A kit and its products for apoptosis detection

Abstract

The present invention relates to the technical field of cell apoptosis detection, and particularly relates to a kit and its products for cell apoptosis detection. The present invention provides that Annexin V-FITC can well achieve the detection of cell apoptosis, has an obvious linear correlation with the cell apoptosis rate, and can achieve highly sensitive detection of the occurrence of cell apoptosis. The method and kit for detecting cell apoptosis constructed by the present invention can effectively identify phosphatidylserine exposed on the outer surface of apoptotic cells, and have more detection advantages compared with the full length of Annexin V. The detection method of the present invention has a good linear relationship between sensitivity and cell apoptosis rate, and has great application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of apoptosis detection, and particularly to a kit and its products for apoptosis detection. Background Art

[0002] Apoptosis, or programmed cell death, is one of the built-in defense mechanisms of cells and plays an important role in the normal development and disease resistance of organisms. Abnormalities in the apoptosis process and pathways can lead to various diseases, including tumors. As an important part of the cell life cycle, a series of biomolecular and cell morphological changes occur during apoptosis.

[0003] The flipping of phosphatidylserine (PS) in the lipid bilayer of the cell membrane from the inner layer to the outer layer, and thus its exposure on the cell surface, is an early event of apoptosis, occurring before the morphological changes of apoptotic cells and also being the initial event of the apoptotic cascade reaction. The externalization of PS, as a marker for apoptotic cells to be recognized by phagocytes, will further cause phenomena such as cytoplasmic shrinkage, chromatin condensation, and nuclear DNA degradation. Therefore, the externalized PS, as a target for apoptosis detection, has high timeliness and can detect cell apoptosis at an early stage. AnnexinV is a member of the intracellular protein annexin family and binds to phosphatidylserine (PS) in a calcium-dependent manner. At the early stage of apoptosis, membrane asymmetry is lost and PS translocates to the cell surface. Fluorescently labeled AnnexinV can specifically bind to it, indicating that the cell is an apoptotic cell.

[0004] However, currently, although apoptosis detection methods based on fluorescently labeled Annexin V have been reported, there are significant problems with their detection sensitivity and detection limit. Most detection effects are average and cannot accurately determine whether cells are apoptotic. Summary of the Invention

[0005] Aiming at the technical problems existing in the prior art, the present invention provides a kit and its products for apoptosis detection. The present invention is based on the active binding domain of Annexin V, involves fluorescently labeled Annexin V, and can thus well achieve the detection of apoptosis, has an obvious linear correlation with the apoptosis rate, can achieve highly sensitive detection of apoptosis occurrence, and has great application prospects.

[0006] Specifically, the present invention first provides a kit for apoptosis detection, and the kit contains a fluorescently labeled active binding domain of Annexin V.

[0007] Preferably, the active binding domain of Annexin V contains the amino acid sequence shown in SEQ ID NO.1;

[0008] Preferably, the Annexin V active binding domain is the amino acid sequence shown in SEQ ID NO.1;

[0009] Furthermore, the present invention also provides a fluorescently labeled Annexin V active binding domain, and the Annexin V active binding domain is labeled with a fluorescent group.

[0010] Preferably, the fluorescent label provided by the present invention is a fluorescent group label such as FITC, FAM, Rhodamine B, TAMRA, Cy3, Cy5, etc.

[0011] Preferably, the fluorescent label provided by the present invention is FITC.

[0012] Furthermore, the present invention also provides an article for detecting apoptosis, and the article is the fluorescently labeled Annexin V active binding domain.

[0013] Furthermore, the present invention also provides a method for fluorescent detection of apoptosis for non-diagnostic purposes, and this method includes contacting the cell lysate of the sample to be tested with the kit or the fluorescently labeled Annexin V active binding domain or the article mentioned above. After the incubation ends, observe the result under a fluorescence microscope or read the fluorescence value with an enzyme-linked immunosorbent assay reader.

[0014] Furthermore, the present invention also provides the application of the above-mentioned fluorescently labeled Annexin V active binding domain in the preparation of a kit for detecting apoptosis.

[0015] The advantages of the present invention are as follows: The Annexin V-FITC provided by the present invention can well achieve the detection of apoptosis, has an obvious linear correlation with the apoptosis rate, and can achieve highly sensitive detection of apoptosis. The method and kit for detecting apoptosis constructed by the present invention can effectively recognize phosphatidylserine exposed on the outer surface of apoptotic cells, and have more detection advantages compared with the full length of Annexin V. The sensitivity of the detection method of the present invention has a good linear relationship with the apoptosis rate and has great application prospects. Specific embodiments

[0016] The following combines specific embodiments to further elaborate on the present invention in detail, so that those skilled in the art can understand the present invention more clearly.

[0017] The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection scope of the present invention.

[0018] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well-known to those skilled in the art.

[0019] Example 1 Preparation of Annexin V-FITC

[0020] Screening of the active binding site of Annexin V: Taking Annexin V (GenPept: AAB60648.1) included in NCBI as the target, analyzing its active binding domain for binding to phosphatidylserine (PS), and comprehensively considering aspects such as the hydrophilicity, charge, polarity, and length of the active binding domain. Finally, the active binding domain sequence was selected as vvgdtsgyyq rmlvvllqan (SEQ ID NO.1).

