A fluorescently labeled polypeptide, an apoptosis detection kit and a detection method thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a fluorescently labeled polypeptide, an apoptosis detection kit and a detection method thereof. The amino acid sequence of the polypeptide in the fluorescently labeled polypeptide is as follows: YGRKKRRQRRRYYAMKGAGTD. The fluorescently labeled polypeptide or the detection kit provided by the present application can accurately detect apoptotic cells, the detection result is similar to that of a commercially available product, and the detection method of the present application does not depend on the presence of Ca 2+ , overcoming the disadvantages of dependence on the presence of Ca 2+ in FITC-Annexin V detection, and being suitable for apoptosis evaluation and detection of various cell types.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a fluorescently labeled polypeptide, a cell apoptosis detection kit and detection method thereof, specifically to a fluorescently labeled polypeptide for detecting cell apoptosis and its preparation method, a cell apoptosis detection kit and its detection method thereof. Background Technology

[0002] Apoptosis, or programmed cell death, is one of the cell's unique defense mechanisms, playing a crucial role in normal development and disease resistance. Abnormalities in the apoptosis process and pathways can lead to various diseases, such as tumors and cardiovascular diseases.

[0003] Currently, there are more than ten methods for detecting apoptosis, mainly through three aspects: cell morphology, biological function, and biochemical markers. Among them, one of the most commonly used methods is the marker detection method based on flow cytometry. The main principle of this method is as follows: Phosphatidylserine (PS) is normally located on the inner side of the cell membrane, but in the early stage of apoptosis, PS can flip from the inner side of the cell membrane to the surface, exposing it to the extracellular environment. Annexin-V is a Ca2+ cell with a molecular weight of 35-36 kDa. 2+ Annexin-V is a phospholipid-dependent protein that binds specifically to PS with high affinity. Labeling Annexin-V with fluorescein (FITC, PE) or biotin, and using the labeled Annexin-V as a fluorescent probe, allows for the detection of apoptosis using flow cytometry or fluorescence microscopy. However, this method is dependent on Ca2+. 2+ The presence of Ca, i.e., Ca in the cell or environment. 2+ It can be an important factor affecting the test results.

[0004] Therefore, it is desirable to provide a non-Ca 2+ Methods for detecting apoptosis-dependent cell death. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a fluorescently labeled peptide, a cell apoptosis detection kit, and a detection method thereof. The fluorescently labeled peptide or detection kit provided by this invention can accurately detect apoptotic cells, and the detection results are similar to those of commercially available products. Furthermore, the detection method of this invention does not depend on Ca2+. 2+ The existence of this technology overcomes the dependence on Ca in FITC-Annexin V detection. 2+ The existing drawbacks make it unsuitable for apoptosis assessment and detection in various cell types.

[0006] In a first aspect, the present invention provides a fluorescently labeled polypeptide for detecting apoptosis, wherein the amino acid sequence of the polypeptide is shown below:

[0007] YGRKKRRQRRRYYAMKGAGTD (Tat-3, SEQ ID NO. 1).

[0008] After analyzing the molecular characteristics of Annexin-V binding to PS, the applicant preliminarily identified three non-Ca molecules that mediate the binding of Annexin-V to PS. 2+ Further analysis of the structural features and in vitro simulated binding characteristics of Annexin-V in the dependent region identified three key sites, yielded their amino acid sequences, and specifically revealed that Tat-3, when labeled with fluorescein, achieved similar detection results to existing commercially available products for apoptosis detection, and was independent of Ca2+. 2+ The existence of this technology overcomes the dependence on Ca in FITC-Annexin V detection. 2+ The existing drawbacks make it unsuitable for apoptosis assessment and detection in various cell types.

[0009] In a preferred embodiment of the present invention, the fluorescein in the fluorescently labeled polypeptide is bonded to the tyrosine residue at the end of the polypeptide.

[0010] As a preferred embodiment of the present invention, the fluorescein includes dansyl chloride.

[0011] As a preferred embodiment of the present invention, the molecular formula of the fluorescently labeled polypeptide is:

[0012] .

