Protein fluorescein labeling method based on dynamic response and application thereof

Abstract

The invention relates to the field of biochemistry, and discloses a protein fluorescein labeling method based on dynamic response and application thereof, and the method comprises the following steps: step 1, selecting fluorescein modifiers of various response types, and step 2, respectively labeling the fluorescein modifiers of different response types with target protein molecules. And step 3, monitoring a dynamic response fluorescence signal of the labeled protein along with the change of a biological environment in a cell culture process. The invention provides a dynamic response type marking technology which can respond to a specific signal in a biological environment, so that the marked fluorescence intensity is changed, the behavior and state of protein in a biological system can be more accurately reflected, and the dynamic response type marking technology has huge advantages in the fields of real-time monitoring, biosensors and the like. A plurality of response type labeling agents are integrated into one labeling system, so that the labeling system is endowed with wider applicability and flexibility, and particularly has important value in application in the fields of cell imaging, disease monitoring and the like.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to a protein fluorescent labeling method. More specifically, the present invention provides a protein fluorescent labeling method based on dynamic response (such as pH, temperature and enzyme activity) for achieving controllability and sensitivity of protein labeling, which is widely used in the fields of biomarkers, cell imaging, disease diagnosis and biosensors. Background Art

[0002] The principles and schemes of protein-coupled fluorescein are crucial in biochemical research and are widely used in cell imaging, molecular labeling, and biological research. These methods are usually used to label molecules such as antibodies, enzymes, and proteins for subsequent analysis, imaging, and detection. The detection signals of proteins themselves mainly come from the ultraviolet absorption of the protein itself. Some proteins also have weak fluorescence, but the intensity of these detection signals is weak, which makes the detection sensitivity low. For some trace amounts of proteins, it is difficult to achieve effective detection through the protein's own signal.

[0003] Common protein labeling methods include isothiocyanate fluoresceins, such as FITC (fluorescein isothiocyanate), Cy3, Cy5, etc., which are the most commonly used fluorescein modification reagents. This type of fluorescein can react with the amino (–NH2) group of the protein to form a stable covalent bond. NHS (N-Hydroxysuccinimide) ester method is another common protein labeling method, which is widely used in different types of modification reagents. NHS ester reaction usually reacts with the amino group in the protein to form a stable amide bond. Disulfide Bond Crosslinking, this method is usually used to couple modification reagents with sulfhydryl (–SH) groups (such as BODIPY, Fluorescein thiocyanate, etc.). Through disulfide bond bridging, a stable covalent bond can be formed between two proteins or between a protein and fluorescein. Affinity Chromatography Method, this method combines with fluorescein through an affinity tag (such as His tag, GST tag), which is suitable for labeling recombinant proteins containing tags.

[0004] Protein labeling methods mainly rely on static labeling, which fixes the fluorescent properties of the label and cannot respond to changes in the biological environment. However, in many practical applications, the biological environment (such as pH, temperature, enzyme activity, etc.) affects the structure and function of the protein, and traditional static labeling methods cannot adapt to such changes. Therefore, there is an urgent need for an innovative method that can respond to changes in the biological environment and achieve "dynamic labeling" or "controllable labeling." Summary of the invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a protein fluorescent labeling method based on dynamic response, comprising the following steps:

[0006] Step 1, selecting multiple response type fluorescent modifiers;

[0007] Step 2: Label the target protein molecules with the fluorescent modifiers of different response types respectively.

[0008] Step 3: Monitor the dynamic response fluorescence signal of the labeled protein during cell culture as the biological environment changes.

[0009] Furthermore, the responsive type fluorescein modifier includes a pH responsive type fluorescein modifier, a temperature responsive type fluorescein modifier and an enzyme activity responsive type fluorescein modifier.

[0010] Furthermore, the pH-responsive fluorescein modifier is fluorescein with an imidazole group.

[0011] Furthermore, the temperature-responsive fluorescein modifier is fluorescein with a thermosensitive group.

[0012] Furthermore, the enzyme activity responsive fluorescein modifier is a fluorescein containing a protease recognition site.

[0013] Furthermore, as the pH value increases, the fluorescence intensity gradually decreases.

[0014] Furthermore, as the temperature increases, the fluorescence intensity gradually increases.

