8: 2 fluorotelomeric alcohol-target protein interaction analysis method based on confocal fluorescence imaging

Abstract

The invention relates to the technical field of molecular biology, in particular to an 8: 2 fluorotelomer-target protein interaction analysis method based on confocal fluorescence imaging. According to the method, Fmoc-Glu (OtBu) OH is used as a linker reagent and reacts with a straight-chain alkane pollutant 8: 2FTOH to generate a 8: 2FTOH-Glu (OtBu)-FITC compound with relatively strong green fluorescence, and then the interaction strength between the 8: 2FTOH and a target protein (AHR) is observed and analyzed through a confocal microscope. The method is simple, visual, high in economic applicability and easy and convenient to operate; compared with the conventional molecule-protein interaction technology, the method has the advantages that the test conditions are simple, and additional instruments and equipment are not needed. Meanwhile, the interaction analysis method provided by the invention can be applied to analysis of multi-organ inflammation and injury mediated by 8: 2FTOH through activation of AHR.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to an 8:2 fluoromodulin alcohol-target protein interaction analysis method based on confocal fluorescence imaging. Background Art

[0002] The linear alkane pollutant 8:2 fluorotelomer alcohol (8:2FTOH) is a new type of persistent organic pollutant that can be enriched in the food chain and is widely used in commercial and industrial products such as paints, polishes, coatings, adhesives and electronic products. The wide application of 8:2FTOH makes it ubiquitous in the environment. 8:2FTOH can enter animals and humans through multiple pathways such as air, soil and food, and has the risks of hepatotoxicity, nephrotoxicity, developmental toxicity, immunotoxicity and endocrine disruption. Although the level of 8:2FTOH in the environment and the health risks it causes are worrying, there are few studies on the mechanisms of tissue damage and inflammation induced by 8:2FTOH.

[0003] The aryl hydrocarbon receptor (AHR) is a highly conserved ligand-activated transcription factor, a central sensor involved in a variety of environmental responses, and an important participant in the biological toxicity induced by environmental pollutants. In recent years, studies have reported that PFAS can activate AHR and mediate oxidative stress and inflammation through this protein. However, the reported studies only used virtual docking technology to illustrate that PFAS can bind to AHR and cause an increase in the expression of CYP1A1, a gene related to the AHR pathway. The reported AHR ligands are all aromatic planar hydrocarbons, and at this stage, no studies have confirmed that 8:2FTOH is a direct ligand of AHR.

[0004] At present, the analysis of the interaction between small molecules and proteins mainly relies on the following four technologies: fluorescence polarization immunoassay (FPIA), isothermal calorimetric titrator (ITC) and surface plasmon resonance (SPR). However, these methods have disadvantages such as expensive instruments and reagents, complex operation, difficulty in obtaining proteins, difficulty in protein fixation and non-specific binding. FITC fluorescence imaging observation has the characteristics of simple experimental conditions, no need for additional instruments and equipment, and easy operation. However, there are no reports on the analysis of the interaction between 8:2FTOH and AHR by FITC fluorescence imaging. Summary of the invention

[0005] The purpose of the present invention is to provide a method for analyzing the interaction between 8:2 fluoromodulin alcohol and target protein based on confocal fluorescence imaging to solve the problems existing in the above-mentioned prior art. The present invention uses Fmoc-Glu (OtBu) OH as a linker reagent and a straight-chain alkane pollutant 8:2FTOH to produce a 8:2FTOH-Glu (OtBu) -FITC complex with strong fluorescence, and then observes and analyzes the strength of the interaction between 8:2FTOH and the target protein through a confocal microscope. The present invention is simple and intuitive, highly economical and easy to operate; compared with previous molecule-protein interaction technologies, the experimental conditions are simple and no additional instruments and equipment are required.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for analyzing the interaction between 8:2 fluoromodulin and target protein based on confocal fluorescence imaging, comprising the following steps:

[0008] After 8:2FTOH, Fmoc-Glu(OtBu)OH and the catalyst are mixed and reacted, washing, drying with anhydrous magnesium sulfate and first vacuum drying are performed in sequence to obtain 8:2FTOH-Fmoc-Glu(OtBu);

[0009] The Fmoc group of the 8:2FTOH-Fmoc-Glu(OtBu) is removed, a second vacuum drying is performed, fluorescein is added for fluorescence reaction, and purification is performed to obtain 8:2FTOH-Glu(OtBu)-FITC;

[0010] The 8:2FTOH-Glu(OtBu)-FITC was co-incubated with cells, and the interaction between the compound and the target protein was determined by fluorescence localization analysis.