[0013] In a second aspect, the present invention provides a method for preparing the fluorescently labeled polypeptide described in the first aspect, the method comprising the following steps:

[0014] (1) Fmoc-Asp(otbu)-Wang resin was placed in a sand core reaction column for swelling and deprotection reaction;

[0015] (2) According to the amino acid sequence from the C-terminus to the N-terminus of the peptide chain, the corresponding amino acids with protecting groups and condensing agents are added to the reaction column, and a coupling reaction is carried out in the presence of an activator. Then, a deprotection reaction is carried out. The coupling reaction and deprotection reaction are repeated until the peptide chain is synthesized.

[0016] (3) After the peptide chain is synthesized, fluorescein is coupled to tyrosine residues to obtain the fluorescently labeled polypeptide.

[0017] In a preferred embodiment of the present invention, the condensing agent is TBTU.

[0018] As a preferred embodiment of the present invention, the activator is N -Methylmorpholine.

[0019] As a preferred embodiment of the present invention, the fluorescein is dansyl chloride.

[0020] Thirdly, the present invention provides a cell apoptosis detection kit, the detection kit comprising the fluorescently labeled peptide described in the first aspect.

[0021] The detection kit provided by this invention can accurately detect apoptotic cells, and its detection results are similar to those of commercially available products. Furthermore, the detection method of this invention does not depend on Ca. 2+ It exists and overcomes the dependence on Ca in FITC-Annexin V detection. 2+ The existing drawbacks make it unsuitable for apoptosis assessment and detection in various cell types.

[0022] As a preferred embodiment of the present invention, the detection kit further includes PBS solvent.

[0023] Fourthly, the present invention provides a method for detecting apoptosis using the fluorescently labeled peptide described in the first aspect or the detection kit described in the third aspect, the method comprising: identifying apoptotic cells by specifically binding the fluorescently labeled peptide to phosphatidylserine, and then detecting the occurrence of apoptosis using flow cytometry or fluorescence microscopy.

[0024] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0025] The fluorescently labeled peptides or detection kits provided by this invention can accurately detect apoptotic cells, and the detection results are similar to those of commercially available products. Furthermore, the detection method of this invention does not depend on Ca2+. 2+ The existence of this technology overcomes the dependence on Ca in FITC-Annexin V detection. 2+ The existing drawbacks make it unsuitable for apoptosis assessment and detection in various cell types. Attached Figure Description

[0026] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0028] Figure 1 Flow cytometry images of renal cell carcinoma cells after apoptosis induction in the Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group.

[0029] Figure 2 Flow cytometry images of colon cancer cells after apoptosis induction in the Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group.

[0030] Figure 3 Flow cytometry images of Annexin V-FITC, Tat-3-Dansyl, and Dansyl groups after neuronal apoptosis was induced.

[0031] Figure 4 For Ca 2+ Flow cytometry images of Annexin V-FITC group, Tat-3-Dansyl group and Dansyl group are present;

[0032] Figure 5 For Ca 2+ Flow cytometry plots for the Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group were not available.

[0033] Figure 6 For Ca 2+ Imaging flow cytometry of the Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group with and without presence. Detailed Implementation

[0034] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0035] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0036] The sources and functions of some of the reagents involved in the following examples are shown in Table 1 below:

[0037] Table 1

[0038]

[0039] Preparation Example 1

[0040] This preparation example provides a fluorescently labeled polypeptide and its synthesis method. The amino acid sequence of the fluorescently labeled polypeptide is YGRKKRRQRRRYYAMKGAGTD (Tat-3, SEQ ID NO.1), and the fluorescein is dansyl chloride, which is bonded to the tyrosine residue at the end of the polypeptide Tat-3, and is denoted as Tat-3-Dansyl.

