A probe system for detecting cholesterol and its application

Abstract

The present invention belongs to the technical field of cholesterol detection, and specifically relates to a probe system for detecting cholesterol and an application thereof, comprising a SNAP-D4 fusion protein expressed by mammalian cells and a lyotropic fluorescent dye Acrylodan-BG, wherein the SNAP-D4 fusion protein expressed by mammalian cells comprises a SNAP tag, and the cysteine ​​at the active site of the SNAP tag is covalently bound to BG to form a fluorescent probe system. The Acrylodan-BG / SNAP-D4 probe system of the present invention has the characteristics of high specificity and strong operability in identifying cholesterol, and cell fluorescence imaging shows that lipid raft cholesterol emits blue fluorescence, while cholesterol in the cell membrane exposed to the aqueous phase, namely active membrane cholesterol, shows green fluorescence. The present invention provides a method for distinguishing and detecting lipid raft cholesterol from active membrane cholesterol.

Description

Technical Field

[0001] This invention belongs to the field of cholesterol detection technology, specifically relating to a probe system for detecting cholesterol and its application. Background Technology

[0002] Cholesterol is a major component of mammalian cell membranes, influencing cell membrane fluidity, permeability, and the function of various enzymes, transporters, and receptors. Mammalian cell membranes contain up to 80% cholesterol of total cellular cholesterol. Lipid rafts are cholesterol- and sphingomyelin-rich microdomains on the cell membrane. Cholesterol in lipid rafts is generally located within the lipid phase of the cell membrane's phospholipid bilayer. Cholesterol in lipid rafts can trigger various cellular signaling events by recruiting specific molecules to the rafts. Active membrane cholesterol, on the other hand, refers to cholesterol with higher chemical activity (escape tendency) when the cholesterol content in lipid rafts exceeds a certain threshold, surpassing the complexing capacity of phospholipids. A portion of this active membrane cholesterol is exposed to the aqueous phase surrounding the cell membrane. Active membrane cholesterol in the inner lobe of the cell membrane can induce endoplasmic reticulum reactions and mitochondrial oxidative sterol reactions. Simultaneously, active membrane cholesterol can affect intracellular cholesterol transport and the function of various ion transport channels or ligand-gated channels. Therefore, monitoring cell membrane lipid raft cholesterol and active membrane cholesterol is of great significance for understanding the physiological and pathological processes of cell membrane function, intracellular cholesterol transport, and cell signal transduction.

[0003] In recent years, cholesterol labeling methods based on isotopes, electrochemical sensors, and fluorescent probes have been developed. However, isotope tracing and electrochemical sensors require specialized environments, expensive experimental equipment, and highly trained operators, thus limiting their widespread use. Fluorescent labeling is a favorable option and is the most intuitive, economical, and easy to implement. However, an effective fluorescent labeling method is currently lacking that can selectively and effectively distinguish between lipid raft cholesterol and active membrane cholesterol. Summary of the Invention

[0004] To address the problems in the prior art, this invention provides a probe system for detecting cholesterol and its application, achieving more specific and convenient cell membrane cholesterol labeling for the fluorescence imaging detection and differentiation of lipid raft cholesterol and active membrane cholesterol in the inner lobes of the cell membrane.

[0005] By modifying the bacterial perforation toxin protein Perfringolysin O (PFO), which specifically binds to cholesterol, and extracting only its D4 domain, PFO lost its cytotoxicity and could be used for cholesterol labeling of live cell membranes. The serine T490 and leucine L491 residues in the D4 domain of Perfringolysin O are sites that specifically recognize cholesterol and may interact with hydroxyl groups. Using the fusion expression of the Perfringolysin O D4 domain with mCherry fluorescent protein in the mCherry fluorescent carrier, cholesterol on the cytoplasmic side of the cell membrane could be successfully labeled. However, the monochromatic fluorescent mCherry-tag fusion protein could not distinguish between lipid raft cholesterol in the lipid phase and active membrane cholesterol in the aqueous phase, thus lacking specificity.

[0006] Further optimization involved hydrophobically modifying the D4 domain of Perfringolysin O (Y415A / D434W / A463WYDA mutation, capable of labeling 1% mol cholesterol in vitro), purifying it in vitro, and then coupling it with the lyochromic group Acrylodan. This fluorescent probe was then microinjected into cells to label cholesterol, thus distinguishing between cholesterol in the lipid phase (blue) and cholesterol in the near-aqueous phase of the membrane (green), which can, to some extent, solve the problem of specific labeling of intracellular cholesterol. While this method utilizes the principle of lyochromic reaction to more specifically detect membrane cholesterol, it requires specialized microinjection equipment, making the operation cumbersome and unsuitable for widespread application in laboratories lacking such equipment.

[0007] Further optimization involved fusing the SNAP tag with the D4 domain of PFO, which recognizes cholesterol-binding proteins, to create the SNAP-D4 protein. This SNAP-D4 was then overexpressed in mammalian cells via a lentiviral vector. Simultaneously, the lyochromic fluorescent dye Acrylodan-BG (acrylodan-benzyloxyguanine) was synthesized. The cysteine ​​residue at the active site of the SNAP tag covalently binds to BG, forming the Acrylodan-BG / SNAP-D4 fluorescent probe system. Since Acrylodan-BG fluoresces in different colors in polar and nonpolar environments—blue in the lipid phase and green in the aqueous phase—the Acrylodan-BG / SNAP-D4 fluorescent probe system can sense cholesterol in the lipid phase of the cell membrane and cholesterol partially exposed in the aqueous phase, which were identified as lipid raft cholesterol and active membrane cholesterol, respectively.

[0008] The technical problem solved by this invention is achieved by the following technical solution:

[0009] The present invention aims to provide a probe system for detecting cholesterol, comprising a SNAP-D4 fusion protein expressed by mammalian cells and a lyochromic fluorescent dye Acrylodan-BG. The SNAP-D4 fusion protein expressed by mammalian cells contains a SNAP tag, and the cysteine ​​residue at the active site of the SNAP tag covalently binds to BG to form a fluorescent probe system.

[0010] Furthermore, the SNAP-D4 fusion protein expressed by mammalian cells contains the amino acid sequence SEQ ID NO.3.

