently screen the expression level and / or structural state of the target protein in different chassis cells.

2. The method according to claim 1, characterized in that: The chassis cells are one or more of HEK 293T, HeLa, Escherichia coli, yeast or Bacillus subtilis; Alternatively, gene editing and / or transformation techniques may be used to modify and transform the genome of the chassis cells to add the desired target gene, so that the target protein is overexpressed, thereby obtaining a large amount of the target protein; The above process can provide reliable technical support for biological research, drug screening or protein function research.

3. The method according to claim 1, characterized in that: The cross-linking agent has two or more different or identical reactive groups, and the reactive groups are one or more of benzenesulfonyl fluoride group, phenoxysulfonyl fluoride group, N-hydroxysuccinimide group, o-phthalaldehyde group, and diaziridine group; The reactive groups are connected by a spacer arm, which is composed of one or more of an alkyl chain, a polyethylene glycol chain, and an amide chain, and has a length of The conformation of the spacer arm can change with the distance to the cross-linked sites, which can be achieved through the free rotation and bending of the spacer arm molecule. When the cross-linked sites are close, the conformation of the spacer arm shrinks, and when the cross-linked sites are far away, the conformation of the spacer arm stretches. The targeting of the cross-linking agent is achieved by adding one or more of the structure of the inhibitor of the target protein, the structure of the affinity group of the target protein, the antibody of the target protein, and the opposite charge of the target protein to the spacer arm skeleton; The fluorescent property of the cross-linking agent is achieved by adding a fluorescent molecule structure to the spacer arm skeleton, such as one or more derivatives of rhodamine, fluorescein, and Alexa Fluor series molecules.

4. The method according to claim 1, characterized in that: The cross-linking agent is water-soluble and membrane-permeable, the reaction time between the cross-linking agent and the intracellular protein is 1s-1h, the reaction efficiency is 20%-100%, it can target the target protein, and has fluorescence characteristics.

5. The method according to claim 1 or 2, characterized in that: The cells on the culture dish are obtained by cell scraping or trypsin digestion, and then centrifuged and washed to obtain a chassis cell sample, wherein the solution for washing the chassis cells is one or more of ammonium bicarbonate buffered saline solution, phosphate buffered saline solution, tris(hydroxymethyl)aminomethane buffered saline solution or 4-hydroxyethylpiperazineethanesulfonic acid buffered saline solution with a pH of 7.0-10.

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6. The crosslinking agent solution according to claim 1, 2, 3 or 4, characterized in that: The chemical cross-linking reaction process is to prepare the cross-linking agent into a solution with a concentration of 0.1mM-1M with an organic solvent, and mix it with a buffer solution containing target protein cells to make the final concentration of the cross-linking agent 0.1-100mM; Wherein, the organic solvent is one or more of organic alcohols (e.g., methanol, ethanol, isopropanol), aprotic polar solvents (e.g., dimethyl sulfoxide, N,N-dimethylformamide, acetone), organic bases (e.g., triethylamine, potassium tert-butoxide, n-butyl lithium), and organic acids (e.g., tartaric acid, oxalic acid, salicylic acid); The buffer solution is one or more of ammonium bicarbonate buffer solution, phosphate buffer solution, tris(hydroxymethyl)aminomethane buffer solution or 4-hydroxyethylpiperazineethanesulfonic acid buffer solution with a pH of 7.0-10.

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7. The method according to any one of claims 1 to 6, characterized in that: A cell buffer solution containing a crosslinker is added to the chassis cells overexpressing the target protein for co-incubation, wherein the volume ratio of the crosslinker solution to the chassis cells is 100:1-1:1, the temperature is set at 20-50°C, the crosslinking reaction time is 1s-1h, and the crosslinking reaction is completed.

8. The method according to any one of claims 1 to 7, characterized in that: After the cross-linking agent cross-links the target protein in different chassis cells, based on the specific effects of the target protein's own conformation and the chemical microenvironment on the cross-linker backbone fluorescence, the cross-linker presents a corresponding conformational state and emits a fluorescent signal of a specific color and intensity, such as one or more of red, yellow or blue.

9. The method according to any one of claims 1 to 8, characterized in that: The protein conformation detection in the chassis cells adopts one or more of a variety of methods such as fluorescence resonance energy transfer technology, fluorescence activated cell sorting technology, fluorescence microscopy sorting and fluorescence live cell imaging sorting, It is used to monitor the fluorescence signals emitted by the cross-linker backbone in different conformational states, thereby obtaining information on the structural state of the cross-linker target protein in chassis cells.

10. The method according to any one of claims 1 to 9, characterized in that: The screening of chassis cells utilizes one or more of fluorescence resonance energy transfer technology, fluorescence activated cell sorting technology, fluorescence microscope sorting, and fluorescence live cell imaging sorting to detect the strength of the fluorescence signal emitted by the cross-linker skeleton, so as to obtain the expression level and / or structural state of proteins in different chassis cells, thereby accurately screening out chassis cells with different functional states.

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