[0041] The synthesis method includes the following steps:

[0042] Weigh 1.2 g of Fmoc-Asp(otbu)-Wang Resin into a sand core reaction column, add an appropriate amount of DCM and soak for 5 min. Remove the solution using a vacuum pump, add decapping solution (DMF:piperidine = 4:1), react for 10 min, remove the solution again, add decapping solution again, and react for another 10 min to remove Fmoc. Wash 9 times with DMF, and test the color; it should be blue. Add the corresponding amounts of Fmoc-Thr(tbu)-OH amino acid, condensing agent TBTU, and... N After reacting with methylmorpholine for 1 h, the reaction mixture was removed and washed 6 times with DMF. If the mixture was colorless and transparent, the reaction was complete. If color was observed, the reaction was incomplete and required re-injection until the mixture was colorless and transparent. This process was repeated, following the amino acid sequence from the C-terminus to the N-terminus of the peptide chain. Fmoc was removed, and amino acids were added until the last tyrosine residue, Fmoc-Tyr(tbu)-OH, was added and Fmoc protection was removed. Dansyl chloride was then added, and the reaction was carried out for 2 h. After washing 6 times with DMF, the mixture was dried with methanol and then lysed with a lysis buffer (TFA:H2O:p-cresol:DODT, volume ratio 90:2.5:2.5:5) to obtain the fluorescently labeled peptide Tat-3-Dansyl.

[0043] Example 1

[0044] This embodiment is used to investigate the effect of the fluorescently labeled peptide Tat-3-Dansyl on the detection of apoptosis in renal cell carcinoma cells.

[0045] (1) Oxygen-glucose deprivation experiment (OGD): First, 1×10⁻⁶ g of oxygen-glucose deprivation solution was inoculated into each well of a 6-well plate. 6Cells of renal cell carcinoma (ACHN) were cultured in DMEM complete medium for 24 h. Then, the DMEM complete medium in the renal cell carcinoma cells was replaced with sugar-free DMEM complete medium. The wells were placed in a sealed chamber, and the entire chamber was filled with anaerobic gas (95% N2 / 5% CO2). The sealed chamber was then incubated at 37°C for 4 h. Afterward, the sugar-free DMEM complete medium was replaced again with DMEM complete medium, and the cells were reoxygenated for 24 h. The cells were then digested with 0.25% trypsin solution for 5 min, centrifuged at 1100 rpm for 5 min, collected with PBS, washed twice with ice-cold PBS, resuspended in 100 μL PBS, and 10 μL of 10x binding buffer was added for later use.

[0046] (2) Solution preparation: 5 mg Tat-3-Dansyl was placed in 2 mL PBS solvent to obtain Tat-3-Dansyl solution; 100 μg Dansyl was placed in 100 μL acetonitrile (to promote Dansyl dissolution) and 0.9 mL PBS solvent to obtain Dansyl solution.

[0047] (3) Add 5 μL Annexin V-FITC + 5 μL 7AAD, 5 μL Tat-3-Dansyl solution + 5 μL 7AAD, and 5 μL Dansyl solution + 5 μL 7AAD to the resuspension obtained in step (1), respectively, and incubate at room temperature for 15 min. These groups are designated as Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group, respectively. After binding, each group of cells is washed once with 1.5 mL PBS to remove non-specifically bound fluorescent dyes, and then resuspended in 100 μL PBS. Flow cytometry is then used for detection.

[0048] Test results are as follows Figure 1 As shown, Figure 1 Flow cytometry images of renal cell carcinomas after apoptosis induction in the Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group. Figure 1 It can be observed that both Tat-3-Dansyl and Annexin V-FITC can detect apoptosis in renal cell carcinoma cells, and 7AAD can detect dead renal cell carcinoma cells with similar results, while dansyl chloride alone cannot detect apoptosis in renal cell carcinoma cells.

[0049] Example 2

[0050] This embodiment is used to investigate the effect of the fluorescently labeled peptide Tat-3-Dansyl on the detection of apoptosis in colon cancer cells.

[0051] (1) Oxygen-glucose deprivation experiment (OGD): First, 1×10⁻⁶ g of oxygen-glucose deprivation solution was inoculated into each well of a 6-well plate. 6 One colon cancer cell line (MC38) was cultured in DMEM complete medium for 24 h. Then, the DMEM complete medium in the colon cancer cell line was replaced with sugar-free DMEM complete medium. The wells were placed in a sealed chamber, and the entire chamber was filled with anaerobic gas (95% N2 / 5% CO2). The sealed chamber was then incubated at 37°C for 4 h. Afterward, the sugar-free DMEM complete medium was replaced again with DMEM complete medium, and the cells were reoxygenated for 24 h. The cells were then digested with 0.25% trypsin solution for 5 min, centrifuged at 1100 rpm for 5 min, collected with PBS, washed twice with ice-cold PBS, resuspended in 100 μL PBS, and 10 μL of 10x binding buffer was added for later use.