[0011] Furthermore, the SNAP-D4 fusion protein encoding mammalian cell expression contains the nucleotide sequence SEQ ID NO.4.

[0012] Furthermore, the cells encoding the SNAP-D4 fusion protein expressed in mammalian cells are Huh-7 cells or L02 cells.

[0013] Furthermore, the SNAP-D4 fusion protein contains the amino acid sequence SEQ ID NO.1.

[0014] Furthermore, the nucleotide sequence encoding the SNAP-D4 fusion protein is SEQ ID NO.2.

[0015] Furthermore, the expression strain encoding the SNAP-D4 fusion protein is one of BL21(DE3)-pLysS, Rosetta(DE3), or BL21-CodonPlus(DE3)-RIL.

[0016] Furthermore, the SNAP-D4 fusion protein is a fusion protein in which the SNAP tag is fused with the D4 domain of PFO, and the D4 domain is a Y415A / D434W / A463W YDA mutant.

[0017] Furthermore, the structure of the lyochromic fluorescent dye Acrylodan-BG is as follows:

[0018]

[0019] An application of a probe system for detecting cholesterol in distinguishing between lipid-phase cholesterol and aqueous-phase cholesterol, specifically for differentiating between lipid raft cholesterol and active membrane cholesterol using fluorescence imaging.

[0020] The preparation method of the probe system for cholesterol detection is as follows:

[0021] 1. A method for expressing and purifying SNAP-D4 protein in E. coli, comprising the following steps:

[0022] a) The SNAP gene sequence was fused with the D4 domain (Y415A / D434W / A463W YDA mutant) nucleotide sequence of PFO and then ligated into the pET-22b cloning vector to construct the E. coli expression plasmid pET-22b-SNAP-D4 expressing the SNAP-D4 protein.

[0023] b) Transform Escherichia coli expression plasmid pET-22b-SNAP-D4 into strains such as BL21(DE3)-pLysS, Rosetta(DE3), and BL21-CodonPlus(DE3)-RIL;

[0024] c) Inducing E. coli to express SNAP-D4 protein by IPTG, collecting inclusion body proteins for denaturation, renaturation and purification to obtain purified SNAP-D4 protein;

[0025] d) The purified SNAP-D4 protein can be used to bind cholesterol in vitro.

[0026] 2. The method for constructing Huh-7 and L02 cell lines stably expressing SNAP-D4 includes the following steps:

[0027] a) The nucleotide sequence of SNAP-D4 was ligated into the pCMV-HIV lentiviral cloning vector (from Heyuan Biotechnology) to construct the lentiviral vector plasmid pCMV-HIV-SNAP-D4 expressing the SNAP-D4 gene;

[0028] b) The lentiviral vector plasmid pCMV-HIV-SNAP-D4 and packaging plasmids psPAX2 and pMD2.0G were co-transfected into 293T cells to package them into lentiviruses carrying the SNAP-D4 encoding gene.

[0029] c) Huh-7 cells and L02 cells were infected with lentiviruses carrying the SNAP-D4 encoding gene, and stable SNAP-D4 Huh-7 cell lines or SNAP-D4 L02 cell lines were obtained after G418 selection.

[0030] d) Cell lines that stably express SNAP-D4 can bind intracellular cholesterol.

[0031] 3. The synthesis of SNAP-tag lyochromic fluorescent probes includes the following steps:

[0032] a) The structural formula is as follows:

[0033]

[0034] b) The synthetic route is as follows:

[0035]

[0036] c) The synthesis steps are as follows:

[0037] 6-Acryloyl-2-dimethylaminonaphthalene (Acrylodan, compound 1) and O6-[4-(aminomethyl)benzyl]guanine (BG-NH2, compound 2) were dissolved in methanol and reacted at 50 °C for 24 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, purified by silica gel column chromatography and vacuum distillation to obtain the yellow-green solid product 3-((4-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)amino)-1-(6-(dimethylamino)naphth-2-yl)prop-1-one, i.e., Acrylodan-BG (compound 3);

[0038] Acrylodan-BG has lyochromic properties and can covalently bind to SNAP-D4 protein in vivo and in vitro. The Acrylodan-BG / SNAP-D4 probe system can distinguish between lipid raft cholesterol and active membrane cholesterol.

[0039] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0040] 1. The Acrylodan-BG / SNAP-D4 probe system of this invention features high specificity and ease of operation in cholesterol identification. Cell fluorescence imaging shows that lipid raft cholesterol emits blue fluorescence, while cholesterol exposed to the aqueous phase in the cell membrane, i.e., active membrane cholesterol, shows green fluorescence. This invention provides a method for distinguishing between lipid raft cholesterol and active membrane cholesterol.

[0041] 2. The SNAP-D4 fusion protein in this invention has the function of binding cholesterol. It can bind cholesterol in vitro after expression in E. coli and purification by inclusion bodies, and can bind cholesterol in vivo when stably expressed in mammalian cells by lentiviral vector.

[0042] 3. The lyochromic fluorescent dye Acrylodan-BG in this invention has the advantages of low-cost raw materials and simple synthesis method.

[0043] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0044] Figure 1 This is a schematic diagram illustrating the principle of labeling intracellular cholesterol using the Acrylodan / SNAP-D4 probe system of this invention.

[0045] Figure 2 This is a map of the prokaryotic expression plasmid pET-22b-SNAP-D4 of the present invention.

[0046] Figure 3 The image shows the results of SNAP-D4 protein purification and in vitro cholesterol binding verification in this invention.

[0047] Figure 4 This is a map of the pCMV-HIV-SNAP-D4 mammalian cell stable expression plasmid of the present invention.

[0048] Figure 5 This is a diagram from an experimental verification test showing that SNAP-D4 expressed in cells can only label cholesterol in the inner leaf of the cell membrane.

[0049] Figure 6 This is a schematic diagram illustrating the synthesis and characterization of Acrylodan-BG according to the present invention.

[0050] Figure 7 This is the infrared spectrum of Acrylodan-BG and SNAP-D4 bound together by the -SH group in this invention.

[0051] Figure 8 The image shows the results of labeling cholesterol in the cell membrane of Huh-7 cells using the Acrylodan-BG / SNAP-D4 probe system of this invention.