[0021] Synthetically encode the sequence of SEQ ID NO.1 and use it to construct a prokaryotic expression plasmid pET-N-GST-PreScission (Beijing BioLabo Technology Co., Ltd., product number YT971) to express the Annexin V recombinant protein with a GST tag at the N-terminus in Escherichia coli DH5α. The recombinant protein was purified by glutathione agarose gel affinity chromatography, and then the GST tag was removed by enzymatic cleavage with PreScission, and the Annexin V short peptide was collected. After detection by SDS-PAGE electrophoresis and identification by Western blot, it was confirmed that the target protein Annexin V short peptide was correctly expressed. The protein concentration was adjusted to 15 mg / mL with PBS solution for standby.

[0022] Take 100 μg / mL Annexin V solution, dialyze it overnight in 0.5 mol / L carbonate buffer with pH 9.5, and add DMSO containing FITC according to the mass ratio of FITC to antibody of 10:1; place the antibody solution on a magnetic stirrer and gently stir it so that no foam is generated. Add the FITC-DMSO solution dropwise to the monoclonal antibody solution according to the above ratio, and stir it at 4°C in the dark for 12 h with a magnetic stirrer; after adding, measure the pH value of the stirred solution with test paper at any time. If it is lower than 9.0, it should be adjusted with sodium carbonate solution. Stir at 4°C for 12 h. Filter Annexin V-FITC with a Sephadex G25 column to remove free fluorescein.

[0023] Use an ultraviolet spectrophotometer to measure the OD values of Annexin V-FITC at wavelengths of 280 nm and 495 nm. According to the formula F / P = (2.87 × OD 495 ) / (OD 280 - 0.35 × OD495 1) Calculate the binding rate of the fluorescent dye to Annexin V to evaluate the labeling effect of Annexin V-FITC.

[0024] The results showed that the optical density value of Annexin V-FITC measured by ultraviolet spectrophotometer at a wavelength of 280 nm was 0.856, and the optical density value at a wavelength of 495 nm was 0.469. According to the formula, the F / P value of the fluorescent antibody was calculated to be 1.946. It indicated that there was no over-labeling phenomenon of Annexin V-FITC in this experiment.

[0025] Example 2 Cell apoptosis detection based on Annexin V-FITC

[0026] Construction of in vitro cell apoptosis model: Taking L-02 cells as an example, 1×10 5 cells were inoculated into RPMI-1640 medium containing 10% FBS. After overnight culture for 24 h, the supernatant was aspirated, the culture medium was replaced and 0.6 mM H2O2 was added. After acting for 1 h, the culture supernatant in the 24-well plate was collected to detect the AST activity and MDA content, and at the same time, the cell apoptosis in the culture plate was observed under a microscope. The results showed that the AST activity and MDA content detected in the culture supernatant of the model group were significantly increased, and the cell apoptosis reached 85.2% under the microscope, confirming the successful construction of the in vitro cell apoptosis model of the present invention.

[0027] Cell apoptosis detection method based on Annexin V-FITC: Lysates of L-02 cells treated with different concentrations of H2O2 (6 mM, 0.6 mM, 0.06 mM, 6 μM, 0.6 μM, 0.06 μM and 0 μM) were collected respectively, and the Annexin V-FITC with a concentration of 50 μg / mL was added, incubated at 37 °C for 60 min, washed 3 times with PBS, and recorded under an enzyme-labeling instrument, with an excitation wavelength of 460 nm and an emission wavelength of 520 nm.

[0028] The control group was based on the full length of Annexin V, and FITC-labeled Annexin V was prepared by the method described in Example 1 of the present invention.

[0029] The results are shown in Table 1 below.

[0030] Table 1: Test results

[0031]

[0032] According to the analysis of fluorescence detection data, it can be seen that the Annexin V-FITC provided by the present invention can well achieve the detection of cell apoptosis, has an obvious linear correlation with the cell apoptosis rate, and can achieve highly sensitive detection of the occurrence of cell apoptosis. In the control group, the detection rate of cell apoptosis is low, and its fluorescence labeling effect is not as good as that of the experimental group. It is further proved that the Annexin V active binding fragment provided by the present invention has more advantages than the full length in detecting cell apoptosis.

[0033] It is necessary to point out here that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not further limitations on the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A kit for detecting cell apoptosis, characterized in that: The kit contains a fluorescently labeled Annexin V active binding domain, and the Annexin V active binding domain has an amino acid sequence as shown in SEQ ID NO.

1.

2. The kit according to claim 1, characterized in that The fluorescent label includes but is not limited to one or more of FITC, FAM, Rhodamine B, TAMRA, Cy3, and Cy5.

3. The kit according to claim 2, characterized in that The fluorescent marker is FITC.

4. A fluorescently labeled Annexin V active binding domain, characterized in that: The Annexin V active binding domain is labeled with a fluorescent group, wherein the Annexin V active binding domain has an amino acid sequence as shown in SEQ ID NO.

1.

5. The fluorescently labeled Annexin V active binding domain according to claim 4, characterized in that The fluorescent label includes but is not limited to one or more of FITC, FAM, Rhodamine B, TAMRA, Cy3, and Cy5.

6. The fluorescently labeled Annexin V active binding domain according to claim 5, characterized in that The fluorescent marker is FITC.

7. A product for detecting cell apoptosis, characterized in that: The product comprises the fluorescently labeled Annexin V active binding domain according to any one of claims 4-6.

8. A non-diagnostic method for detecting apoptosis by fluorescence, characterized in that: The detection method comprises contacting the test kit according to any one of claims 1 to 3 or the fluorescently labeled Annexin V active binding domain according to any one of claims 4 to 6 or the product according to claim 7 with the cell lysate of the sample to be tested, and after the incubation, observing the result under a fluorescence microscope or reading the fluorescence value with an enzyme-labeled instrument.

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