[0015] Furthermore, after adding a specific enzyme, the fluorescence intensity increased.

[0016] An application of the protein fluorescent labeling method based on dynamic response is applied to biological labeling, cell imaging, disease diagnosis and biosensors.

[0017] The present invention proposes a dynamic response labeling technology that can respond to specific signals in the biological environment (such as pH, temperature, enzyme activity, etc.), so that the fluorescence intensity of the label changes, and can more accurately reflect the behavior and state of proteins in the biological system, especially in the fields of real-time monitoring and biosensors. Integrating multiple responsive labeling agents (for example, pH responsive, temperature responsive, enzyme activity responsive) into a labeling system gives the labeling system a wider range of applicability and flexibility, especially in multi-field applications such as cell imaging and disease monitoring. DETAILED DESCRIPTION

[0018] The present invention will be further described below.

[0019] Example 1: pH-responsive protein fluorescent labeling

[0020] Experimental objectives

[0021] By designing pH-responsive fluorescein modifiers, pH-dependent regulation of protein labeling can be achieved, and the effect of pH changes on protein fluorescence signals can be detected.

[0022] Experimental procedures

[0023] 1. Selection of modifier: Select a pH-responsive fluorescein (such as fluorescein with an imidazole group), which exhibits strong fluorescence in an acidic environment and weakens in an alkaline environment.

[0024] 2. Protein modification: Dissolve the target protein in PBS buffer and modify the protein using NHS azidate ester to introduce azide groups onto the protein.

[0025] 3. Fluorescein coupling reaction: The pH-responsive fluorescein is subjected to a click chemistry reaction (azide-alkyne reaction) with the azide-modified protein, and the labeling process is carried out in a buffer solution at pH 7.4.

[0026] 4. Fluorescence detection: Measure the changes in fluorescence intensity of the labeled protein under different pH conditions (such as pH 5.0, 7.4 and 9.0).

[0027] Experimental Results

[0028] At pH 5.0, the fluorescence intensity was higher.

[0029] At pH 7.4, the fluorescence intensity was moderate.

[0030] At pH 9.0, the fluorescence intensity decreased significantly.

[0031] The specific results are shown in Table 1.

[0032] pH Fluorescence intensity (relative value) 5.0 1.00 7.4 0.60 9.0 0.20

[0033] Table 1 Relative values ​​of fluorescence intensity at different pH values.

[0034] Example 2: Temperature-responsive protein fluorescent labeling

[0035] Experimental objectives

[0036] By designing temperature-responsive fluorescent modifiers, the effects of temperature changes on protein-labeled fluorescent signals are studied, especially the effects of labeling temperature changes on proteins in cell experiments.

[0037] Experimental procedures

[0038] 1. Selection of modifiers: Select fluorophores with thermosensitive groups (such as hydrophilic groups and hydrophobic groups) that can cause changes in fluorescence signals when the temperature changes.

[0039] 2. Protein modification: Dissolve the target protein in PBS buffer and modify the protein using NHS azidate ester to add azide groups.

[0040] 3. Fluorescein coupling reaction: The temperature-responsive fluorescein modifier is coupled to the protein through a click chemistry reaction (azide-alkyne reaction).

[0041] 4. Temperature change monitoring: Detect the changes in fluorescence intensity of the labeled protein at different temperatures (such as 25°C, 37°C, 42°C).

[0042] Experimental Results

[0043] At low temperature (25°C), the fluorescence intensity of the labeled protein is low.

[0044] At 37°C, the fluorescence intensity increased significantly.

[0045] At 42°C, the fluorescence intensity was further enhanced.

[0046] The specific results are shown in Table 2.

[0047] Temperature(℃) Fluorescence intensity (relative value) 25 0.30 37 0.80 42 1.20

[0048] Table 2 Relative values ​​of fluorescence intensity at different pH values.

[0049] Example 3: Enzyme activity responsive protein fluorescent labeling

[0050] Experimental objectives

[0051] By designing enzyme activity-responsive luciferin modifiers, the effects of specific enzymes (such as proteases or phosphatases) on protein labeling are detected, and the enzyme activity is dynamically monitored by utilizing the labeling changes triggered by enzyme activity.