[0052] Figure 9 This image shows the verification results of the Acrylodan-BG / SNAP-D4 probe system of this invention in distinguishing cholesterol in lipid rafts within the cell membrane.

[0053] Figure 10 This is a diagram showing the verification results of the Acrylodan-BG / SNAP-D4 probe system of this invention in detecting cholesterol in active membranes exposed to the aqueous phase. Detailed Implementation

[0054] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0055] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0056] Example 1:

[0057] Expression and purification of SNAP-D4 protein in Escherichia coli and verification of its in vitro cholesterol binding.

[0058] 1. Methods and Materials:

[0059] The pET22b-SNAP-D4 fusion protein was synthesized and constructed by Shanghai Sangon Biotech Co., Ltd. The codon-optimized SNAP-D4 gene sequence from *E. coli* was constructed between the NdeI / XhoI restriction sites of the pET22b(+) empty vector. D4 was fused to the C-terminus of SNAP, without a linker sequence. The amino acid sequence of the prokaryotic SNAP-D4 fusion protein, SEQ ID NO. 1, is as follows:

[0060] MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVS SSGAVGGYEGGLAVKEWLLAHEGHRLGKPGLGKGKINLDHSGAYVAQFEVAWDEVSADKEGNEVLTHKTWDGNYQWKTAHYSTVIPLEANARNIRIKARECTGLWWEWWRDVISEYDVPLTNNINVSIWGTTLYPGSSITYNLEHHHHHH

[0061] The gene sequence SEQ ID NO.2 is as follows:

[0062] atggacaaagattgcgaaatgaaacgtaccaccctggatagcccgctgggcaaactggaactgagcggctgcgaacagggcctgcatgaaattaaactgctgggtaaaggcaccagcgcggccgatgcggttgaagttccggccccggccgccgtgctgggtggtccggaaccgctgatgcaggcgaccgcgtggctgaacgcgtattttcatcagccggaagcgattgaagaatttccggttccggcgctgcatcatccggtgtttcagcaggagagctttacccgtcaggtgctgtggaaactgctgaaagtggttaaatttggcgaagtgattagctatcagcagctggcggccctggcgggtaatccggcggccaccgccgccgttaaaaccgcgctgagcggtaacccggtgccgattctgattccgtgccatcgtgtggttagctctagcggtgcggttggcggttatgaaggtggtctggcggtgaaagagtggctgctggcccatgaaggtcatcgtctgggtaaaccgggtctgggaAAAGGCAAAATCAACCTGGATCACAGCGGCGCGTACGTTGCGCAGTTCGAAGTTGCGTGGGATGAAGTTAGCGCGGATAAAGAAGGCAACGAAGTTCTGACCCACAAAACCTGGGATGGTAACTACCAGTGGAAAACCGCGCACTACAGCACCGTTATCCCGCTGGAAGCGAACGCGCGTAACATCCGTATCAAAGCGCGTGAATGCACCGGCCTGTGGTGGGAATGGTGGCGTGATGTTATCAGCGAATACGATGTTCCGCTGACCAACAACATCAACGTTAGCATCTGGGGTACCACCCTGTACCCAGGCAGCAGCATCACCTACAACctcgagcaccaccaccaccaccactga

[0063] The constructed pET22b-SNAP-D4 plasmid was transformed into *E. coli* Rosetta (DE 3) cells. After overnight incubation on plates, one colony was picked and transferred to 5 mL of Luria-Bertani (LB) medium (containing 100 μg / mL ampicillin) and cultured at 37°C and 220 rpm until turbidity was reached. The bacterial culture was then transferred to 2000 mL of LB medium containing 100 μg / mL ampicillin for amplification until the OD 600 reached approximately 0.6. Then, 0.1–0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and SNAP-D4 expression was induced at 30°C for 8 h. The induced culture was centrifuged at 8000 rpm for 3 min. Resuspend the protein in 50 mL of cold NTA lysis buffer (20 mM Tris, 500 mM sodium chloride, 10% glycerol, 0.1 mg / mL lysozyme, pH 8.0), sonicate, and centrifuge at 16000 rpm for 50 min. Remove the supernatant protein and resuspend the precipitate in 3 mL of inclusion body lysis buffer (6 M guanidine hydrochloride, 20 mM Tris, 5 mM dithiothreitol, pH 8.0). After centrifugation to remove the precipitate, dilute the supernatant with 6 mL of dilution buffer (3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA·2NA, pH 4.2). Then, add the supernatant dropwise to 200 mL of refolding buffer (0.5 mM oxidized glutathione, 5 mM reduced glutathione, 2 mM EDTA·2NA, 100 mM Tris, pH 8.0) and stir for 48 h. After complete renaturation of inclusion bodies, the protein solution was transferred to a dialysis bag, concentrated to 10-20 mL in PEG 20000, and dialyzed in NTA lysis buffer. The protein was then bound to Ni NTA agarose resin (Invitrogen, USA). The resin was washed with NTA lysis buffer, and the protein was eluted with NTA lysis buffer containing 20-500 mM imidazole. SDS-PAGE gel electrophoresis and R-250 staining were used to analyze the SNAP-D4 purified protein.

[0064] The purified SNAP-D4 protein was validated in vitro for its cholesterol binding using enzyme-linked immunosorbent assay (ELISA). Each well of a 96-well plate was coated with 30 μL of 1% agarose (IXA type, Sigma-Aldrich, USA) and microwaved until completely melted. The gel was dried in an incubator at 37°C. Cholesterol and its analogues (including bile acids, dexamethasone, dehydrocholesterol, estrone, ergosterol, estradiol, progesterone, and methyltestosterone) were dissolved in ethanol to a final concentration of 2 mM. 50 μL of the lipid was added to each well of the 96-well plate, and the plate was completely dried in a fume hood. The plate was then incubated for 2 hours at room temperature with either unpurified supernatant containing SNAP-D4 or purified SNAP-D4 protein diluted with 2% BSA-PBS. After washing twice with PBS, the plate was incubated with rabbit anti-His-tagged antibody (1:2000 dilution, Wuhan Sanying Pharmaceutical Co., Ltd.) for 2 hours at room temperature. After washing twice with PBS, the sample was incubated for 2 hours at room temperature with biotin-conjugated goat anti-rabbit IgG and horseradish peroxidase (HRP)-conjugated streptavidin (1:10000, both diluted with 2% BSA-PBS, Wuhan Sanying Company). The sample was then washed twice with PBS and developed with TMB solution. The absorbance was measured at 450 nm using a microplate reader (Biotek, USA).