[0052] (2) Solution preparation: 5 mg Tat-3-Dansyl was placed in 2 mL PBS solvent to obtain Tat-3-Dansyl solution; 100 μg Dansyl was placed in 100 μL acetonitrile and 0.9 mL PBS solvent to obtain Dansyl solution.

[0053] (3) Add 5 μL Annexin V-FITC + 5 μL 7AAD, 5 μL Tat-3-Dansyl solution + 5 μL 7AAD, and 5 μL Dansyl solution + 5 μL 7AAD to the resuspension obtained in step (1), respectively, and incubate at room temperature for 15 min. These groups are designated as Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group, respectively. After binding, each group of cells is washed once with 1.5 mL PBS to remove non-specifically bound fluorescent dyes, and then resuspended in 100 μL PBS. Flow cytometry is then used for detection.

[0054] Test results are as follows Figure 2 As shown, Figure 2 Flow cytometry images of colon cancer cells after apoptosis induction in the Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group. Figure 2 It can be found that Tat-3-Dansyl and Annexin V-FITC can both detect apoptosis in colon cancer cells, 7AAD can detect dead colon cancer cells, and the detection results are similar, while dansyl chloride alone cannot detect apoptosis in colon cancer cells.

[0055] Example 3

[0056] This embodiment is used to investigate the effect of the fluorescently labeled peptide Tat-3-Dansyl on the detection of apoptosis in primary neurons.

[0057] (1) Oxygen-glucose deprivation experiment (OGD): First, 1×10⁻⁶ g of oxygen-glucose deprivation solution was inoculated into each well of a 6-well plate. 6 Primary neurons were cultured in NeuroBasal complete medium for 7 days. Then, the NeuroBasal complete medium in the primary neurons was replaced with sugar-free DMEM complete medium. The wells were placed in a sealed chamber, and the entire chamber was filled with anaerobic gas (95% N2 / 5% CO2). The sealed chamber was incubated at 37°C for 2 hours. Afterward, the DMEM was replaced with NeuroBasal complete medium again, and the cells were reoxygenated for 24 hours. The cells were then digested with 0.25% trypsin solution for 5 minutes, centrifuged at 1100 rpm for 5 minutes, and collected with PBS. The cells were washed twice with ice-cold PBS, resuspended in 100 μL of PBS, and 10 μL of 10x binding buffer was added for later use.

[0058] (2) Solution preparation: 5 mg Tat-3-Dansyl was placed in 2 mL PBS solvent to obtain Tat-3-Dansyl solution; 100 μg Dansyl was placed in 100 μL acetonitrile and 0.9 mL PBS solvent to obtain Dansyl solution.

[0059] (3) Add 5 μL Annexin V-FITC + 5 μL 7AAD, 5 μL Tat-3-Dansyl solution + 5 μL 7AAD, and 5 μL Dansyl solution + 5 μL 7AAD to the resuspension obtained in step (1), respectively, and incubate at room temperature for 15 min. These groups are designated as Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group, respectively. After binding, each group of cells is washed once with 1.5 mL PBS to remove non-specifically bound fluorescent dyes, and then resuspended in 100 μL PBS. Flow cytometry is then used for detection.

[0060] Test results are as follows Figure 3 As shown, Figure 3 Flow cytometry images of Annexin V-FITC, Tat-3-Dansyl, and Dansyl groups after neuronal apoptosis induction. Figure 3 It can be observed that both Tat-3-Dansyl and Annexin V-FITC can detect neuronal apoptosis, 7AAD can detect dead cells of progenitor neurons, and the detection results are similar, while dansyl chloride alone cannot detect neuronal apoptosis.

[0061] Example 4

[0062] This embodiment is used to investigate Ca. 2+ The impact on the detection results of apoptosis in primary neurons.