[0011] Preferably, the target protein is aryl hydrocarbon receptor.

[0012] Preferably, the molar concentration ratio of the 8:2FTOH, Fmoc-Glu(OtBu)OH and the catalyst is 1:1.2:1.

[0013] Preferably, the mixing reaction time is 22 hours and the temperature is 20°C.

[0014] Preferably, the preparation used for removing the Fmoc group is 50% polyvinyl pyrrolidone.

[0015] Preferably, the catalyst is 4-dimethylaminopyridine.

[0016] Preferably, the fluorescein is isothiocyanate.

[0017] Preferably, the purification column used in the purification is a G25 fast desalting column.

[0018] Preferably, the temperature of the first vacuum drying is 60° C. and the time is 0.5 h;

[0019] And / or, the temperature of the second vacuum drying is 60° C. and the time is 2 hours.

[0020] Preferably, the temperature of the fluorescence reaction is 4°C and the time is 4 hours.

[0021] The present invention discloses the following technical effects:

[0022] The present invention establishes a method for observing and analyzing the interaction between 8:2FTOH and aromatic hydrocarbon receptor in cells based on confocal fluorescence imaging. The method is simple and intuitive, and has high economic applicability. Specifically: the present invention uses Fmoc-Glu (OtBu) OH as a linker reagent and a linear alkane pollutant 8:2FTOH to produce a 8:2FTOH-Glu (OtBu) -FITC complex with strong green fluorescence, and then the strength of the interaction between 8:2FTOH and a target protein (AHR) can be observed and analyzed by a confocal microscope. The present invention is simple and intuitive, has high economic applicability, and is easy to operate; compared with previous molecule-protein interaction technologies, the experimental conditions are simple and no additional instruments and equipment are required.

[0023] At the same time, the present invention discovered and reported for the first time that the straight-chain alkane pollutant 8:2FTOH is a direct ligand of AHR. Therefore, the interaction analysis method provided by the present invention can be used for the analysis of 8:2FTOH mediating multi-organ inflammation and damage by activating AHR. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The synthesis process of 8:2FTOH-Glu(OtBu)-FITC;

[0026] Figure 2 It is the mass spectrum of 8:2FTOH-Glu(OtBu);

[0027] Figure 3 It is the mass spectrum of 8:2FTOH-Glu(OtBu)-FITC;

[0028] Figure 4The confocal fluorescence images of 8:2FTOH-Glu(OtBu)-FITC and AHR at different incubation times; among them, 8:2FTOH+FITC refers to the treatment using 8:2FTOH-Glu(OtBu)-FITC; FITC refers to the treatment using only FITC. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 18: Establishment of Synthesis Conditions of 2FTOH-Fmoc-Glu(OtBu)

[0035] (1) Optimization of the synthesis ratio of 8:2FTOH-Fmoc-Glu(OtBu)

[0036] The synthesis ratio was optimized according to Table 1, with the yield of 8:2FTOH-Fmoc-Glu(OtBu) as the selection criterion.

[0037] The specific steps are as follows:

[0038] According to the ratio 1 and ratio 2 in Table 1, accurately weigh the corresponding mass of 8:2FTOH, Fmoc-Glu(OtBu)OH and catalyst DMAP (4-dimethylaminopyridine) in a 50mL round-bottom flask containing 15mL dichloromethane (DCM). After complete dissolution, stir at room temperature 25°C for 24h. After the reaction is completed, add an equal volume of DCM and wash twice with an equal volume of distilled water. After washing, dry with anhydrous magnesium sulfate and vacuum dry at 60°C for 0.5h to obtain 8:2FTOH-Fmoc-Glu(OtBu), and weigh to calculate the yield.