[0065] 2. Results:

[0066] We constructed the SNAP-D4(Y415A / D434W / A463W YDA mutant) pET22b(+) vector, and the plasmid map is shown below. Figure 2 .

[0067] After transforming E. coli SNAP-D4 into Rosseta (DE3 cells), it was expressed as inclusion bodies. After refolding and purification, the purity of SNAP-D4 was approximately 95%, and the protein molecular weight was approximately 35 kDa as expected. Figure 3 A) SNAP-D4 expression was low in the supernatant of E. coli lysate. This may be due to multiple mutations in the hydrophobic tryptophan of the D4 protein, making it more sensitive to cholesterol binding. Therefore, the protein's hydrophobicity makes it more prone to forming inclusion bodies.

[0068] To verify whether SNAP-D4 binds cholesterol in vitro, we used an ELISA method. We found that native SNAP-D4 expressed in the supernatant (unpurified, as it was poorly expressed) could bind to cholesterol. Figure 3 B). We then examined the purified and refolded SNAP-D4 inclusion body protein and found that it could also bind cholesterol in a concentration-dependent manner, unlike the bovine serum albumin (BSA) control group. Figure 3C) indicates that the inclusion bodies successfully refolded and remained active, demonstrating that SNAP-D4 can bind cholesterol in vitro, and the optimal SNAP-D4 concentration for cholesterol binding is 50-100 μg / mL. Figure 3 C). Furthermore, we examined the sensitivity and specificity of SNAP-D4 binding to cholesterol. For example... Figure 3 As shown in Figure D, SNAP-D4 (50 μg / mL) can bind different doses of cholesterol in a Langmuir-type saturation mode, rather than an S-type, indicating the high affinity of SNAP-D4 for cholesterol binding. SNAP-D4 binds cholesterol in a linear range of 0-60 μM. Figure 3 E) indicates the sensitivity and linearity of SNAP-D4 binding to cholesterol. Furthermore, SNAP-D4 binds only to cholesterol and not to cholesterol analogs, demonstrating its specificity in cholesterol binding. Figure 3 F).

[0069] Figure 3 SNAP-D4 protein purification and in vitro cholesterol binding validation. A shows the purified SNAP-D4 protein, expressed in pET22b-SNAP-D4 transformed *E. coli* Rosseta (DE3) cells, after inclusion body refolding. B shows the binding of unpurified, active *E. coli* supernatant containing SNAP-D4 protein to cholesterol (2 mM). C shows the binding of different concentrations of purified inclusion body refolded SNAP-D4 protein (0-100 μg / mL) to cholesterol (2 mM). DE shows the binding of inclusion body refolded SNAP-D4 protein (50 μg / mL) to different concentrations of cholesterol (0-2 mM) and the linear range of cholesterol binding (0-60 μM). F shows the specificity of SNAP-D4 binding to cholesterol. N≥3, ***P<0.001, mean ± SD.

[0070] Example 2:

[0071] Construction of Huh-7 and L02 cell lines stably expressing SNAP-D4 and validation of their binding to intracellular cholesterol.

[0072] 1. Methods and Materials:

[0073] The D4 (YDA mutant) gene, after optimization according to mammalian cell codons, was synthesized by Shanghai Sangon Biotech Co., Ltd. and constructed into the pSNAPf vector (NEB), resulting in the pSNAP-D4f plasmid containing the eukaryotic gene SNAP-D4. D4 is fused to the C-terminus of SNAP and does not contain a linker. Finally, the SNAP-D4 gene was constructed into the pCMV-HIV lentiviral vector (Heyuan Biotechnology) to obtain the pCMV-HIV-SNAP-D4 mammalian cell expression plasmid. Plasmid construction was performed according to the instructions of Novizan 2x Phanta Max MasterMix PCR high-fidelity enzyme and Hanheng Biotechnology's seamless cloning protocol. The amino acid sequence of SNAP-D4 expressed in mammalian cells, SEQ ID NO. 3, is as follows:

[0074] MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVVPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCH RVVQGDLDVGGYEGGLAVKEWLLAHEGHRLGKPGLGKGKINLDHSGAYVAQFEVAWDEVSADKEGNEVLTHKTWDGNYQWKTAHYSTVIPLEANARNIRIKARECTGLWWEWWRDVISEYDVPLTNNINVSIWGTTLYPGSSITYN

[0075] The gene sequence SEQ ID NO.4 is as follows:

[0076] ATGGACAAAGACTGCGAAATGAAGCGCACCACCCTGGATAGCCCTCTGGGCAAGCTGGAACTGTCTGGGTGCGAACAGGGCCTGCACCGTATCATCTTCCTGGGCAAAGGAACATCTGCCGCCGACGCCGTGGAAGTGCCTGCCCCAGCCGCCGTGCTGGGCGGACCAGAGCCACTGATGCAGGCCACCGCCTGGCTCAACGCCTACTTTCACCAGCCTGAGGCCATCGAGGAGTTCCCTGTGCCAGCCCTGCACCACCCAGTGTTCCAGCAGGAGAGCTTTACCCGCCAGGTGCTGTGGAAACTGCTGAAAGTGGTGAAGTTCGGAGAGGTCATCAGCTACAGCCACCTGGCCGCCCTGGCCGGCAATCCCGCCGCCACCGCCGCCGTGAAAACCGCCCTGAGCGGAAATCCCGTGCCCATTCTGATCCCCTGCCACCGGGTGGTGCAGGGCGACCTGGACGTGGGGGGCTACGAGGGCGGGCTCGCCGTGAAAGAGTGGCTGCTGGCCCACGAGGGCCACAGACTGGGCAAGCCTGGGCTGGGTAAAGGCAAGATCAATCTCGACCATTCAGGGGCATATGTGGCCCAATTTGAAGTGGCCTGGGATGAAGTGAGCGCGGACAAGGAAGGCAATGAAGTGCTTACACACAAGACATGGGACGGTAATTATCAGTGGAAAACAGCACACTACTCCACTGTAATCCCTCTTGAAGCCAACGCACGCAACATTAGGATTAAAGCTCGGGAATGTACCGGCCTGTGGTGGGAATGGTGGAGGGACGTGATCTCTGAGTACGATGTGCCGCTCACCAACAACATAAATGTAAGCATCTGGGGCACCACCTTGTACCCGGGATCTAGCATCACCTACAACTGA