[0052] Experimental procedures

[0053] 1. Selection of modifiers: Select a fluorescein modifier with a specific enzyme recognition sequence, such as a fluorescein containing a protease recognition site (such as a trypsin site).

[0054] 2. Protein modification: Dissolve the target protein in PBS or Tris-HCl buffer, and modify the protein using NHS azidate ester to add azide groups.

[0055] 3. Fluorescein coupling reaction: Use click chemistry (azide-alkyne reaction) to couple the enzyme activity-responsive fluorescein modifier to the protein.

[0056] 4. Enzyme treatment and detection: Add specific enzymes (such as trypsin) for treatment and observe the changes in the fluorescence intensity of the labeled protein before and after enzyme treatment.

[0057] Experimental Results

[0058] The fluorescence intensity of untreated protein was the baseline value (0.50).

[0059] After adding trypsin, the fluorescence intensity changes significantly, reflecting the hydrolysis of the enzyme.

[0060] The specific results are shown in Table 3.

[0061] Enzyme treatment status Fluorescence intensity (relative value) Unprocessed 0.50 Trypsin treatment 1.00

[0062] Table 3 Relative values ​​of fluorescence intensity under different enzyme treatment conditions.

[0063] Example 4: Real-time biosignal monitoring with integrated dynamic markers

[0064] Experimental objectives

[0065] Combining pH, temperature and enzyme activity responsive labeling technology, the dynamic changes of proteins in different biological environments are studied, and real-time tracking of protein functional status is achieved through dynamic monitoring.

[0066] Experimental procedures

[0067] 1. Select three responsive luciferin modifiers (responding to pH, temperature and enzyme activity respectively).

[0068] 2. Label the target protein molecules with three different types of fluorescent modifiers.

[0069] 3. Monitor the fluorescence signal of the labeled protein during cell culture as it changes with pH, ​​temperature and enzyme activity through a real-time fluorescence detection system.

[0070] 4. Use a multi-channel fluorescence microscope to simultaneously detect changes in three signal channels.

[0071] Experimental Results

[0072] During cell culture, the fluorescence signal gradually increases or decreases as the pH changes.

[0073] When the temperature increases, the fluorescence signal also shows an increasing trend.

[0074] After the addition of protease, changes in labeling caused by enzyme activity are reflected in changes in the fluorescence signal.

[0075] The specific results are shown in Table 4.

[0076]

[0077] Table 4 Relative values ​​of fluorescence intensity under different biological environments.

Claims

1. A protein fluorescent labeling method based on dynamic response, characterized in that The steps include: Step 1, selecting multiple response type fluorescent modifiers; Step 2: Label the target protein molecules with the fluorescent modifiers of different response types respectively.

2. Step 3: Monitor the dynamic response fluorescence signal of the labeled protein during cell culture as the biological environment changes.

3. The protein fluorescent labeling method based on dynamic response according to claim 1, characterized in that: The responsive type luciferin modifiers include pH responsive type luciferin modifiers, temperature responsive type luciferin modifiers and enzyme activity responsive type luciferin modifiers.

4. The protein fluorescent labeling method based on dynamic response according to claim 2, characterized in that: The pH-responsive fluorescein modifier is fluorescein with an imidazole group.

5. The protein fluorescent labeling method based on dynamic response according to claim 2, characterized in that: The temperature-responsive fluorescein modifier is fluorescein with a thermosensitive group.

6. The protein fluorescent labeling method based on dynamic response according to claim 2, characterized in that: The enzyme activity responsive fluorescein modifier is a fluorescein containing a protease recognition site.

7. The protein fluorescent labeling method based on dynamic response according to claim 2, characterized in that: As the pH value increases, the fluorescence intensity gradually decreases.

8. The protein fluorescent labeling method based on dynamic response according to claim 2, characterized in that: As the temperature increases, the fluorescence intensity gradually increases.

9. The protein fluorescent labeling method based on dynamic response according to claim 1, characterized in that: After adding a specific enzyme, the fluorescence intensity increases.

10. An application of the protein fluorescent labeling method based on dynamic response according to any one of claims 1 to 8, characterized in that: Applications: biomarkers, cell imaging, disease diagnosis and biosensors.

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