[0063] (1) Following the steps (1) described in Example 3, primary neurons were subjected to oxygen-glucose deprivation (OGD) experiments, washed with PBS, and then resuspended in 100 μL PBS. One group was given 10 μL of 10x binding buffer, while the other group was not given 10 μL of 10x binding buffer.

[0064] (2) Solution preparation: 5 mg Tat-3-Dansyl was placed in 2 mL PBS solvent to obtain Tat-3-Dansyl solution; 100 μg Dansyl was placed in 100 μL acetonitrile and 0.9 mL PBS solvent to obtain Dansyl solution.

[0065] (3) Add 5 μL Annexin V-FITC + 5 μL 7AAD, 5 μL Tat-3-Dansyl solution + 5 μL 7AAD, and 5 μL Dansyl solution + 5 μL 7AAD to the two resuspensions obtained in step (1), respectively, and incubate at room temperature for 15 min. Record the results as Ca 2+ The Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group exist, as well as Ca 2+ The Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group were not present. After binding, each group of cells was washed once with 1.5 mL PBS to remove non-specifically bound fluorescent dye, and then resuspended in 100 μL PBS before flow cytometry analysis.

[0066] Test results as follows Figure 4-6 As shown. Among them, Figure 4 For Ca 2+ Flow cytometry plots of Annexin V-FITC group, Tat-3-Dansyl group and Dansyl group are available. Figure 5 For Ca 2+ Flow cytometry plots for the Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group were not available. Figure 6 For Ca 2+ Imaging flow cytometry of the Annexin V-FITC group, Tat-3-Dansyl group, and Dansyl group with and without presence.

[0067] Depend on Figure 4-6 It can be found that Annexin V-FITC's effect on cell apoptosis detection depends on Ca. 2+ The presence of Tat-3-Dansyl, and its non-Ca2+-dependent effects on cell apoptosis detection results. 2+ Dependency.

[0068] In summary, the fluorescently labeled peptides provided by this invention showed similar detection results to those of commercially available Annexin V-FITC apoptosis detection in the detection of renal cell carcinoma cells, colon cancer cells, and primary neurons. Furthermore, the apoptosis detection results of the fluorescently labeled peptides provided by this invention exhibited non-Ca2+ characteristics. 2+ This overcomes the dependence of Annexin V-FITC apoptosis detection on Ca2+. 2+ The testing requirements.

[0069] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0070] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fluorescently labeled polypeptide for detecting apoptosis, characterized in that, The amino acid sequence of the fluorescently labeled polypeptide is shown below: YGRKKRRQRRRYYAMKGAGTD.

2. The fluorescently labeled polypeptide according to claim 1, characterized in that, The fluorescein in the fluorescently labeled polypeptide is bonded to the tyrosine residue at the end of the polypeptide.

3. The fluorescently labeled polypeptide according to claim 2, characterized in that, The fluorescein includes dansyl chloride.

4. The fluorescently labeled polypeptide according to claim 3, characterized in that, The molecular formula of the fluorescently labeled polypeptide is: 。 5. The method for preparing the fluorescently labeled polypeptide according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) Fmoc-Asp(otbu)-Wang resin was placed in a sand core reaction column for swelling and deprotection reaction; (2) According to the amino acid sequence from the C-terminus to the N-terminus of the peptide chain, the corresponding amino acids with protecting groups and condensing agents are added to the reaction column, and a coupling reaction is carried out in the presence of an activator. Then, a deprotection reaction is carried out. The coupling reaction and deprotection reaction are repeated until the peptide chain is synthesized. (3) After the peptide chain is synthesized, fluorescein is coupled to tyrosine residues to obtain the fluorescently labeled polypeptide.

6. A cell apoptosis detection kit, characterized in that, The detection kit includes the fluorescently labeled polypeptide according to any one of claims 1-4.

7. The detection kit according to claim 6, characterized in that, The test kit also includes PBS solvent.

8. A method for detecting apoptosis for non-diagnostic or therapeutic purposes using the fluorescently labeled polypeptide according to any one of claims 1-4 or the detection kit according to claim 6 or 7, characterized in that, The method includes: recognizing apoptotic cells by specifically binding the fluorescently labeled peptide to phosphatidylserine, and then detecting the occurrence of apoptosis using flow cytometry or fluorescence microscopy.

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