[0077] The PCR primer design is as follows:

[0078]

[0079] The target gene (SNAP-D4) was amplified using 2×phanta Max master mix, and the reaction system is as follows:

[0080] 2x Phanta Max Master Mix 25μL forward primer 2μL reverse primer 2μL pSNAP-D4f template 50ng-100ng ddH2O 20μL Approximately 50μL

[0081] The reaction conditions are as follows:

[0082] step temperature Duration 1 101℃ Hot cap 2 95℃ 3min 3 95℃ 15s 4 50℃ 15s 5 72℃ 50s, proceed to step 3, repeat 34 times. 6 72℃ 5min 7 4℃ cool down

[0083] The linearized pCMV-HIV vector backbone (purchased from Heyuan Biotechnology) was digested with XbaI+NotI enzyme (purchased from Yugong Biotechnology) under the following conditions: PCR incubation at 37℃ for 2 hours. The digestion system is as follows:

[0084] 10×Cut buffer 3μL pCMV-HIV vector 1.5μg DNA XbaI enzyme 1.5μL NotI enzyme 1.5μL ddH2O water Make up to 30μL total 30μL

[0085] After double digestion, the restriction enzymes were inactivated at 85℃ for 5 min. Identification was performed by gel electrophoresis. Seamless clones were ligated under the following conditions: 50℃ for 60 min (PCR bath). The ligation system is as follows:

[0086] 2×HB infusion enzyme 10μL Linearized carrier skeleton 2 μL (total 135 ng / μL) Target gene 1 μL (total 80-100 ng / μL) ddH2O 7μL total 10μL

[0087] The ligation product was transformed into DH5α. After picking single clones and shaking for about 4-5 hours, the bacterial culture was identified by PCR according to the instructions of the Colony 2x T5 superPCR mix kit. Based on the PCR results, 150 μL of each of the two positive recombinant cultures were selected and sent to Colony Biotechnology for sequencing. The sequencing results were correct.

[0088] The pCMV-HIV-SNAP-D4 mammalian expression plasmid was transformed into DH5α bacteria. After bacterial amplification in 300 mL LB, endotoxin-free plasmids were extracted and transfected according to the Goldhi endofree plasmid maxi kit (Kangwei Reagent Company) instructions. The OD260 concentration of the extracted plasmids was measured, and they were identified by agarose gel electrophoresis. Four days after passage of 293T cells to 10 cm⁻¹, when the cells reached approximately 80% confluency, three plasmids were co-transfected using the JetPrime kit as follows: 15 μg DNA was added to each 10 cm⁻¹ dish, i.e., 10 μL of 500 ng / μL pspax2 plasmid (laboratory stock) + 10 μL of 500 ng / μL pMD2.G plasmid (laboratory stock) + 50 μL of 100 ng / μL pCMV-HIV-SNAP-D4 + 750 μL buffer + 45 μL reagent mixture. Each virus was packaged into three 10 cm⁻¹ dishes, each containing 12 mL of culture medium. After 48 hours, collect the virus-containing culture medium, add fresh medium, and collect again the next day. Weigh approximately 40g (accurate to 0.001g) each in a 38.6mL ultracentrifugation tube (open-top type, pre-sterilized with 75% alcohol) using a precision balance. Place the tube in the corresponding SW 28 rotor and centrifuge using a Beckman floor-standing ultracentrifuge at 14000g (rcf) for 1 hour and 45 minutes at 4°C, with an acceleration of 5 and a deceleration of 5. After ultracentrifugation, release the vacuum, remove the centrifuged liquid, and pour the supernatant into a 1:5 dilution of 84 disinfectant. Resuspend the precipitate in 500μL of culture medium and filter through a 0.45μm filter. After 72 hours, collect the supernatant again and concentrate the lentivirus using a 30kd ultrafiltration centrifuge tube. Filter and set aside. Take 100μL of the concentrated virus solution into a 24-well plate containing Huh7 cells or other cell lines (grown to 50%-80% density). After 48 hours, passage to a 6cm dish or 6-well plate. After 96 hours, add G418 at 0.75 mg / mL-1 mg / mL for selection, changing the medium every 2 days. Select for at least 20-30 days to obtain stable expression cell lines.

[0089] The successfully established SNAP-D4 stably expressing Huh-7 cell line was stained with the commercially available SNAP-tag fluorescent dye tetramethylrhodamine-benzyloxyguanine (TMR-BG, NEB). Cells were treated with proteinase K (50 μg / ml) and incubated with or without digoxigenin (20 μM) for 5 minutes, and observed under a confocal microscope. Proteinase K is used to digest proteins in the outer leaves of the cell membrane, while digoxigenin permeates the cell membrane, allowing proteinase K to digest proteins in the inner leaves.

[0090] 2. Results:

[0091] The pCMV-HIV-SNAP-D4 mammalian cell expression plasmid was successfully constructed after sequencing alignment. The plasmid map is shown below. Figure 4As shown, we successfully constructed a Huh7 cell line stably expressing SNAP-D4, and confirmed that SNAP-D4 expressed in the cells can only label cholesterol in the inner leaf of the cell membrane. Figure 5 We used the reported EGFP-D4 protein to label cholesterol in the extracellular leaf. When proteinase K was added to cells expressing SNAP-D4, the amount of EGFP-D4 protein bound to the extracellular leaf decreased, while the fluorescence signal of the SNAP-D4 / TMR-BG probe system did not decrease. Both EGFP-D4 and SNAP-D4 were degraded by proteinase K after digoxin permeation. These observations confirm that SNAP-D4 expressed in cells can only label cholesterol in the intracellular leaf.