[0039] Table 1 Compound ratio

[0040]

[0041] The results showed that when the molar concentration ratio of 8:2FTOH, Fmoc-Glu(OtBu)OH and DMAP was 1:1.2:1, the yield of 8:2FTOH-Fmoc-Glu(OtBu) was the highest (Table 1).

[0042] (2) Optimization of synthesis temperature and time of 8:2FTOH-Fmoc-Glu(OtBu)

[0043] Further optimization was performed based on the temperature and synthesis time of the previous step, and the ratio of 8:2FTOH, Fmoc-Glu(OtBu)OH and DMAP was 1:1.2:1. The temperature and synthesis time were optimized according to Table 2, and the yield of 8:2FTOH-Fmoc-Glu(OtBu) was used as the selection criterion.

[0044] The specific steps are as follows:

[0045] Accurately weigh the corresponding mass of 8:2FTOH, Fmoc-Glu(OtBu)OH and catalyst DMAP in a 50mL round-bottom flask containing 15mL dichloromethane (DCM) at a molar concentration ratio of 1:1.2:1. After complete dissolution, stir at 20℃, 25℃ and 30℃ for 22, 24 and 26h respectively. After the reaction is completed, add an equal volume of DCM and wash twice with an equal volume of distilled water. After washing, dry with anhydrous magnesium sulfate and vacuum dry at 60℃ for 0.5h to obtain 8:2FTOH-Fmoc-Glu(OtBu), and weigh to calculate the yield.

[0046] Table 2 Yield (%) of 8:2FTOH-Fmoc-Glu(OtBu) at different temperatures and synthesis times

[0047] Temperature (℃) / Synthesis time (h) 22 24 26 20 94 88 84 25 89 77 76 30 82 75 77

[0048] The results showed that the yield of 8:2FTOH-Fmoc-Glu(OtBu) was highest when stirred at 20°C for 22 h (Table 2).

[0049] Example 28: Establishment of Synthesis Conditions of 2FTOH-Glu(OtBu)-FITC

[0050] Screening of de-Fmoc reagents: The specific types of de-Fmoc reagents are shown in Table 3, and the yield of 8:2FTOH-Glu(OtBu)-FITC is used as the selection criterion.

[0051] The synthesis process of 8:2FTOH-Glu(OtBu)-FITC is as follows Figure 1 As shown, the specific steps are as follows:

[0052] Accurately weigh the corresponding mass of 8:2FTOH, Fmoc-Glu(OtBu)OH and catalyst DMAP in a 50mL round-bottom flask containing 15mL dichloromethane (DCM) at a molar concentration ratio of 1:1.2:1. After complete dissolution, stir at 20°C for 22h. After the reaction is completed, add an equal volume of DCM and wash twice with an equal volume of distilled water. After washing, dry with anhydrous magnesium sulfate and vacuum dry at 60°C for 0.5h to obtain 8:2FTOH-Fmoc-Glu(OtBu). 8:2FTOH-Fmoc-Glu(OtBu) was stirred at room temperature for 30min with 50% polyvinylpyrrolidone solution, acetone and diethylamine to remove the Fmoc group, and vacuum dried at 60°C for 2h to obtain 8:2FTOH-Glu(OtBu). The above product was dissolved in dimethylformamide (DMF), and 0.1mM fluorescein isothiocyanate (FITC) was added and reacted at 4°C for 4h. The reaction product was purified by G25 fast desalting column to obtain 8:2FTOH-Glu(OtBu)-FITC, and then vacuum dried at 60°C and weighed to calculate the yield. At the same time, 8:2FTOH-Glu(OtBu) and 8:2FTOH-Glu(OtBu)-FITC were detected by mass spectrometry. The mass spectrum of 8:2FTOH-Glu(OtBu) is shown in Figure 2 As shown, the mass spectrum of 8:2FTOH-Glu(OtBu)-FITC is as follows Figure 3 shown.