[0092] Figure 5 SNAP-D4 expressed in cells cannot cross the plasma membrane. SNAP-D4 is stained with TMR-BG. Red fluorescence indicates SNAP-D4 / TMR-BG, and green fluorescence indicates EGFP-D4 protein. Scale bar = 10 μm.

[0093] Example 3:

[0094] Synthesis and characterization of the lyochromic fluorescent probe Acrylodan-BG.

[0095] 1. Methods and Materials:

[0096] 50 mg (1 eq) of 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan) and 119 mg (2 eq) of O6-[4-(aminomethyl)benzyl]guanine (BG-NH2,O6-[4-(aminomethyl)benzyl]guanine) were weighed into a reaction flask. 10 mL of methanol was added, and the reaction was carried out at 50 °C for 24 h under nitrogen protection. The filtered solution was extracted three times with dichloromethane (DCM), and purified by thin-layer chromatography (CH2Cl2:CH3OH / 10:1). After rotary evaporation, the yellow-green product 3-((4-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)amino)-1-(6-(dimethylamino)naphth-2-yl)prop-1-one (compound 5), i.e., Acrylodan-BG, was obtained with a yield of 79.8%.

[0097] 2. Results:

[0098] Synthesis process and final structural formula are as follows Figure 6 As shown in Figure A, the Acrylodan-BG mass spectrum is as follows: Figure 6 As shown in B, HPLC-MS (ESI) m / z: 518 {[M+Na+]}. Its NMR spectrum is as follows. Figure 6As shown in C, the specific proton NMR data are as follows: 1H NMR (600MHz, CDCl3-d6) δ: 8.24 (s, 1H), 7.62 (m, 3H), 7.27 (s, 4H), 5.16 (s, 1H), 4.16 (s, 1H), 3.10 (s, 1H), and 3.02 (d, 2H). Figure 6 As shown in Figure D, Acrylodan-BG has an absorption peak at 380 nm. The fluorescence properties of Acrylodan-BG in different polar and nonpolar solvents were measured using a fluorescence spectrophotometer. Figure 6 As shown in Figure EG, the emission spectra of Acrylodan-BG differ in different solvents under 380 nm excitation. In polar solvents such as water, methanol, and ethanol, the emission peak of Acrylodan-BG is located at 502-548 nm, mainly exhibiting green fluorescence; while in dioxane (… Figure 6 In nonpolar solvents such as EF, dichloromethane (DCM), dimethyl sulfoxide (DMSO), and dimethylformamide (DMF), the emission peak is located at 472-489 nm, mainly exhibiting blue fluorescence. SNAP-D4 protein or cholesterol itself does not affect the lyochromic properties of Acrylodan-BG. Figure 6 F). This result indicates that the newly synthesized Acrylodan-BG possesses lyotropic properties. Meanwhile, as... Figure 7 As shown in the figure, infrared spectroscopy reveals that Acrylodan-BG can couple with purified SNAP-D4 protein in vitro via the thiol group of the SNAP-tag protein, indicating that Acrylodan-BG can bind to SNAP-D4 protein in vitro. After staining with Acrylodan-BG, the SNAP-D4 protein in Huh-7 cells stably expressing SNAP-D4, after SDS-PAGE electrophoresis of the cell lysate, emitted Acrylodan-BG fluorescence under ultraviolet light, indicating that Acrylodan-BG can also bind to SNAP-D4 protein in vivo. Figure 6 H).

[0099] Figure 6Synthesis and characterization of Acrylodan-BG. A shows the synthetic route of Acrylodan-BG. B shows the 1H NMR spectrum of Acrylodan-BG. C shows the mass spectrometry analysis of Acrylodan-BG. D shows the UV absorption spectrum of Acrylodan-BG. E, G, shows the fluorescence emission spectrum of Acrylodan-BG in the organic solvent dioxane (EF) under 380 nm excitation light and the fluorescence emission spectrum in other solvents (G). H shows the SDS-PAGE electrophoresis verification of SNAP-D4 binding to Acrylodan-BG in Huh-7 cells. The left arrow indicates the binding of SNAP-D4 to Acrylodan-BG under UV irradiation. The right arrow indicates the SNAP-D4 protein purified by Coomassie Brilliant Blue staining, indicating the location of SNAP-D4.

[0100] Example 4:

[0101] Validation of intracellular cholesterol detection using the Acrylodan-BG / SNAP-D4 probe system.

[0102] 1. Methods and Materials:

[0103] Cytotoxicity assays were performed using the CCK-8 assay kit. Huh-7 cells were seeded in 96-well plates (1×10⁶ cells / wells). 4After culturing for 48 h, different concentrations of Acrylodan-BG were added to the wells (n=4), and incubated for 4 h. Then, 10 μL of CCK-8 stock solution (10% w / w) was added to each well. After incubation at 37 °C for 1 hour, the absorbance of each well was measured at 450 nm using a microplate reader. Cholesterol was prepared by sonication in 0.5% BSA in DMEM (10 min each time), filtered through a 0.22 μm membrane filter, and the stock solution (10 mM) was stored at -20 °C. Huh-7 cells stably expressing SNAP-D4 were seeded in 35 mm laser scanning confocal microscope (LSCM) culture dishes and stained with TMR-BG (3 μM) or Acrylodan-BG (0.5-5 μM) at 37 °C and 5% CO2 for 30 min. The cells were washed three times with culture medium and incubated in fresh culture medium for 30 min to allow unreacted substrate to diffuse out of the cells. Image acquisition was performed using a Nikon AX inverted confocal microscope, a Plan Apoλ60x oil objective, and NIS software (Nikon). The excitation wavelength for TMR-BG was 561 nm, and the emission wavelength was set to 571-605 nm. The excitation wavelength for Acrylodan-BG was 405 nm, and the emission wavelengths were set to 429-474 nm (blue channel) and 505-544 nm (green channel), respectively. Image analysis was performed using the MorphoLibJ plugin in ImageJ software (NIH). First, the cell membrane region was selected using the polygon tool, and a mask was created. Then, the membrane region was further divided into outer and inner circles in the MorphoLibJ plugin. The average fluorescence intensity of the cell membrane was calculated as (fluorescence intensity of the outer circle - fluorescence intensity of the inner circle) / (area of ​​the outer circle - area of ​​the inner circle).

[0104] 2. Results:

[0105] We found that Acrylodan-BG was non-toxic to Huh-7 cells in the range of 0-200 μM. Figure 8 A). Huh-7 cells stably expressing SNAP-D4 were treated overnight with 2.5 mM cholesterol and stained with Acrylodan-BG (0.5 μM). Figure 8 Both green and blue fluorescence in the cell membrane of BC cells increased. Simultaneously, treatment of Huh-7 cells with the cholesterol-consuming agent methyl β-cyclodextrin (MβCD) weakened or even eliminated both blue and green fluorescence on the cell membrane of Acrylodan-BG / SNAP-D4. Some fluorescence also translocated to the cytoplasm. These results are consistent with the fluorescence changes observed in the positive control SNAP-D4 / TMR-BG. Figure 8These results demonstrate that Acrylodan-BG / SNAP-D4 can label cholesterol in cell membranes. Since the blue and green fluorescence eventually disappears in the cell membrane after cholesterol depletion, there are few nonspecific cholesterol-sensing proteins in the cell membrane. Acrylodan-BG has a significant advantage over the commercial substrate TMR-BG, which emits only a single red fluorescence, due to its lyochromic properties, emitting predominantly blue light in nonpolar solvents and predominantly green light in polar solvents, thus aiding in the differentiation between lipid-phase and aqueous-phase cholesterol in cell membranes.

[0106] Figure 8 Acrylodan-BG / SNAP-D4 labeling of cholesterol in Huh-7 cell membranes. A shows the cytotoxicity of Acrylodan-BG. B shows fluorescence imaging of Huh-7 cells stably expressing SNAP-D4 after Acrylodan-BG staining and cholesterol treatment. Blue channel, λex: 405nm, λem: 429-474nm. Green channel, λex: 405nm, λem: 505-544nm. Scale bar = 5μm. C shows... Figure 8 Quantitative analysis of cell membrane fluorescence in B. D is a time-lapse imaging of Huh7 cells stably expressing SNAP-D4 after treatment with MβCD (10 μM). Cells were stained with TMR-BG (3 μM) or Acrylodan-BG (0.5 μM) for 30 min, washed with PBS, and incubated in culture medium for 30 min. Cells were stained with the nuclear dye Hoechst 33342. TMR-BG staining images were obtained by combining the red channel (λex: 561 nm, λem: 571-605 nm) and the blue channel (λex: 405 nm, λem: 429-474 nm). Scale bar = 5 μm. EG. is Figure 8 Quantitative analysis of D cell membrane fluorescence. N = 10, mean ± standard deviation. ***P < 0.001. TMR-BG, tetramethylrhodamine-benzyloxyguanine; MβCD, methyl β-cyclodextrin.

[0107] Example 5:

[0108] The lipid phase cholesterol in the cell membrane detected by the Acrylodan-BG / SNAP-D4 probe system was lipid raft cholesterol.

[0109] 1. Methods and Materials:

[0110] Huh-7 cells stably expressing SNAP-D4 were stained with Acrylodan-BG at 37℃ for 30 min and washed three times with PBS. 4 × 10⁴ cells were collected. 8Cells were lysed in TNE buffer (150 mM sodium chloride, 2 mM EDTA, 50 mM Tris-HCl, pH 7.4) containing protease inhibitors and sonicated for 5 seconds each time, 15 times in total, using a Bandelin (Germany). The lysis buffer was treated with 1% Triton X-100 and incubated on ice for 30 min. After centrifugation at 5000g for 10 min, the supernatant was transferred to an MLS 50 centrifuge tube containing 1.2 ml of TNE buffer with a final concentration of 45% sucrose. Then, 2.6 ml of 35% sucrose was added, followed by 1.2 ml of 5% sucrose. The tube was centrifuged at 268000g for 18 h in an MLS 50 rotor (Beckman Coulter, USA). The fractions were separated from the top, 0.4 ml each, for a total of 12 fractions. Each fraction was subjected to Western blotting with Flotillin-1 rabbit polyclonal antibody (1:2000 dilution, Wuhan Sanying). The emission of each component was detected at 470 nm (blue) and 550 nm (green) under 405 nm excitation light. Cell membrane integrity was detected using a lactate dehydrogenase (LDH) activity assay kit (Nanjing Jiancheng Bioengineering Institute). After Huh-7 cells were treated with cholesterol or MβCD, the cell culture supernatant was collected, centrifuged at 1000 g for 3 min, dead cells were removed, and the LDH released in the cell culture supernatant was detected.

[0111] 2. Results:

[0112] Using the lipid raft marker protein Flotillin-1, we found that lipid rafts are mainly present in fractions 3-5, and the blue fluorescence intensity emitted by the Acrylodan-BG / SNAP-D4 probe system in this fraction is significantly greater than that of the green fluorescence. Figure 9 AD). Furthermore, after exogenous cholesterol stimulation increases lipid raft cholesterol or MβCD consumes lipid raft cholesterol, the blue fluorescence in the lipid raft components increases or decreases accordingly, and the area under the blue fluorescence curve (AUC) of components 3-5 significantly increases or decreases. Figure 9 E), the change in green fluorescence was not obvious. This suggests that Acrylodan-BG / SNAP-D4 can detect lipid raft cholesterol and shows it as blue fluorescence. Cholesterol can increase the rigidity of lipid rafts, thereby reducing cell membrane permeability and increasing cell membrane integrity. After adding exogenous cholesterol or supplementing or consuming cholesterol with MβCD, the release of LDH in the cell culture supernatant indicates an increase or decrease in cell membrane integrity. Figure 9 E). This physiological change is consistent with the blue fluorescence change of the Acrylodan-BG / SNAP-D4 probe system, further confirming the feasibility and physiological significance of this probe system in detecting lipid raft cholesterol.