[0053] Table 3 Yield (%) of 8:2FTOH-Glu(OtBu)-FITC under different Fmoc removal reagents

[0054] Removal of Fmoc reagent Yield (%) 50% Polyvinylpyrrolidone 82 acetone 50 Diethylamine 66

[0055] The results showed that the yield was the highest when 50% polyvinyl pyrrolidone was used as the de-Fmoc reagent (Table 3).

[0056] Example 38: Establishment of 2FTOH-Glu-FITC and Cell Incubation Duration

[0057] Accurately weigh the corresponding mass of 8:2FTOH, Fmoc-Glu(OtBu)OH and catalyst DMAP in a 50mL round-bottom flask containing 15mL dichloromethane (DCM) at a molar concentration ratio of 1:1.2:1. After complete dissolution, stir at 20°C for 22h. After the reaction is completed, add an equal volume of DCM and wash twice with an equal volume of distilled water. After washing, dry with anhydrous magnesium sulfate and vacuum dry at 60°C for 0.5h to obtain 8:2FTOH-Fmoc-Glu(OtBu). 8:2FTOH-Fmoc-Glu(OtBu) was stirred with 50% polyvinylpyrrolidone solution at room temperature for 30min to remove the Fmoc group, and vacuum dried at 60°C for 2h to obtain 8:2FTOH-Glu(OtBu). The above product was dissolved in dimethylformamide (DMF), and 0.1mM FITC was added and reacted at 4°C for 4h. The reaction product was purified by G25 fast desalting column to obtain 8:2FTOH-Glu(OtBu)-FITC. After incubating 8:2FTOH-Glu(OtBu)-FITC with cells for 1 and 2 hours, the culture medium containing the complex was removed and the immunofluorescence test of AHR was performed. The colocalization of 8:2FTOH-Glu(OtBu)-FITC (green fluorescence) and AHR (red fluorescence) was observed under a confocal microscope. The results are shown in Figure 2. Figure 4 The results showed that the imaging effect of 8:2FTOH-Glu(OtBu)-FITC and AHR was the best when incubated for 2 hours, and the co-localization between 8:2FTOH and AHR could be observed.

[0058] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for analyzing the interaction between 8:2 fluoromodulin and target protein based on confocal fluorescence imaging, characterized in that: The following steps are involved: After 8:2 fluorotelomer alcohol (8:2 FTOH), Fmoc-Glu (OtBu) OH and a catalyst are mixed and reacted, washing, drying with anhydrous magnesium sulfate and first vacuum drying are performed in sequence to obtain 8:2 FTOH-Fmoc-Glu (OtBu); The Fmoc group of the 8:2FTOH-Fmoc-Glu(OtBu) is removed, a second vacuum drying is performed, fluorescein is added for fluorescence reaction, and purification is performed to obtain 8:2FTOH-Glu(OtBu)-FITC; The 8:2FTOH-Glu(OtBu)-FITC was co-incubated with cells, and the interaction between the compound and the target protein was determined by fluorescence localization analysis.

2. The interaction analysis method according to claim 1, characterized in that: The target protein is the aryl hydrocarbon receptor.

3. The interaction analysis method according to claim 1, characterized in that: The molar concentration ratio of the 8:2FTOH, Fmoc-Glu(OtBu)OH and the catalyst is 1:1.2:

1.

4. The interaction analysis method according to claim 1, characterized in that: The mixing reaction was carried out for 22 hours at a temperature of 20°C.

5. The interaction analysis method according to claim 1, characterized in that: The preparation used for removing the Fmoc group is 50% polyvinyl pyrrolidone.

6. The interaction analysis method according to claim 1 or 3, characterized in that: The catalyst is 4-dimethylaminopyridine.

7. The interaction analysis method according to claim 1, characterized in that: The fluorescein is isothiocyanate.

8. The interaction analysis method according to claim 1, characterized in that: The purification column used in the purification is a G25 fast desalting column.

9. The interaction analysis method according to claim 1, characterized in that: The first vacuum drying temperature is 60°C and the time is 0.5h; And / or, the temperature of the second vacuum drying is 60° C. and the time is 2 hours.

10. The interaction analysis method according to claim 1, characterized in that: The temperature of the fluorescence reaction is 4° C. and the time is 4 hours.

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