[0113] Figure 9Acrylodan-BG / SNAP-D4 helps distinguish cholesterol in intracellular lipid rafts. A shows lipid rafts extracted from Huh-7 cells stably expressing SNAP-D4. Immunoblotting of Flotillin-1 proteins suggests that lipid rafts are mainly present in fractions 3-5. B and D show semi-quantitative results of Flotillin-1 immunoblotting and fluorescence of lipid raft components in Huh-7 cells treated with or without cholesterol (3mM) or MβCD (10mM) using a fluorescence microplate reader. E is... Figure 9 The under-curve fluorescence area (AUC) of 3-5 components in BD and the relative cell membrane integrity indicated by LDH detection in cell culture supernatant. N≥3, *P<0.05, **P<0.01, ***P<0.001, mean ± standard deviation.

[0114] Example 6:

[0115] The intracellular aqueous cholesterol detected by the Acrylodan-BG / SNAP-D4 probe system is active membrane cholesterol.

[0116] 1. Methods and Materials:

[0117] Huh-7 cells stably expressing SNAP-D4 were treated with cholesterol oxidase liposomes, or L02 cells stably expressing SNAP-D4 were treated with a cholesterol gradient. The cells were then incubated in ultrapure water (hypotonic) for 1 hour until swelling and rupture. Cell lysates were collected, centrifuged at 700g for 2 min to remove the pellet, and then centrifuged at 14000g for 10 min to collect the pellet. The cell membrane pellet was resuspended in physiological saline. Protein concentration was determined using a BCA assay kit. + -K + ATPase activity was determined using an ATPase assay kit (Nanjing Jiancheng Bioengineering Institute). Each group had at least three replicate wells. For liposome preparation, the lipid mixture (POPC:POPS:POPE 50:35:15 mol%) was dried and hydrated to a final concentration of 1 mM lipid, with or without cholesterol oxidase (30 U / mL). Liposomes were repeatedly squeezed using a 10 ml syringe and a 30-gauge needle, and sonicated for 3 min until liposome formation was observed under a microscope.

[0118] 2. Results:

[0119] It has been reported that cholesterol oxidase reacts only with active membrane cholesterol in the aqueous phase. To verify that the Acrylodan-BG / SNAP-D4 system can detect active membrane cholesterol, we prepared cholesterol oxidase liposomes to facilitate its entry into Huh-7 cells. Studies have reported that active membrane cholesterol can regulate Na+. + -K +ATPase activity was also observed; we found that after cholesterol oxidase consumed cholesterol from the active membrane, Na+... + -K + ATPase activity also decreased. Figure 10 A) Prove that active membrane cholesterol and Na + -K + ATPase activity is closely related. After treatment with cholesterol oxidase for several minutes, the green fluorescence of Acrylodan-BG / SNAP-D4 in Huh-7 cells significantly decreased, while the blue fluorescence did not decrease significantly. Figure 10 (BC). No change in fluorescence was observed after treatment with blank liposomes, ruling out the possible influence of liposomes on fluorescence changes. The results suggest that the green fluorescence of the Acrylodan-BG / SNAP-D4 probe system may indicate the presence and physiological significance of active membrane cholesterol.

[0120] To further verify the activity of membrane cholesterol, we stimulated the L02 cell line, which stably expresses SNAP-D4 and contains very little intracellular cholesterol, with exogenous cholesterol. It has been reported that Na+ ionization occurs when cholesterol concentration exceeds a threshold (10-20 mol% cholesterol). + -K + ATPase activity changed drastically. We found that when L02 cells were treated with approximately 4 mM cholesterol, Na... + -K + ATPase activity increases suddenly, showing an S-shaped upward curve. Figure 10 D). Simultaneously, both blue and green fluorescence of Acrylodan-BG / SNAP-D4 increased upon cholesterol treatment. However, the blue fluorescence increased with a Langmuir-type saturation curve, while the green fluorescence increased with an S-shaped curve, showing a significant change upon treatment with 4 mM cholesterol. Figure 10 E), and Na + -K + The pattern of increased ATPase activity is consistent with that of other methods. These results further demonstrate that the green fluorescence of Acrylodan-BG / SNAP-D4 is associated with active membrane cholesterol and its physiological effects, further proving the feasibility and significance of the Acrylodan-BG / SNAP-D4 probe system for detecting active membrane cholesterol.

[0121] Figure 10 The Acrylodan-BG / SNAP-D4 probe system is helpful for detecting active membrane cholesterol exposed to the aqueous phase. A is blank liposomes or liposomes containing cholesterol oxidase (30 U / mL) after treatment of Huh-7 cells with Na+. + -K +ATPase activity. B shows the changes in blue and green fluorescence of Acrylodan-BG / SNAP-D4 cells after treatment with blank liposomes or cholesterol oxidase liposomes, respectively. C is... Figure 10 B represents the area under the curve (AUC) of green and blue fluorescence. D represents the Na+ levels after treating L02 cells with different concentrations of cholesterol. + -K + ATPase activity. E represents the fluorescence changes of blue and green fluorescence in L02 cells treated with different concentrations of cholesterol. N≥3, **P<0.01, mean ± standard deviation, ns, no statistical significance.

[0122] Figure 1 This diagram illustrates the principle of labeling intracellular cholesterol with Acrylodan / SNAP-D4. In the diagram, A represents the reaction between the cysteine-containing thiol group at the SNAP-tag active site of SNAP-D4 and the O-6-benzylguanine group of Acrylodan-BG. B shows the Acrylodan / SNAP-D4 probe system distinguishing between lipid-phase cholesterol (lipid raft cholesterol) and partially aqueous-exposed membrane cholesterol (active membrane cholesterol) within the cell.

[0123] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0124] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A probe for detecting cholesterol, characterized in that: SNAP-D4 fusion protein expressed in mammalian cells and lyotropic fluorescent dye Acrylodan-BG, the SNAP-D4 fusion protein expressed in mammalian cells contains a SNAP tag, and the cysteine ​​at the active site of the SNAP tag is covalently bound to BG of the lyotropic fluorescent dye Acrylodan-BG to form a fluorescent probe system; The SNAP-D4 fusion protein expressed by mammalian cells has the amino acid sequence SEQ ID NO.3; The structure of the solvatochromic fluorescent dye Acrylodan-BG is as follows: 。 2. The use of a cholesterol detection probe as claimed in claim 1 in distinguishing and detecting lipid phase cholesterol and aqueous phase cholesterol, characterized in that: Used to distinguish and detect lipid raft cholesterol and active membrane cholesterol by fluorescence imaging.

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